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白介素-31促进系统性硬化症的纤维化和T辅助2极化

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发表时间:2021-10-13 09:37作者:武汉新启迪Xinqidibio

摘要

系统性硬化症(SSC)是一种以纤维化和自身免疫为特征的慢性多系统疾病。白细胞介素(IL)-31参与了肝纤维化和T辅助(Th)2免疫反应,两者都是SSC的特征。IL-31在SSC发病中的确切作用尚不清楚.我们在SSC患者皮肤成纤维细胞(DFS)中显示IL-31和IL-31受体A(IL-31RA)的过表达。我们阐明了IL-31在SSC中的双重作用,其中IL-31直接促进DFS中的胶原生成,并通过增加DFS中前Th2细胞因子的表达间接增强Th2免疫应答。此外,用抗IL-31RA抗体阻断IL-31可明显改善SSC小鼠模型的纤维化和Th2极化。因此,除了将IL-31作为SSC中纤维化和Th2免疫反应的中介物外,我们的研究还为靶向IL-31/IL-31RA轴治疗SSC提供了理论依据。

导言

系统性硬化症(SSC)是一种结缔组织疾病,其特点是皮肤和内脏细胞外基质沉积过多。1,2。由此产生的纤维化会导致组织功能障碍和器官衰竭,从而导致衰弱和生命危险。虽然SSC的发病机制尚不清楚,但也有各种免疫异常的报道,提示了SSC的自身免疫特性。特别是T细胞的活化和极化在患者和SSC动物模型中都得到了广泛的研究。3。例如,CD4+T细胞可在SSC早期浸润皮肤。4。这些组织浸润的T细胞显示活化标记物的表达增加。5。外周血中的T细胞也在SSC中被激活。6。此外,活化的CD4+SSC中的T细胞主要倾向于T助手(Th)2,这与组织纤维化有关。3,7。事实上,主要的Th2细胞因子如白介素(IL)-4和白细胞介素-13(IL-13)在SSC患者的皮肤和血清中表达过高。8,9。在博莱霉素诱导的ssc小鼠模型中,Th2细胞因子也与皮肤和肺纤维化有关,这是一种建立良好的ssc实验模型。10,11。机械作用下,IL-4和IL-13直接诱导成纤维细胞产生胶原。12,13。此外,IL-4推动了天真的CD4的分化。+T细胞转化成分泌IL-4的Th2细胞,从而使Th2和促纤维反应永久化。14。增强Th2免疫反应的细胞因子和趋化因子也在SSC中起重要作用。例如,ssc患者中过表达的IL-6通过促进Th2分化和促进成纤维细胞胶原的产生而促进sSC的发展。15,16,17,18。综上所述,这些研究表明Th2优势是SSC的一个重要的免疫学特征,它直接或间接地促进纤维化。

IL-31是IL-6细胞因子家族的一员,最初被描述为小鼠皮炎的诱导剂。19。IL-31主要由Th2细胞产生,在多种细胞中表达,包括成纤维细胞、角质形成细胞和巨噬细胞。20,21。IL-31信号的胞内传递是由一个由IL-31受体A(IL-31RA)和癌胚素M受体(OSMR)组成的异二聚体受体介导的。IL-31RA是IL-31受体的唯一受体,而OSMR是由一种肿瘤素M受体复合物所共有的。19。在这两个受体亚单位中,IL-31主要与IL-31RA结合.IL-31与IL-31受体复合物的结合激活JAK/STAT、PI3K/AKT和其他信号通路。22,23,24导致广泛的免疫反应。

IL-31与Th2显性疾病密切相关。事实上,先前的研究已显示IL-31在过敏性哮喘、特应性皮炎及皮肤T细胞淋巴瘤等以Th2为主的疾病中的表达增加,而IL-31的高表达与Th2反应有关。25,26,27,28,29,30。值得注意的是,功能性阻断抗IL-31RA单克隆抗体(MAb)的线虫病在第二阶段试验中已被证明能改善特应性皮炎的皮肤表现,提示IL-31作为治疗靶点的潜力。31。此外,最近的研究表明IL-31与肝硬化有关。32。在ssc的作用下,blm-sc小鼠肺纤维化组织中IL-31的表达增加。33。此外,Yaseen等人。结果显示ssc患者IL-31和IL-31 RA表达上调。34。他们还证实了IL-31对人真皮成纤维细胞(DFS)和SSC模型小鼠的促纤维化作用。这些背景促使我们进一步探索IL-31在SSC中的作用及其作为治疗靶点的潜力。

在此,我们发现SSC患者的DFS中IL-31和IL-31RA均过表达,IL-31促进胶原和Th2诱导细胞因子的表达。此外,我们还证明,用抗IL-31RA单抗抑制IL-31信号转导可以改善BLM-SSC小鼠的纤维化和Th2极化,为靶向IL-31治疗SSC提供了理论依据。

结果

血清IL-31水平与SSC纤维化及Th2极化的关系

首先,我们用酶联免疫吸附试验(ELISA)检测了74例SSC患者和14例健康对照者血清中IL-31水平。与健康对照组相比,SSC患者血清IL-31水平显著升高(图一)。1A)。值得注意的是,在弥漫性皮肤SSC(DcSSc)患者中,循环IL-31较局限性皮肤SSC(LcSSc)患者更为丰富。其次,根据血清白细胞介素-31(IL-31)水平的截断值,将患者分为两组(健康对照组平均+2sd;表)。1)。血清IL-31水平升高的SSC患者,其dcSSc、肺纤维化、食管受累率均高于IL-31正常患者,均为纤维化的临床后果。事实上,IL-31水平升高的ssc患者以严重的皮肤和肺纤维化为特征,改良的rodnan皮肤总厚度评分较高。35(MRSS),肺活量预测值(%VC)和一氧化碳弥散量(%DLCO)预测值较低。此外,IL-31水平升高的SSC患者血清IL-4、IL-6和IL-13水平较高,而IL-13是与Th2反应相关的重要细胞因子,在SSC中表达过高。3。血清IL-31水平与MRSS呈正相关,与%VC、%DLCO呈负相关,与血清IL-4、IL-6、IL-13呈正相关(图一)。1B)。因此,SSC患者血清IL-31过度分泌与纤维化和Th2上调有关,这是本病的主要特征。

图1:血清IL-31水平与SSC纤维化及Th2极化相关.
figure1

a测定dcSSc患者血清IL-31水平(n=55),LcSSc患者(n=19),以及健康对照者(n=14)用一种特殊的ELISA试剂盒。水平线代表平均值。断线表示截止值(健康对照组的平均值+2sd).**p < 0.01 and ***p < 0.001 vs. healthy controls. Significance was evaluated by two-tailed Mann–Whitney U测试一下。精确性p值=0.00002(sc对ctrl)、0.000008(dcssc对ctrl)、0.008(lcssc对ctrl)。健康对照者b本文分析了SSC患者血清IL-31水平与以下指标的相关性。nMRSS、%VC、%DLCO、血清IL-4、IL-6、IL-13水平。采用Spearman秩相关检验进行相关分析。精确性p值=0.000000005(MRSS);0.005(%VC);0.033(%DLCO);0.012(IL-4);0.00003(IL-6);0.026(IL-13)。源数据作为源数据文件提供。

