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Increased serum soluble programmed death ligand 1(sPD-L1) is associated with the presence of interstitial lung disease in rheumatoid arthritis: A monocentric cross-sectional study

  • Author Footnotes
    1 Xinyu Wu and Li Xu have equally contributed to this work.
    Xinyu Wu
    Footnotes
    1 Xinyu Wu and Li Xu have equally contributed to this work.
    Affiliations
    Department of Rheumatology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China
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  • Author Footnotes
    1 Xinyu Wu and Li Xu have equally contributed to this work.
    Li Xu
    Footnotes
    1 Xinyu Wu and Li Xu have equally contributed to this work.
    Affiliations
    Department of Rheumatology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China

    Department of Medical, Jiaxing University Affiliated Women and Children Hospital, Jiaxing, 314000, China
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  • Qi Cheng
    Affiliations
    Department of Rheumatology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China
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  • Liuyan Nie
    Affiliations
    Department of Rheumatology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China
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  • Songzhao Zhang
    Affiliations
    Department of Clinical Laboratory, The Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, 310009, China
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  • Yan Du
    Correspondence
    Corresponding author.
    Affiliations
    Department of Rheumatology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China
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  • Jing Xue
    Correspondence
    Corresponding author.
    Affiliations
    Department of Rheumatology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, 310009, China
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  • Author Footnotes
    1 Xinyu Wu and Li Xu have equally contributed to this work.
Open ArchivePublished:March 28, 2020DOI:https://doi.org/10.1016/j.rmed.2020.105948

      Highlights

      • Serum sPD-L1 levels were increased in RA-ILD.
      • SPD-L1 levels were negatively associated with pulmonary function in RA-ILD.
      • SPD-L1 was independently associated with the presence of RA-ILD.

      Abstract

      Objective

      This paper is to examine the relationship between serum soluble programmed death ligand 1(sPD-L1) levels and the development of interstitial lung disease (ILD) in patients with rheumatoid arthritis (RA).

      Method

      Serum sPD-L1 were measured by enzyme-linked immunosorbent assay. sPD-L1 levels in RA with ILD, RA without ILD and healthy controls were compared. Associations between ILD and various markers including sPD-L1 and confounding factors were investigated by logistic regression analysis. Diagnostic values of sPD-L1 for the presence of ILD were investigated using receiver operating characteristics curve analysis.

      Results

      Serum sPD-L1 levels were higher in RA patients with ILD than RA patients without ILD and healthy controls (23.7 ± 9.8 vs. 18.0 ± 7.7 pg/mL, P = 0.01 and 23.7 ± 9.8 vs. 2.9 ± 1.5 pg/mL, P < 0.0001). sPD-L1 levels were positively correlated with RF titer (r = 0.245, P = 0.03), CRP (r = 0.265,P = 0.01), HRCT score (r = 0.265, P = 0.04) and Ferritin (r = 0.442, P = 0.01), but negatively associated with FVC% (r = −0.359, P = 0.01) and DLCO% (r = −0.399, P = 0.008). sPD-L1 and anti-CCP antibody status were independently associated with the presence of ILD during multivariate logistic regression analysis. Sensitivity and specificity of sPD-L1 levels for the detection of ILD in RA patients were 51.7% and 79.3%, respectively (area under the curve = 0.661).

      Conclusion

      Serum sPD-L1 levels were increased in RA patients with ILD. Increased sPD-L1 levels were associated with the presence of ILD.

