Colonic Dendritic Cells Research Articles

Exosomes derived from cancer cells relieve inflammatory bowel disease in mice

Exosome therapy has garnered significant attention due to its natural delivery capabilities, low toxicity, high biocompatibility, and potential for personalised treatment through engineering modifications. Recent studies have highlighted the ability of tumour cell-derived exosomes (TDEs) to interact with immune cells or modify the immune microenvironment to suppress host immune responses, as well as their unique homing ability to parental cells. The core question of this study is whether this immunomodulatory property of TDEs can be utilised for the immunotherapy of inflammatory diseases. In our experiments, we prepared exosomes derived from murine colon cancer cells CT26 (CT26 exo) using ultracentrifugation, characterised them, and conducted proteomic analysis. The therapeutic potential of CT26 exo was evaluated in our dextran sulphate sodium salt (DSS)-induced inflammatory bowel disease (IBD) mouse model. Compared to the control and 293 T exo treatment groups, mice treated with CT26 exo showed a reduction in the disease activity index (DAI) and colon shortening rate, with no noticeable weight loss. Haematoxylin and eosin (H&E) staining of colon paraffin sections revealed reduced inflammatory infiltration and increased epithelial goblet cells in the colons of CT26 exo-treated group. Furthermore, we conducted preliminary mechanistic explorations by examining the phenotyping and function of CD4+ T cells and dendritic cells (DCs) in the colonic lamina propria of mice. The results indicated that the ameliorative effect of CT26 exosomes might be due to their inhibition of pro-inflammatory cytokine secretion by colonic DCs and selective suppression of Th17 cell differentiation in the colon. Additionally, CT26 exo exhibited good biosafety. Our findings propose a novel exosome-based therapeutic approach for IBD and suggest the potential application of TDEs in the treatment of inflammatory diseases.

TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

Successful treatment of chronic inflammatory diseases integrates both the cessation of inflammation and the induction of adequate tissue repair processes. Strikingly, targeting a single proinflammatory cytokine, tumor necrosis factor (TNF), induces both processes in a relevant cohort of inflammatory bowel disease (IBD) patients. However, the molecular mechanisms underlying intestinal repair following TNF blockade during IBD remain elusive. Using a novel humanized model of experimental colitis, we demonstrate that TNF interfered with the tissue repair program via induction of a soluble natural antagonist of IL-22 (IL-22Ra2; IL-22BP) in the colon and abrogated IL-22/STAT3-mediated mucosal repair during colitis. Furthermore, membrane-bound TNF expressed by T cells perpetuated colonic inflammation, while soluble TNF produced by epithelial cells (IECs) induced IL-22BP expression in colonic dendritic cells (DCs) and dampened IL-22-driven restitution of colonic epithelial functions. Finally, TNF induced IL-22BP expression in human monocyte-derived DCs and levels of IL22-BP correlated with TNF in sera of IBD patients. Thus, our data can explain how anti-TNF therapy induces mucosal healing by increasing IL-22 availability and implicates new therapeutic opportunities for IBD.

Gut Bacteria Induce Granzyme B Expression in Human Colonic ILC3s In Vitro in an IL-15-Dependent Manner.

Group 3 innate lymphoid cells (ILC3s) in the gut mucosa have long been thought to be noncytotoxic lymphocytes that are critical for homeostasis of intestinal epithelial cells through secretion of IL-22. Recent work using human tonsillar cells demonstrated that ILC3s exposed to exogenous inflammatory cytokines for a long period of time acquired expression of granzyme B, suggesting that under pathological conditions ILC3s may become cytotoxic. We hypothesized that inflammation associated with bacterial exposure might trigger granzyme B expression in gut ILC3s. To test this, we exposed human colon lamina propria mononuclear cells to a panel of enteric bacteria. We found that the Gram-negative commensal and pathogenic bacteria induced granzyme B expression in a subset of ILC3s that were distinct from IL-22-producing ILC3s. A fraction of granzyme B+ ILC3s coexpressed the cytolytic protein perforin. Granzyme B expression was mediated, in part, by IL-15 produced upon exposure to bacteria. ILC3s coexpressing all three IL-15R subunits (IL15Rα/β/γ) increased following bacterial stimulation, potentially allowing for cis presentation of IL-15 during bacterial exposure. Additionally, a large frequency of colonic myeloid dendritic cells expressed IL-15Rα, implicating myeloid dendritic cells in trans presentation of IL-15 to ILC3s. Tonsillar ILC3s minimally expressed granzyme B when exposed to the same bacteria or to rIL-15. Overall, these data establish the novel, to our knowledge, finding that human colonic ILC3s can express granzyme B in response to a subset of enteric bacteria through a process mediated by IL-15. These observations raise new questions about the multifunctional role of human gut ILC3s.

