Mesenteric Lymph Node CD4 Research Articles

Lactococcus lactis subsp. cremoris YRC3780 modifies function of mesenteric lymph node dendritic cells to modulate the balance of T cell differentiation inducing regulatory T cells.

The intestinal immune system plays a pivotal role in the induction of immune responses against food. In the case of T cell response, dendritic cells (DCs) are especially important. However, the regulation of immune responses to food by intestinal DCs has been poorly described. In this study, we analyzed the effect of Lactococcus lactis subsp. cremoris YRC3780, a lactic acid bacterial strain isolated from kefir, a traditional fermented milk product, on the immune responses induced by antigen presentation by intestinal DCs to T cells as well as the mechanism of action of these immunomodulatory effects. It has been shown that L. cremoris YRC3780 ameliorates the symptoms of pollinosis in both animal and human studies. CD11c+ cells from mesenteric lymph nodes (MLNs) of BALB/c mice were cultured as MLN DCs with L. cremoris YRC3780 and expression of genes inducing regulatory T cells (Tregs) was examined by qPCR. In addition, MLN DCs were cocultured with CD4+ T cells from DO11.10 transgenic mice expressing an ovalbumin (OVA)-specific TCR and the OVA antigen peptide and L. cremoris YRC3780. Induction of Tregs was examined by flow cytometry, gene expression was analyzed by DNA microarray and qPCR, and the production of cytokines was measured by ELISA. MLN DCs from TLR2-deficient mice and components of L. cremoris YRC3780 were used to examine the recognition of YRC3780 by MLN DCs. L. cremoris YRC3780 enhanced the expression of genes involved in Treg induction in MLN DCs and induced Foxp3+CD4+T cells in an MLN DC and CD4+ T-cell co-culture system. The effect on MLN DCs was likely mediated by receptors other than TLR2. Together with microarray analyses of CD4+ T cell gene expression and cytokine ELISA, it was demonstrated that L. cremoris YRC3780 promoted the induction of Th1 and Tregs, and regulated the balance of Th1/Th2 and Treg/Th17 cells involving multiple genes via the antigen-presentation of MLN DCs. Our findings provide insights into the modulation of intestinal immune responses mediated by DCs and the antiallergic effects of lactic acid bacteria.

Opposing Roles of DCs and iNKT Cells in the Induction of Foxp3 Expression by MLN CD25+CD4+ T Cells during IFNγ-Driven Colitis

We have previously shown that a deficiency of CD1d-restricted invariant natural killer T (iNKT) cells exacerbates dextran sulfate sodium (DSS)-induced colitis in Yeti mice that exhibit IFNγ-mediated hyper-inflammation. Although iNKT cell-deficiency resulted in reduced Foxp3 expression by mesenteric lymph node (MLN) CD4+ T cells in DSS-treated Yeti mice, the cellular mechanisms that regulate Foxp3 expression by CD25+CD4+ T cells during intestinal inflammation remain unclear. We found that Foxp3-CD25+CD4+ T cells expressing Th1 and Th17 phenotypic hallmarks preferentially expanded in the MLNs of DSS-treated Yeti/CD1d knockout (KO) mice. Moreover, adoptive transfer of Yeti iNKT cells into iNKT cell-deficient Jα18 KO mice effectively suppressed the expansion of MLN Foxp3-CD25+CD4+ T cells during DSS-induced colitis. Interestingly, MLN dendritic cells (DCs) purified from DSS-treated Yeti/CD1d KO mice promoted the differentiation of naive CD4+ T cells into Foxp3-CD25+CD4+ T cells rather than regulatory T (Treg) cells, indicating that MLN DCs might mediate Foxp3+CD25+CD4+ T cell expansion in iNKT cell-sufficient Yeti mice. Furthermore, we showed that Foxp3-CD25+CD4+ T cells were pathogenic in DSS-treated Yeti/CD1d KO mice. Our result suggests that pro-inflammatory DCs and CD1d-restricted iNKT cells play opposing roles in Foxp3 expression by MLN CD25+CD4+ T cells during IFNγ-mediated intestinal inflammation, with potential therapeutic implications.

