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.

Apremilast interferes with the TGFß1-induced transition of human skin fibroblasts into profibrotic myofibroblasts: in vitro study.

OBJECTIVES: Fibroblast-to-myofibroblast transition and extracellular matrix overproduction represent progressive events in chronic inflammatory and fibrotic diseases, in which TGFß1 is one of the key mediators. Phosphodiesterase 4 (PDE4) acts as a proinflammatory enzyme through the degradation of cyclic adenosine monophosphate and it is overexpressed in skin fibroblasts. The study investigated how apremilast (a PDE4 inhibitor) interferes with the intracellular signalling pathways responsible for the TGFß1-induced fibroblast-to-myofibroblast transition and profibrotic extracellular matrix protein synthesis. METHODS: Cultured human skin fibroblasts were stimulated with TGFß1 (10 ng/ml) alone or combined with apremilast (1 and 10 µM) for 4, 16 and 24 h. Other aliquots of the same cells were previously stimulated with TGFß1 and then treated with apremilast (1 and 10 µM) for 4, 16 and 24 h, always under stimulation with TGFß1. Gene and protein expression of αSMA, type I collagen (COL1) and fibronectin were evaluated, together with the activation of small mothers against decapentaplegic 2 and 3 (Smad2/3) and extracellular signal-regulated kinase (Erk1/2) proteins. RESULTS: Apremilast reduced the TGFß1-induced increase in αSMA, COL1 and fibronectin gene expression at 4 and 16 h, and protein synthesis at 24 h of treatment in cultured fibroblasts, even for cells already differentiated into myofibroblasts by way of a previous stimulation with TGFß1. Apremilast inhibited the TGFß1-induced Smad2/3 and Erk1/2 phosphorylation at 15 and 30 min. CONCLUSION: Apremilast seems to inhibit in vitro the fibroblast-to-myofibroblast transition and the profibrotic activity induced by TGFß1 in cultured human skin fibroblasts by downregulating Smad2/3 and Erk1/2 intracellular signalling pathways.

Altered expression of RXFP1 receptor contributes to the inefficacy of relaxin-based anti-fibrotic treatments in systemic sclerosis.

OBJECTIVES: Relaxin is a potent anti-fibrotic hormone that has been tested to ameliorate fibrosis in systemic sclerosis (SSc), but with controversial results. The aim of the study is to sequence relaxin receptor gene RXFP1 and to assess its mRNA expression and protein levels in the skin of SSc patients and healthy subjects. METHODS: Fibroblasts were isolated from unaffected/affected skin samples of (n=16) limited-cutaneous-SSc-(LcSSc) and from affected ones of (n=4) diffuse-cutaneous-SSc-(DcSSc) patients. Fibroblasts from healthy subjects were used as controls. Sequencing of exonic target regions of interest for RXFP1 gene was performed, coupled with mRNA transcript variant analysis. RXFP1 mRNA and protein levels were assessed by quantitative-real-time-PCR-(qRT-PCR) and by immunocytochemistry-(ICC). Alpha-smooth-muscle-actin-(α-SMA) synthesis induced by transforming-growth-factor-beta-1-(TGF-ß1) stimulation was investigated in all fibroblasts with and without pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the receptor RXFP1). RESULTS: Sequencing of RXFP1 gene showed no relevant mutations in all fibroblast populations. The analysis of mRNA transcripts revealed the presence of 13 different mRNA isoforms of RXFP1 (7 coding and 6 non-coding) upregulated in LcSSc/DcSSc-affected samples and not in LcSSc-unaffected and in healthy ones. On the contrary, ICC demonstrated the absence of RXFP1 in LcSSc/DcSSc-affected fibroblasts and the presence in LcSSc-unaffected and in healthy ones. To prove these findings, serelaxin pre-incubation was unable to counteract TGF-ß1-driven upregulation of α-SMA in LcSSc/DcSSc-affected fibroblasts only, but not in LcSSc-unaffected and healthy ones. CONCLUSIONS: The absence/altered expression of relaxin receptor RXFP1 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease.

