Mucosal Immunity in Mycobacterial Infections

Anna Tjärnlund

Stockholm University

All previously published papers were reproduced with permission from the publishers. © Anna Tjärnlund, Stockholm 2007 ISBN 91-7155-388-6 Printed in Sweden by Universitetsservice AB, Stockholm 2007 Distributor: Stockholm University Library

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Integrity without knowledge is weak and useless,

and knowledge without integrity is dangerous and dreadful.

Samuel Johnson (1709-1784) English author, critic, & lexicographer

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SUMMARY

More than a century after the identification of the tubercle bacillus and the first attempts at

vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious

diseases. TB, caused by the bacterium Mycobacterium tuberculosis, is typically a disease of

the lung, which serves both as the port of entry and as the major site of disease manifestation.

The currently used vaccine, BCG, is administered parenterally and induces a systemic

immune response. However, it fails to protect against pulmonary TB, thereby raising the

question whether vaccination targeting the mucosal immunity in the lungs could be more

favourable.

The respiratory mucosal surfaces represent the first line of defense against a multitude of

pathogens. Secretory IgA in mucosal secretions has an important function by blocking the

entrance of pathogenic organisms and preventing infections. Additionally, a role for IgA in

the modulation of immune responses is currently being revealed. In this work, we investigated

the relevance of mucosal IgA in the protection against mycobacterial infections using mice

deficient for IgA and the polymeric Ig receptor, which is the receptor responsible for mucosal

secretions of IgA. Gene-targeted mice were more susceptible to mycobacterial infections in

the respiratory tract and displayed reduced production of proinflammatory, and protective,

factors such as IFN-γ and TNF-α in the lungs. The mechanisms explaining the defective

proinflammatory responses in the lungs of deficient mice might involve impaired signalling

through Fcα receptors, or homologous receptors, which could lead to an inadequate activation

of pulmonary macrophages. This could subsequently result in suboptimal induction and

production of cytokines and chemokines important for the attraction and migration of immune

cells to the site of infection.

An induction of optimal adaptive immune responses to combat mycobacterial infections

requires prompt innate immune activation. Toll-like receptors (TLRs) are vital components of

the innate branch of the immune system, ensuring early recognition of invading pathogens.

Using TLR-deficient mice we demonstrated an important role for TLR2, and partly TLR4, in

the protection against mycobacterial infection in the respiratory tract. TLR2-deficient mice

failed to induce proper proinflammatory responses at the site of infection, and macrophages

derived from the knockout mice displayed impaired anti-mycobacterial activity.

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Experimental evidence has concluded that the immune response upon an infection can

influence the outcome of succeeding infections with other pathogens. Concurrent infections

might additionally interfere with responses to vaccinations and have deleterious effects. We

developed an in vitro model to study the effect of a malaria infection on a successive M.

tuberculosis infection. Our results demonstrate that a malaria blood-stage infection enhances

the innate immune response to a subsequent M. tuberculosis infection with a Th1 prone

profile. Reduced infectivity of malaria-exposed dendritic cells implies that a malaria infection

could impose relative resistance to ensuing M. tuberculosis infection. However, a prolonged

Th1 response may interfere with malaria parasite control.

The outcome of this work emphasizes the importance of generating effective immune

responses in the local mucosal environment upon respiratory mycobacterial infections. It

furthermore puts new light on the immunological interaction between parasites and

mycobacteria, which could have implications for future vaccine research.

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ORIGINAL ARTICLES

This doctoral thesis is based on the following papers, which are referred to by their Roman

numerals in the text:

I. Rodríguez, A*., Tjärnlund, A*., , Ivanyi, J., Singh, M., García, I., Williams, A.,

Marsh, P. D., Troye-Blomberg, M., and Fernández, C. (2005). Role of IgA in the

defense against respiratory infections. IgA deficient mice exhibited increased

susceptibility to intransal infection with Mycobacterium bovis BCG. Vaccine

23(20): 2565-2572.

II. Tjärnlund, A*., Rodríguez, A*., Cardona, P-J., Guirado, E., Ivanyi, J., Singh, M.,

Troye-Blomberg, M., and Fernández, C. (2006). Polymeric Ig receptor knockout

mice are more susceptible to mycobacterial infections in the respiratory tract. Int.

Immunol. 18(5):807-816.

III. Tjärnlund, A., Guirado, E., Julian, E., Cardona, P-J., and Fernández, C. (2006).

Determinant role for TLR signalling in acute mycobacterial infection in the

respiratory tract. Microbes Infect. 8(7):1790-1800.

IV. Tjärnlund, A., Troye-Blomberg, M., Pawlowski, A. (2007). The effect of malaria

parasite-derived materials on dendritic cell susceptibility and response to

subsequent Mycobacterium tuberculosis infection. Submitted.

* These authors contributed equally

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CONTENTS SUMMARY 5 ORIGINAL ARTICLES 7 ABBREVIATIONS 11 INTRODUCTION 13 TUBERCULOSIS 13 MYCOBACTERIAL INFECTIONS 13

Immune evasion 14 IMMUNE RESPONSE TO MYCOBACTERIAL INFECTIONS 15

Innate immunity 15 Macrophages 16 Dendritic cells 17 Neutrophils 18 Toll-like receptors 19 γδT cells 20 Adaptive immunity 21 Cellular immunity 22 CD4+ T cells 22 CD8+ T cells 24 Humoral immunity 25 Granuloma formation 25

TB: THE DISEASE 26 Diagnosis 27 Tuberculin skin test 27 Microscopy 27 Cultivation 28 Molecular methods 28 Treatment 28 The current BCG vaccine 29 New vaccine candidates 30

ANIMAL MODELS OF TB 31 MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT 33 SPECIFIC IMMUNE RESPONSES 33 SECRETORY IgA 35 FUNCTIONS OF IgA 36 IgA RECEPTORS 37 IgA DEFICIENCY 39 MALARIA 40 PARASITE LIFE CYCLE 40 IMMUNITY TO BLOOD-STAGE MALARIA 41

Adaptive immunity 42

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Humoral immunity 42 Cellular immunity 43 DCs in malaria 43

CO-INFECTION BETWEEN TB AND MALARIA 45 ANIMAL STUDIES 46 PRESENT STUDY 47 AIMS 47 MATERIALS AND METHODS 48 RESULTS AND DISCUSSION 48 PAPER I 48 PAPER II 51 PAPER III 54 PAPER IV 58 CONCLUDING REMARKS 62 ACKNOWLEDGEMENTS 64 REFERENCES 68

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ABBREVIATIONS

APC Antigen-presenting cell

BAL Broncho-alveolar lavage

BCG Mycobacterium bovis Bacillus Calmette-Guérin

CFU Colony forming unit

CR Complement receptor

CT Cholera toxin

DC Dendritic cell

DC-SIGN DC-specific intercellular adhesion molecule-3 grabbing

nonintegrin

dIgA Dimeric immunoglobulin A

DOTS Directly observed treatment short-course

FcαR Fc receptor for immunoglobulin A

FcR Fc receptor

GPI Glycosylphosphatidylinositol

HIV Human immunodeficiency virus

Hz Hemozoin

Ig Immunoglobulin

i.n. Intranasal

iNOS Inducible nitric oxide synthase

IFN-γ Interferon-gamma

IL Interleukin

IRAK Interleukin-1 receptor associated kinases

i.v. Intravenous

M cell Microfold cell

MALT Mucosa-associated lymphoid tissue

MDR Multidrug-resistant

MHC Major histocompatibility complex

MR Mannose receptor

MyD88 Myeloid differentiation factor 88

NO Nitric oxide

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pIgA Polymeric immunoglobulin A

pIgR Polymeric immunoglobulin receptor

PPD Purified protein derivative

PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1

PfRBC Plasmodium falciparum-infected red blood cell

RANTES Regulated upon activation normal T-cell sequence

RBC Red blood cell

RNI Reactive nitrogen intermediate

SC Secretory component

SIgA Secretory immunoglobulin A

SIgAD Selective immunoglobulin A deficiency

TB Tuberculosis

TCR T-cell receptor

TGF-β Transforming growth factor-beta

Th Helper-T cell

TLR Toll-like receptor

TNF-α Tumor necrosis factor-alpha

TST Tuberculin skin test

XDR Extensively drug-resistant

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INTRODUCTION

TUBERCULOSIS

Tuberculosis (TB) remains one of the leading infectious diseases and causes high mortality in

humans, resulting in almost 2 million deaths annually (WHO, 2007). The increasing global

health burden of TB is due both to the synergistic pathogenesis of co-infection with the

human immunodeficiency virus (HIV), as well as to the continued dissemination of

multidrug-resistant (MDR) Mycobacterium tuberculosis strains (Toossi, 2003; Coker, 2004).

Despite this alarming health challenge, the capacity for treating and preventing TB remains

limited, and a uniformly effective vaccine is lacking.

The M. tuberculosis complex, the cause of TB, is comprised of M. tuberculosis, M. bovis, M.

africanum, M. microti and M. canetti. Although all members can cause TB, M. tuberculosis is

the most prevalent. The natural reservoir of M. tuberculosis and M. canetti is limited to

humans and that of M. microti is mainly limited to small rodents (Kremer et al., 1998). In

contrast, the host range of M. bovis is very broad and this species can cause disease among a

wide range of wild and domestic animals, as well as in humans (Ayele et al., 2004). M.

africanum has been isolated from humans and various animal species (Thorel, 1980;

Alfredsen and Saxegaard, 1992). All members in the complex are slow-growing organisms,

with generation times ranging from 12 to 24 hours depending on environmental and microbial

variables.

MYCOBACTERIAL INFECTIONS

The causative agent of infectious TB, M. tuberculosis, is a rod-shaped, obligate aerobic

bacillus, which is shielded by a unique wax-rich cell wall composed of long-chain fatty acids,

glycolipids and other components (reviewed in Kaufmann, 2001). This robust cell wall of the

bacteria contributes to intracellular survival in host phagocytes.

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TB can manifest itself at any tissue site, but the lungs represent both the main port of entry

and the most important site of disease manifestation. Droplets containing bacilli are expelled

from individuals with active pulmonary TB, and subsequently inhaled into the respiratory

tract (Riley et al., 1995) and phagocytosed by alveolar macrophages. Only particles less than

5 μm in diameter can gain access to the alveoli (Hatch, 1942), where macrophages, resident

within the alveolar space, can phagocytose the bacillus. Most of the bacteria engulfed by

alveolar macrophages will be eliminated from the body through mucocilliary movements, and

a few bacteria will be transported by macrophages into the lung interstitium.

Once M. tuberculosis has entered the lungs, one of four potential fates is possible

(Dannenberg, 1994):

1. The initial host response can effectively kill and eliminate the bacilli. These

individuals will not develop TB at any time point in the future.

2. The bacilli can grow and multiply immediately after infection, thereby causing clinical

disease known as primary TB.

3. The bacilli may become dormant and never cause disease, resulting in a latent

infection that is manifested only as positive tuberculin skin test (TST).

4. The dormant bacilli can eventually begin to grow with resultant clinical disease known

as reactivation TB.

Immune evasion

In spite of the targeting macrophages, whose function is the elimination of microbes, M.

tuberculosis can remain viable after phagocytosis due to different strategies evolved to evade

host immune responses. The use of non-activating complement receptors (CR) may be

advantageous for the bacterium, since engagement of these receptors does not induce the

release of cytotoxic reactive oxygen intermediates (Wright and Silverstein, 1983). Moreover,

it is well known that mycobacteria can prevent the normal phagosome-lysosome fusion

resulting in persistence of the bacteria within the host cell (Armstrong and Hart, 1975). In this

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way the bacteria not only remain viable, but bacterial antigens are prevented from being

presented to T cells. Mycobacteria appear to increase the retention of the tryptophan-aspartate

containing coat (TACO) protein on the surface of the mycobacterial phagosome, thereby

preventing phagosome-lysosome fusion (Ferrari et al., 1999). Moreover, characterization of

mycobacterial phagosomes has revealed the presence of the transferrin receptor (CD71), rab5,

and early phagosome Ag 1, all markers of early endosomes, while late endosomal proton-

ATPases are blocked from recruitment to mycobacteria-harbouring phagosomes (Sturgill-

Koszycki et al., 1994; Clemens and Horwitz, 1995; Sturgill-Koszycki et al., 1996). In

addition, the expression of major histocompatibility complex (MHC) class II molecules is

decreased in M. tuberculosis-infected macrophages (Noss et al., 2000). Other immune evasion

mechanisms are the secretion of proteins such as superoxide dismutase and catalases by M.

tuberculosis, which are antagonistic to reactive oxygen intermediates (Andersen et al., 1991),

and the inhibition of macrophage apoptosis (Fratazzi et al., 1999). Macrophages infected with

M. tuberculosis produce inhibitory cytokines, such as transforming growth factor (TGF)-β

and interleukin (IL)-10, which reduce macrophage activation, thereby leading to decreased

clearance of bacteria (Barnes et al., 1992; Toossi et al., 1995).

IMMUNE RESPONSE TO MYCOBACTERIAL INFECTIONS

Innate immunity

The initial response to an infection is mediated by components of the innate immunity that

serves primarily to restrict the multiplication and dissemination of the pathogens, as well as to

initiate the ensuing adaptive response. In addition to macrophages, M. tuberculosis also

interacts with epithelial cells in the alveolar space of the lung and is able to invade and

replicate in this cell type (Bermudez and Goodman, 1996; Garcia-Perez et al., 2003).

However, the role of alveolar epithelium in mycobacterial infections has not been fully

elucidated. In addition to forming a physical barrier, alveolar epithelial cells can express

adhesion molecules and release cytokines and chemokines, such as IL-8 and monocyte

chemotactic protein-1, and thereby modulate the local immune response (Lin et al., 1998). M.

tuberculosis infection has also been shown to induce the expression of inducible nitric oxide

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synthase (iNOS) mRNA by epithelial cells and the production of nitric oxide (NO) (Roy et

al., 2004), and, more recently, interferon (IFN)-γ (Sharma et al., 2007).

Through the presentation of mycobacterial antigens, and the expression of costimulatory

molecules and cytokines, phagocytic cells play an important role in the initiation and direction

of the adaptive immunity.