      Keywords

      1. Introduction

      Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation, which can lead to irreversible articular damage, a decrease in physical functioning, and radiologic progression [
      • Harris Jr., E.D.
      Rheumatoid arthritis. Pathophysiology and implications for therapy.
      ,
      • Kim K.W.
      • Kim H.R.
      Macrophage migration inhibitory factor: a potential therapeutic target for rheumatoid arthritis.
      ]. RA-related interstitial lung disease (ILD) is observed in 4.5–7.7% of RA patients [
      • Kelly C.A.
      • Saravanan V.
      • Nisar M.
      • Arthanari S.
      • Woodhead F.A.
      • Price-Forbes A.N.
      • Dawson J.
      • Sathi N.
      • Ahmad Y.
      • Koduri G.
      • Young A.
      N. British Rheumatoid Interstitial Lung, Rheumatoid arthritis-related interstitial lung disease: associations, prognostic factors and physiological and radiological characteristics--a large multicentre UK study.
      ,
      • Bongartz T.
      • Nannini C.
      • Medina-Velasquez Y.F.
      • Achenbach S.J.
      • Crowson C.S.
      • Ryu J.H.
      • Vassallo R.
      • Gabriel S.E.
      • Matteson E.L.
      Incidence and mortality of interstitial lung disease in rheumatoid arthritis: a population-based study.
      ] and is characterized by dyspnea, dry cough and characteristic findings on chest imaging or pulmonary function tests [
      • Kim E.J.
      • Collard H.R.
      • King Jr., T.E.
      Rheumatoid arthritis-associated interstitial lung disease: the relevance of histopathologic and radiographic pattern.
      ].
      Immune checkpoint PD-1 is one of the important inhibitory molecules for controlling inflammatory response to injury the normal tissues. Programmed death protein (PD-1)/programmed death ligand 1(PD-L1) axis represents a relevant negative feedback loop for maintaining immune homeostasis, but it is also of crucial importance for restricting tumor immunity [
      • Chen D.S.
      • Mellman I.
      Elements of cancer immunity and the cancer-immune set point.
      ,
      • Santarpia M.
      • Gonzalez-Cao M.
      • Viteri S.
      • Karachaliou N.
      • Altavilla G.
      • Rosell R.
      Programmed cell death protein-1/programmed cell death ligand-1 pathway inhibition and predictive biomarkers: understanding transforming growth factor-beta role.
      ]. Checkpoint inhibitor agents targeting PD-1/PD-L1 liberate antitumor T cells from the blockade enabling their further activation, proliferation and killing tumor cells. This cancer immunotherapy has revolutionized treatment and outcomes in several cancers.
      Both PD-1 and PD-L1 also have their soluble forms, the finding contributing to the complexity and multiplicity of the PD-1/PD-L1 immunomodulation pathway [
      • Zhu X.
      • Lang J.
      Soluble PD-1 and PD-L1: predictive and prognostic significance in cancer.
      ,
      • Sorensen S.F.
      • Demuth C.
      • Weber B.
      • Sorensen B.S.
      • Meldgaard P.
      Increase in soluble PD-1 is associated with prolonged survival in patients with advanced EGFR-mutated non-small cell lung cancer treated with erlotinib.
      ,
      • Okuma Y.
      • Hosomi Y.
      • Nakahara Y.
      • Watanabe K.
      • Sagawa Y.
      • Homma S.
      High plasma levels of soluble programmed cell death ligand 1 are prognostic for reduced survival in advanced lung cancer.
      ]. Abundant mouse and human data released recently clearly point that checkpoint regulatory molecules are essential in some autoimmune diseases, not only in cancer [
      • Zhang Q.
      • Vignali D.A.
      Co-stimulatory and Co-inhibitory pathways in autoimmunity.
      ,
      • Dai S.
      • Jia R.
      • Zhang X.
      • Fang Q.
      • Huang L.
      The PD-1/PD-Ls pathway and autoimmune diseases.
      ]. Recent study further indicates that, serum sPD-1/PD-L1 is easily detected in clinical practice and should be further evaluated as a potential prognostic or/and predictive biomarker in idiopathic pulmonary fibrosis [
      • Jovanovic D.
      • Roksandic Milenkovic M.
      • Kotur Stevuljevic J.
      • Markovic J.
      • Ceriman V.
      • Kontic M.
      • Skodric Trifunovic V.
      Membrane PD-L1 expression and soluble PD-L1 plasma levels in idiopathic pulmonary fibrosis-a pilot study.
      ].
      In this study we aimed to elucidate the possible dysregulation of the sPD-L1 in RA and to investigate the relationship with RA-ILD.

