A Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis.

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic ß cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ T cells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin ß7-, perforin-, and major histocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood T cells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic T cells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.

Macrophage immunotherapy: overcoming impediments to realize promise.

As an essential component of immunity, macrophages have key roles in mammalian host defense, tissue homeostasis, and repair, as well as in disease pathogenesis and pathophysiology. A source of fascination and extensive research, in this Opinion we challenge the utility of the M1-M2 paradigm, and discuss the importance of accurate characterization of human macrophages. We comment on the application of single cell analytics to define macrophage subpopulations and how this could advance therapeutic options. We argue that human macrophage cell therapy can be used to alleviate many diseases, and offer a viewpoint on the knowledge gaps that must be filled to render such a therapeutic approach a reality and, ideally, a common future practice in precision medicine.

Neuroimmunophysiology of the gastrointestinal tract.

Gut physiology is the epicenter of a web of internal communication systems (i.e., neural, immune, hormonal) mediated by cell-cell contacts, soluble factors, and external influences, such as the microbiome, diet, and the physical environment. Together these provide the signals that shape enteric homeostasis and, when they go awry, lead to disease. Faced with the seemingly paradoxical tasks of nutrient uptake (digestion) and retarding pathogen invasion (host defense), the gut integrates interactions between a variety of cells and signaling molecules to keep the host nourished and protected from pathogens. When the system fails, the outcome can be acute or chronic disease, often labeled as "idiopathic" in nature (e.g., irritable bowel syndrome, inflammatory bowel disease). Here we underscore the importance of a holistic approach to gut physiology, placing an emphasis on intercellular connectedness, using enteric neuroimmunophysiology as the paradigm. The goal of this opinion piece is to acknowledge the pace of change brought to our field via single-cell and -omic methodologies and other techniques such as cell lineage tracing, transgenic animal models, methods for culturing patient tissue, and advanced imaging. We identify gaps in the field and hope to inspire and challenge colleagues to take up the mantle and advance awareness of the subtleties, intricacies, and nuances of intestinal physiology in health and disease by defining communication pathways between gut resident cells, those recruited from the circulation, and "external" influences such as the central nervous system and the gut microbiota.

Perturbed mitochondrial dynamics, an emerging aspect of epithelial-microbe interactions.

Mitochondria exist in a complex network that is constantly remodeling via the processes of fission and fusion in response to intracellular conditions and extracellular stimuli. Excessive fragmentation of the mitochondrial network because of an imbalance between fission and fusion reduces the cells' capacity to generate ATP and can be a forerunner to cell death. Given the critical roles mitochondria play in cellular homeostasis and innate immunity, it is not surprising that many microbial pathogens can disrupt mitochondrial activity. Here we note the putative contribution of mitochondrial dysfunction to gut disease and review data showing that infection with microbial pathogens can alter the balance between mitochondrial fragmentation and fusion, preventing normal remodeling (i.e., dynamics) and can lead to cell death. Current data indicate that infection of epithelia or macrophages with microbial pathogens will ultimately result in excessive fragmentation of the mitochondrial network. Concerted research efforts are required to elucidate fully the processes that regulate mitochondrial dynamics, the mechanisms by which microbes affect epithelial mitochondrial fission and/or fusion, and the implications of this for susceptibility to infectious disease. We speculate that the commensal microbiome of the gut may be important for normal epithelial mitochondrial form and function. Drugs designed to counteract the effect of microbial pathogen interference with mitochondrial dynamics may be a new approach to infectious disease at mucosal surfaces.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis.

Patients with rheumatoid arthritis experience chronic pain, depression and fatigue, even when inflammation of the joints is well controlled. To study the relationship between arthritis, depression, and sustained pain when articular inflammation is no longer observed, we tested the hypothesis that brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis. The murine model of antigen-induced arthritis (AIA) was used to evaluate the effects of knee inflammation on sustained pain and depression-like behavior. We measured joint pain using an automated dynamic plantar algesiometer and depression-like behavior with the tail suspension test. Cytokines were measured by Luminex assay and ELISA. TNF in the brain was blocked by intracerebroventricular injection of anti-TNF antibodies. Histological damage and elevated levels of cytokines were observed in the knee 24 h after antigen treatment, but not at 13 days. Reduced pain thresholds were seen 24 h and 13 days after treatment. Depression-like behavior was observed on day 13. Treatment with the antidepressant imipramine reduced both depression-like behavior and persistent pain. However, blocking joint pain with the analgesic dipyrone did not alter depression-like behavior. Elevated levels of TNF, CCL2, and CXCL-1 were observed in the hippocampus 24 h after treatment, with TNF remaining elevated at day 13. Intracerebroventricular infusion of an anti-TNF antibody blocked depression-like behavior and reduced persistent pain. We have demonstrated that depression-like behavior and pain is sustained in AIA mice after the resolution of inflammation. These changes are associated with elevated levels of TNF in the hippocampus and are dependent upon brain TNF. The findings reveal an important mechanistic link between the expression of chronic pain and depression in experimental arthritis. Furthermore, they suggest treating depression in rheumatoid arthritis may positively impact other debilitating features of this condition.