Macrophages

Macrophages are regarded as phagocytic cells that initially ingest M. tuberculosis. Thus, they

provide an important cellular niche during infection. The macrophages are considered to be

the main cellular host for mycobacteria, and their major role is the rapid killing of the

invading organism through the release of toxic reactive oxygen and nitrogen intermediates, or

killing by lysosomal enzymes following fusion with the bacterial phagosome. The receptors

that have been implicated in the uptake of mycobacteria include, the mannose receptor (MR),

that recognizes mannose residues on mycobacteria (Schlesinger, 1993; Schlesinger et al.,

1996), Fc receptors (FcRs) binding antibody-coated bacteria, CR1, CR3, and CR4, which bind

complement factor C3-opsonized bacilli (Schlesinger et al., 1990; Hirsch et al., 1994; Aderem

and Underhill, 1999), surfactant receptors (Downing et al., 1995), and scavenger receptors

(Zimmerli et al., 1996).

Upon infection, macrophages have been shown to secrete proinflammatory cytokines, such as

tumor necrosis factor (TNF)-α, IL-1, and IL-6, which are believed to be important for the

recruitment of cells to the site of infection (Giacomini et al., 2001). Furthermore, the secretion

of TNF-α may also aid in the activation of macrophages to produce reactive oxygen and

nitrogen intermediates, and help granuloma formation (Flynn et al., 1995; Roach et al., 2002).

The significance of these toxic nitrogen oxides in the host defense against M. tuberculosis has

been well documented, both in vitro and in vivo, particularly in the murine system

(MacMicking et al., 1997; Shiloh and Nathan, 2000). In the mouse, reactive nitrogen

intermediates (RNIs) play a protective role in both the acute and chronic persistent infection

(MacMicking et al., 1997; Flynn et al., 1998). More importantly, accumulating evidence

supports a role for these reactive molecules in the host defense against human TB (Nicholson

et al., 1996; Wang et al., 1998), although this still remains controversial.

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The mechanisms by which NO and other RNIs may affect antimicrobial activity, could be

through the modification of bacterial DNA, proteins and lipids (reviewed in Chan et al.,

2001). NO can deaminate, as well as directly damage bacterial DNA, and has been

demonstrated to induce apoptosis. RNIs also have the potential to disrupt signalling pathways,

Dendritic cells

While dendritic cells (DC) do not display effective anti-microbial activity upon mycobacterial

encounters, their secretion of cytokines and expression of costimulatory molecules help in

modulating the adaptive immune response, supporting a helper T cell (Th) 1 biased T-cell

response. The major role of DCs during mycobacterial infections appears to be that of an

antigen-presenting cell (APC). It was recently shown that DCs, but not macrophages, infected

with M. tuberculosis were capable of driving Th 1 polarization of naïve CD4+ T cells

(Hickman et al., 2002). Activation of human monocyte-derived DCs with the 19 kDa

mycobacterial lipoprotein results in the preferential secretion of IL-12, a key player in host

defense against M. tuberculosis (Thoma-Uszynski et al., 2000). An additional property of

DCs that contributes to their effectiveness in initiating immune responses is their ability to

migrate from peripheral tissues to secondary lymphoid tissues after acquiring antigens. Naïve

T cells are thereby activated via antigen-presenting-, and costimulatory-molecules in the

presence of polarizing cytokines such as IL-12.

DCs and macrophages appear to have different roles during infection with mycobacteria. For

instance, activated macrophages, but not DCs, have the ability to kill intracellular M.

tuberculosis (Bodnar et al., 2001). The different intracellular behaviour of M. tuberculosis in

macrophages and DCs may reflect differences in the receptors involved in bacterial uptake in

the two cell types. DCs have lectin-surface receptors, such as the recently identified DC-

specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) (Geijtenbeek et

al., 2000), that facilitate antigen uptake (Engering et al., 2002) and phagocytosis of

mycobacteria by DCs (Geijtenbeek et al., 2003; Tailleux et al., 2003). Although CR3 and MR

are also expressed by human DCs, they seem to be less important for the uptake of the

tubercle bacillus (Tailleux et al., 2003). Ligation of DC-SIGN with the M. tuberculosis-

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derived lipoarabinomanan induces IL-10 secretion in DCs, and thereby suppresses their

function (Geijtenbeek et al., 2003).

Neutrophils

It has been suggested that neutrophils participate in the host defense against mycobacterial

infections since circulating neutrophils become activated and are recruited to the lungs early

in infection. They can be found at the infection nidus at the onset of infection, as well as

several days after the initial reponse (Pedrosa et al., 2000; Fulton et al., 2002). In vivo

depletion of neutrophils prior to mycobacterial infection enhances bacterial growth in the

lungs of infected mice, whereas local treatment with the neutrophil chemoattractant

macrophage-inflammatory protein-2 enhances neutrophil recruitment and decreases

mycobacterial growth (Appelberg et al., 1995; Fulton et al., 2002). The mechanisms by which

neutrophils exert their anti-mycobacterial function are not completely resolved, although

several hypotheses have been proposed. These include the secretion of chemokines (Riedel

and Kaufmann, 1997; Seiler et al., 2003), the induction of granuloma formation (Seiler et al.,

2003), and macrophage uptake of neutrophil-specific molecules such as myeloperoxidase

(Hanker and Giammara, 1983), and lactoferrin (Silva et al., 1989). Another mechanism

whereby neutrophils indirectly contribute to the killing of mycobacteria was recently

demonstrated by Tan et al. Mycobacteria-infected macrophages acquire the contents of

neutrophil granules and their anti-microbial molecules by the uptake of apoptotic neutrophil

debris, which is trafficked to endosomes and colocalize with the intracellular bacteria (Tan et

al., 2006). The involvement of neutrophils in direct killing of mycobacteria has been a matter

of controversy. In vitro studies have demonstrated the ability of neutrophils to kill virulent M.

tuberculosis (Brown et al., 1987; Jones et al., 1990), although this has been questioned

(Denis, 1991; Aston et al., 1998). Kisich et al. showed that neutrophils in human pulmonary

lesions contained intracellular M. tuberculosis, thereby demonstrating a phagocytic role for

neutrophils in human TB. Human neutrophils were furthermore able to kill virulent M.

tuberculosis in vitro (Kisich et al., 2002).

Neutrophils are clearly important in the early immunity to bacterial infections. They respond

rapidly to chemotactic stimuli released by the bacteria or inflammed epithelium and, thus,

arrive early at the site of infection. Besides their anti-mirobial role, neutrophils have been

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implicated in the modulation of the adaptive immune response by the release of

chemoattractants, which recruit other immune cells, such as T cells, monocytes, macrophages

and DCs to the site of infection (Kasama et al., 1993; Yang et al., 2000; Scapini et al., 2001).

It was recently demonstrated that neutrophils and DCs interact physically through DC-SIGN

expressed on DCs and Mac-1 expressed on neutrophils (van Gisbergen et al., 2005;

Megiovanni et al., 2006). This interaction enables neutrophils to induce maturation of DCs

via TNF-α secretion and a preferential production of IL-12 by the matured DCs (van

Gisbergen et al., 2005), which in turn results in an enhanced activation of T cells (Megiovanni

et al., 2006).

Toll-like receptors

Besides phagocytosis, the recognition of M. tuberculosis or mycobacterial products is also

crucial for an effective host response. Central to the immune defense against microbial

pathogens are pattern recognition receptors, such as the toll-like receptors (TLRs). There are

13 members of the TLR family known today, of which TLR1-10 are found in humans

(Ulevitch, 2004). Besides microbial products, TLRs also recognize endogenous ligands, such

as heat shock proteins (Ohashi et al., 2000; Vabulas et al., 2002), extracellular matrix

breakdown products (Termeer et al., 2002; Guillot et al., 2002), and intracellular contents

from necrotic cells (Gallucci et al., 1999; Li et al., 2001). Ligation of TLRs initiates a signal

transduction pathway that culminates in the activation of NF-κB and induction of several

immuno-related genes, including cytokines and chemokines (Hoffmann et al., 1999; Aderem

and Ulevitch, 2000). TLR activation is therefore an important link between innate cellular

responses and the subsequent activation of adaptive immune response to microbial pathogens.

DCs express the broadest repertoire of TLRs through which they can recognize a plethora of

microbial compounds. Upon TLR triggering, immature DCs, apart from cytokine secretion,

undergo the process of maturation, resulting in an augmented expression of T-cell

costimulatory molecules, such as CD80 and CD86, along with antigen-presentation

molecules, such as MHC class II (Tsuji et al., 2000; Hertz et al., 2001; Michelsen et al.,

2001).

Emerging evidence suggests that TLRs play an important role in the activation of immune

cells by pathogens, including M. tuberculosis. TLR2, TLR4, and more recently, TLR1/TLR6

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that heterodimerise with TLR2, have been implicated in the recognition of mycobacterial

antigens (Bulut et al., 2001; Hajjar et al., 2001). Predominantly, a role for TLR2 in the

immune recognition of M. tuberculosis has been demonstrated. Mycobacterial products have

been shown to induce secretion of TNF-α and NO by macrophages via interactions with

TLRs, as well as inducing apoptosis in the host cell (Aliprantis et al., 1999; Brightbill et al.,

1999).

Infection studies using TLR gene-disrupted mice have, however, provided conflicting data,

depending on the experimental settings, for instance the dose of bacteria used. TLR2-/- mice

have been demonstrated to be more susceptible to M. tuberculosis infection than wild-type

mice, while others have reported a redundant role for TLR2 in this context (Reiling, et al.,

2002; Sugawara et al., 2003; Drennan et al., 2004). In addition, outcomes of infection studies

with TLR4-deficient mice show disparity (Reiling et al., 2002; Kamath et al., 2003; Shim et

al., 2003). Signal transduction by most TLRs, with the exception of TLR3, requires the

adaptor molecule myeloid differentiation factor 88 (MyD88) (Medzhitov et al., 1998; Adachi

et al., 1998; Kawai et al., 1999). MyD88 is an intracellular adaptor protein in the IL-1

receptor/IL-1 receptor associated kinases (IRAK) pathway that links TLR recognition with the

activation of IRAK and TNF receptor associated factor (TRAF), translocation of NF-κB, and

gene transcription (Akira et al., 2003). Mice deficient in MyD88 fail to generate

proinflammatory responses when stimulated through TLRs (Adachi et al., 1998; Kawai et al.,

1999), and demonstrate high susceptibility to several infectious agents, including

mycobacteria (Muraille et al., 2003; Mun et al., 2003; Feng et al., 2003; Fremond et al.,

2004).

In this way, TLRs contribute to the innate immune system by the induction of antimicrobial

effector molecules, upon ligation. In addition, the recognition of mycobacterial products by

TLRs induces secretion of cytokines, chemokines, and upregulation of immunostimulatory

molecules, and subsequently the modulation of the adaptive immune response.

γδ T cells

T cells that express the γδ T-cell receptor (TCR) participate in immunity against M.

tuberculosis (Kaufmann, 1996), and are believed to play a role in the early immune response

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(Izzo and North, 1992). Dieli et al. demonstrated an early accumulation of γδ T cells in the

lungs of pulmonary BCG-infected mice, reaching a peak 3 weeks before αβ T cells, where

they produce IFN-γ, exert cytotoxic activity against BCG-infected macrophages, and play a

regulatory role in the induction of CD8+ T cells in the lungs (Dieli et al., 2003). In addition,

studies using γδ TCR knockout mice indicate that γδ T cells may be involved in the regulation

of granuloma formation, which is critical for the control of mycobacteria (D’Souza et al.,

1997). These results indicate that γδ T cells might be important for the control of

mycobacterial infection in the period between innate and adaptive immunity. Human γδ T

cells, predominantly the Vγ9/Vδ2 TCR subset, which represents a major peripheral blood T

cell subset, have also been demonstrated to respond to M. tuberculosis antigens (Kabelitz et

al., 1991), and monocytes infected with live M. tuberculosis were seen to be particularly

effective in expanding this subset of γδ T cells (Havlir et al., 1991).

The effector functions of γδ T cells in the immune response to M. tuberculosis appear to be

both cytokine secretion and cytotoxicity. Studies with M. tuberculosis antigen-activated γδ T

cell clones or primary cells demonstrated IFN-γ production, as well as TNF-α production, in

response to phosphate antigens (reviewed in Boom, 1999). Likewise, cytotoxicity mediated

by γδ T cells has been confirmed and was dependent upon activation through the TCR (Munk

et al., 1990; Dieli et al., 2003). M. tuberculosis-reactive γδ T cells from the peripheral blood

of TST positive subjects were cytotoxic for monocytes pulsed with mycobacterial antigens.

Adaptive immunity

M. tuberculosis is a classic example of a pathogen against which the protective immune

response relies on cell-mediated immunity. The initial interaction in the lungs is with alveolar

macrophages, but after this first encounter DCs and monocyte-derived macrophages, recruited

to the site of infection, also take part in the phagocytic process (Henderson et al., 1997;

Thurnher et al., 1997). Infected DCs mature and migrate to draining lymph nodes to prime

naïve T cells via processed antigens. Inflammation in the lungs provides the signals that direct

the effector T lymphocytes back to the site of infection where granulomas are formed. The

anatomic affinity of these cells appears to be mainly determined by site-specific integrins,

“homing receptors”, on their surface and complementary mucosal tissue-specific receptors,

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“addressins”, on vascular endothelial cells (Kunkel and Butcher, 2003). In addition,

chemokines produced in the local microenvironment promote chemotaxis toward mucosal

tissues and regulate integrin expression on mucosal lymphocytes, thereby controlling cell

migration (Champbell et al., 2003).

Cellular immunity

CD4+ T cells

It is well established that CD4+ T cells are of utmost importance for protective immunity

against M. tuberculosis. CD4+ T cells recognize peptide antigens from mycobacteria,

degraded in the phago-lysosomal compartments and complexed with MHC class II molecules

(Davis and Björkman, 1988). Murine studies with antibody depletion of CD4+ T cells (Muller

et al., 1987), adoptive transfer (Orme and Collins, 1984), or the use of gene-deficient mice

(Caruso et al., 1999), have demonstrated that CD4+ T cell subsets are required for the control

of the infection. The primary effector function of CD4+ T cells is the production of cytokines;

first and foremost IFN-γ, which is crucial for the induction of microbicidal activities by

macrophages, but also TNF-α. Production of these cytokines by CD4+ T cells is important for

the control of TB (Flynn et al., 1993; Flynn et al., 1995), and studies using IFN-γ gene

depleted mice demonstrate that these mice are highly susceptible to virulent M. tuberculosis

infection, with defective macrophage activation and uncontrolled bacillar growth (Cooper et

al., 1993). Furthermore, it is known that humans, defective in genes for IFN-γ or the IFN-γ

receptor, are prone to serious mycobacterial infections, including M. tuberculosis (Ottenhof et

al., 1998). TNF-α in turn plays a key role in the granuloma formation (Kindler et al., 1989;

Senaldi et al., 1996). It induces macrophage activation, and has immunoregulatory properties

(Orme and Cooper, 1999; Tsenova et al., 1999), and is also important for the containment of

latent infection in granulomas, both in mice (Mohan et al., 2001) and humans (Ehlers, 2003).

