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INVESTIGATION OF INHALED NITRIC OXIDE AND MESENCHYMAL STROMAL CELLS AS NOVEL
THERAPEUTIC STRATEGIES TO IMPROVE CLINICAL OUTCOME IN EXPERIMENTAL SEVERE INFLUENZA
by
Ilyse Darwish
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science University of Toronto
© Copyright by Ilyse Darwish (2012)
ii
Investigation of Inhaled Nitric Oxide and Mesenchymal Stromal Cells as
Novel Therapeutic Strategies to Improve Clinical Outcome in
Experimental Severe influenza
Ilyse Darwish
Masters of Science
Institute of Medical Science University of Toronto
2012
Abstract
Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-
inflammatory cytokine production, results in approximately 250–500 thousand deaths annually
worldwide. Current influenza research is focused on therapeutics to target the influenza virus or
modulate influenza virus-induced inflammation as potential treatment options to improve clinical
outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to
evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the
lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating
deleterious host responses to influenza infection. Here, we report that MSCs and iNO,
administered alone either prophylactically or post-influenza virus infection, fail to modulate host
inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve
survival of infected mice.
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Acknowledgments
I am grateful to my supervisor Dr. W. Conrad Liles for his mentorship, guidance and support, and
to my committee members Dr. Kevin Kain, Dr. Samira Mubareka, and Dr. David Kelvin, for their
guidance and support. I am very thankful to the Kain lab for creating a supportive learning
environment which allowed me to excel throughout my graduate training, with particular thanks to
Dr. Hani Kim, Dr. Laura Erdman, Dr. Constance Finney, Dr. Lena Serghides and Beata Majczrak
for their patient teaching. Thank you to my parents, my sister and my partner for their support
throughout my studies.
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Table of Contents
Page
Chapter 1: Literature Review
1.1 Global burden of influenza 1
1.2 Structure and life cycle of the influenza virion 2
1.3 Uncomplicated and severe influenza 5
1.4 Viral determinants of pathogenesis 6 1.4.1 Hemagglutinin…………………..…………………………………………………..... 6 1.4.2 Polymerase proteins including PB1-F2 peptide……………………………………… 7 1.4.3 Other viral determinants…………………..………………………………………….. 8
1.5 Antiviral Therapy 9
1.5.1. Neuraminidase inhibitors, M2 ion channel inhibitors and other antivirals…….......... 9 1.6 Investigational antiviral therapy – inhaled nitric oxide 13
1.7 Host determinants of pathogenesis
1.7.1 Cytokine dysregulation ………..……………………………………………............... 15
1.8 Immunomodulatory therapy 19 1.8.1 Glucocorticoids…………………..……………………………………………........... 21 1.8.2 Macrolides…………………..……………………………………………………...... 22 1.8.3 TNF inhibitors…………………..…………………………………………………... . 23 1.8.4 Cyclooxygenase-2 inhibitors…………………..…………...……………………….... 24 1.8.5 HMG-CoA reductase inhibitors…………………..………………...………………... 26 1.8.6 High mobility group box 1 antagonists……………………………….…………...…. 27 1.8.7 Peroxisome proliferator activated receptor agonists.………………..……………….. 28 1.8.8 AMP-activated protein kinase agonists……………………………..………………... 30 1.9 Investigational immunomodulatory therapy – mesenchymal stromal cells (MSCs) 31 1.9.1 MSCs and the immune system……………………………………………..………... 32 1.9.2 MSC duality – immunostimulant or immunosuppressant? …………………………. 36 1.9.3 Effects of MSCs in pre-clinical models……………...……………………………… 38 1.9.4 MSC derived paracrine mediators…………………………………………………… 39 1.10 Mouse models of influenza 41
1.11 Research aims and objectives 44
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Chapter 2: Inhaled nitric oxide antiviral therapy f or severe influenza
2.1 Introduction………………………………………………………………………………... 46
2.2 Materials and methods………………………………………………………..……………. 48
2.3 Results……………………………………………………………………………………… 50
2.4 Discussion..…………………………………………………………………………………. 55
Chapter 3: Mesenchymal stromal cell immunomodulatory therapy for severe influenza
3.1 Introduction……………………………………………………………………………........ 57
3.2 Materials and methods……………………………………………………………………... 59
3.3 Results……………………………………………………………………………………… 63
3.4 Discussion………………………………………………………………………………...... 81
Chapter 4: Summary and future directions
4.1 Summary ………………...………………………………..…………………………..…… 91
4.2 Future Directions……...………………………………………………………………........ 93
4.3 Final Considerations………………………………………………………………….......... 96
Chapter 5: References 98
vi
List of Tables
Table 1.1. Summary of investigational immunomodulatory agents for treatment 20
of influenza
vii
List of Figures
Chapter 1
Fig. 1.1. Structure and life cycle of influenza A virus 4
Fig. 1.2. Mesenchymal stem cell immune cell targets 34
Chapter 2
Fig. 2.1. Prophylactic iNO therapy increased weight loss and decreased survival 51
of C57Bl/6 mice infected with influenza A/WSN/33 virus
Fig. 2.2. Prophylactic and post-infection intermittent iNO does not alter weight 53
loss kinetics or survival of C57Bl/6 mice infected with influenza A/WSN/33 virus
Fig. 2.3. Intermittent high dose iNO prophylactic therapy failed to decrease viral 54
load of C57Bl/6 mice infected with influenza A/WSN/33 virus
Chapter 3
Fig. 3.1. Male C57BL/6 mice infected with 425 EID50 influenza A/PR/8 virus develop 63
increased morbidity and mortality
Fig. 3.2. Male C57BL/6 mice infected with 425 EID50 influenza A/PR/8 virus develop 65
lung inflammation and acute lung injury
Fig. 3.3. hMSCs fail to improve survival, alter weight loss kinetics or affect lung 67
viral load in experimental severe influenza
Fig. 3.4. hMSCs fail to alter lung inflammation in experimental severe influenza 69
Fig. 3.5. hMSCs fail to alter acute lung injury in experimental severe influenza 70
Fig. 3.6. hMSCs fail to alter weight loss kinetics in experimental sub-lethal 71
influenza
Fig. 3.7. Cytokine stimulated hMSC prophylaxis fails to alter weight loss kinetics 73
or improve survival in experimental severe influenza
Fig. 3.8. hMSC adjunctive therapy may improve survival in experimental influenza 75
A/PR/8 virus infection but not influenza A/Mex/4108 virus infection and hMSC
adjunctive therapy does not alter weight loss kinetics in both models of
experimental severe influenza
Fig. 3.9. hMSCs do not decrease BAL fluid or lung parenchymal inflammation when 77
used as adjunctive therapy in mice infected with influenza A/PR/8 virus
viii
Fig. 3.10. hMSCs fail to decrease acute lung injury when used as adjunctive 78
therapy in mice infected with influenza A/PR/8 virus
Fig. 3.11. hMSCs do not decrease BAL fluid or lung parenchymal inflammation 79
when used in adjunctive to antiviral therapy in mice infected with influenza
A/Mex/4108 virus
Fig. 3.12. hMSCs fail to decrease acute lung injury when used as adjunctive 80
therapy in mice infected with influenza A/Mex/4108 virus
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List of Abbreviations
A/Mex/4108: A/Mexico/4108/09
A/PR/8: A/PuertoRico/8/34
A/WSN/33: A/Wisconsin/33
AICAR: aminoimidazole carboxamide ribonucleotide
ALI: acute lung injury
AMPK: AMP-activated protein kinase
Ang-1: angiopoietin-1
APC: antigen presenting cell
ARDS: acute respiratory distress syndrome
BAL: bronchoalveolar lavage
CFU: colony forming unit
COX: cyclooxygenase
CV-N: cyanovirin-N
DC: dendritic cell
eNOS: endothelial nitric oxide synthase
FDA: Food and Drug Administration
GFP: green fluorescent protein
GvHD: graft-vs-host disease
HA: hemagglutinin
HLA: human leukocyte antigen
HMGB-1: high mobility group box 1
HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A
HPAIV: high pathogenic avian influenza
IDO: indoleamine 2,3-dioxygenase
IFN: interferon
IL: interleukin
ILI: influenza-like-illness
IL-1R: interleukin-1 receptor
iNO: inhaled nitric oxide
x
iNOS: inducible nitric oxide synthase
KGF: keratinocyte growth factor
LPAIV: low pathogenic avian influenza
LPS: lipopolysaccharide
MDCK cell: Madin-Darby canine kidney cell
MHC: major histocompatability complex
M1: matrix 1
M2: matrix 2
M2 macrophage: alternatively activated macrophage
NA: neuraminidase
NAI: neuraminidase inhibitor
NEP: nuclear export protein
NF-κB: nuclear factor kappa B
NK cell: natural killer cell
NO: nitric oxide
NOS: nitric oxide synthase
NP: nucleoprotein
NSAID: non-steroidal anti-inflammatory
NS1: non-structural protein 1
NS2: non-structural protein 2
PA: polymerase acidic protein
PB1: polymerase basic protein 1
PB2: polymerase basic protein 2
PB1-F2: polymerase basic protein 1 – F2
PFU: plaque forming unit
PGE2: prostaglandin E2
PPAR: peroxisome proliferator activated receptor
ROS: reactive oxygen species
SARS: severe acute respiratory syndrome
siRNA: short interfering RNA
SNAP: S-nitroso-N-acetylpenicillamine
sTNFR: soluble TNF receptor
xi
TGF-β: transforming growth factor beta
TNF: tumour necrosis factor
TSG-6: TNF inducible protein 6
VCAM-1: vascular cell adhesion molecule-1
vRNP: viral ribonucleoprotein
WHO: world health organization
1
Chapter 1 : Literature Review
1.1 Global burden of severe influenza
Influenza viruses A, B, and C belong to the Orthomyxoviridae family of single stranded
negative-sense RNA viruses. Influenza B viruses are known to infect only humans and seals,
while influenza A viruses can infect a wide range of avian and mammalian hosts. Influenza B
viruses can cause mild to severe respiratory tract infections and can account for a significant
proportion of laboratory confirmed cases of influenza; however, they do not cause pandemics
[299]. This is likely due to their host range, which limits the generation of new strains by
reassortment. Influenza C viruses are known to infect both humans and pigs and may cause mild
upper respiratory tract illness in humans which can rarely lead to infection of the lower
respiratory tract; nearly all adults have been infected with these viruses [300].
While wild aquatic birds are the primary reservoir for influenza A viruses, periodic reassortment
between avian and mammalian influenza viruses or adaptation to novel host species can give rise
to influenza epidemics or pandemics [1]. During annual influenza epidemics in humans,
approximately 5–15% of the population will be infected with influenza A virus [2]. Of those
infected, an estimated 3–5 million of these cases are classified as severe illness, leading to 250–
500 thousand deaths each year [2]. In the United States alone, seasonal influenza accounts for
more than 226,000 hospital admissions annually [3]. The spectre of an influenza pandemic,
however, poses a drastically worse scenario.
2
Up to 50% of the population can be infected with influenza A virus during a pandemic year [4].
The most devasting pandemic to date, the 1918 Spanish influenza, was estimated to have killed
50 to 100 million people [5], with an estimated case fatality rate of 2–3% [6]. Case fatality rates
for the 1957/1958 Asian flu and the 1968 Hong Kong flu pandemics were estimated to be
approximately 0.2% [6], while the most recent pandemic, the 2009 swine origin influenza virus
(H1N1) pandemic, had a relatively low case fatality rate of 0.026% [7].
Most recently, 602 cases of avian influenza virus (H5N1) have been reported to the World
Health Organization (WHO) between 2003 and April 2012, of which 355 cases resulted in
fatalities [8]. However, the WHO employs stringent criteria to ascertain H5N1 disease
excluding asymptomatic individuals. A recent meta-analysis which calls the WHO H5N1
disease criteria into question reveals that 1–2% of study participants from 20 studies were
seropositive for H5N1 [10]. Therefore, the existence of mild or subclinical H5N1 infection
means the true case fatality rate is likely to be less than 60% as reported by WHO.
Furthermore, individuals with limited access to healthcare may not receive a laboratory-
confirmed diagnosis of H5N1 influenza virus infection [9].
1.2 Structure and life cycle of the influenza virion
The influenza virus genome is comprised of eight single-stranded negative-sense viral RNA
segments which encode the ten following gene products: hemagglutinin (HA), neuraminidase
(NA), matrix 2 (M2), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2),
polymerase acidic protein (PA), nucleoprotein (NP), matrix 1 (Ml), non-structural protein 1
3
(NS1) and non-structural protein 2 (NS2). An alternate reading frame in the PB1 polymerase
gene segment which encodes the protein PB1-F2 may also be generated in select influenza A
viruses [33].
The eight influenza viral segments are encapsulated by a host-derived lipid bilayer envelope in
which viral encoded glycoproteins HA and NA, as well as M2 are embedded [11]. Currently
there are 17 known subtypes of HA and 10 known subtypes of NA, which can be found in a
number of combinations on the influenza A virus surface. Subtypes H1N1, H2N2, and H3N2 are
known to circulate in humans while other subtypes are commonly found in other species
including 9 different subtypes of H5, H7, and H9 found mainly in wild birds and poultry. H7N7
influenza A viruses can infect horses and H3N8 viruses can infect both horses and dogs. Other
subtypes have been isolated from bats, pigs, marine mammals and guinea pigs [301,302].
Each influenza A viral RNA segment is structurally bound to NP to form viral ribonucleoprotein
(vRNPs), which are associated with the 3 viral polymerase proteins that form the polymerase
complex (PB1, PB2, and PA) located at the ends of the nucleocapsids. Finally, M1 forms a shell
surrounding the virion nucleocapsids, underneath the virion envelope [1].
4
Figure 1.1. Structure and life cycle of influenza A virus (© Richard Burke Squires, courtesy of the Biology Image Library, ref. [12].)
The process by which influenza A virus infects host cells begins with the binding of HA to sialic
acid on host epithelial cells. The HA0 precursor protein undergoes post-translational cleavage
into the HA1-HA2 complex, a process critical to influenza virulence [13]. The virus is then
internalized to form a host cell endosome containing each RNA segment. Hydrogen ions in the
endosome are pumped through the opening of a proton pore, the M2 ion channel, which results
in endosomal acidification and subsequent release of vRNPs into the cytoplasm. Via facilitated
translocation with the aid of at least two nuclear localization sequences (NLS) contained by the
NP known as NLS1 and NLS2, the vRNPs are then imported into the nucleus of the host cell
where RNA replication, transcription and assembly of progeny vRNPs occurs [291]. Newly-
made vRNPs assemble in the nucleus and are exported into the cytoplasm. HA, NA and M2
5
migrate via the golgi apparatus and anchor in the plasma membrane forming the new virion
membrane. Viral segments are then arranged so that each of the eight segments is included in a
newly formed virion. The process by which this occurs is not well understood however it is
thought to involve several protein-protein interactions between the cytoplasmic tails of the viral
integral membrane proteins, the matrix protein and the vRNPs [297]. Finally, sialylated progeny
virions gather at the host cell membrane where their sialic acid is cleaved by NA which frees the
newly assembled infectious influenza progeny virions from the host cell [11]. This process of
influenza respiratory tract virus replication peaks within 24–72 hours after illness onset [88].
1.3 Uncomplicated and severe influenza
Most influenza virus infections are uncomplicated and resolve within 7 – 10 days. Symptoms of
acute mild infection are sudden onset of high fever, coryza, cough, headache, malaise and
transient tracheo-bronchitis [4]. Individuals who are more susceptible to developing severe
disease include the elderly, children, pregnant women, immunocompromised individuals and
individuals with known underlying health conditions including asthma, chronic obstructive
airway disease, cardiovascular disease, obesity, and diabetes [4,14]. Clinically, severe influenza
is characterized by dyspnea, cyanosis, bloody (or coloured) sputum, chest pain, persistent fever
(duration > 3 days), altered mental status, and/or hypotension [15]. Severe influenza can lead to
diffuse alveolar damage with histopathologic features of acute lung injury (ALI) and the acute
respiratory distress syndrome (ARDS) [16–20], which directly contributes to influenza-
associated morbidity and mortality. ARDS is characterized by increased permeability of the
microvascular endothelium and disruption of the alveolar-capillary barrier, leading to a
proteinaceous exudate in the alveolar airspaces, accompanied by neutrophil, macrophage, and
6
erythrocyte infiltration [21,22]. Post-mortem examination of the lungs of patients who
succumbed to infection with the 2009 pandemic H1N1 influenza virus revealed diffuse alveolar
damage with or without a hemorrhagic component or necrotizing bronchiolitis; severe
neutrophilic inflammation was noted in a substantial number of cases [23]. Severe infection may
also result in renal dysfunction and multi-organ failure, and secondary bacterial pneumonia may
also cause substantial illness and death, as was observed with the 1918 influenza pandemic.
1.4 Viral determinants of influenza pathogenesis 1.4.1 Hemagglutinin
High and low pathogenic avian influenza viruses (HPAIV and LPAIV, respectively) differ in
their HA protein cleavage sequence. HPAIV possesses a polybasic cleavage site
(QRERRRKKR/G) while LPAIV possesses one to two basic amino acids at this site [24–26].
The multibasic cleavage motif found in HPAIV is recognized by subtilisen-like endoproteases,
such as furin, which are present in all tissues, while the LPAIV lacking this motif is recognized
by trypsin-like enzymes preferentially expressed at the surface of respiratory and gastrointestinal
epithelia [23]. Therefore, LPAIV, although infectious, replicates in a limited number of cell
types and is therefore restricted in its ability to spread in the host, while HPAIV is able to
replicate in all tissues leading to widespread lethal systemic infection [23,26]. Hatta et al. also
found that high cleavability of HA was essential for lethal murine H5N1 infection as conversion
of the multibasic amino acid HA site to the sequence typical of avirulent avian influenza viruses
highly attenuated an H5N1 modified virus [27].
The importance of the avian HA glycoprotein in the induction of pro-inflammatory cytokines has
been demonstrated in vitro [28]. The importance of influenza virus HA in immunomodulation is
7
also illustrated by the 1918 influenza pandemic, in which virus containing the 1918 HA/NA was
associated with enhanced pulmonary infiltrates and production of IFN-γ, TNF, MIP-2, and CCL3
[29].
