In vivo synthesis of bacterial amyloid curli contributes to joint inflammation during S. Typhimurium infection.

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.

IgM Plasma Cells Reside in Healthy Skin and Accumulate with Chronic Inflammation.

Antibodies are key to cutaneous host defense and inflammation. Despite their importance, the mechanisms by which skin antibodies are sustained are poorly described. Here, we identified that, in addition to antibody production in lymphoid tissues, plasma cells reside in healthy mouse and human skin. In naïve mice, IgM was the predominant isotype produced in skin. Skin plasma cells developed independently of T cells and microbiota. Importantly, chronic skin inflammation promoted the massive accumulation of IgM-secreting cells, and cutaneous immunization directed both T cell-dependent and -independent antigen-specific IgM-secreting cells into skin. Unlike their counterparts in lymphoid tissues, cutaneous IgM-secreting cells were completely dependent on survival factors such as a proliferation-inducing ligand or B cell-activating factor, which were constitutively expressed and upregulated during inflammation in skin. Our data support a model in which skin plasma cells supply natural and adaptive IgM to the cutaneous environment, thereby supporting homeostatic skin barrier functions and providing defense against pathogen intrusion. Our results are also of potential relevance for manipulation of cutaneous plasma cells in inflammatory skin diseases or cutaneous plasma cell malignancies.

STAT2 dependent Type I Interferon response promotes dysbiosis and luminal expansion of the enteric pathogen Salmonella Typhimurium.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2-/- mice, we observed decreased hypoxia in the gut tissue of Stat2-/- mice. Neutrophil numbers were similar in wild-type and Stat2-/- mice, yet Stat2-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium.

Precision editing of the gut microbiota ameliorates colitis.

Inflammatory diseases of the gastrointestinal tract are frequently associated with dysbiosis, characterized by changes in gut microbial communities that include an expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Here we show that a dysbiotic expansion of Enterobacteriaceae during gut inflammation could be prevented by tungstate treatment, which selectively inhibited molybdenum-cofactor-dependent microbial respiratory pathways that are operational only during episodes of inflammation. By contrast, we found that tungstate treatment caused minimal changes in the microbiota composition under homeostatic conditions. Notably, tungstate-mediated microbiota editing reduced the severity of intestinal inflammation in mouse models of colitis. We conclude that precision editing of the microbiota composition by tungstate treatment ameliorates the adverse effects of dysbiosis in the inflamed gut.

The Functional Amyloid Curli Protects Escherichia coli against Complement-Mediated Bactericidal Activity.

Escherichia coli strains may be beneficial or pathogenic. Many E. coli strains that cause human disease, especially those responsible for bacteremia and sepsis, express virulence factors that impart resistance to the complement system. The bacterial amyloid curli functions in bacterial adherence and enhances the formation of biofilms. Survival of curli-producing parental and curli-deficient mutant E. coli in the context of a human complement response was evaluated using an in vivo murine model of bacteremia. Results showed that curli production enhanced E. coli survival, which suggests that curli defends against complement-mediated killing. This observation was supported by the results of in vitro assays comparing bacterial survival in human serum. Experiments in which the classical or alternative complement pathways were blocked indicated that the classical pathway is the major contributor to complement activation and that curli inhibits this activity. Our analyses indicate that curli does not appear to play a role in protecting E. coli against alternative pathway complement activation. We found that curli increases binding of E. coli cells to complement component Complement component 1q (C1q) but does not affect Complement component 3b (C3b) binding. We conclude that curli defends E. coli against complement-mediated killing via inhibition of the classical complement pathway.

Amyloid-DNA Composites of Bacterial Biofilms Stimulate Autoimmunity.

Research on the human microbiome has established that commensal and pathogenic bacteria can influence obesity, cancer, and autoimmunity through mechanisms mostly unknown. We found that a component of bacterial biofilms, the amyloid protein curli, irreversibly formed fibers with bacterial DNA during biofilm formation. This interaction accelerated amyloid polymerization and created potent immunogenic complexes that activated immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in systemic lupus erythematosus (SLE). When given systemically, curli-DNA composites triggered immune activation and production of autoantibodies in lupus-prone and wild-type mice. We also found that the infection of lupus-prone mice with curli-producing bacteria triggered higher autoantibody titers compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity.

Biofilm-associated bacterial amyloids dampen inflammation in the gut: oral treatment with curli fibres reduces the severity of hapten-induced colitis in mice.

