Cytokine-induced alterations of α7 nicotinic receptor in colonic CD4 T cells mediate dichotomous response to nicotine in murine models of Th1/Th17- versus Th2-mediated colitis

Abstract

Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4(+)CD62L(+) T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory α7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in regulating colonic inflammation.

Figure 1. Clinical and histological correlations in mice with experimental colitides treated with nicotine

A. Representative images of colons dissected from control BALB/c mice treated i.r. with 47.5% ethanol in saline (Control), mice with oxazolone-induced colitis either untreated (Ox) or treated with 7.5 mg/kg/day of nicotine (Ox+N), and untreated (T) vs. nicotine-treated (T+N) mice with TNBS colitis. The treatment with nicotine started from the day before instillation of a haptenating agent (i.e., at the day “zero”) and ended on the 4^th day of experiment, i.e., the day before mice were sacrificed. Note: the colon from untreated mouse with oxazolone colitis has several necrotic areas, whereas in the nicotine-treated animal the inflammation is visible only in the distal segment. While the colon of mouse with TNBS colitis is diffusely hyperemic, it lacks overt necrosis. In contrast, the mouse with TNBS colitis treated by nicotine has developed necrotic areas, as seen in the proximal segment of colon. B. Clinical evaluation of the effects of nicotine treatment on the severity of colitis in mice with oxazolone- and TNBS-induced colitides. The DAI values were computed as described in detail in Materials and Methods (Table 1). In oxazolone colitis, the differences are statistically significant (p<0.05) on days 4 and 5. In TNBS colitis, the statistical differences between the DAI values of nicotine-treated vs. untreated mice are significant (p<0.05) starting from the 3^rd day after instillation of the haptenating agent. Each point represents the mean ± SD of data obtained in 20 mice. The DAI values in control mice instilled with the ethanol solution without haptenating agents were equal or close to zero (data not shown). C. The representative images of the histologic findings in nicotine-treated vs. untreated mice with oxazolone- and TNBS-indiceds colities. The photomicrographs of H&E stained 5 μm sections of matching segments of colons were made at 10x magnification. Abbreviations are the same as as in panel “A”. Note: the extensive goblet cell depletion with submucosal involvement and lymphocytic infiltrations characteristic of oxazolone colitis were not present in mice treated with nicotine. In contrast, nicotine treatment aggravated colonic inflammation in TNBS colitis, leading to crypt destruction, goblet cells depletion and extensive lymphocytic infiltration. D. Histopathologic analysis of the effects of treatment with nicotine on the colonic inflammation in mice with oxazolone- and TNBS-induced cotises. The HAI values were computed as described in detail in Materials and Methods (Table 2). Nicotine treatment protected the colons from inflammation induced by oxazolone, as can be judged from an approximately 2-fold decrease of HAI. Vice versa, the HAI value computed in mice with TNBS colitis treated by nicotine significantly increased, indicating that nicotine worsened TNBS-induced colonic inflammation. Asterisk = p<0.05 compared to the HAI value computed in colons of the nicotne-untreated mice with the respective form of colitis.

Figure 2. The effects of nicotine treatment of mice with oxazolone- and TNBS-induced colitides on the peripheral and colonic populations of T lymphocytes

The FCM assays with T cell marker antibodies were performed using splenocytes and LPMCs freshly isolated from intact (control) and nicotine-treated and untreated mice with experimental colitides at the end of nicotine treatments, as detailed in the Materials and Methods section. The cells were triple stained for CD3, CD4 and CD8 and examined by flow cytometry. The contour plots were generated after gating on lymphocytes (by forward and side scatter) for CD3 staining or gating on CD3 T cells during the analyses of CD4/CD8 staining. Shown on the graphs are representative dot blots for the CD3 T cell population of spleen MCs (A) and the CD4 and CD8 T cell subpopulations of CD3 T cells from spleen (B) and colon (C). Note: treatment with nicotine decreased the percentage of spleen CD3 and CD4 T cells to the normal levels in mice with oxazolone colitis, and further elevated the percentage of CD3 T cells without altering the already elevated numbers of spleen CD4 T cells in mice with TNBS colitis.

Figure 3. The effects of nicotine treatment of mice with experimental IBD on the percentage of Tregs and Th17 cells among colonic CD4 T cells

The cryosections of colons from mice with oxazolone- and TNBS-induced colitides treated with nicotine were subjected to a double or triple immunofluorescence staining for CD4/CD25, CD4/CD25/Foxp3 and CD4/Th17 on day 5 after hapten treatment, as described in Materials and Methods. The results are expressed as means ± SD of the percentage of CD25 (A), CD25/Foxp3 (B), and IL-17 (C) positive cells among colonic CD4 T cells, taken as 100%. The data was computed in at least 10 different microscopic fields at magnification 60x in the triplicates of tissue sections from three mice in each group. Asterisk = p<0.05 compared to nicotine-untreated mice with the respective form of colitis.

