m6A RNA methylation controls salivary gland epithelial cell function and has a protective role in Sjögren's disease.

OBJECTIVES: The RNA epitranscriptomic modification known as N6-methyladenosine (m6A) represents a novel mechanism of gene regulation that is poorly understood in human autoimmune diseases. Our research explores the role of this RNA m6A modification in salivary gland epithelial cells (SGEC) and its impact on the pathogenesis of Sjögren's disease (SjD). METHODS: SGECs from SjD patients and controls were analysed for m6A writers METTL3 and METTL14 expression using RNA-seq, quantitative PCR and immunohistochemistry. Functional assays assessed the impact of METTL3 knockdown or pharmacological inhibition on proinflammatory gene expression and immune cell interactions (using transwell and coculture systems). Mechanistic studies examined METTL3-mediated m6A modifications in double-stranded RNA (dsRNA) formation through immunofluorescence. Unsupervised clustering identified patterns of interferon activation in salivary glands and their correlation with m6A writers. RESULTS: METTL3 and METTL14 were elevated in SGEC from SjD patients in comparison to controls. Paradoxically, inhibiting METTL3 increased proinflammatory gene expression, enhancing SGEC's ability to attract immune cells and activate B cells. Conversely, inhibiting the eraser FTO had the opposite effect. METTL3-mediated m6A modifications prevented dsRNA formation and IFN signalling activation. SGEC from SjD showed insufficient METTL3 upregulation compared with controls in response to inflammatory triggers, indicating a limited capacity to regulate the inflammatory response. SjD patients with elevated disease activity and higher interferon signature exhibit reduced METTL3 expression. CONCLUSIONS: Impairment of m6A modifications in SGEC in response to inflammatory triggers favour the formation of dsRNA, potentially amplifying the interferon loop and contributing to SjD pathogenesis.

Hyperosmolar environment and salivary gland epithelial cells increase extra-cellular matrix remodeling and lymphocytic infiltration in Sjögren's syndrome.

Salivary gland epithelial cells (SGECs) play an active role in primary Sjogren's syndrome (pSS) pathogenesis. Quantitative and qualitative abnormalities of saliva might expose SGECs to chronic hyperosmolarity. We aimed to decipher the links between hyperosmolar stimulation of SGECs and lymphocytic infiltration of the salivary glands (SG) observed in pSS. RNAseq was performed on NS-SV-AC cells stimulated with hyperosmolar media containing NaCl (100 mM) or sucrose (200 mM), or with iso-osmolar (Iso) medium. RNAseq was performed on primary cultured SGECs from pSS and controls, in the presence or not of B cells. Hyperosmolar stimulation of NS-SV-AC-cells identified an upregulation of interferon-induced (MX1, IFIT2) and MMPs genes. Enrichment analysis revealed an over-representation of fibrosis pathway. In parallel, RNAseq of SGECs comparing pSS to controls identified an over-representation of a pathway involving MMPs. Given the unexpected upregulation of collagen (COL3A1, COL1A2) and ADAMTS genes in pSS SGECs, we hypothesized that SGECs might undergo epithelial-mesenchymal transition. ZEB2 was upregulated and SLUG was down regulated in SGECs from pSS versus controls. MMP24 and ZEB2 were higher in SGECs from pSS with a focus score ≥1 versus <1. Lastly, SGECs cocultured with B cells expressed higher levels of COL1A2. These results suggest the existence of a vicious circle. Alteration of SGECs in pSS participates in the establishment of a hyperosmolar microenvironment, which in turn promotes SGECs transcriptomic modifications. These modifications include extracellular matrix remodeling and promote SG lymphocytic infiltration.

Monocyte/Macrophage Abnormalities Specific to Rheumatoid Arthritis Are Linked to miR-155 and Are Differentially Modulated by Different TNF Inhibitors.