      2. Methods and materials

      2.1 Human subjects

      Blood samples of 58 clinical diagnosed RA-ILD patients and 29 clinical diagnosed RA patients without a pulmonary complication were collected from the Department of Rheumatology, the Second Affiliated Hospital of Zhejiang University School of Medicine from January 2014 to December 2018. The RA was diagnosed according to the Guideline of American College of Rheumatology (ACR) 1987 criteria for the diagnosis of RA [
      • Arnett F.C.
      • Edworthy S.M.
      • Bloch D.A.
      • McShane D.J.
      • Fries J.F.
      • Cooper N.S.
      • Healey L.A.
      • Kaplan S.R.
      • Liang M.H.
      • Luthra H.S.
      • et al.
      The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.
      ]. Patients with ILD and IPF due to other causes, complications and/or chronic pulmonary diseases and infectious diseases such as chronic obstructive pulmonary disease (COPD), pulmonary infection, tuberculosis, and tumors in the lung were excluded from this study. Patients with severe heart, lung and renal dysfunction were also excluded. All blood samples were collected in tubes with heparin, and serum were isolated and frozen in 100 μL aliquots at −80 °C until analyzed. There was no genetic relationship among these individuals. 45 healthy controls were recruited from healthy staff members of the hospital. Prior to enrollment, all participants signed the informed consent to donate their blood samples and their clinical information was de-identified for research. Our study was approved by the ethics committee of the Second Affiliated Hospital of Zhejiang University School of Medicine.

      2.2 Clinical assessment of patients

      The demographic feature, serologic features, current treatment and clinical features, including medication history, the 28 joint Disease Activity Score (DAS28), current medication at baseline, serum levels of rheumatoid factors (RF), anti-cyclic peptide containing citrulline (Anti-CCP), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Ferritin, respiratory manifestations and related measurement including the forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLCO), were conducted for all patients. DLCO values of, 80% of predicted normal values and FVC values of, 80% of predicted normal values were considered abnormal. were also recorded.

      2.3 Clinical assessment of patients and ILD diagnosis

      ILD was diagnosed according to high-resolution computed tomography (HRCT) and pulmonary function tests. The HRCT findings were scored on a scale of 1–6, according to the classification method proposed by Kazuya et al. [
      • Ichikado K.
      • Suga M.
      • Muranaka H.
      • Gushima Y.
      • Miyakawa H.
      • Tsubamoto M.
      • Johkoh T.
      • Hirata N.
      • Yoshinaga T.
      • Kinoshita Y.
      • Yamashita Y.
      • Sasaki Y.
      Prediction of prognosis for acute respiratory distress syndrome with thin-section CT: validation in 44 cases.
      ], with the variables as follows: (1) normal attenuation, (2) ground glass attenuation (GGA) without traction bronchiectasis or bronchiolectasis, (3) consolidation without traction bronchiectasis or bronchiolectasis, (4) GGA with traction bronchiectasis or bronchiolectasis, (5) consolidation with traction bronchiectasis or bronchiolectasis, and (6) honeycombing. An overall CT score was obtained by adding the six averaged scores (three zones in each lung) assigned by a radiologist.

      2.4 Detection of serum sPD-L1 by enzyme-linked immunosorbent assay (ELISA)

      The concentration of serum sPD-L1 was measured using commercially available ELISA kit per manufacturer's instruction. The ELISA kit for sPD-L1 was a product of Invitrogen (American). For detection of sPD-L1 protein, the serum was directly detected with stock suspension, and its concentration was presented as pg/mL through a standard curve.

      2.5 Statistical analysis

      Statistical analysis of data was performed using PRISM (version 5) (GraphPad Software, La Jolla, CA, USA) and/or SPSS for Windows (version 18.0) (SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed by Student's t-test. Comparisons of categorical variables were conducted using Pearson chi-square tests. For nonparametric data, results were expressed as median (range) values, and the differences between groups were analyzed by the Mann-Whitney U test. Spearman's correlation coefficient was applied to detect the correlation between two groups. Univariate logistic regression analysis was performed to determine the factors associated with the presence of ILD. Multivariate logistic regression analysis was performed by including the confounding factors that were found to be significantly associated with the univariate analyses. The factors were selected in a stepwise manner owing to the small number of events in the logistic model. ROC curve was used to determine the best cut off value and validity of certain variable. Data was presented as mean ± standard deviation (SD). A p value of less than 0.05 was considered statistically significant.

