Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice

Celiac disease (CD) is frequently diagnosed in patients with type 1 diabetes (T1D), and T1D patients can exhibit Abs against tissue transglutaminase, the auto-antigen in CD. Thus, gliadin, the trigger in CD, has been suggested to have a role in T1D pathogenesis. The objective of this study was to investigate whether gliadin contributes to enteropathy and insulitis in NOD-DQ8 mice, an animal model that does not spontaneously develop T1D. Gliadin-sensitized NOD-DQ8 mice developed moderate enteropathy, intraepithelial lymphocytosis, and barrier dysfunction, but not insulitis. Administration of anti-CD25 mAbs before gliadin-sensitization induced partial depletion of CD25(+)Foxp3(+) T cells and led to severe insulitis, but did not exacerbate mucosal dysfunction. CD4(+) T cells isolated from pancreatic lymph nodes of mice that developed insulitis showed increased proliferation and proinflammatory cytokines after incubation with gliadin but not with BSA. CD4(+) T cells isolated from nonsensitized controls did not response to gliadin or BSA. In conclusion, gliadin sensitization induced moderate enteropathy in NOD-DQ8 mice. However, insulitis development required gliadin-sensitization and partial systemic depletion of CD25(+)Foxp3(+) T cells. This humanized murine model provides a mechanistic link to explain how the mucosal intolerance to a dietary protein can lead to insulitis in the presence of partial regulatory T cell deficiency.     

Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+ and Foxp3-CD25- CD4+ regulatory T cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (T(reg)). We found that total T(reg), KJ1-26+ T(reg) and CTLA-4+ T(reg) were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by co-administering cholera toxin or by treatment with anti-TGF-beta mAb. CD25+ T(reg), but also CD25-CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ T(reg) from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ T(reg). Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3-CD25-CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ T(reg) cells, which expressed Foxp3 more strongly than naturally developing T(reg) and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25- T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ T(reg) together with the generation of both Foxp3+ and Foxp3-CD25- CD4+ T(reg).     

pConsensus peptide induces tolerogenic CD8+ T cells in lupus-prone (NZB x NZW)F1 mice by differentially regulating Foxp3 and PD1 molecules

Systemic lupus erythematosus is an autoimmune disease caused primarily by autoantibodies (including IgG anti-DNA) and immune complexes that cause tissue damage. After tolerization with an artificial peptide (pConsensus, pCons) based on murine anti-DNA IgG sequences containing MHC class I and class II T cell determinants, lupus-prone (NZB x NZW)F(1) female (BWF(1)) mice develop regulatory CD4+CD25+ T cells and inhibitory CD8+ T cells, both of which suppress anti-DNA Ig production and immune glomerulonephritis. In the present work, we show that splenocytes from BWF(1) mice treated with pCons had significant expansion of primarily CD8+ T cells. CD4+ T cells and B cells were each directly suppressed by CD8+ T cells from tolerized mice in a contact-independent manner. Both pCons-induced CD8+CD28+ and CD8+CD28- T cells suppressed production of anti-DNA in vitro. Silencing with small interfering RNA of Foxp3 abrogated the suppression mediated by both CD8+ T cell subsets. Additionally, CD8+ T cells from tolerized mice were weakly cytotoxic against syngeneic B cells from old anti-DNA-producing mice, but not from young mice. Importantly, pCons treatment had dual effects on CD8+ suppressor T cells from tolerized mice, increasing the intracellular expression of Foxp3 while decreasing the surface expression of PD1 molecules. Blocking PD1/PDL1 interactions in the CD8+ T cells from tolerized mice reduced their expression of Foxp3 and their ability to suppress CD4+CD25- proliferation. In contrast, blocking PD1/PDL1 in naive T cells increased Foxp3 expression. Our data suggest that tolerization with pCons activates different subsets of inhibitory/cytotoxic CD8+ T cells whose targets are both CD4+CD25- effector T cells and B cells.     

TNFα regulates the localization of CD40 in lipid rafts of glioma cells

Resistance of glioblastoma multiforme (GBM) to TNFα induced apoptosis is attributed to NFκB activation. As TNF-receptor family member CD40 regulates NFκB activation, we investigated the role of CD40 in NFκB activation in GBM. We observed elevated CD40 levels in human glioma samples as compared to the surrounding normal tissue. Treatment with TNFα elevated CD40 levels in glioma cells and inhibition of CD40 signaling failed to abrogate TNFα induced NFκΒ activity. While TNFα increased the interaction between TRAF2/6, IκBα, IKKα/β in the CD40 signalosome, the level of CD40 in the signalosome remained unaffected upon TNFα treatment. Interestingly, TNFα decreased the spatial localization of CD40 and increased TRAF2/6 co-localization with lipid raft marker Caveolin. As localization of CD40 signalosome in lipid raft is crucial for NFκB activation, TNFα mediated decreased clustering of CD40 in lipid rafts could have possibly contributed to its non-involvement in NFκB activation.     