表1 SSC患者的临床和实验室特征。

IL-31和IL-31 RA在ssc dfs中均过表达。

接下来,我们评估了IL 31IL31RA应用实时聚合酶链反应(PCR)对10例SSC患者和6例健康对照者的皮肤进行实时聚合酶链反应(PCR)。SSC皮损的mRNA水平明显升高。IL 31IL31RA与健康对照者的皮肤相比较(见图)。2A)。我们接下来进行双免疫荧光染色,以评估IL-31和IL-31RA蛋白在SSC皮损中的表达和定位(图1)。2B)。IL-31和IL-31 RA与成纤维细胞特异性蛋白1(FSP-1)共同定位于SSC离体皮肤,表明IL-31和IL-31 RA在SSC DFS中的表达。我们没有在健康对照者皮肤中检测IL-31或IL-31RA与FSP-1的共定位。在SSC皮损中,IL-31也与CD4(补充图)共同定位.1A),这与以前的研究表明IL-31在Th2细胞中的表达是一致的。19,20。在SSC分组内,IL-31+CD4+T细胞在dcSSc患者的皮肤中较lcSSc患者的皮肤中丰富(附图)。1B)。在培养的SSC DFS中,IL-31和IL-31RA的表达在mRNA和蛋白水平上均显著高于健康对照组。2C,d)。此外,IL 31IL-4增强了表达,IL31RAIL-4和IL-13在SSC DFS中的表达增强(图1)。2E提示Th2优势可能与SSC DFS中IL-31和IL-31RA的过度表达有关。我们还检测到IL-4(补充图)刺激的SSC DFS分泌IL-31的增加。2A)。IL-31和IL-31RA在SSC DFS中的表达均上调,提示IL-31/IL-31RA轴在SSC纤维化中起一定作用。

图2:SSC患者皮肤和DFS中IL-31和IL-31RA表达上调.
figure2

a相对mRNA表达水平IL 31IL31RA对SSC患者皮肤进行实时PCR分析(n=10)和健康对照组(n=6)。精确p值=0.0002(IL 31); 0.0002 (IL31RA)。相对折差=2.37(IL 31); 3.89 (IL31RA). bSSC患者和健康对照组(标度bar=20m)皮肤中IL-31(顶部,红色),IL-31RA(底部,红色),FSP-1(绿色)和细胞核(DAPI,蓝色)的典型双免疫荧光图像。结果表明,这是三个独立实验的代表,具有相似的结果。c, d。相对mRNA水平(c)和蛋白质水平(d)SSC患者和健康对照组DFS中IL-31和IL-31 RA的表达(n分别用实时PCR法和ELISA法测定。精确p值=0.007(IL 31); 0.0006 (IL31RA);0.004(IL-31);0.0006(IL-31RA)。相对折差=2.05(IL 31); 3.13 (IL31RA)。E.相对mRNA表达水平IL 31IL31RA用实时PCR法检测IL-4或IL-13刺激的SSC DFS(n分别=5)。精确p值(IL-40.1ng/ml与介质、IL-41ng/ml与介质、IL-410ng/ml与介质、IL-130.1ng/ml与媒体、IL-131ng/ml与媒体相比)=0.841、0.008、0.008、0.151、0.841、0.548(IL 31); 0.310, 0.008, 0.008, 0.999, 0.008, 0.008 (IL31RA)。相对倍差(IL-40.1ng/ml、IL-41ng/ml、IL-410ng/ml、IL-130.1ng/ml、IL-131ng/ml、IL-1310ng/ml)=1.02、2.92、6.78、1.13、0.98、1.08(IL 31); 1.16, 5.36, 13.91, 1.01, 3.15, 4.76 (IL31RA)。数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001 vs. healthy controls (a, c,和d)或媒体(e)。意义是由两尾曼恩-惠特尼决定的。U测试一下。健康对照者。源数据作为源数据文件提供。

IL-31促进SSC DFS中胶原和前Th2细胞因子的产生

为了探讨IL-31在SSC DFS中的作用,我们随后用重组人IL-31刺激SSC患者和健康对照组的DFS。在ssc dfs中,rhIL-31显著且剂量依赖性地增加了mrna的表达。Col1a1Col1a2(无花果)3A),编码I型胶原蛋白,这是皮肤主要的细胞外基质成分。36。相比之下,在健康对照dfs中,rhIL-31对胶原表达的影响不大,Col1a1Col1a2只有在高浓度rhIL-31(100 ng/ml)刺激下,水平才会升高。同样,rhIL-31以剂量依赖性的方式促进SSC DFS产生I型胶原,而在健康对照DFS中,I型胶原生成仅在高浓度rhIL-31(100 ng/ml;图1)诱导下产生。3B)。这些结果提示IL-31是SSC DFS胶原合成的直接诱导剂,IL-31RA在SSC DFS中高表达。我们还观察到,SSC DFS的条件培养基通过IL-31信号通路显着地增加了SSC DFS中I型胶原的生成(补充图)。2B,c)。此外,rhIL-31增加了转化生长因子-β1的表达,降低了SSCDFS中MMP-1、MMP-3和MMP-9的表达。3C)。因为转化生长因子-β1促进成纤维细胞胶原生成,而基质金属蛋白酶促进成纤维细胞降解。37,38IL-31可能与促进SSC DFS过度胶原沉积的细胞因子环境有关。RhIL-31刺激还可使α-平滑肌肌动蛋白(α-SMA)在mRNA和蛋白水平上表达增加(如图所示)。3D,e表明IL-31促进SSC DFS向成纤维细胞的转分化。此外,rhIL-31还能促进SSC的增殖和迁移,抑制SSC DFS的凋亡。3F-h),从而增强了SSC DFS的特性,从而导致了SSC过度纤维化。39。总体而言,IL-31增强了SSC样表型,促进了SSC DFS中胶原的产生.

