N-acetylglucosamine-6-O sulfation on intestinal mucins prevents obesity and intestinal inflammation by regulating gut microbiota.

Intestinal mucins play an essential role in the defense against bacterial invasion and the maintenance of gut microbiota, which is instrumental in the regulation of host immune systems; hence, its dysregulation is a hallmark of metabolic disease and intestinal inflammation. However, the mechanism by which intestinal mucins control the gut microbiota as well as disease phenotypes remains nebulous. Herein, we report that N-acetylglucosamine (GlcNAc)-6-O sulfation of O-glycans on intestinal mucins performs a protective role against obesity and intestinal inflammation. Chst4-/- mice, lacking GlcNAc-6-O sulfation of the mucin O-glycans, showed significant weight gain and increased susceptibility to dextran sodium sulfate-induced colitis as well as colitis-associated cancer accompanied by significantly reduced immunoglobulin A (IgA) production caused by an impaired T follicular helper cell-mediated IgA response. Interestingly, the protective effects of GlcNAc-6-O sulfation against obesity and intestinal inflammation depend on the gut microbiota, evidenced by the modulation of the gut microbiota by cohousing or microbiota transplantation reversing disease phenotypes and IgA production. Collectively, our findings provide insight into the significance of host glycosylation, more specifically GlcNAc-6-O sulfation on intestinal mucins, in protecting against obesity and intestinal inflammation via regulation of the gut microbiota.

Luminal bacteria coated with IgA and IgG during intestinal inflammation as a new and abundant stimulus for colonic macrophages.

In the gut, secretory immunoglobulin A is the predominant humoral response against commensals, although healthy hosts also produce microbiota-specific IgG antibodies. During intestinal inflammation, the content of IgG in the lumen increases along with the proportion of commensal bacteria coated with this antibody, suggesting signalling through the IgG-CD64 axis in the pathogenesis of inflammatory bowel diseases. In this work, we evaluated day by day the frequency of faecal bacteria coated with IgA and IgG during the development of DSS colitis. We studied the phenotype and phagocytic activity of F4/80+ CD64+ colonic macrophages, as well as the production of cytokines and nitric oxide by lamina propria or bone marrow-derived macrophages after stimulation with IgA+ , IgG+ and IgA+ IgG+ bacteria. We found that the percentage of faecal IgA+ IgG+ double-coated bacteria increased rapidly during DSS colitis. Also, analysis of the luminal content of mice with colitis showed a markedly superior ability to coat fresh bacteria. IgA+ IgG+ bacteria were the most potent stimulus for phagocytic activity involving CD64 and Dectin-1 receptors. IgA+ IgG+ bacteria observed during the development of DSS colitis could represent a new marker to monitor permeability and inflammatory progression. The interaction of IgA+ IgG+ bacteria with CD64+ F4/80+ macrophages could be part of the complex cascade of events in colitis. Interestingly, after stimulation, CD64+ colonic macrophages showed features similar to those of restorative macrophages that are relevant for tissue repair and healing.

PTH induces bone loss via microbial-dependent expansion of intestinal TNF+ T cells and Th17 cells.

Bone loss is a frequent but not universal complication of hyperparathyroidism. Using antibiotic-treated or germ-free mice, we show that parathyroid hormone (PTH) only caused bone loss in mice whose microbiota was enriched by the Th17 cell-inducing taxa segmented filamentous bacteria (SFB). SFB+ microbiota enabled PTH to expand intestinal TNF+ T and Th17 cells and increase their S1P-receptor-1 mediated egress from the intestine and recruitment to the bone marrow (BM) that causes bone loss. CXCR3-mediated TNF+ T cell homing to the BM upregulated the Th17 chemoattractant CCL20, which recruited Th17 cells to the BM. This study reveals mechanisms for microbiota-mediated gut-bone crosstalk in mice models of hyperparathyroidism that may help predict its clinical course. Targeting the gut microbiota or T cell migration may represent therapeutic strategies for hyperparathyroidism.

Erythroid differentiation regulator-1 induced by microbiota in early life drives intestinal stem cell proliferation and regeneration.

