The rpoS gene confers resistance to low osmolarity conditions in Salmonella enterica serovar Typhi.

Salmonella enterica serovars Typhimurium and Typhi are enteropathogens that differ in host range and the diseases that they cause. We found that exposure to a combination of hypotonicity and the detergent Triton X-100 significantly reduced the viability of the S. Typhi strain Ty2 but had no effect on the S. Typhimurium strain SL1344. Further analysis revealed that hypotonicity was the critical factor: incubation in distilled water alone was sufficient to kill Ty2, while the addition of sodium chloride inhibited killing in a dose-dependent manner. Ty2's loss of viability in water was modified by culture conditions: bacteria grown in well-aerated shaking cultures were more susceptible than bacteria grown under less aerated static conditions. Ty2, like many S. Typhi clinical isolates, has an inactivating mutation in the rpoS gene, a transcriptional regulator of stress responses, whereas most S. Typhimurium strains, including SL1344, have the wild-type gene. Transformation of Ty2 with a plasmid expressing wild-type rpoS, but not the empty vector, significantly increased survival in distilled water. Moreover, an S. Typhi strain with wild-type rpoS had unimpaired survival in water. Inactivation of the wild-type gene in this strain significantly reduced survival, while replacement with an arabinose-inducible allele of rpoS restored viability in water under inducing conditions. Our observations on rpoS-dependent differences in susceptibility to hypotonic conditions may be relevant to the ability of S. Typhi and S. Typhimurium to tolerate the various environments they encounter during the infectious cycle. They also have implications for the handling of these organisms during experimental manipulations.

RXRα Regulates the Development of Resident Tissue Macrophages.

Resident tissue macrophages (RTMs) develop from distinct waves of embryonic progenitor cells that seed tissues before birth. Tissue-specific signals drive a differentiation program that leads to the functional specialization of RTM subsets. Genetic programs that regulate the development of RTMs are incompletely understood, as are the mechanisms that enable their maintenance in adulthood. In this study, we show that the ligand-activated nuclear hormone receptor, retinoid X receptor (RXR)α, is a key regulator of murine RTM development. Deletion of RXRα in hematopoietic precursors severely curtailed RTM populations in adult tissues, including the spleen, peritoneal cavity, lung, and liver. The deficiency could be traced to the embryonic period, and mice lacking RXRα in hematopoietic lineages had greatly reduced numbers of yolk sac and fetal liver macrophages, a paucity that persisted into the immediate postnatal period.

Helminth-Induced and Th2-Dependent Alterations of the Gut Microbiota Attenuate Obesity Caused by High-Fat Diet.

BACKGROUND & AIMS: Epidemiological and animal studies have indicated an inverse correlation between the rising prevalence of obesity and metabolic syndrome and exposure to helminths. Whether helminth-induced immune response contributes to microbiota remodeling in obesity remains unknown. The aim of this study is to explore the immune-regulatory role of helminth in the prevention of HFD-induced obesity through remodeling gut microbiome. METHODS: C57BL/6J WT and STAT6-/- mice were infected with Heligmosomoides polygyrus and followed by high fat diet (HFD) feeding for 6 weeks. The host immune response, body weight, and fecal microbiota composition were analyzed. We used adoptive transfer of M2 macrophages and microbiota transplantation approaches to determine the impact of these factors on HFD-obesity. We also examined stool microbiota composition and short chain fatty acids (SCFAs) concentration and determined the expression of SCFA-relevant receptors in the recipient mice. RESULTS: Helminth infection of STAT6-/- (Th2-deficient) mice and adoptive transfer of helminth-induced alternatively activated (M2) macrophages demonstrated that the helminth-associated Th2 immune response plays an important role in the protection against obesity and induces changes in microbiota composition. Microbiota transplantation showed that helminth-induced, Th2-dependent alterations of the gut microbiota are sufficient to confer protection against obesity. Collectively, these results indicate that helminth infection protects against HFD-induced obesity by Th2-dependent, M2 macrophage-mediated alterations of the intestinal microbiota. CONCLUSION: Our findings provide new mechanistic insights into the complex interplay between helminth infection, the immune system and the gut microbiota in a HFD-induced obesity model and holds promise for gut microbiome-targeted immunotherapy in obesity prevention.