Although IFN-γ production by CD4+ T cells is a very important effector function, CD4+ T

cells have most likely other roles in controlling M. tuberculosis infection. In MHC class II-/--

or CD4-/-mice, the levels of IFN-γ were severely diminished early in infection, but returned to

wild-type levels later on (Tascon et al., 1998; Caruso, et al., 1999). Nevertheless, the gene-

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deficient mice were not rescued by this later IFN-γ production and succumbed to infection.

Both CD4+ T cell clones and mycobacterial-antigen-expanded CD4+ T cells have been shown

to exhibit cytolytic effector functions against mycobacterial-antigen pulsed or mycobacteria-

infected macrophages (Orme et al., 1992).

Active TB is characterized by a profound and prolonged suppression of M. tuberculosis-

specific T-cell responses, demonstrated by decreased production of IL-2 and IFN-γ (Toossi et

al., 1986; Huygen et al., 1988; Zhang et al., 1994; Torres et al., 1994; Hirsch et al., 1999).

Overproduction of immunosuppressive cytokines, such as IL-10 and TGF-β, by mononuclear

phagocytes has been implicated in decreased T cell function during TB (Hirsch et al., 1996;

Gong et al., 1996; Hirsch et al., 1997; Hirsch et al., 1999). However, while IL-10 and TGF-β

levels return to normal after anti-TB treatment, M. tuberculosis-stimulated production of IFN-

γ remains depressed beyond completion of the treatment (Hirsch et al., 1996; Hirsch et al.,

1999).

Regulatory-T cells constitute a key component of peripheral tolerance suppressing potentially

autoreactive T cells and preventing autoimmune diseases (Sakaguchi et al., 1995; Sakaguchi,

2005). Different subsets of regulatory-T cells have been described, such as IL-10-secreting

(Tr1) or TGF-β-secreting regulatory T cells (TH3), and CD4+CD25+ regulatory T cells

(O’Garra and Vieira, 2004; Sakaguchi, 2005; Belkaid and Rouse, 2005). The latter cells share

common markers with conventional, activated CD4+ T cells, and are now identified by the

molecular marker FoxP3 (Sakaguchi, 2005; Fontenot and Rudensky, 2005; Roncador et al.,

2005). Involvement of IL-10-secreting regulatory-T cells has been proposed in TB anergic

patients (Boussiotis et al., 2000). Recent reports indicate a role for CD4+CD25+ regulatory T

cells in the negative modulation of anti-TB immune responses. An increased frequency of

CD4+CD25+FoxP3+ regulatory-T cells in the blood and at the site of infection was associated

with M. tuberculosis infection (Chen et al., 2007), corroborating a previous study showing

enhanced frequency of CD4+CD25+ T cells during active TB and sustained enhancement after

completion of anti-TB treatment (Ribeiro-Rodrigues et al., 2006).

23

CD8+ T cells

Despite the residence of the bacteria within phagosomes, CD8+ T cells take part in the

immunity against mycobacterial infections (reviewed in Smith and Dockrell, 2000).

Mycobacterial antigen-specific CD8+ T cells are restricted by either MHC class I or CD1

molecules. CD1 molecules are nonpolymorphic molecules that present lipids or glycolipids to

T cells, as opposed to peptide epitopes presented by MHC molecules (reviewed in Porcelli

and Modlin, 1999). Mice genetically disrupted in the genes for β2-microglobulin or the

transporter of antigen processing, and therefore deficient in MHC class I and non-classical

MHC class Ib molecules and, thus, unable to activate CD8+ T cells, display an increased

susceptibility to M. tuberculosis infection compared to wild-type mice (Flynn et al., 1992;

Behar et al., 1999; Sousa et al., 1999), indicating a protective role for these cells.

The mechanism by which mycobacterial proteins gain access to the MHC class I molecules is

not clear. Mycobacteria-induced pores or breaks in the phagosomal membrane have been

proposed as mechanisms (Myrvik et al., 1984; Mazzaccaro et al., 1996; Teitelbaum et al.,

1999). The bacteria could use this as a way of gaining access to cytosolic nutrients and

introducing toxic molecules into the cytoplasm. Additionally, mycobacterial antigens would

be allowed to enter the cytoplasm of infected cells and hence the MHC class I pathway.

Two principal effector functions for CD8+ T cells have been suggested: lysis of infected cells

and production of cytokines, explicitly IFN-γ, although the relative contributions of these

functions are not yet established. CD1- and MHC class I-restricted CD8+ T cells, specific for

mycobacterial antigens, have been shown to induce lysis of infected human DCs and

macrophages, resulting in reduced intracellular bacterial numbers (Stenger et al., 1997; Cho et

al., 2000). This was dependent on perforin, which is required for pore formation (Stenger et

al., 1997), while granulysin was seen to be responsible for killing the intracellular bacteria

(Stenger, et al., 1998). Moreover, antigen-specific CD8+ T cells can target and kill infected

macrophages, and also induce growth inhibition of M. tuberculosis, through apoptotic

mechanisms (Oddo et al., 1998). The role of IFN-γ in mycobacterial infections, on the other

hand, is considered to be through the activation of macrophages. CD8+ T cells from lungs of

infected mice have been shown to be primed for IFN-γ production, although this production

appears to be limited in the lungs (Serbina and Flynn, 1999).

24

Humoral immunity

While the essential role for T cells in the control of M. tuberculosis infection is well

established, the role of B cells and antibodies is less well understood. Although neglected for

a long time, their role has recently been reappreciated. In individuals infected with M

tuberculosis, a vigorous humoral response is always present, in addition to the well-defined

cellular immune response. Infection studies performed using B cell-deficient mice have

revealed conflicting data. One study reported an increase in viable bacilli in B cell-deficient

mice compared to wild-type mice (Vordermeier et al., 1996), whereas results from another

study suggested that absence of B cells, and hence antibodies, does not affect the outcome of

infection with M. tuberculosis (Johnson et al., 1997). Yet another study demonstrated that B

cell deficiency results in a reduced recruitment of neutrophils, macrophages, and CD8+ T cells

to the lungs upon M. tuberculosis infection (Bosio et al., 2000), suggesting that B cells may

influence the cellular composition at this site by regulating chemokines and adhesion

molecules. An additional role for B cells as APCs has also been suggested (Vordermeier et

al., 1996).

Passive immunization with monoclonal antibodies has been reported to protect mice against

mycobacterial infection. It was recently shown that IgA antibodies specific for the α-

crystallin antigen of M. tuberculosis significantly reduced the number of colony forming units

(CFUs) in lungs of infected mice (Williams et al., 2004). These results corroborate other

studies, reporting a protective role for monoclonal antibodies and passive immunization in

experimental mycobacterial infections (Teitelbaum et al., 1998; Pethe et al., 2001; Chambers

et al., 2004; Hamasur et al., 2004).

Granuloma formation

Granuloma formation is the hallmark of M. tuberculosis infection. Granulomas are formed in

response to chronic local antigenic stimulation, and can be observed in many different

infectious diseases, including schistosomiasis, leprosy, and leishmaniasis (Reyes-Flores,

1986; Modlin and Rea, 1988; Palma and Saravia, 1997; Rumbley and Phillips, 1999; Boros,

1999). The structure and composition of granulomas vary depending on the organism. A

tuberculous granuloma is observed concomitantly with a highly activated cell-mediated

25

immune response, which generally mediates the control of mycobacterial numbers in the

lungs. The granuloma is composed of many different cells, including macrophages, CD4+-,

and CD8+-T cells, and B cells (Gonzales-Juarrero et al., 2001). These cells control the

infection by providing a local environment for cellular interactions, leading to an effective

immune response where cytokine production, macrophage activation and CD8+ T cell-effector

functions lead to killing of the mycobacteria. Granulomas also provide a way of containing

the bacilli by walling off and preventing the spread of infection. Altogether, these actions lead

to an inhibition of growth, or to the death of M. tuberculosis. However, they also result in

inflammatory pathology that contributes to the damage of the host tissue.

TB: THE DISEASE

TB is primarily a pulmonary infectious disease. Following infection with M. tuberculosis

there is an early transient influx of granulocytes, but the hallmark of mycobacterial infections

is the development of granulomatous lesions (Rook and Bloom, 1994). Although granuloma

formation provides a mean of containing the infection, granulomas may displace and destroy

adjacent tissues. Initially well-formed granulomas may gradually develop central caseation

that may lead to extensive fibrosis or cavity formation in the lungs.

TB can involve any organ system in the body. While pulmonary TB is the most common

clinical manifestation, extrapulmonary TB is also an important clinical problem. The bacilli

can spread from the initial site of infection, in the lung, through the lymphatics or blood to

other parts of the body and cause extrapulmonary TB of the pleura, lymphatics, bone,

urogenital system, meninges, peritoneum, or skin. Before the HIV pandemic, and in studies

involving immunocompetent adults, it was observed that extrapulmonary TB constituted

about 10-20% of all TB cases (Weir and Thornton, 1985; Fanning, 1999;). In HIV-positive

patients, extrapulmonary TB accounts for more than 50% of all TB cases (Theuer et al.,

1990). The most common extrapulmonary sites in HIV-positive individuals are the lymph

nodes.

Disseminated, or miliary, TB refers to the involvement of two or more organs simultaneously,

and can occur during primary infection or after reactivation of a latent infection, as well as

after reinfection.

26

Diagnosis

An early confirmation of the diagnosis of TB is a challenging problem. The established

methods have limitations in speed, sensitivity and specificity. In general, it is more difficult to

diagnose extrapulmonary TB than pulmonary TB, since this often requires invasive

procedures to obtain diagnostic specimens for histological or bacteriological confirmation.

Tuberculin skin test

The TST is currently the only widely used method for identifying latent infection with M.

tuberculosis in asymptomatic individuals. This test is based on the fact that infection with M.

tuberculosis produces a delayed-type hypersensitivity reaction to certain mycobacterial

components in the extracts of culture filtrates called “tuberculins”. The test, also known as the

Mantoux method, is administered by intradermal injection of the tuberculin purified protein

derivative (PPD), which produces a wheal of the skin. The visible induration (in mm) is

measured 48 and 72 hours after injection. There are, however, concerns regarding the TST.

Several factors may contribute to false-negative results, such as age, poor nutrition and

general health, overwhelming acute illness, or immunosuppression, such as medications or

HIV infection (American Thoracic Society, 2000). In addition, false-positive results can occur

in individuals who have been infected with other mycobacteria, including vaccination with

BCG. Because of its low sensitivity, TST cannot be used to rule out the possibility of active

TB.

Microscopy

The microscopal detection of acid-fast bacilli in stained sputum smears is the first

bacteriological evidence of the presence of mycobacteria in clinical specimens. Acid-fast

staining procedure depends on the ability of mycobacteria to retain dye when treated with

mineral acid or an acid-alcohol solution. Smear examination is rapid, inexpensive, technically

simple, and highly specific for acid-fast bacilli, such as M. tuberculosis. Additionally, it gives

a quantitative estimation of the number of bacilli being excreted. The identification of smear

positive patients is of major importance since only smear positive pulmonary TB patients are

27

regarded as highly infectious to others. However, smear microscopy cannot discriminate

between M. tuberculosis and other mycobacteria and, in addition, lacks sensitivity, since

5000-10000 bacteria/ml in the sample are needed for a positive result (American Thoracic

Society, 2000).

Cultivation

Mycobacterial growth in cultures is the ultimate proof of mycobacterial infection and is often

used as the reference method due to its high sensitivity and specificity (Schirm et al., 1995;

Walker, 2001). With this method, as few as 10 bacteria/ml within a sample are necessary for

bacterial detection (American Thoracic Society, 2000). Also, the cultivation of the etiological

agent has been essential for species identification, drug susceptibility testing and monitoring

the response to therapy. Nevertheless, the slow growth rate of M. tuberculosis and most other

mycobacterial pathogens complicates the use of cultivation as a diagnostic technique.

Molecular methods

The use of nucleic acid amplification for the diagnosis of TB is rapidly evolving. These

technologies allow for the amplification of specific target sequences of nucleic acids that can

be detected through the use of a nucleic acid probe, and both RNA and DNA amplification

systems are commercially available (Cohen et al., 1998).

Treatment

Although TB can be cured; current treatment is complex and long lasting, involving four

drugs for 2 months and two drugs for at least another 4 months. The combination of different

drugs is neccessary in order to avoid development of resistant and MDR TB. Isoniazid,

rifampicin, pyrazinamide and streptomycin constitute the first line of TB drugs that are used

and predominantely target actively growing bacteria through the inhibition of cell wall

synthesis, DNA replication and protein synthesis. The long duration for chemotherapy is due

28

to the fact that M. tuberculosis is a slow growing organism and, following the initial killing of

the majority of bacteria, persistent bacteria can revert to the state of latency.

Through the WHO sponsored, directly observed treatment short-course (DOTS) program,

effective TB therapy is now available to around 70% of the world’s population. Yet, the

likelihood of DOTS therapy resulting in the eradiction of TB is limited by the large reservoir

of latently infected individuals, as well as by delays in diagnosis. The treatment regime is

additionally demanding for the patient, labour intensive for health staff and is compromised in

settings where health services are poorly accessible.

In 1993 the WHO declared TB a global health emergency. One reason for this extraordinary

declaration was the increase in MDR TB, which was reaching epidemic proportions. The

management of MDR TB is a challenging problem, given that treatment is less effective,

more toxic and much more expensive compared to the treatment of patients with drug

susceptible TB. In the last few years the fluoroquinolone group of drugs has been added to the

chemotherapy of resistant forms of TB, and have been used as a part of regimens to treat

patients with MDR TB.

During the last year, alarming findings of what has been named extensively drug-resistant

(XDR) TB has been reported. XDR TB is caused by strains of M. tuberculosis resistant to

virtually all second-line drugs. Inappropriate treatment regimens have probably contributed to

the development of XDR TB, which raises the concern for a future epidemic of untreatable

TB.