Correlation between circulating fibrocytes and dermal thickness in limited cutaneous systemic sclerosis patients: a pilot study.

The objective is to detect any possible correlation between the modified Rodnan skin score (mRSS) and dermal thickness (DT) measured by skin high-frequency ultrasound (US) and the percentage of circulating fibrocytes in patients with limited cutaneous systemic sclerosis (lcSSc). Eight lcSSc patients and five healthy subjects (control group, CNT) were enrolled. The skin involvement was evaluated by mRSS and US (18 and 22 MHz probes) in all 13 subjects in the 17 standard skin areas evaluated by mRss. Circulating fibrocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of all lcSSc patients and the CNT group to analyze their percentage at baseline time (T0) when the experiments started with PBMCs' isolation and collection and after 8 days of culture (T8). Non-parametric tests were used for the statistical analysis. A positive correlation between the percentage of circulating fibrocytes at T0, mRSS (p = 0.04 r = 0.96), and DT-US, evaluated by the 22 MHz and the 18 MHz probes (p = 0.03, r = 0.66 and p = 0.05, r = 0.52, respectively), was observed in lcSSc patients. Conversely, at T8, there was no correlation (p > 0.05) between these parameters in lcSSc group. In the CNT group, no correlations between mRSS or DT-US and the percentage of circulating fibrocytes were observed both at T0 and T8. The study shows the presence of a significant relationship between the percentage of circulating fibrocytes and DT, as evidenced by both mRSS and US, in limited cutaneus SSc. This observation may well suggest the reasonable hypothesis of a crucial contribution of circulating fibrocytes to skin fibrosis progression, which might be considered as further biomarkers.

A circulating cell population showing both M1 and M2 monocyte/macrophage surface markers characterizes systemic sclerosis patients with lung involvement.

BACKGROUND: Systemic sclerosis (SSc) is a disorder characterized by immune system alterations, vasculopathy and fibrosis. SSc-related interstitial lung disease (ILD) represents a common and early complication, being the leading cause of mortality. Monocytes/macrophages seem to have a key role in SSc-related ILD. Interestingly, the classically (M1) and alternatively (M2) activated monocyte/macrophage phenotype categorization is currently under revision. Our aim was to evaluate if circulating monocyte/macrophage phenotype could be used as biomarker for lung involvement in SSc. To this purpose we developed a wide phenotype characterization of circulating monocyte/macrophage subsets in SSc patients and we evaluated possible relations with lung involvement parameter values. METHODS: A single centre cross-sectional study was performed in fifty-five consecutive SSc patients, during the year 2017. All clinical and instrumental tests requested for SSc follow up and in particular, lung computed tomography (CT) scan, pulmonary function tests (PFTs), Doppler echocardiography with systolic pulmonary artery pressure (sPAP) measurement, blood pro-hormone of brain natriuretic peptide (pro-BNP) evaluation, were performed in each patient in a maximum one-month period. Flow cytometry characterization of circulating cells belonging to the monocyte/macrophage lineage was performed using specific M1 (CD80, CD86, TLR2 and TLR4) and M2 surface markers (CD204, CD163 and CD206). Non-parametric tests were used for statistical analysis. RESULTS: A higher percentage of circulating CD204+CD163+CD206+TLR4+CD80+CD86+ and CD14+CD206+CD163+CD204+TLR4+CD80+CD86+ mixed M1/M2 monocyte/macrophage subsets, was identified to characterize patients affected by SSc-related ILD and higher systolic pulmonary artery pressure. Mixed M1/M2 monocyte/macrophage subset showed higher percentages in patients positive for anti-topoisomerase antibody, a known lung involvement predictor. CONCLUSIONS: The present study shows for the first time, through a wide flow cytometry surface marker analysis, that higher circulating mixed M1/M2 monocyte/macrophage cell percentages are associated with ILD, sPAP and anti-topoisomerase antibody positivity in SSc, opening the path for research on their possible role as pathogenic or biomarker elements for SSc lung involvement.

Dissecting the inflammatory response in polymyalgia rheumatica: the relative role of IL-6 and its inhibition.