图3:IL-31诱导DFS中的SSC样表型。
figure3

a, bDcSSc患者和健康对照组的dfs培养24h,用rhIL-31(0,10,25,50或100 ng/ml)进行检测。Col1a1Col1a2MRNA表达a)和I型胶原蛋白的产生(b)分别采用实时PCR法和ELISA法。精确性p值(Ctrl+IL-31 10 ng/ml vs Ctrl,Ctrl+IL-31 25 ng/ml vs Ctrl,Ctrl+IL-31 50 ng/ml vs Ctrl,Ctrl+IL-31 100 ng/ml vs Ctrl,SSC+IL-31 10 ng/ml vs SSC,SSC+IL-31 25 ng/ml vs SSC,SSC+IL-31 50 ng/ml vs SSC,SSC+IL-31 100 ng/ml vs SSC)=0.690,0.095,0.056,0.008,0.032,0.016,0.008,0.008(Col1a1); 0.421, 0.421, 0.222, 0.016, 0.008, 0.008, 0.008, 0.008 (Col1a20.508,0.127,0.841,0.008,0.008,0.008,0.008,0.008(I型胶原蛋白)。相对差异(Ctrl+IL-31 10 ng/ml,Ctrl+IL-31 25 ng/ml,Ctrl+IL-31 50 ng/ml,Ctrl+IL-31 100 ng/ml,SSC+IL-31 10 ng/ml,SSC+IL-31 50 ng/ml,SSC+IL-31 50 ng/ml,SSC+IL-31 100 ng/ml)=1.10,1.16,1.19,1.53,1.47,2.10,2.24,2.71,2.86(Col1a1); 1.11, 1.10, 1.31, 1.54, 1.83, 2.35, 2.54, 2.75, 3.25 (Col1a2). c用ELISA法检测rhIL-31(50 ng/ml)处理的dcSSc DFS上清液中TGF-β1、CTGF、MMP-1、MMP-3和MMP-9的蛋白水平。精确p值分别为0.008(TGF-β1)、0.587(MMP-1)、0.008(MMP-1)、0.016(MMP-3)、0.024(MMP-9).dRhIL-31(50 ng/ml,左)对dcSScDFS中α-SMA的代表性Westernblot分析。β-肌动蛋白表达的定量(右)。精确性p值=0.008。e相对mRNA表达水平Acta 2用实时PCR方法检测rhIL-31(50 ng/ml)对dcSSc型DFS的影响。精确性p值=0.008。相对褶皱差=2.85。f用ELISA法测定rhIL-31(50 ng/ml)处理的dcSSc DFS的BrdU掺入量。在450 nm处测定吸光度。精确性p值=0.008。g用rhIL-31(50 ng/ml)治疗dcSSc患者的DFS,流式细胞仪检测细胞凋亡情况。有代表性的点图显示(左)。附件五+,7-AAD细胞被认为是早期凋亡细胞,Annexin-V+,7-AAD+细胞分别被认为是晚期凋亡细胞(右)。精确p值(IL-31 vs培养基)=0.008(早期凋亡);0.008(晚期凋亡).hDcSSc患者和健康对照组的DFS用丝裂霉素C预处理,划伤造成伤口,单独用rhIL-31(50 ng/ml)或培养基孵育24h。显示具有代表性的显微图像(左,标度为500 m)。红线显示伤口的边界。创面闭合表示为创面缩小的百分比(右)。精确p值(Ctrl+媒体与Ctrl+IL-31,Ctrl+媒体与SC+媒体,Ctrl+媒体与SSC+IL-31,Ctrl+IL-31与SC+IL-31,Ctrl+IL-31 vs SC+IL-31,SC+媒体对SC+IL-31)=0.005,0.0000001,0.00000000006,0.00008,0.000000004,0.00002。i, j用rhIL-31(50 ng/ml)或单纯培养24h,检测dcSSc患者血清中IL-6、IL-33和ccl 2的表达水平。i)和ELISA(j)。精确p值=0.008(IL6); 0.032 (IL 33); 0.008 (CCL 2);0.008(IL-6);0.032(IL-33);0.008(CCL 2)。相对折差=1.99(IL6); 1.37 (IL 33); 2.39 (CCL 2)。N=5。数据以平均±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001 vs. unstimulated Ctrl or SSc fibroblasts (a, b)或媒体(C-g, i,和j)。意义是由两尾曼恩-惠特尼决定的。U试验(阿格, i,和j)和单向方差分析,然后是Tukey的事后比较检验(H)。Ctrl,健康对照;OD,光密度。源数据作为源数据文件提供。

除了过度的胶原合成外,sscdfs还与导致疾病免疫异常的细胞因子和趋化因子有关。40,41,42。因此,为了探讨白细胞介素-31(IL-31)对SSC DFS细胞因子和趋化因子表达的影响,我们用rhIL-31刺激SSC DFS,并检测了以下细胞因子在dfs中的表达和Th2免疫应答:IL-6、IL-33和CC趋化因子配体(Ccl)2。40,41,42。如图所示。3i,jRhIL-31在SSC DFS的mRNA和蛋白水平上均显著增加IL-6、IL-33和CCL 2的表达。因此,IL-31也促进了SSC DFS中前Th2细胞因子的表达.

随后,我们探讨了IL-31对SSC DFS产生影响的信号通路.Westernblot分析显示rhIL-31诱导SSC DFS中STAT 3磷酸化。4A),这与Yaseen等人的研究是一致的。34。RhIL-31刺激也可诱导SSC DFS中STAT 1和STAT 5的磷酸化,但低于STAT 3。因为以前的一项研究已经证明,STAT 3与Col1a2对SSC成纤维细胞过度产生胶原有重要作用。43,我们假设有一条通路涉及IL-31,STAT 3,和Col1a2将促进SSC DFS中胶原的产生。为了验证这一假说,我们进行了染色质免疫共沉淀(芯片)试验。如先前报道的那样,在HS4区域设置了STAT 3结合位点。43。如图所示。4B,STAT 3与HS4军团的绑定Col1a2在SSC DFS中观察到增强子。

图4:IL-31通过IL-31RA和STAT 3发挥作用.
figure4

aRhIL-31处理的dcSSc DFS中STAT 1、STAT 3、STAT 5及其磷酸化形式的代表性westernblot分析(50 ng/ml,左)。β-肌动蛋白表达的定量(右)。精确p值(10 min vs 0 min,20 min vs 0 min)=0.008,0.008(pSTAT 1/STAT 1);0.008,0.008(pSTAT 3/STAT 3);0.016,0.040(pSTAT 5/STAT 5)。相对折叠差(10 min,20 min)=3.63,3.34(pSTAT 1/STAT 1);2.11,7.23(pSTAT 3/STAT 3);1.36,1.35(pSTAT 5/STAT 5)。b具有代表性的dcSSc DFS芯片检测磷酸化STAT 3与Col1a2增强剂(左)免疫沉淀染色质用特异性引物进行实时PCR分析。Col1a2增强剂(右)精确性p值=0.029。n=4项独立于生物的实验。c用ELISA法检测转染IL-31RA siRNA并经rhIL-31(50 ng/ml)处理的dcSSc DFS上清液中I型胶原、IL-6、IL-33和CCL 2蛋白水平。精确p值=0.00000009,0.111,0.0000008(I型胶原);0.0000009,0.202,0.000007(IL-6);0.002,0.766,0.005(IL-33);0.000003,0.222,0.00002(CCL 2)。dELISA法检测转染OSMR siRNA或STAT 1抑制剂(STAT 1-I:fludarabine;50 M)、STAT 3(STAT-Ⅰ:Stattic;5 M)或STAT 5(STAT 5-I:CaS 285986-31-4;50 M)的dcSSc DFS上清液中Ⅰ型胶原蛋白水平,然后用rhIL-31(50 ng/ml)处理。精确性p值=0.238(IL-31+OSMR siRNA对IL-31),0.175(IL-31+STAT 1-I vs IL-31),0.008(IL-31+STAT 3-I vs IL-31),0.548(IL-31+STAT5-I vs IL-31)。n=5生物独立实验,除非另有说明。数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001. Significance was determined using two-tailed Mann-Whitney U试验(a, b,和d)和单向方差分析,然后是Tukey的后特别比较检验(c)。PSTAT 1,磷酸化STAT 1;pSTAT 3,磷酸化STAT 3;pSTAT 5,磷酸化STAT 5。源数据作为源数据文件提供。

