Flagellin FliC Research Articles

Hybrid flagellin as a T cell independent vaccine scaffold.

BackgroundTo extend the potency of vaccines against infectious diseases, vaccines should be able to exploit multiple arms of the immune system. One component of the immune system that is under-used in vaccine design is the subset of B cells known to be capable of responding to repetitive antigenic epitopes and differentiate into plasma cells even in the absence of T cell help (T-independent, TI).ResultsTo target vaccine responses from T-independent B cells, we reengineered a bacterial Flagellin (FliC) by replacing its exposed D3 domain with a viral envelope protein from Dengue virus (DENV2). The resulting hybrid FliC protein (hFliC) was able to form stable filaments decorated with conformationally intact DENV2 envelope domains. These filaments were not only capable of inducing a T cell-dependent (TD) humoral antibody response, but also significant IgM and IgG3 antibody response in a helper T cell repertoire-restricted transgenic mouse model.ConclusionsOur results provide proof-of-principle demonstration that a reengineered hybrid FliC could be used as a platform for polymeric subunit vaccines, enhancing T cell-dependent and possibly inducing T-independent antibody responses from B-1 B cells as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0194-0) contains supplementary material, which is available to authorized users.

Improvement in T helper 1-related immune responses in BALB/c mice immunized with an HIV-1 gag plasmid combined with a chimeric plasmid encoding interleukin-18 and flagellin.

Both flagellin (fliC) and IL-18 (INF-γ-inducing factor) have been developed as adjuvants for improving immunogenicity in DNA-vaccinated hosts. An HIV-1 gag plasmid encodes a protein harboring broad epitopes for cytotoxic T-lymphocytes. In this study, the immunogenicity of BALB/c mice immunized with an HIV-1 gag plasmid (pVAX/gag) combined with a chimeric plasmid encoding IL-18 fused to flagellin (pcDNA3/IL-18_fliC) or a single plasmid encoding IL-18 (pcDNA3/IL-18) and/or flagellin (pcDNA3/fliC) was assessed. Through in vitro transcription and translation, it was demonstrated that both mRNA and protein were appropriately expressed by each construct. The IL-18 and flagellin fusion protein, which could be detected in supernatants from transfected cells, was effective in inducing IFN-γ by lymphocytes. Following i.m. immunization, expressions of flagellin or IL-18 were detected in muscle cells by immunohistochemistry analysis from 72 hr. At 12 weeks post-immunization, both gag-specific IgG in sera and spleen cell proliferation were high in all murine groups. However, the IgG2a/IgG1 ratio, Th1 cytokine (IL-2 and IFN-γ) production and proportion of gag-specific CD3(+) CD8(+) IFN-γ-secreting cells were significantly higher in the murine group co-immunized with pVAX/gag plasmid and pcDNA3/IL-18_fliC than in the mice immunized with pVAX/gag plasmid combined with either pcDNA3/fliC or pcDNA3/IL-18 plasmid or both. These findings suggest that a chimeric plasmid encoding IL-18 fused to flagellin can be used as an adjuvant-like plasmid to improve the Th1 immune response, particularly for induction of CD3(+) CD8(+) IFN-γ-secreting cells in gag plasmid-vaccinated mice.

The O-Antigen Capsule of Salmonella enterica Serovar Typhimurium Facilitates Serum Resistance and Surface Expression of FliC.

Group IV polysaccharide capsules are common in enteric bacteria and have more recently been described in nontyphoidal Salmonella species. Such capsules are known as O-antigen (O-Ag) capsules, due to their high degree of similarity to the O-Ag of the lipopolysaccharide (LPSO-Ag). Capsular polysaccharides are known virulence factors of many bacterial pathogens, facilitating evasion of immune recognition and systemic dissemination within the host. Previous studies on the O-Ag capsule of salmonellae have focused primarily on its role in bacterial surface attachment and chronic infection; however, the potential effects of the O-Ag capsule on acute pathogenesis have yet to be investigated. While much of the in vivo innate immune resistance of Salmonella enterica serovar Typhimurium is attributed to the high-molecular-weight LPS, we hypothesized that the O-Ag capsule may enhance this resistance by diminishing surface expression of pathogen-associated molecular patterns, such as flagella, and increasing resistance to host immune molecules. To test this hypothesis, O-Ag capsule-deficient mutants were constructed, and the loss of O-Ag capsular surface expression was confirmed through microscopy and immunoblotting. Loss of O-Ag capsule production did not alter bacterial growth or production of LPS. Western blot analysis and confocal microscopy revealed that O-Ag capsule-deficient mutants demonstrate reduced resistance to killing by human serum. Furthermore, O-Ag capsule-deficient mutants produced exclusively phase I flagellin (FliC). Although O-Ag capsule-deficient mutants did not exhibit reduced virulence in a murine model of acute infection, in vitro results indicate that the O-Ag capsule may function to modify the antigenic nature of the bacterial surface, warranting additional investigation of a potential role of the structure in pathogenesis.