The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava

Background

The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses.

Methods

To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included ³H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA).

Results

SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3⁺ mature T cells and CD45R/B220⁺ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG_1a, and IgG_2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP.

Conclusion

These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways.

General significance

The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.     

Bifidobacterium CECT 7765 improves metabolic and immunological alterations associated with obesity in high‐fat diet‐fed mice

Objectives: To evaluate the effects of administration of Bifidobacterium pseudocatenulatum CECT 7765 on metabolic and immune alterations in obese mice. Design and Methods: Adult male wild‐type C57BL‐6 mice were fed a standard diet or high‐fat diet (HFD), supplemented or not with B. pseudocatenulatum CECT 7765 for 7 weeks. The assessments included biochemical and immunological parameters, insulin resistance, glucose tolerance, histology of liver, white‐adipose and intestinal tissues, immunocompetent cell functions, and microbiota‐related features. Results: B. pseudocatenulatum CECT 7765 reduced serum cholesterol, triglyceride, and glucose levels and decreased insulin resistance and improved glucose tolerance in obese mice. This strain reduced serum levels of leptin, interleukin (IL)‐6 and monocyte chemotactic protein‐1, while increased those of IL‐4 in HFD‐fed mice. B. pseudocatenulatum CECT7765 reduced liver steatosis and the number of larger adipocytes and number of fat micelles in enterocytes of obese mice. The strain also improved the function of macrophages and dendritic cells in relation to phagocytosis, cytokine production, and induction of T‐lymphocyte proliferation. The strain administration increased bifidobacteria and reduced enterobacteria and the inflammatory properties of the gut content in HFD‐fed mice. Conclusion: B. pseudocatenulatum CECT 7765 was shown to ameliorate both metabolic and immunological dysfunctions related to obesity in HFD‐fed mice.     

Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice

Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25(high) cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25-Foxp3+ or CD25(low)Foxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25(high)Foxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25-Foxp3+ but not from the conversion of peripheral Foxp3-) cells. CD25(high)Foxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25-Foxp3+ cells and/or conversion of CD4+CD25-Foxp3- cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-beta is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.     

Gliadin fragments induce phenotypic and functional maturation of human dendritic cells

Celiac disease is a chronic inflammatory disease developing in genetically predisposed individuals. Ingested gliadin, the triggering agent of the disease, can cross the epithelial barrier and elicit a harmful T cell-mediated immune response. Dendritic cells (DC) are supposed to play a pivotal role in shaping the immune response. The direction of the immune response toward immunity or tolerance depends on the stage of maturation and the functional properties of the DC. DC become fully functional APC upon maturation by various stimuli. We investigated the effect of a peptic digest of gliadin on the maturation of human monocyte-derived DC. Stimulation of cells with gliadin, in contrast with other tested food proteins, led to enhanced expression of maturation markers (CD80, CD83, CD86, and HLA-DR molecules) and increased secretion of chemokines and cytokines (mainly of IL-6, IL-8, IL-10, TNF-alpha, growth-related oncogene, MCP-1, MCP-2, macrophage-derived chemokine, and RANTES). Maturation was accompanied by a greater capacity to stimulate proliferation of allogeneic T cells and significantly reduced endocytic activity. Furthermore, gliadin-induced phosphorylation of members of three MAPK families (ERK1/2, JNK, and p38 MAPK) was demonstrated. The largest contribution of p38 MAPK was confirmed using its inhibitor SB203580, which markedly down-regulated the gliadin-triggered up-regulation of maturation markers and cytokine production. Gliadin treatment also resulted in increased NF-kappaB/DNA binding activity of p50 and p65 subunits. Taken together, gliadin peptides can contribute to overcoming the stage of unresponsiveness of immature DC by inducing phenotypic and functional DC maturation, resulting in more efficient processing and presentation of gliadin peptides to specific T lymphocytes.     

Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kappaB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kappaB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to na?ve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kappaB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.     

Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)γ and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation and mucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-γ and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection.     

High TCR stimuli prevent induced regulatory T cell differentiation in a NF-κB-dependent manner

The concentration of Ag or mitogenic stimuli is known to play an important role in controlling the differentiation of naive CD4(+) T cells into different effector phenotypes. In particular, whereas TCR engagement at low Ag doses in the presence of TGF-β and IL-2 can promote differentiation of Foxp3-expressing induced regulatory T cells (iTregs), high levels of Ag have been shown in vitro and in vivo to prevent Foxp3 upregulation. This tight control of iTreg differentiation dictated by Ag dose most likely determines the quality and duration of an immune response. However, the molecular mechanism by which this high-dose inhibition of Foxp3 induction occurs is not well understood. In this study, we demonstrate that when cells are in the presence of CD28 costimulation, TCR-dependent NF-κB signaling is essential for Foxp3 inhibition at high doses of TCR engagement in mouse T cells. Prevention of Foxp3 induction depends on the production of NF-κB-dependent cytokines by the T cells themselves. Moreover, T cells that fail to upregulate Foxp3 under iTreg-differentiating conditions and high TCR stimulation acquire the capacity to make TNF and IFN-γ, as well as IL-17 and IL-9. Thus, NF-κB helps T cells control their differentiation fate in a cell-intrinsic manner and prevents peripheral iTreg development under conditions of high Ag load that may require more vigorous effector T cell responses.     