此外,我们还探讨了IL-31对SSC DFS的影响是否是通过IL-31RA介导的。用小干扰RNA(SiRNA)敲除IL-31RA可显著降低IL-31诱导的I型胶原、IL-6、IL-33和CCL 2的表达。4C)。因为mRNA的表达OSMR,另一个受体亚单位组成IL-31受体复合物,在SSC(补充图)中升高。3)我们还研究了OSMR信号通路是否和在多大程度上参与了IL-31诱导的SSC DFS胶原表达的增加。如图所示。4D在rhIL-31刺激下,OSMR基因敲除对SSC DFS中I型胶原的表达无明显影响。因此,IL-31RA介导的信号转导对SSC DFS中I型胶原的诱导作用比通过OSMR信号转导更为重要。我们还观察到选择性STAT 3抑制剂显著降低rhIL-31诱导的SSC DFS胶原生成。4D)。STAT 1或STAT 5的选择性抑制剂对胶原的产生无明显影响。因此,通过STAT 3的信号转导在IL-31诱导胶原的过程中确实起着重要的作用。这些结果提示IL-31对SSC DFS的影响主要是由IL-31RA和STAT 3介导的。

IL-31对BLM-SSC小鼠皮肤和肺纤维化的促进作用

为探讨白细胞介素-31(IL-31)对小鼠体内纤维化的影响,我们用重组小鼠白细胞介素-31(rmIL-31)皮下注射BLM-SSC小鼠和PBS处理的对照组小鼠。5A)。RmIL-31能明显加重BLM-SSC小鼠的皮肤和肺纤维化。5B,c)。RmIL-31处理的blm-sc小鼠的表达明显增强。Col1a1, Col1a2,将I型胶原与假手术组BLM-SSC小鼠进行比较(图1)。5D,e)。与BLM-SSC小鼠相比,rmIL-31对PBS处理的对照组小鼠皮肤和肺纤维化作用明显,但在一定程度上低于BLM-SSC小鼠。我们还观察了IL-31和IL-31RA在BLM-SSC小鼠纤维皮肤和肺组织中的上调作用。5F和补充图。4)。因此,rmIL-31对BLM-SSC小鼠皮肤和肺纤维化的诱导作用可能与IL-31RA在这些组织中的大量表达有关。其次,我们探讨了肺组织中表达IL-31的细胞类型(附图)。5A)。在BLM-SSC小鼠中,IL 31主要由成纤维细胞表达,用siRNA抑制IL-31,减少I型胶原的释放(补充图)。5B)。因此,自分泌的表达IL 31成纤维细胞可能在BLM-SSC小鼠肺胶原过度生成中起重要作用。类似的结果IL31RA,也主要由BLM-SSC小鼠肺成纤维细胞表达,而成纤维细胞的抑制则降低了胶原的释放(补充图)。5C,d)。这些结果表明IL-31和IL-31RA在BLM诱导的肺纤维化中的表达与成纤维细胞的功能相关。

图5:IL-31增强BLM-SSC小鼠皮肤和肺纤维化。
figure5

a空白对照组(PBS-Ctrl)和空白对照组(PBS-Ctrl)分别于第1~14天给予rmIL-31或生理盐水作为假手术组,并于第15天进行分析。b用苏木精和伊红染色(左)和马尾松三色染色(右)显示典型的皮肤和肺组织切片(皮肤水平比例尺=100μm,肺染色20 m)。带有箭头的垂直条表示真皮厚度。组织学检查皮肤厚度和肺纤维化评分。确切p值(PBS-Ctrl+Sham vs PBS-Ctrl+IL-31,PBS-Ctrl+Sham vs BLM-SSC+Sham,PBS-Ctrl+Sham vs BLM-SSC+IL-31,PBS-Ctrl+IL-31 vs BLM-SSC+Sham,PBS-Ctrl+IL-31 vs BLM-SSC+IL-31,BLM-SSC+SSC-31)=0.003,0.00001,0.0000000005,0.049,0.00000006,0.000004(真皮厚度);0.009,0.0000003,0.00000000004,0.0001,0.000000001,0.000003(肺纤维化评分)。c皮肤和肺组织羟脯氨酸含量。确切p值(PBS-Ctrl+Sham vs PBS-Ctrl+IL-31,PBS-Ctrl+Sham与BLM-SSC+Sham,PBS-Ctrl+Sham vs BLM-SSC+IL-31,PBS-Ctrl+IL-31 vs BLM-SSC+Sham,PBS-Ctrl+IL-31 vs BLM-SSC+IL-31,BLM-SSC+SSC-31)=0.036,0.00007,0.0000001,0.027,0.00001,0.004(皮肤);0.015,0.00003,0.0000001,0.025,0.00002,0.011(肺)。d相对mRNA表达水平Col1a1Col1a2对皮肤和肺组织进行实时PCR检测。精确性p=0.004,0.000000002,0.00000000004,0.0000004,0.000000003,0.005=0.004,0.000000002,0.00000000004,0.0000004,0.005(Col1a1,0.033,0.000000002,0.000000000002,0.00000006,0.00000000002,0.000005(Col1a2,0.046,0.00004,0.00000001,0.011,0.0000006,0.0003(Col1a1,0.006,0.000002,0.000000007,0.002,0.000001,0.004(Col1a2、肺)。相对折叠差(PBS-Ctrl+IL-31,BLM-SSC+Sham,BLM-SSC+IL-31)=1.86,3.78,4.61(Col1a1、1.86、4.87、7.03(Col1a2,1.47,2.05,2.91(Col1a1、1.54、2.14、2.69(Col1a2、肺)。e采用酶联免疫吸附试验(ELISA)检测皮肤和肺组织I型胶原蛋白水平。确切p值(PBS-Ctrl+Sham vs PBS-Ctrl+IL-31,PBS-Ctrl+Sham与BLM-SSC+Sham,PBS-Ctrl+Sham vs BLM-SSC+IL-31,PBS-Ctrl+IL-31 vs BLM-SSC+Sham,PBS-Ctrl+IL-31 vs BLM-SSC+IL-31,BLM-SSC+SSC-31)=0.048,0.00004,0.00000001,0.011,0.0000006,0.0003(皮肤);0.029,0.000005,0.000000004,0.002,0.0000002,0.0004(肺)。f相对mRNA表达水平IL 31IL31RA对皮肤和肺组织进行实时PCR检测。精确p值=0.008(IL 31,皮肤);0.008(IL31RA,皮肤);0.008(IL 31,肺);0.008(IL31RA、肺)。相对褶皱差=5.49(IL 31,皮肤);3.48(IL31RA,皮肤);2.51(IL 31、肺);1.98(IL31RA、肺)。n数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001. Significance was determined using one-way analysis of variance followed by Tukey’s post hoc comparison test (be)和两尾曼恩-惠特尼U试验(F)结果表明,这是三个独立实验的代表,具有相似的结果。PBS-Ctrl,PBS处理对照。源数据作为源数据文件提供。

