The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis.

In the present study, we define the relation between TGF-beta and IL-10 in the regulation of the Th1-mediated inflammation occurring in trinitrobenzene sulfonic acid (TNBS)-colitis. In initial studies, we showed that the feeding of trinitrophenol-haptenated colonic protein to SJL/J mice induces CD4(+) regulatory T cells that transfer protection from induction of TNBS-colitis, and that such protection correlates with cells producing TGF-beta, not IL-10. Further studies in which SJL/J mice were fed haptenated colonic protein, and then administered either anti-TGF-beta or anti-IL-10 at the time of subsequent TNBS administration per rectum, showed that while both Abs abolished protection, anti-TGF-beta administration prevented TGF-beta secretion, but left IL-10 secretion intact; whereas anti-IL-10 administration prevented both TGF-beta secretion and IL-10 secretion. Thus, it appeared that the protective effect of IL-10 was an indirect consequence of its effect on TGF-beta secretion. To establish this point further, we conducted adoptive transfer studies and showed that anti-IL-10 administration had no effect on induction of TGF-beta producing T cells in donor mice. However, it did inhibit their subsequent expansion in recipient mice, probably by regulating the magnitude of the Th1 T cell response which would otherwise inhibit the TGF-beta response. Therefore, these studies suggest that TGF-beta production is a primary mechanism of counter-regulation of Th1 T cell-mediated mucosal inflammation, and that IL-10 is necessary as a secondary factor that facilitates TGF-beta production.     

CD4+ T cells from IL-10-deficient mice transfer susceptibility to NSAID-induced Rag colitis

Products of arachidonic acid metabolism are important for mucosal homeostasis, because blockade of this pathway with an NSAID triggers rapid onset of severe colitis in the IL-10 knockout (IL-10(-/-)) model of IBD. Rag mice do not make T or B cells. This study determined whether reconstitution of Rag mice with T cells from IL-10(-/-) mice transferred NSAID colitis susceptibility. Rag mice were reconstituted by intraperitoneal injection with splenocytes from wild-type (WT) or IL-10(-/-) animals. Colitis was induced by using piroxicam and was graded histologically. Isolated lamina propria mononuclear cells (LPMC), lamina propria T cells, and LPMC depleted of T cells from reconstituted Rag mice were studied for cytokine production. Only animals reconstituted with IL-10(-/-) CD4(+) T cells and administered piroxicam developed severe colitis. LPMC from these colitic animals made IFN-gamma, whose production was dependent on T cells. Some IL-10 was produced but only from non-T cells. LPMC from the healthy Rag mice that were reconstituted with WT T cells and were piroxicam resistant made much more IL-10. This was mostly T cell dependent. In conclusion, only CD4(+) T cells from IL-10(-/-) animals leave Rag mice susceptible to NSAID-induced, Th1 colitis. Lamina propria T cells normally make large quantities of IL-10, suggesting that IL-10 from T cells may be protective.     

Colitogenic Th1 cells are present in the antigen-experienced T cell pool in normal mice: control by CD4+ regulatory T cells and IL-10

CD4(+) regulatory T cells have been shown to prevent intestinal inflammation; however, it is not known whether they act to prevent the priming of colitogenic T cells or actively control these cells as part of the memory T cell pool. In this study, we describe the presence of colitogenic Th1 cells within the CD4(+)CD45RB(low) population. These pathogenic cells enrich within the CD25(-) subset and are not recent thymic emigrants. CD4(+)CD45RB(low) cells from germfree mice were significantly reduced in their ability to transfer colitis to immune deficient recipients, suggesting the presence of commensal bacteria in the donor mice drives colitogenic T cells into the Ag-experienced/memory T cell pool. This potentially pathogenic population of Ag-experienced T cells is subject to T cell-mediated regulation in vivo by both CD4(+)CD25(+) and CD4(+)CD25(-) cells in an IL-10-dependent manner. Furthermore, administration of an anti-IL-10R mAb to unmanipulated adult mice was sufficient to induce the development of colitis. Taken together, these data indicate that colitogenic Th1 cells enter into the Ag-experienced pool in normal mice, but that their function is controlled by regulatory T cells and IL-10. Interestingly, IL-10 was not absolutely required for CD4(+)CD25(+) T cell-mediated inhibition of colitis induced by transfer of naive CD4(+)CD45RB(high) cells, suggesting a differential requirement for IL-10 in the regulation of naive and Ag-experienced T cells.     

Pretreatment with the probiotic VSL#3 delays transition from inflammation to dysplasia in a rat model of colitis-associated cancer

Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.     