BACKGROUND/OBJECTIVES: A disruption of epithelial barrier function can lead to intestinal inflammation. Toll-like receptor (TLR) 2 activation by microbial products promotes intestinal epithelial integrity and overall gut health. Several bacterial species, including enteric bacteria, actively produce amyloid proteins as a part of their biofilms. Recognition of amyloid fibres found in enteric biofilms, termed curli, by the Toll-like receptor (TLR)2/1 complex reinforces barrier function. Here, we investigated the effect of purified curli fibres on inflammation in a mouse model of acute colitis. METHODS: Bone marrow-derived macrophages as well as lamina propria cells were treated with curli fibres of both pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli Nissle 1917 biofilms. Mice were given 0.1 or 0.4 mg of purified curli orally 1 day post administration of 1% 2,4,6-trinitrobenzene sulphonic acid (TNBS) enema. Histopathological analysis was performed on distal colonic tissue taken 6 days post TNBS enema. RNA extracted from colonic tissue was subjected to RT-PCR. RESULTS: Here we show that curli fibres of both pathogenic and commensal bacteria are recognised by TLR2 leading to the production of IL-10, immunomodulatory cytokine of intestinal homeostasis. Treatment of mice with a single dose of curli heightens transcript levels of Il10 in the colon and ameliorates the disease pathology in TNBS-induced colitis. Curli treatment is comparable to the treatment with anti-tumour necrosis factor alpha (anti-TNFα) antibodies, a treatment known to reduce the severity of acute colitis in humans and mice. CONCLUSION: These results suggest that the bacterial amyloids had a role in helping to maintain immune homeostasis in the intestinal mucosa via the TLR2/IL-10 axis. Furthermore, bacterial amyloids may be a potential candidate therapeutic to treat intestinal inflammatory disorders owing to their remarkable immunomodulatory activity.

Toll-like receptor 2 and NLRP3 cooperate to recognize a functional bacterial amyloid, curli.

Amyloids are proteins with cross-ß-sheet structure that contribute to pathology and inflammation in complex human diseases, including Alzheimer's disease, Parkinson's disease, type II diabetes, and secondary amyloidosis. Bacteria also produce amyloids as a component of their extracellular matrix during biofilm formation. Recently, several human amyloids were shown to activate the NLRP3 inflammasome, leading to the activation of caspase 1 and production of interleukin 1ß (IL-1ß). In this study, we investigated the activation of the NLRP3 inflammasome by bacterial amyloids using curli fibers, produced by Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we show that curli fibers activate the NLRP3 inflammasome, leading to the production of IL-1ß via caspase 1 activation. Investigation of the underlying mechanism revealed that activation of Toll-like receptor 2 (TLR2) by curli fibers is critical in the generation of IL-1ß. Interestingly, activation of the NLRP3 inflammasome by curli fibers or by amyloid ß of Alzheimer's disease does not cause cell death in macrophages. Overall, these data identify a cross talk between TLR2 and NLRP3 in response to the bacterial amyloid curli and generation of IL-1ß as a product of this interaction.

The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

CXCR4 is dispensable for T cell egress from chronically inflamed skin via the afferent lymph.

T cell recirculation through extralymphoid tissues is essential to immune surveillance, host defense and inflammation. In this process, T cells enter the tissue from the blood and subsequently leave via the afferent lymph. In the absence of inflammation, T cells require CCR7 expression to egress from the skin or lung, which is consistent with the constitutive expression of the CCR7 ligand CCL21 on lymphatic endothelium. However, during chronic inflammation alternative chemoattractants come into play, allowing Ccr7-deficient (Ccr7-/-) T cells to egress efficiently from affected skin. As T cell egress from inflamed sites is a potential control point of the inflammatory response, we aimed to determine alternative T cell exit receptors using a mouse and a sheep model. We show that CCR7+ and CCR7- T cells exiting from the chronically inflamed skin were highly responsive to the CXCR4 ligand CXCL12, which was induced in the lymphatics in the inflamed site. Based on these findings, we hypothesized that CXCR4 mediates T cell egress from inflamed skin. However, pharmacological inhibition of CXCR4 did not affect the tissue egress of wildtype or Ccr7-/- CD4 and CD8 T cells after adoptive transfer into chronically inflamed skin. Similarly, adoptively transferred Cxcr4-/- Ccr7-/- and Ccr7-/- T cells egressed from the inflamed skin equally well. Based on these data, we conclude that, while CXCR4 might play an essential role for other cell types that enter the afferent lymphatics, it is dispensable for T cell egress from the chronically inflamed skin.

Ovine skin-recirculating γδ T cells express IFN-γ and IL-17 and exit tissue independently of CCR7.