采用实时PCR和ELISA法检测rmIL-31对皮肤和肺细胞因子产生的影响。IL-4、IL-6、IL-10、IL-17A、肿瘤坏死因子(TNF)、转化生长因子-β1和干扰素(IFN)-γ在BLM-SSC小鼠皮肤和肺组织中的表达明显高于PBS处理的对照组(见图)。6a,b,补充图。6),如先前报告所述10。RmIL-31可进一步提高BLM-SSC小鼠皮肤和肺中IL-4、IL-6、IL-10和TGF-β1的表达水平,促进这些组织中Th2和促纤维细胞因子的过度表达。RmIL-31对BLM-SSC小鼠Th1细胞因子IFN-γ、Th17细胞因子IL-17A和肿瘤坏死因子(TNF)的表达无明显影响。因此,IL-31通过增加硬化性皮肤和肺组织中Th2和促纤维化细胞因子的表达而增强BLM诱导的纤维化。我们没有观察到rmIL-31对皮肤或肺组织CTGF表达的显著影响。6C,d),这与在SSC DFS中得到的结果是一致的。3C)。此外,rmIL-31还显著降低了rmIL-31的mRNA水平。Mmp3, MMP 9,和Mmp13在BLM-SSC小鼠肺组织中,其mRNA水平显著升高。TIMP 1, Timp2,和TIMP 3(无花果)6E)。因为基质金属蛋白酶表达减少和TIMPs表达增加与SSC胶原降解受损有关。38IL-31进一步增强BLM-SSC小鼠肺组织中MMPs和TIMPs的表达,这可能是导致胶原过度沉积的原因之一。综合起来,IL-31增强了BLM诱导的纤维化,增加了与纤维化和Th2反应相关的细胞因子的表达。

图6:IL-31上调Th2和纤维化相关细胞因子对BLM-SSC小鼠的影响。

a相对mRNA表达水平IL4, IL6, IL 10, IL17A, 肿瘤坏死因子, Tgfb 1,和IFNG对皮肤和肺组织进行实时PCR检测。精确p值(pbs-ctrl+sham vs pbs-ctrl+IL-31,pbs-ctrl+sham vs blm-ssc+sham,pbs-ctrl+sham vs bm-ssc-sc-31,pbs-ctrl+IL-31 vs blm-ssc+sham,pbs-ctrl+IL-31 vs blm-ssc+IL-31,bbs-ctrl+sham-ssc+IL-31)=0.866,0.039,0.00006,0.162,0.0003,0.021(IL4,0.403,0.00000000006,0.0000000000002,0.0000000003,0.0000000000004,0.000001(IL6,0.034,0.004,0.000003,0.699,0.0007,0.007(IL 10,0.926,0.000007,0.0000005,0.00002,0.000001,0.344(IL17A,0.977,0.012,0.063,0.005,0.029,0.832(肿瘤坏死因子,0.572,0.0001,0.00000001,0.001,0.00000008,0.0001(Tgfb 1,0.428,0.00007,0.002,0.001,0.040,0.361(IFNG,0.00008,0.003,0.0000002,0.286,0.008,0.0002(IL4,0.023,0.00001,0.0000001,0.007,0.00002,0.038(IL6,0.002,0.227,0.00003,0.085,0.231,0.002(IL 10,0.192,0.000008,0.000001,0.0004,0.00005,0.686(IL17A,0.641,0.013,0.041,0.127,0.321,0.937(肿瘤坏死因子,0.967,0.0002,0.0000005,0.0005,0.000001,0.014(Tgfb 1,0.973,0.00003,0.0001,0.00007,0.0003,0.868(IFNG、肺)。相对折叠差(PBS-Ctrl+IL-31,BLM-SSC+Sham,BLM-SSC+IL-31)=1.55,3.12,5.45(IL4、2.14、13.09、19.11(IL6、2.59、3.15、5.16(IL 10,1.22,3.63,4.24(IL17A、0.89、1.97、1.74(肿瘤坏死因子、1.34、2.57、4.12(Tgfb 1、1.68、3.71、2.98(IFNG、3.59、2.81、5.20(IL4、5.56、10.92、15.12(IL6、2.29、1.56、2.85(IL 10、2.35、5.74、6.45(IL17A,1.27,1.81,1.68(肿瘤坏死因子、1.16、2.98、4.22(Tgfb 1、1.22、4.34、3.95(IFNG、肺)。bELISA法检测转化生长因子-β1的蛋白水平.确切p值(PBS-Ctrl+Sham vs PBS-Ctrl+IL-31,PBS-Ctrl+Sham与BLM-SSC+Sham,PBS-Ctrl+Sham vs BLM-SSC+IL-31,PBS-Ctrl+IL-31 vs BLM-SSC+Sham,PBS-Ctrl+IL-31 vs BLM-SSC+IL-31,BLM-SSC+SSC-31)=0.924,0.00006,0.0000003,0.0002,0.0000007,0.022(皮肤);0.805,0.00003,0.0000004,0.0002,0.000002,0.076(肺)。c, d相对mRNA水平和蛋白质水平CTGF用实时聚合酶链反应(PCR)进行评价。c)和ELISA(d)分别。精确p值(pbs-ctrl+sham vs pbs-ctrl+IL-31,pbs-ctrl+sham vs blm-ssc+sham,pbs-ctrl+sham vs bm-ssc-sc-31,pbs-ctrl+IL-31 vs blm-ssc+sham,pbs-ctrl+IL-31 vs blm-ssc+IL-31,bbs-ctrl+sham-ssc+IL-31)=0.912,0.000000003,0.0000000005,0.000000007,0.000000001,0.391(CTGF,0.967,0.00000007,0.000000009,0.0000001,0.00000002,0.393(CTGF;0.972,0.00006,0.00002,0.00003,0.00001,0.941(CTGF,皮肤);0.933,0.005,0.006,0.017,0.019,0.999(CTGF,肺)。相对折叠差(PBS-Ctrl+IL-31,BLM-SSC+Sham,BLM-SSC+IL-31)=1.14,3.79,4.13(皮肤);1.12,3.86,4.30(肺)。e相对mRNA表达水平Mmp3, MMP 9, Mmp13, TIMP 1, Timp2,和TIMP 3采用实时PCR技术对BLM-SSC小鼠肺组织进行检测.精确p值=0.008(Mmp3); 0.016 (MMP 9); 0.008 (Mmp13); 0.008 (TIMP 1); 0.008 (Timp2); 0.008 (TIMP 3)。相对折差=0.79(Mmp3); 0.72 (MMP 9); 0.68 (Mmp13); 2.06 (TIMP 1); 1.42 (Timp2); 1.26 (TIMP 3). n数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001. Significance was determined using one-way analysis of variance followed by Tukey’s post hoc comparison test (a-d) and two-tailed Mann–Whitney U试验(e)。结果表明,这是三个独立实验的代表,具有相似的结果。PBS-Ctrl,PBS处理对照。源数据作为源数据文件提供。