Peroxisome proliferator-activated receptor gamma is required for regulatory CD4+ T cell-mediated protection against colitis

Peroxisome proliferator-activated receptor (PPAR) gamma activation has been implicated in the prevention of immunoinflammatory disorders; however, the mechanisms of regulation of effector and regulatory CD4+ T cell functions by endogenously activated PPAR-gamma remain unclear. We have used PPAR-gamma-deficient CD4+ T cells obtained from tissue-specific PPAR-gamma null mice (i.e., PPAR-gamma fl/fl; MMTV-Cre+) to investigate the role of endogenous PPAR-gamma on regulatory T cell (Treg) and effector CD4+ T cell function. Overall, we show that the loss of PPAR-gamma results in enhanced Ag-specific proliferation and overproduction of IFN-gamma in response to IL-12. These findings correlate in vivo with enhanced susceptibility of tissue-specific PPAR-gamma null mice to trinitrobenzene sulfonic acid-induced colitis. Furthermore, the transfer of purified PPAR-gamma null CD4+ T cells into SCID recipients results in enteric disease. To test the assertion that the deficiency of PPAR-gamma in Treg impairs their ability to prevent effector T cell-induced colitis, we performed cotransfer studies. These studies demonstrate that PPAR-gamma-expressing, but not PPAR-gamma null Treg, prevent colitis induced by transfer of naive CD4+ T cells into SCID recipients. In line with these findings, the production of IFN-gamma by spleen and mesenteric lymph node-derived CD4+ T cells was down-regulated following transfer of PPAR-gamma-expressing, but not PPAR-gamma null, Treg. In conclusion, our data suggest that endogenous PPAR-gamma activation represents a Treg intrinsic mechanism of down-regulation of effector CD4+ T cell function and prevention of colitis.     

CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4

Regulatory T cells are critical in regulating the immune response, and therefore play an important role in the defense against infection and control of autoimmune diseases. However, a therapeutic role of regulatory T cells in an established disease has not been fully established. In this study, we provide direct evidence that CD4(+)CD25(+) regulatory T cells can cure an established, severe, and progressive colitis. SCID mice developed severe colitis when adoptively transferred with naive CD4(+)CD25(-) T cells and infected with the protozoan parasite Leishmania major. The disease development can be completely halted and symptoms reversed, with a healthy outcome, by transferring freshly isolated or activated CD4(+)CD25(+) T cells from syngeneic donors. The therapeutic effect of the regulatory T cells was completely blocked by treatment of the recipients with anti-IL-10R, anti-CTLA4, or anti-TGF-beta Ab. However, the resurgence of colitis under these treatments was not accompanied by the reactivation of Th1 or Th2 response nor was it correlated to the parasite load. These results therefore demonstrate that CD4(+)CD25(+) T cells are therapeutic and that the effect is mediated by both IL-10/TGF-beta-dependent and independent mechanisms. Furthermore, colitis can manifest independent of Th1 and Th2 responses.     

Expression of HAb18G in non-small lung cancer and characterization of activation, migration, proliferation, and apoptosis in A549 cells following siRNA-induced downregulation of HAb18G

VSL#3 probiotics can be effective on induction and maintenance of the remission of clinical ulcerative colitis. However, the mechanisms are not fully understood. The aim of this study was to examine the effects of VSL#3 probiotics on dextran sulfate sodium (DSS)-induced colitis in rats. Acute colitis was induced by administration of DSS 3.5 % for 7 days in rats. Rats in two groups were treated with either 15 mg VSL#3 or placebo via gastric tube once daily after induction of colitis; rats in other two groups were treated with either the wortmannin (1 mg/kg) via intraperitoneal injection or the wortmannin + VSL#3 after induction of colitis. Anti-inflammatory activity was assessed by myeloperoxidase (MPO) activity. Expression of inflammatory related mediators (iNOS, COX-2, NF-κB, Akt, and p-Akt) and cytokines (TNF-α, IL-6, and IL-10) in colonic tissue were assessed. TNF-α, IL-6, and IL-10 serum levels were also measured. Our results demonstrated that VSL#3 and wortmannin have anti-inflammatory properties by the reduced disease activity index and MPO activity. In addition, administration of VSL#3 and wortmannin for 7 days resulted in a decrease of iNOS, COX-2, NF-κB, TNF-α, IL-6, and p-Akt and an increase of IL-10 expression in colonic tissue. At the same time, administration of VSL#3 and wortmannin resulted in a decrease of TNF-α and IL-6 and an increase of IL-10 serum levels. VSL#3 probiotics therapy exerts the anti-inflammatory activity in rat model of DSS-induced colitis by inhibiting PI3K/Akt and NF-κB pathway.     