Gut microbiota and their metabolites are instrumental in regulating intestinal homeostasis. However, early-life microbiota associated influences on intestinal development remain incompletely understood. Here we demonstrate that co-housing of germ-free (GF) mice with specific-pathogen free (SPF) mice at weaning (exGF) results in altered intestinal gene expression. Our results reveal that one highly differentially expressed gene, erythroid differentiation regulator-1 (Erdr1), is induced during development in SPF but not GF or exGF mice and localizes to Lgr5+ stem cells and transit amplifying (TA) cells. Erdr1 functions to induce Wnt signaling in epithelial cells, increase Lgr5+ stem cell expansion, and promote intestinal organoid growth. Additionally, Erdr1 accelerates scratch-wound closure in vitro, increases Lgr5+ intestinal stem cell regeneration following radiation-induced injury in vivo, and enhances recovery from dextran sodium sulfate (DSS)-induced colonic damage. Collectively, our findings indicate that early-life microbiota controls Erdr1-mediated intestinal epithelial proliferation and regeneration in response to mucosal damage.

"Western Diet"-Induced Adipose Inflammation Requires a Complex Gut Microbiota.

BACKGROUND & AIMS: Consumption of a low-fiber, high-fat, Western-style diet (WSD) induces adiposity and adipose inflammation characterized by increases in the M1:M2 macrophage ratio and proinflammatory cytokine expression, both of which contribute to WSD-induced metabolic syndrome. WSD-induced adipose inflammation might result from endoplasmic reticulum stress in lipid-overloaded adipocytes and/or dissemination of gut bacterial products, resulting in activation of innate immune signaling. Hence, we aimed to investigate the role of the gut microbiota, and its detection by innate immune signaling pathways, in WSD-induced adipose inflammation. METHODS: Mice were fed grain-based chow or a WSD for 8 weeks, assessed metabolically, and intestinal and adipose tissue were analyzed by flow cytometry and quantitative reverse transcription polymerase chain reaction. Microbiota was ablated via antibiotics and use of gnotobiotic mice that completely lacked microbiota (germ-free mice) or had a low-complexity microbiota (altered Schaedler flora). Innate immune signaling was ablated by genetic deletion of Toll-like receptor signaling adaptor myeloid differentiation primary response 88. RESULTS: Ablation of microbiota via antibiotic, germ-free, or altered Schaedler flora approaches did not significantly impact WSD-induced adiposity, yet dramatically reduced WSD-induced adipose inflammation as assessed by macrophage populations and cytokine expression. Microbiota ablation also prevented colonic neutrophil and CD103- dendritic cell infiltration. Such reduced indices of inflammation correlated with protection against WSD-induced dysglycemia, hypercholesterolemia, and liver dysfunction. Genetic deletion of myeloid differentiation primary response 88 also prevented WSD-induced adipose inflammation. CONCLUSIONS: These results indicate that adipose inflammation, and some aspects of metabolic syndrome, are not purely a consequence of diet-induced adiposity per se but, rather, may require disturbance of intestine-microbiota interactions and subsequent activation of innate immunity.

Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice.

Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.

Early-Life Microbiota Exposure Restricts Myeloid-Derived Suppressor Cell-Driven Colonic Tumorigenesis.

Gut microbiota and their metabolites are instrumental in regulating homeostasis at intestinal and extraintestinal sites. However, the complex effects of prenatal and early postnatal microbial exposure on adult health and disease outcomes remain incompletely understood. Here, we showed that mice raised under germ-free conditions until weaning and then transferred to specific pathogen-free (SPF) conditions harbored altered microbiota composition, augmented inflammatory cytokine and chemokine expression, and were hyper-susceptible to colitis-associated tumorigenesis later in adulthood. Increased number and size of colon tumors and intestinal epithelial cell proliferation in recolonized germ-free mice were associated with augmented intratumoral CXCL1, CXCL2, and CXCL5 expression and granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation. Consistent with these findings, CXCR2 neutralization in recolonized germ-free mice completely reversed the exacerbated susceptibility to colitis-associated tumorigenesis. Collectively, our findings highlight a crucial role for early-life microbial exposure in establishing intestinal homeostasis that restrains colon cancer in adulthood.

Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Autotaxin (ATX or ENPP2) is a secreted lysophospholipase D that produces lysophosphatidic acid (LPA), a pleiotropic lipid mediator acting on specific GPCRs. ATX and LPA have been implicated in key (patho)physiologic processes, including embryonic development, lymphocyte homing, inflammation, and cancer progression. Using LPA receptor knockout mice, we previously uncovered a role for LPA signaling in promoting colitis and colorectal cancer. Here, we examined the role of ATX in experimental colitis through inducible deletion of Enpp2 in adult mice. ATX expression was increased upon induction of colitis, whereas ATX deletion reduced the severity of inflammation in both acute and chronic colitis, accompanied by transient weight loss. ATX expression in lymphocytes was strongly reduced in Rag1-/- and µMT mice, suggesting B cells as a major ATX-producing source, which was validated by immunofluorescence and biochemical analyses. ATX secretion by B cells from control, but not Enpp2 knockout, mice led to ERK activation in colorectal cancer cells and promoted T cell migration. We conclude that ATX deletion suppresses experimental colitis and that B cells are a major source of ATX in the colon. Our study suggests that pharmacological inhibition of ATX could be a therapeutic strategy in colitis.-Lin, S., Haque, A., Raeman, R., Guo, L., He, P., Denning, T. L., El-Rayes, B., Moolenaar, W. H., Yun, C. C. Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment.

