High serum C-X-C motif chemokine ligand 10 (CXCL10) levels may be associated with new onset interstitial lung disease in patients with systemic sclerosis: evidence from observational, clinical, transcriptomic and in vitro studies

Abstract

Background: Systemic sclerosis-interstitial lung disease (SSc-ILD) is the leading cause of death in patients with SSc. There is an unmet need for predictive biomarkers to identify patients with SSc at risk of ILD. Previous studies have shown that interferon (IFN) pathways may play a role in SSc. We assessed the use of C-X-C motif chemokine ligand 10 (CXCL10) as a predictive biomarker for new onset of ILD in patients with SSc.

Methods: One-hundred-sixty-five (Female, N = 130) patients with SSc (SSc-ILD, N = 41) and 13 (Female, N = 8) healthy controls were investigated retrospectively. CXCL10 protein levels were measured by ELISA. We performed log rank analysis with baseline CXCL10 serum levels. CXCL10 nanoString data from lung tissues obtained from transplanted patients with SSc-ILD were extracted. Fifteen (Female, N = 10) patients with SSc (SSc-ILD, N = 7) were recruited for bronchoalveolar lavage (BAL) procedure. Lung fibroblasts were treated with BAL-fluid or serum from patients with SSc with or without ILD. Inflammatory/fibrotic genes were assessed.

Findings: Serum CXCL10 levels were higher in patients with SSc-ILD compared to SSc patients without ILD [Median (IQR):126 pg/ml (66-282.5) vs. 78.5 pg/ml (50-122), P = 0.029, 95% CI: 1.5 × 10^-6 to 0.4284]. Survival analysis showed that baseline CXCL10 levels >78.5 pg/ml have a 2.74-fold increased risk of developing new onset of ILD (Log-rank: P = 0.119) on follow-up. CXCL10 levels in BAL supernatant were not different in patients with SSc-ILD compared to SSc without ILD [76.1 pg/ml (7.2-120.8) vs. 22.3 pg/ml (12.1-43.7), P = 0.24, 95% CI: -19.5 to 100]. NanoString showed that CXCL10 mRNA expression was higher in inflammatory compared to fibrotic lung tissues [4.7 (4.2-5.6) vs. 4.3 (3.6-4.7), P = 0.029]. Fibroblasts treated with SSc-ILD serum or BAL fluids overexpressed CXCL10.

Interpretations: Clinical, transcriptomic, and in vitro data showed that CXCL10 is potentially involved in early SSc-ILD. More research is needed to confirm whether CXCL10 can be classified as a prospective biomarker to detect patients with SSc at higher risk of developing new onset ILD.

Funding: This collaborative project is co-financed by the Ministry of Economic Affairs and Climate Policy of the Netherlands utilizing the PPP-allowance made available by the Top Sector Life Sciences & Health to stimulate public-private partnerships (PPP-2019_007). Part of this study is financially supported by Sanofi Genzyme (NL8921).

Keywords: Biomarker; CXCL10; Fibrosis; ILD; Inflammation; Systemic sclerosis.

Fig. 1

Schematic overview of the performed studies. (Study-1) is a retrospective cohort that has followed 165 patients with SSc and 13 healthy controls. (Study-2) is a cross-sectional study that recruited 15 patients with SSc for bronchoalveolar lavage. (Study-3) is a transcriptomic study on SSc-ILD lung tissue sections. (Study-4) is an in vitro study where lung fibroblasts were stimulated with local or systemic SSc/SSc-ILD fluids. Created by Biorender.com.

Flowchart 1

Inclusion and exclusion criteria for study-1.

Flowchart 2

Inclusion and exclusion criteria for study-2.

Fig. 2

Systemic (serum) levels of CXCL10 in SSc without ILD (N = 124), SSc-ILD (N = 41) and healthy controls (N = 13). Patients with SSc with or without interstitial lung disease have statistically significant higher CXCL10 levels compared to healthy controls (Mann–Whitney U test). Patients with SSc-ILD had statistically significant higher levels of CXCL10 compared to SSc without ILD (Mann–Whitney U test). Measurements are shown as median values (dashed) with IQR (dotted) in pg/ml.