Proinflammatory macrophages and miR-155 are increased in patients with rheumatoid arthritis (RA). We studied membrane TNF (mTNF) expression on blood monocytes, polarization into macrophages, miR-155 expression, and the effect of anti-TNF on these biomarkers in RA patients. Sixty-seven RA patients and 109 controls (55 healthy, 54 with spondyloarthritis and connective tissue diseases) were studied. Monocytes were isolated and differentiated into macrophages with or without anti-TNF. mTNF expression was increased on monocytes from RA patients, but not from other inflammatory diseases, correlated with disease activity. Under human serum AB or M-CSF, only monocytes from RA had a defect of differentiation into M2-like macrophages and had a propensity for preferential maturation toward M1-like macrophages that contributed to synovial inflammation. This defect was correlated to mTNF expression and was partially reversed by monoclonal anti-TNF Abs but not by the TNF soluble receptor. miR-155 was increased in M2-macrophages except in adalimumab-treated patients. Transfection of healthy monocytes with miR-155 induced a decrease in M2-like markers, and transfection of RA monocytes with antagomir-155 allowed restoration of M2-like polarization. Defect in differentiation of monocytes into M2-like-macrophages linked to increased miR-155 and correlated with increased mTNF on monocytes could play a key role in RA pathogenesis. Monoclonal anti-TNF Abs but not the TNF soluble receptor partially restored this defect.

Salivary gland epithelial cells from patients with Sjögren's syndrome induce B-lymphocyte survival and activation.

OBJECTIVE: Primary Sjögren's syndrome (pSS) is characterised by chronic hyperactivation of B lymphocytes. Salivary gland epithelial cells (SGECs) could play a role in promoting B-lymphocyte activation within the target tissue. We aimed to study the interactions between SGECs from patients with pSS or controls and B lymphocytes. METHODS: Patients had pSS according to 2016 European League Against Rheumatism/American College of Rheumatology criteria. Gene expression analysis of SGECs and B lymphocytes from pSS and controls isolated from salivary gland biopsies and blood was performed by RNA-seq. SGECs from pSS and controls were cocultured with B-lymphocytes sorted from healthy donor blood and were stimulated. Transwell and inhibition experiments were performed. RESULTS: Gene expression analysis of SGECs identified an upregulation of interferon signalling pathway and genes involved in immune responses (HLA-DRA, IL-7 and B-cell activating factor receptor) in pSS. Activation genes CD40 and CD48 were upregulated in salivary gland sorted B lymphocytes from patients with pSS. SGECs induced an increase in B-lymphocyte survival, which was higher for SGECs from patients with pSS than controls. Moreover, when stimulated with poly(I:C), SGECs from patients with pSS induced higher activation of B-lymphocytes than those from controls. This effect depended on soluble factors. Inhibition with anti-B-cell activating factor, anti-A proliferation-inducing ligand, anti-interleukin-6-R antibodies, JAK1/3 inhibitor or hydroxychloroquine had no effect, conversely to leflunomide, Bruton's tyrosine kinase (BTK) or phosphatidyl-inositol 3-kinase (PI3K) inhibitors. CONCLUSIONS: SGECs from patients with pSS had better ability than those from controls to induce survival and activation of B lymphocytes. Targeting a single cytokine did not inhibit this effect, whereas leflunomide, BTK or PI3K inhibitors partially decreased B-lymphocyte viability in this model. This gives indications for future therapeutic options in pSS.

Role of the IL-12/IL-35 balance in patients with Sjögren syndrome.

BACKGROUND: An interferon signature is involved in the pathogenesis of primary Sjögren syndrome (pSS), but whether the signature is type 1 or type 2 remains controversial. Mouse models and genetic studies suggest the involvement of TH1 and type 2 interferon pathways. Likewise, polymorphisms of the IL-12A gene (IL12A), which encodes for IL-12p35, have been associated with pSS. The IL-12p35 subunit is shared by 2 heterodimers: IL-12 and IL-35. OBJECTIVE: We sought to confirm genetic association of the IL12A polymorphism and pSS and elucidate involvement of the IL-12/IL-35 balance in patients with pSS by using functional studies. METHODS: The genetic study involved 673 patients with pSS from 2 French pSS cohorts and 585 healthy French control subjects. Functional studies were performed on sorted monocytes, irrespective of whether they were stimulated. IL12A mRNA expression and IL-12 and IL-35 protein levels were assessed by using quantitative RT-PCR and ELISA and a multiplex kit for IL-35 and IL-12, respectively. RESULTS: We confirmed association of the IL12A rs485497 polymorphism and pSS and found an increased serum protein level of IL-12p70 in patients with pSS carrying the risk allele (P = .016). Serum levels of IL-12p70 were greater in patients than control subjects (P = .0001), especially in patients with more active disease (P = .05); conversely, IL-35 levels were decreased in patients (P = .0001), especially in patients with more active disease (P = .05). In blood cellular subsets both IL12p35 and EBV-induced gene protein 3 (EBI3) mRNAs were detected only in B cells, with a trend toward a lower level among patients with pSS. CONCLUSION: Our findings emphasize involvement of the IL-12/IL-35 balance in the pathogenesis of pSS. Serum IL-35 levels were associated with low disease activity, in contrast with serum IL-12p70 levels, which were associated with more active disease.

