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Age-associated B cells (ABC) accumulate with age and are associated with autoimmunity and chronic infection. However, their contributions to acute infection in the aged and their developmental pathways are unclear. We find that the response against influenza A virus infection in aged mice is dominated by a Fas

Age-associated B cells (ABC) accumulate with age and are associated with autoimmunity and chronic infection. However, their contributions to acute infection in the aged and their developmental pathways are unclear. We find that the response against influenza A virus infection in aged mice is dominated by a Fas+GL7- effector B cell population we call infection-induced-ABC (iABC). Most iABC express IgM and include antibody-secreting cells in the spleen, lung and bone marrow. We find that in response to influenza, IgD+CD21-CD23-ABC are the precursors of iABC. These IgD+ABC develop in germ free mice, so are independent of foreign antigen recognition, suggesting they arise from an intrinsic age-associated developmental program. The response of ABC to influenza infection, resulting in iABC, is T cell independent and requires both extrinsic TLR7 and TLR9 signals. In response to influenza infection, IgD+ABC can induce a faster recovery of weight and higher total anti-influenza IgG and IgM titers that can neutralize virus. Immunization with whole inactivated virus also generates iABC in aged mice. Thus, in unimmunized aged mice, whose other B and T cell responses have waned, IgD+ABC are likely the naïve B cells with the potential to become Ab-secreting cells and to provide protection from infection in the aged.

As mice age their adaptive immune system changes dramatically, leading to weakened responses to newly encountered antigens and poor efficacy of vaccines. A shared pattern emerges in the aged, with both CD4 T and B cell responses requiring higher levels of pathogen recognition. Moreover, in aged germ-free mice we find accumulation of the same novel age-associated T and B cell subsets that we and others have previously identified using mice maintained in normal laboratory animal housing conditions, suggesting that their development follows an intrinsic program.

BACKGROUND: Adherent and invasive Escherichia coli (AIEC) is preferentially associated with ileal Crohn’s disease (CD). The role of AIEC in the development of inflammation and its regional tropism is unresolved. The presence of long polar fimbriae (LPF) in 71% of ileal CD AIEC suggests a role for LPF in the tropism and virulence of AIEC. The aim of our study is to determine if AIEC, with or without LpfA, induces intestinal inflammation in monoassociated IL-10(-/-) mice. METHODS: We compared murine AIEC strains NC101 (phylogroup B2, LpfA-) and CUMT8 (phylogroup B1, LpfA+), and isogenic mutant CUMT8 lacking lpfA(154), with a non-AIEC (E. coli K12), evaluating histologic inflammation, bacterial colonization, mucosal adherence and invasion, and immune activation. RESULTS: IL-10(-/-) mice monoassociated with AIEC (either CUMT8, CUMT8:ΔlpfA, or NC101) but not K12 developed diffuse small intestinal and colonic inflammation. There was no difference in the magnitude and distribution of inflammation in mice colonized with CUMT8:ΔlpfA compared with wild-type CUMT8. Bacterial colonization was similar for all E. coli strains. Fluorescence in situ hybridization revealed mucosal adherence and tissue invasion by AIEC but not K12. Production of the cytokines IL-12/23 p40 by the intestinal tissue and IFN-γ and IL-17 by CD4 T cells correlated with inflammation. CONCLUSIONS: IL-10(-/-) mice monoassociated with murine AIEC irrespective of LpfA expression developed chronic inflammation accompanied by IL-12/23 p40 production in the small and large intestines and IFN-γ/IL-17 production by CD4 T cells that model the interplay between enteric pathosymbionts, host susceptibility, and enhanced immune responses in people with IBD.

Interleukin-10 (IL-10) is a key regulator of mucosal homeostasis. In the current study we investigated the early events after monoassociating germ-free (GF) wild type (WT) mice with an E. coli strain that we isolated previously from the cecal contents of a normal mouse housed under specific pathogen free (SPF) conditions. Our results show that IFN-γ secreted by mesenteric lymph node (MLN) cells from both IL-10 deficient mice and WT mice, stimulated ex vivo with E. coli lysate, was dramatically higher at day 4 after monoassociation compared to IFN-γ secreted by cells from GF mice without E. coli colonization. Production of IFN-γ rapidly and progressively declined after colonization of WT but not IL-10 deficient mice. E. coli lysate-stimulated WT MLN cells also produced IL-10 that peaked at day 4 and subsequently declined, but not as precipitously as IFN-γ. WT cells that express CD4, CD8, and NKp46 produced IFN-γ; WT CD4-positive cells and B cells produced IL-10. Recombinant IL-10 added to E. coli-stimulated MLN cell cultures inhibited IFN-γ secretion in a dose-dependent fashion. MLN cells from WT mice treated in vivo with neutralizing anti-IL-10 receptor antibody produced more IFN-γ compared with MLN cells from isotype control antibody-treated mice. These findings show that a resident E. coli that induces chronic colitis in monoassociated IL-10 deficient mice rapidly but transiently activates the effector immune system in normal hosts, in parallel with induction of protective IL-10 produced by B cells and CD4(+) cells that subsequently suppresses this response to mediate mucosal homeostasis. This article is protected by copyright. All rights reserved.

Background & Aims Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis , in the development of colitis in mice. Methods Germ-free, interleukin-10–deficient (IL-10 −/− ) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10 −/− and TNF ΔARE/Wt mice. GelE specificity was shown with the MMP inhibitor marimastat. Results Histologic analysis (score 0–4) of E faecalis monoassociated IL-10 −/− mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10 −/− and TNF ΔARE/Wt mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohn's disease and ulcerative colitis. Conclusions The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10 −/− and TNF ΔARE/Wt mice) by impairing epithelial barrier integrity.