Effects of Different Routes and Forms of Vitamin D Administration on Mesenteric Lymph Node CD4+ T Cell Polarization and Intestinal Injury in Obese Mice Complicated with Polymicrobial Sepsis.

This study compared the efficacies of enteral cholecalciferol and/or intravenous (IV) calcitriol administration on mesenteric lymph node (MLN) cluster-of-differentiation-4-positive (CD4+) T cell distribution and intestinal barrier damage in obese mice complicated with sepsis. Mice were fed a high-fat diet for 16 weeks and then sepsis was induced by cecal ligation and puncture (CLP). Mice were divided into the following sepsis groups: without vitamin D (VD) (S); with oral cholecalciferol 1 day before CLP (G); with IV calcitriol 1 h after CLP (V); and with both cholecalciferol before and IV calcitriol after CLP (GV). All mice were sacrificed at 12 or 24 h after CLP. The findings show that the S group had a higher T helper (Th)17 percentage than the VD-treated groups at 12 h after CLP. The V group exhibited a higher Th1 percentage and Th1/Th2 ratio than the other groups at 24 h, whereas the V and GV groups had a lower Th17/regulatory T (Treg) ratio 12 h post-CLP in MLNs. In ileum tissues, the VD-treated groups had higher tight junction protein and cathelicidin levels, and higher mucin gene expression than the S group at 24 h post-CLP. Also, aryl hydrocarbon receptor (AhR) and its associated cytochrome P450 1A1 and interleukin 22 gene expressions were upregulated. In contrast, levels of lipid peroxides and inflammatory mediators in ileum tissues were lower in the groups with VD treatment after CLP. These results suggest that IV calcitriol seemed to have a more-pronounced effect on modulating the homeostasis of Th/Treg subsets in MLNs. Both oral cholecalciferol before and IV calcitriol after CLP promoted cathelicidin secretion, alleviated intestinal inflammation, and ameliorated the epithelial integrity in obese mice complicated with sepsis possibly via VD receptor and AhR signaling pathways.

Intravenous calcitriol administration modulates mesenteric lymph node CD4+ T-cell polarization and attenuates intestinal inflammation in obese mice complicated with polymicrobial sepsis.

Sepsis is a lethal syndrome with T-cell dysregulation, imbalanced inflammatory reactions, and gastrointestinal dysfunction. Obesity coexistent with sepsis can cause more-deleterious disease outcomes. Vitamin D is a nutrient with immunomodulatory ability and helps maintain intestinal homeostasis. This study investigated treatment with calcitriol on mesenteric lymph node (MLN) CD4+ T-cell polarization and intestinal injury in obese mice with sepsis. Mice received a high-fat diet for 10 weeks; then, mice were separated into an obese control group without sepsis and sepsis groups that underwent cecal ligation and puncture (CLP). Septic mice were subdivided into a group that was injected with saline (SS group) or a group that was injected with calcitriol (SD group) via a tail vein 1 h after CLP. Obese mice with sepsis were euthanized at 12 or 24 h post CLP. Sepsis resulted in increased percentages of type 2 T helper (Th2), Th17, and regulatory T (Treg) cells in MLNs. Also, inflammation-associated genes were upregulated and tight junction genes downregulated in the intestines after CLP. Compared with the SS group, the SD group exhibited reduced Th2, Th17, and Treg percentages in MLNs. Also, intestinal inflammatory chemokine expressions were reduced, whereas MUC2, ZO-1, and occludin had increased after CLP. Lower inflammatory cytokine levels in peritoneal lavage fluid in the ileum were also noted in the SD group. Intravenous calcitriol treatment after sepsis can elicit more-balanced CD4 T-cell subsets in lymph nodes near the intestines and alleviate intestinal inflammation and injury in obese mice complicated with sepsis.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Inducing a stressed phenotype in healthy recipient mice by adoptively transferring CD4+ lymphocytes from mice undergoing chronic psychosocial stress

