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.

Effects of Selenium Supplementation on the Indices of Disease Activity, Inflammation and Oxidative Stress in Patients with Rheumatoid Arthritis: a Randomized Clinical Trial.

The aim of study was to evaluate the effect of selenium supplementation on disease activity, inflammation, and oxidative stress in patients with rheumatoid arthritis (RA). This study was a randomized double-blind placebo-controlled trial on 59 patients with RA. Participants were randomly divided to receive 200 µg/day of selenium or a placebo for 12 weeks. The disease activity score (DAS.CRP and DAS.ESR), erythrocyte sedimentation rate (ESR), serum levels of C-reactive protein (CRP), fasting blood glucose, lipids, antibodies to cyclic citrullinated protein (anti-CCP), nitric oxide, glutathione, and total antioxidant capacity were assessed. The mean of DAS.CRP and DAS.ESR decreased significantly within both study groups after the intervention. However, the between-group comparisons revealed no significant differences. The CRP levels decreased significantly in the selenium group, and this decrease was near the significance level compared to the placebo (P = 0.05). However, after adjusting for baseline values, the observed difference between groups did not remain significant. In addition, the values of ESR and anti-CCP decreased significantly within the selenium group. Although, between-group comparison did not statistically significant, the change in ESR and anti-CCP in the selenium group was small clinically relevant compared to the placebo [the effect size (95% CI) for ESR: 0.38 (- 0.14, 0.89), and for anti-CCP: 0.32 (- 0.2, 0.83)]. Our study showed that selenium caused a small clinically relevant improvement in some RA biomarkers such as ESR and anti-CCP. Future studies that evaluate the effects of novel forms of supplements such as selenium nanoparticles on the clinical symptoms and biomarkers of RA are suggested. Trial Registration: At www.irct.ir as IRCT20190924044869N1 on 2020-06-14.

Increased gene expression of CCR6 and RORγt in peripheral blood cells of rheumatoid arthritis patients and their correlation with anti-cyclic citrullinated peptide and disease activity.

OBJECTIVES: The significance of T helper 17 (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated in many studies. Retinoic acid receptor-related orphan receptor γt (RORγt) is a transcription factor that is specifically involved in the generation of Th17 cells. Besides, the chemokine receptor CCR6, the receptor for CCL20, is characteristically expressed by these cells. Considering the pivotal roles of Th17 cells in RA pathogenesis, in this study, we assessed the gene expression of CCR6 and RORγt in the peripheral blood leukocytes of new case RA patients. Also, we evaluated their association with anticyclic citrullinated peptide (anti-CCP) antibodies and disease activity. METHODS: Forty-five new case RA patients and 45 healthy persons have been recruited in this investigation. The gene expression of CCR6 and RORγt was evaluated by quantitative real-time PCR (qRT-PCR), and anti-CCP antibodies plasma levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Disease activity was measured according to the disease activity score-28 (DAS-28) formula. RESULTS: The gene expression of CCR6 and RORγt increased remarkably in new case RA patients compared to healthy controls (p < .05 and p < .01, respectively). Moreover, there was a positive correlation between RORγt gene expression and parameters, including gene expression of CCR6 (p = .001, r = .461), plasma levels of CCL20 (p = .0009, r = .477), ESR (p = .004, r = .419), DAS-28 (p = .006, r = .402), anti-CCP (p = .019, r = .346), and RF (p = .001, r = .451). Also, CCR6 gene expression was positively associated with the DAS-28 (p = .037, r = .310), plasma levels of anti-CCP (p = .037, r = .312), and ESR (p = .029, r = .327). CONCLUSION: Increased gene expression of CCR6 and RORγt in peripheral blood leukocytes of new case RA patients may contribute to the exacerbation and pathogenesis of RA.

Using Treg, Tr1, and Breg Expression Levels to Predict Clinical Responses to csDMARD Treatment in Drug-naive Patients With Rheumatoid Arthritis.

