| Food allergy is defined as a disorder in which antigenic food proteins elicit skewed immune response by the abrogation of oral tolerance. The prevalence of food allergy is estimated to be about3%to4%in adults and approximately6%to8%in young children and infants. Commonly, food allergy is the immunoglobulin E (IgE)-mediated reactions to food antigens, such as cow’s milk, eggs, peanuts, soybeans, wheat, fish and tree nuts, with a skewed T helper2(Th2) response. The clinical symptoms of food allergy vary from mild abdominal-irritating to lifethreatening anaphylactic shock. So far, no definitive therapies are available for food allergy; the only effective remedy is to avoid ingesting the offending food. Yet, our knowledge is still limited about how food allergy is initiated. A hapten is a small molecule that cannot elicit an immune response alone, but it does when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself. In recent years, with the huge increase in atopic diseases, there has been a noted increase in dietary hapten exposure through processed food, formula milk, and oral antibiotic and drug use. In fact, there is an established association between hapten and several atopic conditions such as allergic dermatitis. Therefore, haptens may be also involved in the pathogenesis of food allergy, which yet remains to be investigated.The T-cell immunoglobulin and mucin domain (TIM)-1is expressed by activated helper T cells. TIM4is the ligand of TIM1and is expressed by activated dendritic cells (DCs). The interaction of TIM1and TIM4plays a major role in the initiation of an aberrant Th2response. Exposure to some microbial products (such as staphylococcal enterotoxin B) can induce the expression of TIM4in DCs. DCs can be activated by trinitrobenzene sulfonic acid (TNBS);yet, whether TNBS-activated DCs can express TIM4is unclear.T regulatory cells (Treg) are one of the major cell populations to fulfill the immune suppressor function during immune responses. Two major types of Tregs are proposed, natural Tregs and inducible Tregs. The natural Tregs develop in the thymus while the inducible Tregs are generated in the peripheral system in response to suitable stimuli such as transforming growth factor-β,interleukin (IL)-10, indoleamine2,3-dioxygenase, etc. The plasticity of CD4+T cells has been recognized in recent years, which are not at the final stage of their differentiation and have the potential to be converted into different subsets of CD4+T cells depending on the cytokine environment. For example, IL-6can drive natural Tregs to express the retinoid-acid receptor-related orphan receptor gamma t (RORyt) and IL-17; some CD4+Foxp3+T cells express proinflammatory cytokines IL-17and interferon (IFN)-y. The already developed Th17cells can be converted into IFN-y-producing Thl cells or IL-4-producing Th2cells when stimulated by IL-12or IL-4, respectively.The T helper (Th) cells include Th1, Th2, Th17cells that play a critical role in the immunity of the body. Thl cells produce the signature cytokine IFN-γ to eliminate the invading microbes. Th2cells secrete IL-4that initiates the humoral and adaptive immunity. Th17cells produce IL-17A (IL-17, in short) that has chemotactic effect to neutrophils and mononuclear cells to attract these cells to inflammatory sites. However, overproduction of these cytokines can induce inflammation such as the high levels of IL-4induce the IgE class switching in B cells that plays a critical role in allergic diseases. Over production of IFN-y and IL-17is responsible for the pathogenesis of a number of disorders such as inflammatory bowel disease. The balance of the production of cytokines from T helper cells is important in maintaining the homeostasis in the body; yet, the orchestrating mechanism is not fully understood.The B-cell lymphoma6protein (Bcl-6) is encoded by the BCL6gene in humans. It is an evolutionarily conserved zinc finger transcription factor. It is also called the Bcl-6transcriptional repressor that is the most commonly involved oncogene in B-cell lymphomas and is a critical regulator of normal B cell differentiation. Recent studies indicate that Bcl-6also plays a critical role in the immune regulatory function in a subset of regulatory T cells. Bcl-6can interfere with the gene transcriptional processes of IL-4. Whether