Background: We have reported that commensal luminal bacterial components induce an active in vitro IFN-γ response in mesenteric lymph node (MLN) and intestinal cells from specific pathogen-free (SPF) HLA-B27 transgenic (TG) rats with chronic colitis but not in cells from non-diseased SPF non-TG, germ-free (GF) non-TG or GF TG rats. Methods: The study examined IL-12 stimulation of MLN IFN-γ responses to luminal bacteria and regulation of these responses by suppressive cytokines. Results: Exogenous IL-12 significantly increased the bacterial lysate-induced IFN-γ response in SPF TG MLN cells, while bacterial lysate and IL-12 synergistically induced IFN-γ from low baseline levels in cells obtained from both SPF and GF non-TG rats, and in GF TG cells. TGF-β fully counteracted the effects of IL-12 and bacterial lysate on non-TG cells by almost completely inhibiting IFN-γ production. In contrast, TG cells were less responsive to TGF-β-mediated downregulation with a substantial residual IFN-γ response to IL-12 plus bacterial lysate. Further experiments showed that CD4+/CD25+ cells had no inhibitory effect on the IFN-γ production and were not required for TGF-β-mediated suppression. Addition of exogenous IL-10 also partially inhibited IFN-γ production by non-TG cells but did not affect TG cells. Conversely, exogenous IL-12 preferentially suppressed bacterial lysate-induced TGF-β and IL-10 production in TG rat cells. Conclusions: An attenuated response to regulatory signals leads to uncontrolled potentiated induction of effector IFN-γ responses to commensal bacteria in HLA-B27 TG rats that spontaneously develop chronic intestinal inflammation. (Inflamm Bowel Dis 2008)

The intestinal epithelium of the adult gut supports a complex, dynamic microbial ecosystem and expresses highly fucosylated glycans on its surface. Uncolonized gut contains little fucosylated glycan. The transition toward adult colonization, such as during recovery from germ-free status or from antibiotic treatment, increased expression of fucosylated epitopes in the colonic epithelium. This increase in fucosylation is accompanied by induction of fut2 mRNA expression and α1,2/3-fucosyltransferase activity. Colonization stimulates ERK and JNK signal transduction pathways, resulting in activation of transcription factors ATF2 and c-Jun, respectively. This increases transcription of fut2 mRNA and expression of α1,2/3-fucosyltransferase activity, resulting in a highly fucosylated intestinal mucosa characteristic of the adult mammalian gut. Blocking the ERK and JNK signaling cascade inhibits the ability of colonization to induce elevated fut2 mRNA and fucosyltransferase activity in the mature colon. Thus pioneer-mutualist symbiotic bacteria may utilize the ERK and JNK signaling cascade to induce the high degree of fucosylation characteristic of adult mammalian colon, and we speculate that this fucosylation facilitates colonization by adult microbiota.

HLA-B27/beta2 microglobulin transgenic (TG) rats spontaneously develop T-cell-mediated colitis when colonized with normal commensal bacteria, but remain disease-free under germ-free conditions. We investigated regulation of in vitro T-cell responses to enteric bacterial components. Bacterial lysates prepared from the caecal contents of specific pathogen-free (SPF) rats stimulated interferon-gamma (IFN-gamma) production by TG but not non-TG mesenteric lymph node (MLN) cells. In contrast, essentially equivalent amounts of interleukin-10 (IL-10) were produced by TG and non-TG cells. However, when cells from MLNs of non-TG rats were cocultured with TG MLN cells, no suppression of IFN-gamma production was noted. Both non-TG and TG antigen-presenting cells (APC) pulsed with caecal bacterial lysate were able to induce IFN-gamma production by TG CD4+ cells, although non-TG APC were more efficient than TG APC. Interestingly, the addition of exogenous IL-10 inhibited non-TG APC but not TG APC stimulation of IFN-gamma production by cocultured TG CD4+ lymphocytes. Conversely, in the presence of exogenous IFN-gamma, production of IL-10 was significantly lower in the supernatants of TG compared to non-TG APC cultures. We conclude that commensal luminal bacterial components induce exaggerated in vitro IFN-gamma responses in HLA-B27 TG T cells, which may in turn inhibit the production of regulatory molecules, such as IL-10. Alterations in the production of IFN-gamma, and in responses to this cytokine, as well as possible resistance of TG cells to suppressive regulation could together contribute to the development of chronic colitis in TG rats.

Summary Interleukin-2-deficient (IL-2−/−) mice develop a spontaneous, progressive, CD4+ T-cell-mediated colitis with an age-related decrease in the number of B lymphocytes. The aim of this study was to determine the mechanisms of B-cell loss in IL-2−/− mice. Serum immunoglobulin G1 (IgG1) levels in 8-week-old IL-2−/− mice were above normal but then decreased dramatically with advancing age. Between 8 and 11 weeks of age, the number of B-cell progenitors (B220+ IgM−) in the bone marrow of IL-2−/− mice was less than half of those in IL-2+/+ littermates. By 22 weeks of age, very few progenitor cells remained in the bone marrow of most mice, and spleens were almost devoid of B cells. Likewise, B1 cells were not present in the peritoneal cavity of aged IL-2−/− mice. Flow cytometry analysis of B-cell differentiation in the bone marrow suggested a progressive loss of B cells from the most mature to the least mature stages, which was not dependent on IL-2 receptor-α (IL-2Rα) expression. B cells transferred from normal animals had similar survival rates in IL-2−/− and wild-type mice. We conclude that conventional B cells in older IL-2−/− mice are lost by attrition owing to a derangement in B-cell development. Because B1 cells are less dependent on the bone marrow, a separate mechanism for their loss is suggested.