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

Helminth Antigen-Conditioned Dendritic Cells Generate Anti-Inflammatory Cd4 T Cells Independent of Antigen Presentation via Major Histocompatibility Complex Class II.

A recently identified feature of the host response to infection with helminth parasites is suppression of concomitant disease. Dendritic cells (DCs) exposed to antigens from the tapeworm Hymenolepis diminuta significantly reduce the severity of dinitrobenzene sulfonic acid-induced colitis in mice. Here we elucidate mechanisms underlying this cellular immunotherapy. We show a requirement for Ccr7 expression on transferred H. diminuta antigen-treated (HD)-DCs, suggesting that homing to secondary lymphoid tissues is important for suppression of colitis. Furthermore, sodium metaperiodate-sensitive helminth-derived glycans are required to drive the anti-colitic response in recipient mice. Induction of Th2-type cytokines and Gata-3+Cd4+ cells in secondary lymphoid tissues is dependent on major histocompatibility complex class II (MHC II) protein expression on transferred DCs, although remarkably, transfer of MHC II-/- HD-DCs still attenuated dinitrobenzene sulfonic acid-induced colitis in recipient mice. Moreover, transfer of Cd4+ splenic T cells retrieved from mice administered MHC II-/- HD-DCs suppressed dinitrobenzene sulfonic acid-induced colitis in recipient mice. Our studies reveal that HD-DCs can suppress colitis via an alternative MHC II-independent pathway that involves, in part, mobilization of T-cell responses. These data support the utility of HD-DCs in blocking colitis, revealing a requirement for Ccr7 and providing for HD-DC autologous immunotherapy for disease in which MHC II expression and/or function is compromised.

Young mice expel the tapeworm Hymenolepis diminuta and are protected from colitis by triggering a memory response with worm antigen.

Infection with helminth parasites reduces the severity of concomitant inflammatory disease in adult mice. There is an alarming increase of inflammatory bowel disease (IBD) in children. It is important to determine whether helminth therapy would be of value in pediatric IBD and whether triggering immunological memory to the worm would be anticolitic. Three-week-old (young) and eight-week-old (adult) Balb/c mice were infected with H. diminuta, and infectivity and T helper 2 (Th2) immunity were assessed. Other mice received H. diminuta with or without a crude worm extract ( HdE) 28-42 days postinfection (dpi) with or without dinitrobenzene sulphonic acid [DNBS, 1.5 mg (young) or 3 mg (adults), ir], and colitis was assessed 72 h later. Infected young mice developed Th2 immunity and expelled H. diminuta; expulsion was delayed by ~2 days compared with adult mice. Colitis, as gauged by macroscopic disease and histopathology scores, was less severe in young mice infected 10 days, but not 8 days, before DNBS. Protection against DNBS-induced colitis was accompanied by an increased capacity to make interleukin (IL)-4 and IL-10. Mice infected with H. diminuta were not protected from DNBS-colitis when challenged 28 days later; however, injection of these mice with HdE coincident with DNBS resulted in less disease and increased splenic IL-4 and IL-10. Using a boost (500 µg HdE, 28 dpi) and repeat HdE (100 µg, 42 dpi) regimen with infected mice suppressed DNBS-colitis, as did adoptive transfer of splenic CD4+ T cells from infected mice with low-dose HdE challenge. Should these data translate to IBD, then helminth therapy could be of value in pediatric-onset IBD, and defining the antigen(s) that elicit antihelminth immunological memory could serve as an anticolitic approach in previously infected individuals. NEW & NOTEWORTHY This study demonstrates that juvenile mice are protected from colitis by infection with the tapeworm Hymenolepis diminuta and that using worm antigen to trigger an immunological memory response in previously infected mice can be used to limit the severity of colitis.

IL-22 Restrains Tapeworm-Mediated Protection against Experimental Colitis via Regulation of IL-25 Expression.