ATG16L1 negatively regulates RICK/RIP2-mediated innate immune responses.

Polymorphisms in the autophagy-related protein 16 like 1 (ATG16L1) and nucleotide-binding oligomerization domain 2 (NOD2) genes are associated with Crohn's disease (CD). Impaired interaction between ATG16L1 and NOD2 underlies CD immunopathogenesis. Although activation of the receptor-interacting serine-threonine kinase (RICK, also known as RIP2), a downstream signaling molecule for NOD2 and multiple toll-like receptors (TLRs), plays a pathogenic role in the development of inflammatory bowel disease, the molecular interaction between ATG16L1 and RICK/RIP2 remains poorly understood. In this study, we examined the physical interaction between ATG16L1 and RICK/RIP2 in human embryonic kidney 293 cells and human monocyte-derived dendritic cells (DCs) expressing excessive and endogenous levels of these proteins, respectively. We established that ATG16L1 binds to RICK/RIP2 kinase domain and negatively regulates TLR2-mediated nuclear factor-kappa B (NF-κB) activation and pro-inflammatory cytokine responses by inhibiting the interaction between TLR2 and RICK/RIP2. Binding of ATG16L1 to RICK/RIP2 suppressed NF-κB activation by down-regulating RICK/RIP2 polyubiquitination. Notably, the percentage of colonic DCs expressing ATG16L1 inversely correlated with IL-6 and TNF-α expression levels in the colon of CD patients. These data suggest that the interaction between ATG16L1 and RICK/RIP2 maintains intestinal homeostasis via the down-regulation of TLR-mediated pro-inflammatory cytokine responses.

Bacteroides thetaiotaomicron-derived outer membrane vesicles promote regulatory dendritic cell responses in health but not in inflammatory bowel disease

BackgroundBacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here, we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn’s disease (CD) or ulcerative colitis (UC).ResultsIn healthy individuals, Bt OMVs stimulated significant (p < 0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p < 0.001 and p < 0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103+ DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p < 0.01 and p < 0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD).ConclusionsOverall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients.A3C6cuutcZR_V1iQMS6TL7Video

Commensal microbiota drive the functional diversification of colon macrophages

Mononuclear phagocytes are a heterogeneous population of leukocytes essential for immune homeostasis that develop tissue-specific functions due to unique transcriptional programs driven by local microenvironmental cues. Single cell RNA sequencing (scRNA-seq) of colonic myeloid cells from specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice revealed extensive heterogeneity of both colon macrophages (MPs) and dendritic cells (DCs). Modeling of developmental pathways combined with inference of gene regulatory networks indicate two major trajectories from common CCR2+ precursors resulting in colon MP populations with unique transcription factors and downstream target genes. Compared to SPF mice, GF mice had decreased numbers of total colon MPs, as well as selective proportional decreases of two major CD11c+CD206intCD121b+ and CD11c−CD206hiCD121b− colon MP populations, whereas DC numbers and proportions were not different. Importantly, these two major colon MP populations were clearly distinct from other colon MP populations regarding their gene expression profile, localization within the lamina propria (LP) and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations at the molecular level and highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.

Regulating colonic dendritic cells by commensal glycosylated large surface layer protein A to sustain gut homeostasis against pathogenic inflammation.

Microbial interaction with the host through sensing receptors, including SIGNR1, sustains intestinal homeostasis against pathogenic inflammation. The newly discovered commensal Propionibacterium strain, P. UF1, regulates the intestinal immunity against pathogen challenge. However, the molecular events driving intestinal phagocytic cell response, including colonic dendritic cells (DCs), by this bacterium are still elusive. Here, we demonstrate that the glycosylation of bacterial large surface layer protein A (LspA) by protein O-mannosyltransferase 1 (Pmt1) regulates the interaction with SIGNR1, resulting in the control of DC transcriptomic and metabolomic machineries. Programmed DCs promote protective T cell response to intestinal Listeria infection and resist chemically induced colitis in mice. Thus, our findings may highlight a novel molecular mechanism by which commensal surface glycosylation interacting with SIGNR1 directs the intestinal homeostasis to potentially protect the host against proinflammatory signals inducing colonic tissue damage.

