M. Vomero

Role of autophagy in immunity and autoimmunity, with a special focus on systemic lupus erythematosus

Autophagy is a lysosome‐mediated catabolic process that allows cells to degrade unwanted cytoplasmic constituents and to recycle nutrients. Autophagy is also involved in innate and adaptive immune responses, playing a key role in interactions against microbes, in antigen processing for major histocompatibility complex (MHC) presentation, and in lymphocyte development, survival, and proliferation. Over recent years, perturbations in autophagy have been implicated in a number of diseases, including autoimmunity. Systemic lupus erythematosus (SLE) is a multifactorial disease characterized by autoimmune responses against self‐antigens generated by dying cells. Genome‐wide association studies have linked several single‐nucleotide polymorphisms (SNPs) in the autophagy‐related gene Atg5 to SLE susceptibility. Loss of Atg5‐dependent effects, including clearance of dying cells and cell antigen presentation, might contribute to the autoimmunity and inflammation associated with SLE. Moreover, activation of the mammalian target of rapamycin (mTOR), a key player in the autophagy regulation, has recently been demonstrated in SLE, confirming an altered autophagy pathway in this disease. In the present review, we summarize the autophagy mechanisms, their molecular regulation, and their relevance in immunity and autoimmunity. The potential of targeting autophagy pathway in SLE, by developing innovative therapeutic approaches, has finally been discussed.—Pierdominici, M., Vomero, M., Barbati, C., Colasanti, T., Maselli, A., Vacirca, D., Giovannetti, A., Malorni, W., Ortona, E. Role of autophagy in immunity and autoimmunity, with a special focus on systemic lupus erythematosus. FASEB J. 26, 1400‐1412 (2012). www.fasebj.org

Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases

BackgroundAntibodies against carbamylated proteins (anti-CarP) have been recently identified in the sera of patients with rheumatoid arthritis (RA). The objective of the study was to evaluate the prevalence, sensitivity and specificity of anti-CarP compared to anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF), replicating the existing data in a large cohort of Italian patients with RA and extending the evaluation to other autoimmune rheumatic diseases (AIRDs).MethodsSerum samples (n = 607) from 309 patients with RA, 200 disease controls and 98 normal healthy subjects (NHS) were evaluated. Anti-CarP were detected using carbamylated fetal calf serum as the antigen. ACPAs were detected using second-generation ELISA and IgM RF was assessed as part of routine analysis.ResultsAnti-CarP antibodies were detected in 117 patients with RA (34.4%), ACPA in 190 patients (61.4%) and RF in 202 patients (65.3%). Two (2.04%) of the NHS were positive for anti-CarP, one NHS (1.02%) was positive for ACPA and three NHS were positive for RF (3.06%). Among disease controls, anti-CarP antibodies were detected in 33 patients (16.5%), ACPA in 29 patients (14.5%) and RF in 64 patients (32%). In particular, 16.8% of patients with systemic lupus erythematosus and 31.1% of patients with Sjögren syndrome were positive for anti-CarP. The sensitivity of anti-CarP, ACPA and RF was 46.8%, 61.8% and 64.4%, respectively and specificity was 91.95%, 89.93% and 76.51%, respectively.ConclusionsThe present study extends the knowledge of anti-CarP antibodies, confirming previous data on the diagnostic accuracy of anti-CarP in RA in a large cohort of Italian patients. Anti-CarP antibodies demonstrated relatively low sensitivity and slightly higher specificity compared to ACPA and RF. Even if predominantly present in RA, anti-CarP was detected in a variable percentage of patients with other autoimmune rheumatic diseases and their generation could be attributed to the inflammatory status; the clinical relevance of anti-CarP antibodies in these latter patients should be further determined.

Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders

Autophagy represents a key mechanism of cytoprotection that can be activated by a variety of extracellular and intracellular stresses and allows the cell to sequester cytoplasmic components and damaged organelles, delivering them to lysosomes for degradation and recycling. However, the autophagy process has also been associated with the death of the cell. It has been demonstrated to be constitutive in some instances and inducible in others, and the idea that it could represent a pathogenetic determinant as well as a possible prognostic tool and a therapeutic target in a plethora of human diseases has recently been considered. Among these, cancer represents a major one. In this review, we recapitulate the critical implications of autophagy in the pathogenesis, progression, and treatment of lymphoproliferative disorders. Leukemias and lymphomas, in fact, represent paradigmatic human diseases in which advances have recently been made in this respect.—Pierdominici, M., Barbati, C., Vomero, M., Locatelli, S. L., Carlo‐Stella, C., Ortona, E., Malorni, W. Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders. FASEB J. 28, 524–535 (2014). www.fasebj.org

Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes

T lymphocytes from patients with Systemic Lupus Erythematosus (SLE) display multiple abnormalities, including increased cell activation, abnormal cell death by apoptosis and impairment of autophagy pathway. In the present study we report the presence of specific antibodies to D4GDI, a small GTPase family inhibitor, in a significant percentage (46%) of SLE patient sera. We also found a significant association between the presence of these autoantibodies and hematologic manifestations occurring in these patients. Investigating the possible implication of anti-D4GDI autoantibodies in SLE pathogenesis or progression, we found that these antibodies were capable of binding D4GDI expressed at the lymphocyte surface and triggering a series of subcellular events, including Rho GTPase activation. These antibodies were also able to induce autophagy in T cells from both healthy donors and SLE patients, but only those negative to these antibodies. We can conclude that anti-D4GDI autoantibodies could be capable of triggering important responses in T cells such as cytoskeleton remodeling and autophagy pathway and that, in SLE patients, the chronic exposure to these specific autoantibodies could lead to the selection of autophagy-resistant T cell clones contributing to the pathogenesis of the disease.

The role of dietary sodium intake on the modulation of T helper 17 cells and regulatory T cells in patients with rheumatoid arthritis and systemic lupus erythematosus

We aimed at investigating whether the frequency and function of T helper 17 (Th17) and regulatory T cells (Treg) are affected by a restriction of dietary sodium intake in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We enrolled RA and SLE patients not receiving drugs known to increase urinary sodium excretion. Patients underwent a dietary regimen starting with a restricted daily sodium intake followed by a normal-sodium daily intake. The timepoints were identified at baseline (T0), after 3 weeks of low-sodium dietary regimen (T3), after 2 weeks of normal-sodium dietary regimen (T5). On these visits, we measured the 24-hour urinary sodium excretion, the frequency and function of Th17 and Treg cells in the peripheral blood, the serum levels of cytokines. Analysis of urinary sodium excretion confirmed adherence to the dietary regimen. In RA patients, a trend toward a reduction in the frequencies of Th17 cells over the low-sodium dietary regimen followed by an increase at T5 was observed, while Treg cells exhibited the opposite trend. SLE patients showed a progressive reduction in the percentage of Th17 cells that reached a significance at T5 compared to T0 (p = 0.01) and an increase in the percentage of Treg cells following the low-sodium dietary regimen at both T1 and T3 compared to T0 (p = 0.04 and p = 0.02, respectively). No significant apoptosis or proliferation modulation was found. In RA patients, we found a reduction at T5 compared to T0 in serum levels of both TGFβ (p = 0.0016) and IL-9 (p = 0.0007); serum IL-9 levels were also reduced in SLE patients at T5 with respect to T0 (p = 0.03). This is the first study investigating the effects of dietary sodium intake on adaptive immunity. Based on the results, we hypothesize that a restricted sodium dietary intake may dampen the inflammatory response in RA and SLE patients.