the expression of Bcl-6in Th2cells can be regulated is unclear.Mast cells are a critical effector cell population in immune inflammation, such as in the immediate allergic reactions, in a number of immune inflammations and in the pathogenesis of cancer. However, mast cells also play important roles acting as initiators and effector cells of innate immunity. Published data indicate that mast cells are actively involved in the immune regulation, combat with bacteria, initiate the adaptive immunity by releasing a set of chemical mediators. Among the mediators derived from mast cells, the serine proteases, including tryptase in human mast cells, rat mast cell protease, mouse mast cell protease-6, have a strong immune regulatory capacity. The serine proteases activate the protease-activated receptors (PAR)1and PAR2to modulate the activities of target cells. During immune reactions, mast cells are mixed with other immune cells, such as Th2cells, on the sites of inflammation. These immune cells have an opportunity to interact each other. Th2cells express PAR2and Bcl-6. Whether mast cell-derived serine protease activates PAR2to modulate the expression of Bcl-6in Th2cells is unclear.In this study, we induced a food antigen-related intestinal Th2inflammation by treating mice with a mixture of a model hapten, TNBS, and ovalbumin (OVA), a model food allergen, without any other adjuvants. This suggests that haptens might be an environmental triggering factor in the pathogenesis of food allergy, we also found that mast cell-derived proteases activated PAR2on Th2cells to upregulate the expression of Bcl-6to suppress the Th2cytokines, and converted the Th2cells to Tregs, which contributed to the self-restriction of the skewed antigen-specific Th2responses.Methods1Generation of Bone Marrow-Derived Dendritic CellsBone marrow (BM) cells were obtained by flushing the femurs of BALB/c mice with phosphate-buffered saline. The BM cells were resuspended in lysis buffer for2to4minutes to lyse red blood cells. The remaining BM cells were washed2times in RPMI1640medium. BM cells were then cultured at1×106cells/mL in6-well plates in RPMI1640culture medium supplemented with200ng/mL of recombinant murine Flt3L. The culture medium was changed on days3and5, and replaced with fresh medium supplemented with Flt3L. The cells were harvested on day7. The purity of CD11c+DCs was>95%, as assessed by flow cytometry.2Effect of TNBS on the Properties of DCsBone marrow-derived dendritic cells (BmDCs) were cultured in96-well plates (1×105/well) in the presence of TNBS at graded doses (from0to200ng/mL) for3days. The supernatants were collected and analyzed by ELISA for the levels of TIM4, CD80and IL-12p70. In addition, CD11c+DCs were isolated from the mice jejunum (see following). Total proteins were extracted from the DCs and analyzed by Western blot analysis for the levels of TIM4, CD80and IL-12p70.3A Murine Model of Hapten-Facilitated Intestinal AllergyMale BALB/c mice (6-8weeks old) were fed with a mixture of TNBS (1mg per mouse) and OVA (100mg per mouse) in0.3mL of saline, TNBS alone, or OVA alone on days0,1,2,3and4. The mice were challenged with OVA (1mg per mouse) in0.3mL of saline intragastrically on days9,11and13using a ball-ended feeding needle. Control groups were treated with normal saline. Twenty-four hours after the last challenge, the mice were killed by cervical dislocation and the jejunal segments collected for analysis.4Assessment of Antigen-Specific Th2ResponseLamina propria mononuclear cells (LPMCs) were isolated from the small intestine following our routine procedure. Jejunal segments were opened and washed with phosphatebuffered saline; mucus was removed by incubation with predigestion buffer. After grinding the tissue between2sterile glass slides to obtain a single cell suspension, the cells were passed through a cell strainer (100mm). The cells were centrifuged over a Percoll gradient for mononuclear cell enrichment. LPMCs (13106/well) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cultured in the presence or absence of OVA for4days. The cells were collected at the end of culture and stained with fluorescence-labeled antibodies of CD3, CD4and IL-4. The cells were analyzed first by CFSE dilution; then, the gated proliferating cells were further analyzed to determine the frequency