BACKGROUND/AIM: Regulatory T cells (Treg) play a crucial role in maintaining immune tolerance and preventing autoimmune diseases. Recent data also indicate that type 1 regulatory T (Tr1) and regulatory B (Breg) cells play an inhibitory (i.e., protective) role in autoimmune diseases. Conventional synthetic disease-modifying antirheumatic drugs (csDMARD) are a first-line therapy for rheumatoid arthritis (RA), and our aim was to predict clinical responses of this treatment using immunophenotyping. MATERIALS AND METHODS: We first detected the presence of immune cells in fresh blood from 16 healthy controls (HC) and 26 patients with RA (14 drug-naive and 12 csDMARD-experienced). Then, we recorded immunophenotypic changes in 14 drug-naive RA (naive RA) patients prior to csDMARD treatment (i.e., day 0) and after receiving treatment for 6 months. The observed changes were also compared with other clinical indicators, including the presence of anti-citrullinated peptide antibodies (anti-CCP), rheumatoid factor (RF) levels, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. RESULTS: Naive RA patients had significantly lower Tregs than HC and csDMARD-experienced patients (both p<0.0001) and the number of Tregs correlated with the diagnosis of RA and therapeutic efficacy of csDMARD treatment. Furthermore, lower baseline levels of Treg, memory Treg, Tr1, and higher PD-1+ Marginal B, Breg cells were significantly associated with decreased development of the 28-joint Disease Activity Score (DAS28) (all p<0.05), revealing better medical response. Multiple regression and principal component analysis identified Treg, Tr1, and Breg as potential predictors of csDMARD responses (Area under curve: 0.9; Accuracy: 92.86%). Furthermore, elevated Treg, Tr1, and Breg cells were associated with decreased DAS28, ESR, and CRP (all p<0.05); changes in Treg and Breg cell expression were also more pronounced among double negative anti-CCP and RF in RA patients with better outcomes (p<0.05). CONCLUSION: Immunophenotyping can be an adjunct clinical tool to identify patients who are poor candidates for csDMARD therapy. Alternative therapeutic interventions in the early stages of disease should be formulated for these patients.

Porphyromonas gulae and PPAD antibodies are not related to citrullination in rheumatoid arthritis.

INTRODUCTION: Porphyromonas gulae have the enzyme PPAD, as P. gingivalis, which is responsible for citrullination related to the pathophysiology of rheumatoid arthritis and periodontitis; this implies the presence of two species of PPAD-producing bacteria in the mouth as well as the presence of citrullinated proteins. There are no previous reports or studies investigating an association between P. gulae PPAD in rheumatoid arthritis (RA). OBJECTIVE: To assess the presence of P. gulae and anti-citrullinated peptide antibodies of P. gulae PAD in patients with RA and their possible relationship with clinical activity markers. SUBJECTS AND METHODS: A total of 95 patients with RA and 95 controls were included. Erythrocyte sedimentation rate (ESR), C-reactive protein, anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) were measured. Activity index-28 (DAS28) and SCDAI. The periodontal diagnosis was established. Presence of P. gulae and P. gingivalis. An ELISA was used to determine antibodies against citrullinated peptides of P. gulae PAD. RESULTS: A P. gulae frequency of 15.8% was observed in the RA group and 9.5% in the control group. Higher levels of ACPA were found in the P. gulae-positive patients of the RA group, finding no significant difference, but if in patients positive for P. gingivalis with statistical significance (p = 0.0001). The frequency of anti-VDK-cit and anti-LPQ-cit9 antibodies to PPAD of P. gulae was higher in the RA group than in the control group without significant difference. No relationship was found with the clinical variables despite the presence of P. gulae and anti-citrullinated peptide antibodies of P. gulae PPAD in patients with RA CONCLUSIONS: It was not possible to establish a connection with clinical variables in RA and P. gulae; as a result, the presence of P. gingivalis continues to contribute significantly to the increase in antibodies against citrullinated proteins/peptides from exogenous sources of citrullination in RA and periodontitis.

Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens: Clonal Diversity and Inter-Individual Variation.

BACKGROUND: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. METHODS: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. RESULTS: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. CONCLUSIONS: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.

Identification of three members of the multidomain adhesion CCp family in Babesia gibsoni.