      3. Results

      3.1 Clinical and demographic features

      87 RA patients (58 with ILD and 29 without ILD) and 45 healthy controls (HC) with matched age and gender were recruited in this study. The RA patients had median disease duration of 72 months ranging from 6 to 360. The mean DAS28-CRP score was 4.9 ± 1.3. To study whether sPD-L1 levels are increased in RA patients with ILD, patients were divided into the RA with ILD group (n = 58, RA-ILD) and the RA without ILD group (n = 29, RA-non-ILD). Comparisons of the collected data for the RA-ILD and RA-non-ILD are shown in Table 1. Female frequency, smoking history, anti-CCP antibody titer, use of biologics, use of MTX, use of glucocorticoids and dose of glucocorticoids showed significant differences in the RA groups with and without ILD.
      Table 1Characteristics of rheumatoid arthritis (RA) patients with or without interstitial lung disease (ILD).
      RA-ILDRA-non-ILD
      (n = 58)(n = 29)P
      sPD-L1 (pg/mL)23.7 ± 9.818.0 ± 7.70.01
      Age (years)65.7 ± 9.461.8 ± 9.20.096
      Female36 (62.1)25 (86.2)0.02
      Disease duration (months)60 (1–360)72 (6–360)0.772
      Smoking history15 (25.9)1 (3.4)0.011
      DAS28-ESR4.8 ± 1.54.7 ± 1.20.739
      Sharp/van der Heijde score18.4 ± 22.229.8 ± 24.70.099
      Anti-CCP positive54 (93.1)21 (75.0)0.008
      Anti-CCP titer696.9 ± 531.4429.3 ± 555.80.004
      RF positive51 (87.9)22 (75.9)0.148
      RF titer615.8 ± 1186.5213.8 ± 194.90.289
      ESR54.2 ± 34.347.1 ± 30.60.716
      CRP32.8 ± 44.339.2 ± 35.50.109
      Ferritin213.4 ± 128.7188.2 ± 180.50.341
      Use of biologics11 (18.9)0 (0)0.012
      Use of MTX10 (17.2)18 (62.1)<0.0001
      Use of GCs45 (77.6)16 (55.2)0.03
      Dose of GCs (mg/day)13.9 ± 12.87.6 ± 7.60.024
      sPD-L1, soluble programmed death ligand 1; DAS28, 28-joint Disease Activity Score; CRP, C-reactive protein; MTX, methotrexate; GCs, glucocorticoids (in prednisolone equivalent); anti-CCP, anticitrullinated peptide antibody; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate. Continuous variables were compared using the Mann–Whitney U test and are expressed as mean ± sd. Categorical variables were compared by the chi-squared test and are expressed as numbers (%) unless otherwise mentioned.

      3.2 Serum sPD-L1 was elevated in RA especially in RA-ILD patients

      Serum sPD-L1 levels were significantly higher in patients with RA-ILD (23.7 ± 9.8 pg/mL) than RA without ILD (18.0 ± 7.7 pg/mL, P = 0.01, Fig. 1A) and healthy controls (2.9 ± 1.5 pg/mL, P < 0.0001, Fig. 1A). The concentrations of serum sPD-L1 were identified to be weak positively correlated with RA serological marker RF (r = 0.245, P = 0.03, Fig. 1B). Further correlation analysis was performed for determination of the correlation coefficients of sPD-L1 with other clinical serological biomarkers, including ESR, CRP and anti-CCP. The correlation coefficients between sPD-L1 and ESR, sPD-L1 and CRP, and sPD-L1 and anti-CCP were respective r = 0.142 (P = 0.19), r = 0.265 (P = 0.01) (Fig. 1C) and r = −0.083 (P = 0.45). In addition, we also find that serum sPD-L1 were positively associated with HRCT scores (r = 0.265, P = 0.04) and Ferritin levels (r = 0.442, P = 0.01), but negatively associated with FVC% (r = −0.359, P = 0.01) and DLCO% (r = −0.399, P = 0.008) (Fig. 2).
      Fig. 1
      Fig. 1Concentrations of sPD-L1 in patients with rheumatoid arthritis (RA) and Healthy controls (HC) (A) and their relationship between rheumatoid factor (RF) (B) and C-reaction protein (CRP) (C).
      Fig. 2
      Fig. 2Correlation between the serum levels of sPD-1 levels with the high-resolution computed tomography score (HRCT score), serum ferritin, diffusing capacity for carbon monoxide percent predicted values (DLCO%) and forced vital capacity percent predicted values (FVC%) in RA-ILD.