Development and evaluation of a Salmonella typhimurium flagellin based chimeric DNA vaccine against infectious bursal disease of poultry

Infectious bursal disease (IBD) is an acute immunosuppressive disease of young chicks, caused by a double-stranded RNA virus. VP2 being the major capsid protein of the virus is an ideal vaccine candidate possessing the neutralizing epitopes. The present study involves the use of flagellin (fliC) as a genetic adjuvant to improve the immune response of VP2 based DNA vaccine against IBD. Our findings revealed that birds immunized with plasmid pCIVP2fliC showed robust immune response than pCIVP2 immunized groups. Further, challenge study proved that genetic fusion of fliC and VP2 can provide a comparatively higher level of protection against vvIBDV challenge in chickens than VP2 alone. These results thus indicate that Salmonella flagellin could enhance the immune responses and protection efficacy of a DNA vaccine candidate against IBDV infection in chickens, highlighting the potential of flagellin as a genetic adjuvant in the prevention of vvIBDV infection.

The flagellin D2/D3 domain induces TLR5- and inflammasome- independent antibody responses (INM2P.357)

Abstract The detailed understanding of the molecular mechanisms underlying the biological activity adjuvants is critical for rational design of more effective vaccines. Bacterial flagellin, a potent immunogen, is being developed as a new adjuvant and platform for human vaccines. We have demonstrated that the conserved D0 and D1 domains of flagellin promote antibody responses through activation of their respective innate immune receptors, but are not required for Salmonella flagellin (FliC) induced antibody responses. We hypothesized that additional flagellin domains also influence specific aspects of flagellin-induced B cell responses. Mice were immunized with a mixture of FliC mutants proteins and ovalbumin, and sera were collected for quantification of anti-flagellin and anti-ovalbumin specific antibody responses. The results from our studies demonstrate that the D2/D3 domain of the FliC is also required for flagellin-induced primary antibody responses and robust secondary responses. D2/D3 domain is capable of inducing antibody responses independent of TLR5 and the inflammasome. Our data indicate the flagellin D2/D3 domain synergizes with the TLR5 (D1) and inflammasome (D0) activating domains to induce potent antibody responses, and that the D2/D3 domain also functions as an adjuvant to promote antibody responses against co-administered antigens. Future studies will elucidate the contribution of the FliC D2/D3 domain to B cell responses and its utility in vaccines.

Flagellin has differential and route-dependent adjuvant effects when encoded in heterologous systemic or mucosal gene-based prime-boost vaccines (VAC4P.1107)

Abstract Flagellin has been tested as a protein-based adjuvant, with most studies focused on antibody responses. In this study, we have evaluated the adjuvant activity of flagellin for cellular and humoral immune responses in gene-based immunizations. DNA and adenovirus (Ad) vectors were constructed to encode mycobacterial Ag85B and/or flagellin (FliC) of S. typhimurium. DNA-encoded flagellin given to mice via the IM route enhanced antigen-specific splenic CD4+ and CD8+ T cell responses. Boosting either IM or intranasally (IN) with Ad vectors expressing Ag85B without flagellin enhanced antigen-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with improved protection against pulmonary challenge with Mtb. However, inclusion of flagellin in both DNA prime and Ad boosting vaccines induced localized pulmonary inflammation and transient weight loss, along with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in mucosal CD4+ and CD8+ T cell responses following IM DNA / IN Ad prime-boosting, although antibody responses were not diminished. These findings suggest that flagellin has differential and route-dependent adjuvant activity when included in systemic or mucosal gene-based prime-boost immunization. Adjuvant activity for both T and B cell responses and safety were optimal when flagellin was included only in the DNA vaccine priming phase of systemic or mucosal DNA/Ad prime-boosting.

