Escherichia coli flagellin stimulates pro-inflammatory immune response

ORIGINAL PAPER

Escherichia coli flagellin stimulates pro-inflammatory immuneresponse

Ayaid Khadem Zgair

Received: 30 October 2011 / Accepted: 1 February 2012 / Published online: 12 February 2012

� Springer Science+Business Media B.V. 2012

Abstract Flagellin, a principal component of bacterial

flagella, is a virulence factor that is recognized by the

innate immune system. Recognition of flagellin by innate

immune receptors stimulates the production of cytokines

necessary for the development of effective immunity. Here,

we demonstrated that the intranasal (i.n.) instillation of

different amount of Escherichia coli K-12 flagellin prepa-

ration (0.5, 1, 2, 4 lg) in BALB/c mice induced pro-

inflammatory immune response. Instillation i.n. of 1 lg of

flagellin induced the maximum expression of interleukin 1

beta (IL-1b), tumor necrosis factor alpha (TNF-a) and

interleukin 6 (IL-6) mRNA and production of pro-inflam-

matory cytokines (IL-1b, TNF-a and IL-6) in mice lungs.

The same dose of flagellin induced neutrophil polymor-

phonuclear cells infiltration in peribronchial and perivas-

cular regions. High number of neutrophil in

bronchoalveolar lavage fluid was found at 24 h after i.n.

instillation of flagellin (1 lg). These findings were con-

comitant with the maximum production of myeloperoxi-

dase and nitric oxide in mice lungs. Present study showed

that the maximum pro-inflammatory mediator levels were

found when mice instilled i.n. with 1 lg E. coli flagellin.

The amount of flagellin of E. coli K-12 that achieve the

maximum stimulation of mucosal pro-inflammatory

immune response in mice lungs was explored in this study.

Keywords E. coli flagellin � Pro-inflammatory cytokines �Nitric oxide � Neutrophil activity

Introduction

Flagellin, the protein monomer, that builds up bacterial

flagella is a pathogen-associated molecular pattern (PAMP)

universally recognized by plants and animals (Felix et al.

1999; Gewirtz et al. 2001). Monomeric flagellin is recog-

nized by Toll-like receptor 5 (TLR5); TLRs represent a

major family of pattern recognition receptors (PRRs).

TLRs are playing a front-line role in host defenses by

inducing innate immune responses through nuclear factor

kappa B (NF-kB) signaling (Coudriet et al. 2010) as well as

through members of the mitogen-activated protein kinase

(MAPK) family (Zhang et al. 2007; Dowling et al. 2008).

Various cells of the pulmonary tract, including the epi-

thelial, macrophages and neutrophil cells, express TLR5

(Lo’pez-Boado et al. 2005; Ramos et al. 2004; Hayashi

et al. 2001). Mucosal administration of flagellin induces

MyD88-dependent signaling, characterized by the swift

production of various pro-inflammatory cytokines [inter-

leukin 1 beta (IL-1b), tumor necrosis factor alpha (TNF-a)

and interleukin 6 (IL-6)], chemokines (interleukin 8 (IL-8))

and nitric oxide (NO) (Steiner et al. 2000; Eaves-Pyles

et al. 2000; Honko and Mizel 2004; Ramos et al. 2004;

Feuillet et al. 2006; Balloy et al. 2007; Nempont et al.

2008; Janot et al. 2009; Zgair and Chhibber 2010; Coudriet

et al. 2010; Huang et al. 2009) and heavy neutrophil

infiltration into the airways (Feuillet et al. 2006; Andersen-

Nissen et al. 2007; Janot et al. 2009; Zgair and Chhibber

2010).

Previous studies have established that flagellin induces

systemic inflammatory responses when administered

intraperitoneally or intravenously (Honko and Mizel 2005).

The impact of flagellin on innate and adaptive immunity in

the lung is clearly important, given the role of flagellin as a

virulence factor (Feldman et al. 1998; Wolfgang et al.