Heligmosomoides polygyrus bakeri Induces Tolerogenic Dendritic Cells that Block Colitis and Prevent Antigen-Specific Gut T Cell Responses

Immunological diseases such as inflammatory bowel disease (IBD) are infrequent in less developed countries, possibly because helminths provide protection by modulating host immunity. In IBD murine models, the helminth Heligmosomoides polygyrus bakeri prevents colitis. It was determined whether H. polygyrus bakeri mediated IBD protection by altering dendritic cell (DC) function. We used a Rag IBD model where animals were reconstituted with IL10(-/-) T cells, making them susceptible to IBD and with OVA Ag-responsive OT2 T cells, allowing study of a gut antigenic response. Intestinal DC from H. polygyrus bakeri-infected Rag mice added to lamina propria mononuclear cells (LPMC) isolated from colitic animals blocked OVA IFN-γ/IL-17 responses in vitro through direct contact with the inflammatory LPMC. DC from uninfected Rag mice displayed no regulatory activity. Transfer of DC from H. polygyrus bakeri-infected mice into Rag mice reconstituted with IL10(-/-) T cells protected animals from IBD, and LPMC from these mice lost OVA responsiveness. After DC transfer, OT2 T cells populated the intestines normally. However, the OT2 T cells were rendered Ag nonresponsive through regulatory action of LPMC non-T cells. The process of regulation appeared to be regulatory T cell independent. Thus, H. polygyrus bakeri modulates intestinal DC function, rendering them tolerogenic. This appears to be an important mechanism through which H. polygyrus bakeri suppresses colitis. IFN-γ and IL-17 are colitogenic. The capacity of these DC to block a gut Ag-specific IFN-γ/IL-17 T cell response also is significant.     

Comparative Effect of the I3.1 Probiotic Formula in Two Animal Models of Colitis

Use of probiotic therapy is an active area of investigation to treat intestinal disorders. The clinical benefits of the I3.1 probiotic formula (Lactobacillus plantarum (CECT7484, CECT7485) and P. acidilactici (CECT7483)) were demonstrated in irritable bowel syndrome (IBS) patients in a randomized, double-blind, placebo-controlled clinical trial. The aim of this study was to evaluate the therapeutic effects of I3.1 in two experimental models of colitis, a dextran sulfate sodium (DSS)-induced colitis model and an interleukin (IL)-10-deficient mice model. Colitis was induced in 32 8-week-old Balb/c mice by administering 3% (w/v) DSS in drinking water for 5 days. Probiotics were administered orally (I3.1 or VSL#3, 1 × 10⁹ CFU daily) for 10 days before the administration of DSS. Also, probiotics (I3.1 or VSL#3, 1 × 10⁹ CFU daily) were administered orally to 36 6-week-old C57B6J IL-10(?/?) mice for 10 weeks. Body weight was recorded daily. Colon samples were harvested for histological examination and cytokine measurements. Body weight after DSS administration did not change in the I3.1 group, whereas the VSL#3 group had weight loss. Also, I3.1 normalized IL-6 to levels similar to that of healthy controls and significantly increased the reparative histologic score. In the IL-10-deficient model, both VSL#3 and I3.1 reduced the severity of colitis compared to untreated controls, and I3.1 significantly reduced the levels of IFN-γ compared to the other two groups. In conclusion, I3.1 displays a protective effect on two murine models of experimental colitis. Results suggest that the mechanism of action could be different from VSL#3.     

IL-10 controls ultraviolet-induced carcinogenesis in mice

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10(+/+), IL-10(+/-), and IL-10(-/-) mice were chronically irradiated with UV. IL-10(+/+) and IL-10(+/-) mice developed skin cancer to similar extents, whereas IL-10(-/-) mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10(-/-) mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10(-/-) mice showed impaired suppressor function when cocultured with conventional CD4+CD25- T cells. CD4+CD25- T cells from IL-10(-/-) mice produced increased amounts of IFN-gamma and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10(-/-) mice, suggesting strong Th1-driven immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10(-/-) mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10(-/-) animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.     