Interleukin (IL)-22, an immune cell-derived cytokine whose receptor expression is restricted to non-immune cells (e.g. epithelial cells), can be anti-inflammatory and pro-inflammatory. Mice infected with the tapeworm Hymenolepis diminuta are protected from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Here we assessed expulsion of H. diminuta, the concomitant immune response and the outcome of DNBS-induced colitis in wild-type (WT) and IL-22 deficient mice (IL-22-/-) ± infection. Interleukin-22-/- mice had a mildly impaired ability to expel the worm and this correlated with reduced or delayed induction of TH2 immunity as measured by splenic and mesenteric lymph node production of IL-4, IL-5 and IL-13 and intestinal Muc-2 mRNA and goblet cell hyperplasia; in contrast, IL-25 increased in the small intestine of IL-22-/- mice 8 and 12 days post-infection compared to WT mice. In vitro experiments revealed that H. diminuta directly evoked epithelial production of IL-25 that was inhibited by recombinant IL-22. Also, IL-10 and markers of regulatory T cells were increased in IL-22-/- mice that displayed less DNBS (3 mg, ir. 72h)-induced colitis. Wild-type mice infected with H. diminuta were protected from colitis, as were infected IL-22-/- mice and the latter to a degree that they were almost indistinguishable from control, non-DNBS treated mice. Finally, treatment with anti-IL-25 antibodies exaggerated DNBS-induced colitis in IL-22-/- mice and blocked the anti-colitic effect of infection with H. diminuta. Thus, IL-22 is identified as an endogenous brake on helminth-elicited TH2 immunity, reducing the efficacy of expulsion of H. diminuta and limiting the effectiveness of the anti-colitic events mobilized following infection with H. diminuta in a non-permissive host.

Reduced intestinal epithelial mitochondrial function enhances in vitro interleukin-8 production in response to commensal Escherichia coli.

Uncoupling of oxidative phosphorylation in epithelial mitochondria results in decreased epithelial barrier function as characterized by increased internalization of non-invasive Escherichia coli and their translocation across the epithelium. We hypothesized that the increased burden of intracellular commensal bacteria would activate the enterocyte, with the potential to promote inflammation. Treatment of human colon-derived epithelial cell lines in vitro with dinitrophenol (DNP) and commensal E. coli (strains F18, HB101) provoked increased production of interleukin (IL-8), which was not observed with conditioned medium from the bacteria, lipopolysaccharide or inert beads. The IL-8 response was inhibited by co-treatment with cytochalasin-D (blocks F-actin rearrangement), chloroquine (blocks phagosome acidification) and a MyD88 inhibitor (blocks TLR signaling), consistent with TLR-signaling mediating IL-8 synthesis subsequent to bacterial internalization. Use of the mitochondria-targeted antioxidant, mitoTEMPO, or U0126 to block ERK1/2 MAPK signalling inhibited DNP+E. coli-evoked IL-8 production. Mutations in the NOD2 (the intracellular sensor of bacteria) or ATG16L1 (autophagy protein) genes are susceptibility traits for Crohn's, and epithelia lacking either protein displayed enhanced IL-8 production in comparison to wild-type cells when exposed to DNP + E coli. Thus, metabolic stress perturbs the normal epithelial-bacterial interaction resulting in increased IL-8 production due to uptake of bacteria into the enterocyte: this potentially pro-inflammatory event is enhanced in cells lacking NOD2 or ATG16L1 that favor increased survival of bacteria within the enterocyte. We speculate that by increasing epithelial permeability and IL-8 production, reduced mitochondria function in the enteric epithelium would contribute to the initiation, pathophysiology, and reactivation of inflammatory disease in the gut.

Infliximab restores colonic barrier to adherent-invasive E. coli in Crohn's disease via effects on epithelial lipid rafts.

