Critical role of intestinal interleukin-4 modulating regulatory T cells for desensitization, tolerance, and inflammation of food allergy

<div><p>Background and objective</p><p>The mechanism inducing either inflammation or tolerance to orally administered food allergens remains unclear. To investigate this we analyzed mouse models of food allergy (OVA23-3) and tolerance (DO11.10 [D10]), both of which express ovalbumin (OVA)-specific T-cell receptors.</p><p>Methods</p><p>OVA23-3, recombination activating gene (RAG)-2-deficient OVA23-3 (R23-3), D10, and RAG-2-deficient D10 (RD10) mice consumed a diet containing egg white (EW diet) for 2–28 days. Interleukin (IL)-4 production by CD4⁺ T cells was measured as a causative factor of enteropathy, and anti-IL-4 antibody was used to reveal the role of Foxp3⁺ OVA-specific Tregs (aiTreg) in this process.</p><p>Results</p><p>Unlike OVA23-3 and R23-3 mice, D10 and RD10 mice did not develop enteropathy and weight loss on the EW diet. On days 7–10, in EW-fed D10 and RD10 mice, splenic CD4⁺ T cells produced significantly more IL-4 than did those in the mesenteric lymph nodes (MLNs); this is in contrast to the excessive IL-4 response in the MLNs of EW-fed OVA23-3 and R23-3 mice. EW-fed R23-3 mice had few aiTregs, whereas EW-fed RD10 mice had them in both tissues. Intravenous injections of anti-IL-4 antibody recovered the percentage of aiTregs in the MLNs of R23-3 mice. On day 28, in EW-fed OVA23-3 and R23-3 mice, expression of Foxp3 on CD4⁺ T cells corresponded with recovery from inflammation, but recurrence of weight loss was observed on restarting the EW diet after receiving the control-diet for 1 month. No recurrence developed in D10 mice.</p><p>Conclusions</p><p>Excessive IL-4 levels in the MLNs directly inhibited the induction of aiTregs and caused enteropathy. The aiTregs generated in the attenuation of T cell-dependent food allergic enteropathy may function differently than aiTregs induced in a tolerance model. Comparing the two models enables to investigate their aiTreg functions and to clarify differences between inflammation with subsequent desensitization versus tolerance.</p></div

Weight changes of EW-re-fed OVA23-3 mice after given the control-diet for 1 month.

<p><b>A</b>, Weight changes (relative to day 0 [100%]) of OVA23-3ECE mice (re-administration of EW diet after 1-month interval of CN diet; diet throughout experimental period, EW→CN→EW diet; ●, n = 3), OVA23-3CCE mice (started EW diet on day 60; diet throughout experimental period: CN→CN→EW diet; ○, n = 3), and OVA23-3CCC mice (received control [CN] diet throughout experimental period, CN→CN→CN diet; △: n = 2). <b>B</b>, Weight changes of OVA23-3ECE (●, n = 2), D10ECE (○, n = 3), D10CCE (◆, n = 3), and D10CCC (◇, n = 3) mice. <b>C,</b> Protocol for re-administrating EW diet (↑, change from EW diet to CN diet on day 28; ↑↑, re-administration of EW diet on day 60). These data are representative of two independent experiments.</p

Treatment with anti-IL-4 mAb upregulates Foxp3 expression on MLN OVA-specific CD4⁺ T cells of EW-fed R23-3 mice.

<p><b>A,</b> Protocol for anti-IL-4 mAb (αIL-4) or control antibody (RatIgG) injection (↓) and EW diet. <b>B,</b> Percentages of OVA-specific Foxp3⁺ CD4⁺ T cells among total OVA-specific CD4⁺ T cells from the spleen and MLNs of mice treated with control antibody (RatIgG) or αIL-4, <b>C,</b> Weight change (relative to day 0 [100%]). D, H&E staining of the jejunum (n = 3 per group). These data are representative of two independent experiments.</p

Proliferation and cytokines production of naïve OVA-specific CD4⁺ T cells from R23-3 and RD10 mice.

<p>Naïve OVA-specific CD4⁺ T cells purified from R23-3 (●) and RD10 (○) mice (n = 3 each) were stimulated with both anti-CD3 mAb and anti-CD28 mAb. <b>A</b>, Proliferation of OVA-specific CD4⁺ T cells. <b>B,</b> IL-4 (upper panel) and IFN-γ (lower panel) production in culture supernatants of OVA-specific CD4⁺ T cells. These results are representative of two independent experiments; *, <i>P</i>< 0.05; **, <i>P</i> < 0.01.</p

Peyer’s Patches and Mesenteric Lymph Nodes Cooperatively Promote Enteropathy in a Mouse Model of Food Allergy

<div><p>Background and Objective</p><p>To 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.</p><p>Methods and Results</p><p>OVA23-3 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.</p><p>Conclusions</p><p>PPs 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.</p></div

Lack of weight loss and enteropathy in EW-fed D10 mice.

<p><b>A</b>, Weight change (relative to day 0 [100%]). The loss was solely indicated in EW-fed OVA23-3 group. <b>B</b>, H&E-stained histology of the jejunum of mice fed with control diet (CN) or fed with EW diet (EW) for 10 or 28 days. Severe inflammation (crypt elongation, villous atrophy, thickness of muscular layer, goblet cell hyperplasia) was present in day 10 of EW-fed OVA23-3 mice but not in that of EW-fed D10 mice (10 days). Furthermore, EW-feeding of OVA23-3 mice for 28 days (28 days) shows severe villous blunting, indicating moderate degree of inflammation under mucosal repair. These results are representative of three independent experiments using CN-fed D10 mice (n = 4), EW-fed D10 mice (n = 4) CN-fed OVA23-3 mice (n = 2), and EW-fed OVA23-3 mice (n = 2).</p

The spleen is dispensable for the establishment of enteropathy in EW-fed OVA23-3 mice.

<p>Weight change (left panel) and jejunal sections (right panel, day 7) of splenectomized EW-fed (SPlackEW, n = 5), splenectomized CN-fed (SPlackCN, n = 3), and sham-operated EW-fed (Mock EW, n = 4) OVA23-3 mice. *, Significant (<i>P</i><0.05) difference between values for splenectomized CN-fed and EW-fed mice.</p