Although chronic stress is an acknowledged risk factor for the development of somatic and affective disorders, the cellular and molecular mechanisms underlying stress-induced pathologies are not fully understood. Interestingly, rodent studies involving immune cell transfer suggest that CD4+ T cells might be at least in part involved in reactivation of a chemically-induced colitis by stress. However, until now evidence is lacking that these immune cell types are indeed involved in the development of a “stressed phenotype”.The aim of the present study was, therefore, to assess the effects of adoptively transferring total mesenteric lymph node cells (mesLNCs) and CD4+ mesLNCs isolated from chronically-stressed mice into healthy recipient mice on various physiological, immunological and behavioral parameters. To induce chronic psychosocial stress in donor mice we employed the chronic subordinate colony housing (CSC) paradigm.Our data indicate that transfer of total or CD4+ mesLNCs from CSC mice, compared with respective cells from single-housed control (SHC) mice, promoted splenomegaly and interferon (IFN)-γ secretion from in vitro anti-CD3-stimulated mesLNCs in naïve recipient mice. This effect was independent of recipient mice additionally being administered with dextran sulfate sodium (DSS) or not. Transfer of CD4+ mesLNCs additionally increased adrenal weight and secretion of IL-6 from in vitro anti-CD3 stimulated mesLNCs in recipients administered with DSS. Importantly, transfer of neither cell type from CSC vs. SHC donor mice affected anxiety-related behavior of recipient mice in the light-dark box.Taken together, our data demonstrate that typical physiological and immunological, but not behavioral, effects of chronic stress can be induced in naïve recipient mice by adoptively transferring mesLNCs, in particular CD4+ mesLNCs, from chronically stressed donor mice.

CD55 Is Essential for CD103+ Dendritic Cell Tolerogenic Responses that Protect against Autoimmunity

Recent studies traced inflammatory bowel disease in some patients to deficiency of CD55 [decay-accelerating factor (DAF)], but the mechanism underlying the linkage remained unclear. Herein, we studied the importance of DAF in enabling processes that program tolerance in the gut and the eye, two immune-privileged sites where immunosuppressive responses are continuously elicited. Unlike oral feeding or ocular injection of ovalbumin in wild-type (WT) mice, which induced dominant immune tolerance, identical treatment of DAF-/- mice or DAF-/- to WT bone marrow chimeras did not. While 10% to 30% of mesenteric and submandibular lymph node CD4+ cells became robust T-regulatory cells (Tregs) in WT forkhead box P3 (Foxp3)-green fluorescent protein mice, few in either site became Tregs with little suppressor activity in DAF-/- Foxp3-green fluorescent protein mice. Phenotyping of CD103+ dendritic cells (DCs) from the ovalbumin-fed DAF-/- mice showed impaired expression of inducer of costimulation (ICOS) ligand, programmed death receptor 1-ligand 1 (PD1-L1), CxxxC chemokine receptor 1 (Cx3CR1), CCR7, and CCR9. Analyses of elicited DAF-/- Foxp3+ Tregs showed reduced expression of interferon regulatory factor 8 (IRF-8)/aldehyde dehydrogenase 1 family member A2 (Aldh1a2) and glycoprotein A repetitions predominant/latency-associated protein associated with Treg transforming growth factor-β production and presentation, as well as integrin β6/integrin β8 associated with Treg and CD103+ DC transforming growth factor-β release. Thus, DAF is required for the properties of CD103+ DCs and their naïve CD4+ cell partners that together program tolerance.

Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential.

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer’s patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer’s patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαβ. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αβ+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.