The roles of the interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) produced during natural killer (NK) cell interaction with macrophages (M phi) were investigated as the basis for the induction of immunoglobulin G2a (IgG2a) anti-bovine serum albumin (BSA) responses by high molecular weight dextran conjugated to BSA (HMW-DEX-BSA). BALB/c mice immunized with HMW-DEX-BSA produced significantly higher levels of both IgG1 and IgG2a anti-BSA than did mice immunized with BSA alone. Both IgG1 and IgG2a anti-BSA levels were higher in mice immunized with BSA conjugated to dextran of molecular weight (MW) 5 000 000-40 000 000 compared with dextran of MW 10,000-60,000. The enhancement of anti-BSA IgG2a levels but not of anti-BSA IgG1 levels was inhibited when free BSA was added to the HMW-DEX-BSA conjugate. NK cell depletion during HMW-DEX-BSA immunization of mice resulted in significantly lower anti-BSA IgG2a levels without affecting anti-BSA IgG1 levels. Naive splenocytes or M phi + NK cell co-cultures incubated with HMW-DEX or HMW-DEX-BSA produced higher IFN-gamma levels than splenocytes or co-cultures incubated with BSA alone. HMW-DEX stimulated both IFN-gamma and IL-12 production by M phi + NK cell co-cultures in a dose-dependent manner. DEX-induced IFN-gamma production by NK cells was dependent upon the presence of IL-12, and IL-12 production by M phi was dependent upon the presence of IFN-gamma in these co-cultures. Both M phi and NK cells bound DEX to their surfaces. These data demonstrate that BSA linked to HMW-DEX enhanced both T-helper-1- and T-helper-2-associated antibody responses to BSA. The results also indicate an IL-12-dependent positive feedback interaction between NK cells and M phi that supports a NK cell/IFN-gamma-dependent mechanism for enhancement of anti-BSA IgG2a antibody responses in mice immunized with HMW-DEX-BSA protein conjugates.

Helicobacter hepaticushas been reported to induce colitis, hepatitis, and hepatocellular carcinoma in several different murine models. The aim of this study was to determine ifH. hepaticuswill cause colitis in monoassociated mice lacking the interleukin-10 gene (IL-10−/−mice) and potentiate colitis in specific-pathogen-free (SPF) IL-10−/−mice. Germfree IL-10−/−mice on either a mixed (C57BL/6 × 129/Ola) or inbred (129/SvEv) genetic background were monoassociated withH. hepaticusATCC 51448 by oral feeding and rectal enemas. In a second experiment, germfree IL-10−/−mice were colonized with stool from SPF mice that harbored or did not harbor endogenousH. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was isolated for histology and IL-12 secretion, and mesenteric lymph node cells were assessed for T-cell activation markers. It was found that IL-10−/−mice monoassociated withH. hepaticusfor up to 16 weeks showed almost no histologic colitis or increased IL-12 production. SPF IL-10-knockout mice had no significant difference in weight loss, mortality rate, histologic scores, colonic IL-12 secretion, or T-cell activation with or withoutH. hepaticus. We conclude thatH. hepaticusdoes not induce or potentiate disease in our IL-10−/−mice and therefore is not required to induce colitis in genetically susceptible hosts.

Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation. IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions. We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice. Breeding colonies were maintained under SPF and germfree (GF) conditions. Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion. GF IL-2 −/− mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 −/− mice led to their death between 28 and 32 wk. Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization. Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 −/− mice in both GF and SPF environments. In contrast to other genetically engineered rodents, IL-2 −/− mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.

We report our initial characterization of the immune system in C3H/HeJBir mice that spontaneously develop perianal ulceration. Analysis of lymphokine production by mucosal and systemic lymphoid cells obtained from these mice revealed higher levels of interleukin (IL)4 in supernatants of in vitro stimulated Peyer's patch and mesenteric lymph node cells from C3H/HeJBir mice with severe perianal lesions than from C3H/HeJBir mice with mild perianal lesions or from normal C3H/HeJ mice. Supernatants that contain the highest amounts of IL-4 contain the lowest amounts of interferon (IFN)-γ. Peyer's patches and mesenteric lymph nodes of C3H/HeJBir mice with severe perianal lesions contain approximately four-fold fewer cells; however, inguinal lymph nodes contain up to 25-fold more cells compared with C3H/HeJ mice. These inguinal lymph node cells secrete IL-6, and T cells that produce IL-2 or IFN-γ can be demonstrated. Serum IgE, IgGl, and IgG2a are all increased in C3H/HeJBir mice with severe perianal ulceration. These results show that C3H/HeJBir mice with grossly evident inflammation have activated lymphocytes in both mucosal and systemic lymphoid organs. Understanding mechanisms of lymphocyte activation and regulation in these mice may provide insights into the pathogenesis of perianal ulceration that is often associated with Crohn's disease.