      3.3 Serum sPD-L1 is associated with the presence of ILD in RA

      We performed univariate and multivariate logistic analyses to determine the factors associated with ILD in RA patients. sPD-L1, female, smoking history, anti-CCP antibody titer, use of MTX, use of glucocorticoids and dose of glucocorticoids were associated with the presence of ILD according to univariate analysis. On multivariate analysis, after selecting the factors in the multivariate model in a stepwise manner, sPD-L1, anti-CCP antibody status and use of MTX remained independent factors that were associated with the presence of ILD (Table 2).
      Table 2Univariate and multivariate logistic regression analyses for factors associated with the presence of interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients.
      UnivariateMultivariate
      OR (95% CI)POR (95% CI)P
      sPD-L1 (pg/mL)1.08 (1.02–1.15)0.0111.15 (0.91–1.11)0.014
      Age (years)1.05 (0.99–1.10)0.073
      Female0.27 (0.08–0.88)0.0290.17 (0.01–5.02)0.305
      Disease duration (months)1.00 (0.95–1.06)0.977
      Smoking history9.77 (1.22–78.14)0.0322.23 (0.04–128.01)0.697
      DAS28-ESR1.05 (0.77–1.44)0.768
      Sharp/van der Heijde score0.86 (0.69–1.07)0.171
      Anti-CCP titer1.01 (1.01–1.03)0.0141.01 (1.00–1.01)0.374
      Anti-CCP positive4.38 (1.16–16.45)0.02922.6 (1.08–473.2)0.045
      RF titer1.00 (0.99–1.01)0.175
      RF positive1.94 (0.59–6.41)0.278
      ESR1.01 (0.99–1.02)0.497
      CRP1.00 (0.99–1.01)0.498
      Ferritin1.00 (0.99–1.01)0.641
      Use of biologics5.71 (0.70–47.01)0.105
      Use of MTX0.13 (0.05–0.34)<0.00010.04 (0.01–0.27)0.001
      Use of GCs2.61 (1.01–6.73)0.055.55 (0.48–63.7)0.169
      Dose of GCs (mg/day)1.06 (1.01–1.12)0.0211.00 (0.91–1.11)0.939
      sPD-L1, soluble programmed death ligand 1; DAS28, 28-joint Disease Activity Score; CRP, C-reactive protein; MTX, methotrexate; GCs, glucocorticoids (in prednisolone equivalent); anti-CCP, anticitrullinated peptide antibody; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate.

      3.4 Serum sPD-L1 levels can be a disease marker for detection of RA with ILD

      To evaluate the significance of the sPD-L1 protein in clinical settings, we analyzed the sensitivities and specificities of these circulating factors in identified RA patients with ILD (Fig. 3). The area under the curve (AUC) was 0.661 (SE: 0.061; range: 0.542–0.780; sensitivity: 0.517; specificity: 0.793) for sPD-L1 protein. According to the cutoff value, RA-ILD were divided into sPD-L1 positive and negative groups. Comparisons of the collected data for the sPD-L1 positive and sPD-L1 negative are shown in Table 3. FVC% and DLCO% showed significant differences in the RA groups with sPD-L1 positive and sPD-L1 negative. These results thus imply that the serum sPD-L1 protein may be an independent biomarker for the identification of ILD in RA patients, which can be an independent biomarker candidate for evaluating disease severity and progression of ILD in RA patients.
      Fig. 3
      Fig. 3Area under the curve (AUC) and sensitivity and specificity of sPD-1 levels in RA patients using receiver operating characteristics curve analysis for the detection of RA with ILD.
      Table 3Characteristic of RA-ILD patients according to sPD-L1 protein status.
      sPD-L1 positive (n = 30)sPD-L1 negative (n = 28)P-value
      Age (mean ± SD) (years)66.0 ± 9.865.5 ± 9.40.691
      Gender (female%)19 (63.3)18 (64.3)0.940
      RA duration (months)66 (6–360)60 (1–300)0.377
      Current/ever-smoker9 (30.0)5 (17.9)0.280
      RF (IU/ml)607.2 ± 1220.3465.7 ± 863.50.301
      Anti-CCP (+) number27 (90.0)27 (96.4)0.334
      ESR (mm/h)50.6 ± 29.550.9 ± 36.60.72
      CRP (mg/dl)41.7 ± 51.324.2 ± 34.60.06
      DAS28 score4.7 ± 1.54.8 ± 1.60.674
      HRCT score6.2 ± 3.45.4 ± 3.30.259
      FVC%79.8 ± 20.195.3 ± 14.70.004
      DLCO%54.2 ± 15.068.8 ± 17.60.006
      Cough20 (66.7)11 (39.3)0.037
      Dyspnea7 (23.3)2 (7.1)0.147
      Chest congestion13 (43.3)7 (25.0)0.142
      Glucocorticoids22 (73.3)22 (78.6)0.641
      MTX6 (20.0)3 (10.7)0.473
      sPD-L1, soluble programmed death ligand 1; DAS28, 28-joint Disease Activity Score; CRP, C-reactive protein; MTX, methotrexate; GCs, glucocorticoids (in prednisolone equivalent); anti-CCP, anticitrullinated peptide antibody; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate; HRCT: high-resolution computed tomography; FVC: forced vital capacity; DLCO: diffusing capacity for carbon monoxide.