1.4.2 Polymerase proteins including PB1-F2 peptide
PB1-F2 is a critical influenza virulence determinant for some influenza virus strains in certain
hosts [30]. The PB1-F2 peptide, encoded in the +1 reading frame of the PB1 segment of the
trimeric viral RNA-dependant RNA polymerase complex, is expressed in the highly virulent
1918 pandemic influenza virus, highly virulent murine-adapted influenza A/Puerto Rico/8/34
(A/PR/8; H1N1) virus and HPAIV [31]. It is pro-apoptotic [32] and has been associated with
increased pathogenicity and mortality in a mouse model [33]. PB1-F2 has also been implicated
in copathogenesis with bacterial pathogens such as Streptococcus pneumonia; the C-terminal
portion of the peptide contributed to acute lung injury, even in the absence of viral replication
[34]. The PB1-F2 peptide of the 2009 pandemic H1N1 influenza virus is truncated due to the
presence of several stop codons in the PB1 gene [35,36]. This may partially account for the
relatively low virulence associated with the 2009 pandemic virus. However, 2009 pandemic
H1N1 influenza virus genetically modified to express PB1-F2 had similar virulence compared to
wild-type 2009 pandemic H1N1 influenza virus in mouse and ferret models, despite elevated
levels of IFN-γ, CCL2, CCL4, and CCL5 [36]. Since the initial viral strains were sequenced,
several isolates circulating during the pandemic were found to express a 57 amino acid PB1-F2
due to a stop-to-leucine substitution at position 12. This appeared to increase virus replication in
cell culture but not pathogenicity in a mouse model [37].
8
Expression of PB1-F2 was also shown to confer increased pathogenicity without altering virus
replication [38] when a serine amino acid is present at position 66 [39]. Influenza viruses with
the N66S mutation in PB1-F2 have been shown to cause enhanced production of cytokines and
associated cellular infiltrates [40]. Thus, the presence of PB1-F2 influenza virus expression is an
important contributor to influenza A virulence and is involved in immunopathology. PB2
mutations have also been noted to promote virulence and transmission, including D701N and
E627K [27,41,42]. More recently, the PB2-E158G mutation was associated with increased viral
replication and mortality in 2009 pandemic H1N1 influenza virus-infected mice as well as H5N1
virus-infected chickens [43].
1.4.3 Other viral determinants
Other virulence determinants, including influenza virus NS1 and NA, have been associated with
increased virus replication as well as inflammation [44,45]. NS1 in particular has been well-
studied, and an NS1-truncated live attenuated virus has been proposed as a vaccine candidate
[46–48]. NS1 is a multifunctional protein and several reviews outlining its role in immune
evasion, viral replication, and host cell modulation have been published [49–52]. Interferon
antagonism is a primary function of the NS1 protein, acting at both the pre- and post-
transcriptional levels of type I interferon production [53–55]. NS1 has also been shown to
directly inhibit the actions of antiviral proteins [56,57]. NS1 activity and virulence varies
depending on viral strain. For example, the NS1 in a highly pathogenic H5N1 virus from an
outbreak involving humans was shown to play a central role to the high mortality associated with
this virus [58]. Finally, NS1 has been implicated in host cell signaling, whereby the C-terminal
9
domain of avian influenza viruses contains a PDZ ligand capable of binding host cell proteins
and leading to decreased survival and increased lung damage in a mouse model [59,60].
1.5 Antiviral therapy
1.5.1 Neuraminidase inhibitors, M2 ion channel inhibitors and other antivirals
While influenza vaccines may only provide moderate protection against influenza [292], annual
immunization remains the primary means of protection against influenza. However, in the event
of a pandemic, strain-specific vaccine production can take up to six months. During this time,
entire unvaccinated populations remain susceptible to a novel pandemic strain. In the interim,
antiviral drugs are important for patient management and the abrogation of transmission.
The United States Food and Drug Administration (FDA) has approved two classes of antiviral
drugs for the prevention and treatment of influenza. These include the M2 ion channel
inhibitors, also known as the adamantanes (amantadine and rimantadine) and the neuraminidase
inhibitors (NAIs) (oseltamivir and zanamivir). The first class of drugs, the adamantanes, block
the influx of H+ ions through the M2 ion channel, preventing the acid-triggered fusion reaction
and thus interfering with viral endosomal release inside the cell [61]. The second class of drugs,
the NAIs, competitively bind to the highly conserved enzyme NA active site by mimicking sialic
acid, the natural substrate of NA. This inhibits the enzyme’s key function by destroying
neuraminic acid-containing receptors, which prevents newly assembled virion cleavage and
release of progeny virions from infected cells [61].
10
Because the licensure of NAIs was obtained based on studies in healthy adults with
uncomplicated seasonal influenza, it is not clear that these drugs are effective for treatment of
severe disease. A major Cochrane review which amalgamated two large reviews of oseltamivir
use for influenza in healthy adults and children, respectively, found that oseltamivir shortens the
duration of symptoms by less than a day in people with influenza-like illness (ILI) but there is no
evidence of an effect on hospitalizations [62]. It is uncertain whether oseltamivir has an effect on
complications. However, treatment with oseltamivir has been associated with a reduction in
mortality in a number of studies [91,294,295].
Because influenza A virus possesses an error-prone RNA polymerase, a high incidence of
mutation occurs and variants resistant to antiviral drugs may be selected; permissive mutations
restoring viral fitness ensure ongoing transmission and infection [65,66]. With the development
of viral resistance, the utility of adamantanes has been all but eliminated. Since 2005, nearly all
influenza A (H3N2) viruses isolated globally were resistant to adamantanes [67,68]. All 2009
pandemic influenza A (H1N1) viruses isolated globally from September 2010 to January 2011
were also resistant to adamantanes [68]. As the continued use of adamantanes further exerts
selective pressure on the development of drug resistance, the use of these drugs as mono-therapy
should be eliminated from current clinical practice.
Unlike adamantanes, the frequency of resistance to NAIs has been minimal, until recently. Since
2007, a significant increase in seasonal influenza A (H1N1) virus mutations conferring resistance
to oseltamivir have been observed [69]. The majority of these mutations have been characterized
by a histidine to tyrosine mutation at residue 274 of the NA; the glutamine at position 276 is
pushed away by the bulkier tyrosine substitute, thus disrupting the hydrophobic pocket which
11
otherwise accommodates the pentyloxy portion of the oseltamivir molecule [69,70]. During the
2008–2009 influenza season, nearly 100% of tested seasonal influenza A/H1N1 virus strains in
the United States and in over a dozen other countries were found to be oseltamivir-resistant
[71,72]. As of October 2010, 313 cases of oseltamivir-resistant 2009 pandemic H1N1 influenza
virus have been identified, and these cases were mostly linked to prophylactic or therapeutic use
of oseltamivir [68,73]. Oseltamivir resistance has also been found in H5N1 virus-infected and
oseltamivir-treated patients [74,75]. However, the majority of 2009 pandemic H1N1 influenza
viruses tested from September 2010 to January 2011 remained susceptible to oseltamivir [68].
Oseltamivir is restricted to oral administration, though a phase I trial is planned by Hoffmann La
Roche for an intravenous (IV) formulation [76]. Clinical trials have also demonstrated the safety
of peramivir, an NAI developed for IV administration. Japan has recently licensed peramivir
under the name Rapiacta [77,78].
Another licensed NAI, zanamivir, has a low incidence of resistance and is currently an
alternative treatment strategy for oseltamivir-resistant strains [79]. Zanamivir’s low resistant rate
may be attributable to a low prescription rate as well as differences in the way it binds to NA
[61]. However, zanamivir is delivered to the lungs by a dry powder inhaler which may pose
difficulties for patients on mechanical ventilation [80], individuals with severe cough or dyspnea,
small children and the elderly [81]. There is also concern of sub-optimal delivery to sites of
infection in influenza virus-infected patients with pneumonia or pulmonary disease [16,80,82].
Therefore, H5N1 virus-infected patients who are viremic may not respond to NAIs as the
antiviral is unlikely to reach the necessary end-target sites, as has been observed where patients
12
shed virus for many days despite combined NAI treatment [293]. Nebulization of zanamivir is
not recommended due to the formulation’s potential to cause mechanical ventilator obstruction,
which may result in death [83]. Zanamivir is currently undergoing phase II trials for parenteral
I.V. use [84,85]. CS-8958 (laninamivir prodrug), an NAI similar to zanamivir but with a
prolonged half-life, is also being studied for use by inhalation and is currently commercially
available as Inavir in Japan [86,87].
In addition to the development of influenza virus drug resistance, another important caveat to
consider with respect to antivirals includes limitations in the timing of administration [88–92].
Initiation of oseltamivir treatment within the first 48–72 hours after symptom onset is associated
with lower mortality and greater antiviral efficacy compared with treatment initiated beyond the
first 48 hours of symptom onset for H1N1, H3N2 and H5N1 infected patients [89,90]. This may
be attributed to the peak in influenza respiratory tract viral replication at 24–72 hours after illness
onset [88]. Furthermore, influenza patients in the intensive care unit or those who died from
severe influenza were less likely to have received antiviral treatment within 48 hours after the
onset of symptoms [91]. Late antiviral treatment may also be unable to prevent the development
of ARDS; 2009 pandemic H1N1 influenza virus-infected patients with ARDS who died began
oseltamivir treatment an average of 5 days after the onset of symptoms compared to infected
patients who survived without ARDS and those with mild-disease, where treatment was initiated
an average of 4 and 2 days post symptom onset, respectively [92]. However, late antiviral
treatment is still associated with a significant reduction in mortality compared to no treatment
[294].
13
Other caveats include limited efficacy in cases of H5N1 virus infection [75] and limited access to
stockpiles during a pandemic [93,94]. More effective, affordable and accessible drugs to treat
severe influenza are urgently required. Other antiviral agents which may target different stages of
the viral life cycle are currently being investigated, including the polymerase inhibitor favipiravir
(T-705), HA inhibitor prokaryotic cyanovirin-N (CV-N), short interfering RNA (siRNA), and a
sialic acid receptor inhibitor, fludase (DAS-181) [95].
1.6 Investigational antiviral therapy - inhaled nitric oxide
Nitric oxide (NO) is an important cellular signaling molecule synthesized from L-arginine by
NO synthase (NOS). In the airways, NOS is present in a variety of cells, including macrophages,
vascular endothelial cells, airway epithelial cells and neurons, where NOS activity is known to
mediate neurotransmission, smooth muscle contraction and mucin secretions. There are three
types of NOS: constituent and calcium-dependant isoforms (both principally present in
endothelial cells and neuronal cells), and the inducible or calcium-independent isoform (iNOS)
[96]. NO is also a well known biological mediator in the host response to infection [96,97].
Various inflammatory stimuli such as lipopolysaccharide (LPS) and cytokines including IFN-γ
and TNF can cause high and sustained NO production by iNOS. Depending on the species,
strain, infection dose and pathogen entry route, iNOS activity can result in pro- or anti-
inflammatory responses, cytotoxicity, or cytoprotection [reviewed in 96].
In vitro, NO antimicrobial activity has been demonstrated against a variety of viruses including
ectromilia virus, vaccinia virus, herpes simplex type 1 viruses, coronavirus, and influenza A and
B viruses [98–102]. In these studies, administration of the NO donor S-nitroso-N-
14
acetylpenicillamine (SNAP) to virus-infected cells significantly reduced viral burden. Lin et al.
reported that SNAP did not have any direct anti-viral capability since pre-treatment of viral
stocks with SNAP for 1 hour failed to affect virus infectivity, but rather biochemical analysis
revealed that NO exerts its antiviral effects by profound inhibition of viral RNA synthesis, viral
protein accumulation, and viral release from infected cells [103].
Severe cases of influenza are often associated with multisystem organ failure and hypoxemic
respiratory failure, including ALI/ARDS requiring ventilatory support [104,105]. Affected
individuals may receive ‘rescue’ therapies, including inhaled nitric oxide (iNO), in an attempt to
improve outcome [105]. However, iNO administration for ARDS secondary to viral pneumonia
has not been specifically reported to improve clinical outcome [104,105].
iNO therapy is currently FDA approved for the treatment of term and near-term neonates with
hypoxemic respiratory failure associated with clinical or echocardiographic evidence of
pulmonary arterial hypertension [106,107]. Variable findings have been reported for iNO
efficacy when administered at 1 ppm and up to 80 ppm. For its indicated use, iNO has been
found to increase vasodilation, transiently improve arterial oxygenation, reduce length of
mechanical ventilation, reduce oxygen requirement, and decrease length of stay in the intensive
care unit [107–109]. One study found iNO, at 30 ppm or less, decreased the spread and intensity
of lung infiltrates and improved arterial oxygen saturation in patients with severe acute
respiratory syndrome (SARS) [110]. However, systematic reviews and meta-analysis of
randomized controlled trials have shown that iNO, when used therapeutically in the management
of ARDS, does not reduce mortality [111–115]. Moreover, iNO therapy for ARDS may increase
15
the risk of iNO treated patients developing renal dysfunction [114,115]. Despite this, 39% of
critical care specialists surveyed reported using iNO for the management of patients with ARDS
in Ontario, Canada [116].
1.7 Host determinants of pathogenesis
1.7.1 Cytokine dysregulation
Various host factors can influence susceptibility to influenza and progression to severe disease
including immune status, underlying health conditions and age. Recently, it has become clear
that one of the most important factors contributing to influenza-related morbidity and mortality is
inflammation. Severe influenza can be characterized as a disease which induces hyper-
production of inflammatory cytokines during host inflammation. In physiologic conditions, anti-
inflammatory cytokines are immunoregulatory molecules that control pro-inflammatory
responses. In pathological conditions, insufficient control of anti-inflammatory responses over
pro-inflammatory responses may occur and the net response of this imbalance determines
individual patient outcome. Specifically, the extent of the host inflammatory response triggered
by various influenza virus strains is positively correlated with clinical severity of influenza
including HPAIV infection and infection with reconstructed 1918 pandemic H1N1 influenza
virus in animal models. Importantly, the degree or magnitude of dysregulated cytokine
production fails to correlate with viral titers in both animal models [117–123] and in humans
[123,124].
16
Cytokine dysregulation has repeatedly been observed in human influenza virus infection from
the earliest H5N1 outbreaks in 1997 onward [13,123,124]. H5N1-induced hypercytokinemia has
also been observed in mouse models [120] and in cell culture [125]. In these H5N1 studies,
levels of cytokines, including CXCL10, CCL5, CXCL9, CCL2, IL-8, IL-10, IL-6, IFN-γ, TNF,
CCL3 and soluble IL-2, were elevated compared to other influenza virus strains.
Influenza caused by seasonal H1N1 and H3N2 virus strains and the recent 2009 pandemic H1N1
influenza virus have not been associated with cytokine dysregulation, which may partially
account for the low case fatality rate of 2009 pandemic H1N1 influenza virus [7,126–128]. In
vitro experiments with seasonal H3N2 influenza virus and other non-pandemic H1N1 influenza
viruses showed no evidence of hypercytokinemia [126,127]. In vitro [128] and ex vivo [127]
experiments did not reveal cytokine dysregulation induced by 2009 pandemic H1N1 influenza
virus infection. However, To et al. found higher levels of pro-inflammatory cytokines in patients
with 2009 pandemic H1N1 influenza virus infection and ARDS [92]. This often resulted in
death compared to patients who survived without ARDS or those who suffered mild forms of the
disease, independent of viral load amongst the three groups. Taken together, these studies
suggest that 2009 pandemic H1N1 influenza virus may be associated with cytokine dysregulation
in the setting of ARDS.
The significance of each elevated cytokine in influenza virus-induced cytokine dysregulation is
not well defined. For example, Peiris et al. found significantly elevated levels of CXCL10 and
CXCL9 but no significant differences in levels of CCL2, CCL5 and IL-8 in patients with H5N1
virus infection compared to patients infected with other influenza viruses [123], while Perrone et
17
al. did not observe significantly elevated levels of CXCL10 or CXCL9, but rather significantly
elevated levels of CCL3, KC (mouse IL-8), IL-1α, IFN-γ, and IL-6, in HPAIV infected mice
[120].
In order to elucidate which cytokines are most important in mediating lung pathology,
investigators have taken advantage of mice with targeted deletions of specific cytokine or
cytokine receptor genes (i.e., cytokine ‘knock-out’ mice). Research involving cytokine knock-
out mice has revealed protective or deleterious roles of certain cytokines in the host response to
influenza, which seems to vary depending on the specific strain or subtype of influenza virus
studied. CCR5-/- mice (receptor for CCL3, CCL4 and CCL5) developed massive inflammation
and severe pulmonary damage associated with decreased survival when infected with influenza
A/PR/8 virus [117]. Similarly, mortality was significantly increased or accelerated in IL-1R1-/-
mice (receptor for IL-1α and IL-1β) compared to wild-type mice when infected with influenza
A/PR/8 virus or reconstructed 1918 virus, respectively [129,130]. This suggests signaling
through IL-1R1 is protective and these observations indicate that a proper balance in immune-
mediated inflammation is necessary to achieve eradication of influenza virus while avoiding
deleterious host-mediated inflammatory tissue injury. Other research has also found similar
morbidity and mortality in CCL3-/-, IL-1R-/-, IL-6-/-, TNFR1-/-, IL-6-/-, TNF-/- or CCL2-/- mice
inoculated with avian influenza virus H5N1 compared to wild-type mice [131,132].
To the contrary, other studies have shown improved outcome in cytokine or cytokine receptor
knock-out mice. CCR2-/- mice infected with influenza A/PR/8 virus had reduced lung injury and
inflammation and mortality compared to wild-type controls [117]. The chemokine receptor
18
CCR2 binds its ligand CCL2 to cause the migration of monocytes and the development of
monocyte-derived inflammatory cells in the lungs which has been associated with influenza virus
induced lung injury and subsequent mortality [117,122,133]. This finding is in contrast to that
observed with CCL2-/- mice reported by Salomon et al. [132]. A further study by Lin et al.
showed reduced lung pathology and an 89% survival increase of CCR2-/- mice compared to
influenza virus-infected wild-type controls [133]. However, Aldridge et al. showed CCR2-/- mice
infected with A/PR/8 had comparable morbidity and mortality to infected wild-type controls
[134]. Subsequent studies using a CCR2 inhibitor, PF-04178903, administered prophylactically
and twice daily post-infection resulted in 100% survival of influenza A/PR/8 virus-infected mice
compared to 25% survival and decreased pulmonary immune pathology in untreated mice [122].
Therefore, CCR2 inhibition may be effective in reducing influenza virus-induced lung pathology
and overall mortality.
