Andrew T. Gewirtz

Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking Toll-Like Receptor 5

Debugging Metabolic Disease Obesity, now officially recognized as an epidemic in many developed nations, is a key component of “metabolic syndrome,” an array of metabolic disturbances that increase an individuals risk of developing diabetes and heart disease. The rise in obesity rates has been largely attributed to the growing imbalance between food intake and energy expenditure, but recent provocative work has suggested a possible link between obesity and the composition of microbes residing within the gut. Vijay-Kumar et al. (p. 228, published online 4 March; see the Perspective by Sandoval and Seeley) now find that mutant mice deficient in a component of the innate immune system (which defends the body against microbial pathogens) develop hallmark features of metabolic syndrome, accompanied by changes in gut microbiota. Notably, transfer of gut microbiota from the mutant mice to wild-type mice conferred several features of metabolic syndrome to the recipients. Thus, the development of metabolic syndrome may indeed be influenced by gut microbes that are regulated by the innate immune system. The innate immune system may promote metabolic health through effects on gut microbes. Metabolic syndrome is a group of obesity-related metabolic abnormalities that increase an individual’s risk of developing type 2 diabetes and cardiovascular disease. Here, we show that mice genetically deficient in Toll-like receptor 5 (TLR5), a component of the innate immune system that is expressed in the gut mucosa and that helps defend against infection, exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity. These metabolic changes correlated with changes in the composition of the gut microbiota, and transfer of the gut microbiota from TLR5-deficient mice to wild-type germ-free mice conferred many features of metabolic syndrome to the recipients. Food restriction prevented obesity, but not insulin resistance, in the TLR5-deficient mice. These results support the emerging view that the gut microbiota contributes to metabolic disease and suggest that malfunction of the innate immune system may promote the development of metabolic syndrome.

Cutting Edge: Bacterial Flagellin Activates Basolaterally Expressed TLR5 to Induce Epithelial Proinflammatory Gene Expression

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-κB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.

Cutting Edge: Impaired Toll-Like Receptor Expression and Function in Aging

Toll-like receptors (TLR) are pattern recognition receptors that recognize conserved molecular patterns on microbes and link innate and adaptive immune systems. We investigated whether the enhanced susceptibility to bacterial, yeast, and viral infections and poor adaptive immune responses in aging are a result of diminished expression and function of TLRs. We examined the expression and function of all murine TLRs on macrophages from young and aged mice. Both splenic and activated peritoneal macrophages from aged mice expressed significantly lower levels of all TLRs. Furthermore, macrophages from aged mice secreted significantly lower levels of IL-6 and TNF-α when stimulated with known ligands for TLR1 and 2, 2 and 6,TLR3, TLR4, TLR5, and TLR9 when compared with those from young mice. These results support the concept that increased susceptibility to infections and poor adaptive immune responses in aging may be due to the decline in TLR expression and function.

Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome

The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, especially in metabolism and immune development, disturbance of the microbiota–host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and the group of obesity-associated diseases collectively referred to as metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, thereby allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine. Thus, agents that disrupt mucus–bacterial interactions might have the potential to promote diseases associated with gut inflammation. Consequently, it has been hypothesized that emulsifiers, detergent-like molecules that are a ubiquitous component of processed foods and that can increase bacterial translocation across epithelia in vitro, might be promoting the increase in inflammatory bowel disease observed since the mid-twentieth century. Here we report that, in mice, relatively low concentrations of two commonly used emulsifiers, namely carboxymethylcellulose and polysorbate-80, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host–microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.

Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response

This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.

Deletion of TLR5 results in spontaneous colitis in mice

Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.

Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis

Objective The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)? Methods and results The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10−/− mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls. Conclusions Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology.

Dysregulation of Inflammatory Responses by Chronic Circadian Disruption

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.

Bacterial Flagellin Is an Effective Adjuvant for CD4+ T Cells In Vivo

Flagellin is secreted by many enteric bacteria and, upon reaching the basolateral membrane of the intestinal epithelium, activates Toll-like receptor 5-mediated innate immune signaling pathways. We hypothesized that any flagellin that gets beyond the epithelium might also regulate cells of the adaptive immune system. Here we demonstrate that the clonal expansion of naive DO11.10 CD4 T cells in response to OVA peptide (323–339) was enhanced 3- to 10-fold in the presence of purified bacterial flagellin in vivo. OVA-specific CD4 T cells were also shown to have undergone more cell division in vivo if flagellin was coinjected with OVA. Flagellin administration increased the expression of B7-1 on splenic dendritic cells, and coinjection of CTLA4-Ig, which is known to block B7 function in vivo, completely ablated the adjuvant effect on CD4 T cells. Therefore, a conserved bacterial protein produced by many intestinal microbes can modulate CD4 T cell activation in vivo. Such an adjuvant effect for flagellin has important implications for vaccine development and the generation of CD4 T cell responses to enteric bacteria.

Lipoxin A4 Analogs Attenuate Induction of Intestinal Epithelial Proinflammatory Gene Expression and Reduce the Severity of Dextran Sodium Sulfate-Induced Colitis

The anti-inflammatory eicosanoid lipoxin A4 (LXA4), aspirin-triggered 15-epi-LXA4, and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA4 analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA4 analog pretreatment attenuated induction of ∼50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA4 analog pretreatment is regulated by NF-κB, suggesting that LXA4 analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA4 analog reduced NF-κB-mediated transcriptional activation in a LXA4 receptor-dependent manner and inhibited induced degradation of IκBα. LXA4 analog did not affect earlier stimulus-induced signaling events that lead to IκBα degradation, such as S. typhimurium-induced epithelial Ca2+ mobilization or TNF-α-induced phosphorylation of IκBα. To establish the in vivo relevance of these findings, we examined whether LXA4 analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA4 analog (15-epi-16-para-fluoro-phenoxy-LXA4, 10 μg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA4 analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-κB pathway can be therapeutic for diseases characterized by mucosal inflammation.