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.

Peripheral and ocular microvascular alterations in systemic sclerosis: observations from capillaroscopic assessments, perfusion peripheral analysis, and optical coherence tomography angiography.

To investigate the correlations between finger microvascular morphology and function in patients with systemic sclerosis (SSc) and the status of ocular microcirculation, as detected by nailfold videocapillaroscopy (NVC), laser speckle contrast analysis (LASCA), and optical coherence tomography angiography (OCTA). The enrollment included 32 SSc patients, classified according to the 2013 ACR/EULAR criteria, and 27 sex- and age-matched healthy controls. The participants underwent comprehensive rheumatological and ophthalmological examinations, as well as NVC, LASCA, and OCTA analysis on the same day at a single center from March to October 2022. SSc patients receiving intravenous prostanoids cycles were assessed at least 1 month after infusion. Statistical analysis was conducted using Stata® 15.1. Significant direct correlations were observed between the mean capillary number (at NVC) and the mean perfusion of fingers (at LASCA) with the retinal and choroidal perfusion (at OCTA) (all p < 0.05). In addition, a significantly reduced retinal and choroidal perfusion was detected in SSc patients vs controls (all p < 0.05). Interestingly, diffuse cutaneous SSc (dcSSc) patients exhibited a lower choroidal perfusion (p = 0.03) but an increased choroidal thickness (CT) than limited cutaneous SSc patients (p < 0.001). CT was increased also in patients with positive Scl70 antibodies and with a history of digital ulcers directly correlating with disease duration (r = 0.67, p = 0.001). Finally, the combination of LASCA and OCTA parameters showed a significant discrimination capacity between SSc patients and controls, with an area under the curve of 0.80 [95% CI (0.74, 0.87)]. Peripheral microvascular damage is correlated with impaired ocular microcirculation in SSc. The increased choroidal thickness observed in dcSSc may be related to local sub-endothelial extracellular matrix deposition. The combined analysis of choroidal and fingertip perfusion offers preliminary insights that may complement traditional diagnostic methods for SSc.

Microvascular status and skin thickness in adults with hypermobile Ehlers-Danlos syndrome: a pilot investigation.

OBJECTIVES: Hypermobile Ehlers-Danlos Syndrome (hEDS) is a hereditary connective tissue disorder characterised by joint hypermobility, chronic musculoskeletal pain, and skin abnormalities and easy bruising. Morphological and functional microvascular status has not yet been studied in hEDS, and dermal thickness (DT) has been poorly investigated, therefore the aim of our study was to investigate it. METHODS: We conducted a study to investigate microvascular morphology by nailfold videocapillaroscopy (NVC), peripheral blood perfusion (PBP) by laser speckle contrast analysis (LASCA), and DT by high-frequency skin ultrasound (22 MHz probe) in adults with hEDS compared to sex- and age-matched controls. RESULTS: Microhaemorrhages were found more prevalent and the capillary number per linear millimetre at the nailfold was slightly higher in hEDS patients than in controls, as well as the NVC score for abnormal shaped capillaries was slightly lower (less abnormal shaped capillaries) in hEDS patients than in controls, even if this was not statistically significant. PBP was comparable between hEDS patients and controls. The DT resulted generally lower in hEDS patients than controls with significant values limited to feet and thorax (p=0.04). A statistically significant positive correlation was observed between the Beighton score and the score for microhaemorrhages (r=0.4, p=0.05), as well as between the Beighton score and DT (r≥0.5, p≤0.02) at the level of feet and thorax. CONCLUSIONS: Our study detected in hEDS patients a normal microvascular function at rest and a suitable capillary morphology butwith increased microvascular fragility. The dermal thickness seems thinner in hEDS patients than in controls in most skin areas, with strong statistically significance at the level of feet and thorax.

Understanding the Immune-Endocrine Effects of Vitamin D in SARS-CoV-2 Infection: A Role in Protecting against Neurodamage.