This study was designed to exploit the ability of Peyer's patch M cells to recognize antigen-antibody complexes in the targeted delivery of a model antigen for the induction of mucosal immunity. Sensitized liposomes consisted of an entrapped model antigen, ovalbumin (OVA), and coated with unrelated antigen-antibody complexes. Sensitized liposomes were administered intrajejunally to mice either with or without monophosphoryl lipid A (MLA). Humoral immune responses were monitored in saliva, feces, serum, and bile. Mice which received sensitized liposomes showed up to 4-fold amounts of specific IgA in saliva, feces, and bile compared to controls. Transient increases in anti-OVA IgA and IgG were observed in serum. Formulations including MLA generated positive anti-OVA IgG responses in both serum and bile. In separate experiments, cell proliferation studies were performed with Peyer's patch lymphocytes harvested from mice immunized with OVA in either standard or sensitized liposomes. Lymphocytes from test mice receiving only sensitized liposomes proliferated in the presence of OVA, but not an unrelated antigen. Taken together, these results support the potential application of antigen-antibody complexes in the stimulation of mucosal immune responses and that MLA may play an important role in overcoming OVA tolerogenicity.

Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.

Feline immunodeficiency virus (FIV), a lentivirus similar to HIV, causes an acquired immunodeficiency syndrome in cats. Similar to human immunodeficiency virus (HIV), the pathogenesis of FIV is associated with dysregulation of the cytokine network. While alterations in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression have been reported in HIV-infected patients, changes attributable to HIV and those caused by cofactors such as secondary infections cannot always be readily distinguished. This study evaluated the effect of FIV infection on TNF-α and IL-6 production in cats not exposed to other potential cofactors such as secondary infections. TNF-α and IL-6 activities were evaluated in bronchoalveolar lavage (BAL) cells from FIV-infected and uninfected specific pathogen free (SPF) cats. Supernatants from lipopolysaccharide (LPS)-stimulated BAL cells from uninfected SPF cats had high levels of TNF-α and IL-6 activity, while stimulated BAL cell supernatants from FIV-infected SPF cats had significantly lower levels of TNF-α but unaltered IL-6 activity. Similarly, Con A/phorbol myristate acetate (PMA) stimulated non-adherent (NA-) peripheral blood mononuclear cells (PBMC) from FIV infected cats synthesized less TNF-α than similarly treated NA-PBMC from uninfected cats. Feline immunodeficiency virus could be recovered from the culture supernatants of BAL cells from infected cats by co-cultivation with susceptible lymphocytes. In situ hybridization identified FIV mRNA in a small fraction of alveolar macrophages in the BAL cell cultures. Considering that these SPF cats have been infected with FIV for up to 3 years, yet remain asymptomatic and have no evidence of secondary infections, the present data suggest that the deficiency in TNF-α production by BAL cells and NA-PBMC is an intrinsic characteristic of the FIV infection.

Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli . After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli , the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo , and an increased tendency for spontaneous lysis in vitro . Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

Background: We recently showed that Bifidobacterium animalis is more prevalent within the colons of interleukin (IL)-10-deficient (−/−) mice than in wildtype (WT) animals colonized with the same specific pathogen-free (SPF) fecal contents. Here we tested the ability of this organism to cause T-cell-mediated intestinal inflammation by introducing it into germ-free (GF) IL-10−/− mice. Methods: GF IL-10−/− or WT mice were monoassociated with Bifidobacterium animalis subsp. animalis ATCC (American Type Culture Collection, Manassas, VA) 25527T or with B. infantis ATCC 15697T. Inflammation was measured by blinded histologic scores of the duodenum, cecum, and colon and by spontaneous secretion of IL-12/IL-23 p40 from colonic explants. Bacterial antigen-specific CD4+ mesenteric lymph node (MLN) T-cell recall responses were measured in response to antigen-presenting cells (APC) pulsed with bacterial lysates. Results:B. animalis caused marked duodenal inflammation and mild colitis in monoassociated IL-10−/− mice, whereas the intestinal tracts of WT animals remained free of inflammation. B. infantis colonization resulted in mild inflammation in the duodena of IL-10−/− mice. CD4+ MLN T cells from B. animalis monoassociated IL-10−/− mice secreted high levels of IFN-γ and IL-17 in response to B. animalis lysate. B. animalis equally colonized the different intestinal regions of WT and IL-10−/− mice. Conclusions:B. animalis, a traditional probiotic species that is expanded in experimental colitis in this model, induces marked duodenal and mild colonic inflammation and TH1/TH17 immune responses when introduced alone into GF IL-10−/− mice. This suggests a potential pathogenic role for this commensal bacterial species in a susceptible host. (Inflamm Bowel Dis 2009)