Babesia gibsoni is an intraerythrocytic apicomplexan parasite transmitted by Haemaphysalis longicornis and causes canine babesiosis. Within the tick, the Babesia parasite undergoes sexual conjugation and the sporogony process of its life cycle. To control B. gibsoni infection, prompt and effective treatment of acute infections and curing chronic carriers are urgently needed. Gene disruption of Plasmodium CCps resulted in blocking the transition of sporozoites from the mosquito midgut to the salivary glands, showing that these proteins are potential targets for the development of a transmission-blocking vaccine. In this study, we described the identification and characterization of three members of the CCp family in B. gibsoni, named CCp1, CCp2, and CCp3. The B. gibsoni sexual stages were induced in vitro by exposing parasites to xanthurenic acid (XA), dithiothreitol (DTT), and tris (2-carboxyethyl) phosphine (TCEP) at serial concentrations. Among them, 100 µM XA-exposed and cultured at 27 °C without CO2B. gibsoni presented diverse morphologies, including parasites with long projections, gradually increased free merozoites, and aggregated and round forms, indicative of sexual stage induction. Then, the expression of CCp proteins of induced parasites was confirmed by real-time reverse transcription PCR, immunofluorescence, and western blot. The results showed that BgCCp genes were highly significantly increased at 24 h post-sexual stage induction (p < 0.01). The induced parasites were recognized by anti-CCp mouse antisera and anti-CCp 1, 2, and 3 antibodies weakly reacted with sexual stage proteins of expected molecular weights of 179.4, 169.8, and 140.0 KDa, respectively. Our observations on morphological changes and confirmation of sexual stage protein expression will advance elemental biological research and lay the foundation for the development of transmission-blocking vaccines against canine babesiosis.

Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

AIM: The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. METHODOLOGY: Twenty-five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. RESULTS: Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. CONCLUSIONS: This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Expression of LINC00638 in rheumatoid arthritis patients with damp-heat obstruction syndrome and the regulatory mechanisms for inflammation and oxidative stress. / ç±»é£Žæ¹¿å ³èŠ‚ç‚Žæ¹¿çƒ­ç—¹é˜»è¯æ‚£è€ LINC00638çš„è¡¨è¾¾åŠå ¶è°ƒæŽ§ç‚Žç—‡å’Œæ°§åŒ–åº”æ¿€çš„æœºåˆ¶.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Both inflammatory response and oxidative stress contribute to the pathogenesis of RA. Oxidative damage can induce and aggravate the imbalance of immune inflammation and promote cell and tissue damage. In this study, the expression of long non-coding RNA (lncRNA) LINC00638 in peripheral blood of patients with RA damp-heat arthralgia syndrome was observed, and the correlation between LINC00638 and disease activity, immune inflammation and oxidative stress indicator was investigated. Subsequently, the mechanisms for LINC00638 in regulating the inflammatory response and oxidative stress in RA fibroblast-like synoviocyte (FLS) under the condition of overexpression and interference were further explored. METHODS: In this study, 48 RA patients with damp-heat arthralgia syndrome and 27 normal healthy subjects, who came from Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, were included; and they were divided into a RA group and a control group. The expression of LINC00638 in peripheral blood mononuclear cells (PBMC) from the subjects was detected by real-time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect serum interleukin (IL)-10, IL-17, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2) expression. Spearman method was used to study the relationship between LINC00638 and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (anti-CCP), and to observe the relation between LINC00638 and the Disease Activity Score of 28 Joint (DAS28), Quantitative Score of Damp Heat Syndrome, Visual Analogue Scale (VAS), Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). RA-FLS was induced by RA-PBMC, and the RA in vitro cell experimental model was established. LINC00638 overexpression plasmid and small interfering RNA (siRNA) were constructed and transfected into RA-FLS. The cell experiments were divided into 4 groups: a pcDNA3. 1- control group, a pcDNA3.1-LINC00638 group, a siRNA-control group, and a siRNA-LINC00638 group. The transfection efficiency of overexpression plasmid and siRNA was detected by real-time PCR, the expression of TNF-α and IL-10 was detected by ELISA, and the expression of antioxidant proteins HO-1 and SOD2 was detected by immunofluorescence. RESULTS: Compared with the control group, the expression of LINC00638 in the RA group was lower (P<0.01). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve of LINC00638 was 0.9271. The DAS28 in RA group was 5.70 (5.40-6.50), the Quantitative Score of Damp-heat Syndrome was 20.0 (17.0-23.0), and the VAS score was 7.0 (6.3-8.0). Compared with the control group, the ESR, CRP, RF, anti-CCP, SAS and SDS scores in the RA group were significantly increased (all P<0.01). Spearman correlation analysis showed that: LINC00638 was negatively correlated with ESR (r=-0.532, P<0.01), CRP (r=-0.367, P<0.05), TNF-α (r=-0.375, P<0.01), MDA (r= -0.295, P<0.05), DAS28 (r=-0.450, P<0.01), and which was positively correlated with SOD2 (r=0.370, P<0.05). After the induction of RA-FLS, the expression level of LINC00638 was significantly decreased (P<0.01), indicating that the stimulation of PBMC could effectively reduce the expression of LINC00638 in RA-FLS, so the experimental model of RA-FLS-induced by PBMC was utilized. Compared with the pcDNA3.1-control group, the expressions of LINC00638, IL-10, SOD2, and HO-1 in the pcDNA3.1-LINC00638 group were significantly increased (all P<0.01), and the expression of TNF-α was decreased (P<0.01). Compared with siRNA-control group, LINC00638, IL-10, SOD2 and HO-1 in the siRNA-LINC00638 group were significantly decreased (all P<0.01), and TNF-α was significantly increased (P<0.01). CONCLUSIONS: LINC00638 is down-regulated in the peripheral blood of RA patients with damp-heat arthralgia syndrome, which is correlated with disease activity, immune inflammation and oxidative stress. Overexpression of LINC00638 can down-regulate pro-inflammatory factors, up-regulate anti-inflammatory factors, and increase antioxidant enzyme activity, thereby improving inflammation and oxidative stress in RA. LINC00638 is the differential lncRNA obtained by the research group's previous high-throughput sequencing of the whole transcriptome of peripheral blood PBMCs in RA patients and validation of clinical samples. In order to deepen the molecular biology research of this gene, the microRNA and mRNA targeted by LINC00638 can be further studied from the perspective of competing endogenous RNAs.