We have previously reported that direct injection of dendritic cells (DC) engineered to express the Type-1 transactivator Tbet (i.e., DC.Tbet) into murine tumors results in antitumor efficacy in association with the development of structures resembling tertiary lymphoid organs (TLO) in the tumor microenvironment (TME). These TLO contained robust infiltrates of B cells, DC, NK cells, and T cells in proximity to PNAd+ blood vessels; however, they were considered incomplete, since the recruited B cells failed to organize into classic germinal center-like structures. We now report that antitumor efficacy and TLO-inducing capacity of DC.Tbet-based i.t. therapy is operational in peripheral lymph node-deficient LTA-/- mice, and that it is highly dependent upon a direct Tbet target gene product, IL-36γ/IL-1F9. Intratumoral DC.Tbet fails to provide protection to tumor-bearing IL-36R-/- hosts, or to tumor-bearing wild-type recipient mice co-administered rmIL-1F5/IL-36RN, a natural IL-36R antagonist. Remarkably, the injection of tumors with DC engineered to secrete a bioactive form of mIL-36γ (DC.IL36γ) also initiated therapeutic TLO and slowed tumor progression in vivo. Furthermore, DC.IL36γ cells strongly upregulated their expression of Tbet, suggesting that Tbet and IL-36γ cooperate to reinforce each other's expression in DC, rendering them competent to promote TLO formation in an "immunologically normalized," therapeutic TME.

Macrophage Isolation from the Mouse Small and Large Intestine.

Macrophages play important roles in maintaining intestinal homeostasis via their ability to orchestrate responses to the normal microbiota as well as pathogens. One of the most important steps in beginning to understand the functions of these cells is the ability to effectively isolate them from the complex intestinal environment. Here, we detail methodology for the isolation and phenotypic characterization of macrophages from the mouse small and large intestine.

Cutting Edge: IL-36 Receptor Promotes Resolution of Intestinal Damage.

IL-1 family members are central mediators of host defense. In this article, we show that the novel IL-1 family member IL-36γ was expressed during experimental colitis and human inflammatory bowel disease. Germ-free mice failed to induce IL-36γ in response to dextran sodium sulfate (DSS)-induced damage, suggesting that gut microbiota are involved in its induction. Surprisingly, IL-36R-deficient (Il1rl2(-/-)) mice exhibited defective recovery following DSS-induced damage and impaired closure of colonic mucosal biopsy wounds, which coincided with impaired neutrophil accumulation in the wound bed. Failure of Il1rl2(-/-) mice to recover from DSS-induced damage was associated with a profound reduction in IL-22 expression, particularly by colonic neutrophils. Defective recovery of Il1rl2(-/-) mice could be rescued by an aryl hydrocarbon receptor agonist, which was sufficient to restore IL-22 expression and promote full recovery from DSS-induced damage. These findings implicate the IL-36/IL-36R axis in the resolution of intestinal mucosal wounds.

IL-36γ Transforms the Tumor Microenvironment and Promotes Type 1 Lymphocyte-Mediated Antitumor Immune Responses.

Cytokines play a pivotal role in regulating tumor immunogenicity and antitumor immunity. IL-36γ is important for the IL-23/IL-17-dominated inflammation and anti-BCG Th1 immune responses. However, the impact of IL-36γ on tumor immunity is unknown. Here we found that IL-36γ stimulated CD8(+) T cells, NK cells, and γδ T cells synergistically with TCR signaling and/or IL-12. Importantly, IL-36γ exerted profound antitumor effects in vivo and transformed the tumor microenvironment in favor of tumor eradication. Furthermore, IL-36γ strongly increased the efficacy of tumor vaccination. Moreover, IL-36γ expression inversely correlated with the progression of human melanoma and lung cancer. Our study establishes a role of IL-36γ in promoting antitumor immune responses and suggests its potential clinical translation into cancer immunotherapy.

