Innate and adaptive immunity represent a harmonic counterbalanced system involved in the induction, progression, and possibly resolution of the inflammatory reaction that characterize autoimmune rheumatic diseases (ARDs), including rheumatoid arthritis (RA). Although the immunopathophysiological mechanisms of the ARDs are not fully clarified, they are often associated with an inappropriate macrophage/T-cell interaction, where classical (M1) or alternative (M2) macrophage activation may influence the occurrence of T-helper (Th)1 or Th2 responses. In RA patients, M1/Th1 activation occurs in an inflammatory environment dominated by Toll-like receptor (TLR) and interferon (IFN) signaling, and it promotes a massive production of pro-inflammatory cytokines [i.e., tumor necrosis factor-α (TNFα), interleukin (IL)-1, IL-12, IL-18, and IFNγ], chemotactic factors, and matrix metalloproteinases resulting in osteoclastogenesis, erosion, and progressive joint destruction. On the other hand, the activation of M2/Th2 response determines the release of growth factors and cytokines [i.e., IL-4, IL-10, IL-13, and transforming growth factor (TGF)-β] involved in the anti-inflammatory process leading to the clinical remission of RA. Several subtypes of macrophages have been described. Five polarization states from M1 to M2 have been confirmed in in vitro studies analyzing morphological characteristics, gene expression of phenotype markers (CD80, CD86, TLR2, TLR4, or CD206, CD204, CD163, MerTK), and functional aspect, including the production of reactive oxygen species (ROS). An M1 and M2 macrophage imbalance may induce pathological consequences and contribute to several diseases, such as asthma or osteoclastogenesis in RA patients. In addition, the macrophage dynamic polarization from M1 to M2 includes the presence of intermediate polarity stages distinguished by the expression of specific surface markers and the production/release of distinct molecules (i.e., nitric oxide, cytokines), which characterize their morphological and functional state. This suggests a “continuum” of macrophage activation states playing an important role during inflammation and its resolution. This review discusses the importance of the delicate M1/M2 imbalance in the different phases of the inflammatory process together with the identification of specific pathways, cytokines, and chemokines involved, and its clinical outcomes in RA. The analysis of these aspects could shed a light on the abnormal inflammatory activation, leading to novel therapeutical approaches which may contribute to restore the M1/M2 balance.

Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05). Conclusions CTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

Active vitamin D [1,25(OH)2D3—calcitriol] is a secosteroid hormone whose receptor is expressed on all cells of the immune system. Vitamin D has a global anti-inflammatory effect and its role in the management of a SARS-CoV-2 infection has been investigated since the beginning of the COVID-19 pandemic. In this narrative review, the laboratory and clinical results of a vitamin D supplementation have been collected from both open-label and blinded randomized clinical trials. The results are generally in favor of the utility of maintaining the serum concentrations of calcifediol [25(OH)D3] at around 40 ng/mL and of the absolute usefulness of its supplementation in subjects with deficient serum levels. However, two very recent large-scale studies (one open-label, one placebo-controlled) have called into question the contribution of vitamin D to clinical practice in the era of COVID-19 vaccinations. The precise role of a vitamin D supplementation in the anti-COVID-19 armamentarium requires further investigations in light of the breakthrough which has been achieved with mass vaccinations.

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.