A. K. Zgair (&)

Department of Biology, College of Science, Baghdad University,

Baghdad, Iraq

e-mail: [email protected]

123

World J Microbiol Biotechnol (2012) 28:2139–2146

DOI 10.1007/s11274-012-1019-0

2004) and a potential adjuvant for vaccine therapy

(Ben-Yedidia et al. 1999; Jeon et al. 2002; McDermott

et al. 2000). Many studies reported that mucosal adminis-

tration of flagellin intranasally induced mucosal innate

immunity. Honko and Mizel (2004) proved that intrat-

racheally (i.t.) instillation of soluble recombinant flagellin

of Salmonella enterica serovar Enteritidis induced innate

immunity in mice lungs (Honko and Mizel 2004). This was

characterized by the infiltration of neutrophils and the rapid

production of TNF-a, IL-6, granulocyte colony-stimulating

factor and the chemokines. In another recent study, intra-

nasal (i.n.) administration of flagellin preparation of

Stenotrophomonas maltophilia induced innate immunity in

mouse lung (Zgair and Chhibber 2010). In this study, we

presented that the i.n. delivery of purified Escherichia coli

flagellin stimulated mucosal pro-inflammatory immunity in

mice lungs.

Materials and methods

Bacterial isolate

Escherichia coli K-12 was used in this study. It is a stan-

dard strain, procured from Microbial Technology Institute,

Chandigarh, India. Bacteria were preserved by lyophiliza-

tion and were routinely cultured at 37�C on Luria–Bertani

agar plates. Subcultures were made every week.

Flagellin preparation

Flagellin from E. coli K-12 was isolated and purified

according to the procedure described earlier (Zgair and

Chhibber 2010). LPS was removed from flagellin prepa-

ration by passing it through a polymyxin B column

according to the manufacturer’s instructions (Detoxin-Gel;

Pierce, Rockford, IL, USA), and residual levels in flagellin

preparations were\1 pg/lg, as detected by the quantitative

chromogenic Limulus amebocyte lysate assay (Bio-Whit-

taker) (Moors et al. 2001; Honko and Mizel 2004).

To determine the effects of flagellin on lung immunity,

flagellin was instilled directly by i.n. route. Different doses

of soluble flagellin (0.5, 1, 2, 4 lg) in a total volume of

50 ll of pyrogen-free phosphate-buffered saline (PBS)

(0.2 M, pH 7.2) were instilled intranasally of BALB/c mice

(McDermott et al. 2000).

Animals

Male BALB/c mice of 6–8 weeks old, weighing 20–25 g,

were procured from central animal house of Baghdad

University, Baghdad, Iraq. Animals were kept in clean

polypropylene cages and fed on standard antibiotic-free

diet (JBD agencies, Pvt. Ltd., India). The study was con-

ducted following approval from the animal ethics com-

mittee of Department of Biology, College of Science,

Baghdad University, Baghdad, Iraq.

Experiment

The experimental group: BALB/c mice, divided into four

subgroups depending on the flagellin dose (0.5, 1, 2, 4 lg).

For intranasal immunization, mice were anesthetized with

Avertin [2,2,2-tribromoethanol (Sigma); tert-amyl alcohol

(Fisher)] by intraperitoneal injection and suspended from a

length of wire by their front incisors (Honko et al. 2006).

The control group consisted of mice that were given PBS

(0.2 M, pH 7.2) intranasally (i.n.). Four experimental group

animals were killed at different time intervals post-flagellin

administration. In the control group, three animals were

killed at different time intervals. Lung tissue was sampled

to quantify inflammatory mediators and histopathological

changes in lung tissue.

Pro-inflammatory cytokines

Whole lungs were weighted and then homogenized in 3 ml

lysis buffer containing 0.5% Triton X-100, 150 mM NaCl,

15 mMTris, 1 mM MgCl2 (pH 7.4) (Mohler et al. 2003).

Cytokines assay

Tumor necrosis factor-a, IL-1b and IL-6 levels were

detected in lung homogenate supernatants of samples from

control and immunized animals at different time intervals

post-instillation of flagellin. Mouse ELISA kits were used

to measure all the cytokines. IL-1b, IL-6 (BD OptEIATM

,

San Jose, CA, USA) and TNF-a (Antigenix America Inc.

Hunting sta. NY, USA) kits were used according to the

manufacture’s instructions.

Preparation of total RNA and RT-PCR

Lungs were homogenized in Trizol reagent (Invitrogen,

CA, USA) with an Ultra Turrax homogenizer and were

stored at -80�C. Total RNA was then isolated using RNA

isolation kit (Ultraspec-II; Biotecx, Houston, TX, USA).