Mesenteric lymph node CD4+ T lymphocytes migrate to liver and contribute to non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is characterized by altered intestinal microbiota and intestinal immune disorder. Here we investigated the role of mesenteric lymph node (MLN) CD4+ T lymphocytes in NAFLD. In high fat diet (HFD)-fed mice, the percentage ratios of Th1 to Th2 cells and Th17 to Treg cells were imbalanced in MLNs. Co-culture assays showed MLN CD4+ T lymphocytes from HFD-fed mice tended to migrate to the liver and promoted hepatic inflammation. Adoptive transfer of MLN CD4+ T lymphocytes from NAFLD mice to HFD-fed mice resulted in higher transaminase, worse hepatic inflammation and lipid accumulation. Antibiotics and probiotics were administrated to regulate intestinal microbiota, and the restoration of MLN Th1/Th2 and Th17/Treg cells in alleviated NAFLD were found. In summary, MLNs CD4+ T subtype cells may involve in NAFLD, and the restoration of MLN CD4+ T subtype cells ratio by regulating intestinal bacteria could be the new strategies.

Reduced early life mucosal integrity decreases thymic cell counts and increases local, but not thymic regulatory, T cell recruitment: Gut mucosal integrity breach and thymic T cells

Early life immune gut microbiota contact is critical for regulatory T cell–mediated oral tolerance induction. We induced a mucosal integrity breach with low dextran sulfate sodium dose right after weaning in BALB/c mice along with a standard high dose to study the impact of increased gut microbiota lymphatic tissue contact on the thymus. Both doses increased gut permeability, which caused a short-term generalized thymic involution and regulatory T cell induction in the mesenteric lymph nodes, even in the absence of clinically apparent inflammation in the low-dose group. The thymic regulatory T cells resisted thymic involution. In the low-dose group, we found acutely altered gut mobilization patterns characterized by changed gut-homing marker CD103 expression on mesenteric lymph node CD4+T cells as well as on mature CD8+T cells and developing CD4−/CD8−thymocytes. Furthermore, CD218a (IL-18-receptor-a) expression was acutely decreased on both mature CD8+T cells and regulatory T cells, while increased on the mesenteric lymph node CD8+T cells, indicating a direct link between the thymus and the mesenteric lymph nodes with CD218a in a functional role in thymic involution. Acute and non-persisting regulatory responses in the mesenteric lymph nodes were induced in the form of a relative regulatory T cell increase. We saw no changes in total thymic regulatory T cells and thus the thymus does not seem to play a major role of in the regulatory immunity induced by increased gut microbiota lymphatic tissue contact around weaning, which in our study primarily was located to the gut.

Helicobacter pylori and its secreted immunomodulator VacA protect against anaphylaxis in experimental models of food allergy.

Food allergy is an increasingly common health problem in Western populations. Epidemiological studies have suggested both positive and negative associations between food allergy and infection with the gastric bacterium Helicobacter pylori. The objective of this work was to investigate whether experimental infection with H. pylori, or prophylactic treatment with H. pylori-derived immunomodulatory molecules, affects the onset and severity of food allergy, either positively or negatively. We infected neonatal C57BL/6 or C3H mice with H. pylori or treated animals with H. pylori components (bacterial lysate or the immunomodulator VacA) and subsequently subjected them to four different protocols for food allergy induction, using either ovalbumin or peanut extract as allergens for sensitization and challenge. Readouts included anaphylaxis scoring, quantification of allergen-specific serum IgE and IgG1 and of the mast cell protease MCPT1, as well as splenic T-helper-2 cell-derived cytokine production. Mesenteric lymph node CD4+ FoxP3+ regulatory T cells were subjected to flow cytometric quantification and sorting followed by qRT-PCR, and to DNA methylation analyses of the Treg-specific demethylated region (TSDR) within the FOXP3 locus. Mice that had been infected with H. pylori or treated with H. pylori-derived immunomodulators showed reduced anaphylaxis upon allergen sensitization and challenge, irrespective of the allergen used. Most of the immunologic assays confirmed a protective effect of H. pylori. CD4+ FoxP3+ T cells were more abundant in protected mice and exhibited a stable Treg phenotype characterized by FOXP3 TSDR demethylation. Helicobacter pylori confers protection against the anaphylaxis associated with ovalbumin and peanut allergy and affects the epigenome of T cells, thereby promoting stable Treg differentiation and functionality. Prophylactic treatment with H. pylori-derived immunomodulators appears to be a promising strategy for food allergy prevention.