Identification of RRM2 in peripheral blood mononuclear cells as a novel biomarker for the diagnosis of rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is a common autoimmune disease. However, the positive diagnosis value of the current biomarkers is unsatisfactory. Here, we aimed to identify RA-associated susceptibility genes and explore their potential as novel biomarkers for the diagnosis of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from healthy controls and RA patients. RNA-seq and bioinformatics analyses were performed to identify the hub genes associated with RA. Then, the expression of hub genes was assessed in mRNA expression profiles from GEO datasets. Real time-quantitative PCR (RT-qPCR) was performed to further confirm the expression of the hub genes using the PBMCs that were collected from RA patients (n=47) and healthy controls (n=40). Finally, we evaluated the diagnostic potential of the candidate mRNAs. RESULTS: RNA-seq analyses revealed 178 dysregulated genes measured by changes in mRNAs between the healthy controls and the RA patients. We identified 3 candidate mRNAs, including ASPM, DTL and RRM2, all of which were highly expressed in RA. RRM2 showed a significant higher expression in remissive RA compared with active RA. Significant correlations were observed between DTL and IL-8, TNF-α which were tested in serum by ELISA, between RRM2 and CDAI, DAS-28, tender and swollen joints, respectively. The expression level of RRM2 was significantly higher in RA patients with the Anti-CCP- than with the Anti-CCP+. The AUC (RA vs. OA) value of RRM2 was 0.941 (p<0.0001; sensitivity=0.867; specificity=0.904). CONCLUSIONS: RRM2 showed high diagnosis efficiency for RA patients. Therefore, the findings provided a novel candidate biomarker for the diagnosis of RA.

Peptidylarginine Deiminase 2 in Murine Antiviral and Autoimmune Antibody Responses.