Figure 4. The nicotinergic effects on the development of Tregs from the naïve CD4⁺CD62L⁺ T cells stimulated with anti-CD3/CD28 antibodies

The enriched naïve CD4⁺CD62L⁺ T cells isolated from the spleens of intact BALB/c mice were cultured in growth medium alone (condition “1”) or stimulated with anti-CD3 and anti-CD28 antibodies in the absence (condition “2”) or presence of nicotine (Nic; condition “3”), 10 ng/ml of IL-4, 100 μM of nicotine plus 10 ng/ml of IL-4 or IL-12 or 100 μM of nicotine plus 10 ng/ml of IL-12 as detailed in Materials and Methods. After 5 days in culture, the expression of Foxp3 protein was analyzed by immunoblotting (A) and FCM (B–D). The graph in panel “A” demonstrates changes of Foxp3 concentration after its normalization for the level of the house keeping protein β-actin. In panels “B–D”, the changes in Foxp3 expression are shown as an overlay histogram representing different treatment conditions.

Figure 5. The influence of IL-4 and IL-12 on nicotinergic regulation IL-10, IL-17 and IFN-γ secretion

A. Colons from mice with oxazolone- or TNBS-induced colitides were collected on day 5 after hapten treatment. Total protein was extracted, and tissue levels of IL-4, IL-12, IL-23 and IFN-γ were detected using mouse ELISA Immunoassay kits, as described in Materials and Methods. Asterisk = p<0.05 compared to control mice; pound sign = p<0.05 compared to untreated mice with the respective form of experimental colitis. B. The naïve CD4⁺CD62L⁺ T cells from intact BALB/c mice were stimulated with anti-CD3 and anti-CD28 in the presence or absence of 100 μM of nicotine ± 10 ng/ml of IL-4 or IL-12 for 5 days, after which the cell culture supernatants were collected and analyzed by ELISA for the presence of IL-10, IL-17 and IFN-γ, as described in Materials and Methods. Asterisk = p<0.05 compared to naïve CD4⁺CD62L⁺ T cells stimulated by TCR/CD3 cross-linking without any additions; pound sign = p<0.05 compared to cells stimulated by anti-CD3/anti-C28 in the presence of nicotine given alone; and plus sign = p<0.05 compared to cells stimulated with anti-CD3/anti-C28 in the presence of relevant cytokine without nicotine. C. The naïve CD4⁺CD62L⁺ T cells from intact BALB/c mice were stimulated with anti-CD3 and anti-CD28 in the presence or absence of 100 μM of nicotine ± 10 ng/ml of IL-4 or IL-12 for 5 days, after which the expression of IL-4, IL-10, IL-17 and IFN-γ was analyzed by FCM, as described in Materials and Methods.

Figure 6. Variations in the expression of α4 and α7 nAChRs by CD4 T cells

A. The expression of α4 and α7 nAChRs by colonic CD4 T cells in different forms of experimental IBD. The percentages of CD4 T cells expressing α4 or α7 in the colonic inflammatory infiltrates of BALB/c mice with oxazolone (Ox)- and TNBS (T)-induced colitides were computed in ten microscopic fields of double stained cryostat sections of colons from three mice in each group (n = 3). Asterisk = p<0.05 compared to mice with oxazolone-induced colitis. B. Clinical evaluation of nicotine treatment on the severity of oxazolone colitis in α7 knockout (KO) mice (B6.129S7-Chrna7tm1Bay/J; stock# 003232). The DAI values were computed as described in detail in Materials and Methods. Each point represents the mean ± SD of data obtained in 5 mice. The differences between the nicotine treated and not treated groups are not significant (p>0.05). C. The effects of IL-4 and IL-12 on α7 nAChR expression. The enriched naïve CD4⁺CD62L⁺ T cells from intact BALB/c mice were stimulated with anti-CD3/anti-CD28 antibodies alone (control) or in the presence of 10 ng/ml of either IL-4 or IL-12 for 5 days in culture, and the expression of α7 mRNA and protein was detected by qPCR and ICW, respectively, as detailed in Materials and Methods. The results were expressed as means ± SD of relative receptor mRNA or protein levels in cells exposed to cytokines compared to that in controls, taken as 1. Asterisk = p<0.05 compared to control group, and pound sign = p<0.05 compared to cells exposed to IL-12.