TNF-a, IL-1b, IL-6 and glyceraldehyde-3-phosphate

dehydrogenase (G3PDH) mRNA levels were measured by

reverse transcription-polymerase chain reaction (RT-PCR)

(Jang et al. 2006). Purified total RNA was used as the

template in RT-PCR. cDNA was synthesized using Molo-

ney murine leukaemia virus reverse transcriptase (Pro-

mega) according to the manufacturer’s instructions. PCR

primers used were as described previously for TNF-a,

2140 World J Microbiol Biotechnol (2012) 28:2139–2146

123

IL-1b, IL-6 and G3PDH (Zimova-Herknerova et al. 2008;

Munoz et al. 2010; Hwang et al. 2011). PCR amplification

consisted of 40 cycles of denaturation at 94�C for 30 s,

annealing at 55�C for 30 s and extension at 72�C for 1 min.

Amplified products were separated on 1.8% agarose gels

and stained with ethidium bromide, and band intensity was

measured in a still video system (Eagle Eye II; Stratagene,

La Jolla, CA, USA). G3PDH mRNA expression was

included to indicate that the differences of gene expression

were not due to differences in the concentration of total

RNA templates. The results were expressed as the ratio of

mean count of IL-1b, TNF-a and IL-6 mRNA expression

level to that of G3PDH mRNA expression level.

Myeloperoxidase (MPO)

The standard method of Hirano (1996) was followed to

check the MPO activity in lungs homogenates (Hirano

1996).

Measurement of nitric oxide (NO) production

To confirm the NO production in the lungs, the nitric oxide

reaction was measured in the form of nitrite level in the

lung homogenate by the colorimetric method of Tsai et al.

(1997).

Bronchoalveolar lavage cell counts

Total cell count in bronchoalveolar lavage (BAL) speci-

mens was measured using hemocytometer. The neutrophil

count was determined by examining stained smear with

Leishman stain (Skerrett et al. 2007).

Histology

Lungs were fixed in 4% formalin (Sigma-Aldrich) for 24 h

and were then embedded in paraffin. Lung blocks were

sectioned at a thickness of 5 lm using a Leica microtome

(Wetzlar, Germany) and were adhered to slides. Three

mice per group were analyzed. Five sections from each

mouse were stained with hematoxylin and eosin and were

analyzed under light microscope (Accu-scope 3013 Phase

Trinocular Halogen with 3.2 MP CMOS Digital Micro-

scope Camera, New York Microscope Co.) (Medan et al.

2002; Munoz et al. 2010). The lung sections taken at each

time point were examined. In each time point, fifteen

sections were made, and in each section, around five fields

were examined to check different histological alterations

such as leukocytes infiltration around blood vessels or all

over the field, shape of blood vessels, general morphology

of alveoli, infiltration of PMN leukocytes inside alveoli and

edema. For semiquantitative analysis, the amount of cell

infiltration was stated according to frequency of this

alteration in each section.

Statistical analysis

All values have been taken as a mean value and the stan-

dard error calculated. The differences were analyzed using

Student’s t test employing Origin 8.0 version software. A

value of p \ 0.05 was considered to be statistically

significant.

Results

Pro-inflammatory cytokines production

Figure 1 shows the effects of pretreatment with different

amount of flagellin (0.5, 1, 2, 4 lg per mouse, i.n.) on the

production of different pro-inflammatory cytokines (IL-b,

TNF-a and IL-6) in mice lungs at 4 h and 12 h after fla-

gellin instillation. Significant elevation of IL-1b production

started as early as 4 h post-flagellin instillation. Maximum

production of IL-1b was detected in lung homogenates of

mice instilled with 1 lg of flagellin, while lowest IL-1bproduction was seen in mice that treated with high dose of

pure flagellin (4 lg) (Fig. 1a). A similar trend was seen in

case of IL-6 levels in the lung homogenates (Fig. 1c). Little

difference was found in case of TNF-a levels in lung

homogenates (Fig. 1b). Flagellin at a concentration of 1 lg

was found to be the best inducer of pro-inflammatory

cytokines in mice lungs, whereas a higher concentration

suppressed their production.

Pro-inflammatory cytokine gene expression

To assess the time course of changes in the levels of IL-

1b, TNF-a and IL-6 mRNA expression, we performed

RT-PCR analysis of total RNA of lung homogenates. For

semiquantitative evaluation, mRNA expression of IL-1b,

TNF-a and IL-6 was normalized against those of house-

keeping gene G3PDH (Fig. 2). IL-1b mRNA expression

started as early as 1 h post-flagellin instillation, and the

amount of IL-1b mRNA increased dramatically with time.

