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.

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).

The soluble receptor for advanced glycation end products is potentially predictive of pulmonary arterial hypertension in systemic sclerosis.

Introduction: Pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) are the leading causes of death in systemic sclerosis (SSc). Until now, no prospective biomarker to predict new onset of SSc-ILD or SSc-PAH in patients with SSc has reached clinical application. In homeostasis, the receptor for advanced glycation end products (RAGE) is expressed in lung tissue and involved in cell-matrix adhesion, proliferation and migration of alveolar epithelial cells, and remodeling of the pulmonary vasculature. Several studies have shown that sRAGE levels in serum and pulmonary tissue vary according to the type of lung-related complication. Therefore, we investigated levels of soluble RAGE (sRAGE) and its ligand high mobility group box 1 (HMGB1) in SSc and their abilities to predict SSc-related pulmonary complications. Methods: One hundred eighty-eight SSc patients were followed retrospectively for the development of ILD, PAH, and mortality for 8 years. Levels of sRAGE and HMGB1 were measured in serum by ELISA. Kaplan-Meier survival curves were performed to predict lung events and mortality and event rates were compared with a log-rank test. Multiple linear regression analysis was performed to examine the association between sRAGE and important clinical determinants. Results: At baseline, levels of sRAGE were significantly higher in SSc-PAH-patients (median 4099.0 pg/ml [936.3-6365.3], p = 0.011) and lower in SSc-ILD-patients (735.0 pg/ml [IQR 525.5-1988.5], p = 0.001) compared to SSc patients without pulmonary involvement (1444.5 pg/ml [966.8-2276.0]). Levels of HMGB1 were not different between groups. After adjusting for age, gender, ILD, chronic obstructive pulmonary disease, anti-centromere antibodies, the presence of puffy fingers or sclerodactyly, use of immunosuppression, antifibrotic therapy, or glucocorticoids, and use of vasodilators, higher sRAGE levels remained independently associated with PAH. After a median follow-up of 50 months (25-81) of patients without pulmonary involvement, baseline sRAGE levels in the highest quartile were predictive of development of PAH (log-rank p = 0.01) and of PAH-related mortality (p = 0.001). Conclusions: High systemic sRAGE at baseline might be used as a prospective biomarker for patients with SSc at high risk to develop new onset of PAH. Moreover, high sRAGE levels could predict lower survival rates due to PAH in patients with SSc.

Release of High-Mobility Group Box-1 after a Raynaud's Attack Leads to Fibroblast Activation and Interferon-γ Induced Protein-10 Production: Role in Systemic Sclerosis Pathogenesis.

Raynaud's Phenomenon (RP) leading to repetitive ischemia and reperfusion (IR) stress, is the first recognizable sign of systemic sclerosis (SSc) leading to increased oxidative stress. High-mobility group box-1 (HMGB1) is a nuclear factor released by apoptotic and necrotic cells after oxidative stress. Since HMGB1 can signal through the receptor for advanced glycation end products (RAGE), we investigated whether an RP attack promotes the release of HMGB1, leading to fibroblast activation and the upregulation of interferon (IFN)-inducible genes. A cold challenge was performed to simulate an RP attack in patients with SSc, primary RP (PRP), and healthy controls. We measured levels of HMGB1 and IFN gamma-induced Protein 10 (IP-10) at different time points in the serum. Digital perfusion was assessed by photoplethysmography. In vitro, HMGB1 or transforming growth factor (TGF-ß1) (as control) was used to stimulate healthy human dermal fibroblasts. Inflammatory, profibrotic, and IFN-inducible genes, were measured by RT-qPCR. In an independent cohort, sera were obtained from 20 patients with SSc and 20 age- and sex-matched healthy controls to determine HMGB1 and IP-10 levels. We found that HMGB1 levels increased significantly 30 min after the cold challenge in SSc compared to healthy controls. In vitro stimulation with HMGB1 resulted in increased mRNA expression of IP-10, and interleukin-6 (IL-6) while TGF-ß1 stimulation promoted IL-6 and Connective Tissue Growth Factor (CTGF). In serum, both HMGB1 and IP-10 levels were significantly higher in patients with SSc compared to healthy controls. We show that cold challenge leads to the release of HMGB1 in SSc patients. HMGB1 induces IP-10 expression in dermal fibroblasts partly through the soluble RAGE (sRAGE) axis suggesting a link between RP attacks, the release of HMGB1 and IFN-induced proteins as a putative early pathogenetic mechanism in SSc.

Is skin autofluorescence (SAF) representative of dermal advanced glycation endproducts (AGEs) in dark skin? A pilot study.

AIMS: Non-invasively assessed skin autofluorescence (SAF) measures advanced glycation endproducts (AGEs) in the dermis. SAF correlates with dermal AGEs in Caucasians and Asians, but studies in dark-skinned subjects are lacking. In this pilot we aimed to assess whether SAF signal is representative of intrinsic fluorescence (IF) and AGE accumulation in dark skin. METHODS: Skin biopsies were obtained in 12 dark-skinned subjects (6 healthy subjects, median age 22 years; 6 diabetes mellitus (DM) subjects, 65 years). SAF was measured with the AGE Reader, IF using confocal microscopy, and AGE distribution with specific antibodies. CML and MG-H1 were quantified with UPLC-MS/MS and pentosidine with HPLC and fluorescent detection. RESULTS: SAF correlated with IF from the dermis (405nm, r = 0.58, p < 0.05), but not with CML (r = 0.54, p = 0.07). CML correlated with IF from the dermis (405nm, r = 0.90, p < 0.01). UV reflectance and the coefficient of variation of SAF were negatively correlated (r = -0.80, p < 0.01). CML and MG-H1 were predominantly present around blood vessels, in collagen and fibroblasts in the dermis. CONCLUSION: This proof of concept study is the first to compare non-invasive SAF with AGE levels measured in skin biopsies in dark-skinned subjects. SAF did not correlate with individual AGEs from biopsies, but was associated with IF. However, the intra-individual variance was high, limiting its application in dark-skinned subjects on an individual basis.

Multidiameter single-fiber reflectance spectroscopy of heavily pigmented skin: modeling the inhomogeneous distribution of melanin.

When analyzing multidiameter single-fiber reflectance (MDSFR) spectra, the inhomogeneous distribution of melanin pigments in skin tissue is usually not accounted for. Especially in heavily pigmented skins, this can result in bad fits and biased estimation of tissue optical properties. A model is introduced to account for the inhomogeneous distribution of melanin pigments in skin tissue. In vivo visible MDSFR measurements were performed on heavily pigmented skin of type IV to VI. Skin tissue optical properties and related physiological properties were extracted from the measured spectra using the introduced model. The absorption of melanin pigments described by the introduced model demonstrates a good correlation with the co-localized measurement of the well-known melanin index.