Spheres of Influence: Insights into Salmonella Pathogenesis from Intestinal Organoids.

The molecular complexity of host-pathogen interactions remains poorly understood in many infectious diseases, particularly in humans due to the limited availability of reliable and specific experimental models. To bridge the gap between classical two-dimensional culture systems, which often involve transformed cell lines that may not have all the physiologic properties of primary cells, and in vivo animal studies, researchers have developed the organoid model system. Organoids are complex three-dimensional structures that are generated in vitro from primary cells and can recapitulate key in vivo properties of an organ such as structural organization, multicellularity, and function. In this review, we discuss how organoids have been deployed in exploring Salmonella infection in mice and humans. In addition, we summarize the recent advancements that hold promise to elevate our understanding of the interactions and crosstalk between multiple cell types and the microbiota with Salmonella. These models have the potential for improving clinical outcomes and future prophylactic and therapeutic intervention strategies.

Intestinal microbes influence development of thymic lymphocytes in early life.

The thymus generates cells of the T cell lineage that seed the lymphatic and blood systems. Transcription factor regulatory networks control the lineage programming and maturation of thymic precursor cells. Whether extrathymic antigenic events, such as the microbial colonization of the mucosal tract also shape the thymic T cell repertoire is unclear. We show here that intestinal microbes influence the thymic homeostasis of PLZF-expressing cells in early life. Impaired thymic development of PLZF+ innate lymphocytes in germ-free (GF) neonatal mice is restored by colonization with a human commensal, Bacteroides fragilis, but not with a polysaccharide A (PSA) deficient isogenic strain. Plasmacytoid dendritic cells influenced by microbes migrate from the colon to the thymus in early life to regulate PLZF+ cell homeostasis. Importantly, perturbations in thymic PLZF+ cells brought about by alterations in early gut microbiota persist into adulthood and are associated with increased susceptibility to experimental colitis. Our studies identify a pathway of communication between intestinal microbes and thymic lymphocytes in the neonatal period that can modulate host susceptibility to immune-mediated diseases later in life.

The YrbE phospholipid transporter of Salmonella enterica serovar Typhi regulates the expression of flagellin and influences motility, adhesion and induction of epithelial inflammatory responses.

SALMONELLA ENTERICA: serovar Typhi is the etiologic agent of typhoid fever, a major public health problem in the developing world. Moving toward and adhering to the intestinal epithelium represents key initial steps of infection by S. Typhi. We examined the role of the S. Typhi yrbE gene, which encodes an inner membrane phospholipid transporter, in these interactions with epithelial cells. Disruption of yrbE resulted in elevated expression of flagellin and a hypermotile phenotype. It also significantly reduced the ability of S. Typhi to adhere to the HeLa epithelial cell line and to polarized primary epithelial cells derived from human ileal organoids. Interestingly, the yrbE-deficient strain of S. Typhi induced higher production of interleukin-8 from the primary human ileal epithelial cell monolayers compared to the wild-type bacteria. Deletion of the flagellin gene (fliC) in the yrbE-deficient S. Typhi inhibited motility and attenuated interleukin-8 production, but it did not correct the defect in adhesion. We also disrupted yrbE in S. Typhimurium. In contrast to the results in S. Typhi, the deficiency of yrbE in S. Typhimurium had no significant effect on flagellin expression, motility or adhesion to HeLa cells. Correspondingly, the lack of yrbE also had no effect on association with the intestine or the severity of intestinal inflammation in the mouse model of S. Typhimurium infection. Thus, our results point to an important and serovar-specific role played by yrbE in the early stages of intestinal infection by S. Typhi.

The commensal bacterium Bacteroides fragilis down-regulates ferroportin expression and alters iron homeostasis in macrophages.