为了进一步探讨IL-31在体内的促纤维化作用是否和在何种程度上由Th2反应介导,我们在rmIL-31和BLM的共同作用下,给小鼠注射抗IL-4受体α(IL-4RA)单抗。如附图所示。7a,b抗IL-4RA单抗可明显减轻rmIL-31诱导的皮肤纤维化和肺纤维化。然而,与BLM和SHAM处理的小鼠相比,BLM、rmIL-31和抗IL-4RA单抗均能增加皮肤和肺的纤维化,提示抗IL-4RA单抗虽能降低IL-31的促纤维化作用,但并不能完全阻断其作用。同样,抗IL-4RA单抗也明显减弱,但并不能完全阻止抗IL-4RA单抗的表达增强。IL4, IL6, IL 10,和Tgfb 1RmIL-31致小鼠皮肤和肺损伤的实验研究(附图)。7C)。这些结果提示IL-31对纤维化和细胞因子表达的影响是部分的,但不是完全通过IL-4RA介导的。我们还评价了TGF-β1与rmIL-31和BLM联合使用抗TGF-β1单抗对IL-31介导的纤维化的作用.如附图所示。8a,b抗TGF-β1单抗可抑制IL-31的促纤维化作用,但未完全阻断.在细胞因子表达方面,blm、rmIL-31和抗β-β1 mAb处理的小鼠细胞因子1的表达水平均低于blm和rmIL-31,但其表达水平与rmIL-31相比,差异有显着性(P<0.0 5)。IL4, IL6, IL 10, IL17A, 肿瘤坏死因子,或IFNG抗转化生长因子-β1单抗无明显影响(附图).8C)。这些结果提示IL-31对BLM-SSC小鼠的促纤维化作用至少部分是通过促进转化生长因子-β1的产生来实现的。9)。在blm诱导的纤维化过程中,IL-31和β-β1相互作用的体外实验表明,抗β1单抗可降低抗转化生长因子-β1单抗。IL 31表达增强IL31RABLM-SSC小鼠肺成纤维细胞的表达(附图)。10)。后者的发现与Yaseen等人的研究相一致。34,可能与抗转化生长因子-β1治疗降低IL-31诱导胶原的结果不一致。虽然还需要进一步的研究来充分阐明IL-31和转化生长因子-β1在干细胞发病过程中的相互作用,但有一种可能是IL-31RA在SSC患者和BLM模型中的表达已经升高(如图1所示)。2, 5F抗β1抗体的进一步上调并不一定通过IL-31/IL-31RA轴引起信号增强。总之,转化生长因子-β1和通过IL-4RA的信号传递对IL-31对BLM-SSC小鼠的促纤维化作用是重要的,但还不够。

IL-31对BLM-SSC小鼠Th2极化的促进作用

接下来我们研究了rmIL-31对脾T细胞分化的影响。7A,补充图。11)。BLM-SSC小鼠与PBS对照组相比,Th2和Th17细胞频率增加,Treg细胞频率降低。此外,BLM-SSC小鼠的Th2/Th1、Th17/Treg和Th2/Treg细胞比例均有增加,这与SSC的Th2和Th17极化有关。10。RmIL-31可进一步提高BLM-SSC小鼠Th2细胞频率、Th2/Th1比值和Th2/Treg比值。虽然rmIL-31能显著提高BLM-SSC小鼠Th17细胞的频率,但也能增加Treg细胞的频率。因此,rmIL-31对BLM-SSC小鼠Th17/Treg比值无显著影响。在PBS对照小鼠中,rmIL-31可显著提高Th2细胞频率,降低Treg细胞频率。RmIL-31对PBS对照小鼠Th2/Th1比值或Th17/Treg比值无明显影响,但增加了Th2/Treg比值。肺和肺引流淋巴结T细胞的变化相似,rmIL-31增加了BLM-SSC小鼠的Th2和Th17细胞频率、Th2/Th1比值和Th2/Treg比值(图一)。7b,c)。在血清细胞因子水平上,rmIL-31促进BLM-SSC小鼠IL-4和IL-6的过度生成。7D)。因此,IL-31促进BLM-SSC小鼠Th2极化。我们还观察了rmIL-31对脾脏巨噬细胞分化的影响,但rmIL-31对m1巨噬细胞频率、M2巨噬细胞频率或M2/M1比值无明显影响(补充图)。12).

图7:IL-31增强BLM-SSC小鼠Th2极化。
figure7

ac用流式细胞术检测rmIL-31和假手术(rmIL-31)处理的BLM-SSC和PBS-Ctrl小鼠的Th1、Th2、Th17和Treg细胞频率。a)、肺(b)和肺引流淋巴结(c)。精确性pPBS-Ctrl+Sham vs PBS-Ctrl+IL-31,PBS-Ctrl+Sham vs BLM-SSC+Sham,PBS-Ctrl+Sham与BLM-SSC+IL-31,PBS-Ctrl+IL-31 vs BLM-SSC+SHAM,PBS-Ctrl+IL-31 vs BLM-SSC+IL-31,BLM-Ctrl+SSC+IL-31=0.960,0.417,0.996,0.145,0.841,0.625(Th1,脾脏);0.017,0.0002,0.00000000007,0.342,0.00000001,0.0000003(Th2,脾);0.795,0.005,0.000006,0.059,0.00006,0.041(Th17,脾脏);0.001,0.0000001,0.008,0.002,0.926,0.0003(Treg,脾脏);0.521,0.005,0.00002,0.079,0.0002,0.043(Th2/Th1比值,脾脏);0.206,0.00005,0.0007,0.003,0.044,0.521(Th17/Treg比值,脾脏);0.007,0.011,0.000004,0.996,0.006,0.004(Th2/Treg比值,脾脏);0.966,0.932,0.996,0.999,0.904,0.849(Th1,肺);0.018,0.0003,0.00000002,0.233,0.000003,0.00009(Th2,肺);0.633,0.005,0.00002,0.059,0.0002,0.039(Th17,肺);0.005,0.0000002,0.00002,0.0002,0.050,0.048(Treg,肺);0.031,0.0007,0.0000002,0.264,0.00002,0.0007(Th2/Th1比值,肺);0.363,0.0002,0.000003,0.005,0.00005,0.125(Th17/Treg比率,肺);0.034,0.0002,0.00000005,0.082,0.000004,0.0005(Th2/Treg比值,肺);0.939,0.488,0.961,0.221,0.719,0.772(Th1,肺引流淋巴结);0.038,0.000009,0.000000006,0.002,0.000000001,0.0000003(Th2,肺引流淋巴结);0.839,0.010,0.00001,0.051,0.00005,0.013(Th17,肺引流淋巴结);0.057,0.000001,0.001,0.0002,0.230,0.009(Treg,肺引流淋巴结);0.225,0.00008,0.000000005,0.004,0.00000006,0.00003(Th2/Th1比值,肺引流淋巴结);0.398,0.00003,0.000001,0.0005,0.00001,0.244(Th17/Treg比率,肺引流淋巴结);0.011,0.00000007,0.000000000007,0.00002,0.0000000002,0.0000005(Th2/Treg比值,肺引流淋巴结)。d采用酶联免疫吸附试验(ELISA)检测血清IL-4和IL-6水平.精确性pPBS-Ctrl+Sham vs PBS-Ctrl+IL-31,PBS-Ctrl+Sham与BLM-SSC+Sham,PBS-Ctrl+Sham与BLM-SSC+IL-31,PBS-Ctrl+IL-31 vs BLM-SSC+Sham,PBS-Ctrl+IL-31 vs BLM-SSC+IL-31,BLM-Ctrl+SSC+IL-31=0.129,0.001,0.000002,0.103,0.0001,0.019(-4);0.085,0.032,0.00007,0.954,0.012,0.033(IL-6)。n数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001. Significance was determined using one-way analysis of variance followed by Tukey’s post hoc comparison test. The results shown are representative of three independent experiments with similar results. PBS-Ctrl, PBS-treated control. Source data are provided as a Source Data file.