Calcitriol and hydroxyderivatives of lumisterol and tachisterol are secosteroid hormones with immunomodulatory and anti-inflammatory properties. Since the beginning of the COVID-19 pandemic, several studies have correlated deficient serum concentrations of vitamin D3 (calcifediol) with increased severity of the course of SARS-CoV-2 infection. Among systemic complications, subjective (anosmia, ageusia, depression, dizziness) and objective (ischemic stroke, meningoencephalitis, myelitis, seizures, Guillain-Barré syndrome) neurological symptoms have been reported in up to 80% of severe COVID-19 patients. In this narrative review, we will resume the pathophysiology of SARS-CoV-2 infection and the mechanisms of acute and chronic neurological damage. SARS-CoV-2 can disrupt the integrity of the endothelial cells of the blood-brain barrier (BBB) to enter the nervous central system. Invasion of pro-inflammatory cytokines and polarization of astrocytes and microglia cells always in a pro-inflammatory sense together with the pro-coagulative phenotype of cerebral endothelial cells in response to both SARS-CoV-2 and immune cells invasion (immunothrombosis) are the major drivers of neurodamage. Calcitriol and hydroxyderivatives of lumisterol and tachisterol could play an adjuvant role in neuroprotection through mitigation of neuroinflammation and protection of endothelial integrity of the BBB. Dedicated studies on this topic are currently lacking and are desirable to confirm the link between vitamin D3 and neuroprotection in COVID-19 patients.

Microvascular damage in autoimmune connective tissue diseases: a capillaroscopic analysis from 20 years of experience in a EULAR training and research referral centre for imaging.

OBJECTIVE: Nailfold videocapillaroscopy (NVC) allows the detection of microvascular damage in autoimmune connective tissue diseases (CTDs). The prevalence of the morphological capillary findings was retrospectively evaluated in a wide cohort of patients with Raynaud's phenomenon secondary to a CTD at the time of the first single NVC, independently from their current treatment, autoantibody profile and comorbidities. METHODS: One-thousand-one-hundred-eighty-one patients affected by CTDs were included from 2001 to 2021. The considered CTDs were systemic sclerosis (SSc), undifferentiated connective tissue disease (UCTD), mixed connective tissue disease (MCTD), dermatomyositis (DM), systemic lupus erythematosus, Sjögren's syndrome and primary antiphospholipid syndrome (aPS). The capillaroscopic parameters were distinguished between scleroderma patterns and non-scleroderma patterns. RESULTS: Giant capillaries were significantly more frequent in SSc, DM and MCTD than in other CTDs (respectively, in 73%, 73% and 61% of patients, p<0.001 when comparing each rate vs the other CTDs). The mean capillary count was significantly lower in SSc, DM and MCTD (respectively, 7.04±0.18 vs 6.5±0.75 vs 7.7±2 capillaries/linear mm) compared with the other CTDs (p<0.001 for each rate vs the other CTDs). The non-specific abnormalities of capillary morphology were significantly more frequent in SSc, MCTD and aPS (respectively, in 48%, 41% and 36% of cases, all p<0.001 vs each other CTDs). CONCLUSION: This large size sample of patients with CTDs, collected over 20 years of analysis, confirms the highest prevalence of specific capillaroscopic alterations in patients with SSc, DM and MCTD, when compared with other CTDs.

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.

Temporomandibular disorders in immune-mediated rheumatic diseases of the adult: A systematic review.

OBJECTIVE: To systematically review the literature concerning temporomandibular disorders (TMDs) in immune-mediated rheumatic diseases (IMRDs) of the adult. The temporomandibular joint (TMJ) outcomes used in clinical studies, the prevalence of TMDs in IMRDs and the risk factors for their development were qualitatively synthetized. METHODS: A literature search on PubMed Central, Embase and Cochrane Library databases was performed for studies including TMJ outcomes in IMRDs patients compared with healthy controls, other rheumatic diseases or in the assessed IMRDs patients after follow-up and treatment. Among the IMRDs of the adult, original articles investigating TMJ involvement in inflammatory polyarthritides and/or autoimmune connective tissue diseases were considered. The quality of the studies was scored using the Newcastle-Ottawa scale (NOS). RESULTS: Of the 3259 screened abstracts, 56 papers were included in the systematic review. Most of the papers (77%) investigated TMDs in rheumatoid arthritis (RA) with a prevalence of signs and symptoms varying from 8% to 70%. The risk factors for TMDs development in RA were female sex, younger age, anti-citrulline peptide autoantibodies (ACPA) positivity, higher disease activity, cervical spine involvement, cardiovascular and neuropsychiatric comorbidities. Ten papers (18%) evaluated TMDs in spondylarthritides (SpA) reporting a prevalence of symptoms and signs in 12%-80% of patients with higher TMDs prevalence in patients with radiographic spine involvement, skin psoriasis and HLADRB1×01 positivity. Among autoimmune connective tissue diseases (CTDs), systemic sclerosis (SSc) displayed the highest evidence of TMDs patient-reported outcomes (PROs) and clinical findings (20-93%), followed by systemic lupus erythematosus (SLE) in 18-85%, primary Sjogren's syndrome (24-54%) and idiopathic inflammatory myopathies (4-26%). In SSc and SLE, TMDs were more frequent in patients with higher disease activity and duration, correlating with the extent of skin fibrosis in SSc and with renal involvement in SLE. CONCLUSION: TMDs in IMRDs display a significant relevance in the rheumatological clinical practice even if often misdiagnosed. This burden is epidemiologically important in terms of PROs and clinical findings which correlate with disease activity in RA, SpA, SSc and SLE. The early recognition and multidisciplinary management of TMDs is warranted and should be aimed at hindering the TMJ structural damage maximizing the quality of life of patients.

