Tumor Necrosis Factor Alpha Contributes to Inflammatory Pathology in the Placenta during Brucella abortus Infection.

Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites, Brucella abortus evades innate immunity, which results in low-level inflammation and chronic infection of phagocytes. In contrast, the host response in the placenta during infection is characterized by severe inflammation and extensive extracellular replication of B. abortus. Despite the importance of reproductive disease caused by Brucella infection, our knowledge of the mechanisms involved in placental inflammation and abortion is limited. To understand the immune responses specifically driving placental pathology, we modeled placental B. abortus infection in pregnant mice. B. abortus infection caused an increase in the production of tumor necrosis factor alpha (TNF-α), specifically in the placenta. We found that placental expression levels of Tnfa and circulating TNF-α were dependent on the induction of endoplasmic reticulum stress and the B. abortus type IV secretion system (T4SS) effector protein VceC. Blockade of TNF-α reduced placental inflammation and improved fetal viability in mice. This work sheds light on a tissue-specific response of the placenta to B. abortus infection that may be important for bacterial transmission via abortion in the natural host species.

Editing of the gut microbiota reduces carcinogenesis in mouse models of colitis-associated colorectal cancer.

Chronic inflammation and gut microbiota dysbiosis, in particular the bloom of genotoxin-producing E. coli strains, are risk factors for the development of colorectal cancer. Here, we sought to determine whether precision editing of gut microbiota metabolism and composition could decrease the risk for tumor development in mouse models of colitis-associated colorectal cancer (CAC). Expansion of experimentally introduced E. coli strains in the azoxymethane/dextran sulfate sodium colitis model was driven by molybdoenzyme-dependent metabolic pathways. Oral administration of sodium tungstate inhibited E. coli molybdoenzymes and selectively decreased gut colonization with genotoxin-producing E. coli and other Enterobacteriaceae. Restricting the bloom of Enterobacteriaceae decreased intestinal inflammation and reduced the incidence of colonic tumors in two models of CAC, the azoxymethane/dextran sulfate sodium colitis model and azoxymethane-treated, Il10-deficient mice. We conclude that metabolic targeting of protumoral Enterobacteriaceae during chronic inflammation is a suitable strategy to prevent the development of malignancies arising from gut microbiota dysbiosis.

Utilization of Host Polyamines in Alternatively Activated Macrophages Promotes Chronic Infection by Brucella abortus.

Treatment of intracellular bacterial pathogens with antibiotic therapy often requires a long course of multiple drugs. A barrier to developing strategies that enhance antibiotic efficacy against these pathogens is our poor understanding of the intracellular nutritional environment that maintains bacterial persistence. The intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs); however, knowledge of the metabolic adaptations promoting exploitation of this niche is limited. Here we show that one mechanism promoting enhanced survival in AAMs is a shift in macrophage arginine utilization from production of nitric oxide (NO) to biosynthesis of polyamines, induced by interleukin 4 (IL-4)/IL-13 treatment. Production of polyamines by infected AAMs promoted both intracellular survival of B. abortus and chronic infection in mice, as inhibition of macrophage polyamine synthesis or inactivation of the putative putrescine transporter encoded by potIHGF reduced both intracellular survival in AAMs and persistence in mice. These results demonstrate that increased intracellular availability of polyamines induced by arginase-1 expression in IL-4/IL-13-induced AAMs promotes chronic persistence of B. abortus within this niche and suggest that targeting of this pathway may aid in eradicating chronic infection.

Iron acquisition pathways and colonization of the inflamed intestine by Salmonella enterica serovar Typhimurium.

Salmonella enterica serotype Typhimurium is able to expand in the lumen of the inflamed intestine through mechanisms that have not been fully resolved. Here we utilized streptomycin-pretreated mice and dextran sodium sulfate (DSS)-treated mice to investigate how pathways for S. Typhimurium iron acquisition contribute to pathogen expansion in the inflamed intestine. Competitive infection with an iron uptake-proficient S. Typhimurium strain and mutant strains lacking tonB feoB, feoB, tonB or iroN in streptomycin pretreated mice demonstrated that ferric iron uptake requiring IroN and TonB conferred a fitness advantage during growth in the inflamed intestine. However, the fitness advantage conferred by ferrous iron uptake mechanisms was independent of inflammation and was only apparent in models where the normal microbiota composition had been disrupted by antibiotic treatment.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

The flagellar regulator TviA reduces pyroptosis by Salmonella enterica serovar Typhi.