The intestinal microbiota has several effects on host physiology. Previous work from our laboratory demonstrated that the microbiota influences systemic iron homeostasis in mouse colitis models by altering inflammation-induced expression of the iron-regulating hormone hepcidin. In the present study, we examined the impact of the gut commensal bacterium Bacteroides fragilis on the expression of the iron exporter ferroportin, the target of hepcidin action, in macrophages, the cell type that plays a pivotal role in iron recycling. Mouse bone marrow-derived macrophages were exposed to B. fragilis and were analyzed by quantitative real-time polymerase chain reaction and Western blotting. We found that B. fragilis down-regulated ferroportin transcription independently of bacterial viability. Medium conditioned by the bacteria also reduced ferroportin expression, indicating the involvement of soluble factors, possibly Toll-like receptor ligands. Consistent with this idea, several of these ligands were able to down-regulate ferroportin. The B. fragilis-induced decrease in ferroportin was functionally important since it produced a significant increase in intracellular iron concentrations that prevented the effects of the iron chelator deferoxamine on Salmonella-induced IL-6 and IL-1ß production. Our results thus reveal that B. fragilis can influence macrophage iron handling and inflammatory responses by modulating ferroportin expression.

Iron and inflammation - the gut reaction.

Anemia is a frequent complication of many inflammatory disorders, including inflammatory bowel disease. Although the pathogenesis of this problem is multifactorial, a key component is the abnormal elevation of the hormone hepcidin, the central regulator of systemic iron homeostasis. Investigations over the last decade have resulted in important insights into the role of hepcidin in iron metabolism and the mechanisms that lead to hepcidin dysregulation in the context of inflammation. These insights provide the foundation for novel strategies to prevent and treat the anemia associated with inflammatory diseases.

Antibiotic Treatment Induces Long-lasting Changes in the Fecal Microbiota that Protect Against Colitis.

BACKGROUND: The interplay between host genetics, immunity, and microbiota is central to the pathogenesis of inflammatory bowel disease. Previous population-based studies suggested a link between antibiotic use and increased inflammatory bowel disease risk, but the mechanisms are unknown. The purpose of this study was to determine the long-term effects of antibiotic administration on microbiota composition, innate immunity, and susceptibility to colitis, as well as the mechanism by which antibiotics alter host colitogenicity. METHODS: Wild-type mice were given broad-spectrum antibiotics or no antibiotics for 2 weeks, and subsequent immunophenotyping and 16S rRNA gene sequencing-based analysis of the fecal microbiome were performed 6 weeks later. In a separate experiment, control and antibiotic-treated mice were given 7 days of dextran sulfate sodium, 6 weeks after completing antibiotic treatment, and the severity of colitis scored histologically. Fecal transfer was performed from control or antibiotic-treated mice to recipient mice whose endogenous microbiota had been cleared with antibiotics, and the susceptibility of the recipients to dextran sulfate sodium-induced colitis was analyzed. Naive CD4 T cells were transferred from control and antibiotic-treated mice to immunodeficient Rag-1 recipients and the severity of colitis compared. RESULTS: Antibiotics led to sustained dysbiosis and changes in T-cell subpopulations, including reductions in colonic lamina propria total T cells and CD4 T cells. Antibiotics conferred protection against dextran sulfate sodium colitis, and this effect was transferable by fecal transplant but not by naive T cells. CONCLUSIONS: Antibiotic exposure protects against colitis, and this effect is transferable with fecal microbiota from antibiotic-treated mice, supporting a protective effect of the microbial community.

Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis.

Commensal Bacteria-induced Interleukin 1ß (IL-1ß) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway.

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1ß. Moreover, purified IL-1ß increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1ß activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1ß-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1ß-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1ß and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1ß up-regulates hepcidin.

Intestinal Inflammation Leads to a Long-lasting Increase in Resistance to Systemic Salmonellosis that Requires Macrophages But Not B or T Lymphocytes at the Time of Pathogen Challenge.