The peptidylarginine deiminases (PADs) and the citrullinated proteins that they generate have key roles in innate immunity and rheumatoid arthritis, an inflammatory arthritis with antibodies that target citrullinated proteins. However, the importance of PADs, particularly PAD2, in the adaptive immune response, both normal and pathogenic, is newly emerging. In this study, we evaluated a requirement for PAD2 in the antibody response in collagen-induced arthritis (CIA), a T and B cell-driven murine model of rheumatoid arthritis, and in the protective antibody response to murine influenza infection. Using PAD2-/- and PAD2+/+ mice on the DBA/1J background, we found that PAD2 is required for maximal anti-collagen antibody levels, but not collagen-specific plasma cell numbers, T cell activation or polarization, or arthritis severity in CIA. Also, we found that PAD2 is required not just for normal levels of persistent hemagglutination inhibiting antibodies but also for full protection from lethal influenza rechallenge. Together, these data provide evidence for a novel modest requirement for PAD2 in a normal antiviral antibody response and in an abnormal autoantibody response in inflammatory arthritis.

The current status of anti-citrullinated protein antibodies and citrullinated protein-reactive B cells in the pathogenesis of rheumatoid arthritis.

Anti-citrullinated protein antibodies are a hallmark of rheumatoid arthritis. It is widely acknowledged that the presence of ACPAs is the result of the interaction of genes, the environment and epigenetic modifications. The mechanism by which the factors, especially citrullination and ACPA glycosylation, affect ACPAs is still unclear. In this article, we review the presence of the ACPAs in RA and their relationship with clinical manifestations. The pathogenicity of ACPAs and B cells in RA was also summarized. A growing body of evidence has shown that ACPA-positive patients have more serious bone erosion and destruction and poor clinical prognosis than ACPA-negative patients. Recently, with the direct study of citrullinated protein-reactive B cells, their role in the development of rheumatoid arthritis has been further understood. It indicates that further understanding of the mechanism of ACPAs and CP-reactive B cells would beneficial in the prevention and treatment of RA.

Expression of LINC00638 in rheumatoid arthritis patients with damp-heat obstruction syndrome and the regulatory mechanisms for inflammation and oxidative stress / 中南大学学报(医学版)