Use of recombinant flagellin in oil-in-water emulsions enhances hemagglutinin-specific mucosal IgA production and IL-17 secreting T cells against H5N1 avian influenza virus infection

Researchers are currently involved in a strong effort to find a safe and effective vaccine against highly pathogenic avian influenza H5N1 viruses. Toward that goal, we obtained soluble recombinant flagellin (FliC) of Salmonella typhimurium to be used as a mucosal adjuvant for H5HA subunit vaccine development. Intranasal immunization of H5HA antigen with recombinant FliC protein in an oil-in-water emulsion increased H5HA-specific IgG and IgA titers in sera, bronchoalveolar lavage fluids (BALFs), and nasal washes. Use of FliC adjuvant for intranasal immunization further augmented B-cell responses in mucosal environments via increased IgA titers in BALFs and nasal washes. Increases in IgA and IgG titers through the use of FliC adjuvant in intranasal immunization correlated with higher neutralizing antibody titers in sera and BALFs and higher numbers of IgG- and IgA-secreting B cells in spleen and cervical lymph nodes. High levels of IL-17A cytokine production were also found in stimulated T cells of spleen and cervical lymph node cells, only by intranasal immunization particularly with the use of FliC adjuvant in oil-in-water emulsions. These findings may provide useful information toward the development of H5HA mucosal influenza vaccines.

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System.

The opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa. The inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Oncolytic tanapoxvirus expressing FliC causes regression of human colorectal cancer xenografts in nude mice.

Colorectal cancers are significant causes of morbidity and mortality and existing therapies often perform poorly for individuals afflicted with advanced disease. Oncolytic virotherapy is an emerging therapeutic modality with great promise for addressing this medical need. Herein we describe the in vivo testing of recombinant variants of the tanapoxvirus (TPV). Recombinant viruses were made ablated for either the 66R gene (encoding a thymidine kinase), the 2L gene (encoding a TNF-binding protein), or both. Some of the recombinants were armed to express mouse chemotactic protein 1 (mCCL2/mMCP-1), mouse granulocyte-monocyte colony stimulating factor (mGM-CSF), or bacterial flagellin (FliC). Tumors were induced in athymic nude mice by implantation of HCT 116 cells and subsequently treated by a single intratumoral injection of one of the recombinant TPVs. Histological examination showed a common neoplastic cell type and a range of immune cell infiltration, necrosis, and tumor cell organization. Significant regression was seen in tumors treated with virus TPV/Δ2L/Δ66R/fliC, and to a lesser extent the recombinants TPV/Δ2L and TPV/Δ66R. Our results suggest that oncolytic recombinants of the TPV armed with activators of the innate immune response may be effective virotherapeutic agents for colorectal cancers in humans and should be explored further to fully realize their potential.

Molecular gene profiling of Clostridium botulinum group III and its detection in naturally contaminated samples originating from various European countries.

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and the flagellin (fliC) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC-I to fliC-V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC-I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC-II was rarely detected, no sample was recorded as fliC-III or fliC-V, and only C. botulinum type D/C samples tested positive for fliC-IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC-I to fliC-III are genetically related (87% to 92% sequence identity), whereas fliC-IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC-I to fliC-III have evolved in a common environment and support a different genetic evolution for fliC-IV. A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.

Rapid Preparation of Unsheathed Bacterial Flagella

(Abstract) The flagellum is required for bacterial swimming and swarming motility. In the biphasic Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellar filament is build up by two distinct monomeric subunits, flagellin FliC and FljB. S. Typhimurium has the ability to switch between two flagellins, FliC and FljB, in a phase-variable manner. The switch to FliC is called phase H1 and considered important for bacterial growth and survival in the spleen in a murine infection model of typhoid fever. Flagellin is secreted as monomeric subunits, but the majority of flagellin is polymerized upon secretion as the flagellar filament. Salmonella flagellin has traditionally been isolated through a process involving multiple steps of centrifugation and acid treatment. Here, we delineate a simplified protocol for preparing Salmonellas flagellin for analytical purpose to determine the amount of flagellin without the aid of antibodies. The growth conditions used were stationary phase, logarithmic phase and a low oxygen and high salt condition mimicking the gastrointestinal tract. Flagellin expression of other source organisms, such as other serovars of Salmonella enterica and Escherichia coli, including flagellar phase- or genetic variants can be analysed. Flagellin expression analysis complements flagella-associated phenotype analysis such as swimming and swarming behaviour.

A Porcine Reproductive and Respiratory Syndrome Virus Vaccine Candidate Based on PRRSV Glycoprotein 5 and the Toll-Like Receptor 5 Agonist Salmonella typhimurium Flagellin

Glycoprotein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV) is a key inducer of neutralizing antibodies. A truncated GP5 gene lacking the signal peptide and transmembrane sequences was amplified via an overlap PCR method and inserted into prokaryotic expression vectors, pET32a or pGEX-6p-1, to add an His or GST tag, respectively. His-tagged GP5 was induced with IPTG, verified by SDS-PAGE and Western blotting, and purified to serve as an immunogen accompanied with the Salmonella typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. Levels of TLR5 and cytokine mRNAs in spleens of mice following injection with FliC were detected by qRT-PCR to verify the activation of innate immunity. FliC was used as an adjuvant and administered with the GP5 to C57BL/6 mice via intraperitoneal injection. Coadministration of GP5 with FliC induced a significantly enhanced GP5-specific IgG and IFN-γ response compared with administration of GP5 alone, and the GP5-specific titer in the GP5 + FliC coadministration group was elevated almost twofold after the third immunization. These results indicate that FliC is an effective adjuvant, increasing the induction of antibodies against GP5 with the induction of both humoral and cellular immune responses.