It is important to bear in mind that conclusions based on experiments employing mice with
specific targeted genetic deletions (‘knock-out’) may be misleading as other downstream factors
may be modified and ‘hidden from view’. Functional redundancy amongst signaling pathways
may result in host compensation which could mask the immunomodulatory benefits of single
cytokine depletion. To account for such host responses, Perrone et al. examined TNF-R1, TNF-
R2, and IL-1R1 triple knock-out mice [121]. When infected with a lethal dose of H5N1, these
mice exhibited decreased inflammatory cell infiltrates and cytokine levels, reduced morbidity,
and a significant delay in mortality compared to wild-type controls [121]. Taken together, this
research suggests that reducing overall inflammation rather than targeting individual cytokines
may prove to be a superior strategy for decreasing influenza-related morbidity and mortality.
19
1.8 Immunomodulatory therapy
The outcome of influenza virus infection is determined by both viral and host factors; therefore,
it would be logical to investigate the efficacy of therapeutic strategies which target the host in
combination with antiviral therapy. Such therapeutic strategies could potentially target either
signaling pathways to reduce viral replication or host-mediated inflammation in an effort to
dampen deleterious tissue/organ dysfunction and injury. Importantly, several experimental
studies in mice show that mortality is reduced by immunomodulatory agents without
concomitant antiviral treatment and without any decrease in virus replication, implying
modulation of host inflammation alone may be sufficient to improve influenza virus-infected
patient outcome or provide additional benefit when used in combination with antiviral agents.
A number of FDA-approved drugs have received investigational interest as immunomodulatory
strategies for the treatment of influenza (described in sections 1.8.1-1.8.8). All of these drugs,
with the exception of CCR-2 antagonist PF-04178903, are produced as generic agents and, thus,
would be relatively inexpensive and available for deployment in the event of a pandemic. In
addition, these agents could potentially circumvent waning efficacy in the face of increasing
antiviral resistance. Chapter 1.8 will focus on immunomodulatory agents for the treatment of
severe influenza which target dysregulated host inflammatory responses (summarized in Table
1.1).
20
Table 1.1. Summary of investigational immunomodulatory agents for treatment of influenza (reprinted with permission from © Expert Reviews Ltd, ref. [135].)
21
1.8.1 Glucocorticoids
Glucocorticoids are a group of corticosteroids which exert anti-inflammatory activity via binding
to cytoplasmic receptors that subsequently interact with glucocorticoid response elements to
regulate transcription of a number of genes [136]. Clinical trials using prolonged low-to-
moderate dose glucocorticoid therapy for ARDS, a prominent feature of severe influenza, have
demonstrated that glucocorticoid therapy enhances the anti-inflammatory activity of the
glucocorticoid-activated glucocorticoid receptor; a major regulator of inflammation in ARDS
[reviewed in 137]. Consistent with these clinical observations, glucocorticoids suppress cytokine
levels [138] and IFN-γ induced pro-inflammatory gene expression including cyclooxygenase-2
(COX-2) expression in human bronchial epithelial cells in vitro [136].
Evidence to date supporting the use of glucocorticoids for the treatment of severe influenza is
inconclusive. In a rat model of experimental H3N2 influenza, daily doses of the glucocorticoid
triamcinolone acetonide combined with an NAI suppressed IFN-γ induction, resulting in
decreased macrophage activation and pulmonary inflammation [139]. However, survival
benefits were not reported in this study, and a study of experimental H5N1 influenza in mice
found no difference in mortality between daily corticosterone-treated mice and controls [132]. In
humans, glucocorticoid therapy has yielded equivocal H5N1 infection with uncertain effects. In a
randomized trial in Vietnam, all 4 H5N1 virus-infected patients who received glucocorticoids
died [15]. Moreover, in a clinical series reported from Thailand, only 2 of 8 H5N1-infected
patients treated with methylprednisolone survived [140]. In contrast, a recent study, while
lacking an ‘influenza infected with ARDS’ control group, reported that glucocorticoid treatment
improved ALI in individuals with ARDS secondary to severe 2009 pandemic H1N1 influenza
22
[141]. In view of these mixed results, the use of glucocorticoids as adjunctive therapy to
antiviral drugs for severe influenza may warrant further study in the form of a randomized
clinical trial, but there is currently insufficient evidence to endorse its routine use.
1.8.2 Macrolides
Macrolides, such as erythromycin, azithromycin, and clarithromycin, are bacteriostatic protein
synthesis inhibitors that exert broad immunomodulatory activities including downregulation and
upregulation of pro- and anti-inflammatory cytokines, respectively, modulation of leukocyte
recruitment, and improvement of macrophage phagocytic function, but without causing global
immunosuppression [142–144]. In vitro, clarithromycin reduces cytokine content in culture
supernatant of seasonal H3N2 influenza virus-infected tracheal epithelial cells [145].
Erythromycin treatment of H2N2 influenza virus-infected mice increased survival from 14% to
57%, and reduced inflammatory cell counts [146].
Secondary bacterial pneumonia is frequently observed during influenza pandemics, resulting in
increased influenza viral titers and lung inflammation [147,148]. As non-lytic antibiotics,
macrolides may be important in clearing influenza bacterial co-infection in addition to
decreasing host inflammation. Ampicillin, a β-lactam antibiotic targeting the bacterial cell wall
has been shown to clear bacterial infection from influenza virus-infected lungs, however
ampicillin use did not alter mortality rate in a mouse model of severe pneumococcal pneumonia
with bacteremia following influenza [149]. This prompted investigation into the use of
azithromycin, which significantly improved outcome of a milder bacteria-influenza co-infection
compared to ampicillin by dampening inflammatory responses caused by ampicillin-induced
23
bacterial lysis, due to their non-lytic antimicrobial activity [150]. Therefore, macrolides may be
advantageous when used in secondary bacterial pneumonia following influenza, and clinical
trials are required to demonstrate a benefit from macrolide use in severe influenza. However, a
note of caution is warranted because of the risk that widespread routine use of antibiotics for
treatment of individuals with influenza may promote the development of problematic
antimicrobial resistance in the long-term [151]. Also, safety concerns including increased risk of
cardiovascular death have been associated with macrolides [296].
1.8.3 TNF inhibitors
Researchers have explored the use of therapeutic agents to lower host inflammation by directly
targeting specific cytokines. TNF-neutralizing monoclonal antibodies and soluble TNF-receptor
fusion proteins are important in managing inflammation caused by immune-mediated disorders,
such as inflammatory bowel disease and rheumatoid arthritis [152,153] and increasing survival
of LPS injected mice [154]. In contrast, anti-TNF strategies have not been beneficial in the
treatment of other inflammatory conditions such as sepsis [155].
Experimental use of anti-TNF therapy in mouse models of influenza has shown inconsistent
results. Few studies have reported that TNF neutralizing strategies for severe influenza in mouse
models reduce lung lesion severity, pulmonary inflammatory cell infiltrates, and T-cell cytokine
production, however with no survival benefit reported [131,132,156,157]. TNFR-1-/- mice
survived longer than wild-type mice when infected with reconstructed 1918 influenza virus,
suggesting that anti-TNF therapy might provide some therapeutic benefit in severe influenza.
However, it should be noted that all mice died in both groups [130].
24
Anti-TNF therapy for severe influenza in humans has not been studied in a controlled clinical
trial, and its failure for the treatment of sepsis casts doubt on its potential benefit in severe
clinical influenza. Additionally, increased risk of bacterial infection and infections with rare
intracellular pathogens are well-described complications of anti-TNF therapy [158]. Thus,
overall experimental evidence to support the clinical use of anti-TNF therapy for severe
influenza is currently lacking, especially when considering the attendant risks.
1.8.4 Cyclooxygenase-2 inhibitors
Cyclooxygenase (COX) enzymes, which catalyze the conversion of arachidonic acid into
prostaglandins, play important roles in modulating immune responses and inflammation
[118,159,160]. Widely used clinically for their analgesic, anti-inflammatory and antipyretic
properties, the COX inhibitors consist of the well known non-selective non-steroidal anti-
inflammatory drugs (NSAIDs), such as ibuprofen, which inhibit both COX-1 and COX-2, and
the selective COX-2 inhibitors, such as celecoxib, which target COX-2 specifically [118].
In vitro but not necessarily in vivo data support the use of COX-2 inhibitors for severe influenza.
Pronounced COX-2 upregulation has been reported following in vitro infection of macrophages
with H5N1 virus and in lung tissue at autopsy from patients who died of H5N1 virus-associated
disease [161]. In vitro, COX-2 inhibitors, including celecoxib, have been shown to suppress
influenza virus-induced inflammatory cytokine production [128,161]. In mouse models of H3N2
influenza, inflammation was reduced and survival increased in COX-2-/- compared to wild-type
mice [118], suggesting a benefit from COX-2 deficiency. However, experiments in knock-out
mice may yield misleading results secondary to effects on unidentified downstream pathways. In
25
murine models of influenza, the use of the pharmacological COX-2 inhibitor celecoxib did not
confer any survival advantage [162,163]. These studies suggest that COX-2 inhibitors alone
may not be effective agents for severe influenza therapy; however, retrospective studies to
determine whether COX-2 inhibitor use was associated with improved clinical outcome may still
be warranted.
Research examining the use of COX-2 inhibitors in combination with anti-viral drugs has shown
promising results. Combination therapy including zanamivir, celecoxib and the anti-
inflammatory drug mesalazine significantly improved survival of mice infected with H5N1
influenza virus compared to treatment with zanamivir alone [162]. Importantly, the triple
combination therapy reduced levels of IL-6, IFN-γ, TNF and MIP-1 in infected mice compared
to zanamivir treatment alone. Single daily use of either immunomodulator or the mesalazine and
celecoxib combination failed to confer any survival advantage. It should also be noted that
combination therapy of mesalazine and celecoxib was not investigated further in this study. The
experimental data suggests that COX-2 inhibitors may provide additional benefit when used
adjunctively to existing antiviral strategies. Further research is necessary to strengthen the
evidence in support of combined celecoxib, mesalazine, and antiviral therapy use for severe
influenza.
Animal models of ALI have shown COX-2-derived mediators to exert a protective role against
inflammation-induced lung injury, in part via enhanced lipoxin signaling [164]. Lipoxins are a
unique class of arachidonic acid-derived lipid pro-inflammatory mediators of inflammation
which display in vivo inhibition of polymorphonuclear cell activation, cytokine release and
26
angiogenesis [reviewed in 165]. Inhibition of lipoxin signaling has been associated with
enhanced lethality of H5N1 virus infection in mice [166]. This finding, when considering the
unconvincing in vivo evidence to support the use of COX-2 inhibitors as monotherapy, provides
indication of the potential use for lipoxin agonists to reduce inflammation in severe influenza and
improve patient outcome.
1.8.5 HMG-CoA reductase inhibitors
A group of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors,
commonly known as statins [167], were proposed as a potential therapeutic strategy to reduce
influenza virus-induced inflammation in 2005 [93]. Statins have been shown to modulate host
immunity by inhibiting activation of immune effector cells, such as macrophages, and antigen
presentation by macrophages to T cells, as well as affecting endothelial function through the
inhibition of reactive oxygen species (ROS) production and upregulation of endothelial nitric
oxide synthase (eNOS) [168, reviewed in 169]. Statins also antagonize high mobility group box
1 protein (HMGB-1), a potent pro-inflammatory cytokine, and suppress CCR2 gene expression
[170–173]. Advantages of this class of drug include its widespread availability, relatively
inexpensive cost, and long shelf life [174].
No study has reported statin reduced mortality in murine models of influenza. In humans, a
cohort study conducted during the 1996–2006 influenza seasons revealed a 16% reduction in 30
day pneumonia mortality with the use of statins, although interpretation of these results may
have been complicated by confounding factors [94,174]. Among patients admitted with
laboratory-confirmed influenza, those who used statins before or during hospital stay had lower
27
odds of dying within 30 days and after adjusting for other variables they reported a 41%
reduction in mortality [175]. It is important to note that adverse drug reactions, such as
autoimmune hepatitis and myopathy, are associated with statin therapy. Taken together, there is
currently insufficient evidence to recommend routine clinical use of statins for the management
of influenza. A randomized clinical trial of acute statin therapy in hospitalized statin-naïve,
influenza virus-infected patients may be warranted [176].
1.8.6 High mobility group box 1 protein antagonists
High mobility group box 1 (HMGB-1) is a 215 amino acid protein that functions intracellularly
as a transcription factor and is highly conserved among species [177]. When released either
passively from dying, necrotic cells or actively from macrophages, HMGB-1 functions in the
extracellular milieu as a signal of tissue injury/damage [178]. Implicated in the pathogenesis of
severe sepsis [179,180] and arthritis [181], HMGB-1 is a potent mediator of pro-inflammatory
activity in the presence of inflammatory cytokines such as IL-1β, IFN-γ or TNF [171,172].
Given these properties, HMGB-1 antagonists could potentially prove useful in the treatment of
‘the cytokine storm’ in severe influenza.
However, HMGB-1 is characterized as a relatively late mediator of inflammation in sepsis
[171,180,182] and, thus, its inhibition may not impact clinical outcome in severe influenza. For
example, plasma levels of HMGB-1 in H2N2 influenza virus-infected mice peaked at day 9 post-
infection (P.I.), a full 2 days after peak mortality was reached [183]. Furthermore, treatment
with the HMGB-1 antagonist ethyl pyruvate failed to reduce mortality [183]. Further studies of
28
other HMGB-1 antagonists will provide a better understanding of the role of HMGB-1 in severe
influenza.
Glycyrrhizin, a compound extracted from liquorice root, has been reported to bind and inhibit
HMGB-1 [184,185], increase T-cell IFN-γ production, and reduce endocytic viral uptake [186–
188]. Glycyrrhizin treatment increased survival in murine models of influenza when
administered prophylactically 1 day prior to infection and continued on days 1 and 4 P.I. [187].
However, protection against LPS induced lung injury in mice receiving glycyrrhizin pre-
treatment has been attributed in part to suppression of COX-2 and iNOS expression [189].
Therefore, additional study is needed to determine whether inhibition of HMGB-1 is in fact the
mechanism by which glycyrrhizin increases survival in experimental influenza. Metformin and
statins also antagonize HMGB-1 [170,190]. Molecular modeling of the HMGB-1-glycyrrhizin
interaction may aid in the development of potent anti-HMGB-1 specific drugs which could
potentially be useful as adjunctive therapy to antiviral drugs in the treatment of severe influenza.
1.8.7 Peroxisome proliferator activated receptor agonists
Peroxisome proliferator activated receptors (PPARs) are a family of lipid activated transcription
factors, consisting of PPAR-α, PPAR-δ, and PPAR-γ, which are key regulators of inflammation
and lipid metabolism [reviewed in 191]. PPAR activation antagonizes crucial inflammatory
pathways such as NF-κB, AP1 and STAT [reviewed in 192], which results in a reduction of
inflammatory cytokines such as IL-6 and IFN-γ [193].
29
Gemfibrozil, a PPAR-α agonist belonging to the fibrate family of drugs, was discovered over 40
years ago to reduce plasma lipid and cholesterol level and is widely used as treatment for
hypertriglyceridemia [194,195]. Gemfibrozil reduces the release of inflammatory cytokines
including TNF, IL-6 and IFN-γ [196,197] and has been proposed for the treatment of severe
influenza [194,196]. Daily injections of gemfibrozil administered in a treatment strategy
following the onset of experimental H2N2 influenza in mice increased survival from 26% to
52% [194]. In contrast, gemfibrozil failed to increase survival of H5N1 influenza virus-
infected mice when administered 48 hours post-inoculation [162]. These studies suggest the need
to conduct further research on the use of gemfibrozil for severe influenza.
Thiazolidinediones, also known as glitazones, are a class of PPAR-γ agonists, including
pioglitazone and rosiglitazone. Pioglitazone has been shown to suppress inflammation in sepsis
[198] and improve insulin sensitivity in patients with impaired glucose tolerance [199] by
reducing CCL2 expression, which is the primary ligand for CCR2 and an important mediator of
host inflammation [199,200]. In experimental murine models, prophylactic administration of
pioglitazone to A/PR/8 influenza virus-infected mice has been reported to increase survival by
20–40% [134,201]. Aldridge et al. found the benefits of pioglitazone administration pre- and
post-influenza virus inoculation in mice to be independent of pulmonary viral load [134].
Therefore, the benefit of pioglitazone therapy can be attributed to modulation of host
inflammatory responses.
Rosiglitazone has also recently been shown to increase survival in experimental murine
influenza. Specifically, Moseley et al. reported 100% survival of A/PR/8 influenza virus-
30
infected mice treated with rosiglitazone compared to 30% survival of influenza virus-infected
controls [201]. Moseley et al. also reported 20% survival of influenza A/California/04/09 (a
2009 pandemic H1N1 strain) virus-infected mice treated with rosiglitazone compared to 100%
fatality of infected controls. These results are by far the most promising reported to date in the
search for an effective immunomodulatory strategy to improve clinical outcome in severe
influenza. Because of the encouraging results observed with PPAR agonists in the treatment of
influenza, biochanin A, a component of red clover with both PPAR-α and PPAR-γ agonistic
properties, has been proposed as a potentially effective immunomodulatory agent for the
treatment of influenza [195].
1.8.8 AMP-activated protein kinase agonists
Increases in the intracellular AMP-to-ATP ratio trigger the activation of AMP activated protein
kinase (AMPK), an enzyme which exerts anti-inflammatory effects upon activation [202].
Aminoimidazole carboxamide ribonucleotide (AICAR) is an AMP-activated protein kinase
activator. In a murine model of LPS-induced ALI, administration of AICAR resulted in AMPK
activation in the lungs which diminished the severity of lung injury as shown by decreased
pulmonary edema, and reduced bronchoalveolar lavage fluid levels of IL-6 and TNF [202].
Moseley et al. reported a 40% increase in survival of A/PR/8 influenza virus-infected mice
treated with AICAR while combination therapy including AICAR and pioglitazone improved
survival by 60% [201].
Metformin, another AMPK agonist and known HMGB-1 antagonist, is routinely used in
combination with pioglitazone for patients with diabetes mellitus and has been shown to improve
31
survival in murine endotoxemia [190]. Metformin and pioglitazone have been shown to have
synergistic effects in type 2 diabetes [203]. This compound is also commercially available.