P054 CD103+SIRPα+ DC are specifically decreased in the inflamed colon from patients with ulcerative colitis but not with Crohn’s disease

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the human gastrointestinal (GI) tract. Intestinal dendritic cells (DC) are essential to maintain the balance between immunity against pathogens and tolerance towards nutrients and commensals. However, there is not much information about DC composition in the human GI tract both in health and IBD. Human GI biopsies were obtained from healthy controls and IBD patients (including UC and CD; both active and quiescent). Tissue was disaggregated and lamina propria mononuclear cells (LPMC) characterised by flow cytometry. Human intestinal DC were identified within singlet viable leucocytes as CD14-CD64-HLA-DR+CD11c+. Type 1 DC were defined as CD103+SIRPα- while type 2 DC were identified as SIRPα+ and further divided into subsets based on the expression of CD103. The proportion of total DC displayed a gradient throughout the healthy human gut as it was higher in the colon (either distal or proximal) compared with the ileum. DC proportion was further decreased in the duodenum. Type 1 (minority) and type 2 (majority) conventional DC did not change their proportion throughout the healthy gut. However, CD103+SIRPα+ DC were the main subset in the duodenum as opposed to CD103-SIRPα+ DC which were predominant in the colon and the ileum. Compared with their CD103-SIRPα+ type 2 counterparts, CD103+SIRPα+ had higher levels of HLA-DR, CD40, CD86, CCR7, CD137L, ICOSL and PD-L1. CD103+SIRPα+ were also more phagocytic and had lower expression of blood-related markers like CLA and CCR2, suggesting that they are derived from CD103-SIRPα+ DC following mucosal conditioning. Indeed, CD103+SIRPα+ numbers were increased following LPMC culture, although this process was reverted in the presence of pro-inflammatory LPS. CD103+SIRPα+ DC displayed an enhanced production of IL-10, both in resting conditions and in the presence of LPS. In IBD, type 2 DC constitutively displayed lower expression of SIRPα irrespectively of IBD-type (CD or UC) or condition (active or quiescent). Nevertheless, the inflamed colon from UC patients, but not from CD, specifically displayed lower numbers of tolerogenic CD103+SIRPα+ DC. These results were in agreement with the colonic cytokine milieu, which was much more pro-inflammatory in UC patients compared with CD. Tolerogenic PD-L1 expression and IL-10 production was associated with CD103+SIRPα+ DC, confirming therefore their tolerogenic phenotype. Human colonic DC from IBD patients constitutively display lower levels of SIRPα. The specific reduction of CD103+SIRPα+ DC in the inflamed mucosa from UC, but not CD, suggests the presence of different pathogenic mechanisms occurring in IBD.

Protein kinase p38α signaling in dendritic cells regulates colon inflammation and tumorigenesis

Dendritic cells (DCs) play pivotal roles in maintaining intestinal homeostasis, but how the DCs regulate diverse immune networks on homeostasis breakdown remains largely unknown. Here, we report that, in response to epithelial barrier disruption, colonic DCs regulate the differentiation of type 1 regulatory T (Tr1) cells through p38α-dependent IL-27 production to initiate an effective immune response. Deletion of p38α in DCs, but not in T cells, led to increased Tr1 and protected mice from dextran sodium sulfate-induced acute colitis and chronic colitis-associated colorectal cancer. We show that higher levels of IL-27 in p38α-deficient colonic cDC1s, but not cDC2s, were responsible for the increase of Tr1 cells. Moreover, p38α-dependent IL-27 enhanced IL-22 secretion from intestinal group 3 innate lymphoid cells and protected epithelial barrier function. In p38α-deficient DCs, the TAK1-MKK4/7-JNK-c-Jun axis was hyperactivated, leading to high IL-27 levels, and inhibition of the JNK-c-Jun axis suppressed IL-27 expression. ChIP assay revealed direct binding of c-Jun to the promoter of Il27p28, which was further enhanced in p38α-deficient DCs. In summary, here we identify a key role for p38α signaling in DCs in regulating intestinal inflammatory response and tumorigenesis, and our finding may provide targets for the treatment of inflammatory intestinal diseases.

