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.

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.

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.

Tumor necrosis factor α inhibits expression of the iron regulating hormone hepcidin in murine models of innate colitis.

BACKGROUND: Abnormal expression of the liver peptide hormone hepcidin, a key regulator of iron homeostasis, contributes to the pathogenesis of anemia in conditions such as inflammatory bowel disease (IBD). Since little is known about the mechanisms that control hepcidin expression during states of intestinal inflammation, we sought to shed light on this issue using mouse models. METHODOLOGY/PRINCIPAL FINDINGS: Hepcidin expression was evaluated in two types of intestinal inflammation caused by innate immune activation-dextran sulfate sodium (DSS)-induced colitis in wild-type mice and the spontaneous colitis occurring in T-bet/Rag2-deficient (TRUC) mice. The role of tumor necrosis factor (TNF) α was investigated by in vivo neutralization, and by treatment of a hepatocyte cell line, as well as mice, with the recombinant cytokine. Expression and activation of Smad1, a positive regulator of hepcidin transcription, were assessed during colitis and following administration or neutralization of TNFα. Hepcidin expression progressively decreased with time during DSS colitis, correlating with changes in systemic iron distribution. TNFα inhibited hepcidin expression in cultured hepatocytes and non-colitic mice, while TNFα neutralization during DSS colitis increased it. Similar results were obtained in TRUC mice. These effects involved a TNFα-dependent decrease in Smad1 protein but not mRNA. CONCLUSIONS/SIGNIFICANCE: TNFα inhibits hepcidin expression in two distinct types of innate colitis, with down-regulation of Smad1 protein playing an important role in this process. This inhibitory effect of TNFα may be superseded by other factors in the context of T cell-mediated colitis given that in the latter form of intestinal inflammation hepcidin is usually up-regulated.