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by arthritis and a range of extra-articular organ involvement. Altered microcirculation is an important contributor to the inflammatory reaction characterizing RA and is involved in synovitis and systemic organ manifestations[1]. Nailfold video capillaroscopy (NVC) is a reliable, safe and highly sensitive method for evaluating the morphology of microcirculation and detailed alterations[2].ObjectivesThe objective of the study was to evaluate the microcirculation in adult RA patients by using NVC and to identify morphological and quantifiable NVC parameters with possible correlations with clinical and laboratory findings.MethodsNVC was performed at baseline on 8 fingers of both hands of 25 RA patients (EULAR/ACR 2010 criteria) (mean age 68.57±13.47 years, mean disease duration 14.6±8.6 years)[3]. Different NVC parameters indicating microvascular damage were detected and analyzed, such as microvascular architecture, capillary morphology and loss of capillaries (linear/mm). The results were categorized into “non-scleroderma” pattern, such as “normal” and “non-specific alterations”, as well as “scleroderma” and “scleroderma-like” pattern. The RA patients were treated (at the time of the analysis) with low dose glucocorticoids, NSAIDs and cDMARDs (see Table 1). Statistical analysis was performed by nonparametric tests.ResultsThe patient’s mean age at the time of NVC was 62.68±12.68 years and they consisted mostly of women (84%). The caucasian cohort was composed of 56% seropositive RA patients, all of which were RF positive (100%) and 71.43% also presented anti-CCP positivity. Nine patients (36%) were affected by Raynaud phenomenon.Eighty-four percent of RA patients (84%) presented together with normal capillaries a varying degree of microvascular nonspecific findings, like tortuosity, capillary crossing as well as some abnormal shapes (ramified capillaries).Interestingly, “Early” scleroderma NVC pattern was observed in 1 patient (4%), showing giant capillaries and microhaemorrhages. “Scleroderma-like” capillaroscopic pattern was found in 2 patients (8%) with the following features: dilatations, giant capillaries, microhaemorrhages, abnormal capillary shape with ramifications and reduced mean capillary number per linear mm. NVC observation of “Early” scleroderma pattern and “Scleroderma-like” pattern in 3 patients (12%), suggested the coexistence of an overlap syndrome (most likely MCTD), also supported by clinical and autoantibodies specificities. No specific RA NVC pattern was detectable.No statistically significant correlations were found between different NVC parameters and autoantibodies or specific RA clinical features.ConclusionThe results of present study confirm in sample size that a specific NVC pattern is not detectable in RA, even in early RA patients. The presence of specific patterns, like “Early” scleroderma NCV pattern and “scleroderma-like” pattern, suggest in those “RA patients” the presence of an overlap syndrome. The inclusion of the NVC analysis in patients affected by autoimmune connective tissue diseases (like RA) may contribute to a better definition of the diagnosis, especially in presence of complex clinical symptomatology (overlap syndromes).References[1] Angeloudi E et al. Mediterr J Rheumatol. 2022.[2] Cutolo M et al. Nat Rev Rheumatol. 2021.[3] Aletaha D et al. Arthritis Rheum. 2010.Table 1.Demographic and clinical characteristics of RA patientsn. 25female/male21/4 (5.25:1)current age (years, mean±SD)68.56 (13.47)age at diagnosis (years, mean±SD)53.96 (14.48)seropositive RA14 (56%)FR+14/14 (100%)ACPA+10/14 (71.43%)ANA+7 (28%)ENA+2 (8%)Raynaud phenomenon9 (12%)NVC performed at diagnosis5 (20%)Therapy at NVC baselineNSAIDs2 (8%)glucocorticoids19 (76%)cDMARDs19 (76%)NSAIDs: non-steroidal anti-inflammatory drugs;cDMARDs: conventional disease-modifying antirheumatic drugs.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

BackgroundNailfold videocapillaroscopy (NVC) safely allows the detection of microvascular damage which can vary in terms of extent and severity (validated NVC patterns) in patients with Raynaud’s phenomenon (RP) secondary to autoimmune connective tissue diseases (CTDs).ObjectivesThe prevalence of the morphological capillary findings was retrospectively evaluated in a wide cohort of patients with RP secondary to a CTD at the time of the first single NVC analysis, independently from their current treatment, autoantibody profile and comorbidities.MethodsOne-thousand-one-hundred-eighty-one (1181) patients affected by CTDs (1065 females, mean age 54.1 ± 16.2, mean disease duration 4.5 years ± 3) were analysed from 2001 to 2021. The considered CTDs, diagnosed through the classification criteria available at the time of the enrollment, included: systemic sclerosis (SSc, 51%), undifferentiated connective tissue disease (UCTD, 26%), mixed connective tissue disease (MCTD, 6%), dermatomyositis (DM, 3%), systemic lupus erythematosus (SLE, 9 %), Sjögren’s syndrome (SS, 3%) and primary anti-phospholipid syndrome (aPS, 2%). The capillaroscopic parameters were classified according to the CAP Fast Track Algorithm and distinguished between scleroderma-pattern (specific NVC alterations) and non-scleroderma patterns (non-specific NVC alterations) [1]. The presence of specific NVC findings detectable with a progressive microangiopathy in SSc patients (“early”, “active”, “late” patterns) have been searched also in other CTDs, beyond SSc. The “scleroderma-like pattern” was defined when a concomitant mix of the specific abnormalities of the SSc-patterns (namely: giant capillaries, loss of capillaries, capillary dilations, microhaemorrhages, abnormal shapes) was detected without fitting the single definition of “Early”, “Active” or “Late” pattern [2].ResultsAmong CTDs, the mean capillary density (1 linear mm) was significantly lower in SSc, DM and MCTD (respectively, 7.04 ± 0.18 vs 6.5 ± 0.75 vs 7.7 ± 0.48) compared with other CTDs. “Early”, “Active” and “Late” NVC scleroderma patterns were detected in 34%, 38% and 16% of SSc patients, whereas the scleroderma-like pattern was found significantly more frequent in DM (63 %) and MCTD (37%).Giant capillaries, capillary dilations and microhaemorrages were significantly more frequent in SSc and DM compared with other CTDs (respectively in 73%, 99% and 70% of SSc patients e in 73%, 96% e 70% of DM patients, Figure 1). The non-specific abnormalities of capillary morphology, such as the ramifications (abnormal shapes as expression of neoangiogenesis) were significantly more frequent in SSc, MCTD and APS among all the CTDs (respectively, in 48%, 41% and 36% of cases). Moreover, giant capillaries and abnormal shapes were detected in 61% and 41% of MCTD patients. In APS, the most significant prevalence of microhaemorrages (50%) was observed compared with other CTDs, to the exclusion of SSc and DM being detectable in 70% of cases. No significant damages were observed in SS and SLE patients (Figure 1).ConclusionThis large sample size of CTDs patients, collected over 20 years of analysis, confirms the highest specificity and severity of the NVC microvascular damage respectively in SSc and DM patients, when compared to other CTDs. Those data will be used to implement easy algorithms to distinguish scleroderma patterns, from non-scleroderma and scleroderma-like patterns.Reference[1]Smith V et al. Autoimmun Rev 2019 [2] Cutolo M et al. Nature Rev Rheumatol 2021Figure 1.Frequency of capillary density, giant capillaries, abnormal shapes and microhemorrhages across different CTDs (see text)AcknowledgementsElvis Hysa, Silvia Sammorì and Carmen Pizzorni equally contributed to this work.Disclosure of InterestsNone Declared.