The efficacy of tocilizumab (TCZ), a monoclonal antibody to the interleukin (IL)-6 receptor, in suppressing disease activity in glucocorticoid-naïve patients with new-onset polymyalgia rheumatica (PMR) was studied. Its effect on a panel of cytokines and growth factors was evaluated. Three patients, fulfilling the PMR ACR/EULAR criteria, received TCZ at the dosage of 8 mg/kg every 4 weeks for three times followed by prednisone 0.2 mg/kg in case of inefficacy. Concentrations of IL-10, IL-6, tumour necrosis factor (TNF)-α, IL-1ß, IL-10, IL-17, interferon (IFN)-γ, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and leukaemia inhibitory factor (LIF) were measured at baseline, after 72 h of the first TCZ infusion and then at weeks 1, 4, 5, 8, 9, 12, 13, 14, 16, and 22. A slight clinical improvement was seen only after the first TCZ infusion, but was largely inferior to that of conventional doses of GC administered subsequently. An ischaemic visual accident suggestive of GCA occurred in one patient during TCZ treatment. IL-6 was increased at baseline compared to controls, further increased after the first TCZ infusion, and was suppressed by GC. IL-17 production decreased during TCZ treatment and reverted to pre-treatment levels after GC. VEGF e PDGF showed a less constant pattern, but an increase of VEGF concentration antedated visual symptoms. The other cytokines were not detectable in patients and controls. In our small sample, TCZ was not able to suppress inflammation at the same degree as GC. As a result, monotherapy with TCZ in PMR cannot be recommended, although its efficacy as adjunctive treatment in GC-resistant patients should be further evaluated.

Dual endothelin receptor antagonists contrast the effects induced by endothelin-1 on cultured human microvascular endothelial cells.

OBJECTIVES: To evaluate the ability of dual endothelin (ET) receptor antagonists (ETA/ETB -ETA/BRAs) to contrast the ET-1-induced effects on cultured human microvascular endothelial cells (HMVECs). METHODS: Some cultured HMVECs were untreated, or treated with ET-1 (100nM) or transforming growth factor ß1 (TGFß1, 10ng/mL) alone for 6 days, in order to induce the endothelial-to-mesenchymal transition (EndoMT). Other cultured HMVECs were pre-treated for 1hr with ETA/BRAs bosentan (10µM) or macitentan (1µM, 10µM) before the stimulation with ET-1 for 6 days. At the end of treatments, a mechanical injury was induced to cultured HMVECs (by scratching the cell monolayer with a sterile tip), and then the cell ability to re-fill the damaged area was determined after 24hrs. EndoMT phenotype markers and monocyte chemoattractant protein-1 (MCP-1) were evaluated by qRT-PCR and Western blotting. Statistical analysis was performed using Mann-Whitney-U non-parametric test. RESULTS: Both ET-1 and TGFß1 induced EndoMT and the MCP-1 over-expression in cultured HMVECs, as well as reduced the process of endothelial cell damage repair. Pre-treatment with ETA/BRAs let cultured HMVECs to significantly restore the in vitro damage of the cell monolayer and antagonised the EndoMT process as well as the MCP-1 over-expression (range p<0.05 - p<0.001). Conversely, untreated or TGFß1-treated HMVECs were found unaffected by the ETA/BRAs treatments. CONCLUSIONS: The treatment with dual ETA/BRAs seems to partially restore the altered cell function induced by ET-1 in cultured endothelial cells, and might justify their therapeutic efficiency in clinical conditions characterised by increased concentrations of ET-1.

Effects of combined treatments with CTLA4-IG (abatacept), dexamethasone and methotrexate on cultured human macrophages.