Maximum gene expression of IL-1b was found at 4 h

post-instillation with 1 lg of flagellin, followed by

instillation with 0.5 lg at the same time point (4 h).

However, the minimum level of IL-1b mRNA expression

was found when mouse instilled with high concentration

of pure flagellin (4 lg) (Fig. 2a, d). Similarly, maximum

TNF-a and IL-6 mRNA expression was seen when mice

instilled with 1 lg of flagellin i.n. followed by instillation

with 2 lg. Minimum expression of these genes (TNF-aand IL-6 mRNA) was detected in lung homogenates of

World J Microbiol Biotechnol (2012) 28:2139–2146 2141

123

mice group that treated with high concentration of fla-

gellin (4 lg) (Fig. 2b, c, e, f). In the present study, the

best upregulation of pro-inflammatory cytokine genes was

observed when mice instilled with 1 lg of flagellin i.n.

and the expression was observed as early as 1 h after

flagellin instillation (i.n.).

0

500

1000

1500

2000

2500

3000

3500

*

*

**

*

*

*

IL-1

β (p

g/g

lung

tiss

ue)

*

4 h 12 h 4 h 12 h0

500

1000

1500

2000

*

**

**

*

*

TN

F- α

(pg/

g lu

ng ti

ssue

)

*

4 h 12 h0

200

1200

1600

2000

2400

2800

**

*

*

*

*

*

IL-6

(pg/

g lu

ng ti

ssue

)

*

Time after flagellin instillation

ba c

Fig. 1 Time-course of changes in IL-b (a), TNF-a (b) and IL-6

(c) levels in lung homogenates of mouse following i.n. instillation of

different amount of E. coli K-12 flagellin: 0 lg (PBS, control)

(hatched bars), 0.5 lg (white bars), 1 lg (black bars), 2 lg (gray

bars) and 4 lg (light horizontal bars). Each value represents the

mean ± SE of four mice each. Asterisks indicate a significant

difference from the untreated control group (p \ 0.05, Student’s

t test)

Fig. 2 Time-course of changes in IL-1b (a, d), TNF-a (b, e) and IL-6

(c, f) mRNA expression in mouse lung homogenates analyzed by RT-

PCR. The lungs were sampled at different time intervals (0, 1, 2, 4 h)

after i.n. instillation of different concentrations of E. coli K-12

flagellin: 0 lg (PBS, control) (hatched bars), 0.5 lg (white bars),

1 lg (black bars), 2 lg (gray bars) and 4 lg (light horizontal bars).

a–c Represent agarose gel electrophoresis of mRNA expression of IL-

1b, TNF-a and IL-6, respectively. d–f Represent densitometric

analysis of IL-1b, TNF-a and IL-6 mRNA expression levels relative

to the expression of the housekeeping gene G3PDH. Each value

represents the mean ± SE of four mice each. Asterisks indicate a

significant difference from the untreated control group (p \ 0.05,

Student’s t test)


123

Neutrophil infiltration and MPO activity levels

Figure 3a shows neutrophil count in BAL fluid (BALF)

obtained from mice lungs post-instillation of different

amounts of flagellin (i.n.). Significant elevation of neutro-

phil count in BALF started at 12 h with maximum at 24 h

post-instillation (i.n.) of flagellin; thereafter, neutrophil

count declined dramatically. Maximum neutrophil infil-

tration was observed in BALF that obtained from mice

instilled with 1 lg of flagellin (i.n.). MPO activity levels in

lung homogenates are summarized in Fig. 3b. Similar to

previous results of neutrophil infiltration, significant

increase in MPO activity was found at 12 h post-flagellin

instillation. Flagellin at a concentration of 1 lg was found

to be the best stimulator of MPO activity in mice lungs.

Nitric oxide (NO) production in mice lungs

In order to show the kinetics of NO production after fla-

gellin instillation i.n., different amounts of pure flagellin

were administrated i.n. to produce inflammatory events in

the lungs. Nitrite levels in lung homogenates were signif-

icantly elevated above naive values throughout the time

course (Fig. 4). Without the instillation of flagellin (control

group), low nitrite accumulation was observed. Significant

elevation of nitrite levels was observed at 4 h post-instil-

lation of 1 lg of flagellin (i.n.). Levels of nitrite increased

gradually thereafter to reach a maximum value at 24 h,

while significant elevation of nitrite following instillation

of 0.5, 2, 4 lg of flagellin was observed at 12 h post-fla-

gellin instillation. Present study proved that an instillation

of 1 lg of flagellin i.n. achieved the maximum accumula-

tion of nitrite level in mouse lung.