OBJECTIVE: Infliximab is important in the therapeutic arsenal of Crohn's disease (CD). However, its effect on mucosal barrier function is not fully understood. Adherent-invasive Escherichia coli (AIEC) are important in CD pathophysiology, but the transmucosal uptake routes are partly unknown. We investigated effects of infliximab on uptake of colon-specific AIEC HM427 across CD colonic mucosa. MATERIALS AND METHODS: Endoscopic biopsies from non-inflamed colon of seven patients with CD, before and after two infliximab infusions, and eight non-inflammation controls, were mounted in Ussing chambers. Paracellular permeability (51Cr-EDTA) and transmucosal passage of GFP-expressing HM427 were studied. Mechanisms of HM427 transepithelial transport were investigated in Caco-2 monolayers treated with TNF, in the presence of infliximab and/or endocytosis inhibitors. RESULTS: Before infliximab treatment, colonic passage of HM427 [CD: 2475 CFU (450-3000); controls 1163(225-1950)] and 51Cr-EDTA permeability were increased in CD (p < .05), but were restored to control levels by infliximab (CD: 150 (18.8-1069)). In TNF-exposed Caco-2 monolayers HM427 transport and lipid rafts/HM427 co-localization was decreased by infliximab. The lipid raft inhibitor methyl-ß-cyclodextrin decreased HM427 transport. CONCLUSION: Infliximab restored the colonic barrier to AIEC in CD; an effect partially mediated by blocking lipid rafts in epithelial cells. This ability likely contributes to infliximab's clinical efficacy in colonic CD.

Absence of the NOD2 protein renders epithelia more susceptible to barrier dysfunction due to mitochondrial dysfunction.

Irregular mitochondria structure and reduced ATP in some patients with IBD suggest that metabolic stress contributes to disease. Loss-of-function mutation in the nucleotide-binding oligomerization domain (NOD)-2 gene is a major susceptibility trait for IBD. Hence, we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with noninvasive Escherichia coli + DNP (16 h). Increased intracellular bacteria in wild-type (WT) and NOD2 knockdown (KD) cells and colonoids from NOD2-/- mice were mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by cotreatment with the antioxidant mitoTEMPO and the ERK inhibitor U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared with WT cells, there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus, in a two-hit hypothesis, decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently, greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations have compromised macrophage and Paneth cell responses to bacteria.NEW & NOTEWORTHY Increased internalization of bacteria by epithelia with dysfunctional mitochondria (reduced ATP) is potentiated if the cells lack nucleotide-binding oligomerization domain 2 (NOD2), mutations in which are inflammatory bowel disease-susceptibility traits. Uptake of bacteria was dependent on reactive oxygen species and MAP-kinase activity, and the increased viable intracellular bacteria in NOD2-/- cells likely reflect a reduced ability to recognize and kill bacteria. Thus a significant barrier defect occurs with NOD2 deficiency in conjunction with metabolic stress that could contribute to inflammation.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions.NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.

Splenic B cells from Hymenolepis diminuta-infected mice ameliorate colitis independent of T cells and via cooperation with macrophages.

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19(+) B cells isolated from mice infected 7 [HdBc(7(d))] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate-induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7(d)) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7(d)) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-ß than CD19(+) B cells from controls. DNBS-induced colitis in RAG1(-/-) mice was inhibited by administration of HdBc(7(d)), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7(d)). Thus, in response to H. diminuta, a putatively unique splenic CD19(+) B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-ß and the generation of, or cooperation with, a regulatory macrophage.

Treatment with Cestode Parasite Antigens Results in Recruitment of CCR2+ Myeloid Cells, the Adoptive Transfer of Which Ameliorates Colitis.

Awareness of the immunological underpinnings of host-parasite interactions may reveal immune signaling pathways that could be used to treat inflammatory disease in humans. Previously we showed that infection with the rat tapeworm, Hymenolepis diminuta, used as a model helminth, or systemic delivery of worm antigen (HdAg) significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Extending these analyses, intraperitoneal injection of HdAg dose-dependently suppressed dextran sodium sulfate (DSS)-induced colitis, and this was paralleled by reduced gamma interferon (IFN-γ), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-α) production and increased IL-10 production from mitogen-activated splenocytes. Treatment with HdAg resulted in a CCR2-dependent recruitment of CDllb+ F4/80+ Ly6Chi Gr-1lo monocyte-like cells into the peritoneum 24 h later that were predominantly programmed death ligand 1 (PD-L1) positive and CXCR2 negative. In vitro assays indicated that these cells were unable to suppress T cell proliferation but enhanced IL-10 and IL-4 production from activated T cells. Adoptive transfer of the HdAg-recruited monocytic cells into naive mice blocked DSS-induced colitis. These findings add to the variety of means by which treatment with parasitic helminth-derived antigens can ameliorate concomitant disease. A precise understanding of the mechanism(s) of action of HdAg and other helminth-derived antigens (and a parallel consideration of putative side effects) may lead to the development of novel therapies for human idiopathic disorders such as inflammatory bowel disease.

Butyrate enhances antibacterial effects while suppressing other features of alternative activation in IL-4-induced macrophages.

