Jesse D. Aitken

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.

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.

Dextran sulfate sodium (DSS)-induced colitis in mice.

Inflammatory bowel diseases (IBD), mainly comprising ulcerative colitis and Crohns Disease, are complex and multifactorial diseases with unknown etiology. For the past 20 years, to study human IBD mechanistically, a number of murine models of colitis have been developed. These models are indispensable tools to decipher underlying mechanisms of IBD pathogenesis as well as to evaluate a number of potential therapeutics. Among various chemically induced colitis models, the dextran sulfate sodium (DSS)‐induced colitis model is widely used because of its simplicity and many similarities with human ulcerative colitis. This model has both advantages and disadvantages that must be considered when employed. This protocol describes the DSS‐induced colitis model, focusing on details and factors that could affect DSS‐induced pathology. Curr. Protoc. Immunol. 104:15.25.1‐15.25.14.

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E. coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohns disease-associated E. coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.

Flagellin Treatment Protects against Chemicals, Bacteria, Viruses, and Radiation

Sudden exposure of human populations to chemicals, pathogens, or radiation has the potential to result in substantial morbidity. A potential means of rapidly protecting such populations might be to activate innate host defense pathways, which can provide broad protection against a variety of insults. However, innate immune activators can, by themselves, result in severe inflammatory pathology, which in large part is driven by hemopoietic-derived cytokines such as TNF-α. We reasoned that, because it preferentially activates epithelial cells, the TLR5 agonist flagellin might not induce severe inflammatory pathology and yet be an ideal agent to provide such non-specific protection, particularly at the mucosal surfaces that serve as a front line of host defense. In accordance, we observed that systemic treatment of mice with purified flagellin did not induce the serologic, histopathologic, and clinical hallmarks of inflammation that are induced by LPS but yet protected mice against chemicals, pathogens, and ionizing radiation. Flagellin-elicited radioprotection required TLR5, the TLR signaling adaptor MyD88, and was effective if given between 2 h before to 4 h after exposure to irradiation. Flagellin-elicited radioprotection was, in part, mediated via effects on cells in bone marrow but yet rescued mortality without a pronounced rescue of radiation-induced anemia or leukopenia. Thus, systemic administration of flagellin may be a relatively safe means of providing temporary non-specific protection against a variety of challenges.

TLR5 or NLRC4 is necessary and sufficient for promotion of humoral immunity by flagellin

The fact that some TLR‐based vaccine adjuvants maintain function in TLR‐deficient hosts highlights that their mechanism of function remains incompletely understood. Thus, we examined the ability of flagellin to induce cytokines and elicit/promote murine antibody responses upon deletion of the flagellin receptors TLR5 and/or NLRC4 (also referred to as IPAF) using a prime/boost regimen. In TLR5‐KO mice, flagellin failed to induce NF‐κB‐regulated cytokines such as keratinocyte‐derived chemokine (CXCL1) but induced WT levels of the inflammasome cytokine IL‐18 (IL‐1F4). Conversely, in NLRC4‐KO mice, flagellin induced keratinocyte‐derived chemokine, but not IL‐18, whereas TLR5/NLRC4‐DKO lacked induction of all cytokines measured. Flagellin/ovalbumin treatment resulted in high‐antibody titers to both flagellin and ovalbumin in WT, TLR5‐KO and DKO mice but did not elicit antibodies to either in TLR5/NLRC4‐DKO mice. Thus, flagellins ability to elicit/promote humoral immunity requires a germ‐line‐encoded receptor capable of recognizing this molecule. Such promotion of adaptive immunity can be effectively driven by either TLR5‐mediated activation of NF‐κB or NLRC4‐mediated activation of the inflammasome.

Toll-Like Receptor–Gut Microbiota Interactions: Perturb at Your Own Risk!