OBJECTIVES: To evaluate the anti-inflammatory effect of CTLA4-Ig (abatacept) and dexamethasone (DEX) monotreatment versus their combination and adding methotrexate (MTX) on cultured human macrophages. METHODS: THP-1 cells, activated into macrophages (PMA 0.05 µg/ml), were cultured for 3 and 24 hrs with CTLA4-Ig (500 µg/ml), DEX (10-8 M), MTX (0.05 µg/ml), and CTLA4-Ig combined with DEX or CTLA4-Ig combined with DEX plus MTX. CTLA4-Ig/CD86 interaction was evaluated by FACS analysis. Quantitative real time-PCR (qRT-PCR), immunocytochemistry (ICC) and immunoassay (ELISA) analysis for inflammatory cytokine (IL-1ß, TNF-α, IL-6) expression were performed. RESULTS: FACS analysis showed in macrophages treated with CTLA4-Ig alone, CTLA4-Ig-DEX and CTLA4-Ig-DEX-MTX a CD86 decrease of almost 35%, versus untreated cells (CNT). After 3 hrs, macrophages treated with DEX alone or with CTLA4-Ig-DEX or CTLA4-Ig-DEX-MTX showed a significant reduction (p<0.05) for all cytokines gene expression, that was still significant for IL-1ß after 24 hrs (p<0.05). After 3 hrs, CTLA4-Ig alone significantly (p<0.05) reduced all cytokine genes; however, after 24 hrs still evident only for TNF-α (p<0.05). After 24 hrs CTLA4-Ig-DEX induced a significant decrease of gene expression (p<0.05) for TNF-α and IL-6, whereas CTLA4-Ig-DEX-MTX induced a decrease (p<0.05) limited to IL-6, versus CNT. Finally, ICC showed, after 24 hrs of CTLA4-Ig-DEX or CTLA4-Ig-DEX-MTX treatment a reduction (p<0.05) of IL-1ß and IL-6 expression, versus CNT; DEX alone reduced only IL-1ß (p<0.05). ELISA analysis confirmed these results. CONCLUSIONS: CTLA4-Ig-DEX and CTLA4-Ig-DEX-MTX combined treatments, decreased at any level the inflammatory cytokine expression more efficiently then monotreatments on activated cultured human macrophages.

CTLA4-Ig/CD86 interactions in cultured human endothelial cells: effects on VEGFR-2 and ICAM1 expression.

OBJECTIVES: Previous studies have reported the presence of CD86 (B7.2) costimulatory molecule on endothelial cells (ECs) and recent data have shown that CTLA4-Ig (abatacept), used as a biological agent in rheumatoid arthritis, interacts with CD86 expressed on different cells involved in synovitis. Therefore, the effects of CTLA4-Ig/CD86 interaction on VEGFR-2 (vascular endothelial growth factor receptor 2) and ICAM1 expression, were evaluated in cultured ECs. METHODS: Activated ECs (γIFN 500 U/ml or IL-17 100 ng/ml), treated with CTLA4-Ig (10, 100, 500 µg/ml) were analysed for CD86, VEGFR-2 and ICAM1 expression, by flow cytometry (FACS), by western blot (WB) and quantitative real time PCR (qRT-PCR). RESULTS: Following CTLA4-Ig treatment (10, 100, 500 µg/ml; 24 hrs), activated ECs decreased their CD86-positivity at FACS: 66, 59, 51%, respectively, versus 68% of untreated cells (cnt) (for γIFN-activated cells) and 42, 47, 46% versus 71% (cnt) (for IL-17-activated ECs). Gamma-IFN-activated ECs, treated with CTLA4-Ig, showed a dose-dependent decrease only for ICAM1 fluorescence. Whereas, WB showed a significant decrease (p<0.05) for both ICAM1 and VEGFR-2 after CTLA4-Ig 500 µg/ml (3 and 24 hrs) and for VEGFR-2 also after CTLA4-Ig 100 µg/ml (3 hrs). QRT-PCR showed a significant decrease (p<0.05) for VEGFR-2 after CTLA4-Ig 500 µg/ml (3 and 24 hrs) and after CTLA4-Ig 100 µg/ml (limited at 3 hrs). QRT-PCR for ICAM1 was negative at 3 and 24 hrs, possibly since it was to late to be detected. CONCLUSIONS: Results support a CTLA4-Ig/CD86 interaction on γIFN and IL-17 activated ECs modulation, in the expression of VEGFR-2 and ICAM1, both relevant for inflammatory and angiogenetic processes, suggesting ECs as a further target for abatacept.