Histopathological examination

To assess whether the expression of pro-inflammatory

genes and upregulation of pro-inflammatory cytokines

production correlated with inflammation and cellular

infiltration into the airways, we performed histological

analysis of lung tissue obtained 24 h after treatment with

1 lg of E. coli flagellin (i.n.). As shown in Fig. 5, flagellin

induced moderate cellular infiltration in peribronchial and

perivascular areas close to these bronchioles (Fig. 5b, d).

Flagellin treatment induced edema, as well as little

0

2

4

6

8

10

***

**

**

**

*

MP

O (

nmol

/mg

prot

ein)

*

4 h 12 h 24 h 48 h4 h 12 h 24 h 48 h0.0

5

6

7

8

9

**

*

*

**

***

Log 10

neu

trop

hilis

/ml

*


a b

Fig. 3 Time-course of changes in neutrophil count (a) in BALF

obtained from mice lungs following i.n. instillation of different

concentrations of flagellin (0, 0.5, 1, 2, 4 lg). b Represents MPO

activity levels in lung homogenates following i.n. instillation of

different concentrations of flagellin: 0 lg (PBS, control) (hatchedbars), 0.5 lg (white bars), 1 lg (black bars), 2 lg (gray bars) and

4 lg (light horizontal bars). Samples were collected at different time

intervals (4, 12, 24, 48 h) after i.n. instillation of E. coli K-12

flagellin. Each value represents the mean ± SE of four mice each.

Asterisks indicate a significant difference from the untreated control

group (p \ 0.05, Student’s t test)

4 h 12 h 24 h 48 h0

20

40

60

80

100

**

**

*

*

*

*

**

**

*

Nitr

ite (

μg/ m

l)


*

Fig. 4 Time-course of changes in nitrite levels in lung homogenates

following i.n. instillation of different concentrations of flagellin: 0 lg

(PBS, control) (hatched bars), 0.5 lg (white bars), 1 lg (black bars),

2 lg (gray bars) and 4 lg (light horizontal bars). Each value

represents the mean ± SE of four mice each. Asterisks indicate a

significant difference from the untreated control group (p \ 0.05,

Student’s t test)


123

infiltration of cells affecting not only perivascular and

peribronchial regions but also some areas of the sur-

rounding lung parenchyma (Fig. 5c). These results suggest

that flagellin induces a transient inflammatory response

without causing any drastic collapse of bronchi and blood

vessel wall.

Discussion

Gram-negative flagellin, a major PAMP of Gram-negative

bacteria recognized by TLR5, is a potent inducer of innate

immune effectors such as cytokines and nitric oxide

(Hayashi et al. 2001; Mizel et al. 2003; Zgair and Chhibber

2010). In the lung, flagellin induces a localized and tran-

sient innate immune response characterized by neutrophil

infiltration and the production of cytokines and chemo-

kines. Many studies reported that flagellin of different

species of bacteria stimulated mucosal innate immunity in

respiratory tract (Ciacci-Woolwine et al. 1998; Honko and

Mizel 2004; Lo’pez-Boado et al. 2005; Munoz et al. 2010;

Zgair and Chhibber 2010). In the present study, we per-

formed that the preparation of flagellin (monomeric pro-

tein) isolated from E. coli K-12 induced mucosal pro-

inflammatory mediators and neutrophil infiltration in

airway of BALB/c mice. We found that the instillation of

1 lg of E. coli flagellin yields abundant levels of pro-

inflammatory cytokines (IL-1b, TNF-a and IL-6). In

addition, this amount of flagellin induced expression of the

pro-inflammatory cytokines mRNA as early as 1 h after

flagellin instillation (i.n.). These findings were concomitant

with maximum neutrophil infiltration and high levels of

NO and MPO production in mice lungs.