Intestinal Antigen-Presenting Cells: Key Regulators of Immune Homeostasis and Inflammation.

The microbiota that populate the mammalian intestine are critical for proper host physiology, yet simultaneously pose a potential danger. Intestinal antigen-presenting cells, namely macrophages and dendritic cells (DCs), are integral components of the mucosal innate immune system that maintain co-existence with the microbiota in face of this constant threat. Intestinal macrophages and DCs integrate signals from the microenvironment to orchestrate innate and adaptive immune responses that ultimately lead to durable tolerance of the microbiota. Tolerance is not a default response, however, because macrophages and DCs remain poised to vigorously respond to pathogens that breach the epithelial barrier. In this review, we summarize the salient features of macrophages and DCs in the healthy and inflamed intestine and discuss how signals from the microbiota can influence their function.

Phenotypic and functional profiling of mouse intestinal antigen presenting cells.

The microbiota that populates the mammalian intestine consists of hundreds of trillions of bacteria that are separated from underlying immune cells by a single layer of epithelial cells. The intestinal immune system effectively tolerates components of the microbiota that provide benefit to the host while remaining poised to eliminate those that are harmful. Antigen presenting cells, especially macrophages and dendritic cells, play important roles in maintaining intestinal homeostasis via their ability to orchestrate appropriate responses to the microbiota. Paramount to elucidating intestinal macrophage- and dendritic cell-mediated functions is the ability to effectively isolate and identify these cells from a complex cellular environment. In this review, we summarize methodology for the isolation and phenotypic characterization of macrophages and DCs from the mouse intestine and discuss how this may be useful for gaining insight into the mechanisms by which mucosal immune tolerance is maintained.

IFNγ-induced suppression of ß-catenin signaling: evidence for roles of Akt and 14.3.3ζ.

The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. ß-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/ß-catenin signaling. Here we show that inhibition of Akt/ß-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated ß-catenin 552 (pß-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits ß-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes ß-catenin transactivation through Akt-dependent C-terminal phosphorylation of ß-catenin to promote its association with 14.3.3ζ. Augmented ß-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/ß-catenin from the nucleus, thereby inhibiting ß-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

Fab'-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis.

Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab'-bearing NPs loaded with TNFα siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.

Lactobacillus rhamnosus (LGG) regulates IL-10 signaling in the developing murine colon through upregulation of the IL-10R2 receptor subunit.

The intestinal microflora is critical for normal development, with aberrant colonization increasing the risk for necrotizing enterocolitis (NEC). In contrast, probiotic bacteria have been shown to decrease its incidence. Multiple pro- and anti-inflammatory cytokines have been identified as markers of intestinal inflammation, both in human patients with NEC and in models of immature intestine. Specifically, IL-10 signaling attenuates intestinal responses to gut dysbiosis, and disruption of this pathway exacerbates inflammation in murine models of NEC. However, the effects of probiotics on IL-10 and its signaling pathway, remain poorly defined. Real-time PCR profiling revealed developmental regulation of MIP-2, TNF-α, IL-12, IL-10 and the IL-10R2 subunit of the IL-10 receptor in immature murine colon, while the expression of IL-6 and IL-18 was independent of postnatal age. Enteral administration of the probiotic Lactobacillus rhamnosus GG (LGG) down-regulated the expression of TNF-α and MIP-2 and yet failed to alter IL-10 mRNA and protein expression. LGG did however induce mRNA expression of the IL-10R2 subunit of the IL-10 receptor. IL-10 receptor activation has been associated with signal transducer and activator of transcription (STAT) 3-dependent induction of members of the suppressors of cytokine signaling (SOCS) family. In 2 week-old mice, LGG also induced STAT3 phosphorylation, increased colonic expression of SOCS-3, and attenuated colonic production of MIP-2 and TNF-α. These LGG-dependent changes in phosphoSTAT3, SOCS3, MIP-2 and TNF-α were all inhibited by antibody-mediated blockade of the IL-10 receptor. Thus LGG decreased baseline proinflammatory cytokine expression in the developing colon through upregulation of IL-10 receptor-mediated signaling, most likely due to the combined induction of phospho-STAT3 and SOCS3. Furthermore, LGG-dependent increases in IL-10R2 were associated with reductions in TNF-α, MIP-2 and disease severity in a murine model of intestinal injury in the immature colon.

A(2B)AR expression in non-immune cells plays an important role in the development of murine colitis.

BACKGROUND: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis. AIMS: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation. METHODS: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. RESULTS: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. CONCLUSIONS: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.