Expression and function of IL-22 in rheumatoid arthritis-associated interstitial lung disease
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摘要:
目的 观察IL-22在类风湿关节炎相关间质性肺病中(RA-ILD)的表达情况，建立动物模型探讨IL-22在肺纤维化中的作用。 方法 选择2021年10月—2022年10月在徐州医科大学附属医院初诊初治的类风湿关节炎合并(RA-ILD组)/未合并(RA-NILD组)间质性肺病患者各33例，同期14位健康体检者为健康对照组，采用ELISA测定3组外周血IL-22水平并比较。采用Pearson检验分析IL-22水平与肺HRCT评分的相关性；采用多因素logistic回归分析研究RA-ILD的危险因素并绘制ROC曲线。生理盐水(NS)和博来霉素(BLM)处理野生小鼠(WT)与IL-22敲基因小鼠(IL-22KO)，建模后予腹腔注射IL-22，HE和Masson染色观察肺组织病理改变，RT-qPCR、Western blotting检测纤维化标志物水平。 结果 RA-NILD组IL-22水平高于RA-ILD组及健康对照组，其水平与HRCT评分呈负相关关系(r=-0.940, P＜0.05)。Logistic回归分析提示IL-22水平是RA-ILD的影响因素，IL-22联合年龄、吸烟、APCA、MCHC诊断RA-ILD的AUC为0.959(95% CI：0.916~1.000)，灵敏度为97.0%，特异度为87.9%。病理染色示BLM组小鼠肺部胶原面积较NS组增多，Szapiel ' s评分和Ashcroft评分升高，纤维化相关基因(Collagen Ⅰ、Vimentin、α-SMA)的mRNA和蛋白质表达也较NS组增高，其中IL-22KO组较WT组增高更显著(P＜0.05)。IL-22治疗后胶原沉积及纤维化基因的mRNA和蛋白表达明显减少(P＜0.05)。 结论 IL-22可能对肺纤维化具有保护作用，未来可能成为潜在的治疗靶点，并可用于临床预测RA-ILD。 -
关键词:
- 类风湿关节炎相关间质性肺病 /
- 间质性肺病 /
- 白介素-22
Abstract:Objective This study aims to observe the expression of IL-22 in patients with rheumatoid arthritis-associated interstitial lung disease(RA-ILD)and establish an experimental model to explore its role in pulmonary fibrosis. Methods We selected 33 patients with rheumatoid arthritis with or without interstitial lung disease, who were newly diagnosed and treated at the Affiliated Hospital of Xuzhou Medical University from October 2021 to October 2022. Additionally, 14 healthy subjects were chosen as controls during the same period. ELISA was used to detect the peripheral blood IL-22 levels in the three groups, and a comparison was made among them. Spearman correlation test was employed to analyze the correlation between IL-22 and lung HRCT scores. Logistic regression was used to analyze risk factors for RA-ILD and to draw ROC curves. Wild mice (WT) and IL-22 knock-out mice (IL-22KO) were treated with normal saline (NS) and Bleomycin (BLM). Following modeling, mice were given intraperitoneal injections of IL-22. HE and Masson staining were used to observe the histopathological changes in the lung tissues. Relevant markers were detected by RT-qPCR and Western blotting. Results The levels of IL-22 were higher in the RA-NILD group compared to the RA-ILD group and the healthy control group. There was a negative correlation between IL-22 levels and lung HRCT scores (r=-0.940, P < 0.05). Logistic regression analysis suggested that a low level of IL-22 was an independent risk factor for RA-ILD. The combined diagnosis of RA-ILD with IL-22, age, smoking, APCA, and MCHC had an AUC of 0.959 (95% CI: 0.916-1.000), with a sensitivity of 97.0% and specificity of 87.9%. HE and Masson staining found that the collagen deposition in the lungs of BLM groups were increased compared with the NS groups, accompanied by Szapiel ' s scores and Ashcroft scores. The mRNA and protein expression of fibrosis-related genes (Collagen Ⅰ, Vimentin, and α-SMA) were increased in the BLM groups compared to the NS group, and the IL-22KO group showed even higher levels (P < 0.05). Treatment with IL-22 resulted in a reduction in collagen deposition, mRNA, and protein expression of fibrosis-related genes (P < 0.05). Conclusion IL-22 exhibits potential as a protective effect against pulmonary fibrosis, suggesting it may be a potential therapeutic target and clinical predictor in the future interventions. -
图 1 RA及RA-ILD患者IL-22水平和HRCT评分的相关性分析