of CD3+CD4+EL-4+T cells. The supernatants were collected to determine the levels of IL-4and IFN-g by ELISA5Preparation of polarized CD4+Th2cellsSpleen cells were obtained from OTII mice, the CD8+T cells were depleted using a magnetic activating cell sorting (MACS) kit. Cells (5×105/ml) were incubated in complete RPMI medium for4days in the presence of OVA323-339peptide (1μg/ml) and IL-4(2ng/ml) plus anti-IFN-γ antibody (5μg/ml) for preparing polarized Th2cells. After4days of incubation, cells were restimulated as before and in the presence of IL-2(10ng/ml) for an additional4days. On Day8, major histocompatibility complex (MHC) Ⅱ+cells were removed using the anti-MHCII antibody MACS kit. The polarized effector Th2cells were collected for further experiments.6Culture and sensitization of mouse bone marrow-derived mast cells (BMMCs)Bone marrow cells from male WBB6F1-Kit+/+(Kit+/+, in short) mice were cultured for up to10weeks in50%enriched medium (RPMI1640containing2mM L-glutamine,0.1mM nonessential amino acids, antibiotics, and10%fetal calf serum) and50%WEHI-3cell conditioned medium as a source of IL-3. After3weeks>98%of the cells were BMMC when checked by flow cytometry. For cell sensitization,106BMMC/ml cells were exposed to anti-OVA IgE (500ng/ml) or anti-DNP IgE in culture overnight.7Adoptive transfer and activation of the Th2cells in vivoThe polarized Th2cells (107cells/mouse; labeled with CFSE) with or without BMMCs (2×106cells/mouse) were injected into the tail veins of mice. The mice were subsequently challenged with feeding OVA (0.5mg/mouse) daily for next3days. The mice were sacrificed on day4. The LPMCs were isolated from the small intestine by gradient density centrifugation.8Real-time quantitative RT-PCR (qRT-PCR)The primers were presented and25ng cDNA were amplified in50μl1×SYBR-Green I PCR master mix, containing200nM primer for Bcl-6. Thermal cycling conditions were:60℃for2min,95℃for10min,30cycles of denaturation at95℃for15s, and annealing/extension at60℃for1min. All the experiments were performed in triplicate, using the qRT-PCR thermal cycler (Bio-Rad), and negative controls were added to each experiment. The quantification was obtained using the relative standard curve method. The standard curve was created by amplification of5μl control total RNA extracted from the cells at different dilutions (1.0-0.025ng/μl by two-fold dilution). The relative amount of each unknown sample was calculated using the linear regression from the respective standard curve. The relative gene expression value was normalized by dividing the target value by the housekeeping gene value.9Flow cytometryCells were fixed with1%paraformaldehyde for1h and stained with fluorescently labeled antibodies (0.5-1μg/ml)(or an isotype IgG using as staining control for matched antibodies) for30min on ice. In intracellular staining, cells were incubated with permealization reagents for30min on ice. The stained cells were analyzed by flow cytometry (FACSCando, BD Bioscience; San Jose, CA).10Generation of Tregs in vitroTh0cells were isolated from the spleen of Kit+/+mice and cultured in anti-CD3/CD28coated plates in the presence of MMCP-6at10nMol and IL-2(2ng/ml) for4days. After washing, the cells were cultured in former conditions for another4-day period. As assessed by flow cytometry, more than90%cells expressed Ecl-6and TGF-β; about10%cells were IL-10+. The cells were used in further experiments of immune suppression assay.11RNA interference (RNAi)The mouse PAR2shRNA and Bcl-6shRNA carried by lentiviral vectors and control shRNA were purchased from Santa Cruz. Cells were transduced with shRNA or control shRNA following the manufacturer’s instruction. As assessed by Western blotting, the knockdown effect was reached to90%at48h after transduction. Transduction with the control shRNA did not affect the expression of any genes.12Confocal microscopyThe jejunal segments were excised from the experimental mice. Cryosections were prepared and fixed with cold acetone for20min. The sections were blocked with1%bull serum albumin (BSA) for30min. First antibodies (0.5-1μg/ml) were added to the sections and incubated overnight at4℃. The fluorescence labeled secondary antibodies were added then and incubated at room temperature for1h. Washing with phosphate buffered saline (PBS) was performed