Immune responses to killed reassorted influenza virus supplemented with natural adjuvants.

In this study, we investigated the immunomodulatory effects of a supplemented killed influenza virus (V) by Echinacea purpurea (E) and Nigella sativa (N) extracts and effect of changing the route of immunization from intramuscular (IM) to intraperitoneal (IP). At the 2nd-, 3rd- and 4th-week post-IM immunizations (WPIMI), the supplemented V with N (VN) induced the most significant IgM response unlike N alone. At the 2nd WPIMI, V or VN induced the highest significant IgG levels. At the 2nd-week post-IP immunization (WPIPI), E and VN induced the most significant IgG levels. Both at the 3rd and 4th WPIMI or WPIPI, various treatments induced significant increases in IgG. At the 4th WPIMI, E, V, and V with E (VE) induced significant increases in the CD4+ thymocytes while all IP treatments caused significant increase in their counts. V and VN induced the most significant IM induction of CD8+ thymocytes while their best IP stimulation was induced by N, VE, and VN. At the 4th WPIMI, various treatments caused significant increases in the mesenteric lymph node (MLN) CD4+, CD8+ counts. WPIPI with V or VE caused significant increases in both the CD4+- and CD8+ MLN cells, whereas VN significantly induced CD8+ MLN cells only. WPIPI with various treatments caused significant increases in the B-cell counts and the peak was obtained by VN.

Forkhead box protein 3 demethylation is associated with tolerance induction in peanut-induced intestinal allergy

Regulatory T (Treg) cells play an essential role in the maintenance of immune homeostasis in allergic diseases. We sought to define the mechanisms underlying induction of tolerance to peanut protein and prevention of the development of peanut allergy. High or low doses of peanut extract were administered to pups every day for 2weeks before peanut sensitization and challenge. After challenge, symptoms, Treg cell numbers, and forkhead box protein 3 (Foxp3), TH2 and TH17 cytokine, and Tgfβ expression in mesenteric lymph node (MLN) CD4+ T cells and jejunum were monitored. Treg cell suppressive activity and Foxp3 methylation in MLN CD4+ T cells were assayed. Feeding high but not low doses of peanut before sensitization induced tolerance, as demonstrated by prevention of diarrhea and peanut-specific IgE responses, increases in the percentage of CD4+CD25+FoxP3+ cells in MLNs, and Foxp3 mRNA and protein expression in CD4+ cells from MLNs or jejunum. Feeding high doses of peanut before sensitization decreased percentages of CD3+CD4+IL-13+ and CD3+CD4+IL-17+ cells in MLNs and decreased Il13 and Il17a and increased Tgfβ mRNA expression in the jejunum; numbers of CD103+ dendritic cells in MLNs were significantly increased. Treg cell suppression was shown to be antigen specific. Foxp3 methylation was increased in peanut extract-sensitized and challenged mice, whereas in tolerized mice levels were significantly reduced. Feeding high doses of peanut to pups induced tolerance to peanut protein. Foxp3 demethylation was associated with tolerance induction, indicating that Treg cells play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis.

Th1/Th2 Balance and Th17/Treg-Mediated Immunity in relation to Murine Resistance to Dextran Sulfate-Induced Colitis.