注：A为所有RA患者(未根据是否合并ILD分组)外周血IL-22与其肺部CT评分的相关性分析；B为RA-ILD患者外周血IL-22与其肺部CT评分的相关性分析。
Figure 1. The correlation between IL-22 levels and ILD score in RA and RA-ILD patients
图 2 血清IL-22以及联合指标(IL-22、年龄、吸烟、APCA和MCHC)诊断RA-ILD的ROC曲线
注：对角线为参考线，绿色线代表IL-22对RA-ILD的分类价值，赋值0：RA-NILD；橘色代表IL-22联合年龄、吸烟、APCA和MCHC对RA-ILD的分类价值，赋值1：RA-ILD。
Figure 2. ROC curves for diagnosing RA-ILD using IL-22 and combination of IL-22, age, smoking, APCA, and MCHC
图 3 各组小鼠肺组织病理染色结果与评分比较
注：A为BLM组与IL-22治疗组小鼠肺组织在第7天的HE染色结果和第21天的Masson染色结果(肺纤维化过程第7天主要为BLM诱导建模后时间，第21天主要为胶原沉积表现)；B为各组小鼠肺组织病理Szapiel's评分、Ashcroft评分和胶原面积(%)的比较(aP＜0.05)。
Figure 3. The results of lung histopathological staining and comparisons of lung inflammation and fibrosis scores in each group
图 4 各组小鼠肺组织纤维化相关蛋白表达
注：各组小鼠肺组织纤维化相关基因Western blotting蛋白表达水平(aP＜0.05)。
Figure 4. Protein expression levels of lung tissue fibrosis-related genes in each group
表 1 引物序列
Table 1. Primer Sequences
引物名称 引物序列 Collagen Ⅰ Forward primer TGACTGGAAGAGCGGAGAGTA Reverse primer CATTAGGCGCAGGAAGGTCA Vimentin Forward primer TTTGCTGACCTCTCTGAGGC Reverse primer CTCCAGGGACTCGTTAGTGC α-SMA Forward primer GTCCCAGACATCAGGGAGTAA Reserve primer TCGGATACTTCAGCGTCAGGA GAPDH Forward primer TTGATGGCAACAATCTCCAC Reverse primer CGTCCCGTAGACAAAATGGT 
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表 2 3组研究对象外周血IL-22水平比较(x±s, pg/mL)
Table 2. Comparisons of IL-22 levels among three groups(x±s, pg/mL)
组别 例数 IL-22 RA-ILD组 33 205.36±44.81a RA-NILD组 33 246.92±41.53bc 健康对照组 14 168.01±37.49 F值 18.876 P值 ＜0.001 注：与对照组比较，aP=0.007；与对照组比较，bP＜0.001；与RA-ILD组比较，cP＜0.001。
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表 3 RA患者发生ILD危险因素的单因素logistic回归分析
Table 3. Univariate analyses for risk factors of RA-ILD
项目 B SE Waldχ2 P值 OR(95% CI) 年龄(年) 0.100 0.028 12.798 ＜0.001 1.105(1.046~1.167) 吸烟(%) 1.760 0.828 4.522 0.033 5.812(1.148~29.436) SJC 0.080 0.034 5.650 0.017 1.083(1.014~1.157) TJC 0.080 0.033 5.884 0.015 1.084(1.016~1.156) DAS28(units) 0.478 0.196 5.951 0.015 1.613(1.098~2.367) IL-22水平(pg/mL) -0.022 0.007 10.646 0.001 0.978(0.965~0.991) aIL-22水平＜243.06(pg/mL) 2.154 0.602 12.789 ＜0.001 8.615(2.647~28.045) ACPA(IU/mL) 0.002 0.001 6.053 0.014 1.002(1.000~1.003) MCHC(g/L) -0.074 0.029 6.353 0.012 0.929(0.877~0.984) ALB(g/L) -0.240 0.077 9.688 0.002 0.786(0.676~0.915) A/G -2.103 0.983 4.582 0.032 0.122(0.018~0.838) CysC(mg/L) 7.379 2.209 11.157 ＜0.001 1 602.199(21.098~1 672.332) 注：SJC为关节压痛数；TJC为关节疼痛数。a为IL-22水平小于截断值243.06，低于此值为危险因素。本表仅列出差异有统计学意义的变量。
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表 4 RA-ILD发生危险因素的多因素logistic回归分析
Table 4. Multivariate analyses for risk factors of RA-ILD
项目 B SE Waldχ2 P值 OR(95% CI) IL-22水平(pg/mL) -0.059 0.019 9.713 0.002 0.943(0.908~0.978) 年龄(年) 0.110 0.052 4.428 0.035 1.116(1.008~1.236) 吸烟(%) 4.288 1.670 6.596 0.010 72.837(2.761~1 921.437) ACPA(IU/mL) 0.006 0.002 9.716 0.002 1.006(1.002~1.009) MCHC(g/L) -0.124 0.043 8.423 0.004 0.884(0.813~0.961)
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表 5 RA-ILD发生危险因素的多因素logistic回归模型
Table 5. Multivariate logistic regression model for risk factors of RA-ILD
项目 B SE Waldχ2 P值 OR(95% CI) aIL-22水平＜243.06(pg/mL) 4.361 1.374 10.068 0.002 78.300(5.297~1 157.474) 年龄(年) 0.107 0.047 5.295 0.021 1.113(1.016~1.220) 吸烟(%) 3.087 1.343 5.282 0.022 21.910(1.575~304.759) ACPA(IU/mL) 0.004 0.001 9.429 0.002 1.004(1.002~1.007) MCHC(g/L) -0.108 0.037 8.601 0.003 0.898(0.835~0.965) 注：a为IL-22水平小于截断值243.06，低于此值为危险因素。
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