OBJECTIVES@#Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Both inflammatory response and oxidative stress contribute to the pathogenesis of RA. Oxidative damage can induce and aggravate the imbalance of immune inflammation and promote cell and tissue damage. In this study, the expression of long non-coding RNA (lncRNA) LINC00638 in peripheral blood of patients with RA damp-heat arthralgia syndrome was observed, and the correlation between LINC00638 and disease activity, immune inflammation and oxidative stress indicator was investigated. Subsequently, the mechanisms for LINC00638 in regulating the inflammatory response and oxidative stress in RA fibroblast-like synoviocyte (FLS) under the condition of overexpression and interference were further explored.@*METHODS@#In this study, 48 RA patients with damp-heat arthralgia syndrome and 27 normal healthy subjects, who came from Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, were included; and they were divided into a RA group and a control group. The expression of LINC00638 in peripheral blood mononuclear cells (PBMC) from the subjects was detected by real-time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect serum interleukin (IL)-10, IL-17, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2) expression. Spearman method was used to study the relationship between LINC00638 and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (anti-CCP), and to observe the relation between LINC00638 and the Disease Activity Score of 28 Joint (DAS28), Quantitative Score of Damp Heat Syndrome, Visual Analogue Scale (VAS), Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). RA-FLS was induced by RA-PBMC, and the RA in vitro cell experimental model was established. LINC00638 overexpression plasmid and small interfering RNA (siRNA) were constructed and transfected into RA-FLS. The cell experiments were divided into 4 groups: a pcDNA3. 1- control group, a pcDNA3.1-LINC00638 group, a siRNA-control group, and a siRNA-LINC00638 group. The transfection efficiency of overexpression plasmid and siRNA was detected by real-time PCR, the expression of TNF-α and IL-10 was detected by ELISA, and the expression of antioxidant proteins HO-1 and SOD2 was detected by immunofluorescence.@*RESULTS@#Compared with the control group, the expression of LINC00638 in the RA group was lower (P<0.01). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve of LINC00638 was 0.9271. The DAS28 in RA group was 5.70 (5.40-6.50), the Quantitative Score of Damp-heat Syndrome was 20.0 (17.0-23.0), and the VAS score was 7.0 (6.3-8.0). Compared with the control group, the ESR, CRP, RF, anti-CCP, SAS and SDS scores in the RA group were significantly increased (all P<0.01). Spearman correlation analysis showed that: LINC00638 was negatively correlated with ESR (r=-0.532, P<0.01), CRP (r=-0.367, P<0.05), TNF-α (r=-0.375, P<0.01), MDA (r= -0.295, P<0.05), DAS28 (r=-0.450, P<0.01), and which was positively correlated with SOD2 (r=0.370, P<0.05). After the induction of RA-FLS, the expression level of LINC00638 was significantly decreased (P<0.01), indicating that the stimulation of PBMC could effectively reduce the expression of LINC00638 in RA-FLS, so the experimental model of RA-FLS-induced by PBMC was utilized. Compared with the pcDNA3.1-control group, the expressions of LINC00638, IL-10, SOD2, and HO-1 in the pcDNA3.1-LINC00638 group were significantly increased (all P<0.01), and the expression of TNF-α was decreased (P<0.01). Compared with siRNA-control group, LINC00638, IL-10, SOD2 and HO-1 in the siRNA-LINC00638 group were significantly decreased (all P<0.01), and TNF-α was significantly increased (P<0.01).@*CONCLUSIONS@#LINC00638 is down-regulated in the peripheral blood of RA patients with damp-heat arthralgia syndrome, which is correlated with disease activity, immune inflammation and oxidative stress. Overexpression of LINC00638 can down-regulate pro-inflammatory factors, up-regulate anti-inflammatory factors, and increase antioxidant enzyme activity, thereby improving inflammation and oxidative stress in RA. LINC00638 is the differential lncRNA obtained by the research group's previous high-throughput sequencing of the whole transcriptome of peripheral blood PBMCs in RA patients and validation of clinical samples. In order to deepen the molecular biology research of this gene, the microRNA and mRNA targeted by LINC00638 can be further studied from the perspective of competing endogenous RNAs.

Association between inflammatory cytokines and immune-checkpoint molecule in rheumatoid arthritis.

BACKGROUND: Anti-citrullinated peptide antibodies (ACPA) and inflammatory cytokines play important roles in the development of rheumatoid arthritis (RA). T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is an immune-checkpoint molecule involved in inhibitory signaling. Galectin-9 (Gal-9) mediated ligation of TIM-3 induces the amelioration of autoimmune diseases. TIM-3 is expressed in synovial osteoclasts and involved in the rheumatoid bone destruction. The aim of this study was to investigate the relationships between inflammatory cytokines and immune-checkpoint molecules in RA patients. METHODS: Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), soluble TIM-3 (sTIM-3) and Gal-9 were determined by ELISA. Patients were stratified into two groups based on ACPA titers: low-medium ACPA (ACPA <200 U/mL) and high ACPA (ACPA ≥200 U/mL). Serum levels of cytokines or immune-checkpoint molecules were evaluated between RA patients with low-medium ACPA titers and high ACPA titers. RESULTS: Elevated serum levels of inflammatory cytokines were correlated with DAS28-ESR in RA patients. Although serum levels of sTIM-3 were elevated in RA patients, significant correlations between sTIM-3 and cytokines (IL-6 or TNF-α) were observed exclusively in RA patients with low-medium ACPA titers (<200 U/mL). Serum levels of IL-6 and TNF-α levels were significantly correlated with elevated Gal-9 levels regardless of ACPA status. A significant correlation between IL-6 and Gal-9 was observed in RA patients without advanced joint damage. Conversely, a significant correlation between TNF-α and Gal-9 was observed in RA patients with advanced joint damage. CONCLUSIONS: Our data indicated that there are positive correlations between circulating inflammatory cytokines and checkpoint molecules in RA patients and these interactions can be modulated by ACPA status or joint damage stage.