between steps. The sections were mounted with cover slips and observed under a confocal microscope.13Statistical AnalysisAll values are expressed as mean6standard deviation of at least3independent experiments. The values were analyzed using2-tailed unpaired Student’s t test when data consisted of2groups or by analysis of variance when＄3groups were compared. A P value,0.05was accepted as statistically significant.Results1TNBS Regulated the Expression of TIM4, CD80and IL-12in DCsThe addition of TNBS to the culture of BmDCs increased the expression of TIM4in BmDCs in a dose dependent manner within the range of0to60ng/mL. However, further increase in TNBS concentration in the culture did not increase the expression of TIM4in BmDCs. CD80expression was detected in the culture medium, which increased in response to TNBS stimulation in a dose-dependent manner. We also measured the levels of IL-12p70in the culture medium. Only low levels of IL-12were detected; however, the expression increased sharply after the dose of TNBS exceeded80ng/mL.2The Serum Levels of Antigen-Specific IgE and Histamine Were Increased in Sensitized MiceAntigen-specific IgE and histamine are the most important mediators in intestinal allergic reactions. Therefore, with the same mouse model, we assessed the OVA specific IgE and histamine in the serum of mice. As shown by ELISA, significantly higher levels of OVA-specific IgE and histamine were detected in the serum of mice treated with both OVA and TNBS compared with those treated with either OVA or TNBS alone.3Antigen-Specific Th2Polarization Was Induced in the IntestineThe LPMCs were isolated from the small intestine and labeled with CFSE and cultured for4days in the presence of the specific antigen, OVA. As shown by flow cytometry, marked proliferation of the CFSE-labeled cells was observed in mice treated with both TNBS and OVA, but not in those treated with either OVA or TNBS alone. The proliferated cells were further analyzed by a gating technique. The results show that>90%proliferated cells were CD3+IL-4+T cells. In addition, we also analyzed the cytokine levels of Thl and Th2in the culture medium. The results show that higher levels of IL-4and lower levels of IFN-g were detected in the group treated with both TNBS and OVA as compared with the controls.4Blocking TIM4Inhibits the Antigen-Specific Th2Response in the IntestineA group of mice was pretreated with anti-TIM4antibody before the exposure to the specific antigen and TNBS. As expected, the Th2response in the intestine was similar to that in the control mice.5Adoptive Th2response was stronger in mast cell-deficient mice than that of the wild type littermatesThe CFSE+Th2cells were gated first and further analyzed by CFSE-dilution assay. The OVA-specific Th2cells (labeled with CFSE) proliferated markedly in W/Wv mouse intestine after the challenge with OVA; the proliferation was significantly less in mice received both Th2cells and OVA specific IgE-sensitized BMMC. A group of the mast cell proficient Kit+/+mice were also received CFSE-labeled OVA-specific Th2cells and challenged with OVA. The frequency of proliferated CFSE+T cells was similar to the W/Wv mice received CFSE+Th2cells alone. The gated CFSE+Th2cells were also analyzed for IL-4expression. The results showed the frequency of IL-4+cells was in parallel to the results of CFSE dilution assay. A portion of CFSE+Th2cells was sort out; the total RNA and protein were extracted and analyzed by qRT-PCR and ELISA. Furthermore, the serum levels of IL-4were also in parallel to the results of the CFSE dilution assay. The serum levels of IFN-γ were not changed much in different groups.6Activation of mast cells increased the expression of Bcl-6in Th2cells The expression of Bcl-6was detectable in non-stimulated Th2cells; the activation of mast cells significantly increased the expression of Bcl-6in Th2cells. To see if other mast cell mediators were involved in the mast cell-induced Bcl-6expression in Th2cells, we treated the Th2cells with five different mast cell mediator antagonists respectively in addition to the procedures above. The results showed that those antagonists did not show detectable inhibition in the mast cell-induced Bcl-6expression in Th2cells. In separate experiments, we knocked down the PAR2gene in OTII Th2cells by RNA