Transcriptomic analyses of lung tissues reveal key genes associated with progression of systemic sclerosis-interstitial lung disease (SSc-ILD).

OBJECTIVE: Systemic sclerosis-interstitial lung disease (SSc-ILD) is the leading cause of death in SSc, affecting around 50 % of the patients. Lung tissue of patients with early-stage SSc-ILD is characterized by a predominant inflammatory response with inconspicuous fibrosis, which may progress to honeycombing fibrosis. Hence, a better understanding of the molecular mechanisms underpinning SSc-ILD pathogenesis is needed to improve treatment options and progression prediction. This transcriptomic study aims to reveal the differential gene expression between control (ctrl) lung tissue and inflammatory, prefibrotic and fibrotic lung tissue to capture progression of early to late phase SSc-ILD. METHODS: Twelve explanted lungs from patients with SSc-ILD were used to analyze gene expression from formalin-fixed paraffin-embedded lung tissues with varying stages of ILD (n = 18) and control lung tissue (n = 6). The SSc-ILD tissues were stratified into three ROIs: inflammatory, prefibrotic, and fibrotic using histological assessments to define a longitudinal simulation of early to late phases of SSc-ILD. The nanoString (nS) nCounter Human Fibrosis Panel was used to profile the transcriptome in the regions of interest. Validation of potential targetswas performed with immunohistochemistry in the same tissues that were used for transcriptome analysis. RESULTS: To validate our simulation model, we performed subgroup analysis that showed an incremental increase in pathway scores related to the severity of fibrosis. Ctrl vs SSc-ILD comparison demonstrated 24 differentially expressed genes, two of which had the most pronounced p-values. Cyclin-dependent kinase inhibitor (cdkn2c) was overexpressed (P = 0.00052) in SSc-ILD compared to ctrl, while expression of Pellino E3 ubiquitin-protein ligase 1 (peli1) showed lower expression (P = 0.0012). Additionally, in all four groups, cdkn2c and peli1 gene expression showed an incremental increase and decrease, respectively. Immunohistochemistry of cdkn2c showed consistent results with the nS analysis. CONCLUSION: More cdkn2c and less peli1 expression were associated with more advanced stages of SSc-ILD on histologic assessment. We report the potential of the cell cycle inhibitor and senescence marker, cdkn2c (p18) to be associated with fibrosis progression.

Macrophages as determinants and regulators of fibrosis in systemic sclerosis.

SSc is a multiphase autoimmune disease with a well-known triad of clinical manifestations including vasculopathy, inflammation and fibrosis. Although a plethora of drugs has been suggested as potential candidates to halt SSc progression, nothing has proven clinically efficient. In SSc, both innate and adaptive immune systems are abnormally activated fuelling fibrosis of the skin and other vital organs. Macrophages have been implicated in the pathogenesis of SSc and are thought to be a major source of immune dysregulation. Due to their plasticity, macrophages can initiate and sustain chronic inflammation when classically activated while, simultaneously or parallelly, when alternatively activated they are also capable of secreting fibrotic factors. Here, we briefly explain the polarization process of macrophages. Subsequently, we link the activation of macrophages and monocytes to the molecular pathology of SSc, and illustrate the interplay between macrophages and fibroblasts. Finally, we present recent/near-future clinical trials and discuss novel targets related to macrophages/monocytes activation in SSc.

Self-Reported Systemic Sclerosis-Related Symptoms Are More Prevalent in Subjects with Raynaud's Phenomenon in the Lifelines Population: Focus on Pulmonary Complications.

Puffy fingers and Raynaud's phenomenon (RP) are important clinical predictors of the development of systemic sclerosis (SSc). We aim to assess the prevalence of SSc-related symptoms, explore pulmonary symptoms, and test the usefulness of skin autofluorescence (SAF) as a non-invasive marker for Advanced Glycation Endproducts (AGEs). Subjects from the Lifelines Cohort Study with known connective tissue disease (CTD) were excluded. Patient characteristics, SAF, self-reported pulmonary symptoms, and spirometry were obtained. Subjects (n = 73,948) were categorized into definite RP (5.3%) with and without SSc-related symptoms and non-RP. Prevalence of at least one potential SSc-related symptom (other than RP) was 8.7%; 23.5% in subjects with RP and 7.1% without RP (p < 0.001). Subjects with RP and additional SSc-related symptoms more frequently reported dyspnea at rest, dyspnea after exertion, and self-reported pulmonary fibrosis, and had the lowest mean forced vital capacity compared to the other groups (RP without SSc-related symptoms and no RP, both p < 0.001). In multivariate regression, dyspnea at rest/on exertion remained associated with an increased risk of SSc-related symptoms in subjects with RP (both p < 0.001). SAF was higher in subjects with RP and SSc-related symptoms compared to the other groups (p < 0.001), but this difference was not significant after correction for potential confounders. The prevalence of SSc-related symptoms was approximately three-fold higher in subjects with RP. Pulmonary symptoms are more prevalent in subjects with RP who also reported additional potential SSc-related symptoms. This might suggest that (suspected) early SSc develops more insidiously than acknowledged. According to this study, SAF is no marker for early detection of 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.

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.

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).

Glutathione-responsive cyclodextrin-nanosponges as drug delivery systems for doxorubicin: Evaluation of toxicity and transport mechanisms in the liver.

The potential mammalian hepatotoxicity of a new class of GSH-responsive cyclodextrin-based nanosponges loaded with the anticancer drug doxorubicin (Dox-GSH-NS) was investigated. Previous studies showed that these nanosponges can release medicaments preferentially in cells having high GSH content, a common feature of chemoresistant cells, and showed enhanced anti-tumoral activity compared to free Dox in vitro and in vivo in cells with high GSH content. Following these promising results, we investigated here the Dox-GSH-NS hepatotoxicity in human HepG2 cells (in vitro) and in the organotypic cultures of rat precision-cut liver slices (PCLS, ex vivo), while their accumulation in rat liver was assessed in vivo. Moreover, the transport in Dox uptake, as well as its efflux, was studied in vitro. Overall, benefiting of the integration of different investigational models, a good safety profile of Dox-GSH-NSs was evidenced, and their hepatotoxicity resulted to be comparable with respect to free Dox both in vitro and ex vivo. Furthermore, in vivo studies showed that the hepatic accumulation of the Dox loaded in the NS is comparable with respect to the free drug. In addition, Dox-GSH-NSs are taken up by active mechanisms, and can escape the efflux drug pump, thus, contributing to overcoming drug resistance.