Anti-Carbamylated Fibrinogen Antibodies Might Be Associated With a Specific Rheumatoid Phenotype and Include a Subset Recognizing In Vivo Epitopes of Its γ Chain One of Which Is Not Cross Reactive With Anti-Citrullinated Protein Antibodies.

To identify the targets recognized by anti-carbamylated protein antibodies (anti-CarP) in patients with early Rheumatoid Arthritis (RA), to study the cross-reactivity between anti-CarP and anti-citrullinated protein antibodies (ACPA) and to evaluate their prognostic value. 331 patients (184 RA and 147 other rheumatisms) from the Very Early Arthritis (VErA) French cohort were analyzed. We performed mass spectrometry analysis of RA sera displaying anti-CarP activity and epitope mapping of the carbamylated fibrinogen γ chain to identify immunodominant peptides. The specificity of these targets was studied using competition assays with the major antigens recognized by ACPA. The prognostic value of anti-carbamylated fibrinogen IgG antibodies (ACa-Fib IgG) was compared to that of anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-CarP using an in-house ELISA. Besides the α chain, the γ chain of fibrinogen, particularly one immunodominant epitope that has a specific reactivity, was identified as a circulating carbamylated target in sera. The prevalence of ACa-Fib was 37% at baseline and 10.9% for anti-CCP-negative RA. In anti-CCP-negative patients, ACa-Fib positivity was associated with a more inflammatory and erosive disease at baseline but not with rapid radiological progression, which remains strongly related to anti-CCP antibodies. Fibrinogen seems to be one of the antigens recognized in vivo by the anti-CarP response, particularly 2 epitopes of the γ chain, one of which is not cross reactive with ACPA. This specificity might be associated with a distinct clinical phenotype since ACa-Fib IgG were shown to be linked to systemic inflammation in very early RA but not to rapid radiological progression.

Single-cell sequencing of immune cells from anticitrullinated peptide antibody positive and negative rheumatoid arthritis.

The presence or absence of anti-citrullinated peptide antibodies (ACPA) and associated disparities in patients with rheumatoid arthritis (RA) implies disease heterogeneity with unknown diverse immunopathological mechanisms. Here we profile CD45+ hematopoietic cells from peripheral blood or synovial tissues from both ACPA+ and ACPA- RA patients by single-cell RNA sequencing and identify subsets of immune cells that contribute to the pathogenesis of RA subtypes. We find several synovial immune cell abnormalities, including up-regulation of CCL13, CCL18 and MMP3 in myeloid cell subsets of ACPA- RA compared with ACPA+ RA. Also evident is a lack of HLA-DRB5 expression and lower expression of cytotoxic and exhaustion related genes in the synovial tissues of patients with ACPA- RA. Furthermore, the HLA-DR15 haplotype (DRB1/DRB5) conveys an increased risk of developing active disease in ACPA+ RA in a large cohort of patients with treatment-naive RA. Immunohistochemical staining shows increased infiltration of CCL13 and CCL18-expressing immune cells in synovial tissues of ACPA- RA. Collectively, our data provide evidence of the differential involvement of cellular and molecular pathways involved in the pathogenesis of seropositive and seronegative RA subtypes and reveal the importance of precision therapy based on ACPA status.

Rheumatoid arthritis serotype and synthetic disease-modifying anti-rheumatic drugs in patients with periodontitis: A case-control study.

Patients with rheumatoid arthritis (RA) experience a higher prevalence of periodontitis. This study aimed to examine the variation of periodontitis experienced with different serotypes suffered by RA patients and to examine the relationship between the different medications taken for RA that may influence this relationship. Two hundred and sixty RA and control participants underwent standardized periodontal examinations. Medical, serological and radiological (Sharp/van der Heijde) records were assessed. Functional status was assessed using the administered Health Assessment Questionnaire. Moreover, disease parameters, including disease activity (DAS28-ESR) and anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) seropositivity were evaluated. Periodontitis was higher in RA (71.54%) compared with controls (54.62%). The stage of periodontitis experienced by ACPA-positive participants were higher than APCA-negative participants. The probing pocket depth and recession experienced by RF-positive participants were higher than those who were RF-negative. RA participants on methotrexate had lower clinical attachment loss and lower periodontal probing depth compared with participants on a combination methotrexate and other disease-modifying antirheumatic drugs. Participants taking corticosteroids had lower gingival index scores. The association between seropositivity and the type of medications taken with periodontal health parameters in this group of patients suggests that both seropositivity and medications taken are important modifiers in the relationship between periodontitis and RA.

