Nintedanib downregulates the profibrotic M2 phenotype in cultured monocyte-derived macrophages obtained from systemic sclerosis patients affected by interstitial lung disease.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFß1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients. METHODS: Fourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients' informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFß1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFß1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test. RESULTS: Cultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFß1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFß1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFß1 after 24 h of treatment (p < 0.05 vs. untreated cells). CONCLUSIONS: In cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFß1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

Microvascular status in juvenile Sjögren's disease: the first nailfold videocapillaroscopy investigation.

INTRODUCTION: Juvenile Sjögren's disease (jSjD) is a rare autoimmune disease characterized by exocrine gland involvement and systemic manifestations, including small vessel vasculitis and Raynaud's phenomenon (RP). We aimed to investigate the microvascular status in jSjD patients by nailfold videocapillaroscopy (NVC) and the potential correlations with clinical and serological features. METHODS: Clinical data from thirteen consecutive jSjD patients (11 females and 2 males), with a mean age of 16 ± 4 years, diagnosed before 16 years of age (mean age at diagnosis 12 ± 3) according to the 2016 American College of Rheumatology/EULAR criteria for adult SjD, were collected including age- and sex-matched healthy controls (HCs). Clinical, laboratory, and instrumental data were collected, together with NVC examination. Non-specific and specific NVC parameters were investigated, such as capillary density, capillary dilations, giant capillaries, microhaemorrhages and abnormal shapes. Associations between NVC findings and clinical/serological features were explored and analysed using parametrical and non-parametrical tests. RESULTS: Capillary density reduction correlated significantly with articular involvement (arthralgias) (p = 0.024). Microhaemorrhages correlated with lower C3 levels (p = 0.034). No specific NVC pattern for jSjD was identified, whereas abnormal capillary shapes were significantly higher in jSjD patients than HCs (p = 0.005). NVC abnormalities were not associated with SjD-specific instrumental tests (biopsy, imaging, Schirmer's test). RP was present in 8% of jSjD patients. CONCLUSIONS: The reduction of capillary density, as well as microhaemorrhages at NVC analysis, are significantly associated with some clinical aspects like articular involvement and serum biomarkers (C3 reduction). The NVC is suggested as safe and further analysis in jSjD patients.

Description of Peripheral Blood Perfusion by Laser Speckle Contrast Analysis (LASCA) in 'Early' versus 'Clinically Overt' Systemic Sclerosis in Routine Clinics.

OBJECTIVE: To investigate in an unselected, systemic sclerosis (SSc) cohort if baseline laser speckle contrast analysis (LASCA) peripheral blood perfusion (PBP) measurements differ between 'early' SSc (without skin involvement, or 'limited' SSc-LSSc) and 'clinically overt' SSc (with skin involvement, limited cutaneous SSc-LcSSc and diffuse cutaneous SSc-DcSSc) in routine setting. METHODS: A group of twenty consecutive 'early' SSc patients and forty consecutive 'clinically overt' SSc patients (twenty LcSSc and twenty DcSSc) underwent clinical and LASCA examinations (to assess the peripheral blood perfusion [PBP] of both hands volar). RESULTS: No statistically significant difference in adjusted PBP was found in the 'early' versus the 'clinically overt' group (p = 0.77) when adjusted for possible confounding factors (e.g., vasoactive medication, active smoking, history of DTL and disease duration). A wide variability was noted when observing the individual datapoints of each subset. CONCLUSION: This study with an unselected SSc population in daily routine, non-research setting, showed there was no difference in adjusted PBP at baseline between 'early' SSc and 'clinically overt' SSc when corrected for possible confounding factors. Interestingly a wide variation of individual datapoints were observed in each subset, which emphasizes the heterogeneity of SSc.