The current BCG vaccine

The current vaccine against TB, the attenuated M. bovis bacillus Calmette-Guérin (BCG), was

developed by the French scientists Calmette and Guérin in the first decade of the 20th

century. It was first given to humans in 1921 and has now been given to more people than any

other vaccine (Fine, 1995a). Although it can prevent disseminated and meningeal TB in

young children, its efficacy against the most prevalent form of disease, pulmonary TB in

adults, has been strongly questioned. Data concerning the protective efficacy of BCG in adults

range from 0% in South India to 80% in the UK (Fine, 1995b). The reason for this variation in

29

efficacy might depend on several factors, including variation in the BCG strains used,

vaccination dose, vaccination protocols, or inappropriate handling of the vaccine (Hess and

Kaufmann, 1999). Additionally, studies from animal models suggest that prior exposure to

live environmental mycobacteria primes the host immune system against mycobacterial

antigens shared with BCG, and recall of this immune response upon vaccination results in an

accelerated clearance of BCG and therefore decreased protection against M. tuberculosis

(Kamala et al., 1996; Brandt et al., 2002). Helminth infections have, furthermore, been shown

to have an impact on the immune response against mycobacterial infections, leading to a Th2

shift in the immune profile, resulting in a reduced protective efficacy of BCG vaccination

(Elias et al., 2005a). Moreover, the hypothesis that the protection by BCG vaccination wanes

over time has also been brought up (Sterne et al., 1998).

Another factor underlying the failure of BCG-aquired protection could be the route of

vaccination. BCG is currently administered intradermally, which might not be optimal for

inducing protective immunity in the respiratory tract. Vaccination at the mucosal site has been

believed to be superior to vaccination at other sites for eliciting protective immune responses

against mucosal infectious diseases (Davis, 2001). Falero-Diaz and colleagues reported that

intranasal (i.n.) vaccination with BCG conferred potent protection against airway M.

tuberculosis challenge (Falero-Diaz et al., 2000). Another study demonstrated that a single

i.n. BCG vaccination is superior to the subcutaneous route for the protection against

pulmonary TB in mice (Chen et al., 2004). In addition, i.n. vaccination offers desirable

advantages, such as ease in administration, feasibility, and the ability to trigger both mucosal

and systemic immune activation (Davis, 2001).

Lastly, BCG vaccination is also known to stimulate cell-mediated immunity, and BCG

immunotherapy has been used in the treatment of bladder cancer resulting in improved

survival (Alexandroff et al., 1999).

New vaccine candidates

Over the past several years there has been an intensive effort to develop a new vaccine against

TB. The TB vaccines under development can be divided into two categories: prophylactic

(preexposure) or therapeutic (postexposure) vaccines. Prophylactic vaccines prevent infection

30

and subsequent disease and should be given to uninfected persons. Therapeutic vaccines aim

to prevent or reduce progression to disease, and would be given to individuals already

infected with M. tuberculosis. To date, a number of vaccine candidates have been tested in

animal models. The increasing knowledge of the tubercle proteins and the development of

techniques to help identify the most immunogenic antigens, have generated numerous subunit

vaccine candidates. Another area in which there has been substantial interest and progress is

DNA vaccines, and several mycobacterial antigens have been targeted in this manner

(Huygen, 1998). Whole bacterial vaccines have the advantage of a built-in adjuvanticity, as

well as containing both protein- and non-protein antigens. This strategy includes live

attenuated bacteria, as well as engineering and overexpression of distinct antigens to improve

the immunogenicity.

ANIMAL MODELS OF TB

Experimental animal models of TB are central to vaccine development. As for many

infectious diseases, there is no ideal animal model for TB. The most common models used for

M. tuberculosis infection are the mouse and the guinea pig. In neither of these species does

the disease perfectly match that seen in humans, however, many aspects of immunity are the

same. Nevertheless, caution must be advocated when extrapolating results from animal

infection experiments to human TB.

The mouse is the most widely used species and provides many advantages (reviewed in

Kaufmann, 2003). The mouse genome has been completely sequenced, mice are relatively

inexpensive, there is a wealth of information on their immune system, and techniques and

reagents for mechanistic studies are abundant. Additionally, the availability of genetically

targeted mice makes it possible for in vivo studies to elucidate the relevance of particular cells

and molecules. A large number of gene knockout and knockin mice, both constitutive and

conditional, has been generated. However, the mouse is relatively resistant to M. tuberculosis,

and does not develop the severe pathology seen in some human patients.

The guinea pig is generally considered even more susceptible to M. tuberculosis than humans

and therefore provides a very sensitive model for testing the efficacy of novel vaccine

candidates. Moreover, granulomatous lesions in guinea pigs are very similar to those seen in

31

human TB patients. Finally, the group 1 CD1 molecules, responsible for the presentation of

mycobacterial glycolipids to CD1-restricted T cells, are present in humans and guinea pigs

but absent in mice (Schaible and Kaufmann, 2000; Ulrichs and Kaufmann, 2002).

The non-human primate model is considered the closest match for human disease in terms of

pathology. The immune response in this model is very similar to that seen in humans, and

most reagents for human cells and molecules can be applied to primate studies. Due to ethical

reasons, experiments in non-human primates should be limited to critical experiments used for

final validations directly preceding clinical trials of vaccines and therapeutic agents in

humans.

32

MUCOSAL IMMUNITY IN THE RESPIRATORY TRACT

Mucosal surfaces lining the respiratory-, gastrointestinal-, and urogenital tracts are the major

sites of entry for pathogens. These mucosal surfaces thereby provide the first line of defense

against entrance of various bacteria and viruses. Protection of mucosal membranes against

colonization, possible entry and invasion by microbes is provided by a combination of non-

specific and specific mechanisms. Production of mucus is part of the non-specific

mechanisms that acts as a physical barrier containing substances such as lysozyme,

lactoferrin, collectin and defensins. Ciliary action can force microbes out of the respiratory

tract. The movement of microbes by the ciliae decreases the time available for the adherence

by pathogens to the epithelium. Tight junctions between neighbouring epithelial cells lining

the mucosal membranes also act as a physical barrier against penetration.

SPECIFIC IMMUNE RESPONSES

Mucosal surfaces contain specialized mucosa-associated lymphoid tissues (MALT) necessary

for antigen sampling and induction of mucosal immune responses. The immune system in the

upper and lower respiratory tract can be divided into three parts (Davis, 2001):

1. an epithelial compartment at the surface of the epithelium and the underlying

connective tissue that contains immunocompetent cells

2. the MALT, subdivided according to anatomical location: the nasal-associated

lymphoid tissue (NALT), larynx-associated lymphoid tissue (LALT), and the

bronchus-associated lymphoid tissue (BALT)

3. lymph nodes draining the respiratory system

Mucosal antigen-specific immune responses are elicited in the MALT, where foreign material

from epithelial surfaces can be sampled and transported by microfold (M) cells, and

subsequently be taken up by underlying DCs and macrophages (reviewed in Kiyono and

Fukuyama, 2004). M cells are specialized epithelial cells that transcytose particles across

33

epithelial barriers to an intraepithelial lymphoid pocket created by the basolateral surface of

the M cell (Neutra et al., 1996). The follicles of the MALT contain all immunocompetent

cells, i. e. T cells, B cells and APCs, that are required for the initiation of an immune

response. The MALT, as well as local and regional draining lymph nodes, thereby constitute

the inductive site for the generation of mucosal immunity. The common mucosal immune

system connects these inductive sites with effector sites where antigen-specific lymphocytes

perform their effector functions after extravasation from peripheral blood, directed by the

local profile of vascular adhesion molecules and chemokines (Fig. 1).

Figure 1. The common mucosal immune system. (Modified from Nat. Rev. Immunol. (2004) 4:699-710).

DCs in the lymphoid tissue capture antigens, process and then present them to lymphocytes in

the context of MHC molecules. After antigen-induced priming, proliferation, and partial

differentiation in the MALT, lymphoid memory and effector cells migrate to regional lymph

nodes where further differentiation can take place (Brandtzaeg et al., 1999). The lymphocytes

34

thereafter pass into the peripheral blood circulation whereby extravasation at mucosal effector

sites occurs. These primed cells express adhesion molecules, or “homing receptors”, specific

for corresponding determinants on endothelial cells in mucosal and exocrine glandular tissues

(Butcher and Picker, 1996).

M cells moreover express MHC class II molecules (Allan et al., 1993) and ICAM-1 on their

cell surface (Ueki et al., 1995), indicating that they can initiate an immune response. There

are reports showing that M cells can be found in the lungs as well as in the gut, and that

particulate antigens can be transported through pulmonary M cells (Tenner-Racz et al., 1979).

Teitelbaum et al. demonstrated that transcytosis by pulmonary M cells facilitates delivery of

M. tuberculosis to the broncho-tracheal lymph nodes early after infection, suggesting a role of

M cells in the early development of the local immune response (Teitelbaum et al., 1999).

SECRETORY IgA

Immunoglobulin (Ig) A is the predominant Ig isotype induced at mucosal sites (Brandtzaeg,

1989), where it is believed to mediate defense mechanisms (Mazanec et al., 1993; Lamm,

1997). Monomers of IgA are polymerized through the J chain, which is added just before the

secretion of IgA by plasma cells (Johansen et al, 2000). IgA-producing plasma cells migrate

to the basolateral surface of mucosal epithelial cells, where secreted IgA is transported to the

luminal side by the polymeric Ig receptor (pIgR), expressed at the basolateral side of

epithelial cells lining the mucosal surfaces (Mostov, 1994) (Fig. 2).

Translocation of IgA involves enzymatic cleavage of the pIgR, whereby the extracellular part

of the molecule, the secretory component (SC), in complex with dimeric IgA (dIgA), forms

the secretory IgA (SIgA), that is released into the luminal secretions (Norderhaug et al.,

1999). pIgR-mediated transcytosis does not require the presence of a ligand, resulting in a

continuous release of the SC into external secretions. In human exocrine fluids, 30 to 60% of

SC is normally in a free form (Brandtzaeg, 1973). The constitutive expression of pIgR in

epithelial cells can be further upregulated by certain cytokines, such as IFN-γ (Sollid et al.,

1987; Youngman et al., 1994; Loman et al., 1997), and TNF-α (Kvale et al., 1988).

35

Figure 2. Transport of mucosal IgA. (A) IgA translocation through the epithelium. (B) Structure of SIgA.

FUNCTIONS OF IgA

IgA is thought to be the most important Ig class for lung defense by protecting the mucosal

surfaces from penetration by microorganisms and foreign antigens, as well as by neutralizing

bacterial products such as enzymes and toxins (Mazanec et al., 1993; Lamm, 1997). Other

mechanisms include the ability of IgA to agglutinate microbes and interfere with bacterial

motility by interacting with their flagella. Immune complexes of IgA and encountered

antigens can be transported across epithelial cells from basal to apical surfaces in vitro

(Kaetzel et al., 1991), and in vivo (Robinson et al., 2001). Foreign substances that have

interfered with the mucosal surface can thereby be eliminated from the body by an IgA-

mediated transport back through the epithelium. Additionally, IgA appears to be able to

interact with viral antigens during transcytosis and interfere with viral synthesis and/or

assembly, thereby neutralizing viruses intracellularly (Mazanec et al., 1992). In vitro studies

have demonstrated evidence for such intraepithelial cell interactions with Sendai virus

(Fujioka et al., 1998), measles virus (Yan et al., 2002), influenza virus (Mazanec et al., 1995),

rotavirus (Burns et al., 1996; Feng et al., 2002), and recently against HIV (Huang et al.,

2005).

36

In humans, two subclasses of IgA, termed IgA1 and IgA2, exist, each being the product of a

separate gene, whereas in mice there is only one class of IgA. The major difference between

IgA1 and IgA2 resides in the hinge region, the short region between the two Fab arms and the

Fc region. IgA1 features an extended hinge due to the insertion of a duplicated stretch of

amino acids, which is lacking in IgA2. The longer hinge in IgA1 may have evolved to offer

advantages in antigen recognition by allowing higher avidity bivalent interactions with

distantly spaced antigens (Boehm et al., 1999; Furtado et al., 2004). In secretions, most of the

locally produced IgA is, as described previously, polymeric with a relative increase in the

proportion of IgA2 in human. The reason for the predominance of this subclass might be due

to the increased vulnerability to proteolytic attack that the extended stretch of amino acids

found in IgA1 bring about. Highly specific IgA-cleaving proteases are produced by a number

of important bacterial pathogens that are able to colonize mucosal surfaces and invade

mucosal tissues (Kilian et al., 1996). For instance, Neisseria meningitides, Haemophilus

influenzae, and Streptococcus pneumoniae secrete proteases that specifically cleave the IgA1

hinge region (Senior et al., 1991). IgA2 remains resistant to cleavage since the susceptile

hinge region is missing. Another strategy evolved by bacteria to circumvent effector functions

by host IgA antibodies is the expression of IgA-binding proteins. These are proteins that bind

specifically to IgA and are produced by many strains of group A Streptococcus (Stretococcus

pyogenes) and group B Streptococcus, which are major human pathogens.

IgA is the second most prevalent antibody in serum after IgG, and serum IgA is

predominantely (90%) monomeric IgA1 in humans but mainly dIgA in other animals.

IgA RECEPTORS

IgA has traditionally been viewed as a non-inflammatory antibody. It is a poor activator of

complement and does not activate the classical pathway, although its role in activation of the

alternative pathway remains controversial. Only recently, it has become apparent that dIgA or

polymeric IgA (pIgA), upon binding to mannan-binding lectin, can induce the activation of

the lectin pathway (Roos et al., 2001). The significance of complement activation by IgA in

vivo remains unclear. It can be speculated that in situations where antigen is limited, IgA

could in fact inhibit complement activation by blocking binding of IgG and IgM, which are

more potent activators of complement. The inability of SIgA to fix complement efficiently or

37

to act as an opsonin is an advantage at mucosal sites, where the induction of an inflammatory

reaction would likely affect the integrity of the mucosal surface (Kerr, 1990).

