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Basophils Control T-Cell Responses and Limit Disease Activity in Experimental Murine Colitis

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Basophils Control T-Cell Responses and Limit Disease Activity in Experimental Murine Colitis ARTICLES nature publishing group Basophils control T-cell responses and limit disease activity in experimental murine colitis M Rodriguez Gomez1, Y Talke1, C Hofmann2, I Ketelsen1, F Hermann1, B Reich1, N Goebel1, K Schmidbauer1, N Dunger2, H Bru¨hl2, K Renner1, S-N Syed1 and M Mack1 Basophils have been recognized as important inducers of T helper type 2 (Th2) responses. Using the colitis model of adoptive transfer of CD4 þ CD62L þ T cells into lymphopenic hosts, we have analyzed how basophils regulate T-cell responses and modulate disease activity. Transferred T cells rapidly proliferate, produce large amounts of interleukin (IL)-3, and expand the number of basophils in an IL-3-dependent manner. Depletion of basophils with two different antibodies substantially upregulated Th1 cytokines in transferred T cells at day 8. Increased Th1 cytokine expression persisted until the end of the experiment when basophil-depleted mice showed exacerbation of colitis with more severe loss of weight, histological damage, colonic leukocyte infiltration, and expression of pro-inflammatory cytokines. In vitro, we show that basophil-derived IL-4 and IL-6 downregulates expression of interferon-c, IL-2, and tumor necrosis factor in T cells. These data show a beneficial role of basophils in a T-cell driven model of autoimmunity. INTRODUCTION inflammatory bowel disease.22,23 Adoptive transfer of naive Basophils develop from a common granulocyte–monocyte CD4 þ T cells in T-cell-deficient mice (e.g., SCID or RAG À / À progenitor in the bone marrow and represent the least abundant mice) is a well-established model of colitis24 exhibiting many of leukocyte subset in the circulation (0.1–0.3%).1 The role of the clinical and histological features of human inflammatory basophils for host immunity has long been underestimated bowel disease. Several experiments suggest that colitis is largely owing to difficulties in their identification and modulation. mediated by preferential expansion of colitogenic pro-inflam- Increasing attention is now drawn to their role in regulation of matory Th1 cells expressing interferon (IFN)-g and tumor adaptive immune responses, as basophils readily release large necrosis factor (TNF). Colitis could not be induced by adoptive quantities of interleukin (IL)-4 and IL-6 and support devel- transfer of T cells from IFN-g-deficient mice25 and was opment of T helper type 2 (Th2) responses.2–7 Several efficiently suppressed with monoclonal antibodies against publications provide evidence that basophils cooperate with TNF.26,27 Also IL-12, a major inducer of Th1 and IL-23, was antigen-presenting MHC (major histocompatibility complex)- shown to be critical for development of colitis.28,29 Over- II-positive dendritic cells for induction of Th2 cells.4,8–11 A large expression of the Th1-related transcription factor STAT4 variety of substances have been described to induce cytokine (signal transducer and activator of transcription factor 4) or release by basophils. The two most important pathways for deficiency of IL-10 resulted in spontaneous development of activation of murine basophils are stimulation with IL-3 and colitis.30–32 Recent studies showed that basophils promote Th2 crosslinkage of FceR1 by binding of antigen to surface-bound immunity in a Th2-impaired environment, suggesting that immunoglobulin E.12–15 The main source of IL-3 are CD4 þ T basophils could be beneficial in pro-inflammatory Th1 cells, which support basophil expansion following infec- disorders.33,34 In addition, it was shown that commensal tion.12,16–18 IL-3 activates basophils and is able to prolong microbiota in the gut suppress basophil development indicat- their survival at very low concentrations.19–21 ing a role of the intestine for basophil homeostasis and Crohn’s disease and ulcerative colitis constitute a chronic Th1–Th2 regulation.35 Basophils markedly increase the release inflammation of the intestine and are collectively known as of IL-4, IL-13, and IL-10 from activated CD4 þ T cells in an 1Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany and 2Department of Internal Medicine I, University Hospital Regensburg, Regensburg, Germany. Correspondence: M Mack ([email protected]) Received 18 October 2012; accepted 9 May 2013; published online 12 June 2013. doi:10.1038/mi.2013.38 188 VOLUME 7 NUMBER 1 | JANUARY 2014 | www.nature.com/mi ARTICLES IL-4- and IL-6-dependent manner while Th1 cytokine show that adoptively transferred T cells produce