Tofacitinib Decreases Autophagy of Fibroblast-Like Synoviocytes From Rheumatoid Arthritis Patients.

The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.

Endothelial-to-mesenchymal transition in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by significant vascular alterations and multi-organ fibrosis. Microvascular alterations are the first event of SSc and injured endothelial cells (ECs) may transdifferentiate towards myofibroblasts, the cells responsible for fibrosis and collagen deposition. This process is identified as endothelial-to-mesenchymal transition (EndMT), and understanding of its development is pivotal to identify early pathogenetic events and new therapeutic targets for SSc. In this review, we have highlighted the molecular mechanisms of EndMT and summarize the evidence of the role played by EndMT during the development of progressive fibrosis in SSc, also exploring the possible therapeutic role of its inhibition.

Anti-D4GDI antibodies activate platelets in vitro: a possible link with thrombocytopenia in primary antiphospholipid syndrome.

BACKGROUND: Thrombocytopenia is a manifestation associated with primary antiphospholipid syndrome (PAPS), and many studies have stressed the leading role played by platelets in the pathogenesis of antiphospholipid syndrome (APS). Platelets are highly specialized cells, and their activation involves a series of rapid rearrangements of the actin cytoskeleton. Recently, we described the presence of autoantibodies against D4GDI (Rho GDP dissociation inhibitor beta, ARHGDIB) in the serum of a large subset of SLE patients, and we observed that anti-D4GDI antibodies activated the cytoskeleton remodeling of lymphocytes by inhibiting D4GDI and allowing the upregulation of Rho GTPases, such as Rac1. Proteomic and transcriptomic studies indicate that D4GDI is very abundant in platelets, and small GTPases of the RHO family are critical regulators of actin dynamics in platelets. METHODS: We enrolled 38 PAPS patients, 15 patients carrying only antiphospholipid antibodies without clinical criteria of APS (aPL carriers) and 20 normal healthy subjects. Sera were stored at - 20 °C to perform an ELISA test to evaluate the presence of anti-D4GDI antibodies. Then, we purified autoantibodies anti-D4GDI from patient sera. These antibodies were used to conduct in vitro studies on platelet activation. RESULTS: We identified anti-D4GDI antibodies in sera from 18/38 (47%) patients with PAPS, in sera from 2/15(13%) aPL carriers, but in no sera from normal healthy subjects. Our in vitro results showed a significant 30% increase in the activation of integrin αIIbß3 upon stimulation of platelets from healthy donors preincubated with the antibody anti-D4GDI purified from the serum of APS patients. CONCLUSIONS: In conclusion, we show here that antibodies anti-D4GDI are present in the sera of PAPS patients and can prime platelet activation, explaining, at least in part, the pro-thrombotic state and the thrombocytopenia of PAPS patients. These findings may lead to improved diagnosis and treatment of APS.

Reduction of autophagy and increase in apoptosis correlates with a favorable clinical outcome in patients with rheumatoid arthritis treated with anti-TNF drugs.

BACKGROUND: Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. RESULTS: PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. CONCLUSIONS: Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

Electrochemical Characterization and Surface Analysis of Activated Glassy Carbon Neural Electrodes.

Glassy carbon (GC) neural electrodes have recently gained visibility thanks to their great resistance to corrosion combined to their ability to record and stimulate neuronal activity. To enhance their electrochemical performance, GC electrodes are often subjected to activation, either through electrical or chemical means. In this study, we have compared the activation of GC electrodes performed using electrical biphasic pulses to chemically-induced activation. Because the GC electrodes used for this research are made by pyrolysing SU-8 photoresist - and thus they undergo massive shrinkage during carbonization - 2 electrode diameters were investigated (300 and 50 µm) with the aim of understanding if their surface composition and their ability to get activated change with their geometry. Chemical activation was induced by immersing the electrodes in 2 solutions: A1 and A2, 30 and 150 mM H2O2/PBS (hydrogen peroxide in phosphate buffered saline) respectively. The comparison between activation methods was done by measuring GC electrodes impedance, charge storage capacity (CSC) and by performing surface analysis, before and after the treatments. Results show that impedance drops in all the cases, especially at low frequencies (<; 1 kHz) and that there is always an increase in CSC. Raman spectra and relative intensities of disorder are very similar for both electrode diameters and before and after every treatment. X-Ray photoelectron spectroscopy (XPS) interestingly shows graphite content only on the 300 µm electrodes and a high percentage of graphite only on the pristine one. Apart from oxygen and nitrogen, no other species were present on the electrodes surface. In conclusion, both electrically and chemically-induced activation help improving the electrochemical performance of GC electrodes without harming them.