Background The role of the Th17/Treg balance in the development of experimental colitis remains poorly understood. Methods We exploited the differential response of BALB/c mice and C57BL/6 mice towards drinking water mediated by dextran sulfate sodium (DSS) challenge. Results DSS-resistant BALB/c mice were characterized by low levels of IFN-γ and TNF-α but high levels of IL-4, IL-6, IL-10, IL-17A, IL-17F, and colon lamina propria and mesenteric lymph node (MLN) CD4+CD25+FoxP3+ T cells when compared to C57BL/6 mice. Collectively, these data indicate the propensity of BALB/c mice towards a Th2/Th17/Treg-polarized immunity protecting these animals against DSS challenge, whereas Th1-polarization of C57BL/6 mice confers sensitivity to DSS-induced colitis. Conclusions The intrinsic congenital capacity of mouse strains with respect to T cell proliferation determines sensitivity to experimental colitis.

Physiological Role of K<sup>+</sup> Channels in the Regulation of T Cell Function

Potassium ion (K(+)) channels play an important role in the modulation of calcium ion (Ca(2+)) signaling via control of the membrane potential. In T-lymphocytes, the voltage-gated K(+) channel, KV1.3, and the intermediate-conductance Ca(2+)-activated K(+) channel, KCa3.1, predominantly contribute to K(+) conductance, and are responsible for cell proliferation, differentiation, apoptosis and infiltration. Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, afflicts more than 0.1% of the population worldwide. In the chemically-induced IBD model mouse, an increase in KCa3.1 activity was observed in mesenteric lymph node CD4(+) T-lymphocytes, concomitant with an upregulation of KCa3.1 and a positive KCa3.1 regulator, NDPK-B. Pharmacological blockade of the KCa3.1 K(+) channel by TRAM-34 and/or ICA17043 elicited 1) a significant decrease in IBD severity, as assessed by diarrhea, visible fecal blood, inflammation and crypt damage of the colon; and 2) restoration of the expression levels of KCa3.1 and Th1 cytokines in CD4(+) T-lymphocytes in the IBD model. Recent studies have indicated the impact of K2P5.1 upregulation in T lymphocytes on the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. The K2P5.1 K(+) channel is therefore highlighted as a potent therapeutic target in managing the pathogenesis of autoimmune diseases. Alternatively, pre-mRNA splicing of ion channels is associated with the development and progression of various diseases, including autoimmune diseases. Therefore, mRNA-splicing mechanisms underlying the transcriptional regulation of K2P5.1 K(+) channels may be a new strategic therapeutic target for autoimmune and inflammatory diseases.

Forkhead Box Protein 3 (FoxP3) Demethylation Is Associated with Tolerance Induction in Peanut-Induced Intestinal Allergy

The role of T regulatory cells (Tregs) in the control of peanut sensitivity and tolerance induction is not well-defined. High (0.5 mg/gram body weight) or low doses (0.05 mg/gram body weight) of peanut extract (PE) were administered to pups every day for 2 weeks prior to peanut sensitization and challenge. Anti-CD25 was administered prior to peanut sensitization and challenge. Symptoms, intestinal inflammation, and Treg numbers in mesenteric lymph nodes (MLN) were monitored. FoxP3 methylation in genomic DNA from MLN CD4+T cells was detected by bisulfite pyrosequencing. Feeding high but not low doses of peanut prior to sensitization induced tolerance to peanut protein, prevented diarrhea and intestinal inflammation and increased the percentage of CD4+CD25+FoxP3+cells in MLN. Mice treated with anti-CD25 reversed established tolerance with reappearance of all manifestations of sensitivity. FoxP3 methylation within the intronic 1 region was significantly increased in sham-fed and PE sensitized and challenged mice compared to non-sensitized but challenged controls. In parallel to induction of tolerance, levels of FoxP3 methylation in 7 of the 9 CpG sites in the intronic 1 region were significantly reduced, to baseline levels, in mice fed high doses of peanut prior to PE sensitization and challenge. Tregs play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis. FoxP3 demethylation was associated with tolerance induction to peanut protein.