Therefore, metformin and AICAR might prove effective in the treatment of severe influenza
when used either alone, or in combination with PPAR-γ agonists and/or antiviral drugs.
1.9 Investigational immunomodulatory therapy – mesenchymal stromal cells (MSCs)
In 1968, Friedenstein and colleagues described a murine bone marrow cell population with the
ability to form colonies and differentiate into several cell types [204]. These cells, now referred
to as mesenchymal stromal (stem) cells (MSCs), represent a heterogenous subset of non-
hematopoeitic pluripotent stromal cells with multilineage potential. MSCs can be isolated from
embryonic tissue, adipose tissue, liver, muscle and dental pulp; however, adult bone marrow
remains the most common source of MSCs for pre-clinical and clinical studies [205,206]. A
position paper released by the International Society for Cellular Therapy [207], defines MSCs by
the following 3 minimum criteria:
1. MSC’s must adhere to plastic when maintained in standard culture conditions.
2. MSCs must possess the ability to differentiate into osteoblasts, chondroblasts and
adipocytes in vitro.
3. MSCs must lack several lineage markers including haematopoietic and endothelial
markers (CD34, CD45, CD11b, CD14, CD79α, CD19, and HLA-DR), and must possess
several mature stromal cell markers (CD105, CD73, CD90).
32
While the first two criteria are mostly universally accepted, the third criteria is complicated by
varying MSC markers identified between species and strains of species; therefore, the ability to
identify a unique MSC cell surface marker remains unresolved [208].
Initial clinical interest in MSCs focused on their ability to differentiate into injured cell types as a
means to improve outcome in pre-clinical models of disease. However, more recent pre-clinical
studies demonstrate that while MSCs can reduce injury, engraftment rates are low [209-211].
Rather, MSCs are now thought to exert their effects by controlling inflammatory and
immunological reactions, and secreting multiple paracrine factors.
1.9.1 MSCs and the immune system
In vitro, MSCs have been shown to suppress both innate and adaptive immune responses
including an ability to suppress effector and cytotoxic T-cell functions, impair cytolytic potential
of natural killer (NK) cell functions, induce regulatory T-cells, and modulate dendritic cell (DC)
and macrophage function. A number of studies have reported that the addition of MSCs to
primary mixed lymphocyte co-cultures can suppress T-cell proliferation [212-214]. MSCs may
also inhibit T-cell proliferation indirectly by inhibiting monocytes from maturing into DCs [215].
DCs play a fundamental role in antigen presentation to naïve T-cells following DC maturation,
which can be induced by pro-inflammatory cytokines or pathogen associated molecules [216].
In a study by Jiang et al., the allostimulatory ability of MSC-treated mature DCs on allogeneic T-
cells was impaired [215]. Here, MSCs were shown to induce an immunosuppressive DC
phenotype by inhibiting their production of pro-inflammatory cytokines TNF, IFN-γ, and IL-12
and inducing DC production of anti-inflammatory cytokine IL-10 [215].
33
MSCs may also affect NK cells of the innate immune system. NK cells play an important role in
antiviral and anti-tumor immune responses due to their cytolytic properties and pro-
inflammatory cytokine production. Under low NK-to-MSC ratios, MSCs alter the phenotype of
NK cells and suppress proliferation, cytokine secretion, and cytotoxicity against major
histocompatability complex (MHC) class I expressing targets [217]. MSC-mediated inhibition of
NK cells is related to the downregulation of NK cell-activating surface receptors, while
conversely, cytokine-activated NK cells can kill MSCs in vitro [218]. Some of these effects
require cell-to-cell contact, whereas others are mediated by soluble factors, including
transforming growth factor - β (TGF-β) and prostaglandin E2 (PGE2). This suggests diverse
mechanisms may be responsible for MSC-mediated NK-cell suppression. Neutrophils are also
important cells in innate immunity which are rapidly mobilized and activated to kill
microorganisms during infection. After binding to bacterial products MSCs have been shown to
delay neutrophil apoptosis through an IL-6 dependant mechanism [219].
Finally, MSCs have been shown to alter macrophage phenotype, in vitro [220] and in vivo [221].
Monocytes preferentially differentiate into IL-10 secreting alternatively activated
(immunosuppressive) macrophages (M2 macrophages) in the presence of MSCs in vitro [220].
Macrophages isolated from MSC-treated septic mice produced significantly higher amounts of
anti-inflammatory cytokine IL-10 than those from non-MSC treated mice, suggesting a
temporary reprogramming of monocyte and therefore macrophage function [221].
34
Figure 1.2. Mesenchymal stem cell immune cell targets (reprinted with permission from © Bentham Science Publishers, ref. [222].)
The clinical use of MSC therapy is likely to rely heavily on the use of allogeneic and not
autologous MSCs. This is because autologous cells may require several weeks to expand in
culture before they could be used in the clinic; by contrast, allogeneic cells would be readily
available “off-the-shelf” as they could be manufactured in advance for immediate use in the
treatment of acute disease. Also, allogeneic transplantation would ensure that the cells are
derived from healthy immunocompetent individuals and thus improving their likelihood of
functionality.
35
However, the use of allogeneic MSCs would require “universality” – that is, a low immunogenic
profile so that the cells could evade the recipient’s immune responses. Expression of human
leukocyte antigens (HLAs) – the major histocompatability complex (MHC) molecules I and II –
present antigenic peptides generated in the cytosol to CD8+ T-cells or in intracellular vesicles to
CD4+ T-cells, respectively. Low human MSC expression of MHC Class I and lack of MHC
class II and classic co-stimulatory molecules such as B7-1, B7-2, CD80, CD86, CD40 and
CD40L allow human MSCs to evade host immune responses [223]. Therefore, human MSCs
could potentially be administered to patients without the need for HLA matching. Indeed,
patients who underwent MSC allotransplants experienced no anti-allogeneic MSC antibody
formation or T-cell sensitization [224].
However, under certain stimuli including low level IFN-γ co-culture, MSCs may upregulate
expression of MHC class II to the cell surface (can detect MHC Class II intracellularly in basal
conditions) and therefore act as antigen-presenting cells (APCs) to activate antigen specific
immune responses [225,226]. Studies have also demonstrated that infusion of allogeneic mouse
MSC can elicit a host immune response and lead to MSC rejection [227,228]. Therefore, MSCs
may not be intrinsically immunopriviliged and may be unable to serve as a “universal donor” in
MHC mismatched recipients, but this may be specific to mouse rather than human-derived cells.
Further investigation is necessary to understand the complex interaction between MSCs and the
immune system.
The use of MSCs for therapeutic applications has also attracted attention for their ability to
“home” to sites of inflammation upon IV injection. Homing has been described as the process by
36
which cells migrate to and engraft in the tissue which they will exert their effects. Since MSCs
may localize to the injured tissue, concerns of MSCs inducing systemic immunosuppression -
which could lead to major complications - may be lessened. In a mouse model of multiple organ
failure, green fluorescent protein (GFP)-tagged MSCs homed to sites of injury within numerous
tissues, with MSCs proportionally homing to regions of most severe disease [229]. Akin to
leukocyte trafficking, a key indicator of MSC homing is the increase in inflammatory
chemokines at the site of injury.
In basal conditions, MSCs express several chemokine receptors located on their surface,
including CCR2, CXCR3, CXCR4, CXCR5, and CX3CR1 as well as RNA encoding the
following chemokines – CCL2, CXCL8, SDF-1(CXCL12), and cytokines TGF-β and IL-6 [230-
232]. Studies have shown that MSCs respond to chemoattractant factors including CCL2,
CXCL12, CX3CL1, CCL3, and IL-8 leading to MSC chemotaxis [233,234]. However
chemokine / cytokine and receptor expression varies in the literature, possibly due to different
culture conditions, including passage and the initial heterogeneity of the isolated cells obtained
from bone marrow [232,235,236]. It is not known which chemokines and their receptors are
most critical for MSC migration.
1.9.2 MSC duality – immunostimulant or immunosuppressant?
MSCs are located at the interface of the bone marrow cavity and on the peripheral surface of the
main sinusoidal vessels [237]. This position gives MSCs a unique ability to function as
“gatekeepers” to the marrow by interacting with all cells transiting in and out of the marrow and
by regulating immune responses as the first line of defense. The role of MSCs in immune
37
regulation has been show to be homeostatic, namely, they possess an ability to serve as either
immunostimulatory or immunosuppressive, depending on the environment.
The presence of IFN-γ and other cytokines in the MSC microenvironment play an important role
in induction of immunosuppression and MSC homing. Ren et al. have demonstrated that the
immunosuppressive effect of human MSCs depends on IFN-γ in the co-presence of either TNF,
IL-1α or IL-1β [238]. Under co-stimulation with these cytokines in vitro, human MSCs secreted
large amounts of indoleamine 2,3-dioxygenase (IDO) and chemokines which in turn drove T-cell
migration in proximity with MSCs, whereas high level IDO production suppressed T-cell
function. Aggarwal and Pittenger demonstrated that the level of PGE2 - an important MSC-
secreted protein - is significantly upregulated by MSCs in the presence of TNF [213]. IFN-γ
mediated MSC activation has also been demonstrated in vivo in a model of graft-vs-host disease
(GvHD) where recipients of IFN-γ-/- T-cells did not respond to MSC treatment [239].
Mechanistically, IFN-γ may act directly on MSCs by upregulating B7-H1, an inhibitory
molecule on the surface of MSCs [240].
On the contrary, low levels of IFN-γ or the absence of specific proteins in the MSC
microenvironment may cause MSCs to behave as APCs [241]. Human MSCs may also act as
immune-enhancers when IDO is knocked-down. Therefore, a clinical scenario where MSCs may
encounter insufficient pro-inflammatory cytokines in vivo could potentially lead to MSC-
mediated immunostimulation. It may then be difficult to determine the point in the disease
progression at which administering MSCs could achieve an immunsuppressive response.
38
1.9.3 Effects of MSCs in pre-clinical models
MSC mediated immunopsuppresion after infusion in vivo has been documented in a diverse
array of pre-clinical animal models of disease. MSC-based prophylactic and treatment strategies
have yielded significant therapeutic benefits in pre-clinical models of a variety of inflammatory
diseases, including rheumatoid arthritis [243], sepsis [221,244] and ALI [209,210,245–248].
However, several studies report no MSC-mediated immunosuppression or therapeutic benefit in
pre-clinical models of GvHD, organ transplantation and rheumatoid arthritis [249-251].
Furthermore, MSCs used in combination with the universal immunosuppressant cyclosporine A
for the treatment of GvHD resulted in accelerated graft rejection in mice [251]. The clinical trial
registry at the National Institute of Health indicates over 200 registered clinical trials currently
investigating the use of MSCs in humans as a therapy for diverse diseases including Crohn’s
disease, GvHD, osteogenesis imperfecta and multiple sclerosis [252].
Most importantly, MSCs have been shown to reduce dysregulated inflammatory responses, to
improve alveolar fluid clearance, and to maintain lung epithelial and endothelial integrity in the
lung during pulmonary inflammation and injury in murine models [209,210,245–248, reviewed
in 253]. These models consist of lung injury induced by either: (1) bleomycin - an antitumour
agent known to cause lung injury by inducing oxidative stress and elevated inflammation, or (2)
endotoxin / LPS – a major constituent of the outer cell wall of gram-negative bacteria. In these
murine models of ALI, the beneficial effects of MSC therapy have been attributed to the
secretion of growth factors, cytokines and lipid mediators [reviewed in 253 and discussed in
section 1.9.3].
39
1.9.4 MSC derived paracrine mediators
While the initial driving force behind therapeutic interest in MSCs was for their regenerative
capacity, it is their paracrine properties and ability to modulate host inflammation that markedly
increased the known range of MSC therapeutic applications and attracted even greater scientific
interest in MSC research. MSCs secrete a vast array of soluble mediators and growth factors
which can modulate the host response. Importantly, these findings have been observed
independent of direct MSC-to-cell contact. For example, concentrated MSC-conditioned media
reversed multi-organ dysfunction syndrome in a rat model [254]. While many
immunosuppressive mediators are suspected to be important for treating lung injury, the proteins
under greatest investigation include IL-1 receptor antagonist (IL-1ra) [255], soluble TNF
receptor (sTNFR) [256], IDO [214], IL-10 [221], angiopoeitin-1 (Ang-1)[298], TNF stimulated
gene 6 protein (TSG-6) [258,259] TGF-β [260], PGE2 [213,221], and keratinocyte growth factor
(KGF) [248]. These proteins may exert their positive effects by immunomodulation, improving
alveolar fluid clearance, or reducing lung endothelial permeability.
IL-1ra, sTNFR-1, IL-10, IDO, PGE2 and TSG-6 may be important in MSC-mediated
immunomodulation. In a bleomycin-induced animal model of lung injury, injected MSCs
decreased inflammatory responses and prevented the development of lung fibrosis. These effects
were attributed to MSC secretion of IL-1ra [255]. In another pre-clinical mouse model of
disease, human MSCs administered to LPS induced sepsis in mice attenuated systemic
inflammation by secreting sTNFR1 [256]. sTNFR1 binds to TNF to neutralize its inflammatory
activity. MSC secretion of IL-10 is also important. MSCs can mediate downregulation of pro-
inflammatory cytokines TNF and MIP-2 and upregulation of anti-inflammatory cytokine IL-10
40
in BAL (BAL) fluid of endotoxin induced lung injury in mice [247]. MSC secretion of PGE2
may be implicated in reprogramming macrophages to increase IL-10 production resulting in
reduced mortality and improved organ function in pre-clinical sepsis [221]. Human MSC
released IDO has been attributed to the immunosuppressive properties of MSCs as well [214]. In
vitro, MSC released IDO – an important protein in tryptophan metabolism - inhibits T-cell
proliferation directly and indirectly via IDO induced monocyte differentiation into M2–like
immunosuppressive cells [220]. Improved myocardial infarction and lung injury in MSC treated
mice has also been attributed to MSC expression of TSG-6 [258,259]. TSG-6 is a TNF induced
anti-inflammatory protein.
In addition to MSC secreted immunomodulatory proteins, the effects of MSCs may be mediated
by endothelial/epithelial specific growth factors including Ang-1 and KGF which are potentially
important in maintaining lung-vascular endothelial integrity. The integrity of the lung
microvascular endothelium is essential to prevent the influx of protein-rich fluid from the plasma
which may further aggravate the ability of the lung epithelium to reduce alveolar edema. Ang-1,
a ligand for the endothelial-specific receptor tyrosine kinase Tie2 [261], is a potent mediator of
angiogenesis that functions to prevent leakage and promote blood vasculature quiescence via
strengthening of endothelial cell junctions and downregulation of surface adhesion molecules,
such as vascular cell adhesion molecule-1 (VCAM-1) and E-selectin [262–265]. In vitro, the
effects of MSCs have been shown to be mediated by Ang-1 which resulted in reduced epithelial
protein permeability in cultured human alveolar type II cells [298]. KGF, another important
MSC soluble mediator, has also been shown to reduce lung injury in animal models of
pulmonary edema [266]. KGF may stimulate the proliferation and differentiation of alveolar type
41
II cells, which in turn may promote lung repair during injury. In an ex vivo perfused human
lung, intra-bronchial instillation of human MSCs 1 hour following endotoxin-induced lung injury
restored alveolar fluid clearance, in part by the secretion of KGF [248].
It is important to note that inhibition of most MSC secreted molecules does not result in
complete loss of MSC immunosuppressive function. For example, upon MSC knockdown of
TSG-6, Danchuk et al. reported a decrease but not complete loss of MSC mediated
immunosuppression in vivo [259]. Also the relative importance of each MSC secreted protein is
dramatically varied between studies. Lastly, administering isolated proteins implicated as the
most important MSC soluble factor does not usually fully recapitulate the immunosuppressive
function of MSC administration. For example, MSCs were more effective in mouse model of
bleomycin induced lung injury, than recombinant or virally delivered IL-1ra [255]. Therefore, it
is likely that no single molecule exerts an exclusive role and MSC mediated effects are likely the
result of concerted actions mediated by many important soluble mediators in combination.
1.10 Mouse models of influenza
To date, mouse models of influenza have demonstrated utility in the guidance of novel
therapeutic investigation and intervention for the treatment of severe influenza in humans. As in
humans, influenza virus virulence in mice is dependent on the virus subtype and strain. In
addition, the lethal dose, replication kinetics, and inflammatory response are affected by a
complex and multigenic host component [303]. Symptoms of severe disease are comparable in
mice and humans as both may feature reduced blood oxygen saturation, lung edema, increased
42
lung viral load, pulmonary immune cell infiltrates and elevated cytokine production and
hemorrhage [267]. Also, onset of symptoms positively correlates with viral titers [268].
However, experimental mice may not show signs of fever, and both cyanosis and dyspnea are
not easily detectable in many models aside from H5N1 influenza [267]. The most widely
accepted indicator of disease progression in influenza murine models is weight loss [269,270]
Antiviral compounds as well as several investigational immunomodulatory agents have shown
efficacy against influenza and host inflammation in mice [271, previously discussed].
Advantages of mouse models include well characterized immune responses and a wide
availability of murine immune reagents. Additionally, mice are inbred so experimental results are
usually reproducible; however, results may be misleading due to genetic variability in the human
population. For example, while zanamivir has demonstrated efficacy against H5N1 in mice
[271], antivirals have shown limited efficacy against H5N1 in humans [75] and the efficacy of
oseltamivir is not entirely clear [62]. Conversely, statins have been shown to reduce disease
severity in humans when their use was evaluated in retrospective studies, while statins are
ineffective at improving outcome in murine models (previously discussed). Therefore, while
mouse models are important in guiding the development of novel therapies for use in humans,
results should be interpreted with caution.
Historically, influenza A/PR/8 virus (H1N1) and A/WSN/33 virus (H1N1) are the most
commonly used strains in experimental influenza [272]. While both A/PR/8 and A/WSN/33 are
known to cause severe inflammation, A/WSN/33 is better known for its neurotropism and its
capacity to cause systemic infection [273]. Both influenza A/PR/8 virus and A/WSN/33 virus
have been serially passaged and adapted to mice in order to cause severe disease. Therefore, it is
important to bear in mind that conclusions based on results obtained using these strains may be
43
misleading due to molecular changes acquired during adaptation. Nonetheless, A/PR/8 has been
employed in the evaluation of many immunomodulatory agents (e.g. PPARs) for the treatment of
severe influenza [134,201].