γδ T cells continuously survey extralymphoid tissues, providing key effector functions during infection and inflammation. Despite their importance, the function and the molecules that drive migration of skin-recirculating γδ T cells are poorly described. Here we found that γδ T cells traveling in the skin-draining afferent lymph of sheep are effectors that produce IFN-γ or IL-17 and express high levels of the skin- and inflammation-seeking molecule E-selectin ligand. Consistent with a role for chemokine receptor CCR7 in mediating T cell exit from extralymphoid tissues, conventional CD4 and CD8T cells in skin-draining lymph were enriched in their expression of CCR7 compared to their skin-residing counterparts. In contrast, co-isolated γδ T cells in skin or lymph lacked expression of CCR7, indicating that they use alternative receptors for egress. Skin-draining γδ T cells were unresponsive to many cutaneous and inflammatory chemokines, including ligands for CCR2, CCR4, CCR5, CCR8, CCR10, and CXCR3, but showed selective chemotaxis toward the cutaneously expressed CCR6 ligand CCL20. Moreover, IL-17(+) γδ T cells were the most CCL20-responsive subset of γδ T cells. The data suggest that γδ T cells survey the skin and sites of inflammation and infection, entering via CCR6 and E-selectin ligand and leaving independent of the CCR7-CCL21 axis.

Microbial amyloids induce interleukin 17A (IL-17A) and IL-22 responses via Toll-like receptor 2 activation in the intestinal mucosa.

The Toll-like receptor 2 (TLR2)/TLR1 receptor complex responds to amyloid fibrils, a common component of biofilm material produced by members of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. To determine whether this TLR2/TLR1 ligand stimulates inflammatory responses when bacteria enter intestinal tissue, we investigated whether expression of curli amyloid fibrils by the invasive enteric pathogen Salmonella enterica serotype Typhimurium contributes to T helper 1 and T helper 17 responses by measuring cytokine production in the mouse colitis model. A csgBA mutant, deficient in curli production, elicited decreased expression of interleukin 17A (IL-17A) and IL-22 in the cecal mucosa compared to the S. Typhimurium wild type. In TLR2-deficient mice, IL-17A and IL-22 expression was blunted during S. Typhimurium infection, suggesting that activation of the TLR2 signaling pathway contributes to the expression of these cytokines. T cells incubated with supernatants from bone marrow-derived dendritic cells (BMDCs) treated with curli fibrils released IL-17A in a TLR2-dependent manner in vitro. Lower levels of IL-6 and IL-23 production were detected in the supernatants of the TLR2-deficient BMDCs treated with curli fibrils. Consistent with this, three distinct T-cell populations-CD4(+) T helper cells, cytotoxic CD8(+) T cells, and γδ T cells-produced IL-17A in response to curli fibrils in the intestinal mucosa during S. Typhimurium infection. Notably, decreased IL-6 expression by the dendritic cells and decreased IL-23 expression by the dendritic cells and macrophages were observed in the cecal mucosa of mice infected with the curli mutant. We conclude that TLR2 recognition of bacterial amyloid fibrils in the intestinal mucosa represents a novel mechanism of immunoregulation, which contributes to the generation of inflammatory responses, including production of IL-17A and IL-22, in response to bacterial entry into the intestinal mucosa.

The skin, a novel niche for recirculating B cells.

B cells infiltrate the skin in many chronic inflammatory diseases caused by autoimmunity or infection. Despite potential contribution to disease, skin-associated B cells remain poorly characterized. Using an ovine model of granulomatous skin inflammation, we demonstrate that B cells increase in the skin and skin-draining afferent lymph during inflammation. Surprisingly, skin B cells are a heterogeneous population that is distinct from lymph node B cells, with more large lymphocytes as well as B-1-like B cells that coexpress high levels of IgM and CD11b. Skin B cells have increased MHC class II, CD1, and CD80/86 expression compared with lymph node B cells, suggesting that they are well-suited for T cell activation at the site of inflammation. Furthermore, we show that skin accumulation of B cells and Ab-secreting cells during inflammation increases local Ab titers, which could augment host defense and autoimmunity. Although skin B cells express typical skin-homing receptors, such as E-selectin ligand and α-4 and ß-1 integrins, they are unresponsive to ligands for chemokine receptors associated with T cell homing into skin. Instead, skin B cells migrate toward the cutaneously expressed CCR6 ligand CCL20. Our data support a model in which B cells use CCR6-CCL20 to recirculate through the skin, fulfilling a novel role in skin immunity and inflammation.

The Vi capsular polysaccharide prevents complement receptor 3-mediated clearance of Salmonella enterica serotype Typhi.