BACKGROUND: Intestinal inflammation is associated with systemic translocation of commensal antigens and the consequent activation of B and T lymphocytes. The long-term consequences of such immune activation are not completely understood. METHODS: C57BL/6 mice were subjected to 2 courses of treatment with dextran sulfate sodium (DSS) to induce colitis. Two to 7 weeks after the DSS treatment, the mice were infected intraperitoneally with Salmonella enterica serovar Typhimurium. The outcome of infection was evaluated based on survival and tissue pathogen burden. RESULTS: Mice that had recovered from DSS colitis displayed a significant increase in resistance to S. Typhimurium infection as indicated by improved survival and decreased tissue pathogen numbers. The colitis-induced increase in resistance to systemic salmonellosis lasted for as long as 7 weeks after discontinuing DSS and was dependent on T lymphocytes but not on B cells. Interestingly, depletion of CD4 and CD8 T cells just before the Salmonella infection did not alter the colitis-induced increase in resistance. Mice that had recovered from colitis had evidence of persistent activation of resident peritoneal macrophages and enhanced Salmonella-induced neutrophil recruitment to the peritoneum. Macrophage depletion with clodronate liposomes abrogated the colitis-induced increase in resistance to Salmonella. CONCLUSIONS: Taken together, our results indicate that DSS colitis leads to a long-lasting increase in resistance to Salmonella infection that is initiated in a T cell-dependent manner but is ultimately mediated independently of B and T cells as a result of persistent changes in innate immune cell function.

Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food-borne bacterial pathogens.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.

Colitis induces enteric neurogenesis through a 5-HT4-dependent mechanism.

BACKGROUND: The intestine is known to contain enteric neuronal progenitors, but their precise identity and the mechanisms that activate them remain unknown. Based on the evidence for the neurogenic role of serotonin (5-HT) in the postnatal gut and the observation of enteric neuronal hyperplasia in inflammatory bowel disease, we hypothesized that colitis induces a neurogenic response through 5-HT4 receptor signaling. METHODS: We examined the effects of 5-HT4 agonism on colonic neurogenesis and gliogenesis in vitro and in vivo in adult mice using dextran sodium sulfate to experimentally induce colitis. RESULTS: In vitro, 5-HT4 agonism led to increased neuronal proliferation and density. Induction of experimental colitis in vivo similarly resulted in increased numbers of myenteric neurons, and this was inhibited by 5-HT4 antagonism. Interestingly, both in vitro and in vivo, 5-HT4 signaling increased glial cell proliferation but did not increase glial cell numbers, leading us to hypothesize that glia may give rise to neurons. After induction of colitis in normal, Nestin-GFP and Sox2-GFP transgenic mice, it was revealed that multiple glial markers (Sox2, Nestin, and CD49b) became strongly expressed by enteric neurons. Immunoselected enteric glia were found to give rise to neurons in culture, and this was inhibited in the presence of 5-HT4 blockade. Finally, isolated glia gave rise to a neuronal network upon transplantation into aganglionic embryonic avian hindgut. CONCLUSIONS: These results show that colitis promotes enteric neurogenesis in the adult colon through a serotonin-dependent mechanism that drives glial cells to transdifferentiate into neurons.

Effect of human immunodeficiency virus infection on plasma bactericidal activity against Salmonella enterica serovar Typhimurium.

Individuals with human immunodeficiency virus (HIV) infection have increased susceptibility to invasive disease caused by Salmonella enterica serovar Typhimurium. Studies from Africa have suggested that this susceptibility is related in part to the development of a high level of lipopolysaccharide (LPS)-specific IgG that is able to inhibit the killing of S. Typhimurium by bactericidal antibodies in healthy individuals. To explore this issue further, we examined the bactericidal activity against S. Typhimurium using serum and plasma samples from healthy controls and various clinical subgroups of HIV-infected adults in the United States. We found that the bactericidal activity in the samples from HIV-positive elite controllers was comparable to that from healthy individuals, whereas it was significantly reduced in HIV-positive viremic controllers and untreated chronic progressors. As demonstrated previously for healthy controls, the bactericidal activity of the plasma from the elite controllers was inhibited by preincubation with S. Typhimurium LPS, suggesting that it was mediated by anti-LPS antibodies. S. Typhimurium LPS-specific IgG was significantly reduced in all subgroups of HIV-infected individuals. Interestingly, and in contrast to the healthy controls, plasma from all HIV-positive subgroups inhibited in vitro killing of S. Typhimurium by plasma from a healthy individual. Our results, together with the findings from Africa, suggest that multiple mechanisms may be involved in the HIV-induced dysregulation of humoral immunity to S. Typhimurium.

Coinfection with an intestinal helminth impairs host innate immunity against Salmonella enterica serovar Typhimurium and exacerbates intestinal inflammation in mice.