Fig. 3

CXCL10 levels were negatively correlated with PFTs in patients with SSc-ILD. a) Spearman's correlation between serum CXCL10 and %FVC shows a moderate negative correlation (N = 38, r = −0.43, P = 0.012. b) Spearman's correlation between serum CXCL10 and %DLco indicates no correlation (N = 32). After adjusting for confounders, CXCL10 was negatively associated with %DLco. PFTs: pulmonary function tests, FVC: forced vital capacity, DLco: capacity for carbon monoxide diffusion.

Fig. 4

Higher CXCL10 may be associated with long-term ILD development. Kaplan–Meier survival curve according to baseline median CXCL10 concentration. CXCL10 levels >78.5 pg/ml shows a 2.74-fold increased hazard of new onset of ILD [Log-rank (Mantel-cox) test, P = 0.119].

Fig. 5

Local CXCL10 concentration levels and correlation between local and systemic CXCL10 levels. a) Comparison between CXCL10 concentration levels in BAL supernatant between SSc-without-ILD (N = 8) and patients with SSc-ILD (N = 6) (Mann–Whitney U test, P = 0.23). Measurements are shown as median values (dashed) with IQR (dotted) in pg/ml. b) Spearman's correlation between CXCL10 concentration levels in serum and BAL supernatant in the same patients showing statistical significance P = 0.007 and r = 0.7 (N = 14). Measurements are in pg/ml.

Fig. 6

CXCL10 gene expression in SSc-ILD inflammatory and fibrotic regions. Transcriptomic analysis showed that CXCL10 is 2.3x more highly expressed in inflammatory regions compared with fibrotic regions in patients with SSc-ILD [P = 0.029 (t-test)]. N = 11 (5 inflammatory SSc-ILD regions and 6 fibrotic SSc-ILD regions).

Fig. 7

Stimulation of normal human lung fibroblasts with SSc fluids (a–d), or inflammatory or fibrotic cytokines (e). (a–d) 5% BAL supernatant from SSc without ILD (BAL SSc) or SSc-ILD (BAL SSc-ILD), or 1% serum from SSc without ILD (SSc serum) or SSc-ILD (SSc-ILD serum) or healthy control (Pool serum) was used to stimulate lung fibroblasts. (a) SSc-ILD BAL and serum-treated fibroblasts overexpressed cxcl10 compared to SSc without ILD BAL (Mann–Whitney U test, P = 0.004) and serum (Mann–Whitney U test, P = 0.0087). Also, SSc-ILD BAL-treated fibroblasts had higher cxcl10 levels compared to CTRL (Mann–Whitney U test, P = 0.0022). Fibroblasts treated with SSc-ILD serum significantly overexpressed cxcl10 compared to fibroblasts treated with healthy serum (Mann–Whitney U test, P = 0.0022). SSc-ILD serum-treated lung fibroblasts overexpressed ctgf expression compared to SSc without ILD and pool sera (Mann–Whitney U test, P = 0.026 and P = 0.0087, respectively). (c–d) No statistically significant differences in tgfβ or αsma expression when lung fibroblasts were treated with BAL or serum (Mann–Whitney U test). The results are medians of three technical experiments (n = 3) where 6 patients' biofluids were used (N = 6). (e) IL-6 (10 ng/ml or 100 ng/ml) or TGF-β (10 ng/ml) or both were used to stimulate lung fibroblasts for 4 h. IL-6 stimulated cxcl10 expression in lung fibroblasts in a concentration-dependent manner. The higher concentration of IL-6 (100 ng/ml) showed increase of cxcl10 expression with a trend towards statistical significance of P value = 0.055 vs. CTRL (Mann–Whitney U test). The results are medians with IQR of three technical experiments (n = 3). CTRL: culture media without stimulant.