Protein Citrullination by Peptidyl Arginine Deiminase/Arginine Deiminase Homologs in Members of the Human Microbiota and Its Recognition by Anti-Citrullinated Protein Antibodies.

The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100-200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case-control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal-Wallis test and Mann-Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis.

suPAR and WT1 modify the adhesion of podocytes and are related to proteinuria in class IV lupus nephritis.

Introduction: Lupus nephritis (LN) affects up to 60 % of the patients with Systemic Lupus Erythematosus (SLE) and renal damage progression is associated with proteinuria, caused in part by the integrity of the glomerular basement membrane (GBM) and by podocyte injury. The soluble urokinase plasminogen activator receptor (suPAR) and Wilms Tumor 1 (WT1) have been related to podocyte effacement and consequently with proteinuria which raises questions about its pathogenic role in LN. Objective: Define whether suPAR levels and WT1 expression influence in podocyte anchorage destabilization in LN class IV. Materials and methods: This is a cross-sectional study of cases and controls. We studied patients with SLE without renal involvement (n = 12), SLE and LN class IV with proteinuria ≤0.5 g/24 h (n = 12), LN class IV with proteinuria ≥0.5 g/24 h (n = 12) and compared them with renal tissue control (CR) (n = 12) and control sera (CS) (n = 12). The CR was integrated by cadaveric samples without SLE or renal involvement and the CS was integrated by healthy participants. The expression and cellular localization of WT1, urokinase-type plasminogen activator receptor (uPAR), ac-α-tubulin, vimentin, and ß3-integrin was assessed by immunohistochemistry (IHC). The concentration of suPAR in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). Results: In patients with LN, the activation of anchoring proteins was increased, such as podocyte ß3-integrin, as well as the acetylation of alpha-acetyl-tubulin and uPAR, in contrast to the decrease in vimentin; interestingly, the cellular localization of WT1 was cytoplasmic and the number of podocytes per glomerulus decreased. The concentrations of suPAR was increased in patients with LN. Conclusion: The destabilization of podocyte anchorage modulated by ß3-integrin activation, and tubulin acetylation, associated with decreased WT1 cytoplasmic expression, and increased suPAR levels could be involved in kidney damage in patients with LN class IV.

Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins.

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.

Apoptosis and cell proliferation: the paradox of salivary glands in Sjögren's disease.

AIM: To assess apoptosis and proliferation in salivary glands of patients with primary Sjögren's syndrome. METHODS: Studies were performed in twenty four minor salivary glands from patients with primary Sjögren's syndrome and an equal number of controls. Apoptosis was studied by immunohistochemistry using monoclonal antibodies anti-Fas FasL and Caspase 3 and apoptotic features by TUNEL. Proliferation was assessed with monoclonal anti-PCNA and anti-Ki67 antibodies. RESULTS: All salivary glands from Sjögren's display apoptotic molecules along the epithelia of salivary ducts and in a smaller amount in acinar tissue. The presence of Caspase 3 Fas FasL was concordant with the expression of apoptosis by TUNEL. Proliferation markers were encountered in inflammatory emigrant cells but not in ductal epithelia nor in acini. Control biopsies poorly expressed apoptotic or proliferation markers. CONCLUSION: Present data suggests that the ductal epithelial and acinar cells of salivary glands from Sjögren's disease patients exhibit increased apoptosis. Proliferation was mainly observed in infiltrating lymphoid cells. Both events constitute a biological paradox related to the inflammatory process of salivary glands in Sjögren's disease.

Apoptosis and redistribution of the Ro autoantigen in Balb/c mouse like in subacute cutaneous lupus erythematosus.

In subacute cutaneous lupus eryhematosus (SCLE) the cutaneous antigens constitute the main source of Ro and La autoantigens. The aim of this investigation was to demonstrate if UV light increases the availability of Ro autoantigen in the skin, also the blocking effect of Ac-DEVD-CMK a caspase inhibitor was assessed. For this purpose newborn Balb/c mice were UVB irradiated (5-30 mJ/cm(2)) equivalent to a moderate to severe sunburn. Animals were injected with monoclonal anti-Ro antibodies from SCLE patients. Apoptosis was also induced by anti-Fas antibody injection. Skin samples were examined by direct immunofluoresence, by TUNEL, and the expression of caspase 3 by RT-PCR. Major findings of present studies were: 1. UVB irradiation and anti-Fas induced apoptosis of keratinocytes. 2. Apoptosis redistribute the Ro antigen on cell surface and is better triggered by Ro antibody. 3. The caspase 3 inhibitor Ac-DEVD-CMK decreases the availability of Ro autoantigen in epidermis and prevents deposition of anti-Ro. In conclusion, the caspase pathway would be blocked to avoid anti-Ro deposition along skin; this finding would be a prospect in the treatment of SCLE patients.

Camptothecin induces the transit of FasL trimers to the cell surface in apoptotic HEp-2 cells.

Fas ligand (L) is a membrane protein from the tumor necrosis factor (TNF) family. It induces apoptosis upon contact with its Fas/CD95/APO1 receptor. Trimerization of FasL on the surface of effector cells is essential in the binding of the Fas trimer of the target cells. The receptor then recruits an adaptor and caspase-like proteins which lead apoptosis. This paper reports on the fate of FasL in HEp-2 cells committed to apoptosis by induction with campthotecin. Our main results demonstrated that in non-apoptotic cells, FasL aggregates in the cytoplasm forming trimers of 120 kDa. Apoptosis increases the trimeric FasL species, but also induces its dissociation into monomers of 35 kDa. In conclusion, camptothecin appears to perturb the Fas and FasL segregation in the cytoplasm by promoting the transit of FasL to the cell surface, thus fostering a process of autocrine or paracrine apoptosis. FasL is trimerized prior to Fas/FasL complex formation, and after apoptosis, FasL undergoes an intense turnover.