Whereas the role of SIgA in mucosal immunity is being elucidated, the function of serum IgA

antibodies is mostly unknown. Serum IgA is considered to be a “discrete housekeeper”

because IgA-immune complexes can be removed by the phagocytic system with little or no

resulting inflammation. However, characterization of FcRs for IgA (FcαRs) has challenged

the paradigm of IgA as a non-inflammatory or even anti-inflammatory Ig. The human FcαR

(FcαRI, CD89) is expressed on eosinophils (Monteiro, et al., 1990), neutrophils (Honorio-

Franca et al., 2001), monocytes (Patry et al., 1996), macrophage subsets, Kupffer cells, and

DCs (Geissmann et al., 2001). Whereas the interaction of IgA with the pIgR is

noninflammatory, antigen-complexed binding to CD89 mediates a broad spectrum of

proinflammatory and immunomodulatory effects depending on the cell type involved (Morton

et al., 1996; Monteiro and van de Winkel, 2003). These effects include antibody-dependent

cell-mediated cytotoxicity, phagocytosis, release of cytokines, superoxide generation, calcium

mobilization, degranulation and antigen presentation. Therefore, a second line of defense at

the interface of mucosal and systemic immunity, provided by FcαR-serum IgA interactions

on Kupffer cells, has been proposed (van Egmond et al., 2000). Under physiological

conditions, SIgA inhibits the invasion of pathogens into the mucosal surface, as a first line of

defense, without activating inflammatory responses. Under pathological conditions, the

pathogens can invade the portal circulation due to a disruption of the mucosal barrier, where

they will subsequently be exposed to serum IgA. Concomitantly produced inflammatory

cytokines induce FcαRI expression on Kupffer cells, and FcαRI-positive Kupffer cells can

thereby phagocytose the pathogens that have entered the circulation.

Despite extensive studies on FcαRs in man, there is scarce knowledge about the structure and

function of FcαRs in mice. A CD89 homologue in rats was recently identified (Maruoka et

al., 2004), but no mouse homologue has yet been found. A common Fcα/μR was newly

characterized on human and mouse B cells and macrophages, and in different tissues like

liver, spleen, and intestine (Shibuya et al., 2000). Using the human FcαR probe, transcripts of

two cDNAs, PIR-A and PIR-B (paired Ig receptors A and B), isolated from a mouse splenic

library, were detected (Kubagawa et al., 1997). The PIR-A and PIR-B genes were fund to be

expressed on B cells and cells from the myeloid lineage. IgA has furthermore been shown to

38

bind to the intracellular lectin Gal-3 (Mac-2) expressed on several cell types including

macrophages (Reljic, et al., 2004a). Gal-3 has been implicated in allergic reactions due to its

ability to activate mast cells by crosslinking receptor-bound IgE (Liu et al., 1993).

IgA DEFICIENCY

Selective IgA deficiency (SIgAD), using 0.05 g/l as the upper limit for diagnosis in adults, is

the most common form of primary immunodeficiency in the western world and affects

approximately 1/600 individuals (reviewed in Hammarström et al., 2000). The variability in

the prevalence among different ethnic groups is striking (1/18000 in Japanese and 1/4000 in

Chinese), which suggests a genetic implication for the disorder. Although SIgA has a fairly

clear biological role, SIgAD is a heterogeneous condition with symptoms ranging from none

at all to recurrent respiratory or gastrointestinal diseases, atopy, asthma, and inflammatory or

autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, and

pernicious anaemia (Burks and Steele, 1986; Burrows and Cooper, 1997; Cunningham-

Rundles, 2001). However, most individuals remain asymptomatic. IgA deficiency can be

associated with IgG subclass deficiency (Oxelius et al., 1981), making it, in some cases,

difficult to distinguish between the cause and the symptoms. The fact that most IgA-deficient

individuals are healthy may be partly explained by a compensatory increase in IgM-bearing B

cells and increased secretory IgM in mucosal fluids (Norhagen et al., 1989). However,

secretory IgM does not completely replace SIgA functionally, particularly not in the upper

respiratory tract (Brandtzaeg et al., 1987).

39

MALARIA

Malaria is caused by a protozoan parasite of the genus Plasmodium. There are four species

affecting humans, Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The disease is

associated with a variety of clinical syndromes ranging from asymptomatic to lethal infections

involving anaemia, organ failure, pulmonary and cerebral disease. P. ovale and P. malariae

are relatively infrequent causes of morbidity, while P. vivax is a common cause of severe

illness, especially in Asia and South America, but is rarely fatal. The vast majority of severe

malaria cases and deaths are caused by P. falciparum, which is endemic in most of sub-

Saharan Africa, where the WHO estimates that 90% of malaria cases occur. There are over

500 million clinical malaria cases every year, with 1-3 million malaria-associated deaths per

year, of which the majority are of children under the age of five (Snow et al., 2005). In

addition to these human malaria parasites, there are other Plasmodium species that infect

various animals.

PARASITE LIFE CYCLE

The life cycle of the malaria parasite is complex and involves several developmental stages

with asexual reproduction in the human host and sexual reproduction in the Anopheles

mosquito vector (reviewed by Stevenson and Riley, 2004) (Fig. 3). An infected female

mosquito injects 10-30 sporozoites into the host during a blood meal. These sporozoites are

carried by the blood to the liver, where they invade hepatocytes within 30 minutes of a bite.

Inside the hepatocytes they undergo a process of asexual replication, which gives rise to

schizonts. Up to this point the infection is non-pathogenic and clinically silent. The liver

schizonts eventually rupture and thousands of merozoites are released into the blood, where

they invade red blood cells (RBC). Each merozoite can divide inside the RBC and develop

through different stages, such as ring and trophozoite, into schizontes. As the parasite

develops, adherent ligands are expressed on the cell membrane of the infected RBC, one

being P. falciparum erythrocyte membrane protein 1 (PfEMP1), which enables the parasitized

cell to bind to receptors expressed by endothelial cells lining the blood vessels inside organs,

such as the brain, lungs and the placenta. Upon rupture of the infected RBC, more than 30

merozoites are released that can then reinfect new RBCs. This gives rise to a cyclic blood-

40

stage infection, which takes 48-72 hours to complete, depending on the Plasmodium species.

The RBC stage of the parasite’s life cycle is responsible for the symptoms and pathology of

malaria. A small subset of merozoites differentiates into male and female gametocytes that

can be taken up by an Anopheles mosquito feeding on the host. Inside the midgut of the

mosquito these mature into gametes. This is the sexual stage of the parasite’s life cycle and

upon fertilization a motile zygote is formed, which penetrates the epithelial cell layer of the

midgut and forms an oocyst. After 10-24 days, thousands of sporozoites are released from the

oocyst, which invade the salivary glands of the mosquito and can be injected into a host upon

the mosquito’s blood meal.

Figure 3. Schematic view of the malaria parasite life cycle. (Nat. Rev. Immunol. (2001) 1:117-125.)

IMMUNITY TO BLOOD-STAGE MALARIA

Both innate and adaptive immune responses are critical for establishing clinical immunity and

control of parasitemia during an infection with P. falciparum. Innate immune responses are

important for the control of initial parasitemia, and play a role not only for non-immune

individuals infected for the first time, but also for semi-immune individuals who may be

infected with a parasite variant they have never encountered before (Stevenson and Riley,

2004). The initial response to infection usually involves splenic removal of parasitized RBCs.

41

Upon rupture of schizonts, parasite products are released into the circulation and trigger the

activation of phagocytes, resulting in a release of pro-inflammatory cytokines that cause fever

and mediate other pathological effects. One of these bioactive parasite products is

glycosylphosphatidylinosiol (GPI), which acts as a malaria pathogen-associated molecular

pattern and toxin. GPI induces the production of several factors implicated in malaria

pathogenesis, for example pro-inflammatory cytokines, such as TNF-α, IL-1 and IL-12, as

well as iNOS and various adhesion molecules that are expressed on vascular endothelium and

recognized by PfEMP1 (Carlson et al., 1992; Schofield and Hackett, 1993; Tachado et al.,

1997; Naik et al., 2000).

Adaptive immunity

In endemic areas where there is a continous exposure to the parasite, adaptive immunity is

gradually built up and the severity and incidence of malarial illness decrease with increasing

age. Since the parasites have the capacity to vary their antigens, which are major targets for

protective antibodies, repeated exposure to the parasite is required for a long-lasting

immunity. During the first months of life, infants are protected from malaria, most likely by

antibodies transferred from the immune mother. Experimental evidence for species- and

stage-specific immunity in malaria demonstrates that adaptive immunity is crucial for the

protection against malaria (reviewed in Troye-Blomberg, 1994).

Humoral immunity

Ig levels in individuals, living in highly endemic areas are strongly elevated and the level of

total anti-malarial antibodies increases with age. Antibody-mediated protection against blood-

stage malaria is primarily mediated by cytophilic IgG antibodies and can involve different

mechanisms (Good and Doolan, 1999). Antibodies can inhibit merozoite invasion of RBCs,

prevent sequestration of infected RBC by inhibiting binding to adhesion molecules on the

vascular endothelium, neutralize parasite GPI, and thereby inhibit the induction of the

inflammatory cascade, prevent parasite binding to the placenta, and lastly, enhance the

clearance of infected RBCs by binding to their surface and thus promote phagocytosis and

removal of the immune complex in the spleen.

42

It has been reported that levels of total IgE, as well as anti-malarial IgE antibodies, are

elevated in malaria patients, in parallel to an elevation of TNF-α (Perlmann et al., 1994). A

pathogenic role for IgE has been suggested, whereby crosslinking of IgE receptors on

monocytes by IgE-containing immune complexes leads to a local overproduction of TNF-α.

This has recently been questioned by studies indicating a protective role for IgE (Bereczky et

al., 2004; Dolo et al., 2005).

Cellular immunity

T cells are essential for both generating, as well as regulating, immunity against blood-stage

malaria. The major T cells controlling blood stage infections are CD4+ T cells, of both the Thl

and Th2 subsets. Evidence from numerous studies, such as selective depletion of CD4+ T cells

in vivo (Weinbaum et al., 1978; Suss et al., 1988; Kumar et al., 1989), and adoptive transfer

of CD4+ T cells to immunocompromised mice (Brake et al., 1988; Meding and Langhorne,

1991; Taylor-Robinson et al., 1993; Taylor-Robinson and Phillips, 1993), have revealed the

critical role for CD4+ T cells in protective immunity against blood-stage malaria infection.

Important for the generation of protective immunity is IL-12 production, which induces a

polarized Th1 response with a production of IFN-γ and TNF-α. These cytokines activate

phagocytic cells, which are important for controlling the level of parasitaemia.

The levels of T cells expressing the γδ TCR are elevated in acute malaria infection, and they

are believed to have a protective role by exerting cytotoxicity, as well as by secretion of

cytokines, such as IFN-γ (reviewed in Dieli et al., 2001). Human NK cells, which are also

activated early during malaria infection (Orago and Facer, 1991), can be activated rapidly by

parasites in vitro (Theander et al., 1987), resulting in IFN-γ production (Artavanis-Tsakonas

et al., 2003). Since RBCs are devoid of MHC molecules, antigen-specific cytotoxic CD8+ T

cells are generally not believed to act against the asexual blood stage.

DCs in malaria

Early interactions between blood-stage malaria parasites and innate cells are thought to be

43

important for shaping the adaptive immune response to blood-stage malaria. However, the

role of DCs in the induction of protection against the blood-stage malaria parasite is not well

defined. Studies have shown that P. falciparum-infected red blood cells (PfRBC) can bind

myeloid DCs and modulate the maturation and function of DCs. Adhesion of PfRBCs has

been shown to inhibit the upregulation of maturation makers, costimulatory molecules and

adhesion molecules on DCs upon stimulation with LPS. Moreover, the ability of such DCs to

induce T cell responses was significantly reduced (Urban et al., 1999). The receptors on DCs

mediating this inhibitory effect, CD36 and CD51, are not only receptors for PfRBCs but are

also involved in recognition of apoptotic cells by phagocytes. Ligation of CD36 and/or CD51

with PfRBC or apoptotic cells results in a reduced production of IL-12 and an increased

production of IL-10 (Urban et al., 2001). The malarial pigment, hemozoin (Hz), has

furthermore been shown to have an immunomodulatory effect on phagocytic cells. Hz is the

coordinated polymerized form of heme subunits generated by the parasite during hemoglobin

catabolism (Slater et al., 1991). Malaria parasites degrade host hemoglobin for its use as

source of amino acids, which is accompanied by the release of free heme. Free heme is

oxidatively active and toxic to both the host cell and the parasite, causing parasite death. Since

the malaria parasite is unable to catabolyze heme, it protects itself through the crystallization

of heme into Hz (Sullivan et al., 1996a). During a Plasmodium infection, Hz is released into

the blood through the rupture of PfRBC and is readily taken up, together with PfRBC, by

macrophages and monocytes (Schwarzer et al., 1992; Schwarzer et al., 1998).

Investigations on DC function in murine Plasmodium infections have yielded contradictory

results. In vitro and in vivo studies in murine models have shown that, in contrast to the effect

on human DCs, P. chabaudi-infected RBC induced the maturation and activation of murine

DCs (Seixas et al., 2001; Perry et al., 2004; Leisewitz et al., 2004). In contrast, other studies

have reported a suppressive effect on DCs (Ocana-Morgner et al., 2003; Pouniotis et al.,

2004). However, a general trend can be noted where there is an early phase of DC activation

during a blood-stage infection, with a condition of low parasitemia, and a late phase of

inhibition, when the condition is characterised by high parasitemia. Interestingly, a recent

study demonstrated that overstimulation of DCs via TLRs renders them refractory to further

activation (Perry et al., 2005). Since malaria is associated with an exponential increase in

parasite load, DCs are expected to develop TLR tolerance as infection progresses (Perry et al.,

2005). The consequence would be subsequent dysfunction of DCs at later stages of infection.

However, whether DC function is impaired during malaria infection still remains to be

44

established.

CO-INFECTION BETWEEN TB AND MALARIA

The WHO estimates that 90% of the malaria cases occur in sub-Saharan Africa where the

highest incidence of TB per capita also exists. Considering the overlapping geographical

endemic areas for malaria and TB, co-infection is expected to occur very frequently. Given

the chronic character of infections caused by M. tuberculosis and P. falciparum, it is highly

plausible that an interaction between one of these microorganisms and the host’s immune

system would have an impact on the course of infection and disease caused by the other

microorganism. Investigations have established that the immune response induced upon

infection with a pathogen may influence the outcome of subsequent infections or vaccinations

(Curry et al., 1995; Helmby et al., 1998). For instance, helminth infections have been shown

to have an impact on the immune response against mycobacterial infections owing to a Th2

shift in the immune profile (Elias et al., 2005a; Elias et al., 2005b). However, the mutual

modulation of host immune responses by M. tuberculosis and P. falciparum in concurrent

infections has not been extensively studied.