The well-being of the intestine and its host requires that this organ execute its complex function amid colonization by a large and diverse microbial community referred to as the gut microbiota. A myriad of interacting mechanisms of mucosal immunity permit the gut to corral the microbiota in such a way as to maximize the benefits and to minimize the danger of living in close proximity to this large microbial biomass. Toll-like receptors and Nod-like receptors, collectively referred to as pattern recognition receptors (PRRs), recognize a variety of microbial components and, hence, play a central role in governing the interface between host and microbiota. This review examines mechanisms by which PRR-microbiota interactions are regulated so as to allow activation of host defense when necessary while preventing excessive inflammation, which can have a myriad of negative consequences for the host. Analysis of published studies performed in human subjects and a variety of murine disease models reveals the central theme that PRRs play a key role in maintaining a healthful stable relationship between the intestine and its microbiota. In contrast, although select genetic ablations of PRR signaling may protect against some chronic diseases, the overriding theme of studies performed to date is that perturbations of PRR-microbiota interactions are more likely to promote disease states associated with inflammation.

Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges

Bacterial flagellin is a dominant innate immune activator of the intestine. Therefore, we examined the role of the intracellular flagellin receptor, NLRC4, in protecting the gut and/or driving inflammation. In accordance with NLRC4 acting through transcription-independent pathways, loss of NLRC4 did not reduce the rapid robust changes in intestinal gene expression induced by flagellin administration. Loss of NLRC4 did not alter basal intestinal homeostasis nor predispose mice to development of colitis upon administration of an anti-interleukin (IL)-10R monoclonal antibody. However, epithelial injury induced by dextran sulfate sodium in mice lacking NLRC4 resulted in a more severe disease, indicating a role for NLRC4 in protecting the gut. Moreover, loss of NLRC4 resulted in increased mortality in response to flagellate, but not aflagellate Salmonella infection. Thus, despite not being involved in rapid intestinal gene remodeling upon detection of flagellin, NLRC4-mediated inflammasome activation results in production of IL-1β and IL-18, two cytokines that protect mice from mucosal and systemic challenges.

Lipocalin 2 Deficiency Dysregulates Iron Homeostasis and Exacerbates Endotoxin-Induced Sepsis

Various states of inflammation, including sepsis, are associated with hypoferremia, which limits iron availability to pathogens and reduces iron-mediated oxidative stress. Lipocalin 2 (Lcn2; siderocalin, 24p3) plays a central role in iron transport. Accordingly, Lcn2-deficient (Lcn2KO) mice exhibit elevated intracellular labile iron. In this study, we report that LPS induced systemic Lcn2 by 150-fold in wild-type mice at 24 h. Relative to wild-type littermates, Lcn2KO mice were markedly more sensitive to endotoxemia, exhibiting elevated indices of organ damage (transaminasemia, lactate dehydrogenase) and increased mortality. Such exacerbated endotoxemia was associated with substantially increased caspase-3 cleavage and concomitantly elevated immune cell apoptosis. Furthermore, cells from Lcn2KO mice were hyperresponsive to LPS ex vivo, exhibiting elevated cytokine secretion. Additionally, Lcn2KO mice exhibited delayed LPS-induced hypoferremia despite normal hepatic hepcidin expression and displayed decreased levels of the tissue redox state indicators cysteine and glutathione in liver and plasma. Desferroxamine, an iron chelator, significantly protects Lcn2KO mice from LPS-induced toxicity, including mortality, suggesting that Lcn2 may act as an antioxidant in vivo by regulating iron homeostasis. Thus, Lcn2-mediated regulation of labile iron protects the host against sepsis. Its small size and simple structure may make Lcn2 a deployable treatment for sepsis.

Toll like receptor-5 : protecting the gut from enteric microbes

The intestine is normally colonized by a large and diverse commensal microbiota and is occasionally exposed to a variety of potential pathogens. In recent years, there has been substantial progress made in identifying molecular mechanisms that normally serve to protect the intestine from such enteric bacteria and which may go awry in chronic idiopathic inflammatory diseases of the gut. One specific molecular interaction that appears to play a key role in governing bacterial–intestinal interactions is that of the bacterial protein flagellin with toll-like receptor 5. This article reviews studies performed in vitro, in mice, and in humans that indicate an important role for the flagellin-TLR5 interaction in regulating both the innate and adaptive immune responses in the intestine.