SAT0378 Autophagy is Up-Regulated in the Salivary Glands of Primary Sjogren's Syndrome Patients and Correlates with the Focus Score and Disease Activity

Background Autophagy is now considered as a major regulator in trafficking events that activates innate and adaptive immunity and consistent evidence supports its role in autoimmunity (1). Primary Sjogrens syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of exocrine glands by T and B cells that, producing chemokines and cytokines, coordinate the chronic inflammatory process. No data on the role of autophagy in pSS are available in humans, although studies in mice demonstrated its involvement in the salivary and lacrimal gland homeostasis (2,3). Objectives We investigated the autophagy process in salivary gland tissue and in peripheral T lymphocytes from pSS patients to evaluate its possible implication in the pathogenesis of the disease. Methods 30 patients with pSS, 20 patients with sicca syndrome or non-specific-chronic-sialoadenitis and 30 healthy donors were studied. Peripheral T lymphocytes were isolated by standard procedures. Salivary gland biopsies were evaluated by i) H&E to assess histological pattern, the severity of inflammatory infiltrate and the presence of germinal centers, ii) RT-PCR for the expression of autophagy-related genes and IL-23p19 and IL-21 mRNA. Autophagy-related proteins (LC3, Atg5, p62/SQSTM1) were detected in peripheral T lymphocytes by western blot and in salivary gland by immunohistochemistry and immunofluorescence. IL-21 and IL-23p19 serum levels were measured by ELISA. Results Autophagy is up-regulated in T cells from the salivary glands, but not from the peripheral blood, of pSS patients and it is correlated with disease activity and damage indexes. Autophagy is also correlated with the local expression of the pro-inflammatory cytokines IL-21 and IL-23p19, but not with serum levels of these cytokines. Conclusions Our data show that, in pSS, T cells present high levels of autophagy, which may up-regulate the expression of pro-inflammatory cytokines, providing evidence for a role of this process in the pathogenesis of pSS and identifying a possible therapeutic target. References Pierdominici M, Vomero M, Barbati C et al. FASEB J. 2012; 26: 1400-1412. Morgan-Bathke M, Lin HH, Chibly AM et al. J Dent Res. 2013; 92: 911-917. Seo Y, Ji YW, Lee SM, et al. Cell Death Dis. 2014; 5: e1309. Disclosure of Interest None declared

Autophagy and Rheumatoid Arthritis: Current Knowledges and Future Perspectives

Autophagy is a degradation mechanism by which cells recycle cytoplasmic components to generate energy. By influencing lymphocyte development, survival, and proliferation, autophagy regulates the immune responses against self and non-self antigens. Deregulation of autophagic pathway has recently been implicated in the pathogenesis of several autoimmune diseases, including rheumatoid arthritis (RA). Indeed, autophagy seems to be involved in the generation of citrullinated peptides, and also in apoptosis resistance in RA. In this review, we summarize the current knowledge on the role of autophagy in RA and discuss the possibility of a clinical application of autophagy modulation in this disease.

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.

Diesel exhaust particles induce autophagy and citrullination in Normal Human Bronchial Epithelial cells

A variety of environmental agents has been found to influence the development of autoimmune diseases; in particular, the studies investigating the potential association of systemic autoimmune rheumatic diseases with environmental micro and nano-particulate matter are very few and contradictory. In this study, the role of diesel exhaust particles (DEPs), one of the most important components of environment particulate matter, emitted from Euro 4 and Euro 5 engines in altering the Normal Human Bronchial Epithelial (NHBE) cell biological activity was evaluated. NHBE cells were exposed in vitro to Euro 4 and Euro 5 particle carbon core, sampled upstream of the typical emission after-treatment systems (diesel oxidation catalyst and diesel particulate filter), whose surfaces have been washed from well-assessed harmful species, as polycyclic aromatic hydrocarbons (PAHs) to: (1) investigate their specific capacity to affect cell viability (flow cytometry); (2) stimulate the production of the pro-inflammatory cytokine IL-18 (Enzyme-Linked ImmunoSorbent Assay -ELISA-); (3) verify their specific ability to induce autophagy and elicit protein citrullination and peptidyl arginine deiminase (PAD) activity (confocal laser scanning microscopy, immunoprecipitation, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis -SDS-PAGE- and Western blot, ELISA). In this study we demonstrated, for the first time, that both Euro 4 and Euro 5 carbon particles, deprived of PAHs possibly adsorbed on the soot surface, were able to: (1) significantly affect cell viability, inducing autophagy, apoptosis and necrosis; (2) stimulate the release of the pro-inflammatory cytokine IL-18; (3) elicit protein citrullination and PAD activity in NHBE cells. In particular, Euro 5 DEPs seem to have a more marked effect with respect to Euro 4 DEPs.