We have concentrated here on the lymphokines which might serve to regulate the different pathways of precursor development. We suggest that, as a result of antigenic stimulation, specific precursor cells both proliferate and become committed to develop into either an effector cell, a memory cell or an anergized cell. Anergy has not been dealt with in this review, but it is likely to be one of the options available. The development of an effector population takes 4-7 d (quite analogous to the time it takes for CTLp to become CTL and for resting B to become Ab-forming cells). The effector populations are large, generally IL-2R-positive cells. These cells have upregulated many adhesion molecule systems [e.g., Pgp-1, LFA-1 and ICAM-1 (Swain unpublished)], but downregulated the Mel-14 homing receptor. Effectors are ready to respond to APC such as specific B cells with a rapid synthesis and secretion of lymphokines. The effector population is then quickly downregulated, both by the turn off of lymphokine synthesis/secretion and possibly by its own suicide. This kind of pattern makes teleological sense since the cells making such high titers of lymphokines could have many potent pleitropic effects. It also seems to be the strategy employed in the generation of other terminally differentiated effectors (such as CTL and plasma cells). The requirement for restimulation and the requirement for direct and perhaps prolonged contact between the helper effector and the APC-B cell can be expected to help ensure that these lymphokines are localized (reviewed in Swain & Dutton 1987, Swain & Croft 1990) and effectively delivered to specific responding cells. We postulate that at the same time, or perhaps subsequent to this, another set of signals drives precursors to generate prememory cells. Our studies suggest these emerging memory cells may be phenotypically unique and we postulate that they are specialized to become a "long-lived" population of memory cells that will persist indefinitely as a protective population of increased frequency for the antigen encountered and which is also able to respond more rapidly and effectively. The greater effectiveness of the memory response would thus be due to dramatically increased frequency, to characteristic and stable changes in adhesion molecule expression and to the fact that, in addition to IL-2, resting memory cells also secrete at least low titers of IL-3, IL-4, IFN-gamma and other lymphokines upon initial restimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-10 deficient (IL-10-/-) mice develop chronic T cell-mediated colitis when colonized with normal commensal bacteria, but germ-free (GF) IL-10-/- mice remain disease-free. Antigen presenting cells (APC) secrete regulatory cytokines that help determine T lymphocyte activation or tolerance. CD4(+) T cells from the mesenteric lymph nodes of inflamed IL-10-/- mice secrete more IFN-gamma and IL-17 when cultured with cecal bacterial lysate-pulsed splenic APC from IL-10-/- mice than when cultured with normal control APC. GF IL-10-/- APC induce similar IFN-gamma and IL-17 responses; therefore, the functional difference between normal and IL-10 deficient APC is inherent to the lack of IL-10 and not secondary to inflammation. Bacterial lysate-pulsed normal APC cultured with CD4(+) cells from colitic IL-10-/- mice or with exogenous IFN-gamma secrete higher amounts of IL-10 compared to the same APC cultured with naive T cells. APC enriched for CD11c(+) cells are potent activators of IFN-gamma and IL-17 production by CD4(+) cells from IL-10-/- mice. These APC also produce IL-12/IL-23 p40 and IL-10. Recombinant IL-10 suppressed and anti-IL-10 receptor antibody increased IFN-gamma, IL-17 and IL-12/IL-23 p40 production in bacterial lysate-pulsed APC and plus CD4(+) T cell co-cultures. Taken together, our results show that endogenous IL-10 produced by APC inhibits responses to commensal bacteria and influences the ability of APC to stimulate IFN-gamma-producing effector lymphocytes, which reciprocally, induce IL-10 production by APC. Cytokines produced by APC are an important determinant of pathogenic versus protective mucosal immune responses to colonic bacterial stimulation.

Background: Monoassociating gnotobiotic IL-10-deficient (−/−) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T-cell-mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently. Methods: Germ-free (GF) inbred 129S6/SvEv IL-10−/− and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3–7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL-12/IL-23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4+ cells were stimulated with antigen-presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN-γ and IL-17 levels were measured in the supernatants. Results: Dual-associated IL-10−/− (but not WT) mice developed mild-to-moderate pancolitis by 3 weeks that progressed to severe distal colonic-predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF-κB was activated in the duodenum and colon in dual-associated IL-10−/− × NF-κBEGFP mice. The aggressiveness of intestinal inflammation and the degree of antigen-specific CD4+ cell activation were greater in dual- versus monoassociated IL-10−/− mice. Conclusion: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL-10−/− mice act additively to induce aggressive pancolitis and duodenal inflammation. (Inflamm Bowel Dis 2007)

SummaryHLA-B27 transgenic (TG) rats develop spontaneous colitis when colonized with intestinal bacteria, whereas athymic nude (rnu/rnu) HLA-B27 TG rats remain disease free. The present study was designed to determine whether or not HLA-B27 expression on T cells is required for development of colitis after transfer of mesenteric lymph node (MLN) cells into rnu/rnu HLA-B27 recipients. Athymic nontransgenic (non-TG) and HLA-B27 TG recipients received MLN cells from either TG or non-TG rnu/+ heterozygous donor rats that contain T cells. HLA-B27 TG rnu/rnu recipients receiving either non-TG or TG MLN cells developed severe colitis and had higher caecal MPO and IL-1β levels, and their MLN cells produced more IFN-γ and less IL-10 after in vitro stimulation with caecal bacterial lysate compared to rnu/rnu non-TG recipients that remained disease free after receiving either TG or non-TG cells. Interestingly, proliferating donor TG T cells were detectable one week after adoptive transfer into rnu/rnu TG recipients but not after transfer into non-TG recipients. T cells from either non-TG or TG donors induce colitis in rnu/rnu TG but not in non-TG rats, suggesting that activation of effector T cells by other cell types that express HLA-B27 is pivotal for the pathogenesis of colitis in this model.

Rats transgenic (TG) for the human major histocompatibility complex (MHC) class I HLA-B27 and beta2-microglobulin genes develop chronic colitis under specific pathogen-free (SPF) but not sterile (germ-free, GF) conditions. We investigated the role of antigen-presenting molecules involved in generating immune responses by CD4+ mesenteric lymph node (MLN) cells from colitic HLA-B27 TG rats to commensal enteric micro-organisms. All TG MLN cells expressed HLA-B27. A higher level of MHC class II was expressed on cells from TG rats, both SPF and GF, compared to non-TG littermates. In contrast, rat MHC class I expression was lower on TG than non-TG cells. Both TG and non-TG antigen presenting cells (APC) pulsed with caecal bacterial antigens induced a marked interferon-gamma (IFN-gamma) response in TG CD4+ T lymphocytes but failed to stimulate non-TG cells. Blocking MHC class II on both TG and non-TG APC dramatically inhibited their ability to induce TG CD4+ T cells to produce IFN-gamma. Blocking HLA-B27 on TG APC similarly inhibited IFN-gamma responses. When the antibodies against MHC class II and HLA-B27 were combined, no APC-dependent IFN-gamma response was detected. These data implicate both native rat MHC class II and TG HLA-B27 in CD4+ MLN T-cell IFN-gamma responses to commensal enteric microflora in this colitis model.