Effects on expression levels of surface immune molecules on T cell subsets in spleens and mesenteric lymph node of mice irradiated by 60Co γ-ray lethally total body irradiation

Objective To explore the effects of 60Co γ-ray lethally total body irradiation (TBI) on activated molecules and adhesion molecules of T cells from mice spleens and mesenteric lymph nodes. Methods From July to October 2012, a total of 12 male BALB/c mice of specefic pathogen free (SPF) level were selected as experimental objects. Inclusion criteria of experimental objects were 10 to 12 week-ages and 20 to 25 g weight. The mice were divided randomly into TBI group (accepted lethally TBI) and control group (not accepted irradiation), 6 mice per group. TBI group mice were exposure to 60Co γ-ray for one time (dosage rate was 0.66 Gy/min and total dose was 7.5 Gy). All the mice were killed by broken spines on the 7th days after irradiation and obtained spleens and mesenteric lymph nodes. Spleens and mesenteric lymph nodes were ground and mononuclear cells were collected using density gradient centrifugation. The mononuclear cells were stimulated and cultured, then labelled with antibodies. The expressions of CD44 and CD62L on CD4+ T, CD8+ T, helper T cell (Th) 1 and cytotoxic T cell (Tc) 1 were detected by flow cytometry. And the levels of above immune molecules of two group were analysed by statistical methods. Results On the 7th days after irradiation, in TBI group, percentages of CD44+ CD4+ T cell were (88.1±1.1)% in spleen and (76.7±1.8)% in mesenteric lymph node, which were lower than those in control group [spleen (95.0±1.1)% and mesenteric lymph node (92.8±1.4)%] (t=-11.01, -17.82; P 0.05). In TBI group, percentages of CD44+ Tc1 [spleen (65.5±4.6)% and mesenteric lymph node (26.6±2.8)%] were higher than those in control group [spleen (41.8±1.5)% and mesenteric lymph node (18.7±1.3)%] (t=9.97, 6.51; P<0.05). The expressions of CD62L in spleens and mesenteric lymph nodes CD4+ T and CD8+ T cells of TBI group were lower than those of control group [spleen (0.3±0.0)% vs (66.7±4.7)%, (1.1±0.2)% vs (64.4±5.1)%, t=-24.52, -21.53, P<0.05; mesenteric lymph node (2.6±0.3)% vs (89.7±2.8)%, (9.1±0.5) vs (79.4±3.3)%, t=-53.75, -36.55, P<0.05]. Conclusions T cells in spleens and mesenteric lymph nodes were activated with immunotype changes after TBI, manifesting as increased activated molecule CD44 expression and down regulated adhesion molecule CD62L expression on effector T cells. The expression levels of CD44 and CD62L might be indicators of activation of T cells and reflection of functional status of T cells after TBI. Key words: Whole-body irradiation; CD8-positive T-lymphocytes; CD4-positive T-lymphocytes; T-lymphocyte subsets; Phenotype

Peyer's patches and mesenteric lymph nodes cooperatively promote enteropathy in a mouse model of food allergy.

Background and ObjectiveTo improve the efficacy and safety of tolerance induction for food allergies, identifying the tissues responsible for inducing intestinal inflammation and subsequent oral tolerance is important. We used OVA23-3 mice, which express an ovalbumin-specific T-cell receptor, to elucidate the roles of local and systemic immune tissues in intestinal inflammation.Methods and ResultsOVA23-3 mice developed marked enteropathy after consuming a diet containing egg white (EW diet) for 10 days but overcame the enteropathy (despite continued moderate inflammation) after receiving EW diet for a total of 28 days. Injecting mice with anti-IL-4 antibody or cyclosporine A confirmed the involvement of Th2 cells in the development of the enteropathy. To assess the individual contributions of Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and the spleen to the generation of effector CD4+ T-cells, we analyzed the IL-4 production, proliferation in response to ovalbumin, and CD4+ T-cell numbers of these tissues. EW feeding for 10 days induced significant IL-4 production in PPs, the infiltration of numerous CD4+ T-cells into MLNs, and a decrease in CD4+ T-cell numbers in spleen. On day 28, CD4+ T-cells from all tissues had attenuated responses to ovalbumin, suggesting tolerance acquisition, although MLN CD4+ T-cells still maintained IL-4 production with proliferation. In addition, removal of MLNs but not the spleen decreased the severity of enteropathy and PP-disrupted mice showed delayed onset of EW-induced inflammatory responses. Disruption of peripheral lymphoid tissues or of both PPs and MLNs almost completely prevented the enteropathy.ConclusionsPPs and MLNs coordinately promote enteropathy by generating effector T-cells during the initial and exacerbated phases, respectively; the spleen is dispensable for enteropathy and shows tolerogenic responses throughout EW-feeding. The regulation of PPs may suppress the initiation of intestinal inflammation, subsequently restricting MLNs and inhibiting the progression of food-allergic enteropathy.