interference; the PAR2-deficient Th2cells were polarized by exposure to OVA (in the presence of DC); we then exposed the PAR2-deficient Th2cells to the activated mast cells with the same procedures above. Indeed, the increase in the expression of Bcl-6was abrogated in the Th2cells. In separate experiments, we pretreated the Th2cells with p38MAPK antagonist or ERK1/2antagonist, which abolished the increase in Bcl-6expression induced by activated mast cells.7The autocrine Bcl-6suppressed the expression of IL-4in Th2cellsThe activation of BMMC significantly suppressed the Th2responses; the suppressor effect was much stronger when the mast cells were activated on day1than that activated on day3. The data demonstrate that the activated mast cells can down-regulate the expressions of IL-4and IL-13in polarized Th2cells.8Activated mast cells prevented activated Th2cells from apoptosis and converted polarized Th2cells to TregsAbout4.36%CFSE-labeled apoptotic Th2cells were observed in the intestine not challenged with the antigen, which was slightly more (6.53%) in mice received both antigen specific Th2cells and specific IgE-sensitized mast cells after challenge with the antigen; however, the frequency of apoptotic Th2cells was markedly increased in mice received antigen specific Th2cells and non-sensitized mast cells, or antigen specific Th2cells and DNP-IgE sensitized mast cells.Among the recovered CFSE+cells (the adoptive transferred Th2cells), a small portion (1.44%) of Foxp3+cells was detected in the mice received Th2cells alone, which was significantly increased (18.8%) in mice received both Th2cells and specific IgE-sensitized mast cells, but was not in mice received Th2/non-sensitized mast cells or Th2/DNP-IgE sensitized mast cells. Further analysis showed that the Foxp3+cells expressed high levels of TGF-β and trivial contents of Th2cytokines. The results were further confirmed by confocal microscopy in which the CFSE-labeled Th2cells were localized in the intestine, of which Foxp3+CFSE-labeled cells were observed in mice received the Th2cells and the antigen specific mast cells, but not in other3group mice.9Bcl-6repressed expression of GATA3and promoted expression of Foxp3in Th2cellsThe data from both qRT-PCR and Western blotting showed high levels of GATA3expression in the polarized Th2cells, which was significantly repressed in those cultured with activated mast cells. The Th2cells cultured with non-activated mast cells, or DNP-IgE sensitized mast cells still showed high levels of GATA3. On the other hand, the expression of Foxp3expression was markedly altered in the Th2cells, which was negatively correlated with the alterations of GATA3. Furthermore, no alternations in the expressions of GATA3and Foxp3were observed in the Bcl-6-deficient Th2cells after culturing with activated mast cells. In the absence of Tregs, the polarized CD4+T cell markedly proliferated; the levels of Th2cytokines were significantly increased in the culture. Both the T cell proliferation and the production of Th2cytokines were significantly suppressed in the presence of Tregs The results indicate that the induced Tregs have the immune suppressor function.Conclusions1. Our knowledge is still limited about how food allergy is initiated. This study first indicate that haptens may be an environmental factor in the pathogenesis of food allergy.2. The present data show that coadministration of TNBS and OVA, the food allergen, can induce the skewed Th2inflammation in the intestine. This indicates that TNBS can be a reliable adjuvant in the development of an animal model for food allergy.3. TNBS can induce DCs to produce TIM4, the latter further interacted with TIM1on CD4+T cells and thus induced the skewed Th2response in the intestine.4. This study adds novel information to mast cells’ properties by showing that activated mast cells induce the Th2cells to express Bcl-6. The expression of Bcl-6suppresses the expression of Th2cytokines in Th2cells as well as inhibits the proliferation of Th2cells.5. The present data provide a new pathway in the induction of Tregs in the body by showing that mast cell activation can trigger the development of Tregs. It is noteworthy that the precursors of Tregs in the present study are not ThO cells but Th2cells. |
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