SUMMARYGerm-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-γ, IL-12, TNF, IL-10 and TGF-β production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-γ, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-β. T cell depletion abolished IFN-γ and decreased IL-12 production, but did not affect IL-10 and TGF-β production. Conversely, neither IL-10 nor TGF-β was produced in cultures of B cell-depleted MLN. In addition, CD4+ T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-γ when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-β, but not IFN-γ, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-γ-producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.

Interleukin (IL)-10-deficient (IL-10-/-) mice develop colitis under specific pathogen-free (SPF) conditions and remain disease free if kept sterile (germ free [GF]). We used four different protocols that varied the time-points of oral administration of Lactobacillus plantarum 299v (L. plantarum) relative to colonization with SPF bacteria to determine whether L. plantarum could prevent and treat colitis induced by SPF bacteria in IL-10-/- mice and evaluated the effect of this probiotic organism on mucosal immune activation. Assessment of colitis included blinded histologic scores, measurements of secreted colonic immunoglobulin isotypes, IL-12 (p40 subunit), and interferon (IFN)-gamma production by anti-CD3-stimulated mesenteric lymph node cells. Treating SPF IL-10-/- mice with L. plantarum attenuated previously established colonic inflammation as manifested by decreased mucosal IL-12, IFN-gamma, and immunoglobulin G2a levels. Colonizing GF animals with L. plantarum and SPF flora simultaneously had no protective effects. Gnotobiotic IL-10-/- mice monoassociated with L. plantarum exhibited mild immune system activation but no colitis. Pretreatment of GF mice by colonization with L. plantarum, then exposure to SPF flora and continued probiotic therapy significantly decreased histologic colitis scores. These results demonstrate that L. plantarum can attenuate immune-mediated colitis and suggest a potential therapeutic role for this agent in clinical inflammatory bowel diseases.

Development of effectors from naive CD4 cells occurs in two stages. The early stage involves activation and limited proliferation in response to T cell receptor (TCR) stimulation by antigen and costimulatory antigen presenting cells, whereas the later stage involves proliferation and differentiation in response to growth factors. Using a TCR-transgenic (Tg+) model, we have examined the effect of aging on effector generation and studied the ability of γc signaling cytokines to reverse this effect. Our results indicate that responding naive CD4 cells from aged mice, compared with cells from young mice, make less interleukin (IL)-2, expand poorly between days 3 to 5, and give rise to fewer effectors with a less activated phenotype and reduced ability to produce cytokines. When exogenous IL-2 or other γc signaling cytokines are added during effector generation, the Tg+ cells from both young and aged mice proliferate vigorously. However, IL-4, IL-7, and IL-15 all fail to restore efficient effector production. Only effectors from aged mice generated in the presence of IL-2 are able to produce IL-2 in amounts equivalent to those produced by effectors generated from young mice, suggesting that the effect of aging on IL-2 production is reversible only in the presence of exogenous IL-2.

The recirculation of naive lymphocytes from blood to lymph that is initiated in high endothelial venules (HEV) of secondary lymphoid organs such as lymph nodes and Peyer's patches (PP) is regulated by multiple interactions of adhesion receptor/counter-receptor pairs involving both selectins and integrins. We showed previously that blocking of only L-selectin is sufficient to ablate trafficking of naive CD4 cells and the development of their responses in peripheral lymph nodes but not in PP where alpha4beta7 integrins are thought to primarily regulate entry. However, although antibody to alpha4 integrins partially inhibited homing of naive CD4 cells to PP and not to lymph nodes, there was no effect on the development primary responses in these tissues or spleens. Since previous studies indicate that both alpha4beta7 integrins and L-selectin regulate adhesion of naive cells to PP HEV, we examined the effect a blockade of both adhesion pathways on the recirculation of naive CD4 cells. There was no detectable homing of naive CD4 cells to PP or lymph nodes when interactions with both receptors were inhibited, resulting in a profound depletion of naive CD4 cells and loss of antigen responses in these sites. In contrast, increased numbers of naive CD4 cells and responses of higher magnitude were found in the spleen. The results demonstrate recirculation of naive CD4 cells through tissues where entry is controlled through HEV is essential for the local generation of primary responses.

We showed previously that L-selectin-dependent recirculation of naive CD4 cells is essential for development of primary responses in peripheral lymph nodes. Recent studies suggest that L-selectin is also required for lymphocyte entry into gut mucosal lymphoid tissues that include Peyer's patches and mesenteric lymph nodes. Here we show that anti-L-selectin antibody, MEL-14, inhibited homing of a rigorously purified, homogenous population of naive CD4 cells into both of these tissues as well as peripheral lymph nodes, directly demonstrating a role for this receptor in regulating entry into gut-associated sites. However, in intact animals, treatment with MEL-14 resulted in the loss of naive CD4 cells (CD45RBhi, CD44lo from peripheral lymph nodes but not Peyer's patches, whereas mesenteric lymph nodes were intermediate in this regard. In mice primed by parenteral immunization with keyhole limpet hemocyanin (KLH), primary CD4 responses were readily detected in both. Peyer's patches and mesenteric lymph nodes, and were not affected by exposure to MEL-14. Indeed, similar frequencies of KLH-specific CD4 cells were recovered from both of these tissues irrespective of MEL-14 treatment. The results indicate that interactions with L-selectin can be circumvented to allow entry of naive CD4 cells into Peyer's patches but not peripheral lymph nodes.