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice.

Flagellin is a potent activator of a broad range of cell types that are involved in innate and adaptive immunity. Therefore, it is a good adjuvant candidate for vaccines, and it might function as a biological protectant against both major acute radiation syndrome during cancer radiotherapy and a mitigator of radiation emergencies. However, accumulating evidence has implicated flagellin in the occurrence of some inflammatory diseases, such as acute lung inflammation, cardiovascular collapse and inflammatory bowel disease. The aim of this study was to elucidate whether only flagellin-TLR5 signaling activation plays a role in the pathophysiology of liver or whether some other flagellin activity also contributes to liver injury either via bacterial infections or during clinical applications. Recombinant flagellin proteins with or without TLR5-stimulating activity were used to evaluate the role of flagellin-TLR5 signaling in liver injury in wild-type and TLR5 KO mice. Gross lesions and large areas of hepatocellular necrosis were observed in liver tissue 12 h after the intraperitoneal administration of 100 or 200 µg flagellin (FliC) in a dose- and time-dependent manner in wild-type mice, but not in TLR5 KO mice. Deletion of the N-terminal or TLR5 binding domain of flagellin inhibited flagellin-induced inflammatory responses and the subsequent acute liver function abnormality and damage. These data confirmed that flagellin is an essential determinant of liver injury and demonstrated that the over-activation of TLR5 signaling by high-dose flagellin caused acute inflammatory responses, neutrophil accumulation and oxidative stress in the liver, which contributes to the progression and severity of flagellin-induced liver injury.

Manuka honey reduces the motility of Pseudomonas aeruginosa by suppression of flagella-associated genes.

Manuka honey is a broad-spectrum antimicrobial agent that seems to affect different bacteria in many different ways. It has been shown to be bactericidal against Pseudomonas aeruginosa by destabilizing the cell wall, but we aimed to investigate whether there were further intracellular target sites. In this study inhibitory effects of manuka honey on P. aeruginosa were investigated using hydrophobicity assays, two-dimensional electrophoresis, quantitative RT-PCR, transmission electron microscopy and motility assays. Exposure of P. aeruginosa to manuka honey reduced both swarming and swimming motility. Moreover, this was a consequence of de-flagellation of the bacterial cell, which was correlated with decreased expression of the major structural flagellin protein, FliC, and concurrent suppression of flagellin-associated genes, including fliA, fliC, flhF, fleN, fleQ and fleR. The differential expression of the flagellar regulon in the presence of manuka honey was mapped schematically. Flagella are integral to bacterial adhesion, the initiation of infection and biofilm formation, and swarming has been associated with increased virulence. By limiting motility in vitro, we infer that manuka honey impacts on the virulence of P. aeruginosa. This deduction must now be tested in vivo.

Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate–chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate.

The flagellin FliC of Clostridium difficile is responsible for pleiotropic gene regulation during in vivo infection.

Clostridium difficile is the main agent responsible for hospital acquired antibiotic associated diarrhoea. In recent years, epidemic strains have emerged causing more severe infections. Whilst C. difficile has two major virulence factors, toxins TcdA and TcdB, it is generally accepted that other virulence components of the bacterium contribute to disease. Previously, it has been suggested that flagella expression from pathogenic bacteria might be implicated in virulence. In a recent study, we observed an increased mortality in a gnotobiotic mouse model when animals were colonized with an isogenic fliC mutant constructed in the PCR-ribotype 027 (B1/NAP1) strain R20291, while animals survived when colonized by the parental strain or after colonization by other high-toxin-producing C. difficile strains. To understand the reasons for this increased virulence, we compared the global gene expression profiles between the fliC-R20291 mutant and its parental strain using an in vitro and in vivo transcriptomic approach. The latter made use of the gnotobiotic mouse model. Interestingly, in the fliC mutant, we observed considerable up-regulation of genes involved in mobility, membrane transport systems (PTS, ABC transporters), carbon metabolism, known virulence factors and sporulation. A smaller but significant up-regulation of genes involved in cell growth, fermentation, metabolism, stress and antibiotic resistance was also apparent. All of these genes may be associated with the increased virulence of the fliC-R20921 mutant. We confirmed that the fliC mutation is solely responsible for the observed changes in gene expression in the mutant strain since expression profiles were restored to that of the wild-type strain in the fliC-complemented strain. Thus, the absence of FliC is directly or indirectly involved in the high mortality observed in the fliC mutant infected animals. Therefore, we provide the first evidence that when the major structural component of the flagellum is neutralized, deregulation of gene expression can occur during infection.