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.

Intracellular NF-kB-decrease and IKBα increase in human macrophages following CTLA4-Ig treatment.

OBJECTIVES: The transcription factor NF-kB is involved in the expression of several genes linked to the immune response, including those of pro-inflammatory cytokines. We investigated cytokine production and NF-kB expression following CTLA4-Ig (abatacept) treatment of cultured human macrophages. METHODS: Human THP1 cells, differentiated in macrophages, were treated with CTLA4-Ig (100, 500 µg/ml; 3,12,24 hours). Quantitative RT-PCR analysis (qRT-PCR) of mRNA for NF-kB, IKBα and for IL-6, TNF-α, IL-1ß, was performed after 3 and 12 hours from treatment. Western blot (WB) analysis for NF-kB and IKBα was performed after 24 hours from treatment. RESULTS: NF-kB gene expression was significantly downregulated (p<0.05), at 3 and 12 hours from CTLA4-Ig treatment, vs. untreated cells (cnt). IKBα resulted significantly increased vs. cnt (p<0.05), at 12 hours from CTLA4-Ig [500 µg/ml] treatment. After 3 hours, CTLA4-Ig [100 µg/ml] induced a significant decrease of TNF-α and IL-6 (p<0.05), vs. cnt and CTLA4-Ig [500 µg/ml] further reduced TNF-α (p<0.001), vs. cnt. After 12 hours from CTLA4-Ig treatment, a significant downregulation for IL-6 and IL1ß expression (p<0.001), vs. cnt, was still evident. Results were confirmed by WB. CONCLUSIONS: NF-kB pathway seems to be implicated in the CTLA4-Ig modulation of macrophage cytokine expression. NF-kB expression resulted downregulated while its cytoplasmatic inhibitor IKBα was increased.

A Systematic Review of Aminaphtone from Pathophysiology to Clinical Applications: Focus on New Rheumatological Acquisitions.

Aminaphtone is a chemical drug that has been used for more than thirty years to treat a variety of vascular disorders, with good clinical results and a satisfying safety profile. In the last two decades, multiple clinical studies have reported the efficacy of the drug in different clinical scenarios of altered microvascular reactivity, describing the downregulation of adhesion molecules (i.e., VCAM, ICAM, Selectins), vasoconstrictor peptides (i.e., Endothelin-1), and pro-inflammatory cytokine expression (i.e., IL-6, IL-10, VEGF, TGF-beta) by Aminaphtone. In this review, we summarize the current knowledge concerning Aminaphtone, with particular attention to rheumatological conditions in which microvascular disfunction plays a pivotal role, such as Raynaud's phenomenon and systemic sclerosis. These latter conditions may represent a promising field of application for Aminaphtone, due to the growing pre-clinical, clinical, and instrumental reports of efficacy. However, randomized, double-blind, placebo-controlled clinical trials are lacking and are desirable.

1,25-dihydroxyvitamin D3 downregulates aromatase expression and inflammatory cytokines in human macrophages.