Intestinal Microbiota and Immunity: A Review

Microorganisms establish a symbiotic relationship at all stages of growth in man, beginning from birth to adulthood. They are found in every part of the body, and they participate in the immune system against pathogen-mediated immune responses. Their actions are elicited by secreting microorganism associated-molecular proteins (MAMPs) which they use as signalling molecules to activate a cascade of immunological responses within the host cell. They maintain the barrier function of the intestinal wall as well as prevent colonisation of the intestine by pathogens. Using their MAMPs, they bind to specific pattern recognition receptors (PRR) activating immune mediators in response to pro-inflammation by pathogens. These immune mediators are either induced or suppressed (in the case of overproduced immune response). Some of these symbionts elicit their action by anaerobic fermentation of dietary fibres into byproducts of short-chain fatty acids (SCFAs). These bacterial metabolites functions by binding to G-protein coupled reactions (GPCRs) on colonic macrophages and Dendritic cells (DCs) and contributed to the increased production of interleukin 10 (IL 10) in response to pathogen-mediated immune response. These unique immunological actions of intestinal microbiota, have shown that microorganisms are beneficial to the host as against the widespread belief that they are disease-causing agents.

Gpr109a limits microbiota-induced IL-23 production to constrain ILC3-mediated colonic inflammation and carcinogenesis.

Abstract A set of coordinated interactions between gut microbiota and the immune cells surveilling the intestine play a key role in shaping local immune responses and intestinal health. There are several immunoregulatory mechanisms in place that prevent the needless activation of immune cells against harmless gut bacteria. The dysregulation of interleukin-23 (IL-23) plays a critical role in the induction of several inflammatory diseases including inflammatory bowel disease (IBD). However, the molecular mechanisms regulating IL-23 production are poorly characterized. Gpr109a is a G-protein coupled receptor that is expressed at a very high level on innate immune cells and has been previously shown to play a key role in the induction of colonic Tregs. We show that Gpr109a−/−Rag1−/− mice exhibit spontaneous rectal prolapse and colonic inflammation, as characterized by the presence of an elevated number of IL-17-producing Rorγt+ innate lymphoid cells (ILC3). The genetic deletion of Rorγt ameliorated the spontaneous colonic inflammation in Gpr109a−/−Rag1−/− mice. Gpr109a-deficient colonic dendritic cells promote ILC3 by producing higher amounts of IL-23. Antibiotics treatment to deplete gut microbiota decreased IL-23 production, ILC3, and colonic inflammation in Gpr109a−/−Rag1−/− mice. Administration of niacin, a Gpr109a agonist, suppressed both IL-23 production by colonic DCs and ILC3 count in a Gpr109a-dependent manner. Collectively, our data presents a model suggesting that targeting Gpr109a will be potentially beneficial in the suppression of IL-23 mediated immunopathologies.

Gpr109a Limits Microbiota-Induced IL-23 Production To Constrain ILC3-Mediated Colonic Inflammation.

A set of coordinated interactions between gut microbiota and the immune cells surveilling the intestine play a key role in shaping local immune responses and intestinal health. Gpr109a is a G protein-coupled receptor expressed at a very high level on innate immune cells and previously shown to play a key role in the induction of colonic regulatory T cells. In this study, we show that Gpr109a-/-Rag1-/- mice exhibit spontaneous rectal prolapse and colonic inflammation, characterized by the presence of an elevated number of IL-17-producing Rorγt+ innate lymphoid cells (ILCs; ILC3). Genetic deletion of Rorγt alleviated the spontaneous colonic inflammation in Gpr109a-/-Rag1-/- mice. Gpr109a-deficient colonic dendritic cells produce higher amounts of IL-23 and thereby promote ILC3. Moreover, the depletion of gut microbiota by antibiotics treatment decreased IL-23 production, ILC3, and colonic inflammation in Gpr109a-/-Rag1-/- mice. The ceca of Gpr109a-/-Rag1-/- mice showed significantly increased colonization by members of Bacteroidaceae, Porphyromonadaceae, Prevotellaceae, Streptococcaceae, Christensenellaceae, and Mogibacteriaceae, as well as IBD-associated microbiota such as Enterobacteriaceae and Mycoplasmataceae, compared with Rag1-/- mice, housed in a facility positive for Helicobacter and murine norovirus. Niacin, a Gpr109a agonist, suppressed both IL-23 production by colonic DCs and ILC3 number in a Gpr109a-dependent manner. Collectively, our data present a model suggesting that targeting Gpr109a will be potentially beneficial in the suppression of IL-23-mediated immunopathologies.