Malaria infection affects the host’s T cells, both qualitatively and quantitatively. Since the

containment of a mycobacterial infection and protective immunity against TB depend on an

intact cellular immunity, malaria-induced impairment of the immune system could influence

susceptibility to, or progression of a M. tuberculosis infection. Clinical evidence on the

outcome of infection or disease in individuals with double Plasmodium/M. tuberculosis

infection, as compared to those infected with a single pathogen, is scarce in the literature. One

indirect piece of evidence comes from a study in Guinea-Bissau that demonstrated a lower

risk of death due to malaria in children with a BCG scar (Roth et al., 2005). This supports

earlier suggestions that the presence of a BCG vaccination scar may be associated with a

general non-specific enhancement of the immune responses to some unrelated infections, and

reduced mortality in children (Velema et al., 1991; Kristensen et al., 2000; Roth et al., 2004),

although revaccination with BCG did not reduce morbidity from malaria in African children

(Rodrigues et al., 2007). A statistically significant association between the presence of

antimalarial antibodies and the diagnosis of TB was reported by Adebajo et al., indicating that

malaria could affect the progression of the mycobacterial disease (Adebajo et al., 1994).

45

ANIMAL STUDIES

The effect of malaria on chronic TB has been investigated in a study utilizing a murine model,

showing increased M. tuberculosis loads in the lungs, liver and spleen of co-infected mice

(Scott et al., 2004). Another study examined the effect of TB on malaria in M. tuberculosis-

infected C57BL/6 mice subsequently infected with Plasmodium yoelii. A protective effect of

prior M. tuberculosis infection against P. yoelii was detected and was associated with an

enhanced Th1 type of immune response (Page et al., 2005). In contrast, Th2-prone BALB/C

mice were not protected against malaria infection by prior M. tuberculosis infection. The

results obtained with the C57BL/6, but not BALB/c mice, corroborate previous studies

demonstrating protection against malaria in animals infected with mycobacteria (Bazaz-

Malik, 1973; Clark et al., 1976; Murphy et al., 1981; Stevenson et al., 1984; Matsumoto et

al., 2001).

Although animal models serve as a valuable tool for studying immune responses and

dissecting underlying mechanisms, caution is advised when extrapolating these results to

humans. Animal models of malaria infection suffer from the lack of specificity caused by host

restriction of malaria species. P. falciparum, the clinically most important human malaria

parasite, does not produce disease in mice.

46

PRESENT STUDY

AIMS

The upper respiratory tract is the port of entrance for the mycobacterium, and the first

encounter between the host and the pathogen takes place in the lung. This raises the

hypothesis that protective immunity against mycobacterial infections is highly dependent on a

local respiratory mucosal immunity in the lungs, in addition to cell-mediated immune

responses. In paper I-II we therefore aimed to investigate the importance of IgA, the major Ig

isotype present at mucosal sites, in the respiratory mucosal immunity against infection with

intracellular mycobacteria.

An efficient immune response against a pathogen is critically dependent on rapid detection of

the invading organism by the innate immunity and the activation of the subseqent adaptive

immune response. Emerging evidence supports the concept that innate immune responses are

vital for the host defense against M. tuberculosis. TLRs represent critical pathogen-

recognition receptors whose signals lead to the generation of important effector responses,

including Th1 responses. We therefore studied the role of TLR signalling in early and late

stages of mycobacterial infection in paper III.

Considering the overlapping geographical endemic areas for two of the leading infectious

causes of morbidity and mortality worldwide, malaria and TB, a high frequency of co-

infection can be presumed. Since DCs have a unique capacity to prime naïve T cells, and

subsequently direct the downstream immunological events that occur after the initial

encounter with the pathogens, they provide a critical link between the innate and adaptive

immune responses. In paper IV we developed a model for studying concurrent

Plasmodium/M. tuberculosis infection using DCs. We assessed the consequences of a malaria

blood-stage infection on a subsequent M. tuberculosis infection of DCs.

47

The specific objectives were:

To evaluate the relevance of IgA in the respiratory tract in the protection against

mycobacterial infection using IgA-deficient mice (paper I).

To study the role of actively secreted IgA in the protection against mycobacterial

infection in the respiratory tract using pIgR-deficient mice (paper II).

To assess the role of different TLRs in the defense against primary mycobacterial

infection using TLR-deficient mice (paper III).

To analyse the effect of malaria antigens on DC susceptibility and response to a

subsequent M. tuberculosis infection (paper IV).

MATERIALS AND METHODS

The materials and methods used in this work are described in the individual papers (I-IV).

RESULTS AND DISCUSSION

PAPER I

In paper I we investigated the role of IgA in the protection against i.n. challenge with BCG

using IgA-deficient mice (Harriman et al., 1999). The most characteristic component of

mucosal immunity is SIgA. An induction of IgA responses in the respiratory tract could

provide a protective function against diseases caused by respiratory infections, among them

TB. We therefore wanted to investigate the relevance of IgA in the respiratory mucosal

immunity against mycobacterial infection, after i.n. immunization with a mycobacterial

antigen.

The antigen used for immunization was the endotoxin-free recombinant PstS-1 protein. This

38 kDa lipoprotein is a putative phosphate-transport receptor, and a known B-, and T-cell

48

stimulant that has been suggested as a potential immunodiagnostic reagent (Wilkinson et al.,

1997). As an adjuvant, for targeting the mucosal immune system, the cholera toxin (CT) was

used. CT is a strong mucosal adjuvant that markedly increases antigen presentation by DCs,

macrophages and B cells (reviewed in Holmgren et al., 2005). It acts by an upregulation of

MHC- and costimulatory molecules as well as chemokine receptors on APCs, and by

increasing the permeability of the epithelium leading to enhanced uptake of co-administered

antigens.

Although protective immunity against the intracellular pathogen M. tuberculosis relies on

efficient cell-mediated immune responses, the function of antibodies is being reappreciated.

(Winslow et al., 2000; Edelson and Unanue, 2001; Hellwig et al., 2001; Glatman-Freedman,

2003). In concordance with this, our results demonstrated that IgA-deficient mice, immunized

with our formulation, were more susceptible to BCG infection compared to immunized wild-

type mice, as shown by a higher bacterial load in the lungs and broncho-alveolar lavage

(BAL). This result suggests involvement of IgA in the protection against infection with

mycobacteria in the respiratory tract.

To address the mechanisms underlying the increased susceptibility to BCG infection

displayed by the IgA-deficient mice, we analyzed the induced local immune responses, after

i.n. immunization with PstS-1 formulated with CT. No IgA was detected in either saliva or

BAL from IgA-/- mice. However, they displayed higher levels of both antigen-specific and

total IgM compared to IgA+/+ mice. Moreover, IgA-deficient mice displayed an impaired T

cell response, as revealed by significantly reduced TNF-α- and IFN-γ-production in the lungs,

when compared to wild-type mice.

Previous studies have demonstrated impaired T cell responses in both IgA-deficient and B-

cell-deficient mice (Vordermeier et al., 1996; Arulanandam et al., 2001; Zhang et al., 2002).

However, the mechanistic details behind have not been clarified. One possibility is

involvement of signalling through FcαRs. The human FcαR (CD89) has been well

characterized, whereas no murine homologue has yet been identified, although a common

Fcα/μR, both human and murine, has been identified, which is expressed on B cells and

macrophages (Shibuya et al., 2000; Sakamoto et al., 2001). Moreover, activated mouse T

cells express FcαRs (Sandor et al., 1992). In vitro studies have demonstrated that monomeric

49

and polymeric IgA, but not IgG or IgM, stimulate TNF-α and NO production, as well as

induction of apoptosis in mouse macrophage cell lines (Reljic et al., 2004b). It is therefore

possible that absence of IgA could lead to a reduced or inadequate activation of macrophages

at mucosal sites, and thereby reduced bacterial clearance. This would furthermore affect

subsequent immunological events, since activated macrophages secrete TNF-α and other

molecules with chemoattractant properties for immune cells.

Reljic et al. (2004a) demonstrated that IgA binds to the intracellular lectin Gal-3, expressed

on activated macrophages, indicating a further role for IgA-mediated inhibition of

intracellular mycobacteria. Gal-3 has been shown to accumulate only in those phagosomes

containing live M. tuberculosis through binding to phosphatidylinositol mannosides, and

appears to influence the clearance of infection (Beatty et al., 2002), since bacterial interaction

with the phagosomal membrane is required for mycobacterial inhibition of phago-lysosome

fusion (de Chastellier and Thilo, 1997). It is possible that the interaction between Gal-3 and

its mycobacterial ligand could be further amplified by IgA, which provides additional

crosslinking of Gal-3. This may keep the pathogen tightly fixed to Gal-3 and away from other

membrane components. In other words, IgA antibodies bound to mycobacteria, or

mycobacterial components, may interfere with interactions between opsonised bacteria and

the membrane of phagosomes, and thereby prevent the survival of mycobacteria within

macrophages.

Important in this context is the study by Geissmann et al. showing that human DCs located

beneath the epithelium express the IgA Fc receptor CD89, and cosslinking by IgA

immunocomplexes results in internalization of the ligand, enhanced expression of the

costimulatory molecule CD86, as well as MHC class II molecules, and an increase in the

allostimulatory activity by the cells (Geissmann et al., 2001).

Another indication of an impaired activation of lung macrophages in the absence of IgA

antibodies, comes from studies demonstrating that only lung-derived macrophages from IgA-

deficient mice, and not bone marrow-derived macrophages, display an impaired activation

phenotype and reduced cytokine production upon stimulation, when compared to wild-type

controls (Rodríguez A., personal communication).

50

All together, our results suggest a role for IgA in the protection against mycobacterial

infection in the respiratory tract, most likely by modulating the locally induced

proinflammatory immune responses through macrophage activation. Another possible

mechanism is the opsonisation of bacteria and blocking the entrance of the bacilli into the

lungs.

PAPER II

In addition to investigating a possible role of IgA in the protection against mycobacterial

infection in the respiratory tract, we also addressed the role of actively secreted antibodies,

specifically SIgA. For this purpose we used pIgR-deficient mice (Shimada et al., 1999).

Although B-cell deficient mice have previously been used in infection studies with

mycobacteria (Vordermeier et al., 1996; Boiso et al., 2000; Turner et al., 2001), mice lacking

actively secreted antibodies have never been used. Generation of pIgR-knockout mice

presents a unique opportunity to explore the relative contribution of local secretory antibodies

versus systemic immunity in the protection against mycobacteria, as has been done previously

in virus-infection studies (Asashi et al., 2002).

Initially, the immunological status regarding antibodies, as well as the induced immune

responses upon i.n. immunization with the same formulation as in paper I, namely PstS-1 in

combination with CT, was assessed in the pIgR-/- mice. Interestingly, although no antigen-

specific IgA was detected in saliva from the knockout mice, IgA levels in BAL were

comparable between the two groups. This finding indicates differences between the upper and

lower respiratory tract concerning the mechanisms of IgA transport to mucosal secretions.

The possibility of transport mechanisms other than through the pIgR cannot be ruled out, nor

can leakage through the epithelial membrane, which has been previously reported (Johansen

et al., 1999). Also, their respective contributions may be different in the upper and lower

respiratory tract. Our finding indicates that the transport of IgA in the upper respiratory tract

is to a larger extent pIgR-mediated than in the lower respiratory tract, where contributions

from other transport mechanisms or leakage may have a greater impact.

Protection studies against i.n. infection with BCG revealed that pIgR-/- mice were more

susceptible to mycobacterial infection than wild-type mice, with significantly higher bacterial

51

burden in the lungs. Although these mice had similar levels of IgA antibodies in the BAL

after i.n. immunization as wild-type mice, their higher bacterial load in the lungs after BCG

infection could indicate that the SC plays a role for the protective capacity of SIgA. The SC

has been associated with regulation of innate, non-specific, responses to pathogens, mainly

due to binding of N-glycans of the SC to bacterial and host factors. One such function is the

stabilization and protection of dIgA and pIgA, and although proteolytic degradation is less

extensive in the respiratory tract than in the gut lumen, the absence of pIgR, and hence the

SC, could imply that the IgA antibodies found in the BAL of pIgR-/- mice are more rapidly

degraded and therefore not able to perform their protective function. A further function

attributed to the SC is its binding to bacterial components, such as Clostridium difficile toxin

A (Dallas and Rolfe, 1998) and fimbriae of enterotoxigenic Escherichia coli (de Oliviera et

al., 2001), thereby limiting infection and morbidity. Although no interactions between the SC

and mycobacteria, or mycobacterial components, have been reported, the possibility cannot be

ruled out. Additonally, it was recently demonstrated, in a mouse model, that SIgA was more

protective than pIgA against respiratory infection with Shigella flexneri, due to carbohydrate-

dependent adherence of the SIgA to the mucus lining of the epithelial cell layer (Phalipon et

al., 2002). SIgA autoantibodies found in saliva from normal subjects were moreover shown to

be polyreactive against pathogenic elements, suggesting an innate function of these antibodies

(Quan et al., 1997). This property was recently confirmed for gastrointestinal SIgA in an

infection study using Salmonella typhimurium (Wijburg et al., 2006). In conclusion, these

properties of the SC suggest that dIgA/pIgA antibodies without the SC, which would be found

in pIgR-/- mice, are less protective than SIgA antibodies that are produced in wild-type mice.

In this study, we also saw that BCG-infected pIgR-/- mice displayed a considerable reduction

in TNF-α and IFN-γ production in lung mononuclear cells. Since these cytokines are central

for protective immunity against mycobacterial infections (Cooper et al., 1993; Flynn et al.,

1995), this impaired mycobacterium-induced proinflammatory response most likely

contributed to the higher bacterial load in the lungs of these mice. We demonstrated in paper I

that IgA-deficient mice have a defective proinflammatory immune response, probably due to

an impaired activation of macrophages or other IgA receptor-bearing cells, and thereby

reduced secretion of cytokines and chemokines. Although the pIgR-knockout mice have IgA

antibodies, the lack of pIgR and SC might render these IgA antibodies less functionable, even

in the sense of their immunomodulary effect. Alveolar macrophages and DCs extending out in

the alveoli might therefore not be optimally activated. Interestingly, a recent study by Favre et

52

al. demonstrated that SIgA works as a weak immunopotentiator in the mucosal environment

in the gastrointestinal tract (Favre et al., 2005). SIgA transported from lumen to the

subepithelial compartment via M cells stimulated mucosal immune responses via upregulated

expression of the costimulatory molecules CD80 and CD86 on DCs.