AB0112 Pathogenetic mechanisms in early rheumatoid arthritis: possible correlation between th17 and treg cells and gut microbiota structure: a pilot study

Background In Rheumatoid Arthritis (RA) pathogenesis T helper17 (Th17) and T regulatory cells (Treg) are largely represented.1 Recent studies highlighted the role of intestinal mucosa environment in modulation of T cells function. The composition of gut microbiota influences the Th17/Treg cells balance and the host immune response,2 so the exposure to deranged intestinal microbiota may be crucial in RA. Objectives The aim of the study was to compare Th17 and Treg cells and gut microbiota composition in patients with early RA (ERA) and in a control group (CG) at baseline and after treatment. Methods Currently, 10 ERA patients and 10 subjects belonging to the CG have been enrolled. All ERA patients were evaluated before (T0) and after 3 months (T1) of treatment with methotrexate (MTX) and glucocorticosteroids (GCS). Blood and faecal samples were collected. After PBMC isolation, staining with coniugated mAbs targeting specific surface and intracellular antigens (CD4 and CD25, IL-17 and FoxP3 respectively) have been used in order to distinguish Th17 and Treg cells. The composition of the faecal microbiota has been analysed by Next Generation Sequences on Illumina MiSeq platform, through 16S rDNA V3-V4 targeted sequencing. Results At T0, the percentage of Th17 cells was higher in patients than in the CG (p<0.0001) while Treg cells were higher in the CG (p=0.013). At T1, the total number of CD4 +and Th17 cells was decreased (p=0.007, p=0.027) while the frequency of Treg cells increased (p=0.028). A normalisation of Treg cells, with frequencies comparable to CG, was present after treatment. Regarding gut microbiota, at phylum level no difference between patients at T0 and the CG were present but we observed a tendency to decrease in the frequency of Actinobacteria after therapy. Furthermore, the relative abundance of Actinobacteria correlated positively with the circulating levels of Th17 (p=0,012, r=0,59) and with the Th17/Treg at T0 (p=0,010, r=0,6), while Nitrospirae correlated positively with Treg (p=0,028, r=0,68) at T1. A significant increase of the relative abundance in the Lachnospiraceae family in patients at T1 compared with T0 (p=0,042) and CG (p=0,043) were noticed. Conclusions Our results highlight the presence of an imbalance between Th17 and Treg cells in patients with ERA. In agreement with literature,3 MTX and GCS influence on Th17 decreasing and Treg cells increasing in patients with ERA, so we can hypothesise that part of the clinical response is own to the improvement in T cells balance. Previous data4 reported that Actinobacteria are strongly correlated with the production of IL-17 and a reduction of Nitrospirae has been associated to increased inflammatory responses and to gut permeability in mice.5 Lachnospiraceae family play an important role in the maintenance of intestinal homeostasis.6 The correlation between gut microbiota composition and Th17/Treg axis observed in our patients may suggest the involvement of some bacteria family in Th17/Treg cells balance in the lamina propria of RA patients treated with MTX, even in the early phases of the disease. References [1] Alunno, et al. 2015. [2] Omenetti, Pizarro. 2015. [3] Szalay, et al. 2013. [4] Chen, et al.2016. [5] Serino, et al. 2012. [6] Wu, et al. 2016. Disclosure of Interest None declared