In line with present study, Honko and Mizel (2004)

reported that the monomeric recombinant flagellin from

S. enterica serovar Enteritidis stimulated high production

of pro-inflammatory cytokines. These investigators con-

firmed the role of TLR5 homomeric complexes in the

induction of TNF-a production in response to flagellin but

they also suggested that TLR5/TLR4 heteromeric com-

plexes are required for the production of nitric oxide (NO)

via an Interferon-gamma (IFN-c-) and Signal transducers

and activators of transcription-1-(STAT-1-)-dependent

mechanism (Mizel et al. 2003). Therefore, flagellin require

TLR5 only to induce pro-inflammatory cytokines produc-

tion and required TLR5 and TLR4 to induce NO produc-

tion, that is why, both receptors are important in

intracellular killing of bacterial by AMs, as NO has bac-

tericidal activity (Marletta et al. 1988; Mizel, et al. 2003).

The flagellin serves as a strong proinflammatory stimulus,

Fig. 5 Inflammatory effects of flagellin in the BALB/c lung. Mice

were anesthetized, and their lungs were i.n. instilled with 1 lg of

E. coli K-12 flagellin in a total volume of 50 ll of pyrogen-free PBS.

Lung sections were prepared and stained for histological analysis with

haematoxylin and eosin. B, bronchiole; BV, blood vessel; E, edema;

PMNL, polymorphonuclear leukocyte. a Control section from a

mouse receiving only PBS (bars, 200 lm). b–d Sections were taken

at 24 h after the flagellin instillation (i.n.). Neutrophil PMNLs

infiltration in the peribronchial and perivascular areas indicated by

arrows in panel d. Edema and neutrophil PMNLs infiltration

surrounding the lung parenchyma shown in panel c (bars, 200 lm,

200 lm and 100 lm in panels b–d, respectively)


123

increasing interleukin 8 (IL-8) production via the activation

of TLR5. (Gewirtz 2006). Although numerous intracellular

signaling pathways may be involved such as IkB/NFkB

pathway, previous studies where flagellin induced IL-8

production in immature (H4 cells) and mature (T84 cells)

enterocytes showed that increased production of chemo-

kines was caused by decreased levels of IkB expression

and activation of NFkB nuclear translocation (Claud et al.

2004). The high production of chemokines (IL-8) results

high infiltration of PMNL in immunized area with flagellin,

this going on with the results that achieved in present

study.

Previous study showed that flagella from the enteropath-

ogenic E. coli strain E2348/69, Y. enterocolitica JB580 and

Pseudomonas aeruginosa PAO1, which did not induce sig-

nificant levels of TNF-a production in human promonocytic

cell line U38 U38 cells, were as potent as Salmonella flagella

in terms of TNF-a and interleukin 1b activation in human

peripheral blood mononuclear cells (PBMC) (Ciacci-

Woolwine et al. 1998). That happened, as these investigators

treated macrophages with flagella (polymeric flagellin). The

polymerized form of flagellin (flagella) is approximately

100-fold less effective to stimulate pro-inflammatory

immune response when compare with monomeric flagellin

(Smith et al. 2003). TLR50s (a key of inflammatory immune

response) targeting of the conserved regions of the flagellin

monomer affords this receptor the ability to recognize

flagellins from a wide variety of bacteria including Salmonella,

E. coli, Pseudomonas, Listeria, Legionella, Clostridia and

Vibrio and subsequently stimulates innate immune system

to produce pro-inflammatory mediators (Vijay-Kumar and

Gewirtz 2009).

Our results demonstrated the ability of E. coli flagellin

to stimulate mucosal pro-inflammatory immune response.

This kind of immune response is non-specific so it can be

used to provide protection against wide spectrum of

pathogens that can infect respiratory tract of the patients

suffer from immune deficiency disease. This work is going

on in my laboratory.

References

Andersen-Nissen E, Hawn TR, Smith KD, Nachman A, Lampano AE,

Uematsu S, Akira S, Aderem A (2007) Tlr5_/_ mice are more

susceptible to Escherichia coli urinary tract infection. J Immunol

178:4717–4720

Balloy V, Verma A, Kuravi S, Si-Tahar M, Chignard M, Ramphal R

(2007) The role of flagellin versus motility in acute lung disease

caused by Pseudomonas aeruginosa. J Infect Dis 196:289–296

Ben-Yedidia T, Tarrab-Hazdai R, Schechtman D, Arnon R (1999)

Intranasal administration of synthetic recombinant peptide-based

vaccine protects mice from infection by Schistosoma mansoni.Infect Immun 67:4360–4366

Ciacci-Woolwine F, Blomfield IC, Richardson SH, Mizel SB (1998)