44
1.11 Research aims and objectives
While the vast majority of influenza virus infections resolve without complications, millions of
individuals develop severe disease each year and are at risk of death. Given the importance of
the innate host response in severe influenza – particularly inflammation - adjunctive therapies to
complement anti-viral treatment strategies are in need. In the face of rising antiviral drug
resistance, novel anti-influenza drugs are also sorely in need. The aim of this work was to
investigate two novel therapeutic treatment strategies for severe influenza. This included an
assessment of iNO therapy as an antiviral agent and exploration of mesenchymal stem cell
therapy as an immunomodulatory strategy.
In Chapter 2, we examined whether iNO administration could reduce viral load and improve
survival in a murine model of severe influenza. Our specific aims were as follows:
1. To determine if iNO delivered either continuously or intermittently could improve
survival in an experimental murine model of severe influenza;
2. To establish whether iNO therapy could reduce the influenza viral burden in an
experimental murine model of severe influenza.
We found that iNO therapy failed to improve survival or decrease viral load in experimental
influenza.
In Chapter 3 we examined whether MSC therapy could modulate the host response to infection
in an in vivo mouse model of severe influenza and therefore improve outcome. The specific
aims of this work were as follows:
45
1. To establish and characterize the influenza A/PR/8 virus mouse model of severe
influenza which develops inflammation and acute lung injury;
2. To determine whether MSC therapy can improve survival and/or dampen host
inflammation and lung injury in a mouse model of severe influenza;
3. To assess the ability of MSC prophylaxis, with or without MSCs pre-incubated with
cytokines, to improve survival in experimental influenza model;
4. To test whether combining oseltamivir with MSCs is effective in reducing mortality
and/or dampening host inflammation and lung injury in experimental influenza model.
We found that MSCs were not effective in dampening host inflammation and acute lung injury in
experimental influenza. Thus, MSCs were unable to decrease morbidity and mortality in our
experimental model of influenza.
46
Chapter 2: Inhaled Nitric Oxide Antiviral Therapy f or Severe Influenza
2.1 INTRODUCTION
Further investigation of antiviral strategies which have shown promising results against influenza
A viruses in vitro but have not been investigated in vivo are warranted. One of these agents
which may be effective when used against influenza virus is NO. In vitro, NO has antimicrobial
activity against a wide range of viruses, including influenza A virus; therefore, we hypothesized
that inhaled NO (iNO) would increase survival in vivo by reducing the viral load in C57Bl/6
mice infected with a lethal dose of influenza A/WSN/33 virus (H1N1). iNO delivery would
provide a safer and easier delivery method than administration of NO donors such as SNAP, as
iNO is currently approved for treating term and near-term neonates with hypoxemic respiratory
failure [106,107].
iNO was delivered to influenza virus-infected mice either continuously or intermittently at 80 or
160 ppm, respectively, using both prophylactic and post-infection treatment strategies. These
dosing regimens were chosen for the following reasons:
1. iNO delivery in hypoxemic respiratory failure is already approved up to a dose of 80
parts per million (ppm) [106,107].
2. Gaseous NO at a high dose of no less than 160 ppm and with five hours of continuous
exposure, can elicit a non-specific antimicrobial response against a broad range of
microorganisms in vitro [274].
47
In vivo, 160 ppm iNO treatment would result in NO binding to hemoglobin to form
methemoglobin, resulting in reduced oxygen transport and hypoxemia, as well as the potential
for elevated levels of the harmful NO metabolite NO2. However, Miller et al. have shown that
iNO in an intermittent delivery regimen of 160 ppm for 30 min every 3.5 hours can prevent
methemoglobinemia and reduce the potential of host cell toxicity in vivo [Miller C, personal
communication]. Therefore, we chose to deliver 160 ppm iNO in a similar manner.
We chose to investigate the use of iNO over other antiviral agents for several reasons. Firstly, it
will be available off patent as of 2013 and therefore will be relatively cheap to administer as
treatment for severe influenza. Secondly, it is relatively easy to administer with a breathing
mask in resource poor areas. Lastly, iNO would likely evade the development of influenza drug
resistance that is often associated with antiviral therapy, due to its non-specific antiviral effects
including enzyme nitrosylation, such as nitrosylation of proteases (e.g. reverse transciptases and
ribonucleotide reductase) containing cysteine residues and viral-encoded transcription factors
that are involved in replication [97, 98].
48
2.2 MATERIALS AND METHODS
Murine influenza model
Animal use protocols were reviewed and approved by the University Health Network Ontario
Cancer Institute Animal Care Committee, and all experiments were conducted in accordance
with institutional guidelines in an animal biosafety level 2 facility. Female C57Bl/6 mice, 9–11
weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and maintained
under pathogen-free conditions with a 12-hour light cycle. On day 0, while under light
isofluorane anesthesia, experimental mice were infected via nasal instillation with 1000 plaque
forming units (PFU) of influenza A/WSN/33 virus (H1N1) (stock kindly provided by Dr.
Eleanor Fish, University Health Network/University of Toronto) in 50 µl PBS. Weight was
recorded daily for a maximum of twelve days P.I., and mice were sacrificed when euthanasia
criteria was met (greater than 20% weight loss). Lung tissue was harvested for analysis on day 5
P.I..
In Vivo NO Delivery
Prophylactic or post-infection iNO therapy was initiated either 1 hour prior to or 4 hours P.I.,
respectively. Mice were placed in flow-through chambers with free access to food and water and
received either compressed room air, continuous NO at 80 ppm +/-5ppm mixed with compressed
room air, or intermittent NO for 30 min every 3.5 hours at 160 ppm+/-5ppm mixed with
compressed room air. Soda lime (200 g) was supplied to each chamber, and gas flow was
maintained at 10–12 L/min to scavenge and minimize NO2 levels, respectively. NO2 levels were
49
limited to <2 ppm for continuous iNO therapy and <8 ppm for intermittent iNO therapy. NO and
NO2 levels were measured using an AeroNOX machine (Pulmonox Medical, AB, CA).
Lung influenza viral load analysis
Lungs were harvested and frozen at -80°C. Lungs were thawed, weighed, and homogenized in 1
ml PBS for 30 sec using a Tissue Miser homogenizer (Fisher Scientific, ON, CA). Lung
homogenates were spun at 10,000xg for 10 min, aliquoted, and stored at -80°C for viral yield
titration. Influenza A/WSN/33 viral yield in lung homogenates was quantified by plaque assay in
Madin-Darby canine kidney (MDCK) cells (ATCC, VA, USA). MDCK cells (ATCC, Manassas,
VA, USA) were maintained in Eagle’s MEM (ATCC) supplemented with 10% FBS and 0.25%
gentamicin. All cell lines were cultured at 37oC with 5% CO2. MDCK cells were plated at a
concentration of 8x106 cells/plate in 6 well culture plates. 12–24 hours later, medium was
removed and MDCK cells were washed twice with PBS. 10-fold dilutions of lung homogenates
were added to MDCK cells in 500 µL Eagle’s MEM, in duplicate, and incubated at 37°C with
5% CO2 for 1 hour with plates rocked every 15 min. After incubation, 1 mL of serum-free 2X
Eagle’s MEM supplemented with 8 µl/ml trypsin, 60 µl/ml of 7.5% sodium bicarbonate and 20
µl/ml antibiotics, combined with 1 ml of 1.2% agarose, was added to each well. Once the agarose
set, plates were incubated at 37°C for 42–72 hours until syncytia were observed. Plates were
fixed with Carnoy’s fixative (3:1, methanol:glacial acetic acid) for 30 min then stained with
0.1% crystal violet in 20% ethanol to visualize plaques. Viral load was expressed as plaque
forming units per gram of lung tissue (PFU/g).
50
Statistical Analysis
Logrank tests were performed on Kaplan-Meier survival curves. Significant differences in
weight loss between groups were assessed by two-way analysis of variance (ANOVA) and
Bonferroni post-tests were performed. Student’s t-tests were carried out on viral yield data to
assess significant differences (p ≤ 0.05) between experimental groups.
51
2.3 RESULTS
2.3.1 Continuous iNO at 80 ppm decreased survival and intermittent high dose iNO at 160 ppm did not increase survival of influenza A virus-infected mice
We evaluated the ability of iNO to improve survival of influenza A/WSN/33 virus-infected mice.
Experimental C57Bl/6 mice were inoculated intranasally with an 80–100% lethal dose of
A/WSN/33 virus (1000 PFU). At 5 days P.I., the majority of mice in all experimental groups
experienced weight loss (Figure 2.1A and 2.2A). At 7 days P.I., mice began to reach euthanasia
criteria (≤80% of day 0 weight), and by day 10 P.I., most mice were euthanized (Figure 2.1B and
2.2B). If 20% weight loss was not met by day 10 P.I., the infection typically resolved, and
surviving mice gained weight.
Figure 2.1. Prophylactic iNO therapy increased weight loss and decreased survival of C57Bl/6 mice infected with influenza A/WSN/33 virus. C57Bl/6 male mice were infected with 1000 PFU A/WSN/33 virus and administered continuous NO at 80 ppm or compressed room air starting 1 hour prior to infection (n=17–18/group, 2 independent pooled experiments). (A) Shown are the weight loss data up to the day of first death. Mice receiving iNO displayed a significant reduction in weight compared to infected controls (Two-way ANOVA p < 0.001 with Bonferroni post-tests; **, p < 0.01 on day 6 and 7 P.I.). Error bars represent standard deviations. (B) Survival curve. iNO significantly reduced survival of treated mice compared to infected controls as shown by Kaplan-Meir survival curves; **, logrank test: P < 0.01).
0 2 4 6 880
85
90
95
100
105
110
iNO prophylaxisAIR
**
**
Day P.I.
% o
f o
rig
inal
wei
gh
t (m
ean
±± ±± S
D)
0 2 4 6 8 10 120
20
40
60
80
100
iNO prophylaxisAIR
**
Day P.I.
Per
cen
t su
rviv
al
A B
52
Weight loss over the course of infection was accelerated in mice administered continuous iNO at
80 ppm starting 1 hour prior to inoculation compared to infected control mice administered
compressed room air (P < 0.001) (Figure 2.1A). Continuous iNO administered at 80 ppm starting
1 hour prior to inoculation significantly decreased survival of A/WSN/33 virus-infected mice
compared to infected control mice administered compressed room air (P < 0.01). During the
course of infection, 100% of continuous iNO treated mice were euthanized compared to 80% of
infected control mice (Figure 2.1B). Intermittent iNO administrated at 160 ppm for 30 min
intervals every 3.5 hours starting either 1 hour prior to or 4 hours P.I. resulted in similar weight
loss kinetics (Figure 2.2A) and consequent survival kinetics (Figure 2.2B) of infected mice
compared to infected control mice administered compressed room air.
53
Figure 2.2. Prophylactic and post-infection intermittent iNO does not alter weight loss kinetics or survival of C57Bl/6 mice infected with influenza A/WSN/33 virus. C57Bl/6 male mice were infected with 1000 PFU A/WSN/33 virus and administered NO intermittently at 160 ppm for 30 min intervals every 3.5 hours starting either 1 hour prior to infection or 4 hours P.I.. Infected control mice were administered compressed room air (n=9–10/group). (A) Shown are the weight loss data up to the day of first death. No difference was observed (Two-way ANOVA). Error bars represent standard deviations. (B) Survival curve. No difference was observed (logrank test).
2.3.2 Continuous or intermittent iNO administration does not reduce lung viral load
As NO at high concentrations has been shown to decrease the viral load of infected cells in vitro
(Miller C, personal communication), we examined whether iNO could reduce the viral load of
influenza virus-infected mice. iNO was administered starting 1 hour prior to influenza
A/WSN/33 virus infection and continued either continuously at 80 ppm or intermittently at 160
ppm for 30 min every 3.5 hours until mouse lungs were harvested at peak influenza viral load in
the lungs (determined to be day 5 P.I. based on preliminary studies, data not shown). Since iNO
was administered both prior to and for 5 days P.I., we were able to test whether iNO at
intermediate (80 ppm) or high concentration (160 ppm) could prevent either viral entry or viral
0 2 4 6 870
80
90
100
110iNO prophylaxisiNO post-infection
AIR
Day P.I.
% o
f o
rig
inal
wei
gh
t (m
ean
±± ±± S
D)
0 2 4 6 8 10 120
20
40
60
80
100
AIRiNO prophylaxisiNO post-infection
Day P.I.
Per
cen
t su
rviv
al
A B
54
replication in vivo, and thereby reduce viral load. Continuous iNO administered at 80 ppm,
intermittent iNO administered at 160 ppm, and administered compressed room air yielded
similar lung viral loads of infected mice on day 5 P.I. (Figure 2.3A and B, respectively).
Therefore, iNO administered both continuously and intermittently failed to reduce lung viral load
of infected mice, compared to infected control mice administered compressed room air.
Figure 2.3. Intermittent high dose iNO prophylactic therapy failed to decrease viral load of C57Bl/6 mice infected with influenza A/WSN/33 virus. Lungs were collected 5 days post-influenza A/WSN/33 virus infection (1000 PFU) from mice treated with (A) continuous iNO at 80 ppm or compressed room air starting 1 hour prior to infection (n=5/group) or (B) intermittent iNO at 160 ppm or compressed room air for 30 min intervals every 3.5 hours starting 1 hour prior to infection (n=5/group). Error bars represent standard deviations. Lung viral load was quantified for all experimental groups by plaque assay on MDCK cells.
105
106
107
intermittent iNO AIR
Day 5 P.I.
PF
U/g
lun
g t
issu
e
105
106
107
continuous iNO AIR
Day 5 P.I.
PF
U/g
lun
g t
issu
e
A B
55
2.4 DISCUSSION
In this report, we investigated the application of iNO therapy as an antiviral strategy for
prophylaxis and treatment of severe influenza. We found that iNO was unable to reduce the viral
burden in the lungs of influenza virus infected mice when administered continuously or
intermittently throughout the course of infection. We also showed that iNO, administered either
intermittently or continuously at varying concentration, was unable to reduce morbidity and
mortality in experimental severe influenza.
Typically, iNO is administered at initial doses of 5–20 ppm in randomized controlled trials and
observational studies for neonatal hypoxic respiratory failure [107]. Although FDA-approved at
concentrations up to 80 ppm, no specific dose of iNO has been proven more advantageous than
another [107,113]. Rather, methemoglobinemia, defined as 7% methemoglobin by Davidson et
al. was more likely to occur at higher concentrations [276]. We speculated that
methemoglobinemia may account for the decrease in survival observed in our study with
continuous iNO administration at 80 ppm. However, we could not draw this conclusion as
methemoglobin levels were not measured. NO2 concentrations were measured daily over the
course of infection and kept below 2 ppm as is acceptable in humans, however, lung toxicity may
still explain these results as the toxic threshold in mice may be lower. On the other hand, given
previous in vitro findings by McMullen et al. [274], 80 ppm may also have been too low of a
concentration to provide an antiviral effect.
A high dose of NO at 160 ppm was administered intermittently, not to target the airway vessels
specifically, but rather to induce an antimicrobial effect while avoiding the harmful effects of
high dose continuous iNO delivery. iNO administered to influenza virus-infected mice in this
56
manner, either prophylactically or therapeutically, failed to improve survival of infected mice,
change the course of weight loss, or decrease the lung viral load, compared to control mice
administered compressed air. Therefore, although administration of high dose intermittent iNO
may have reduced the harmful side-effects of NO, antimicrobial activity was not observed in
vivo. However, the variation in weight loss over the course of infection within either the iNO
treated or untreated groups, as indicated by large and overlapping standard deviations, may
suggest that perhaps too few animals were studied to identify a true effect of iNO therapy.
In conclusion, despite the demonstrated antimicrobial activity of NO against influenza A virus in
vitro, the results of this study do not support the use of iNO as a prophylactic or treatment
strategy to reduce viral burden or improve clinical outcome in severe influenza in vivo.
Furthermore, it may be difficult to achieve virucidal concentrations of NO in the airways using
iNO at concentrations that are safe in the living host.
57
Chapter 3: Mesenchymal Stromal Cell Immunomodulatory Therapy for Severe Influenza
3.1 INTRODUCTION
MSCs offer considerable promise as a novel influenza treatment strategy. While previous
strategies to inhibit a specific component of the immune response in influenza have met with all
but failure, a more complex overall immunomodulatory effect demonstrated by MSCs may be
more effective in decreasing influenza associated morbidity and mortality. In addition, MSCs
may be able to reprogramme the immune response to reduce destructive host inflammation while
maintaining a sufficient inflammatory response to control virus replication. Also, MSCs may
enhance the repair of lung injury and restore epithelial and endothelial integrity, either by direct
MSC cell contact or secretion of various paracrine mediators including KGF and Ang-1.
Indeed, several reports have suggested that mesenchymal stem cells (MSCs) could exert a potent
immunosuppressive effect in pre-clinical models of acute lung injury, and thus may have a
therapeutic potential for other inflammatory / lung injury dependent pathologies [209,210,245-
248]. Therefore, we aimed to establish whether MSCs could be used to control overzealous host
inflammatory responses and decrease acute lung injury in experimental severe influenza, a major
cause of morbidity and mortality in severe influenza.
Most experimental evidence to date regarding the use of MSC treatment for ALI comes from
studies that employed mouse MSCs (mMSCs) [209,210,245,247]. However, it is critical that the
58
efficacy and mechanism of human MSCs (hMSCs) be studied in depth before proceeding to
clinical trials so that the human physiological response could be accurately predicted. Evidence
suggests that the mechanism by which hMSCs exert their effects varies from that of mMSCs
[238]. Therefore, MSC investigators have more recently employed hMSCs in pre-clinical study
[256,258,259]. Likewise, we chose to evaluate the use of hMSCs rather than mMSCs in our
experimental model of severe influenza.
As basal levels of MSC chemokines / cytokine expression can be modified by pre-stimulation
with IFN-γ and TNF [231,238], we sought to assess whether cytokine pre-incubated hMSCs
could improve survival in our experimental influenza model. We also examined the effect of
hMSCs in combination with antiviral therapy, oseltamivir, for the two following reasons. Firstly,
any future MSC therapy for severe influenza patients will be administered in combination with
antiviral therapy, as is standard treatment. Secondly, oseltamivir might have important effects on
the host inflammatory environment and therefore effect MSC functionality and
migration/homing. Because inflammation and ALI/ARDS are major determinants of morbidity
and mortality in severe influenza, MSC therapy represents a promising adjunctive
immunomodulatory strategy to improve outcome in severe influenza and warrants investigation.