GPR81, a Cell-Surface Receptor for Lactate, Regulates Intestinal Homeostasis and Protects Mice from Experimental Colitis.

At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation.

Brief Report: Inflammatory Colonic Innate Lymphoid Cells Are Increased During Untreated HIV-1 Infection and Associated With Markers of Gut Dysbiosis and Mucosal Immune Activation.

HIV-1 infection is associated with intestinal inflammation, changes in the enteric microbiota (dysbiosis), and intestinal epithelial cell damage. NKp44 innate lymphoid cells (ILCs) play an important role in epithelial barrier maintenance through the production of interleukin (IL)-22 but also display functional plasticity and can produce inflammatory cytokines [eg, interferon gamma (IFNγ)] in response to cytokine milieu and stimulatory signals. The objective of this pilot study was to enumerate frequencies of IL-22 and IFNγ-expressing colonic NKp44 ILCs during untreated, chronic HIV-1 infection. A cross-sectional study was performed to compare numbers of cytokine-expressing ILCs in colonic biopsies of untreated, chronic HIV-1 infected (n = 22), and uninfected (n = 10) study participants. Associations between cytokine ILC and previously established measures of virological, immunological, and microbiome indices were analyzed. Multicolor flow cytometry was used to measure the absolute number of colonic CD3NKp44CD56 ILCs expressing IL-22 or IFNγ after in vitro mitogenic stimulation. Numbers of colonic NKp44 ILCs that expressed IFNγ were significantly higher in HIV-1 infected versus uninfected persons and positively correlated with relative abundances of dysbiotic bacterial species in the Xanthomonadaceae and Prevotellaceae bacterial families and with colonic myeloid dendritic cell and T-cell activation. Higher numbers of inflammatory colonic ILCs during untreated chronic HIV-1 infection that associated with dysbiosis and colonic myeloid dendritic cell and T-cell activation suggest that inflammatory ILCs may contribute to gut mucosal inflammation and epithelial barrier breakdown, important features of HIV-1 mucosal pathogenesis.

Type I interferon signaling restrains IL-10R+ colonic macrophages and dendritic cells and leads to more severe Salmonella colitis.

Type I interferons (IFNα, IFNβ) are key regulators of innate and adaptive immunity, modulating the severity of both viral and bacterial infections. While type I IFN signaling leads to improved outcomes in viral infections, its role in bacterial infections is more contextual and depends on the specific pathogen and route of infection. Given the limited evidence on whether type I IFN signaling affects enteric bacterial pathogens, we investigated the role of this signaling pathway in Salmonella enterica serovar Typhimurium (S. typhimurium)–induced colitis. Comparing mice deficient in IFNAR1- the common receptor for IFNα and IFNβ- with wild-type mice, we found that type I IFN signaling leads to more rapid death, more severe colonic inflammation, higher serum levels of pro-inflammatory cytokines, and greater bacterial dissemination. Specific ablation of plasmacytoid dendritic cells (pDCs), which are prominent producers of type I IFNs in antiviral responses, did not alter survival after infection. This result established that pDCs do not play a major role in the pathogenesis of S. typhimurium colitis. Flow cytometric analysis of macrophages and conventional dendritic cells (cDCs) during active colitis demonstrated an increase in CD11c- macrophages and CD103+ cDCs in the colon of Ifnar1-/- animals. Interestingly, cells expressing the anti-inflammatory cytokine receptor IL-10R are more abundant within these subsets in Ifnar1-/- than in wild-type mice. Moreover, blockade of IL-10R in Ifnar1-/- mice increased their susceptibility to S. typhimurium colitis, suggesting that altered numbers of these immunoregulatory cells may underlie the difference in disease severity. This cross-talk between type I IFN and IL-10R signaling pathways may represent a key host cellular mechanism to investigate further in order to unravel the balance between pathogenic inflammation and homeostasis of the colon. Taken together, our data clearly demonstrate that type I IFN signaling is pathogenic in S. typhimurium colitis.