OBJECTIVES: Vitamin D deficiency seems to be involved in the development and severity of autoimmune/inflammatory diseases such as rheumatoid arthritis (RA). To evaluate the influence of calcitriol (1,25-dihydroxyvitamin D3, 1,25(OH)2D3) on aromatase expression in cultures of human macrophages, as a new target for vitamin D cell modulation and pro-inflammatory cytokine production. METHODS: Cultures of human monocytic THP-1 cells were activated to macrophages and treated for 24 hours with 1,25(OH)2D3 (10-8M), 17ß-estradiol (E2, 10-8M) both alone and in combination, in order to evaluate the effects on the intracrine estrogen metabolism. Untreated human macrophages were used as controls (basal). P450-aromatase synthesis was evaluated by immunocytochemistry (ICC) and western blot analysis (WB). The expression of P450-aromatase gene (CYP19A1) was investigated by real-time PCR (RT-PCR). Macrophage pro-inflammatory cytokines IL1-ß, IL-6 and TNF-α were evaluated by ELISA and WB. RESULTS: In E2 untreated condition, 1,25(OH)2D3 reduced P450-aromatase synthesis and CYP19A1 gene expression in cultured cells. Moreover, pro-inflammatory cytokine production (IL1-ß, IL-6 and TNF-α) was significantly reduced by 1,25(OH)2D3 treatment (p<0.001 vs. basal for all cytokines). However, 1,25(OH)2D3 was found to significantly downregulate the E2-mediated increase in P450-aromatase synthesis and gene expression (p<0.001 for both vs. E2-treated macrophages), as well as the production of all pro-inflammatory cytokines (p<0.001 vs. E2-treated cells). CONCLUSIONS: Our data suggest that 1,25(OH)2D3 may downregulate the pro-inflammatory cytokine production in human activated macrophages by significantly decreasing the aromatase activity, especially in presence of an estrogenic milieu such as in the RA synovial tissue.

The Role of M1/M2 Macrophage Polarization in Rheumatoid Arthritis Synovitis.

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)-ß] 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.

Vitamin D and COVID-19: Narrative Review after 3 Years of Pandemic.

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.

Identification and quantification of selected inflammatory genes modulated by leflunomide and prednisone treatment in early rheumatoid arthritis patients.

OBJECTIVES: The present study evaluates the effects of combined leflunomide (LEF) and low dose of prednisone therapy, on selected inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) of early rheumatoid arthritis (ERA) patients by gene microarray analysis and quantitative real time-polymerase chain reaction (qRT-PCR). METHODS: Ten ERA patients (mean age 53 ± 10 years) were assigned as untreated (group 1) or pre-treated (group 2) with prednisone (5 mg/day for 3 months) after informed consent and ethics committee approval. Five sex- and age-matched healthy subjects were used as controls (CNT). RNA was extracted by PBMCs, amplified, labelled and hybridised on inflammation DualChip microarray. The expression ratio of 282 inflammatory genes between CNT and ERA patients, before (T0) and after 12 weeks (T1) of combined therapy was detected. qRT-PCR was performed on 7 selected inflammatory RA-related genes (STAT4, MAPK9, HIF1A, MIF, STAT6, NFKB1, TNFRSF1B). RESULTS: At T0, microarray analysis showed 34 altered genes in both ERA groups when compared to CNT (vs. CNT). Seven RA-related genes, investigated in further details, were found up-regulated in group 1 and down-regulated or unchanged in group 2 vs. CNT. At T1, combined therapy induced the down-regulation of these genes in both groups vs. CNT as also confirmed by qRT-PCR performed on selected genes. CONCLUSIONS: Untreated ERA patients seem characterised by up-regulation of specific genes involved both in the resistance/inhibition to apoptosis and in the stimulation of pro-inflammatory cytokine production by immune inflammatory cells. Combined LEF and low dose of prednisone therapy seems to play synergistic effects on down-regulation of these genes.

Influence of Seasonal Vitamin D Changes on Clinical Manifestations of Rheumatoid Arthritis and Systemic Sclerosis.

Vitamin D [1,25(OH)2D-calcitriol] is basically a steroid hormone with pleiotropic biologic effects, and its impact on the regulation of immune system may influence several clinical conditions. Calcidiol (25OHD), as precursor of calcitriol, derives, for the most part (80%), from cutaneous cholesterol (7-dehydrocholesterol) under the action of UV-B (sunlight). Consequently, serum concentrations fluctuate during the year following the circannual rhythm of sun exposition. We will update about the available evidence regarding the complex influence of seasonal vitamin D changes on two different chronic connective tissue diseases, namely rheumatoid arthritis (RA) and systemic sclerosis (SSc). Notably, RA is an emblematic model of autoimmune disease with prevalent joint inflammatory features, while SSc is mainly an autoimmune progressive pro-fibrotic disease. However, in both conditions, low serum concentrations of 25OHD are involved in the pathogenesis of the diseases, and emerging data report their impact on clinical manifestations.