The short-chain fatty acid butyrate is produced by fermentation of dietary fiber by the intestinal microbiota; butyrate is the primary energy source of colonocytes and has immunomodulatory effects. Having shown that macrophages differentiated with IL-4 [M(IL-4)s] can suppress colitis, we hypothesized that butyrate would reinforce an M(IL-4) phenotype. Here, we show that in the presence of butyrate M(IL-4)s display reduced expression of their hallmark markers Arg1 and Ym1 and significantly suppressed LPS-induced nitric oxide, IL-12p40, and IL-10 production. Butyrate treatment likely altered the M(IL-4) phenotype via inhibition of histone deacetylation. Functionally, M(IL-4)s treated with butyrate showed increased phagocytosis and killing of bacteria, compared with M(IL-4) and this was not accompanied by enhanced proinflammatory cytokine production. Culture of regulatory T cells with M(IL-4)s and M(IL-4 + butyrate)s revealed that both macrophage subsets suppressed expression of the regulatory T-cell marker Foxp3. However, Tregs cocultured with M(IL-4 + butyrate) produced less IL-17A than Tregs cocultured with M(IL-4). These data illustrate the importance of butyrate, a microbial-derived metabolite, in the regulation of gut immunity: the demonstration that butyrate promotes phagocytosis in M(IL-4)s that can limit T-cell production of IL-17A reveals novel aspects of bacterial-host interaction in the regulation of intestinal homeostasis.

Cryopreserved Interleukin-4-Treated Macrophages Attenuate Murine Colitis in an Integrin ß7 - Dependent Manner.

The adoptive transfer of alternatively activated macrophages (AAMs) has proven to attenuate inflammation in multiple mouse models of colitis; however, the effect of cryopreservation on AAMs, the ability of previously frozen AAMs to block dinitrobenzene sulfonic acid (DNBS) (Th1) and oxazolone (Th2) colitis and their migration postinjection remains unknown. Here we have found that while cryopreservation reduced mRNA expression of canonical markers of interleukin (IL)-4-treated macrophages [M(IL-4)], this step did not translate to reduced protein or activity, and the cells retained their capacity to drive the suppression of colitis. The anticolitic effect of M(IL-4) adoptive transfer required neither T or B cell nor peritoneal macrophages in the recipient. After injection into the peritoneal cavity, M(IL-4)s migrated to the spleen, mesenteric lymph nodes and colon of DNBS-treated mice. The chemokines CCL2, CCL4 and CX3CL1 were expressed in the colon during the course of DNBS-induced colitis. The expression of integrin ß7 on transferred M(IL-4)s was required for their anticolitic effect, whereas the presence of the chemokine receptors CCR2 and CX3CR1 were dispensable in this model. Collectively, the data show that M(IL-4)s can be cryopreserved M(IL-4)s and subsequently used to suppress colitis in an integrin ß7-dependent manner, and we suggest that these proof-of-concept studies may lead to new cellular therapies for human inflammatory bowel disease.

Adoptive transfer of helminth antigen-pulsed dendritic cells protects against the development of experimental colitis in mice.

Infection with helminth parasites and treatment with worm extracts can suppress inflammatory disease, including colitis. Postulating that dendritic cells (DCs) participated in the suppression of inflammation and seeking to move beyond the use of helminths per se, we tested the ability of Hymenolepis diminuta antigen-pulsed DCs to suppress colitis as a novel cell-based immunotherapy. Bone marrow derived DCs pulsed with H. diminuta antigen (HD-DCs), or PBS-, BSA-, or LPS-DCs as controls, were transferred into wild-type (WT), interleukin-10 (IL-10) knock-out (KO), and RAG-1 KO mice, and the impact on dinitrobenzene sulphonic acid (DNBS)-induced colitis and splenic cytokine production assessed 72 h later. Mice receiving HD-DCs were significantly protected from DNBS-induced colitis and of the experimental groups only these mice displayed increased Th2 cytokines and IL-10 production. Adoptive transfer of HD-DCs protected neither RAG-1 nor IL-10 KO mice from DNBS-colitis. Furthermore, the transfer of CD4(+) splenocytes from recipients of HD-DCs protected naïve mice against DNBS-colitis, in an IL-10 dependent manner. Thus, HD-DCs are a novel anti-colitic immunotherapy that can educate anti-colitic CD4(+) T cells: mechanistically, the anti-colitic effect of HD-DCs requires that the host has an adaptive immune response and the ability to mobilize IL-10.

Targeting mitochondria-derived reactive oxygen species to reduce epithelial barrier dysfunction and colitis.

Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coli with or without mitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)-induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn's disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. In vitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.