Spatial variability of bacteria in the rhizosphere of Elsholtzia splendens under Cu contamination.

Elsholtzia splendens is a well-known Cu-tolerant plant; yet, the impact of Cu-contaminated soil on bacterial community in its rhizosphere is not known. We studied the spatial variability of bacteria in the rhizosphere using Cu-contaminated soil with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR. In the uncontaminated soil, the content of the dissolved organic carbon (DOC) and bacterial diversity gradually increased in the rhizosphere soil along the root growth direction (from the interface zone to the meristematic zone), while for the Cu-contaminated soil, the highest DOC content and the strongest potential bioavailability of Cu were found in the interface zone, which also had the lowest bacteria diversity. Bacteria diversity was positively correlated with DOC in the uncontaminated soil (p < 0.01) but not in the contaminated soil. Compared with uncontaminated soil, some species such as Firmicutes only existed in the rhizosphere of contaminated soil, while the very small amount (if any) of some species exists such as Deinococcus-Thermus, indicating that the contaminated environment altered the bacterial composition. Moreover, spatial variation of the bacterial community was found among different soil zones. Real-time PCR confirmed the spatial variation via the gene expression of flagellin (fliC) and chemotaxis gene (cheA). The spatial characteristics of cheA expression were consistent with that of DOC and bacterial diversity. In conclusion, we demonstrated that the spatial variation of the bacterial community in the rhizosphere was present, independent of Cu contamination. DOC and Cu toxicity may affect specific gene expressions such as fliC and cheA, resulting in bacterial spatial variation.

Effects of cigarette smoke on Toll-like receptor (TLR) activation of chronic obstructive pulmonary disease (COPD) macrophages

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal innate immune response. We have investigated the changes in the innate immune response of COPD alveolar macrophages exposed to both cigarette smoke and Toll-like receptor (TLR) stimulation. COPD and control alveolar macrophages were exposed to cigarette smoke extract (CSE) followed by TLR-2, -4 and -5 ligands [Pam3CSK4, lipopolysaccharide (LPS) and phase I flagellin (FliC), respectively] or non-typeable Haemophilus influenzae (NTHi). CSE exposure suppressed TLR-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and regulated on activation, normal T cell expressed and secreted (RANTES) production in both COPD and control alveolar macrophages, but had no effect on interleukin 8 (CXCL8) production. Similarly, CSE suppressed NTHi-induced TNF-α but not NTHi-induced CXCL8 production in COPD alveolar macrophages. Gene expression analysis showed that CSE suppressed LPS-induced TNF-α transcription but not CXCL8 transcription in COPD alveolar macrophages. The dampening effect of CSE on LPS-induced cytokine production was associated with a reduction in p38, extracellular signal regulated kinase (ERK) and p65 activation. In conclusion, CSE caused a reduced innate immune response in COPD alveolar macrophages, with the exception of persistent CXCL8 production. This could be a mechanism by which alveolar macrophages promote neutrophil chemotaxis under conditions of oxidative stress and bacterial exposure.

Salmonella enterica Serovar Typhi Impairs CD4 T Cell Responses by Reducing Antigen Availability

Salmonella enterica serovar Typhi is associated with a disseminated febrile illness in humans, termed typhoid fever, while Salmonella enterica serovar Typhimurium causes localized gastroenteritis in immunocompetent individuals. One of the genetic differences between both pathogens is the presence in S. Typhi of TviA, a regulatory protein that shuts down flagellin (FliC) expression when bacteria transit from the intestinal lumen into the intestinal mucosa. Here we investigated the consequences of TviA-mediated flagellum gene regulation on flagellin-specific CD4 T cell responses in a mouse model of S. Typhimurium infection. Introduction of the S. Typhi tviA gene into S. Typhimurium suppressed antigen presentation of dendritic cells to flagellin-specific CD4 T cells in vitro. Furthermore, TviA-mediated repression of flagellin expression impaired the activation and proliferation of naive flagellin-specific CD4 T cells in Peyer's patches and mesenteric lymph nodes, which was accompanied by increased bacterial dissemination to the spleen. We conclude that TviA-mediated repression of flagellin expression reduces antigen availability, thereby weakening flagellin-specific CD4 T cell responses.

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.