NFκB regulates expression of Polo-like kinase 4

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.     

Bacteroides uniformis CECT 7771 Ameliorates Metabolic and Immunological Dysfunction in Mice with High-Fat-Diet Induced Obesity

Background

Associations have been made between obesity and reduced intestinal numbers of members of the phylum Bacteroidetes, but there is no direct evidence of the role these bacteria play in obesity. Herein, the effects of Bacteroides uniformis CECT 7771 on obesity-related metabolic and immune alterations have been evaluated.

Methods and Findings

Adult (6–8 week) male wild-type C57BL-6 mice were fed a standard diet or a high-fat-diet HFD to induce obesity, supplemented or not with B. uniformis CECT 7771 for seven weeks. Animal weight was monitored and histologic, biochemical, immunocompetent cell functions, and features of the faecal microbiota were analysed after intervention. The oral administration of B. uniformis CECT 7771 reduced body weight gain, liver steatosis and liver cholesterol and triglyceride concentrations and increased small adipocyte numbers in HFD-fed mice. The strain also reduced serum cholesterol, triglyceride, glucose, insulin and leptin levels, and improved oral tolerance to glucose in HFD fed mice. The bacterial strain also reduced dietary fat absorption, as indicated by the reduced number of fat micelles detected in enterocytes. Moreover, B. uniformis CECT 7771 improved immune defence mechanisms, impaired in obesity. HFD-induced obesity led to a decrease in TNF-α production by peritoneal macrophages stimulated with LPS, conversely, the administration of B. uniformis CECT 7771 increased TNF-α production and phagocytosis. Administering this strain also increased TNF-α production by dendritic cells (DCs) in response to LPS stimulation, which was significantly reduced by HFD. B. uniformis CECT 7771 also restored the capacity of DCs to induce a T-cell proliferation response, which was impaired in obese mice. HFD induced marked changes in gut microbiota composition, which were partially restored by the intervention.

Conclusions

Altogether, the findings indicate that administration of B. uniformis CECT 7771 ameliorates HFD-induced metabolic and immune dysfunction associated with intestinal dysbiosis in obese mice.     

Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells

CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IκBα degradation. Inhibition of NFκB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon γ (IFNγ) levels in the medium of PBMCs. Neutralization of IFNγ and IFNγ receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS-induced CD40 expression on human PBMCs through activation of NFκB and JNK, and partially through the induction of IFNγ production.     

A function for IL-7R for CD4+CD25+Foxp3+ T regulatory cells

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25-Foxp3low T cells. Interestingly, common gamma chain (gammac) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25-Foxp3low subset. Therefore, other gammac-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the gammac-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rbeta and IL-7Ralpha contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the gammac knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rbeta in mice double deficient in IL-2Rbeta and IL-7Ralpha established that IL-2Rbeta is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rbeta-/- mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.     

Prevention or Early Cure of Type 1 Diabetes by Intranasal Administration of Gliadin in NOD Mice

Induction of long-term tolerance to β-cell autoantigens has been investigated both in animal models and in human type 1 diabetes (T1D) in order to prevent the disease. As regards external compounds, the dietary plant protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevent T1D in animal models. Herewith we investigated whether intranasal (i.n.) administration of gliadin or gluten may arrest the diabetogenic process. I.n. administration of gliadin to 4-week-old NOD mice significantly reduced the diabetes incidence. Similarly, the insulitis was lowered. Intranasal gliadin also rescued a fraction of prediabetic 13-week-old NOD mice from progressing to clinical onset of diabetes compared to OVA-treated controls. Vaccination with i.n. gliadin led to an induction of CD4⁺Foxp3⁺ T cells and even more significant induction of γδ T cells in mucosal, but not in non-mucosal lymphoid compartments. This prevention strategy was characterized by an increased proportion of IL-10 and a decreased proportion of IL-2, IL-4 and IFN-γ-positive CD4⁺Foxp3⁺ T cells, and IFN-γ-positive γδ T cells, preferentially in mucosal lymphoid organs. In conclusion, i.n. vaccination with gliadin, an environmental antigen with possible etiological influence in T1D, may represent a novel, safer strategy for prevention or even early cure of T1D.