Salmonella flagellin induces tumor necrosis factor alpha in a

human promonocytic cell line. Infect Immun 66:1127–1134

Claud EC, Lu L, Anton PM, Savidge T, Walker WA, Cherayil BJ

(2004) Developmentally regulated IkappaB expression in intes-

tinal epithelium and susceptibility to flagellin-induced inflam-

mation. Proc Natl Acad Sci USA 101:7404–7408

Coudriet GM, He J, Trucco M, Mars WM, Piganelli JD (2010)

Hepatocyte growth factor modulates Interleukin-6 production in

bone marrow derived macrophages: implications for inflamma-

tory mediated diseases. PLoS One 5:e15384

Dowling D, Hamilton CM, O’Neill SM (2008) A comparative

analysis of cytokine responses, cell surface marker expression

and MAPKs in DCs matured with LPS compared with a panel of

TLR ligands. Cytokine 41:254–262

Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG,

Szabo C, Salzman AL (2000) Flagellin, a novel mediator of

Salmonella-induced epithelial activation and systemic inflam-

mation: IjB degradation, induction of nitric oxide synthase,

induction of proinflammatory mediators, and cardiovascular

dysfunction. J Immunol 166:1248–1260

Feldman M, Bryan R, Rajan S, Scheffler L, Brunnert S, Tang H,

Prince A (1998) Role of flagella in pathogenesis of Pseudomonasaeruginosa pulmonary infection. Infect Immun 66:43–51

Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive

perception system for the most conserved domain of bacterial

flagellin. Plant J 18:265–276

Feuillet V, Medjane S, Mondor I, Demaria O, Pagni PP, Gala0n JE,

Flavell RA, Alexopoulou L (2006) Involvement of Toll-like

receptor 5 in the recognition of flagellated bacteria. Proc Natl

Acad Sci USA 103:12487–12492

Gewirtz AT (2006) Flag in the crossroads: flagellin modulates innate

and adaptive immunity. Curr Opin Gastroenterol 22:8–12

Gewirtz AT, Simon PO, Schmitt CK, Taylor LJ, Hagedorn CH,

O’Brien AD, Neish AS, Madara JL (2001) Salmonella typhimu-rium translocates flagellin across intestinal epithelia, inducing a

proinflammatory response. J Clin Investig 107:99–109

Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR,

Eng JK, Akira S, Underhill DM, Aderem A (2001) The innate

immune response to bacterial flagellin is mediated by Toll-like

receptor 5. Nature 410:1099–1103

Hirano S (1996) Migratory responses of PMN after intraperitoneal

and intratracheal administration of lipopolysaccharide. Am J

Physiol 270:L836–L845

Honko AN, Mizel SB (2004) Mucosal administration of flagellin

induces innate immunity in the mouse lung. Infect Immun

72:6676–6679

Honko AN, Mizel SB (2005) Effects of flagellin on innate and

adaptive immunity. Immunol Res 33:83–101

Honko AN, Sriranganathan N, Lees CJ, Mizel SB (2006) Flagellin is

an effective adjuvant for immunization against lethal respiratory

challenge with Yersinia pestis. Infect Immun 74:1113–1120

Huang L-Y, DuMontelle JL, Zolodz M, Deora A, Mozier NM,

Golding B (2009) Use of toll-like receptor assays to detect and

identify microbial contaminants in biological products. J Clin

Microbiol 47:3427–3434

Hwang JH, Chen JC, Yang SY, Wang MF, Chan YC (2011)

Expression of tumor necrosis factor-a and interleukin-1b genes

in the cochlea and inferior colliculus in salicylate-induced

tinnitus. J Neuroinflammation 8:30

Jang CH, Choi JH, Byun MS, Jue DM (2006) Chloroquine inhibits

production of TNF-a, IL-1b and IL-6 from lipopolysaccharide-

stimulated human monocytes/macrophages by different modes.