BACKGROUND AND AIMS: Interleukin 10 (IL10) inhibits monocyte/macrophage and T lymphocyte effector functions. This study examined the effect of systemically administered IL10 on acute and chronic granulomatous enterocolitis, hepatitis, and arthritis in a rat model. METHODS: Lewis rats were injected intramurally with streptococcal peptidoglycan-polysaccharide (PG-APS) polymers. Beginning 12 hours before PG-APS injection, rats were treated daily with subcutaneous murine recombinant IL10 or vehicle for three or 17 days. RESULTS: IL10 attenuated acute enterocolitis in a dose dependent fashion (p < 0.01). Protective effects were more profound in the chronic granulomatous phase with decreased enterocolitis and markedly inhibited leucocytosis, hepatic granulomas, and chronic erosive arthritis (p < 0.001). IL10 downregulated tissue IL1, IL6, tumour necrosis factor alpha, and interferon gamma gene expression, consistent with the in vitro effects of IL10 on PG-APS-stimulated splenocytes. Caecal IL1 protein concentrations and IL2 and interferon gamma secretion by in vitro stimulated mesenteric lymph nodes were downregulated in IL10 treated animals. CONCLUSIONS: These results indicate that exogenous IL10 can inhibit experimental granulomatous inflammatory responses and suggest that IL10 treatment could be an effective new therapeutic approach in human disorders such as Crohn's disease, rheumatoid arthritis, and sarcoidosis.

The ability to detect feline cytokine expression would allow further characterization of the feline immune system. Bioassays are currently available for the measurement of feline IL2, IL6 and TNF alpha but not for other biologically important cytokines. To detect the expression of other cytokines, a reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed. Since feline cytokine gene sequences other than TNF alpha were not available, mammalian DNA and mRNA sequences for IL2, IFN gamma, IL4, IL6, IL10, IL12 and beta-actin, obtained from the Genbank database were compared and oligonucleotide primers chosen from consensus sequences. To validate the cytokine and beta-actin primers, peripheral blood mononuclear cells from specific pathogen free (SPF) cats were cultured in the presence of Con A for various periods of time (0-72 h). RNA was collected, reverse transcribed into cDNA, and the cDNA was amplified by PCR with each set of cytokine primer pairs. RT-PCR products were hybridized with specific 32P end-labeled internal oligonucleotide probes and then analyzed with the AMBIS imaging system to determine the kinetics of cytokine mRNA production. The beta-actin signal was used to control for sample to sample variation in the quantity of mRNA and variation in the RT and PCR reactions. Peak mRNA expression for most cytokines was found to occur between 2 to 4 h of Con A stimulation. mRNA expression was correlated with cytokine bioactivity for IL2 and IL6. Peak IL2 bioactivity occurred after 8 h of Con A stimulation, 4 h after the mRNA expression had peaked. Although IL6 mRNA expression peaked between 2 and 4 h of stimulation, bioactivity was not detected until 8 h of stimulation and continued to increase over the next 24-48 h.

Background & Aims Mice that are deficient in interleukin (IL)-10 develop colitis, mediated by T-helper (Th)1 and Th17 cells, and IL-10–producing regulatory T (Treg) cells suppress colitis, implicating IL-10 in maintaining mucosal homeostasis. We assessed the relative importance of immunoregulatory IL-10 derived from T cells or from antigen presenting cells (APCs) in development of intestinal inflammation. Methods CD4 + cells from germ-free (GF) or specific pathogen-free (SPF) IL-10 −/− or wild-type mice were injected into IL-10 −/− , Rag2 −/− mice or Rag2 −/− mice that express IL-10. After 6–8 weeks, we evaluated inflammation, spontaneous secretion of cytokines from colonic tissue, and mRNA levels of the transcription factor T-bet and the immunoregulatory cytokine transforming growth factor (TGF)-β. CD4 + T cells were co-cultured with bacterial lysate-pulsed APCs and assayed for cytokine production, FoxP3 expression, and TGF-β-mediated Smad signaling. Results CD4 + cells from GF or SPF IL-10 −/− or wild-type mice induced more severe colitis and higher levels of inflammatory cytokines in IL-10 −/− , Rag2 −/− mice than in IL-10-replete, Rag2 −/− mice. Co-cultures of IL-10 −/− or wild-type CD4 + T cells plus bacterial lysate-pulsed APCs from IL-10 −/− mice contained more interferon (IFN)-γ, IL-12/23p40, and IL-17 than co-cultures of the same T cells plus APCs from wild-type mice. CD11b + APCs were required for these effects. Blocking IL-10 receptors increased production of IFN-γ and IL-12/23p40 whereas exogenous IL-10 suppressed these cytokines. IL-10–producing APCs induced TGF-β-mediated, retinoic acid-dependent, differentiation of FoxP3 + Treg cells, whereas blocking the retinoic acid receptor, in vitro and in vivo, reduced proportions of FoxP3 + Treg cells. Conclusions IL-10 produced by APCs regulates homeostatic T-cell responses to commensal bacteria.