      4. Discussion

      This is the first report on the association between serum sPD-L1 levels and the presence of ILD in RA patients. sPD-L1 levels were increased in the RA-ILD patients. Furthermore, sPD-L1 and anti-CCP antibody were found to be independent disease markers for the detection of ILD in RA patients.
      In the present study, we examined sPD-L1 in serum of RA-ILD patients, and identified that serum sPD-L1 had a strong association with ILD in RA patients. Interestingly, we also found that the increased serum sPD-L1 in RA-ILD patients was positively correlated with rheumatoid factor (RF) and HRCT score. Additionally, a higher level of serum sPD-L1 was negatively associated with FVC% and DLCO% in RA-ILD patients. Multivariate logistic regression analysis suggested that the sPD-L1 was clinically significant risk factors in RA-ILD. ROC curves further demonstrated that the capacity of serum sPD-L1 as a potential biomarker in RA-ILD. This study thus implies that the circulating sPD-L1 may be a valuable biomarker for identifying ILD in RA patients.
      Previous studies have demonstrated that aging, male, smoking [
      • Dawson J.K.
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      Predictors of progression of HRCT diagnosed fibrosing alveolitis in patients with rheumatoid arthritis.
      ,
      • Sathi N.
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      Patients with limited rheumatoid arthritis-related interstitial lung disease have a better prognosis than those with extensive disease.
      ] and several serological markers are the predictors for the development of ILD. For example, Krebs von den Lungen-6 (KL-6) is known as a classical marker for idiopathic and connective tissue disease (CTD)-associated ILD [
      • Kohno N.
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      ]. In addition, lactate dehydrogenase (LDH) [
      • DeRemee R.A.
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      ], anti-cyclic citrullinated peptide (anti-CCP) antibody [
      • Kelly C.A.
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      N. British Rheumatoid Interstitial Lung, Rheumatoid arthritis-related interstitial lung disease: associations, prognostic factors and physiological and radiological characteristics--a large multicentre UK study.
      ,
      • Giles J.T.
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      ] and rheumatoid factor [
      • Turesson C.
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      Severe extra-articular disease manifestations are associated with an increased risk of first ever cardiovascular events in patients with rheumatoid arthritis.
      ] have recently been reported to be markedly increased in patients with ILD. Except for sPD-L1, in this study we also found that anti-CCP antibody and smoking factory were also risk factors and female was a protective t factor for ILD in RA, which were consistent with previous reports [
      • Kelly C.A.
      • Saravanan V.
      • Nisar M.
      • Arthanari S.
      • Woodhead F.A.
      • Price-Forbes A.N.
      • Dawson J.
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      • Young A.
      N. British Rheumatoid Interstitial Lung, Rheumatoid arthritis-related interstitial lung disease: associations, prognostic factors and physiological and radiological characteristics--a large multicentre UK study.
      ,
      • Dawson J.K.
      • Fewins H.E.
      • Desmond J.
      • Lynch M.P.
      • Graham D.R.
      Predictors of progression of HRCT diagnosed fibrosing alveolitis in patients with rheumatoid arthritis.
      ,
      • Sathi N.
      • Urwin T.
      • Desmond S.
      • Dawson J.K.
      Patients with limited rheumatoid arthritis-related interstitial lung disease have a better prognosis than those with extensive disease.
      ,
      • Giles J.T.
      • Danoff S.K.
      • Sokolove J.
      • Wagner C.A.
      • Winchester R.
      • Pappas D.A.
      • Siegelman S.
