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.

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.

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.

Preliminary assessment of the prototype Yfiler® Plus kit in a population study of Northern Italian males.

A total of 150 samples of unrelated males from North of Italy were analyzed with the prototype Yfiler® Plus kit (Thermo Fisher Scientific, Oyster Point, CA). This kit is a short tandem repeat (STR) assay based on six-dye chemistry that amplifies 25 Y-STR loci. Sixteen loci are in common with Yfiler® kit and nine are new (DYS576, DYS627, DYS460, DYS518, DYS570, DYS449, DYS481, DYF387S1a/b, DYS533). In this population study, the improvement of adding additional Y-STR markers increased the discriminatory capacity from 0.787 with the minimum haplotype (MHT) loci to 1 with the prototype Yfiler® Plus kit.

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.

The intervention of macrophages in progressive fibrosis characterizing systemic sclerosis: A systematic review.

BACKGROUND AND AIM: Systemic sclerosis (SSc) is an immune mediated connective tissue disease characterized by microvascular dysfunction, aberrant immune response, and progressive fibrosis. Although the immuno-pathophysiological mechanisms underlying SSc are not fully clarified, they are often associated with a dysfunctional macrophage activation toward an alternative (M2) phenotype induced by cytokines [i.e., IL-4, IL-10, IL-13, and transforming growth factor (TGF-ß)] involved in the fibrotic and anti-inflammatory process. A spectrum of macrophage activation state has been identified ranging from M1 to M2 phenotype, gene expression of phenotype markers, and functional aspects. This systematic review aims to analyze the importance of M2 macrophage polatization during the immune mediated process and the identification of specific pathways, cytokines, and chemokines involved in SSc pathogenesis. Moreover, this review provides an overview on the in vitro and in vivo studies aiming to test therapeutic strategies targeting M2 macrophages. METHODS: A systematic literature review was performed according to the preferred Reported Items for Systematic Reviews and Meta-Analyses (PRISMA). The search encompassed the online medical databases PubMed and Embase up to the 30th of June 2024. Original research manuscripts (in vitro study, in vivo study), animal model and human cohort, were considered for the review. Exclusion criteria encompassed reviews, case reports, correspondences, and conference abstracts/posters. The eligible manuscripts main findings were critically analyzed, discussed, and summarized in the correspondent tables. RESULTS: Out of the 77 screened abstracts, 49 papers were deemed eligible. Following a critical analysis, they were categorized according to the primary (29 original articles) and secondary (20 original articles) research objectives of this systematic review. The data from the present systematic review suggest the pivotal role of M2 macrophages differentiation and activation together with the dysregulation of the immune system in the SSc pathogenesis. Strong correlations have been found between M2 macrophage presence and clinical manifestations in both murine and human tissue samples. Interestingly, the presence of M2 cell surface markers on peripheral blood monocytes has been highlighted, suggesting a potential biomarker role for this finding. Therapeutic effects reducing M2 macrophage activities have been observed and/or tested for existing and for new drugs, demonstrating potential efficacy in modulating the pro-fibrotic immune response for treatment of SSc. CONCLUSIONS: The increased M2 macrophage activation in course of SSc seems to offer new insights on the self-amplifying inflammatory and fibrotic response by the immune system on such disease. Therefore, the revaluation of immunomodulatory and ongoing antifibrotic therapies, as well as novel therapeutical approaches in SSc that contribute to limit the M2 macrophage activation are matter of intense investigations.

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.

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.

CTLA4-Ig treatment induces M1-M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients.

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.

Nintedanib downregulates the transition of cultured systemic sclerosis fibrocytes into myofibroblasts and their pro-fibrotic activity.

BACKGROUND: Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity. METHODS: Circulating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 µM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis. RESULTS: Significantly elevated gene and protein expressions of αSMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70-ILD- patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN. Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance. Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70-ILD- fibrocytes compared to the related untreated cells. CONCLUSIONS: Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+ SSc patients.