independent techniques to evaluate the microbial composition of pouch and afferent limb tissue biopsies from patients with variable pouch outcomes. Methods: Patients with confirmed UC or familial adenomatous polyposis(FAP) who had a colectomy and IPAA, with ileostomy closure ≥1 year prior to study enrollment were recruited from Mount Sinai Hospital at the time of follow-up endoscopy. Retrospective and cross-sectional clinical/endoscopic information was collected at the time of enrollment and patients were classified into 4 groups: FAP/ no pouchitis(FAP), UC/no pouchitis(NP), UC/pouchitis(P) andUC/CD-like phenotype(CDL). P was defined based on the mPDAI. CDL diagnosis was based on presence of afferent limb inflammation or fistula. 2 biopsies were collected and snap-frozen in liquid nitrogen (1 afferent limb and 1 pouch). Microbial DNA was extracted and sequenced using the 454 GS FLX Titanium Sequencing system using primers specific for the V1-V3 and V2-V3 hypervariable regions of 16S rRNA genes in 2 batches. Sequences were quality trimmed and aligned to a SILVA reference database using mothur, with a minimum bootstrap cutoff of 80%. A threshold level of 0.05 was used to assign sequence clusters to a specific genus and patients were considered positive for genera with counts >0. Dichotomized results were analyzed using Fisher's exact test and Bonferroni correction for multiple testing. Results: 245 patients were recruited to the study of whom 79 were selected for analysis with complete data available (18 FAP, 21 NP, 20 P, 20 CDL). Analysis at the genus level showed that Blautia was detected significantly less often in the P group in both afferent limb (p=0.002) and pouch samples (p=0.008) compared to the other groups. Bacteroides and Sutterella were significantly less frequent among P and CDL groups in the pouch samples only (p=0.001 and 0.01 respectively). There was no significant difference in the proportions of these genera between outcome groups in the different batches. Conclusion: Differences in microbial composition are associated with outcomes following IPAA. Specifically, the genera Blautia, Bacteroides and Sutterella were found less frequently in patients with P and CDL compared to those without pouchitis. Further study may allow us to identify the role these organisms play in pouch pathogenesis, and to identify interactions occurring between microorganisms and host endogenous elements such as genotype and development of anti-microbial antibodies.

The immunoprophylaxis of neonates against intestinal pathogens remains a challenge. The approach is generally based on the entero-mammary linkage of the gestationing animal. However, a clear correlation between the virulence of the pathogen and IgA inducing capability exists. Therefore, our investigation aimed at the possibility of inducing mucosal immunity via the anti-idiotype pathway. Recent advances in human MAB production (for review Teng, N.N. 1985) and IgA producing hybridomas (Colwell, D., 1986, Dean, C., 1986) made research on this concept in a veterinary species feasible. PM-1 is a stable, non-Ig producing murine-porcine heteromyeloma capable of high fusion rates and stable porcine MAB production upon fusion with porcine lymphocytes of various sources (triple heterohybrids). PM-1 was obtained by intensive subcloning, in selective medium, of hybridomas resulting from the fusion of the murine myeloma P3x63-Ag8.653 and the porcine lymphoblastoid cell line P-16. Fusions of PM-1 with PBL, MNL, TDL and SL resulted fusion rates of 20–100% (hybridomas/wells inoculated). A total of 359 hybridomas were monoclonal Ig-producers. The majority of those analysed produced the IgM isotype. Only one heteorhybrid, YF 42-1, resulting from a PM-1 x PBL fusion was identified as producing dimeric IgA. Fusion with TDL, obtained from pigs after surgical removal of the mesenteric lymph nodes, resulted in 16 IgM and 2 IgG producing hybridomas. No IgA producing hybridomas were obtained from PM-1 x TDL fusions.

Proteinase-Activated Receptor-4 (PAR-4) is 7-transmembrane domain G protein-coupled receptor equally activated by thmmbin attd trypsin. Since enzymatic activities of potential PAR-4 agonists such as thrombin and trypsin are largely increased into the luminal content of inflamnmtory' bowel disease patients. We hypothesized that local PAR-4 activation caused inflammation and is imphcated in the generation of inflammation in a model of Dextran Sodium Sulfate (DSS)-induced colitis. Methods: PAR-4 expression in mouse and human colon was stud~ed by RT-PCR Groups of mice received into their distal colon either a selective PAR-4 agonist (the PAR-4-activating peptide AYPGKF, 200p.g) or a control peptide (inacuve on PAR-4). Pepducin, a novel cell penetrating palmitoylated peptide, based on the bunmn PAR-4 3rd intracellular loop, acts as a PAR-4 antagonist. We have used this PAR-4 antagonist to prove that the effects of intracolnnic injection of PAR-4 agonists were due to PAR-4 activation, by treating mice that have received AYPGKF, with the PAR-4 antagonist or its vehicle. Then, to ins.estigate the role of PAR-4 in the pathogenesis of colitis, 2.5 % DSS was added to the nmuse drinking water to induce colitis, and these mice were treated twice daily, (50p~g/day; 100p~g/day), with the PAR-4 antagonist or its vehicle. At different times after PAR-4-AP intracolic injection or 7 days after the induction of DSS colitis, mice were sacrificed and inflammatory parameters (myeloperoxidase activity, macroscopic damage score, wall thickness) were measured in the mouse colon, Results: PAR-4 signal was detected both in mouse and human colon, Int racolonic administration of AYPGKF, but not the control peptide, caused an inflammatory reaction characterized by increased granulocyte infiltration and edema. Inflammation which reached a maxinmm 10 h after the intmcolonic injection and was back to normal 48h later, was completely inhibited by' PAR-4 antagonist treatment. Seven days afier the reduction of DSS colitis, increased wall thickness and damage score were significantly reduced, in a dose-dependent manner, in mice treated with the PAR-4 antagomst compared to vehicle-treated mice, but MPO acti~aty was not significantly different in all DSS-treated groups. Conclusion: PAR-4 activation triggers inflammation in mouse colon and plays an active role in the generation of inflammation in the DSS-induced colitis model, However, the pro-inflammatory role of PAR-4 did not seem to be associated with granulocyte recruitment.