We next addressed the role of SIgA in the protection against natural infection with virulent M.

tuberculosis. Viable bacterial counts in the lungs clearly demonstrated that pIgR-knockout

mice were more susceptible than wild-type mice at the early phase of infection. At the late

phase of infection no differences between the two groups could be seen. The higher

susceptibility to M. tuberculosis infection was associated with significantly reduced

expression of important protective factors in the lungs. Expression of the proinflammatory

cytokines IFN-γ and TNF-α was substantially reduced, which corroborates the findings in the

BCG-infected pIgR-deficient mice. Moreover, expression of iNOS was also reduced.

Production of RNIs is an important defense mechanism against microbial pathogens exerted

by macrophages. A reduced level of iNOS in the lungs of infected pIgR-/- mice is therefore in

line with the higher bacterial load in the lungs of these mice. Moreover, reduced expression of

the regulated upon activation normal T-cell sequence (RANTES) was also seen in the lungs of

infected knockout mice. This C-C chemokine is involved in attracting monocytes and

lymphocytes to sites of infection, as well as promoting Th1 type of responses (Taub et al.,

1996; Dairaghi et al., 1998; Chensue et al., 1999). These findings indicate that pIgR-/- mice

display lower expression of factors important for protective immunity against TB and proper

granuloma formation. Indeed, histological analysis of granulomas in the lungs demonstrated a

decreased infiltration of macrophages and lymphocytes, but increased numbers of neutrophils

in the pIgR-knockout mice compared to wild-type mice. In addition, a higher degree of

necrosis and karyorrhexis was evident in the granulomas in the knockout mice. This

qualitative histopathological difference was detected at the early phase of infection, whereas

no major differences in granuloma composition where seen during the late phase. In this

context, it is interesting to note that human SC has been reported to bind and inactivate IL-8

(Marshall et al., 2001), a cytokine important for the attraction of neutrophils. Although no

rodent IL-8 homologue has been identified, a murine IL-8 receptor homologue exists (Cerretti

et al., 1993), and mice deficient for this receptor display impaired neutrophil responses and

reduced leukocyte migration (Lee et al., 1995; Becker et al., 2000; Hang et al., 2000). It is

therefore tempting to speculate that the ligand for this murine receptor homologue could, in a

similar fashion to human IL-8, bind to SC, whereby mucosal secretions from pIgR-/- mice

53

would contain higher levels of the putative IL-8 homologue compared to wild-type mice,

resulting in increased chemotaxis of neutrophils in the knockout mice.

Our findings suggest that mycobacteria-infected pIgR-/- mice display a delayed immune

response, due to reduced expression of RANTES and TNF-α and consequently impaired

attraction of macrophages and lymphocytes to the site of infection, as well as a lower

proinflammatory response. As a consequence, pIgR-deficient mice allow more extensive

bacterial growth in the lungs, which in turn triggers an increased granulomatous infiltration in

order to control the bacilli. At the later phase of infection pIgR-/- mice have managed to

control the bacterial growth, displaying comparable viable bacterial counts in the lungs as the

wild-type mice.

Collectively, our results imply a role for SIgA in the modulation of mycobacteria-induced

proinflammatory immune responses and subsequently a role in the protection against

mycobacterial infections.

PAPER III

Resistance against mycobacterial infection and active TB is dependent on the host’s ability to

generate a Th1 type of immune response. Activation of TLRs is an important link between

innate cellular responses and the subsequent initiation of adaptive immune defenses against

microbial pathogens, including mycobacteria. In paper III we investigated the importance of

TLR2 and TLR4 in early and late stage of infection using TLR2-/- mice (Takeuchi et al.,

2004), and TLR4-/- mice (Hoshino et al., 1999) with a C57BL/6 background.

Studies on the interaction between mycobacterial components and TLRs are extensive as well

as numerous, and have established that the 19 kDa lipoprotein and lipid derivatives of

mycobacterium mainly interact with TLR2 to induce predominantely proinflammatory

responses (Krutzik and Modlin, 2004). Although these in vitro studies are essential for

dissecting interactions at a molecular level, the physiological role of TLRs can only be

evaluated using whole mycobacteria. The engagement of multiple TLRs, as well as other

receptors, may affect the outcome of each single interaction. We therefore initially

investigated the susceptibility of TLR-deficient mice to BCG infection. Given that TB is

54

primarily acquired through inhalation of airborne droplets containing the bacterium, we

infected the experimental animals via the respiratory tract, in order to resemble a natural

infection. Analysis of the bacterial burden in the lungs of infected mice demonstrated an

impaired control of bacterial growth in the lungs of TLR2-/- mice at the early stage of

infection, whereas no difference between wild-type and knockout mice could be detected at

the later stage. The role of TLR4 signalling appears less important in the control of

mycobacterial growth, both at early and late stages of infection. In addition, TLR6- and

TLR9-deficient mice did not differ from wild-type mice in terms of inhibition of bacillary

growth. The fact that an increased susceptibility among TLR2-/- mice was only evident during

early stages of infection, suggests that the absence of signalling through TLR2 could be

compensated for as the infection proceeded. Other danger signals, such as endogenous factors

released upon inflammation-induced tissue pathology, might contribute to evoking an immune

response through other TLRs or additional receptors. Heat shock proteins (Ohashi et al.,

2000; Vabulas et al., 2002), hyaluronan (Termeer et al., 2002) and fibronectin (Smiley et al.,

2001) released from damaged cells have been shown to trigger TLR4 signalling.

When using the intravenous (i.v.) route of infection no differences between any of the

experimental groups could be seen, indicating the importance of the chosen route of infection.

Several studies have addressed the role of TLRs in inducing protective immunity against

mycobacterial infection by infecting experimental animals i.v. Since TLR expression on

resident, as well as attracted immune cells, may differ in various parts of the body, results

from i.v. infections might not be relevant for an airborne bacterium. For instance, human

alveolar epithelial cells express both TLR2 and TLR4 (Armstrong et al., 2004). However,

considering the possibility of dissemination of a mycobacterial infection, i.v. infection can

provide supplementary information. In the light of our results, TLR-dependent effector

mechanisms appear to be especially important in the respiratory tract. Indirect support for this

comes from studies demonstrating TLR-mediated activation of human bronchial epithelial

cells and subsequent secretion of beta defensin-2 (Kagnoff and Eckmann, 1997; Hertz et al.,

2003). Pulmonary epithelial cells have additionally been shown to produce IFN-γ in response

to M. tuberculosis infection (Sharma et al., 2007). Although no receptor interaction was

assessed in that study, this finding implies a role for the alveolar epithelial cells in the innate

immunity against mycobacterial infections. Data on the TLR distribution on murine

pulmonary epithelial cells is, however, lacking.

55

In addition to BCG, we also used virulent M. tuberculosis as the infectious agent. Our results

from aerosol infection of mice were in line with the BCG findings, with the exception that

TLR4-/- mice, in addition to TLR2-/- mice, displayed an impaired control of mycobacterial

growth at the early stage of infection, which was not seen during BCG infection. We believe

the reason for this difference could be due to the different mycobacteria used. Considering

that the latter experiment involved the pathogen causing TB, these findings indicate that

TLR4 signalling, besides TLR2, is of importance for inducing protective immunity against

virulent M. tuberculosis infections. We also included TLR6-/-- and TLR9-/- mice in this

experiment, however, no differences between these mouse strains and wild-type mice were

detected.

Alveolar-resident macrophages within the lung are considered to be the main cellular host for

mycobacteria, and the major role for these cells is rapid killing of the invading pathogen. This

is due to the release of toxic reactive oxygen and nitrogen intermediates, or to the killing by

lysosomal enzymes following fusion of the lysosome with the mycobacteria-containing

phagosome. In addition, cytokine production by these cells is essential for the initiation of

cellular immune responses. Given the important function of macrophages, and the fact that

these cells express TLRs (Punturieri et al., 2004), we evaluated the intracellular growth of

mycobacteria in bone marrow-derived macrophages from TLR-deficient mice, as well as their

cyokine response. Our results suggest that the higher bacterial load in TLR2-/- mice is a result

from impaired activation of macrophages, since they display impaired capacity to control

intracellular growth of BCG, in addition to a reduced secretion of TNF-α upon infection with

mycobacteria. The absence of TLR4 signalling appears less harmful since TLR4-/-

macrophages were not as affected as macrophages from the TLR2-/- mice. Phagosome

maturation has been demonstrated to be regulated by TLR signalling (Blender and Medzhitov,

2004), which could explain the growth advantage of BCG in TLR2-deficient macrophages.

TNF-α produced by activated macrophages is important not only for further activation of

macrophages, but also for attracting other immune cells and granuloma formation via

induction of chemokines (Algood et al., 2004). We moreover detected completely abolished

TNF-α production by MyD88-deficient macrophages, indicating that this signalling pathway

is the principal mediator in initiating anti-mycobacterial responses in macrophages.

Additional experiments revealed a reduced iNOS mRNA expression in TLR2-deficient

56

macrophages upon BCG infection, giving further evidence for the importance of TLR2

signalling for the induction of effector functions against mycobacteria in macrophages.

TLR-deficient mice, particularly TLR2-/-mice, furthermore showed defects in the generation

of proinflammatory and Th1-associated cytokine responses when using an antigen

restimulation assay. This suggests defective T-cell priming in vivo in these mice, which is

supported by a previous study (Heldwein et al., 2003). The absence of T cell activation and

expansion following infection could be a consequence of impaired antigen presentation and

costimulation, involving macrophages as well as DCs. Although an intrinsic defect in the

previously reported TLR2-deficient T cell function could not be ascribed the impaired T cell

priming in vivo (Heldwein et al., 2003), the possibility still remains. Not only classical innate

cells, but also T cells have been shown to express certain TLRs (Kabelitz, 2007). It is

therefore possible that TLRs could act as costimulatory molecules to enhance the proliferation

and cytokine production of TCR-stimulated T cells.

In contrast to in vitro restimulation assays, ex vivo studies investigating the expression of

protective molecules, such as IFN-γ, TNF-α, RANTES and iNOS, in the lungs of M.

tuberculosis-infected mice demonstrated no differences between TLR-knockout mice and

wild-type controls. Since TLR2- and TLR4-deficient mice displayed higher bacterial burdens

in the lungs at the early stage of M. tuberculosis infection, it is possible that an impairment of

the studied molecules, that initially might have caused the increased susceptbility to infection,

had already been compensated for at the time of assessment. Histological evaluation of the

lungs revealed a higher granulomatous response in the lungs of infected TLR-knockout mice

at the later stage of infection, most likely a consequence of the initially higher bacterial load

that triggered a stronger granulomatous infiltration. This response would then sufficient to

control the bacterial growth.

Collectively, our results demonstrate a protective role for TLR2, and partly TLR4, in the host

defense against mycobacterial infection in the respiratory tract. These receptors are

particularly important during the early phase of the infection. A deficiency in mainly TLR2,

but also to some extent TLR4, results in an impaired anti-mycobacterial macrophage activity,

and a defective proinflammatory response.

57

PAPER IV

Malaria, an infectious disease causing around 2 million deaths every year, shares a common

geographical distribution with TB. The chronic character of the infections caused by the two

pahogens, M. tuberculosis and P. falciparum, implies that the interaction of one of these

pathogens with the host’s immune system could have an impact on the course of infection

caused by the other. Such mutual interactions have previously been demonstrated for other

co-infecting pathogens (Curry et al., 1995; Helmby et al., 1998). We therefore established a

model to study the effect of blood-stage malaria on human DC response and the susceptibility

of DCs to a succeeding M. tuberculosis infection. Given the unique qualities of DCs as

inducers and modulators of immune responses upon interactions with microbial substances,

we believe that this model is highly relevant for dissecting the effect of a concurrent M.

tuberculosis and P. falciparum infection. In endemic areas, malaria infection is likely to

precede a M. tuberculosis infection, since Plasmodium infections are aquired during early

childhood, whereas the risk of M. tuberculosis infection increases later in life (Chadha et al.,

2003; Balasubramanian et al., 2004; Kolappan et al., 2004). Our initial purpose was therefore

to assess the effect a malaria blood-stage infection would have on human DCs, in particular

with regard to a succeeding M. tuberculosis infection.

P. falciparum parasites (strain F32, mycoplasma free) were cultured (Trager and Jensen,

1976) in human O+ RBC, and schizont- and late trophozoite stage PfRBCs were harvested for

co-culturing with human DCs. Alternatively, the malarial pigment Hz was used to represent a

malaria blood-stage infection. Hz is the aggregated form of heme polymers, generated after

parasite degradation of host hemoglobin, and released in the blood stream upon rupture of

parasite-infected RBC. To evaluate the effect of Hz, and not contaminating proteins,

membranes or GPI molecules, we used synthetic Hz (Egan et al., 2001). Synthetic Hz (β-

hematin) is structurally (Slater et al., 1991; Sullivan et al., 1996b) and biologically (Sherry et

al., 1995) similar to Hz formed naturally by parasites. These two preparations, PfRBC and

Hz, were used as malaria parasite-derived material (MDM) in the study.

We initially assessed the susceptibility to M. tuberculosis infection of DCs pre-exposed to

either PfRBC or Hz. It has previously been reported that PfRBCs suppress DC function

(Urban et al., 1999; Urban et al., 2001), and that Hz inhibits the differentiation of human

58

monocytes into DCs, and reduces their responsiveness to maturation signals (Skorokhod et

al., 2004). This suggested that DCs exposed to PfRBC or Hz could be more susceptible to M.

tuberculosis infection. However, our results revealed the opposite, since MDM-exposed DCs

were infected by M. tuberculosis to a lower extent as compared to non-exposed controls.

Light microscopic analysis additionally revealed that the two pathogens, or pathogen-derived

substances, could co-exist in the same cell.

In order to elucidate the mechanism of the reduced susceptibility to M. tuberculosis infection,

we analysed the expression of M. tuberculosis uptake-mediating receptors after pre-exposure

of DCs to either PfRBC or Hz. The recently identified C-type lectin DC-SIGN is the major

receptor for M. tuberculosis on DCs (Geijtenbeek et al., 2003; Tailleux et al., 2003). When

evaluating the cell surface expression of DC-SIGN after co-culturing DCs with Hz, we

detected no change in expression as compared to untreated cells. In addition, exposing DCs to

PfRBC induced a slight increase in the expression of DC-SIGN. The same results were

obtained for MR, another M. tuberculosis receptor expressed on DCs, as well as on

macrophages (Schlesinger, 1993). These findings imply that the decreased susceptibility of

DCs to M. tuberculosis infection cannot be attributed to changed uptake of bacteria due to an

altered receptor expression.