Specific Autoantibodies and Microvascular Damage Progression Assessed by Nailfold Videocapillaroscopy in Systemic Sclerosis: Are There Peculiar Associations? An Update.

BACKGROUND: Specific autoantibodies and nailfold videocapillaroscopy (NVC) findings are serum and morphological diagnostic hallmarks of systemic sclerosis (SSc) as well as useful biomarkers which stratify the microvascular progression and prognosis of patients. METHODS: The aim of our narrative review is to provide an update and overview of the link between SSc-related autoantibodies, used in clinical practice, and microvascular damage, evaluated by NVC, by exploring the interaction between these players in published studies. A narrative review was conducted by searching relevant keywords related to this field in Pubmed, Medline and EULAR/ACR conference abstracts with a focus on the findings published in the last 5 years. RESULTS: Our search yielded 13 clinical studies and 10 pre-clinical studies. Most of the clinical studies (8/13, 61.5%) reported a significant association between SSc-related autoantibodies and NVC patterns: more specifically anti-centromere autoantibodies (ACA) were associated more often with an "Early" NVC pattern, whereas anti-topoisomerase autoantibodies (ATA) more frequently showed an "Active" or "Late" NVC pattern. Five studies, instead, did not find a significant association between specific autoantibodies and NVC findings. Among the pre-clinical studies, SSc-related autoantibodies showed different mechanisms of damage towards both endothelial cells, fibroblasts and smooth muscle vascular cells. CONCLUSIONS: The clinical and laboratory evidence on SSc-related autoantibodies and microvascular damage shows that these players are interconnected. Further clinical and demographic factors (e.g., age, sex, disease duration, treatment and comorbidities) might play an additional role in the SSc-related microvascular injury whose progression appears to be complex and multifactorial.

Off-label use of mycophenolate mofetil in the treatment of rare and complex rheumatic connective tissue diseases.

OBJECTIVES: We aimed to investigate the clinical off-label use of mycophenolate mofetil (MMF), including its safety and efficacy in patients with rare and complex rheumatic connective tissue diseases (rCTDs). METHODS: A survey was distributed across experts from ERN-ReCONNET reference centres in order to assess the experience with MMF off-label use. Patient-level data of patients with rCTDs under treatment with MMF was also collected for analysis of safety and efficacy. RESULTS: Twelve experts from eleven centres distributed throughout Europe (7 countries) answered the survey. The experience was concordant in that, despite of its off-label use, experts reported opting frequently for this therapeutic alternative with robust confidence on its efficacy and safety. The analysis of 108 patients with rCTDs under MMF revealed a good safety profile, as well as good clinical outcomes, especially for systemic lupus erythematosus and idiopathic inflammatory myopathies. The presence of interstitial lung disease was, as expected, associated with a worse clinical outcome despite use of MMF. CONCLUSIONS: MMF is widely used in reference centres for rCTDs. Its safety profile and efficacy seem to be recognised by experts and demonstrated with patient-level analysis. While selected rCTDs will likely remain an off-label indication for MMF, robust data seem to support this therapy as an appropriate alternative for safely and effectively treating many manifestations of rCTDs.