Capsular polysaccharides are important virulence factors of invasive bacterial pathogens. Here we studied the role of the virulence (Vi) capsular polysaccharide of Salmonella enterica serotype Typhi (S. Typhi) in preventing innate immune recognition by complement. Comparison of capsulated S. Typhi with a noncapsulated mutant (ΔtviBCDE vexABCDE mutant) revealed that the Vi capsule interfered with complement component 3 (C3) deposition. Decreased complement fixation resulted in reduced bacterial binding to complement receptor 3 (CR3) on the surface of murine macrophages in vitro and decreased CR3-dependent clearance of Vi capsulated S. Typhi from the livers and spleens of mice. Opsonization of bacteria with immune serum prior to intraperitoneal infection increased clearance of capsulated S. Typhi from the liver. Our data suggest that the Vi capsule prevents CR3-dependent clearance, which can be overcome in part by a specific antibody response.

Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms.

Responses to host amyloids and curli amyloid fibrils of Escherichia coli and Salmonella enterica serotype Typhimurium are mediated through Toll-like receptor (TLR) 2. Here we show that TLR2 alone was not sufficient for mediating responses to curli. Instead, transfection experiments with human cervical cancer (HeLa) cells and antibody-mediated inhibition of TLR signalling in human macrophage-like (THP-1) cells suggested that TLR2 interacts with TLR1 to recognize curli amyloid fibrils. TLR1/TLR2 also serves as a receptor for tri-acylated lipoproteins, which are produced by E. coli and other Gram-negative bacteria. Despite the presence of multiple TLR1/TLR2 ligands on intact bacterial cells, an inability to produce curli amyloid fibrils markedly reduced the ability of E. coli to induce TLR2-dependent responses in vitro and in vivo. Collectively, our data suggest that curli amyloid fibrils from enterobacterial biofilms significantly contribute to TLR1/TLR2-mediated host responses against intact bacterial cells.

Responses to amyloids of microbial and host origin are mediated through toll-like receptor 2.

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimer's disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.

The capsule-encoding viaB locus reduces intestinal inflammation by a Salmonella pathogenicity island 1-independent mechanism.

Salmonella enterica serotype Typhimurium elicits acute neutrophil influx in the human intestinal mucosa within 1 or 2 days after infection, resulting in inflammatory diarrhea. In contrast, no overt symptoms are observed within the first 1 or 2 weeks after infection with S. enterica serotype Typhi. Here we show that introduction of the capsule-encoding viaB locus of serotype Typhi reduced the ability of serotype Typhimurium to elicit acute intestinal inflammation in a streptomycin-pretreated mouse model. Serotype Typhimurium requires a functional invasion-associated type III secretion system (type III secretion system 1 [T3SS-1]) to elicit cecal inflammation within 48 h after infection of streptomycin-pretreated mice, and the presence of the viaB locus reduced its invasiveness for human intestinal epithelial cells in vitro. However, a reduced activity of T3SS-1 could not account for the ability of the viaB locus to attenuate cecal inflammation, because introduction of the viaB locus into an invasion-deficient serotype Typhimurium strain (invA mutant) resulted in a significant reduction of pathology and inflammatory cytokine expression in the cecum 5 days after infection of mice. We conclude that a T3SS-1-independent mechanism contributes to the ability of the viaB locus to reduce intestinal inflammation.

Contribution of flagellin pattern recognition to intestinal inflammation during Salmonella enterica serotype typhimurium infection.

Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3alpha and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.

Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.

Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor-deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella.

The viaB locus enables Salmonella enterica serotype Typhi to reduce Toll-like receptor (TLR) dependent cytokine production in tissue culture models. This DNA region contains genes involved in the regulation (tviA), biosynthesis (tviBCDE) and export (vexABCDE) of the Vi capsule. Expression of the Vi capsule in S. Typhimurium, but not expression of the TviA regulatory protein, reduced tumour necrosis factor-alpha (TNF-alpha) and IL-6 production by murine bone-marrow derived macrophages. Production of TNF-alpha and IL-6 was dependent on expression of TLR4 as stimulation of macrophages from TLR4(-/-) mice with S. Typhimurium did not result in expression of these cytokines. Intraperitoneal infection of mice with S. Typhimurium induced expression of TNF-alpha and inducible nitric oxide synthase (iNOS) in the liver. Introduction of the cloned viaB region into S. Typhimurium reduced TNF-alpha and iNOS expression to levels observed after infection with a S. Typhimurium msbB mutant. In contrast, no differences in TNF-alpha expression between the S. Typhimurium wild type and strains expressing the Vi-capsule or carrying a mutation in msbB were observed after infection of TLR4(-/-) mice. We conclude that the Vi capsule prevents both in vitro and in vivo recognition of S. Typhimurium lipopolysaccharide by TLR4.