Rheumatology 45:703–710

Janot L, Sirard JC, Secher T, Noulin N, Fick L, Akira S, Uematsu S,

Didierlaurent A, Hussell T, Ryffel B, Erard F (2009)


123

Radioresistant cells expressing TLR5 control the respiratory

epithelium’s innate immune responses to flagellin. Eur J

Immunol 39:1587–1596

Jeon SH, Ben-Yedidia T, Arnon R (2002) Intranasal immunization

with synthetic recombinant vaccine containing multiple epitopes

of influenza virus. Vaccine 20:2772–2780

Lo’pez-Boado YS, Cobb LM, Deora R (2005) Bordetella bronchi-septica flagellin is a proinflammatory determinant for airway

epithelial cells. Infect Immun 73:7525–7534

Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS (1988)

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric

oxide is an intermediate. Biochemistry 27:8706–8711

McDermott PF, Ciacci-Woolwine F, Snipes JA, Mizel SB (2000)

High affinity interaction between gram-negative flagellin and a

cell surface polypeptide results in human monocyte activation.

Infect Immun 68:5525–5529

Medan D, Wang L, Yang X, Dokka S, Castranova V, Rojanasakul Y

(2002) Induction of neutrophil apoptosis and secondary necrosis

during endotoxin-induced pulmonary inflammation in mice.

J Cell Physiol 191:320–326

Mizel SB, Honko AN, Moors MA, Smith PS, West AP (2003)

Induction of macrophage nitric oxide production by gram-

negative flagellin involves signaling via heteromeric Toll-like

receptor 5/Toll-like receptor 4 complexes. J Immunol 170:

6217–6223

Mohler J, Azoulay-Dupuis E, Amory-River C, Mazoit JX, Bedos JPP,

Rieux V, Moine P (2003) Streptococcus pneumoniae strain-

dependent lung inflammatory responses in a murine model of

pneumonia. Intensive Care Med 29:808–816

Moors MA, Li L, Mizel SB (2001) Activation of interleukin-1

receptor associated kinase by gram-negative flagellin. Infect

Immun 69:4424–4429

Munoz N, Van Maele L, Marque0s JM, Rial A, Sirard JC, Chabalgoity

JA (2010) Mucosal administration of flagellin protects mice from

Streptococcus pneumoniae lung infection. Infect Immun 78:

4226–4233

Nempont C, Cayet D, Rumbo M, Bompard C, Villeret V, Sirard JC

(2008) Deletion of flagellin’s hypervariable region abrogates

antibody-mediated neutralization and systemic activation of

TLR5-dependent immunity. J Immunol 181:2036–2043

Ramos HC, Rumbo M, Sirard JC (2004) Bacterial flagellins:

mediators of pathogenicity and host immune responses in

mucosa. Trends Microbiol 12:509–517

Skerrett SJ, Wilson CB, Liggitt HD, Hajjar AM (2007) Redundant

Toll-like receptor signaling in the pulmonary host response to

Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol

292:L312–L322

Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA,

Barrett SL, Cookson BT, Aderem A (2003) Toll-like receptor 5

recognizes a conserved site on flagellin required for protofilament

formation and bacterial motility. Nat Immunol 4:1247–1253

Steiner TS, Nataro JP, Poteet-Smith CE, Smith JA, Guerrant RL

(2000) Enteroaggregative Escherichia coli expresses a novel

flagellin that causes IL-8 release from intestinal epithelial cells.

J Clin Investig 105:1769–1777

Tsai WC, Strieter RM, Zisman DA, Wilkowski JM, Bucknell KA,

Chen GH, Standiford TJ (1997) Nitric oxide is required for

effective innate immunity against Klebsiella pneumoniae. Infect

Immun 65:1870–1875

Vijay-Kumar M, Gewirtz AT (2009) Flagellin: key target of mucosal

innate immunity. Mucosal Immunol 2:197–205

Wolfgang MC, Jyot J, Goodman AL, Ramphal R, Lory S (2004)

Pseudomonas aeruginosa regulates flagellin expression as part

of a global response to airway fluid from cystic fibrosis patients.

Proc Natl Acad Sci USA 101:6664–6668

Zgair AK, Chhibber S (2010) Stenotrophomonas maltophilia flagellin

induces a compartmentalized innate immune response in mouse

lung. J Med Microbiol 59:913–919

Zhang Z, Reenstra W, Weiner DJ, Louboutin JP, Wilson JM (2007)

The p38 mitogen-activated protein kinase signaling pathway is

coupled to Toll-like receptor 5 to mediate gene regulation in

response to Pseudomonas aeruginosa infection in human airway

epithelial cells. Infect Immun 75:5985–5992

Zimova-Herknerova M, Myslivecek J, Potmesil P (2008) Retinoic

acid attenuates the mild hyperoxic lung injury in newborn mice.

Physiol Res 57:33–40


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