BackgroundIn rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarization into “classically” (M1) or “alternatively activated” (M2) phenotypes and the release of pro-inflammatory cytokines (1). In the active inflammatory phase of RA, circulating intermediate monocytes and synovial tissue macrophages show a M1 phenotype, whereas MerTK+M2 macrophages seem to characterize the synovial tissue of RA patients under remission (2-4). In RA, CTLA4-Ig fusion protein (abatacept) reduces the pro-inflammatory activity of macrophages by interacting with the costimulatory molecule CD86 on surface cell membrane of activated cells, including macrophages (2).ObjectivesThe in vitro study investigated the efficacy of CTLA4-Ig treatment to induce the shift from the M1 phenotype into an M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from active RA patients.MethodsCultured MDMs obtained from peripheral blood mononuclear cells of 5 active RA patients (mean age 54±13 years) and 5 age-matched healthy subjects (HSs) after overnight stimulation with phorbol myristate acetate (5ng/ml), were treated with CTLA4-Ig at the concentrations of 100mg/mL or 500mg/mL for 3, 12, 24 and 48 hours. A part of cultured RA-MDMs as wells as cultured HS-MDMs were maintained in growth medium (RPMI at 10% of fetal bovine serum) without any treatment and used as unstimulated cells. Gene expression of CD80, CD86 and toll-like receptor-4 (TLR4), as M1 markers, as well as macrophage scavenger receptors (CD163, CD204), mannose receptor-1 (CD206), as surface M2 markers, and MerTK (functional M2 marker) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by Wilcoxon t-test.ResultsCultured RA-MDMs showed a high basal gene expression of TLR4, CD80 and CD86 compared to HS-MDMs, confirming to be activated M1 macrophages. In these macrophages, CTLA4-Ig treatment downregulated the gene expression of M1 markers at both concentrations and all timings, but significantly limited to TLR4 and CD80 markers (500mg/mL,12 hours: p<0.05). Conversely, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD163, MerTK and CD206 (p<0.05), whereas only the high concentration of CTLA4-Ig significantly upregulated CD204 gene expression (p<0.05). The protein synthesis of all M2 surface markers was increased after 24 hours of treatment primarily by the high concentration of CTLA4-Ig, and significantly for CD204 and CD206 (p<0.05).ConclusionCTLA4-Ig treatment seems to exert an important anti-inflammatory effect by promoting the shift from a M1 to an M2 phenotype in cultured RA macrophages The results suggest a further mechanism for CTLA4-Ig in the modulation of the RA synovitis (5).References[1]Yang X et al. Cell Prolif. 2020;53:e12854.doi:10.111/cpr.12854.[2]Kumar RA et al. Int. Immunol.2018;65:348-59.[3]Boutet MA et al. Autoimmun Rev.2021;20:102758. doi: 10.1016/j.autrev.2021.102758.[4]Alivernini S et al. Nat Med. 2020;26:1295-306. 5. Cutolo M et al. Arthritis Res Ther. 2009;11:R176; doi: 10.1186/ar2865.Disclosure of InterestsMaurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Celgene, Pfizer, Boehringer Ingelheim, Samuele Tardito: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Rosanna Campitiello: None declared, Sabrina Paolino: None declared, Carmen Pizzorni: None declared, Alberto Sulli Grant/research support from: Laboratories Baldacci, Vanessa Smith Grant/research support from: Boehringer Ingelheim, Janssen-Cilag, Stefano Soldano: None declared

ObjectiveNailfold 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.MethodsOne-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.ResultsGiant 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).ConclusionThis 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.