59
3.2 MATERIALS AND METHODS
Murine influenza model
Animal use protocols were reviewed and approved by the University Health Network Ontario
Cancer Institute Animal Care Committee, and all experiments were conducted in accordance
with institutional guidelines in an animal biosafety level 2 facility. Male C57Bl/6 mice, 8–10
weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and maintained
under pathogen-free conditions with a 12-hour light cycle. On day 0, while under light
isofluorane anesthesia, experimental mice were infected by nasal instillation with 425 EID50 of
influenza A/PR/8 virus (H1N1) (stock kindly provided by Dr. David Kelvin, University Health
Network/University of Toronto) in 50 ul PBS. Weight was recorded daily for a maximum of
twelve days P.I., and mice were sacrificed when euthanasia criteria was met (greater than 20%
weight loss). BAL fluid, lung tissue, and blood were harvested for analysis.
Cells
Human mesenchymal stromal cells (hMSCs) were provided by the Texas A&M Health Science
Center College of Medicine Institute for Regenerative Medicine at Scott & White. hMSCs
(isolated from a 24 year old male donor) were quickly thawed to 37°C and plated for 24 hours in
α-MEM with 2–4 mM L-glutamine supplemented with 100 U/ml penicillin, 100 ug/ml
streptomycin and 16.5% Fetal Bovine Serum (FBS). After 24 hrs, plates were washed with PBS
and cells were lifted by incubation with trypsin/EDTA for 2 min at 37°C and re-plated at 60
cells/cm2. Cells were incubated for 7–10 days for each subsequent passage (P). P3–P4 hMSCs
were lifted as described above and spun at 300xg for 5 min at 4°C with 10 ml of media. Media
60
was then discarded and cells were spun twice at 300xg, 4°C with 10 ml of fresh Dulbecco’s
Phosphate Buffered Saline (PBS; Gibco/Invitrogen, Burlington ON, CA). After last wash, cells
were re-suspended in 1 ml fresh PBS and total cell count was determined via hemocytometry.
hMSCs were re-suspended in PBS at 2.5x 106 cells/ml and 100 ul was injected via the tail vein
into experimental mice on day -2, 0, 2, and 5 P.I.. Injections were performed using 26 1/2 gauge
needles and a typical mouse restrainer.
MDCK cells (ATCC, Manassas, VA, USA) were maintained in Eagle’s MEM (ATCC)
supplemented with 10% FBS and 0.25% gentamycin. All cell lines were cultured at 37oC with
5% CO2.
Reagents
Oseltamivir phosphate was extracted from Tamiflu capsules (Roche, Switzerland) and
administered to mice via oral gavage. Experimental mice were administered 5 mg/kg in 100 ul
ddH20, twice daily, every 12 hours, beginning either 2 or 5 days P.I. for a maximum of 5 days
[as described in 277]. Recombinant human IFN-γ and TNF were purchased from R&D Systems
(Minneapolis, MN, USA) and hMSCs were pre-incubated for 24 hours prior to cell harvest at 10
and 3 ng/ml, respectively [as described in 220].
BAL fluid analysis
BAL fluid of both lungs was obtained by instillation and aspiration of three consecutive 500 ul
aliquots of PBS. BAL fluid was spun at 800xg at 4°C for 5 min. Supernatant from the first
lavage was removed and stored at -80°C for further analysis. Red blood cells in BAL fluid cell
pellet were lysed with 250 ul red blood cell lysis buffer for 5 min then spun again at 800xg for 5
61
min at 4°C. Combined cell pellets from all washes per mouse were re-suspended in 200 ul PBS.
Total cell numbers were determined using a hemocytometer. BAL fluid concentrations of
CCL3, CCL2, CXCL10, CCL5, mouse keratinocyte-derived cytokine (KC) and IFN-γ were
determined by sandwich ELISA (R&D Systems, Minneapolis, MN, USA) as per manufacturer’s
instructions. BAL fluid total protein concentration was measured using a BCA protein assay,
and BAL fluid IgM and albumin concentration was determined by sandwich ELISA (Bethyl
Laboratories, Montgomery, TX, USA) as per manufacturer’s instructions.
Lung homogenate analysis
Lungs were harvested and frozen at -80°C. Lungs were thawed, weighed, and homogenized in 1
ml PBS for 30 sec using a Tissue Miser homogenizer (Fisher Scientific, ON, CA). Lung
homogenates were spun at 10,000xg for 10 min, aliquoted, and stored at -80°C for viral yield
titration. Influenza A/PR/8 viral yield in lung homogenates was quantified by plaque assay in
MDCK cells (ATCC, VA, USA) as described in section 2.2.
Blood Analysis
Infected mice and uninfected controls were anesthetized with intraperitoneal (IP) injection of
ketamine (100 mg/kg) and xylazine (5 mg/kg) on day 7 post infection. Cardiac puncture was
performed where approximately 600 µL of whole blood was isolated from each mouse. Samples
were kept at room temperature for 30 min then stored on ice until centrifugation at 13,000 rpm,
4°C, for 10 minutes. Serum was aliquoted and stored at -80°C for further cytokine analysis.
Cytokine levels were determined as described above.
62
Statistical Analysis
Analysis was performed using GraphPad Prism software. Kaplan-Meier survival curves were
compared using the logrank test. Differences between groups were assessed by one- or two-way
analysis of variance (ANOVA) with Bonferroni post-tests.
63
3.3 RESULTS
3.3.1 Influenza A/PR/8 virus infection is characterized by a high mortality rate, elevated inflammation and acute lung injury
To establish a lethal model of severe influenza, experimental C57Bl/6 mice were infected
intranasally with 425 EID50 influenza A/PR/8 virus. This dose resulted in a mortality rate of
80%, where death was established according to standard euthanasia criteria (≤80% of day 0
weight) (Figure 3.1B). At 6 days P.I., the majority of mice in all experimental groups
experienced weight loss while at 7 days P.I., mice began to reach euthanasia criteria (Figure
3.1A and B). By 10 days P.I., most mice were euthanized. If 20% weight loss was not met by
day 10 P.I., the infection typically resolved, and surviving mice gained weight.
Figure 3.1. Male C57BL/6 mice infected with 425 EID50 influenza A/PR/8 virus develop increased morbidity and mortality. Eight week-old male C57Bl/6 mice were infected with 425 EID50 influenza A/PR/8 virus. (A) Weight loss is expressed in percentage of original weight over the course of infection (n=10). Error bars represent standard deviation. (B) Kaplan-Meir survival curve (n=10).
0 2 4 6 8 10 1270
80
90
100
110
Day P.I.
% o
f ori
gin
al w
eig
ht (
mea
n±± ±±
SD
)
0 2 4 6 8 10 120
20
40
60
80
100
Day P.I.
Per
cen
t su
rviv
al
A B
64
In order to characterize influenza A/PR/8 virus infection as severe influenza which develops
elevated host inflammation, BAL fluid, lung homogenate, and serum were collected from
C57BL/6 mice on days 0, 2, 4, 5, and 6 over the course of infection and examined for total
number of inflammatory cells, as well as cytokine and chemokine protein content. Specifically,
CCL2, CCL5, KC, CXCL10, and IFN-γ levels were quantified, as these proteins have been
shown to be significantly elevated in and most commonly associated with severe influenza
compared to mild or uncomplicated influenza [135]. BAL fluid inflammatory cells began to
increase on day 4 P.I. until 6 days P.I., one day prior to the first death (Figure 3.2A). BAL fluid
levels of CCL2, CCL5, KC and CXCL10 began to rise at approximately 4 days P.I. and
continued to increase until 6 days P.I. (Figure 3.2B). BAL fluid IFN-γ began to increase 6 days
P.I., 2 days following the increase in CCL2, CCL5, KC and CXCL10. Lung homogenate levels
of CCL2, CCL5, KC, CXCL10, and IFN-γ followed a similar trend as is observed in BAL fluid,
while serum levels remained low or below the limit of detection throughout the course of
infection.
In order to characterize influenza A/PR/8 virus infection as severe influenza which develops
acute lung injury (ALI), BAL was performed on C57BL/6 mice on days 0, 2, 4, 5, and 6 P.I.
Total protein, and more specifically, IgM, are indicative of alveolar-capillary membrane barrier
leak and, therefore, were quantified in BAL fluid [18]. Levels of total protein and IgM were
elevated starting 4 days P.I., and continued to rise until the first day when euthanasia criteria was
met (Figure 3.2C).
65
Figure 3.2. Male C57BL/6 mice infected with 425 EID50 influenza A/PR/8 virus develop lung inflammation and acute lung injury. (A) Total inflammatory cell count was measured by haemocytometry in BAL fluid of influenza virus-infected mice on days 0,2,4,5, and 6 P.I. (n=5-7/group). (B) A panel of cytokines and chemokines was examined by ELISA in the serum, lung homogenate and BAL fluid of influenza virus-infected mice on days 0,2,4,5, and 6 P.I. (n=5-7/group). (C) Total protein and IgM, standard markers of acute lung injury, were measured by BCA assay and ELISA, respectively, on days 0,2,4,5, and 6 P.I. (n=5-7/group). Protein levels and cell count are presented as median with interquartile range (IQR).
0 1 2 3 4 5 60
1000
2000
3000 IFNγγγγ
Day P.I.
BA
L/S
eru
m/L
ung
pg
/ml
±± ±± I
QR
0 1 2 3 4 5 60
500
1000
1500
2000
0
1000
2000
3000
4000
5000
6000CXCL10
Day P.I.
BA
L/S
eru
m p
g/m
l±± ±±
IQR
Lu
ng
pg
/ml±± ±± S
D
0 1 2 3 4 5 60
200
400
600
800
0
5000
10000
CCL5
Day P.I.
BA
L/S
eru
m p
g/m
l±± ±±
IQR
Lung
pg/m
l±± ±± SD
0 1 2 3 4 5 60
100
200
300
400
500
600
700
0
250
500
750
1000
1250CCL2
Day P.I.
BA
L/S
eru
m p
g/m
l±± ±±
IQR
Lun
g p
g/m
l + S
D
0 1 2 3 4 5 60
100
200
300
400
500
0
1000
2000
3000
4000
5000KC
Day P.I.
BA
L/S
eru
m p
g/m
l±± ±±
IQR
Lu
ng
pg
/ml +
SD
0 1 2 3 4 5 60
500
1000
1500
2000
2500
Total Protein
Day P.I.
BA
L u
g/m
l±± ±±
IQR
0 1 2 3 4 5 60
500
1000
1500
2000IgM
Day P.I.
BA
L n
g/m
l±± ±±
IQR
CBAL
SerumBAL
Lung Homogenate
BAL
SerumBAL
Lung Homogenate
SerumBAL
Lung Homogenate
SerumBAL
Lung HomogenateSerumBAL
Lung Homogenate
A
0 1 2 3 4 5 60
250
500
750
1000Inflammatory Cell Count
Day P.I.To
tal
# o
f ce
lls
in B
AL
(x1
03 )±± ±±
IQR
B
66
3.3.2 Mesenchymal stem cell therapy fails to improve survival and/or dampen host inflammation and lung injury in experimental severe influenza
2.5x105 hMSCs were administered either 4 hours prior to infection (day 0), early (day 2), or late
(day 5) in the course of infection. A dose of 2.5x105 hMSCs was chosen since this dose was
effective in a report by Mei et al. [210]. MSCs were obtained from the Texas A&M Health
Science Center College of Medicine Institute for Regenerative Medicine at Scott & White and
were reported by the Centre as meeting the MSC defining criteria proposed by the International
Society for Cellular Therapy (207, data not shown).
In the lethal influenza A/PR/8 virus mouse model, hMSCs administered prior to, early, or late in
the course of infection, resulted in similar weight loss kinetics (Figure 3.3A) and consequent
survival kinetics (Figure 3.3B) of infected mice compared to infected control mice administered
PBS. No difference in lung viral titer was observed between experimental groups (Figure 3.3C).
67
Figure 3.3. hMSCs fail to improve survival, alter weight loss kinetics or affect lung viral load in experimental severe influenza. Eight week-old male C57Bl/6 mice were administered 2.5 x 105 hMSCs (passage 3), via the tail vein, either 4 hours prior to influenza A/PR/8 virus infection (425 EID50), 2 days post infection (P.I.), or 5 days P.I. Weight was recorded daily. No significant differences in (A) weight loss kinetics (Two-way ANOVA, n=18-20/group, 2 pooled experiments) or (B) survival (logrank test, n=18-20/group, 2 pooled experiments) were observed. Error bars represent standard deviation. (C) lungs were collected 7 days P.I and quantified via plaque assay on MDCK cells. No significant differences were observed (One-way ANOVA, n=5/group). Error bars represent standard deviation.
hMSC Day 0 P.I.hMSC Day 2 P.I.hMSC Day 5 P.I.PBS
survival mice weight loss
A B
hMSC Day 0 P.I.hMSC Day 2 P.I.hMSC Day 5 P.I.PBS
survival mice weight loss
103
104
105
106
107
Day 7 P.I.
hMSCs Day 0 P.I.hMSCs Day 2 P.I.hMSCs Day 5 P.I.PBS
PF
U/g
lu
ng
tis
sue
+ S
D
C
0 2 4 6 8 10 120
20
40
60
80
100
Day P.I.
Per
cen
t su
rviv
al
0 2 4 6 8 10 1270
75
80
85
90
95
100
105
Day P.I.
% o
f ori
gin
al w
eig
ht (
mea
n±± ±±
SD
)
68
To assess whether hMSCs could dampen lung inflammation, experimental mice administered
hMSCs or PBS were sacrificed on day 7 P.I. and BAL fluid levels of CCL2, CXCL10, KC,
CCL5, and IFN-γ were quantified. All BAL fluid cytokine and chemokine levels were similar
for hMSC treated mice compared to control mice (Figure 3.4). Cytokine and chemokine levels
were below the limit of detection in BAL fluid of uninfected mice. To assess whether hMSCs
could decrease acute lung injury in experimental influenza, we tested the BAL fluid for markers
of acute lung injury. Similarly, there was no difference in BAL fluid total protein, IgM, and
albumin levels on day 7 P.I. between experimental groups (Figure 3.5).
69
Figure 3.4. hMSCs fail to alter lung inflammation in experimental severe influenza. Eight week-old male C57Bl/6 mice infected with 425 EID50 influenza A/PR/8 virus and treated with 2.5x105 hMSCs (passage 3) were sacrificed on day 7 P.I. BAL was performed. No significant difference in BAL fluid level of selected cytokines / chemokines was observed between mice treated early (day 2 P.I.) or late (day 5 P.I.) in the course of infection, compared to infected control mice (One-way ANOVA, n=5/group). Error bars represent standard deviation. ND = nondetectable.
0
200
400
600
800
1000
1200
1400
Day 7 P.I.
CCL2
ND
BA
L
pg
/ml
+
SD
0
250
500
750
1000
Day 7 P.I.
CXCL10
ND
BA
L p
g/m
l +
SD
0
100
200
300
400
500
Day 7 P.I.
CCL5
ND
BA
L p
g/m
l +
SD
0
25
50
75
100
125
150
175
Day 7 P.I.
KC
ND
BA
L p
g/m
l +
SD
0
1000
2000
3000
4000IFNγγγγ
ND
Day 7 P.I.
BA
L p
g/m
l +
S
D
hMSC Day 5 P.I.hMSC Day 2 P.I.PBSUninfected
70
Figure 3.5. hMSCs fail to alter acute lung injury in experimental severe influenza. Eight week-old male C57Bl/6 mice infected with 425 EID50 influenza A/PR/8 virus and treated with 2.5x105 hMSCs (passage 3) were sacrificed on day 7 P.I. BAL was performed. No significant difference in total protein, IgM, or albumin, was observed between mice treated early (day 2 P.I.) or late (day 5 P.I.) in the course of infection, compared to infected control mice (One-way ANOVA, n=5/group). Error bars represent standard deviation. ND = nondetectable.
Next we tested whether hMSCs could decrease morbidity in a non-lethal model of A/PR/8
influenza virus infection. Since a dose of 425 EID50 A/PR/8 virus resulted in 80% mortality, we
hypothesized that administering hMSCs in a milder infection model would allow greater
opportunity for hMSCs to provide a therapeutic effect. Mice were administered a sub-lethal dose
of A/PR/8 virus (150 EID50) and hMSCs were administered to infected mice in both prophylactic
(2 days prior to infection) and therapeutic (2 and 5 days P.I.) experimental groups. Weight loss
over the course of infection was recorded (Figure 3.6). There was no difference in weight loss
between experimental groups.
0
1
2
3
4
5
Day 7 P.I.
Total Protein
BA
L m
g/m
l +
SD
0
100
200
300
400
500
600
700ALBUMIN
Day 7 P.I.
ND
BA
L u
g/m
l +
SD
0
1
2
3
4
5
6 IgM
Day 7 P.I.
NDBA
L u
g/m
l +
SD
hMSC Day 5 P.I.hMSC Day 2 P.I.PBSUninfected
71
Figure 3.6. hMSCs fail to alter weight loss kinetics in experimental sub-lethal influenza. Eight week-old male C57Bl/6 mice were administered 2.5 x 105 hMSCs (passage 3), via the tail vein, 2 days prior to a sub-lethal influenza A/PR/8 virus infection (150 EID50), 2 days post infection (P.I.), or 5 days P.I. Weight was recorded daily. No significant difference in weight loss over the course of infection was observed (Two-way ANOVA, n=11/group). Error bars represent standard deviation.
72
3.3.3 Mesenchymal stem cell prophylaxis, with or without MSC cytokine pre-incubation, fails to improve survival in experimental influenza
Several important findings in MSC biology have centered around the micro-immuno
environment for which hMSCs persist. One key observation is the requirement of IFN-γ in
combination with TNF, IL-1α or IL-1β, to induce human MSC-mediated immunosuppression
[238]. Therefore, we tested whether pre-incubating hMSCs with both IFN-γ and TNF [as
described in 220], could enhance the immunosuppressive properties of hMSCs and therefore
improve survival when administered to infected mice in our experimental model of severe
influenza. 2.5x105 cytokine stimulated or non-stimulated hMSCs (passage 3) were administered
to influenza virus-infected mice as prophylaxis (2 days prior to infection) or therapy (5 days
P.I.). Weight loss kinetics (Figure 3.7A) and consequent survival kinetics (Figure 3.7B) were
unchanged with administration of cytokine stimulated or non-stimulated hMSCs 2 days prior to
infection, compared to infected control mice administered PBS. Similarly, no improvement in
weight loss or survival was observed with cytokine stimulated hMSCs administered on day 5
P.I., compared to infected control mice administered PBS (data not shown).