Sex hormones modulate the effects of Leflunomide on cytokine production by cultures of differentiated monocyte/macrophages and synovial macrophages from rheumatoid arthritis patients.

Immune response is greater in females than in males and lymphocytes/monocytes from female subjects (or tested in vitro with estrogens) show higher immune/inflammatory reactivity. In order to test in vitro the interactions between 17beta-estradiol (E2--10(-9) M), testosterone (T--10(-8) M) and the antiproliferative/immune suppressive drug Leflunomide metabolite A77 1726 (LEF-M--30 microM) employed in rheumatoid arthritis (RA), their combined effects were evaluated on inflammatory cytokine (CK) expression/production in cultures of differentiated macrophages (M) (from activated THP-1 monocytes) and primary cultures of RA synovial macrophages (SM). TNFalpha, IL-6 and TGFbeta were detected by immunocytochemistry (ICC), Western blot analysis (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR). The ICC, WB and RT-PCR showed a significant down-regulation induced by LEF-M on CK expression by cultured M when compared to untreated cells (IL-6 p < 0.01, TNFalpha p < 0.001, TGFbeta p < 0.01). At ICC analysis E2 increased CK expression, whereas T decreased the expression, confirmed by WB and RT-PCR (range between p < 0.05 and p < 0.001). LEF-M treatment significantly downregulated the CK expression in E2/T treated M: the effect was more significant in LEF-M plus T-treated cells versus controls (range between p < 0.01 and p < 0.001). Concerning the RA SM, the results were replicated (range between p < 0.05 and p < 0.001). E2 seems to contrast, but T seems to synergize the LEF-M activity. Results might support a stronger therapeutical efficacy, at least for LEF, in male RA patients, as already reported by clinical evidences.

Effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts from the same systemic sclerosis patients: an in vitro assay.

BACKGROUND: Systemic sclerosis (SSc) is characterized by vasculopathy and progressive fibrosis. CTLA4-Ig (abatacept) is able to interact with the cell surface costimulatory molecule CD86 and downregulate the target cell. The aim of this study was to evaluate the in-vitro effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts isolated from the same SSc patient. METHODS: Circulating fibrocytes and skin fibroblasts were obtained from eight SSc patients with "limited" cutaneous involvement and from four healthy subjects (HSs). Samples were analyzed by fluorescence-activated cell sorter analysis (FACS) at baseline (T0) and after 8 days of culture (T8) for CD45, collagen type I (COL I), CXCR4, CD14, CD86, and HLA-DRII expression. Circulating fibrocytes were treated for 3 h and skin fibroblasts for 24/48 h with CTLA4-Ig (10, 50, 100, 500 µg/ml). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for CD86, COL I, FN, TGFß, αSMA, S100A4, CXCR2, CXCR4, CD11a, and Western blotting was performed for COL I and FN. RESULTS: Using qRT-PCR, the T8-cultured SSc circulating fibrocytes which had not been treated with CTLA4-Ig showed higher gene expression for CD86, αSMA, S100A4, TGFß, and COL I compared with HS circulating fibrocytes. Interestingly, αSMA/COL I gene expression was significantly lower only in the SSc circulating fibrocytes treated with CTLA4-Ig for 3 h (p < 0.01, p < 0.05). On the contrary, no effects were observed for either SSc or HS skin fibroblasts after CTLA4-Ig treatment. COL I and FN protein expression was unchanged in both SSc and HS skin fibroblasts by Western blot. CONCLUSIONS: Circulating fibrocytes seem to be more responsive to CTLA4-Ig treatment than skin fibroblasts from the same SSc patient, likely due to their higher expression of CD86. CTLA4-Ig treatment might downregulate the fibrotic process in SSc patients by downregulating the fibrocytes, circulating progenitor cells.

Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts.

BACKGROUND: Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. METHODS: Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 µM to 0.3 µM) or ACT-333679 (from 10 µM to 0.1 µM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. RESULTS: Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. CONCLUSIONS: Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.

Correction: Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0166433.].