Mucosal biopsy shows immunologic changes of the colon in patients with early MS.

Objective:To investigate immune cells of the colonic mucosa and fecal short-chain fatty acids (SCFAs) in treatment-naive patients with a clinically isolated syndrome (CIS) or early relapsing MS.Methods:In this cross-sectional proof-of-concept study, we obtained mucosal specimens during ileocolonoscopy from 15 untreated patients with CIS/MS and 10 controls. Mucosal immune cells were analyzed by FACS, and gas chromatography-mass spectrometry measurements of stool samples served to determine SCFA.Results:The number of total dendritic cells (DCs), CD103+ tolerogenic DCs, and CD4+25+127–regulatory T cells (Tregs) was significantly reduced in the distal colon of patients with CIS/MS compared with controls, whereas we found no differences in the proximal colon. The patients' fecal samples also showed a substantially lower content of SCFA and especially lower levels of butyrate and acetate.Conclusions:Our findings indicate a disturbed homeostasis of colonic DCs and Tregs in patients with MS which could be associated with colonic SCFA depletion. Although not implying causality, these findings confirm parallel abnormalities of the gut in MS and warrant further research if modulation of the colonic SCFA profile or the colonic Treg pool can serve to modify the course of MS.

P107 Lactobacillus plantarum 06CC2 induces intestinal IL-10–producing dendritic cells to ameliorate acute experimental colitis in mice

Background: Lactobacillus plantarum (LP) 06CC2, isolated from Mongolian traditional food, affects the systemic immune activity of NK cells. Here, we investigated the beneficial anti-colitis effect of LP 06CC2 in dextran sulphate sodium (DSS)-induced colitis in C57BL/6 mice. Methods: Mice treated with 1% DSS were administered PBS only (control group) or heat-killed lyophilised LP 06CC2 (LP group) by oral gavage for 19 consecutive days. We assessed the severity of colitis using a disease activity index, measured colon length and weight, and examined colon tissue macroscopically and histopathologically. We also collected colonic lamina propria mononuclear cells (LPMCs), in which we monitored expression of genes encoding inflammatory cytokines (IFN-γ, IL-6, IL-12, TNF-α, and IL-10). To further characterise the involvement of IL-10 in the effects of LP06CC2, we continuously administered neutralising anti–IL-10 monoclonal antibody to mice with DSS-induced colitis, with or without LP 06CC2 feeding. Results: Reduction in body weight, disease activity index, and pathology score were all significantly lower in the LP group than in the control group (p<0.05). The LP strain prevented shortening of the large intestine: average colon length was significantly shorter in the LP group than in the control group (p<0.01). IL-10 expression in CD11c-positive cells in colonic LPMCs was significantly higher in the LP group than in the control group, although there was no significant difference in expression of IFN-γ or IL-12 between the groups. After administration of anti–IL-10 neutralising antibody, the reduction in body weight in the LP group was the same as that in the control group. Thus, the positive effects of LP, i.e., prevention of body weight loss following administration of DSS, were abolished by neutralisation of IL-10. Conclusions: LP 06CC2 attenuated colon inflammation by inducing IL-10 production in colonic dendritic cells.

The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2 –/–mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2 –/–mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

Effects of gene silence on immune function of colonic dendritic cells in rats with irritable bowel syndrome visceral hypersensitivity