Microparticles and autophagy: a new frontier in the understanding of atherosclerosis in rheumatoid arthritis.

Microparticles (MPs) are small membrane vesicles released by many cell types under physiological and pathological conditions. In the last years, these particles were considered as inert cell debris, but recently many studies have demonstrated they could have a role in intercellular communication. Increased levels of MPs have been reported in various pathological conditions including infections, malignancies, and autoimmune diseases, such as rheumatoid arthritis (RA). RA is an autoimmune systemic inflammatory disease characterized by chronic synovial inflammation, resulting in cartilage and bone damage with accelerated atherosclerosis increasing mortality. According to the literature data, also MPs could have a role in endothelial dysfunction, contributing to atherosclerosis in RA patients. Moreover many researchers have shown that a dysregulated autophagy seems to be involved in endothelial dysfunction. Autophagy is a reparative process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. It has been shown in many works that basal autophagy is essential to proper vascular function. Taking into account these considerations, we hypothesized that in RA patients MPs could contribute to atherosclerosis process by dysregulation of endothelial autophagy process.

Porphyromonas gingivalis in the tongue biofilm is associated with clinical outcome in rheumatoid arthritis patients.

Several studies have suggested a link between human microbiome and rheumatoid arthritis (RA) development. Porphyromonas gingivalis seems involved in RA initiation and progression, as supported by the high occurrence of periodontitis. In this case-control study, we analysed tongue P. gingivalis presence and quantification in a large healthy and RA cohort. We enrolled 143 RA patients [male/female (M/F) 32/111, mean ± standard deviation (s.d.), age 57·5 ± 19·8 years, mean ± s.d. disease duration 155·9 ± 114·7 months); 36 periodontitis patients (M/F 11/25, mean ± s.d., age 56 ± 9·9 years, mean ± s.d. disease duration 25·5 ± 20·9 months); and 57 patients (M/F 12/45, mean ± s.d., age 61·4 ± 10·9 years, mean ± s.d. disease duration 62·3 ± 66·9 months) with knee osteoarthritis or fibromyalgia. All subjects underwent a standard cytological swab to identify the rate of P. gingivalis/total bacteria by using quantitative real-time polymerase chain reaction. The prevalence of P. gingivalis resulted similarly in RA and periodontitis patients (48·9 versus 52·7%, P = not significant). Moreover, the prevalence of this pathogen was significantly higher in RA and periodontitis patients in comparison with control subjects (P = 0·01 and P = 0·003, respectively). We found a significant correlation between P. gingivalis rate in total bacteria genomes and disease activity score in 28 joints (DAS28) (erythrocyte sedimentation rate) (r = 0·4, P = 0·01). RA patients in remission showed a significantly lower prevalence of P. gingivalis in comparison with non-remission (P = 0·02). We demonstrated a significant association between the percentage of P. gingivalis on the total tongue biofilm and RA disease activity (DAS28), suggesting that the oral cavity microbiological status could play a role in the pathogenic mechanisms of inflammation, leading to more active disease.

Anti-carbamylated protein antibodies in systemic lupus erythematosus patients with articular involvement.

Objective Several studies have evaluated the prevalence of rheumatoid factor (RF) and anti-citrullinated proteins antibodies (ACPA) in systemic lupus erythematosus (SLE) patients but no data are available on the anti-carbamylated proteins (anti-CarP), a new biomarker for rheumatoid arthritis (RA). We evaluated the anti-CarP prevalence in SLE patients with joint involvement and the associations with different phenotypes. Methods Seventy-eight SLE patients with joint involvement were enrolled (F/M 73/5; mean ± SD age 47.6 ± 11.2 years; mean ± SD disease duration 214.3 ± 115.6 months). As control groups, we evaluated SLE patients without joint manifestations ( N = 15), RA ( N = 78) and healthy individuals (HS, N = 98). Anti-CarP were assessed by home-made ELISA in all patients and controls, RF and ACPA in SLE patients with joint involvement (commercial ELISA kit). Results The prevalence of anti-CarP in SLE patients with joint involvement was similar to RA ( p = NS) and significantly higher compared with SLE without joint involvement and HS ( p < 0.0001, p < 0.0001, respectively). Four patients were positive for all three antibodies: seventy-five percent of these showed Jaccoud arthropathy. Fourty-five percent of ACPA-ve/RF-ve patients were anti-CarP + ve. Conclusions The evaluation of anti-CarP in SLE joint involvement demonstrated a prevalence of almost 50%, similar to RA and significantly higher than SLE without joint involvement and HS.

Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes.

It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.