      • Connors G.
      • Robinson W.H.
      • Bathon J.M.
      Association of fine specificity and repertoire expansion of anticitrullinated peptide antibodies with rheumatoid arthritis associated interstitial lung disease.
      ].
      A rare adverse effect of MTX is hypersensitivity pneumonitis, which is described in 0.43% patients [
      • Salliot C.
      • van der Heijde D.
      Long-term safety of methotrexate monotherapy in patients with rheumatoid arthritis: a systematic literature research.
      ]. This organ-specific hypersensitivity reaction has led to a creeping concern in routine practice that MTX may also be associated with an increased incidence or exacerbation of the interstitial lung disease (ILD) that is associated with RA and may be a reason to withhold MTX from RA patients with any lung disease. However, in our study, multivariate logistic regression analysis demonstrated that MTX treatment was not associated with an increased risk of RA-ILD diagnosis. On the contrary, evidence suggested that MTX may be a protective factor for RA-ILD and it was consistent with a recent report [
      • Kiely P.
      • Busby A.D.
      • Nikiphorou E.
      • Sullivan K.
      • Walsh D.A.
      • Creamer P.
      • Dixey J.
      • Young A.
      Is incident rheumatoid arthritis interstitial lung disease associated with methotrexate treatment? Results from a multivariate analysis in the ERAS and ERAN inception cohorts.
      ].
      Ferritin was also reported to be associated with CTD-ILD [
      • Shi J.
      • Li S.
      • Yang H.
      • Zhang Y.
      • Peng Q.
      • Lu X.
      • Wang G.
      Clinical profiles and prognosis of patients with distinct antisynthetase autoantibodies.
      ]. In our study, although it showed no association with RA-ILD, maybe due to relatively small sample, the serum ferritin level was positively associated with sPD-L1, thus, association between ferritin and RA-ILD needs future study.
      There are several limitations in our current study. First, the sample size of this study was small, which may lead to potential analytical bias. Further studies with large cohorts are needed. Second, the subjects in our study were from a single institution, and these subjects were homogenous Han Chinese ethnic group. A multicenter study with various ethnic groups is needed to evaluate the generalizability of our results. Third, we only measured serum sPD-L1 expression without evaluating PD-1 and PD-L1 in patient's lung tissue biopsies by immunohistochemistry (IHC) and quantify PD-1 in Peripheral Blood Mononuclear Cells (PBMCs) by real time RT-PCR and correlating to blood leukocytes and DAS28 Scores. Finally, evidence from this study warrants further investigation with prospective study design to help draw more confident conclusions.

      CRediT authorship contribution statement

      Xinyu Wu: Data curation, Methodology, Validation, Funding acquisition. Li Xu: Methodology, Resources, Formal analysis, Software. Qi Cheng: Methodology, Resources, Validation. Liuyan Nie: Methodology, Resources. Songzhao Zhang: Methodology, Validation. Yan Du: Conceptualization, Software, Investigation, Writing - original draft, Project administration, Funding acquisition, Visualization. Jing Xue: Conceptualization, Investigation, Writing - review & editing, Project administration, Supervision.

      Declaration of competing interest

      The authors declare no conflicts of interest regarding this work. The Corresponding Authors have the right to grant on behalf of all authors and do grant on behalf of all authors.

      Acknowledgements

      This work was supported by the National Natural Science Foundation of China (No. 81501388 ), Zhejiang Provincial Public Technology Applied Research Project, China (No. 2015C33177 , No. 2017C33032 ) and National Natural Science Foundation of Zhejiang Province, China (No. LY20H100007 , LQ17H160007 ).

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