73
Figure 3.7. Cytokine stimulated hMSC prophylaxis fails to alter weight loss kinetics or improve survival in experimental severe influenza. Eight week-old male C57Bl/6 mice were administered 2.5 x 105 hMSCs, pre-incubated for 24 hours with or without 10 ng/ml IFN-γ and 3 ng/ml TNF, 2 days prior to infection with 425 EID50 influenza A/PR/8 virus. Weight was recorded daily. No significant difference in (A) weight loss kinetics (Two-way ANOVA, n=10/group) or (B) survival (logrank test, n=10/group) were observed. Error bars represent standard deviation.
3.3.4 Mesenchymal stem cell therapy fails to reduce mortality and/or dampen host inflammation and lung injury when used as an adjunctive therapy in two models of experimental influenza
Influenza virus-infected individuals seeking clinical care will likely be treated with antiviral
drugs upon presentation. Therefore, we tested the hypothesis that the combination of a
neuraminidase inhibitor, oseltamivir, together with hMSCs, could be effective in reducing
mortality. To reflect a common clinical scenario, we delayed combination therapy for 5 days
after challenging C57Bl/6 with 550 EID50 influenza A/PR/8 virus. Mice receiving oseltamivir
and control mice receiving PBS had a survival rate of 18% and 17%, respectively. With the
addition of hMSC therapy to oseltamivir treatment, the survival rate increased to 35% (Figure
0 2 4 6 8 10 12 140
20
40
60
80
100
Day P.I.
Per
cen
t su
rviv
al
0 1 2 3 4 5 6 7 870
80
90
100
110
Day P.I.
% o
f ori
gina
l wei
ght
(mea
n±± ±±
SD
)
hMSC Day -2 P.I.cytokine stimulated hMSC Day -2 P.I.PBSA B
hMSC Day -2 P.I.cytokine stimulated hMSC Day -2 P.I.PBS
74
3.8A right). However, this result was not statistically significant (logrank test; p=0.47). Weight
loss over the course of infection was similar between experimental groups (Figure 3.8A left).
It is important to employ a variety of influenza virus strains to ascertain that the experimental
effects can be applicable to human infection by various strains. Therefore, a similar experiment
to assess hMSCs as an adjunctive treatment strategy was performed with a lethal dose (104
EID50) of influenza A/Mexico/4108/09 (A/Mex/4108) virus, a 2009 pandemic H1N1 human
influenza strain that can directly infect mice without undergoing serial passage (Figure 3.8B).
As the onset of weight loss over the course of infection occurs more rapidly in our influenza
A/Mex/4108 virus model compared to our influenza A/PR/8 virus model, oseltamivir and hMSC
treatment was initiated on day 2 P.I. No difference in weight loss (Figure 3.8B left) or survival
(Figure 3.8B right) between experimental groups was observed.
75
Figure 3.8. hMSC adjunctive therapy may improve survival in experimental influenza A/PR/8 virus infection but not influenza A/Mex/4108 virus infection and hMSC adjunctive therapy does not alter weight loss kinetics in both models of experimental severe influenza. (A) Eight- week old male C57Bl/6 mice infected with 550EID50 influenza A/PR/8 virus were administered 5 mg/kg of oseltamivir in 100 ul ddH2O via gavage, twice daily for 5 days, either with or without hMSC treatment (2.5x105 cells), starting 5 days P.I. Left Weight loss kinetics over the course of infection were measured to assess morbidity in mice. Shown are the weight loss data up to day 8 P.I., when most mice reach euthanasia criteria. No difference between experimental groups was observed (Two-way ANOVA, n=29-32/group; 3 pooled experiments). Error bars represent standard deviation. Right Survival curves. Survival of mice treated with hMSCs and oseltamivir in combination was increased compared to control group; however, this was not significant. (logrank test p=0.47, n=29-32/group; 3 pooled experiments). (B) Eight- week old male C57Bl/6 mice infected with 104 EID50 influenza A/Mex/4108 virus were administered 5 mg/kg of oseltamivir in 100 ul ddH2O via gavage, twice daily for 5 days, either with or without hMSC treatment (2.5x105 cells), starting 2 days P.I. Left Weight loss kinetics over the course of infection were measured to assess morbidity in mice. Shown are the weight loss data up until day 7 P.I., when most mice reach euthanasia criteria. No difference between experimental groups was observed (Two-way ANOVA, n=9-12/group). Error bars represent standard deviation. Right Survival curve. No difference was observed (logrank test, n=9-12/group).
0 1 2 3 4 5 6 7 870
80
90
100
Day P.I. 550 EID50 A/PR/8
% o
f o
rig
inal
wei
gh
t (m
ean
±± ±± S
D)
0 2 4 6 8 10 120
20
40
60
80
100
Day P.I. 550 EID50 A/PR/8
Per
cent
su
rviv
al
0 1 2 3 4 5 6 770
80
90
100
Day P.I. 104 EID50 A/Mex/4108
% o
f o
rig
inal
wei
gh
t (m
ean
±± ±± S
D)
0 2 4 6 80
20
40
60
80
100
Day P.I. 104 EID50 A/Mex/4108
Per
cen
t su
rviv
al
A
B
OseltamivirOseltamivir + hMSC
PBSOseltamivirOseltamivir + hMSC
PBS
OseltamivirOseltamivir + hMSC
PBS OseltamivirOseltamivir + hMSC
PBS
76
In the A/PR/8 experimental model, mice from each experimental group as described above were
sacrificed on day 7 P.I. BAL fluid and lung homogenate levels of cytokines and chemokines
CCL2, CXCL10, KC, CCL5, and IFN-γ were quantified. BAL fluid and lung homogenate
cytokine and chemokine levels were similar for combination therapy treated mice compared to
control mice that received oseltamivir alone or PBS (Figure 3.9). CXCL10 was significantly
decreased in the lung parenchyma of mice that received oseltamivir, in combination with or
without hMSCs (One-way ANOVA, p<0.05). No difference in BAL fluid markers of acute lung
injury (total protein, IgM, and albumin) was observed on day 7 P.I. for hMSC treated mice
compared to control mice that received oseltamivir or PBS alone (Figure 3.10).
In our influenza A/Mex/4108 virus experimental model, mice from each experimental group as
described above were sacrificed on day 5 P.I. BAL fluid level of cytokines and chemokines
CCL2, CXCL10, KC, CCL5, and IFN-γ were quantified and no difference was observed on day
5 P.I. (Figure 3.11). No difference in BAL fluid markers of acute lung injury (total protein, IgM,
and albumin) was observed on day 5 P.I. for hMSC treated mice compared to control mice that
received oseltamivir or PBS alone (Figure 3.12).
77
Figure 3.9. hMSCs do not decrease BAL fluid or lung parenchymal inflammation when used as adjunctive therapy in mice infected with influenza A/PR/8 virus. Eight week-old male C57Bl/6 mice infected with 550 EID50 influenza A/PR/8 virus were sacrificed on day 7 P.I. Mice treated with antivirals were administered 5 mg/kg oseltamivir in 100 ul ddH20 once every 12 hours via gavage on day 5 P.I. for a maximum of 5 days. For the combined treatment group, 2.5x105 hMSCs were also administered via the tail vein, 5 days P.I. BAL was performed and lungs were excised. No significant difference in BAL fluid or lung parenchymal level of selected cytokines / chemokines were observed between hMSC treated mice in combination with oseltamivir treatment, compared to oseltamivir alone (one-way ANOVA, n=7/group). Error bars represent standard deviation. ND = nondetectable.
0
500
1000
1500
2000
2500
0
2
4
6IFNγγγγ
BAL LUNG
ND ND
Day 7 P.I.
BA
L p
g/m
l +
S
D
Lu
ng
ng
/ml +
SD
0
1
2
3
4
5
0
5
10
15
20
BAL LUNG
CCL2
ND
Day 7 P.I.B
AL ng/m
l + S
D
Lung n
g/m
l + S
D
0
300
600
900
1200
0
1
2
3
4p<0.05CXCL10
BAL LUNG
ND
Day 7 P.I.
BA
L p
g/m
l +
S
D
Lu
ng
ng
/ml +
SD
0
100
200
300
400
0
10
20
30
40
50
BAL LUNG
CCL5
ND
Day 7 P.I.
BA
L p
g/m
l +
S
D
Lu
ng
ng
/ml +
SD
PBSOseltamivirOseltamivir + hMSC
Uninfected
0
25
50
75
100
125
150
175
0
50
100
150
200
250
300
350KC
BAL LUNG
BA
L p
g/m
l +
SD
Lu
ng pg
/ml +
SD
Day 7 P.I.
78
Figure 3.10. hMSCs fail to decrease acute lung injury when used as adjunctive therapy in mice infected with influenza A/PR/8 virus. Eight week-old male C57Bl/6 mice infected with 550 EID50 influenza A/PR/8 virus were sacrificed on day 7 P.I. Mice treated with antivirals were administered 5 mg/kg oseltamivir in 100 ul ddH20 once every 12 hours via gavage on day 5 P.I. for a maximum of 5 days. For the combined treatment group, 2.5x105 hMSCs were also administered via the tail vein, 5 days P.I. BAL was performed. No significant difference in BAL fluid total protein, IgM or albumin was observed between hMSC treated mice in combination with oseltamivir treatment, compared to oseltamivir alone (one-way ANOVA, n=7/group). Error bars represent standard deviation. ND = nondetectable.
0
1
2
3
4TOTAL PROTEIN
Day 7 P.I.
BA
L m
g/m
l +
SD
0
300
600
900
1200ALBUMIN
Day 7 P.I.
NDBA
L u
g/m
l +
S
D
0
1
2
3
4
5
6IgM
Day 7 P.I.
NDBA
L u
g/m
l +
S
D
PBSOseltamivirOseltamivir + hMSC
Uninfected
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Figure 3.11. hMSCs do not decrease BAL fluid or lung parenchymal inflammation when used as adjunctive therapy in mice infected with influenza A/Mex/4108 virus. Eight week-old male C57Bl/6 mice infected with 104 EID50 A/Mex/4108 virus were sacrificed on day 5 P.I. Mice treated with antivirals were administered 5 mg/kg oseltamivir in 100 ul ddH20 once every 12 hours via gavage on day 2 P.I. for a maximum of 4 days. For the combined treatment group, 2.5x105 hMSCs were also administered, via the tail vein, 2 days P.I. BAL was performed. No significant difference in BAL fluid or lung parenchymal level of selected cytokines / chemokines was observed between hMSC treated mice in combination with oseltamivir treatment, compared to oseltamivir alone (one-way ANOVA, n=5/group). Error bars represent standard deviation. ND = nondetectable.
0
500
1000
1500
2000
2500 IFNγγγγ
Day 5 P.I.
ND
BA
L p
g/m
l + S
D
0
50
100
150
200
250 KC
ND
Day 5 P.I.
BA
L p
g/m
l + S
D
0
200
400
600
800 CCL2
ND
Day 5 P.I.
BA
L p
g/m
l + S
D
0
100
200
300
400 CCL5
ND
Day 5 P.I.
BA
L p
g/m
l + S
D
0
500
1000
1500 CXCL10
ND
Day 5 P.I.
BA
L p
g/m
l + S
D
PBSOseltamivirOseltamivir + hMSC
Uninfected
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Figure 3.12. hMSCs fail to decrease acute lung injury when used as adjunctive therapy in mice infected with influenza A/Mex/4108 virus. Eight week-old male C57Bl/6 mice infected with 104 EID50 influenza A/Mex/4108 virus were sacrificed on day 5 P.I. Mice treated with antivirals were administered 5 mg/kg oseltamivir in 100 ul ddH20 once every 12 hours via gavage on day 2 P.I. for a maximum of 4 days. For the combined treatment group, 2.5x105 hMSCs were also administered, via the tail vein, 2 days P.I. BAL was performed. No significant difference in BAL fluid total protein, IgM or albumin was observed between hMSC treated mice in combination with oseltamivir treatment, compared to oseltamivir alone (one-way ANOVA, n=5/group). Error bars represent standard deviation. ND = nondetectable.
0
1000
2000
3000
4000 ALBUMIN
ND
BA
L u
g/m
l + S
D
Day 5 P.I.0
250
500
750
1000 IgM
ND
BA
L n
g/m
l +
SD
D ay 5 P .I .0.0
0.5
1.0
1.5
2.0 TOTAL PROTEIN
BA
L m
g/m
l +
SD
Day 5 P.I.
PBSOseltamivirOseltamivir + hMSC
Uninfected
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3.4 DISCUSSION
MSC-based prophylactic and treatment strategies have yielded significant therapeutic benefits in
various pre-clinical models of acute lung injury [209,210,245-248]. These encouraging findings
in which MSCs were efficacious led us to hypothesize that MSCs might dampen host
inflammation, decrease acute lung injury, and improve survival in an experimental model of
severe influenza, characterized by heightened inflammation and acute lung injury. Despite these
positive pre-clinical findings, our study demonstrated that hMSCs were unable to affect
inflammation, lung injury and survival in experimental influenza. This finding is in accordance
with few published reports where MSCs were unable to dampen host inflammation and improve
outcome in vivo [249-251].
Clinical management of severe influenza complicated by ARDS is often guided by the
ALI/ARDS literature. For example, it is recommended that lung-protective ventilations
strategies such as low tidal volume ventilation be used in managing patients with severe
influenza complicated by ARDS, based on recommendations of the ARDSNet trial [278]. Given
the demonstrated utility of drawing from ALI/ARDS literature to inform potential treatment
strategies for influenza, it was reasonable to evaluate the therapeutic potential of MSCs in the
context of severe influenza. Despite similarities in clinical presentation and pathophysiology of
ALI and severe influenza, our negative findings reinforce that viral-induced ALI is not wholly
comparable with other forms and etiologies of ALI, such as bacterial derived ALI.
We first established the influenza A/PR/8 virus mouse model of severe influenza, characterized
by the development of inflammation and acute lung injury, and evaluated the use of hMSCs for
the prevention or treatment of severe disease (Fig. 3.1,3.2). While influenza A/PR/8 virus
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infection induced a host response and lung injury in vivo, we do not know whether the host
response was specifically dysregulated, as we did not compare host responses between various
influenza virus infection models. However, in our model, approximately 80% of mice reached
euthanasia criteria - defined by 20% weight loss - where such weight loss was associated with
elevated inflammation.
The failure of hMSCs to suppress inflammation and decrease lung injury could be attributed to
myriad factors. Firstly, the hMSCs may have rapidly differentiated in culture which rendered
them ineffective as immunosuppresive agents. In this study, hMSCs were cultured per the
“manufacturer’s guidelines”, in terms of low passage number, medium and FBS stocks and
concentrations, and cell harvesting at log phase of growth, as these conditions have been deemed
optimal [279]. Despite these attempts to create optimal conditions, it is possible that the cultures
spontaneously differentiated in vitro rendering them ineffective in vivo. Also, the structure and
function of hMSCs tend to vary between donors. For example, Francois et al. found that the
amount of IDO produced by each hMSC donor varied, thereby influencing their
immunosuppressive potential [220]. Nevertheless, culture-expanded MSCs tested in our
experimental model always exhibited a spindle-shaped fibroblastic morphology and were
deemed to be of high quality as indicated by measuring the number of colony forming units (a
generally accepted indicator of MSC quality [279]) (data not shown).
Our results may also be explained if the hMSCs were unable to home to the lungs in our
experimental model. Varied protocols to isolate and expand MSC populations have been
reported to influence MSC homing capacity. For example, protocols which employ progressive
subculturing are associated with a loss of chemokine receptors and decreased chemotactic
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responses [232,235,236]. MSCs delivered intravenously would reach the pulmonary
microcirculation first; therefore, homing might not be of concern when lungs are the end target
organ. For example, MSCs may be trapped in the lung as microemboli after IV infusion [258].
Mei et al. also found an average 47% of injected MSCs in the lungs 15 minutes after MSC
delivery into LPS-challenged mice [210]. However, we did not test for the presence of MSCs in
the lung after IV delivery in our model, therefore, we can not definitively conclude that the cells
did in fact reach the lungs.
If the cells were indeed delivered to the lungs when administered intravenously in our model of
influenza, must they have remained in the lungs in order to elicit a beneficial effect? Since we
employed allogeneic MSCs, HLA mismatching may have led to rapid rejection after injection.
This result would be consistent with other reports where allogeneic murine MSCs were rejected
when administered in MHC mismatched mice compared to autologous cell transplant [227,228].
Additionally our cells were derived from human donors, rather than extracted from mice, which
could have exacerbated MSC rejection in vivo. This strategy was employed because human
clinical trials involving MSCs employ MSCs derived from human donors and a similar cross-
species strategy was employed by others in an attempt to elucidate the mechanism of human
MSC mediated effects [258,259]. They found that hMSCs attenuated lung inflammation and
injury (similar to mouse MSCs) which suggests that the beneficial effects are not restricted to
mouse MSCs in mice. Furthermore, MHC class II antigens are not expressed on human MSCs
[223] but are constitutively expressed on murine MSCs [227]. Therefore, human MSC rejection
may be less likely than murine MSC rejection. Indeed, patients who underwent MSC
allotransplant experienced no anti-allogeneic MSC antibody formation or T-cell sensitization
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[224], suggesting human MSCs may not be rejected by the recipient and are indeed
immunoprivileged.
Even if the cells were not rejected, their in vivo life span may nonetheless have been too short to
mediate outcome. The majority of MSCs were lost from the lung at 3 and 14 days, respectively,
after delivery to mice with LPS induced lung injury [209-211]. These findings could suggest that
in our experimental model of severe influenza, MSCs did not persist long enough to induce a
beneficial response. However, these studies found MSCs to improve outcome in experimental
lung injury despite low levels of MSC retention in the lungs. Therefore, the reported benefit in
response to cell-based therapy in lung injury models does not necessarily require a high level of
long-term MSC persistence in the lung. Furthermore, MSCs may need not reach the lungs to
exert their effects. For example, intraperitoneal administration of hMSCs attenuated LPS-
induced lung inflammation, despite being less effective than IV delivery or oropharyngeal
aspiration [259].