Salmonella enterica serovar Typhimurium is a Gram-negative food-borne pathogen that is a major cause of acute gastroenteritis in humans. The ability of the host to control such bacterial pathogens may be influenced by host immune status and by concurrent infections. Helminth parasites are of particular interest in this context because of their ability to modulate host immune responses and because their geographic distribution coincides with those parts of the world where infectious gastroenteritis is most problematic. To test the hypothesis that helminth infection may negatively regulate host mucosal innate immunity against bacterial enteropathogens, a murine coinfection model was established by using the intestinal nematode Heligmosomoides polygyrus and S. Typhimurium. We found that mice coinfected with S. Typhimurium and H. polygyrus developed more severe intestinal inflammation than animals infected with S. Typhimurium alone. The enhanced susceptibility to Salmonella-induced intestinal injury in coinfected mice was found to be associated with diminished neutrophil recruitment to the site of bacterial infection that correlated with decreased expression of the chemoattractants CXCL2/macrophage inflammatory protein 2 (MIP-2) and CXCL1/keratinocyte-derived chemokine (KC), poor control of bacterial replication, and exacerbated intestinal inflammation. The mechanism of helminth-induced inhibition of MIP-2 and KC expression involved interleukin-10 (IL-10) and, to a lesser extent, IL-4 and IL-13. Ly6G antibody-mediated depletion of neutrophils reproduced the adverse effects of H. polygyrus on Salmonella infection. Our results suggest that impaired neutrophil recruitment is an important contributor to the enhanced severity of Salmonella enterocolitis associated with helminth coinfection.

Intestinal inflammation modulates expression of the iron-regulating hormone hepcidin depending on erythropoietic activity and the commensal microbiota.

States of chronic inflammation such as inflammatory bowel disease are often associated with dysregulated iron metabolism and the consequent development of an anemia that is caused by maldistribution of iron. Abnormally elevated expression of the hormone hepcidin, the central regulator of systemic iron homeostasis, has been implicated in these abnormalities. However, the mechanisms that regulate hepcidin expression in conditions such as inflammatory bowel disease are not completely understood. To clarify this issue, we studied hepcidin expression in mouse models of colitis. We found that dextran sulfate sodium-induced colitis inhibited hepcidin expression in wild-type mice but upregulated it in IL-10-deficient animals. We identified two mechanisms contributing to this difference. Firstly, erythropoietic activity, as indicated by serum erythropoietin concentrations and splenic erythropoiesis, was higher in the wild-type mice, and pharmacologic inhibition of erythropoiesis prevented colitis-associated hepcidin downregulation in these animals. Secondly, the IL-10 knockout mice had higher expression of multiple inflammatory genes in the liver, including several controlled by STAT3, a key regulator of hepcidin. The results of cohousing and fecal transplantation experiments indicated that the microbiota was involved in modulating the expression of hepcidin and other STAT3-dependent hepatic genes in the context of intestinal inflammation. Our observations thus demonstrate the importance of erythropoietic activity and the microbiota in influencing hepcidin expression during colitis and provide insight into the dysregulated iron homeostasis seen in inflammatory diseases.

Serum-induced up-regulation of hepcidin expression involves the bone morphogenetic protein signaling pathway.

Hepcidin is a peptide hormone that is secreted by the liver and that functions as the central regulator of systemic iron metabolism in mammals. Its expression is regulated at the transcriptional level by changes in iron status and iron requirements, and by inflammatory cues. There is considerable interest in understanding the mechanisms that influence hepcidin expression because dysregulation of hepcidin production is associated with a number of disease states and can lead to iron overload or iron-restricted anemia. In order to shed light on the factors that alter hepcidin expression, we carried out experiments with HepG2 and HuH7, human hepatoma cell lines that are widely used for this purpose. We found that the addition of heat-inactivated fetal calf serum to these cells resulted in a significant dose- and time-dependent up-regulation of hepcidin expression. Serum also activated signaling events known to be downstream of bone morphogenetic proteins (BMPs), a group of molecules that have been implicated previously in hepcidin regulation. Inhibition of these signals with dorsomorphin significantly suppressed serum-induced hepcidin up-regulation. Our results indicate that a BMP or BMP-like molecule present in serum may play an important role in regulating hepcidin expression.