Colonization with Heligmosomoides polygyrus suppresses mucosal IL-17 production

Helminth exposure appears to protect hosts from inappropriate inflammatory responses, such as those causing inflammatory bowel disease. A recently identified, strongly proinflammatory limb of the immune response is characterized by T cell IL-17 production. Many autoimmune type inflammatory diseases are associated with IL-17 release. Because helminths protect from these diseases, we examined IL-17 production in helminth-colonized mice. We colonized mice with Heligmosomoides polygyrus, an intestinal helminth, and analyzed IL-17 production by lamina propria mononuclear cells (LPMC) and mesenteric lymph node (MLN) cells. Colonization with H. polygyrus reduces IL-17A mRNA by MLN cells and inhibits IL-17 production by cultured LPMC and MLN cells. Helminth exposure augments IL-4 and IL-10 production. Blocking both IL-4 and IL-10, but not IL-10 alone, restores IL-17 production in vitro. Colonization of colitic IL-10-deficient mice with H. polygyrus suppresses LPMC IL-17 production and improves colitis. Ab-mediated blockade of IL-17 improves colitis in IL-10-deficient mice. Thus, helminth-associated inhibition of IL-17 production is most likely an important mechanism mediating protection from inappropriate intestinal inflammation.     

Cytokine-induced alterations of α7 nicotinic receptor in colonic CD4 T cells mediate dichotomous response to nicotine in murine models of Th1/Th17- versus Th2-mediated colitis

Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4(+)CD62L(+) T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory α7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in regulating colonic inflammation.     

On the mechanisms of T cell silencing by IL-10 DNA: direct and indirect inhibition of T cell functions

Previously we reported that mucosal IL-10 DNA administration resulted in long-term suppression of virus-induced inflammatory responses by silencing Th1-type CD4+ T cell functions. However, the mechanism by which IL-10 silences the activity of CD4+ T cells was not clear. The present report has shown that mucosal IL-10 DNA administration led to the reduction of reactivity of T cells following TCR stimulation. IL-10 DNA also downregulated APC functions to stimulate T cells but the effect was temporary. Bystander suppression, including that of IL-10 producing regulatory cells, appeared not to be directly involved in the inhibition of T cell reactivity because both anti-IL-10 and anti-IL-10R could not block the suppression of T cell functions. This silenced state could be maintained following adoptive transfer to untreated animals. The nature of the silencing appears to be a reversible anergic state since Ag stimulation in the presence of exogenous IL-2 restored T cell reactivity. Furthermore, IL-10-induced silenced T cells could be induced in vitro by culturing the T cells with rIL-10 in the presence or the absence of antigen stimulation. This state persisted in the absence of rIL-10 and persisted for at least 3 days. A more notable effect, however, was observed when the T cells were incubated with IL-10 in the presence of APC and Ag. These results indicate that IL-10 induced a long-term silenced state in T cells by direct and indirect inhibition of T cell functions.     

Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.     

Nasal anti-CD3 antibody ameliorates lupus by inducing an IL-10-secreting CD4+ CD25- LAP+ regulatory T cell and is associated with down-regulation of IL-17+ CD4+ ICOS+ CXCR5+ follicular helper T cells

Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4(+)CD25(+) regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4(+)CD25(-)latency-associated peptide (LAP)(+) regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-beta-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4(+)CD25(-)LAP(+) regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4(+)ICOS(+)CXCR5(+) follicular helper T cells that are required for autoantibody production. CD4(+)ICOS(+)CXCR5(+) follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4(+)CD25(-)LAP(+) regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.     

Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.     

Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR γ to suppress colitis

Background

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.

Conclusions/Significance

Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis.     

Regulation of the IL-10/IL-12 axis in human dendritic cells with probiotic bacteria

In this study, we have used monocyte-derived dendritic cells (DCs) to design a screening model for the selection of microorganisms with the ability to suppress DC-secreted IL-12p70, a critical cytokine for the induction of T-helper cell type 1 immune responses under inflammatory conditions. By the treatment of DCs with cocktails containing TLR agonists and proinflammatory cytokines, the cells increased the secretion of the Th1-promoting cytokine IL-12p70. Clinically used probiotics were tested for their IL-10- and IL-12p70-stimulating properties in immature DCs, and showed a dose-dependent change in the IL-10/IL-12p70 balance. Lactobacillus acidophilus NCFM(?) and the probiotic mixture VSL#3 showed a strong induction of IL-12p70, whereas Lactobacillus salivarius Ls-33 and Bifidobacterium infantis 35624 preferentially induced IL-10. Escherichia coli Nissle 1917 induced both IL-10 and IL-12p70, whereas the probiotic yeast Saccharomyces boulardii induced low levels of cytokines. When combining these microorganisms with the Th1-promoting cocktails, E. coli Nissle 1917 and B. infantis 35624 were potent suppressors of IL-12p70 secretion in an IL-10-independent manner, indicating a suppressive effect on Th1-inducing antigen-presenting cells. The present model, using cocktail-stimulated DCs with potent IL-12p70-stimulating capacity, may be used as an efficient tool to assess the anti-inflammatory properties of microorganisms for potential clinical use.     

Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.