Alternatively, our ascribed cell dose could have been too low to exert an immunosuppressive
effect, regardless of where the cells localize and/or persist in vivo. We consistently administered
a 2.5x105 dose of hMSCs, via the tail vein, in our experimental influenza model as this dose was
effective in a report by Mei et al. [210]. However, in that report cells were administered via the
jugular vein and not the tail vein, which could potentially account for the observed difference;
cells introduced via the jugular vein have less distance to travel and therefore less potential to be
destroyed before reaching the lungs, compared to cells administered via the tail vein. Indeed,
Kim et al. found that a dose of 1x106 cells resulted in a significantly prolonged life span and a
significantly higher number of motor neurons in mice with amyotrophic lateral sclerosis
compared to untreated mice or mice treated with 1x104 cells [280]. Additionally, several positive
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findings regarding the use of MSC therapy in lung injury models reported administration of 5 x
105 cells and 7.5 x 105 cells [245,247]. Therefore, it is possible that, as opposed to having too few
cells persist in the lung, the problem was created at the outset with an insufficient dosage of
MSCs delivered via the tail vein. This issue could be circumvented by injecting more cells.
However, rats administered 5 million cells at one time point or 2 million cells at two time points
in a transplantation model did not suppress immunity but rather accelerated graft rejection [251].
As alluded to earlier, the route of MSC delivery may have affected our findings. MSCs
delivered directly into the lung airspaces have been shown to decrease cytokines in BAL fluid
and improve survival after endotoxin induced lung injury [247]. However, MSCs delivered
intravenously in a similar model did not reduce BAL inflammatory cytokines [246]. Therefore,
it is possible that direct rather than IV MSC delivery to the lungs of influenza virus-infected mice
might have improved MSC mediated immunosupression and outcome in our experimental
model. However, MSCs administered by various methods in addition to intrapulmonary delivery
- including tail vein, jugular vein, and intraperitoneal delivery - were effective in improving
outcome in experimental lung injury [209,210,245,246,259]. Therefore, while route of delivery
may be important, it is likely insufficient to explain our negative results.
Given the vast amounts of literature describing the immunosuppressive functions of MSCs, it is
reasonable to question whether MSC immunosuppression could diminish the ability of the host
to control virus replication and mediate viral clearance. However, we did not observe any
increase in viral load with hMSC therapy (Fig. 3.3C). To the contrary, recent in vivo studies
have provided evidence that MSCs may enhance antimicrobial immune effector cell function and
reduce microbial burden during infectious challenge [221,244]. For example, Mei et al. reported
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decreased total bacterial counts in septic mice, which was hypothesized to be attributed to
increased splenic monocyte and macrophage phagocytosis [244]. In our model, influenza viral
titer might have been too overwhelming for any anti-microbial effects to have occurred, or the
hMSCs may have been unable to exert anti-viral compared to anti-bacterial effects.
Alternatively, it is possible that the influenza virus in the lung itself interacted with the MSCs in
a manner which prevented the MSCs from exerting their immunosuppressive activity.
Temporary inactivation of MSC mediated immunosuppression induced by viral activation of
toll-like receptors (TLRs) 3 and 4 on MSCs has been described [281]. This finding may
represent a mechanism that allows the immune system to function effectively in the presence of
virus or bacteria. This finding could account for reduced MSC functionality in the course of
infection since influenza RNA can serve as a ligand for TLR3.
As the induction of MSC immunosuppression highly depends on the microenvironment into
which the cells are introduced, when is the ideal time to administer MSCs in severe influenza?
hMSCs were delivered early (day 2 P.I.) or late (day 5 P.I.) in the course of infection when lung
inflammation was minimal or elevated, respectively. In both regimens, MSCs failed to dampen
host lung inflammation, decrease acute lung injury, or improve morbidity and mortality (Fig.
3.3-3.5). Despite many in vitro findings of MSC mediated immunosuppression, the
microenvironment in vivo is much more variable and unpredictable. When applying MSCs to an
animal model of disease, MSC immunosuppression might be easily turned off in vivo, or rather
immunostimulatory, as has been reported in other models of disease [250]. This may account for
the inability of MSCs to dampen host inflammation in our animal model of severe influenza.
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Unfortunately, there is no validated method of measuring MSC bioactivity in vivo, which
generates further challenges [282].
Another discrepancy which may account for observed differences between the effects of MSCs
in our model of lung injury and those with reported MSC therapeutic benefit is the length of time
between introducing the injury-causing agent, and the inflammatory response / onset of disease.
Most models of acute lung injury result in acute lung inflammation which peaks within 24-48
hours then resolves shortly thereafter, such as in the endotoxin lung injury model. In our model
of severe influenza virus-induced lung injury, the host response did not ensue until
approximately 4 days post-influenza virus infection - and mice could remain alive for up to 9
days P.I. before they succumbed to death. This may have important implications for our
findings. MSCs introduced to the lung on day 5 post-influenza virus infection may not have
exerted effects which lasted long enough to affect the outcome of influenza virus-infected mice.
Additionally, MSCs administered prophylactically or early in the course of infection may not
have been sufficiently stimulated by the host microenvironment, as the host inflammatory
response was only detected beginning around 4 days post-influenza virus infection (Fig. 3.2).
Contrary to our hypothesis, we found that MSCs administered prophylactically increased
morbidity of influenza virus-infected mice; however, this finding was not significant (Fig. 3.7).
An explanation for these effects could be that insufficient in vivo levels of pro-inflammatory
cytokines rendered MSCs immune-enhancing, despite MSC ex-vivo pre-stimulation for one
group of MSC treated mice in an attempt to counter this effect. Nevertheless, MSCs administered
concurrently with lung injury or before lung injury in experimental models have been effective
[209,245,246]. In fact, Ortiz et al. reported that MSCs were only effective when administered at
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the same time as bleomycin [245]. Therefore, MSCs can exert their beneficial effects in the
initial stages of injury, albeit in a model of bleomycin induced injury and not viral induced
injury. Although timing of MSC administration is likely to be an important factor, it is difficult
to conclude that the non- or minimally inflammatory environment into which the MSCs were
introduced when administered prophylactically or early in the course of influenza virus infection
is the reason for which the cells lacked efficacy in improving morbidity and mortality.
MSCs administered in combination with oseltamivir also had no effect (Fig. 3.8 – 3.12). Since
the mice treated with oseltamivir only did not show improved survival in our influenza A/PR/8
virus or influenza A/Mex/4108 virus experimental models, we postulated that the infection may
simply have been too severe for any agent to improve outcome. In particular, the absence of
clinical effect could have been because the high levels of inflammation present in severe
influenza may have overcome the immunosuppressive properties of MSCs in vivo. This is
consistent with the absence of clinical effect in experimental GvHD and rheumatoid arthritis,
both diseases characterized by overwhelming inflammation [249,250]. However, this may not
necessarily be the case in our study since MSCs have proven effective in pre-clinical sepsis, a
disease characterized by an extreme systemic inflammatory response [221].
While antiviral treatment was initiated late in the course of infection to re-capitulate the clinical
scenario, the timing of administration was likely too late to inhibit viral replication, since viral
load has been shown to peak in the lungs 24-72 hours after infection [88]. Oseltamivir is also
known to have limited efficacy when administered beyond 48 hours post-onset of symptoms –
mice begin to lose weight around day 5 P.I. which could be deemed the onset of influenza
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symptoms, however, it is difficult to correlate symptom onset in humans with our experimental
animal model.
Despite a lack of efficacy with oseltamivir treatment, we observed decreased levels of IFN-γ,
CCL2, CCL5 and KC in BAL fluid and significantly decreased CXCL10 in lung interstitium of
oseltamivir-treated mice, independent of MSC treatment (Fig. 3.9). Wong et al. noted
oseltamivir could dampen host inflammation independent of an antiviral effect, which may
explain our findings [283]. Similarly, others have also hypothesized that a dampened immune
response, including decreased cytokine production, may be the mechanism of action of
oseltamivir, rather than influenza virus replication inhibition [284]. However, it is difficult to
determine whether our finding was attributable to oseltamivir immunomodulatory effects or to an
indirect effect of oseltamivir on viral replication, as lung viral load was not quantified in this set
of experiments.
Noteworthy, decreased lung inflammation (albeit insignificant) with oseltamivir treatment did
not correlate with improved survival in our experimental model. This may indicate that our
experimental model was too severe, as not even a modest ability to dampen host inflammation
was able to translate into decreased morbidity and mortality. Related, neither oseltamivir or
oseltamivir/MSC combination treatment administered starting day 2 post infection in the 2009
pandemic H1N1 experimental influenza (A/Mex/4108) virus model had any effect. Thus,
although influenza A/Mex/4108 virus was a human strain which is not murine-adapted (thereby
having possibly greater clinical relevance to human influenza virus infection than influenza
A/PR/8 virus) our dose of 104 EID50 influenza A/Mex/4108 virus was probably too
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overwhelming to observe any therapeutic effects of either oseltamivir or MSCs as the influenza
virus dose proved to be 100% lethal regardless of the treatments applied.
Finally, although MSC-secreted proteins have been attributed to MSC-mediated improved
outcome in pre-clinical models of lung injury, they may not have been useful in the treatment of
severe influenza, despite underlying pathophysiology common to lung injury derived from viral
or bacterial derived pneumonia, sepsis, or trauma. For example, althought it may be beneficial to
administer recombinant TSG-6 as treatment for LPS-induced lung injury in mice [259], TSG-6
treatment for severe influenza may be detrimental due to its ability to upregulate COX-2 [285],
as COX-2 upregulation may contribute to increased morbidity and mortality in severe influenza
(discussed in chapter 1). The same reasoning is applicable to MSC upregulation of PGE2, which
is generated by conversion of arachidonic acid via COX-2. Furthermore, IFN-γ and TNF-
induced upregulation of MSC secreted factors may have exacerbated disease severity; this may
explain why MSCs pre-incubated with these cytokines resulted in increased morbidity and
mortality (Fig. 3.7). Further evaluation of individual MSC-secreted proteins for treating severe
influenza may be warranted.
In conclusion, our study was not able to demonstrate the therapeutic potential of MSCs in
experimental severe influenza in mice. Given the overwhelming number of publications
identifying MSCs as an effective treatment in a variety of disease models, we believe that our
findings are valuable to the field of MSC research as they provide a noteworthy contrast to some
of the existing literature in this field. Ongoing clinical trials should reveal the true potential of
these cells and provide insight as to whether further investigation of MSCs for severe influenza
treatment is warranted.
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Chapter 4: Summary and Future Directions
4.1 Summary
Antiviral drugs currently approved for the treatment of influenza have inherent therapeutic
limitations, including unknown efficacy in treating severe disease or complications, timing of
administration (i.e. limited clinical impact when administered beyond the first 48 hours of
symptom onset) and rising antiviral resistance. Therefore, we investigated the use of iNO as a
new antiviral treatment for severe influenza (Chapter 2). Unfortunately, we found that iNO was
unable to dampen the viral load and thus improve outcome in experimental severe influenza.
This may have been due to the instability and short half-life of NO and the toxic effects of NO
metabolites. In future, there may be merit to exploring another vehicle of NO delivery which
could perhaps stabilize nitric oxide without generating toxic effects. Overall, our findings
emphasize the limitations of drawing conclusions from in vitro experimental findings, as the host
response in vivo is much more complex; in vitro findings may not reflect what will be
correspondingly observed in vivo where myriad pathways are activated during infection.
Severe influenza is associated with an overexuberant host-mediated innate immune response,
leading to excessive inflammation, tissue injury and acute lung injury. It is clear that this
deleterious inflammatory host response is a major contributor to influenza-related morbidity and
mortality. Development of novel and innovative adjunctive treatment strategies that target the
host response to the influenza virus may be critically important for clinical outcome in the case
of severe disease. It is clear that severe influenza with acute lung injury is a highly complex
disease process involving many pathophysiologic manifestations. Given this, it is not surprising
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that strategies targeting one aspect of the disease process have yet to prove efficacious. This
suggests that a new multifaceted therapeutic approach, aimed at reducing lung injury while
maintaining a balanced immune response and facilitating lung repair, could be of greatest value.
In this regard, mesenchymal stem cells were an obvious choice warranting investigation as a new
treatment approach to lung injury and enhanced inflammation developed in severe influenza due
to their previous demonstrated effects in pre-clinical models of lung injury (Chapter 3).
However, contrary to our hypothesis, we found that MSCs did not reduce cytokine/chemokine
responses or improve ALI in our murine influenza model. Our experimental findings reinforce
that, despite similarities in clinical presentation and pathobiology of acute lung injury caused by
various sources, beneficial effects in other models of ALI/ARDS may not necessarily be
translated to influenza-virus induced ALI.
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4.2 Future directions
Our main focus was to test whether MSCs, given alone or as adjunctive therapy, could reduce
host inflammation, specifically, inflammatory cytokines correlated with severe disease and
protein level in the lungs - a hallmark of ALI. We were unable to identify any beneficial effect;
therefore, a number of plausible explanations merit further investigation. For example, were
MSCs in fact mediating the host response? We did not provide any indication for MSC
biological activity in vivo and host interaction. To determine this, we could investigate
biomarkers of MSC activity in vivo. Specifically, BAL fluid levels of important MSC-secreted
proteins, including KGF, Ang-1 and PGE2, could be measured following MSC delivery.
In light of the fact that oseltamivir did not decrease morbidity and mortality in our experimental
model, we could not conclude that MSCs were ineffective as an adjunctive therapeutic agent.
Therefore, studies to optimize oseltamivir dosing were recently completed (data not shown).
Further examination of an MSC and oseltamivir combination treatment strategy - with a dose of
oseltamivir that improves survival by ~30% when used alone - is underway.
Further studies could address the hypothesis that insufficient MSC dosing led to sub-optimal in
vivo effects, as the optimal MSC dose necessary to obtain a therapeutic benefit remains
undefined. A report by Kim et al. found MSC mediated effects in vivo to positively correlate
with increasing cell dose [280]. Therefore, we could determine whether provision of 1 million
MSCs to influenza virus-infected mice improves outcome. We could also try intratracheal or
intrapulmonary delivery of cells to influenza virus-infected mice, as there is no standardized
method of MSC delivery that achieves greatest clinical efficacy (as discussed in Chapter 3).
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While it would be favourable to develop a reliable protocol to culture MSCs, it may also be
valuable to assess other in vitro culturing systems. Specifically, MSCs could be grown as 3D
spheroids under the “hanging drop” method as this form of culture has been shown to enhance
MSC mediated immunosuppression, compared to monolayer culture, in a co-culture system with
LPS-activated macrophages and in a mouse model of zymosan induced peritonitis [286]. It may
also be valuable to assess MSC delivery in a more stable form. To protect against MSC
degradation, delivery of hydrogel-encapsulated MSCs as described by Karoubi et al. could
provide enhanced cell survival in the lungs [287]. Alternatively, MSC cultures could potentially
be modified to improve their immunosuppressive properties.
Lastly, if these investigations reveal a role for MSC-mediated improvement in severe disease,
therapeutic strategies based on enhancement of MSC-secreted proteins could be considered. We
have not found any literature that reports a therapeutic benefit with the use of MSC-secreted
mediators in severe influenza. However, single MSC-released mediators have been effective in
improving outcome in pre-clinical models of lung injury. For example, intrapulmonary delivery
of recombinant human TSG-6, a known MSC mediator, reduced LPS-induced inflammation in
the lung [259]. In particular, proteins which could affect the endothelial barrier may be of
interest as inhibition of endothelial activation/dysfunction and associated capillary leak has
recently been shown to increase survival of H5N1 virus-infected mice, without altering cytokine
levels or viral load [288]. Binding of the ligand Slit to its endothelial receptor Robo4 inhibits
hyperpermeability induced by VEGF and promotes cell-surface expression of adherens junction
protein VE cadherin [288,289]. London et al. significantly improved survival in experimental
H5N1 influenza murine models by administering the active fragment of Slit, Slit2N, which is
released via proteolytic cleavage upon binding to Robo4 [288]. This study suggests that
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strengthening the vascular-endothelial barrier may be an important therapeutic target for severe
influenza treatment.
With this in mind, we could evaluate the role of MSC-secreted protein Ang-1, a potent mediator
of angiogenesis that functions to prevent leakage and promote blood vasculature quiescence, in
the course of severe influenza virus infection. Should Ang-1 be dysregulated, we could assess
Ang-1 rescue strategies in restoring endothelial integrity in the lungs, such as administration of
Ang-1 via overexpression in a viral vector. Increasing Ang-1 levels to preserve endothelial
integrity may prove to be an effective strategy to prevent or ameliorate ALI caused by severe
influenza.
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4.3 Final considerations
Antiviral resistance against influenza, including dual (adamantane and oseltamivir) resistance
[290], is likely to continue to increase, posing further public health concerns. Therapeutic
strategies employing antiviral agents alone are unlikely to significantly improve overall clinical
outcome of individuals with severe influenza. Therefore, interest in adjunctive
immunomodulatory strategies for the treatment of influenza needs to grow, and innovative
approaches targeting the host response to influenza need to emerge.
The current, limited experimental evidence to support the use of immunomodulatory therapy
must be strengthened. Promising approaches to combined antiviral and immunomodulatory
therapy for influenza have been demonstrated by Zheng et al. using celecoxib, mesalazine and
zanamivir in pre-clinical models of severe influenza [162]. Further study of such approaches is
clearly warranted. Based on the promising results in pre-clinical models of severe influenza,
combination therapy employing PPAR agonists and/or COX-2 inhibitors with conventional
antiviral agents should be studied in randomized clinical trials of severe influenza.
Infection and subsequent death due to avian influenza A (H5N1) is likely to continue. In the
event that the H5N1 virus develops the ability to transmit from human-to-human, a pandemic
may occur. Furthermore, as evidenced by 2009 pandemic H1N1 influenza, it is not currently
possible to predict which influenza virus strain will give rise to the next pandemic, and how
virulent that strain will be. New adjunctive treatment strategies, in addition to new antiviral
therapy, will likely be required to significantly improve clinical outcome.
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Finally, immunomodulatory strategies that have yielded positive results in pre-clinical and
clinical studies of ALI/ARDS may have therapeutic implications for the management of severe
influenza. Targeted strategies to preserve vascular-endothelial integrity in an effort to prevent
influenza-related end-organ dysfunction/injury, including ALI/ARDS, should emerge as an
innovative approach to improve clinical outcome in severe influenza.
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Chapter 5: References
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