抗IL-31RA单抗对BLM-SSC小鼠纤维化和Th2极化的影响

为探讨IL-31在SSC发生发展中的作用,我们用功能阻断抗IL-31RA单抗(NRA 0049)检测了BLM-SSC小鼠的纤维化和Th2极化。我们每周注射抗IL-31RA单抗,并每日注射BLM,共3周(见图)。8A)。抗IL-31RA单抗能显著降低BLM-SSC小鼠皮肤和肺组织中的真皮厚度、肺纤维化评分和胶原含量。8B-e)。此外,抗IL-31RA单抗还能抑制IL-4、IL-6、IL-10和TGF-β1在BLM-SSC小鼠皮肤和肺中的表达(如图1所示)。8F,补充图。13)。抗IL-31RA单抗也能显著降低BLM-SSC小鼠脾、肺和肺引流淋巴结中Th2细胞比例、Th2/Th1比值和Th2/Treg比值(图1)。9A-c)。此外,抗IL-31RA单抗还能改善BLM-SSC小鼠血清中IL-4和IL-6的过度生成。9d)。抗IL-31RA单抗对BLM诱导的纤维化和Th2极化有明显的抑制作用.

图8:抗IL-31RA抗体可减轻BLM-SSC小鼠的纤维化。
figure8

a分别于第1、8、15天给予抗小鼠IL-31RA抗体(αIL-31RA)或同类型对照抗体(Iso),并于第22天处死,以评价BLM-SSC和PBS处理对照组(PBS-Ctrl)小鼠的免疫功能。b用苏木精和伊红染色(左)和马尾松三色染色(右)显示典型的皮肤和肺组织切片(皮肤水平比例尺=100μm,肺染色20 m)。带有箭头的垂直条表示真皮厚度。组织学检查皮肤厚度和肺纤维化评分。精确p值(pBS-Ctrl+iso vs BLM-sSC+iso,pBS-ctrl+iso与BLM-sSC+αIL-31RA,BLM-sc+iso vsαIL-31RA)=0.00004,0.028,0.004(真皮厚度);0.0000002,0.00002,0.00004(肺纤维化评分)。c皮肤和肺组织中羟脯氨酸的含量。精确性p值(pbs-ctrl+iso vs blm-ssc+iso,pbs-ctrl+iso vs blm-sc+αIL-31 RA,blm-sc+iso vs blm-sc+αIL-31 RA)=0.00005,0.052,0.003(皮肤);0.00004,0.011,0.012(肺)。d相对mRNA表达水平Col1a1Col1a2对皮肤和肺组织进行实时PCR检测。精确性p值(pbs-ctrl+iso vs bm-ssc+iso,pbs-ctrl+iso vs bm-ssc+αIL-31 ra,bbm-sc+iso vs bm-ssc+αIL-31 ra)=0.0000000003,0.00009,0.00000002(Co11a1,0.00000002,0.005,0.0000006(Co11a2,0.0000004,0.014,0.00002(Col1a1,0.0000002,0.001,0.00005(Col1a2、肺)。相对折叠差(BLM-SSC+iso,BLM-SSC+αIL-31RA)=4.21,2.00(Col1a1、皮肤);5.64、2.27(Col1a2,2.62,1.50(Col1a1;3.08,1.86(Col1a2、肺)。e采用酶联免疫吸附试验(ELISA)检测I型胶原蛋白在皮肤和肺组织中的表达。精确p值(pbs-ctrl+iso vs blm-ssc+iso,pbs-ctrl+iso vs blm-sc+αIL-31 RA,blm-sc+iso vs blm-sc+αIL-31 RA)=0.0000002,0.021,0.000007(皮肤);0.0000005,0.026,0.00002(肺)。fMRNA表达IL4, IL6, IL 10, IL17A, 肿瘤坏死因子, Tgfb 1,和IFNG采用实时PCR技术对小鼠皮肤和肺组织进行检测。精确p值(pbs-ctrl+iso vs bm-ssc+iso,pbs-ctrl+iso vs bm-sc+αIL-31 ra,blm-sc+iso vs blm-ssc+αIL-31 ra)=0.00001,0.002,0.015(IL4,0.0000005,0.0003,0.0007(IL6,0.0006,0.083,0.037(IL 10,0.0003,0.001,0.625(IL17A,0.00004,0.00005,0.996(肿瘤坏死因子,0.00000001,0.00006,0.00001(Tgfb 1,0.000003,0.000005,0.848(IFNG,0.0000001,0.0001,0.0001(IL4,0.000003,0.003,0.001(IL6,0.0005,0.251,0.010(IL 10,0.000000007,0.000000009,0.927(IL17A,0.003,0.002,0.997(肿瘤坏死因子,0.0000009,0.001,0.0005(Tgfb 1,0.00004,0.00002,0.943(IFNG、肺)。相对折叠差(BLM-SSC+iso,BLM-SSC+αIL-31RA)=4.19,2.83(IL4、皮肤);18.09、10.01(IL6,皮肤);5.75,3.15(IL 10,皮肤);5.05,4.38(IL17A,皮肤);2.57,2.55(肿瘤坏死因子、3.52、2.14(Tgfb 1,皮肤);4.51,4.30(IFNG;5.00,3.01(IL4、17.72、8.92(IL6,2.56,1.50(IL 10;7.20,7.05(IL17A、2.17、2.19(肿瘤坏死因子、4.14、2.48(Tgfb 1、5.34、5.54(IFNG、肺)。n数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001. One-way analysis of variance followed by Tukey’s post hoc comparison test was used. The results shown are representative of three independent experiments with similar results. PBS-Ctrl, PBS-treated control; αIL-31RA, anti-mouse IL-31RA antibody; iso, isotype control IgG. Source data are provided as a Source Data file.