It is possible that the reduced uptake of M. tuberculosis by DCs after pre-exposure to malaria

antigens is simply a consequence of a diminished phagocytic capacity and not a specific event

for M. tuberculosis. To address this question we assessed the phagocytosis of latex beads after

treatment of DCs with Hz. There was a clear loss in phagocytic ability by DCs exposed to Hz,

which indicates a general loss in phagocytic activity as the cause for the reduced uptake of M.

tuberculosis by these DCs. Immature DCs are efficient in antigen uptake and act as sentinels

in the periphery, where they capture antigens and then migrate to draining lymph nodes to

prime T cells. The process of migration is associated with functional and phenotypical

maturation such that, upon arrival in the lymph node the DC has acquired the capacity to

effectively stimulate naïve T cells. This is related to upregulation of MCH class II- and

costimulatory molecules (Steinman, 1991). The functional consequence of maturation is the

DCs’ loss in phagocytic ability. After co-culturing DCs with PfRBC or Hz, we detected an

upregulation of MHC class II- and the costimulatory molecules CD86 and CD40, which

indicated a maturation of the cells, in line with the finding of their reduced phagocytic ability.

59

Efficient induction of T-cell activation is dependent on the interaction between ligands on T

cells with costimulatory molecules expressed on APCs. Considering the previously reported

inhibition of PfRBC of DC responses to the maturation stimulus LPS (Urban et al., 1999), we

expected that the response of DCs to M. tuberculosis infection would be downmodulated after

prior treatment with the malaria antigens. In contrast to the findings by Urban et al., the M.

tuberculosis-induced maturation of DCs was not affected by exposure to either PfRBC or Hz

prior to M. tuberculosis infection. These discrepant findings might be due to the different

stimuli used, and their divergent signalling pathways. M. tuberculosis interacts with various

receptors, including different TLRs (Geijtenbeek et al., 2003; Quesniaux et al., 2004; Bafica

et al., 2005; Floto et al., 2006), while LPS interacts only with TLR4 (Poltorak et al., 1998). In

addition to serving as receptor for PfRBCs and apoptotic cells, CD36 was recently implicated

in mediating responses to microbial ligands through TLR2, by binding to diacylglycerides and

enhancing signalling thorugh the TLR2/TLR6 heterodimer (Hoebe et al., 2005). In addition,

TLR2 agonists are able to block TLR4-induced cytokine production upon concomitant

engagement of TLR2 and TLR4 in human DCs (Re and Strominger, 2004). Binding to CD36

by PfRBC could thereby recruit TLR2 into a signalling complex and affect the signalling

casade induced by LPS through TLR4 (Stevenson and Urban, 2006). Considering the

complexity in the interactions between mycobacteria and DCs, and subsequently the divergent

signalling pathways engaged, the inhibitory effect of PfRBC may be overcome upon M.

tuberculosis infection. Moreover, the induction of maturation by Hz has been shown to be

dependent on TLR9 (Coban et al., 2005). Since human myeloid DCs do not express this

receptor (Mazzoni and Segal, 2004), this pathway should not be of importance in our model.

Besides TCR-mediated recognition of antigens presented by MHC molecules and interaction

with costimulatory molecules expressed on the surface of APCs, activation of T cells is highly

dependent on production of cytokines by APCs. Analysis of cytokine production by DCs after

M. tuberculosis infection demonstrated an enhanced Th1 type of response after prior

treatment to malaria-derived antigens. Both PfRBC- and Hz-treated DCs displayed increased

secretion of TNF-α and reduced secretion of IL-10, following M. tuberculosis infection. In

addition, pre-exposure to PfRBC also triggered IFN-γ production by M. tuberculosis-infected

DCs, while Hz had no effect in this context. As stated earlier, a Th1-biased immune response,

with initiation of cell-mediated immunity, is crucial for resistance against mycobacterial

infections and development of active TB, with IFN-γ and TNF-α being key components. In

60

light of this, our findings allow for the speculation that malaria infection could impose relative

resistance to M. tuberculosis infection, although the ensuing T-cell reponse was not studied in

our model. Moreover, dissemination of mycobacterial infections can be achieved by migration

of infected DCs, implying that the reduced infectivity of PfRBC- or HZ-exposed DCs could

result in a decreased spread of a M. tuberculosis infection. However, although the protective

effect by TNF-α is well established, an exaggerated production can result in host pathology.

Moreover, IL-10 can reduce the extent of inflammatory-induced injury by its

immunouppressive effect (Bogdan et al., 1991; Cunha et al., 1992; Flesch et al., 1994;

Murray et al., 1997; Marshall et al., 1997; Balcewicz-Sablinska et al., 1998).

It is tempting to speculate that an enhanced innate response to M. tuberculosis infection after

prior exposure to MDM, which we found in this study, could have a harmful effect on the

control of malaria infection. The initial response to malaria infection is characterised by a Th1

type of immunity that eventually shifts to a predominantly Th2 type, in order to initiate

antibody-mediated immune mechanisms important for parasite clearance (Taylor-Robinson

and Smith, 1991; Langhorne et al., 2002). A prolonged proinflammatory response could

potentially interfere with this immunological shift.

Taken together, results of our study demonstrate that malaria-blood stage infection can

influence the function of human DCs and have an impact on the outcome and response to a

concurrent M. tuberculosis infection.

61

CONCLUDING REMARKS

TB remains a major health threat worldwide and is the largest cause of death attributable to a

single infectious agent. The causative agent of the disease, M. tuberculosis, has a particular

tropism for the lungs, making pulmonary TB the most common and dangerous form of the

disease. Mucosal surfaces in the respiratory tract are constantly challenged by massive

amounts of inhaled microorganisms and antigens, and in many cases the main protective

effector function against mucosal infections is the production of pathogen-specific local SIgA

responses, achieved solely via activation of the mucosal immune system. The importance of B

cells and antibodies in protection against the intracellular pathogen M. tuberculosis remains

controversial, although a protective capacity of antibodies has lately been reconsidered.

Based on the fact that TB is transmitted via the respiratory tract, where the infection

additionally is manifested, and that IgA in mucosal secretions is the most characteristic

component of mucosal immunity, we investigated the relevance of IgA in protection against

mycobacterial infection in the respiratory tract. We conclude from our studies that IgA

antibodies play a role in the protection against mycobacterial infection in the respiratory tract,

by modulating the locally induced proinflammatory immune responses and/or by blocking the

entrance of the bacillus into the lungs. The mechanisms explaining the defective

proinflammatory responses in the lungs of IgA- and pIgR-deficient mice might involve

impaired signalling through FcαRs, or homologous receptors, which may lead to inadequate

activation of pulmonary macrophages. Insufficient activation of macrophages could

consequently result in suboptimal production of factors important for attraction or migration

of immune cells to the lungs, as well in reduced killing of the bacteria inside the

macrophages. The SC, being extraordinary abundant at mucosal sites, provide additional

innate-like properties that could be of importance for protection of mucosal surfaces.

Induction of effective immune responses cannot be achived without prior activation of the

innate immunity. Production of cytokines and chemokines by macrophages and DCs is crucial

for initiating protective immune reponses against M. tuberculosis, making early recognition of

the invading pathogen essential for containment of infection. TLRs are key molecules in

bridging innate and adaptive immunity. Since M. tuberculosis infects professional phagocytes

that express a diverse array of TLRs, interest is focused on the function and importance of

62

these receptors in combating mycobacterial infections. Our studies demonstrate a protective

role for TLR2, and partly TLR4, in anti-mycobacterial immune responses. However,

important to consider when evaluating involvement of host factors to immunological

responses against infectious agents is the natural route of infection. This is obvious from

divergent results using different routes of infection.

There is increasing evidence that the immune response against one pathogen can influence the

outcome of subsequent infections with other pathogens. Similarly, unrelated infections can

modulate vaccine-induced protective responses to other pathogens. In the light of alarming

reports on the deleterious interaction of co-infecting HIV and M. tuberculosis with the host,

and the now generally accepted harmful conseqences of co-infection beween HIV and

malaria, studies of concurrent infections with more than one pathogen should be emphasized.

Information from such studies will be of importance for vaccine reseach and drug design.

Little is known about the outome of concurrent M. tuberculosis/P. falciparum infections,

although malaria and TB occur endemically in overlapping geographical areas. We developed

an in vitro model for studying the interaction beween the pathogens causing TB and malaria

and human DCs, and our results suggest that a malaria blood-stage infection can affect the

reponse to and outome of an ensuing M. tuberculosis infection. These findings encourage

further studies on the complex immunological events occurring in individuals simultaneously

infected with these two extremely important pathogens.

For the future perspective, it is important to consider the advantages of efficiently targeting

vaccine antigens to appropriate components within the systemic and mucosal immune

compartments. As TB is primarily an infection of the respiratory tract, a successful vaccine

against TB should stimulate appropriate immunological responses to counteract the tubercle

bacillus in the lungs. The effect of concurrent infectious agents, or potential infections, on

vaccination is moreover an essential question to address.

63

ACKNOWLEDGEMENTS These studies were financially supported by the European Commission specific RTD programs QLK2-CT-1999-

00367 and QLK2-CT-2002-00846, Hjärt- och Lungfonden, the Swedish Agency for Research for Research with

Developing Countries (Sida/SAREC) and EU LSHP-CT-2004 503578.

I wish to express my warm gratitude to everyone who has contributed in any way in making

this thesis a reality and given me support during these intense and fascinating years,

especially:

My supervisors Marita Troye-Blomberg and Andrzej Pawlowski. Marita, my warmest

gratitude for always finding solutions, for believing in me and giving me the trust, strength

and opportunity to carry on and finishing my studies. Thanks for your concern for me and my

situation and for always having my best interest in mind. Andrzej, my deepest gratitude for

dedicating your time and interest in me and my studies, for being a warm, caring, patient, and

helpful person, and for making me grow as a researcher and as a person, and for making me

believe in myself. Thanks for always being willing to answer questions. And for always

knowing the answers!

My previous supervisor Carmen Fernández for your commitment and dedication for my

work and the results we achieved.

Gunilla Källenius for welcoming me to SMI and the TB section and letting me continue my

PhD studies there, and for being a kind and caring person.

My dear friend, former colleague and co-author Ariane Rodríguez for introducing me into

the various techniques and strategies at the department. I am immensely grateful for your

friendship and all the support you have, and continue to give me.

My cherished friend Nancy Vivar for your help, support, patience, laughs, chats at SMI and

MTC. Tanks for being my “ventil” and for your endless patience with me and for your

willingness to teach me everything you know. You inspire me to be a better person!

64

Margareta “Maggan” Hagstedt, the foundation on which the department is built on. My

warmest gratitude to you for all your help and support these years and especially for your help

with our “friends”. I have enjoyed working with you and miss our coffee breaks.

My collaborators and co-authors in Barcelona, Pere-Joan Cardona, Evelyn Guiardo,

Esther Julian, for excellent collaboration and valuable discussions.

Former and present seniors at the department of immunology: Klavs Berzins, Manuchehr

Abedi Valugerdi, Eva Severinson, Eva Sverremark-Ekström and Alf Grandien. Thank

you for sharing your vast knowledge in the field of immunology and for your support and

help with my work.

Gelana Yadeta, for making the department run smoothly and for all your help with one or the

other thing.

Shanie Saghafian Hedengren, I treasure our friendship deeply and I am grateful for all your

support. When the going gets tough, the tough gets going!

Nora Bachmayer and Yvonne Sundström, thanks for your friendship, support and all the

fun we have together! What would I do without you?

Hedvig Perlmann, for your positive and friendly character and for being an inspiration in

science. I would like to recognize late Peter Perlmann whose generous and dedicated

personality has been, and continues to be, an inspiration for researchers in the field of

immunology.

My old roomies Karin Lindroth, Monika Hansson and Masashi Hayano, thanks for your

guidance during my first trembling time at the department, and for being caring and good

friends, even after leaving the department.

Everyone in the TB section at SMI:

Maria Andersson, Solomon Ghebremichael, Melles Haile, Beston Hamasur, Sven

Hoffner, Emma Huitric, Lech Ignatowicz, Pontus Juréen, Tuija Koivula, Lisbeth Klintz,

Jolanta Mazurek, Alexandra Pennhag, Ramona Peterson, Senia Rosales, Juan Carlos

65

Toro and Jim Werngren. Thank you for welcoming me to SMI and making me feel part of

the section. I also want to thank everyone at the Department of Bacteriology, SMI, for

providing a friendly atmosphere where I have enjoyed working. A special thanks to Kristina

Jonas for all your help.

Past and present colleagues at the department of immunology – Petra Amoudruz, John

Arko-Mensah, Nancy Awah, Halima Balogun, Ahmed Bolad, Mounira Djerbi, Salah

Eldin Farouk, Pablo Giusti, Ben Gyan, Ulrika Holmlund, Elisabet Hugosson, Nnaemeka

Iriemenam, Lisa Israelsson, Gun Jönsson, Anna-Karin Larsson, Khosro Masjedi, Jacob

Minang, Amre Nasre, Alice Nyakeriga, Jubayer Rahman, Camilla Rydström, Valentina

Screpanti, Ylva Sjögren, Ankie Söderlund, Khaleda Qazi Rahman, Manijeh Vafa, Nina-

Maria Vasconcelos, Stefania Varani. A special thanks to Ann Sjölund for your warm

personality and “magic hands” with cells.

Francesca Chiodi at MTC, and the students in her group for being kind and helpful whenever

I came asking for something or desperately wondered where Nancy was.

The staff at the animal house, Eva Nygren and Solveig Sundberg, for taking such good care

of my mice and always greeting me with a smile.

My wonderful and fantastic friends who keep me balanced in life, especially:

Anneli and Mats for being the most wonderful people I have ever met! You make my life

brighter and happier. Thanks for your generosity and letting me enjoy Minus, Yoda and

Snufs whenever I want.

Anna H, for being so stable and making me realize that life is more than work and science,

and for help and support whenever I needed it.

My Friskis companion and dear friend Pernilla for putting up with all my delays during the

years. Maybe a gympa-dinner-movie evening soon?

Eva, min allra käraste vän, thank you for always listening to me, being there for me, and

making my life happy.

66

Marie, you are my role model in life. Your fantastic strength inspires me and keeps me

focused on what is import in life. Little Douglas for being the most adorable boy in the world!

Finally, and most of all, I would like to express my deepest and warmest gratitude to my

beloved family, my mother Margareta, father Sigge, sister Karin and grandmother Vera.

Where in this world would I be without you? What would I be? Thank you for your love and

support throughout my life, and especially the last five years.

Simon, thanks for joining the Tjärnlund family, for your warm and generous personality and

for putting up with all the “dos talk”.

67

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