Objective To explore the role and mechanism of protein disulfide isomerase A3 (PDIA3) in abnormal colonic mucosa immune response of irritable bowel syndrome (IBS) rats with visceral hypersensitivity. Methods A total of 48 SD rats were divided into blank control group (n=12), empty virus group (IBS-1) (n=12), PDIA3 silencing group (IBS-2) (n=12) and model control group(IBS-3) (n=12). The expression of CD103 in ileocecus colonic tissues was detected by immunohistochemistry. Dendritic cells (DC) of mesenteric lymph node were isolated by flow cytometry. CD4+ /CD8+ T cells of spleen were separated by immune-magnetic beads sorting technique. DC and CD4+ /CD8+ T cells were co-cultured. The proliferation ability of lymphocytes promoted by DC was measured by methyl thiazolyl tetrazolium (MTT). The secretion levels of interlcukin (IL)-4 and IL-9 of CD4+ /CD8+ T cells stimulated by DC were determined by enzyme linked immunosorbent assay (ELISA) method. Independent sample t-test was performed for statistical analysis. Results The cell number of CD103 positive DC of rats colon of blank control group, IBS-3 group was 6.25±1.14 and 10.83±1.03(t=10.07, P<0.05); that of IBS-2 group was 7.42±0.90, and compared with that of IBS-3 group, the difference was statistically significant (t=-9.25, P<0.05). The MTT value of CD4+ T cells proliferation stimulated by DC of blank control group and IBS-3 group was 0.54±0.01 and 0.60±0.01(t=3.373, P<0.05); that of IBS-2 group was 0.53±0.01, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.139, P<0.05). The MTT value of CD8+ T cells proliferation stimulated by DC was 0.52±0.01 and 0.59±0.00(t=3.539, P<0.01); that of IBS-2 group was 0.54±0.01, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.183, P<0.05). The level of IL-4 secreted by CD4+ T cells promoted by DC of blank control group and IBS-3 group was 10.24±0.09 and 16.61±1.00 (t=3.222, P<0.05); that of IBS-2 group was 11.75±0.54, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.539, P<0.01). The level of IL-9 secreted by CD4+ T cells stimulated by DC of blank control group and IBS-3 group was 15.86±10.19 and 43.51±11.32 (t=4.529, P<0.05); that of IBS-2 group was 29.05±2.09, and compared with that of IBS-3 group, the difference was statistically significant (t=-6.841, P<0.01). The level of IL-4 secreted by CD8+ T cells promoted by DC of blank control group and IBS-3 group was 7.35±0.12 and 13.91±0.57 (t=19.557, P<0.01); that of IBS-2 group was 8.63±0.24, and compared with that of IBS-3 group, the difference was statistically significant (t=-14.782, P<0.01). The level of IL-9 secreted by CD8+ T cells stimulated by DC of blank control group and IBS-3 group was 29.12±5.14 and 60.70±11.02 (t=4.122, P<0.05); that of IBS-2 group was 37.17±2.65, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.255, P<0.05). Conclusions Protein disulfide isomerase A3 may mediate the process of DC activating T lymphocyte levels of related cytokines, cause abnormal immune response of colonic mucosa and promote the genesis of IBS visceral hypersensitivity. Key words: Protein disulfide isomrase A3; Dendritic cells; Interleukin-4; Interleukin-9; CD4-positive T-lymphocytes; CD8-positive T-lymphocytes; Visceral hypersensitivity

Corticotropin-releasing factor augments LPS-induced immune/inflammatory responses in JAWSII cells.

The effect of corticotropin-releasing factor (CRF) on gastrointestinal tract inflammation via modulation of the immune system cells such as dendritic cells (DCs) is not known. We investigated the expression of CRF and its receptors CRFR1 and CRFR2 in a colonic DC model (JAWSII cells) in a pro-inflammatory and anti-inflammatory milieu. The mRNA expression of CRF and protein expression of CRFR1 and CRFR2 was assessed, and lipopolysaccharide (LPS) was used to induce the maturation of JAWSII cells. JAWSII cells were divided into four groups: control, CRF, LPS, and LPS + CRF. The levels of secreted cytokines IL-6, IL-4, TNF-α, and MIP-1α were determined, both in JAWSII cells and in the culture supernatant, by qRT-PCR and ELISA. The expression of CRFR1 and CRFR2 in JAWSII cells was accompanied by a low CRF expression. Compared with control group, CRF group did not affect the expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and MIP-1α, but LPS treatment significantly increased the expression of these cytokines, indicating maturation of JAWSII cells. CRF further augmented the production of IL-6, TNF-α, and MIP-1α in mature JAWSII cells, with no increase in TNF-α mRNA expression. However, the expression of anti-inflammatory factor IL-4 did not change after LPS treatment. CRF treatment decreased the expression of IL-4 in both mature and immature JAWSII cells. JAWSII cells produce low level of CRF and express CRFR1 and CRFR2 surface receptors. CRF promotes immune/inflammatory responses in mature JAWSII cells when induced by LPS treatment.