Understanding interactions between risk factors, and assessing the utility of the additive and multiplicative models through simulations.

Understanding the genetic background of complex diseases requires the expansion of studies beyond univariate associations. Therefore, it is important to use interaction assessments of risk factors in order to discover whether, and how genetic risk variants act together on disease development. The principle of interaction analysis is to explore the magnitude of the combined effect of risk factors on disease causation. In this study, we use simulations to investigate different scenarios of causation to show how the magnitude of the effect of two risk factors interact. We mainly focus on the two most commonly used interaction models, the additive and multiplicative risk scales, since there is often confusion regarding their use and interpretation. Our results show that the combined effect is multiplicative when two risk factors are involved in the same chain of events, an interaction called synergism. Synergism is often described as a deviation from additivity, which is a broader term. Our results also confirm that it is often relevant to estimate additive effect relationships, because they correspond to independent risk factors at low disease prevalence. Importantly, we evaluate the threshold of more than two required risk factors for disease causation, called the multifactorial threshold model. We found a simple mathematical relationship (square root) between the threshold and an additive-to-multiplicative linear effect scale (AMLES), where 0 corresponds to an additive effect and 1 to a multiplicative. We propose AMLES as a metric that could be used to test different effects relationships at the same time, given that it can simultaneously reveal additive, multiplicative and intermediate risk effects relationships. Finally, the utility of our simulation study was demonstrated using real data by analyzing and interpreting gene-gene interaction odds ratios from a rheumatoid arthritis case-control cohort.

Current insights and future prospects for the pathogenesis and treatment for rheumatoid arthritis.

Genetic, environmental, and epigenetic factors simultaneously or serially contribute to immune cells and resident joint tissue cell abnormalities, invariably leading to joint destruction. Understanding the immune cell dysfunction in earlier years has brought forward life-changing therapeutics to patients with rheumatoid arthritis (RA). Further advances in the understanding of the immune and joint tissue-resident cell signaling and metabolic defects should produce additional tools to treat people with RA and foretell those who will respond to each biological or small drug. This review presents the latest evidence on RA pathogenesis and outlines the prospects for achieving precision medicine.

Increased expression of CXCL2 in ACPA-positive rheumatoid arthritis and its role in osteoclastogenesis.

Anti-citrullinated protein/peptide antibodies (ACPA) play important roles in the pathogenesis of rheumatoid arthritis (RA). ACPA-positive (ACPA+ ) and ACPA-negative (ACPA- ) RA were suggested to be different disease subsets, with distinct differences in genetic variation and clinical outcomes. The aims of the present study were to compare gene expression profiles in ACPA+ and ACPA- RA, and to identify novel candidate gene signatures that might serve as therapeutic targets. Comprehensive transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from ACPA+ and ACPA- RA patients and healthy controls was performed via RNA sequencing. A validation cohort was used to further investigate differentially expressed genes via polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Spearman's correlation test was used to evaluate the correlation of differentially expressed genes and the clinical and laboratory data of the patients. The role of differentially expressed genes in osteoclastogenesis was further investigated. Expression of C-X-C motif chemokine ligand 2 (CXCL2) was significantly increased in ACPA+ RA than in ACPA- RA, which was validated in PBMCs and serum. CXCL2 promoted the migration of CD14+ monocytes and increased osteoclastogenesis in RA patients. RAW264.7 macrophages were used to investigate specific mechanisms, and the results suggested that CXCL2 stimulated osteoclastogenesis via extracellular receptor kinase (ERK) mitogen-activated protein kinase (MAPK) and nuclear factor kappa B pathways. In conclusion, CXCL2 was highly expressed in ACPA+ RA than in ACPA- RA. CXCL2 promoted osteoclastogenesis and was related to bone erosion in RA, which suggests that the blockade of CXCL2 might be a novel strategy for the treatment of RA.