MyD88 signaling inhibits protective immunity to the gastrointestinal helminth parasite Heligmosomoides polygyrus.

Helminth parasites remain one of the most common causes of infections worldwide, yet little is still known about the immune signaling pathways that control their expulsion. C57BL/6 mice are chronically susceptible to infection with the gastrointestinal helminth parasite Heligmosomoides polygyrus. In this article, we report that C57BL/6 mice lacking the adapter protein MyD88, which mediates signaling by TLRs and IL-1 family members, showed enhanced immunity to H. polygyrus infection. Alongside increased parasite expulsion, MyD88-deficient mice showed heightened IL-4 and IL-17A production from mesenteric lymph node CD4+ cells. In addition, MyD88−/− mice developed substantial numbers of intestinal granulomas around the site of infection, which were not seen in MyD88-sufficient C57BL/6 mice, nor when signaling through the adapter protein TRIF (TIR domain–containing adapter–inducing IFN-β adapter protein) was also ablated. Mice deficient solely in TLR2, TLR4, TLR5, or TLR9 did not show enhanced parasite expulsion, suggesting that these TLRs signal redundantly to maintain H. polygyrus susceptibility in wild-type mice. To further investigate signaling pathways that are MyD88 dependent, we infected IL-1R1−/− mice with H. polygyrus. This genotype displayed heightened granuloma numbers compared with wild-type mice, but without increased parasite expulsion. Thus, the IL-1R–MyD88 pathway is implicated in inhibiting granuloma formation; however, protective immunity in MyD88-deficient mice appears to be granuloma independent. Like IL-1R1−/− and MyD88−/− mice, animals lacking signaling through the type 1 IFN receptor (i.e., IFNAR1−/−) also developed intestinal granulomas. Hence, IL-1R1, MyD88, and type 1 IFN receptor signaling may provide pathways to impede granuloma formation in vivo, but additional MyD88-mediated signals are associated with inhibition of protective immunity in susceptible C57BL/6 mice.

Upregulation of KCa3.1 K+ channel in mesenteric lymph node CD4+ T lymphocytes from a mouse model of dextran sodium sulfate-induced inflammatory bowel disease

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1/KCNN4 plays an important role in the modulation of Ca(2+) signaling through the control of the membrane potential in T lymphocytes. Here, we study the involvement of KCa3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in a mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5% dextran sulfate sodium for 7 days. Inflammation-induced changes in KCa3.1 activity and the expressions of KCa3.1 and its regulators in MLN CD4(+) T lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp, and flow cytometric analyses. Concomitant with an upregulation of KCa3.1a and nucleoside diphosphate kinase B (NDPK-B), a positive KCa3.1 regulator, an increase in KCa3.1 activity was observed in MLN CD4(+) T lymphocytes in the IBD model. Pharmacological blockade of KCa3.1 elicited the following results: 1) a significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of KCa3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4(+) T lymphocytes. These findings suggest that the increase in KCa3.1 activity induced by the upregulation of KCa3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4(+) T lymphocyte and, therefore, that the pharmacological blockade of KCa3.1 may decrease the risk of IBD.