large amounts production (IL-2, IFN-g) is suppressed.2–7,13 Basophils not of IL-3, which leads to expansion of basophils. Depletion of only alter the phenotype of CD4 þ T cells but also provide basophils before adoptive transfer of T cells increased Th1 important help for humoral memory immune responses.13,36–38 cytokine expression in T cells and resulted in exacerbation of Consequently in murine models of collagen-induced arthritis20 colitis, indicating that basophils have a protective role in a and lupus nephritis,39 basophils contribute to autoantibody Th1-driven model of autoimmunity. production and disease activity. Lymphopenic SCID and RAG À / À mice offer the possibility to investigate the impact RESULTS of basophils on a T-cell driven disease without effects of the Characterization of basophils in lymphopenic mice humoral immune system. We hypothesized that basophils alter Lymphopenic SCID mice are deficient in T and B cells but show the phenotype of T cells during their early expansion in normal numbers of basophils (approximately 25 basophils per lymphopenic hosts and suppress development of colitis. We ml blood). We investigated whether reconstitution of SCID mice Day 7 Day 7 ) No T cells ** 800 –1 ) ** 20 With T cells –1 600 15 ** 400 10 * 200 5 ** Plasma IL-3 (pg ml Plasma cytokines (pg ml 0 0 IL-2 IL-4 IL-6 TNF IFN-γ IL-3 ++ FcR1+ Fc R1 0.02% # 0. 4% Spleen 120 without T cells NS * 80 # Total 40 0.4% 0.09% Spleen 0 with T cells basophils x1,000 per spleen d 7 d 14 d 25 d 37 Day 7 60 # 0.6% 0.3% Spleen with T cells ++ 40 Day 14 R1 # ε Fc 20 ** * 0 Spleen basophils x1,000 per spleen 0.7% 0.7% d 7 d 14 d 25 d 37 with T cells Day 25 WT T cells 200 IL-3–/– T cells l blood No T cells μ 0.5% 0.5% Spleen 100 with T cells ** Day 37 # # * * CD49b Basophils / 0 d 8 d 16 d 29 d 84 100 101 102 103 104 FcεR1 Figure 1 Characterization of basophils after reconstitution of SCID mice with CD4 þ CD62L þ T cells. (a–c) SCID mice (n ¼ 6/group) were reconstituted with 2 Â 106 CD4 þ CD62L þ T cells from BALB/c mice (white bars) or received just phosphate-buffered saline without T cells (grey bars) and analyzed 7 days later. (a) Plasma cytokine levels were quantified by cytometric bead array. (b) The plasma level of interleukin (IL)-3 was measured by in vivo cytokine capture assay. (c) By staining with antibodies against FceR1 and CD49b, the numbers of FceR1 þ and FceR1 þþbasophils were quantified in the spleen of SCID mice 7, 14, 25, and 35 days after reconstitution with CD4 þ CD62L þ T cells from BALB/c mice. (d) SCID mice were adoptively transferred with 0.5 Â 106 CD4 þ CD62L þ T cells from BALB/c mice (white bars) or IL-3-deficient mice (black bars) (nine mice per group). The control group received no T cells (grey bars; 10 mice per group). After 8, 16, 29, and 84 days, basophils were quantified in the peripheral blood by flow cytometry with counting beads. Data are represented as mean±s.e.m. IFN, interferon; TNF, tumor necrosis factor; WT, wild type. MucosalImmunology | VOLUME 7 NUMBER 1 | JANUARY 2014 189 ARTICLES þ with CD4 T cells affects plasma cytokine levels and the Reconstitution: þ frequency of basophils. Reconstitution of mice with CD4 T 1,000 WT T cells cells induced a pro-inflammatory plasma cytokine milieu with ) 800 IL-3–/– T cells –1 increased levels of IL-2, IL-6, TNF, and IFN-g on day 7 600 No T cells ** # (Figure 1a). Also plasma levels of IL-3, which is known to (pg ml 400 activate and expand basophils, are increased more than fivefold 200 # Spleen IL-4 release # after transfer of T cells (Figure 1b). At various time points after 0 reconstitution of SCID mice with CD4 þ CD62L þ T cells, we Total cells Baso– Total cells Baso– quantified the absolute and relative numbers of basophils in the Medium + IL-3 spleen, bone marrow, and peripheral blood. Total basophils and especially a subpopulation of FceR1 þþ basophils were markedly increased after reconstitution with T cells. In the 1,000 * spleen the most pronounced increase of basophils was seen on ) 800 –1 day 25, while in the bone marrow the increase started earlier 600 and was already prominent at day 7 (Figure 1c, Supplementary (pg ml 400 Figure S1). In the peripheral blood, the most pronounced BM IL-4 release 200 ** ** increase of basophils was observed on days 16 and 29 0 Total cells Baso– Total cells Baso– (Figure 1d and Supplementary Figure S2). We also show that reconstitution with T cells from IL-3 À / À mice delays the Medium + IL-3 increase in basophils (day 16), indicating that IL-3 contributes Figure 2 Ex vivo analysis of interleukin (IL)-4 release by basophils after to the increase of basophils in vivo. Reconstitution with wild- reconstitution of SCID mice with CD4 þ CD62L þ T cells.
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