图9:抗IL-31RA抗体抑制BLM-SSC小鼠Th2极化.
figure9

ac用流式细胞术(FCM)检测抗小鼠IL-31RA抗体(αIL-31RA)和同型对照抗体(Iso)对BLM-sSC和pbs-ctrl小鼠Th1、Th2、Th17和Treg细胞的影响。a)、肺(b)和肺引流淋巴结(c)。精确性p值(pbs-ctrl+iso vs blm-ssc+iso,pbs-ctrl+iso vs bm-sSC+αIL-31 RA,blm-sc+iso vs blm-sc+αIL-31 RA)=0.649,0.769,0.978(Th1,脾);0.0006,0.269,0.010(Th2,脾);0.010,0.085,0.463(Th17,脾);0.021,0.026,0.994(脾脏);0.006,0.674,0.027(Th2/Th1比值,脾脏);0.005,0.020,0.712(Th17/Treg比值,脾脏);0.0002,0.042,0.019(Th2/Treg比率,脾脏);0.547,0.830,0.878(Th1,肺);0.000004,0.032,0.0002(Th2,肺);0.002,0.005,0.768(Th17,肺);0.042,0.040,0.999(Treg,肺);0.00001,0.052,0.0006(Th2/Th1比值,肺);0.006,0.010,0.957(Th17/Treg比值,肺);0.0000003,0.003,0.00004(Th2/Treg比率,肺);0.785,0.830,0.996(Th1,肺引流淋巴结);0.000005,0.004,0.002(Th2,肺引流淋巴结);0.015,0.012,0.988(Th17,肺引流淋巴结);0.008,0.007,0.999(Treg,肺引流淋巴结);0.00000005,0.0001,0.00005(Th2/Th1比值,肺引流淋巴结);0.0008,0.0006,0.983(Th17/Treg比值,肺引流淋巴结);0.000002,0.001,0.002(Th2/Treg比率,肺引流淋巴结)。d采用酶联免疫吸附试验(ELISA)检测血清IL-4和IL-6水平.精确性p值(pbs-ctrl+iso vs blm-ssc+iso,pbs-ctrl+iso vs blm-sc+αIL-31 RA,blm-sc+iso vs blm-sc+αIL-31 RA)=0.00001,0.0007,0.031(IL-4);0.0000008,0.0005,0.0009(IL-6)。n数据以平均值±SD表示。*p < 0.05, **p < 0.01, and ***p < 0.001. One-way analysis of variance followed by Tukey’s post hoc comparison test was used. The results shown are representative of three independent experiments with similar results. PBS-Ctrl, PBS-treated control; αIL-31RA, anti-mouse IL-31RA antibody; iso, isotype control IgG. Source data are provided as a Source Data file.

讨论

在本研究中,我们展示了抗IL-31RA单抗在SSC小鼠模型中的作用。虽然以前的研究表明IL-31有促进纤维化的作用。34,44抗IL-31RA单抗在SSC中的作用尚未见报道。在此,我们初步证明SSC患者血清IL-31水平显著升高(图一)。1A)。值得注意的是,血清IL-31水平与皮肤和肺纤维化的严重程度以及血清IL-4、IL-6和IL-13水平呈正相关(图一)。1B和表1),提示IL-31与SSC的纤维化和Th2免疫应答有关。此外,SSC患者的DFS中IL-31和IL-31RA的表达均显著高于健康对照组(图一)。2)。在SSC DFS中,IL-31诱导胶原生成和促进纤维化和Th2极化的细胞因子的表达(见图)。3)。重要的是,这些作用主要由IL-31RA和STAT 3介导。4)。在BLM-SSC小鼠中,外源性给药rmIL-31可导致皮肤和肺纤维化加重,Th2细胞比例增加,与纤维化和Th2反应相关的细胞因子表达增加。57)。这些结果使我们认为IL-31增强了SSC的纤维化和Th2极化。此外,抗IL-31RA单抗能显著减轻BLM-SSC小鼠的纤维化和Th2极化。8, 9),展示了IL-31在SSC发展中的关键作用。总的来说,与Yaseen等人最近的文章保持一致。34这些结果提示IL-31是治疗SSC的一个有希望的靶点。

成纤维细胞过度产生细胞外基质是导致皮肤和内脏进行性纤维化的重要病理特征。45。最近,Yaseen等人。在体外和体内均显示了IL-31的促纤维化作用。34。结果表明,IL-31对SSC DFS和BLM-SSC小鼠的胶原合成均有促进作用.此外,我们还证明IL-31促进了SSC样表型在SSC DFS中的表达,包括向肌成纤维细胞分化的增加(图1)。3).

Th2极化是SSC的主要免疫异常,在SSC患者和动物模型中均有观察。3。越来越多的证据表明Th2细胞是IL-31的主要产生者。19,20。最近的一项研究也报道,IL-4是Th2细胞分泌IL-31所必需的。46。以及Th2细胞因子对IL-31和IL-31RA的上调作用(图二)。2EIL-31在SSC患者中的过度表达可能反映了该病的Th2优势。此外,IL-31增强了SSC DFS中IL-6、IL-33和CCL 2的表达。3i,j)。由于这些细胞因子能促进Th2细胞因子在不同细胞和组织中的表达,这一结果提示IL-31通过上调dfs中这些前Th2细胞因子的表达而促进sSC中Th2细胞因子的表达。40,41,42。的确,体内研究表明,IL-31促进了BLM-SSC小鼠Th2极化。6, 7)。IL-31RA阻断mAb后,抑制了BLM-SSC小鼠Th2细胞因子的表达和Th2细胞的分化。8, 9本研究结果表明,IL-31增强了SSC的Th2极化。

SSC是最具破坏性的自身免疫性疾病之一,发病率和死亡率都很高,这在很大程度上是由于缺乏有效的治疗。因此,在治疗SSC方面,新药的开发是非常迫切的。目前的研究表明,IL-31通过直接诱导成纤维细胞产生胶原和诱导成纤维细胞产生促Th2细胞因子间接促进SSC的发展。Th2极化进一步促进IL-31的产生,通过IL-31/IL-31RA轴形成恶性循环,导致SSC的发展。虽然还需要进一步的研究来阐明IL-31在SSC发病机制中的确切作用,但我们的研究提供了一个关于IL-31在SSC中作用的未知洞察。此外,我们的研究还证明抗IL-31RA单抗能明显改善SSC的纤维化和Th2极化。这些结果为临床应用抗IL-31RA单抗治疗SSC提供了理论依据。


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