To discern virulent from innocuous microbes, the innate immune system senses events associated with bacterial access to immunoprivileged sites such as the host cell cytosol. One such pathway is triggered by the cytosolic delivery of flagellin, the major subunit of the flagellum, by bacterial secretion systems. This leads to inflammasome activation and subsequent proinflammatory cell death (pyroptosis) of the infected phagocyte. In this study, we demonstrate that the causative agent of typhoid fever, Salmonella enterica serovar Typhi, can partially subvert this critical innate immune recognition event. The transcriptional regulator TviA, which is absent from Salmonella serovars associated with human gastroenteritis, repressed the expression of flagellin during infection of human macrophage-like (THP-1) cells. This mechanism allowed S. Typhi to dampen inflammasome activation, leading to reduced interleukin-1ß (IL-1ß) secretion and diminished cell death. Likewise, the introduction of the tviA gene in nontyphoidal Salmonella enterica serovar Typhimurium reduced flagellin-induced pyroptosis. These data suggest that gene regulation of virulence factors enables S. Typhi to evade innate immune recognition by concealing a pathogen-induced process from being sensed by the inflammasome.

PPARγ-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages.

Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator-activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAMs. Glucose uptake was crucial for increased replication of B. abortus in AAMs, and for chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAMs and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAMs, and targeting this pathway may aid in eradicating chronic infection.

CD4+ T cell-derived IL-10 promotes Brucella abortus persistence via modulation of macrophage function.

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4(+)CD25(+) T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25(+)CD4(+) T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.

Sensing of bacterial type IV secretion via the unfolded protein response.

Host cytokine responses to Brucella abortus infection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required for B. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses during B. abortus infection. IMPORTANCE Brucella species are pathogens that require a type IV secretion system (T4SS) to survive in host cells and to maintain chronic infection. By as-yet-unknown pathways, the T4SS also elicits inflammatory responses in infected cells. Here we show that inflammation caused by the T4SS results in part from the sensing of a T4SS substrate, VceC, that localizes to the endoplasmic reticulum (ER), an intracellular site of Brucella replication. Possibly via binding of the ER chaperone BiP, VceC causes ER stress with concomitant expression of proinflammatory cytokines. Thus, induction of the unfolded protein response may represent a novel pathway by which host cells can detect pathogens deploying a T4SS.

Human α-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets.

Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human α-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.

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.

The TviA auxiliary protein renders the Salmonella enterica serotype Typhi RcsB regulon responsive to changes in osmolarity.

In response to osmolarity, Salmonella enterica serotype Typhi (S. Typhi) regulates genes required for Vi capsular antigen expression oppositely to those required for motility and invasion. Previous studies suggest that osmoregulation of motility, invasion and capsule expression is mediated through the RcsC/RcsD/RcsB phosphorelay system. Here we performed gene expression profiling and functional studies to determine the role of TviA, an auxiliary protein of the RcsB response regulator, in controlling virulence gene expression in S. Typhi. TviA repressed expression of genes encoding flagella and the invasion-associated type III secretion system (T3SS-1) through repression of the flagellar regulators flhDC and fliZ, resulting in reduced invasion, reduced motility and reduced expression of FliC. Both RcsB and TviA repressed expression of flhDC, but only TviA altered flhDC expression in response to osmolarity. Introduction of tviA into S. enterica serotype Typhimurium rendered flhDC transcription sensitive to changes in osmolarity. These data suggest that the auxiliary TviA protein integrates a new regulatory input into the RcsB regulon of S. Typhi, thereby altering expression of genes encoding flagella, the Vi antigen and T3SS-1 in response to osmolarity.

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.

RosE represses Std fimbrial expression in Salmonella enterica serotype Typhimurium.

The Salmonella enterica serotype Typhimurium (S. typhimurium) genome contains a large repertoire of putative fimbrial operons that remain poorly characterized because they are not expressed in vitro. In this study, insertions that induced expression of the putative stdABCD fimbrial operon were identified from a random bank of transposon mutants by screening with immuno-magnetic particles for ligand expression (SIMPLE). Transposon insertions upstream of csgC and lrhA or within dam, setB and STM4463 (renamed rosE) resulted in expression of StdA and its assembly into fimbrial filaments on the cell surface. RosE is a novel negative regulator of Std fimbrial expression as indicated by its repression of a std::lacZ reporter construct and by binding of the purified protein to a DNA region upstream of the stdA start codon. Expression of Std fimbriae in the rosE mutant resulted in increased attachment of S. typhimurium to human colonic epithelial cell lines (T-84 and CaCo-2). A rosE mutant exhibited a reduced ability to compete with virulent S. typhimurium for colonization of murine organs, while no defect was observed when both competing strains carried a stdAB deletion. These data suggest that a tight control of Std fimbrial expression mediated by RosE is required during host pathogen interaction.