Liposomal AntagomiR-155-5p Restores Anti-Inflammatory Macrophages and Improves Arthritis in Preclinical Models of Rheumatoid Arthritis.

OBJECTIVE: We previously reported an increased expression of microRNA-155 (miR-155) in the blood monocytes of patients with rheumatoid arthritis (RA) that could be responsible for impaired monocyte polarization to anti-inflammatory M2-like macrophages. In this study, we employed two preclinical models of RA, collagen-induced arthritis and K/BxN serum transfer arthritis, to examine the therapeutic potential of antagomiR-155-5p entrapped within PEGylated (polyethylene glycol [PEG]) liposomes in resolution of arthritis and repolarization of monocytes towards the anti-inflammatory M2 phenotype. METHODS: AntagomiR-155-5p or antagomiR-control were encapsulated in PEG liposomes of 100 nm in size and -10 mV in zeta potential with high antagomiR loading efficiency (above 80%). Mice were injected intravenously with 1.5 nmol/100 µL PEG liposomes containing antagomiR-155-5p or control after the induction of arthritis. RESULTS: We demonstrated the biodistribution of fluorescently tagged PEG liposomes to inflamed joints one hour after the injection of fluorescently tagged PEG liposomes, as well as the liver's subsequent accumulation after 48 hours, indicative of hepatic clearance, in mice with arthritis. The injection of PEG liposomes containing antagomiR-155-5p decreased arthritis score and paw swelling compared with PEG liposomes containing antagomiR-control or the systemic delivery of free antagomiR-155-5p. Moreover, treatment with PEG liposomes containing antagomiR-155-5p led to the restoration of bone marrow monocyte defects in anti-inflammatory macrophage differentiation without any significant functional change in other immune cells, including splenic B and T cells. CONCLUSION: The injection of antagomiR-155-5p encapsulated in PEG liposomes allows the delivery of small RNA to monocytes and macrophages and reduces joint inflammation in murine models of RA, providing a promising strategy in human disease.

Restoration of Default Blood Monocyte-Derived Macrophage Polarization With Adalimumab But Not Etanercept in Rheumatoid Arthritis.

Introduction: We previously reported a specific defect of rheumatoid arthritis (RA) monocyte polarization to anti-inflammatory M2-like macrophages related to increased miR-155 expression in all RA patients except those receiving adalimumab (ADA). In this longitudinal study, we examined whether different tumor necrosis factor inhibitors were able to restore monocyte polarization to M2-like macrophages and their effect on the transcriptomic signature. Methods: M2-like polarization induced by human serum AB was studied in 7 healthy donors and 20 RA patients included in the ABIRA cohort before and 3 months after starting ADA or etanercept (ETA). The differential gene expression of M2- and M1-related transcripts was studied in macrophage-derived monocytes after differentiation. Results: At baseline, RA monocytes showed a defect of polarization to M2-like macrophages as compared with healthy donor monocytes, which was negatively correlated with disease activity. M2-like polarization from circulating monocytes was restored only with ADA and not ETA treatment. The transcriptomic signature demonstrated downregulation of M2-related transcripts and upregulation of M1-related transcripts in active RA. In patients receiving ADA, the transcriptomic signature of M2-related transcripts was restored. Conclusion: This longitudinal study demonstrates that ADA but not ETA is able to restore the M2-like polarization of monocytes that is defective in RA.

Interleukin-7/Interferon Axis Drives T Cell and Salivary Gland Epithelial Cell Interactions in Sjögren's Syndrome.

OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by a lymphocytic infiltration of salivary glands (SGs) and the presence of an interferon (IFN) signature. SG epithelial cells (SGECs) play an active role in primary SS pathophysiology. We undertook this study to examine the interactions between SGECs and T cells in primary SS and the role of the interleukin-7 (IL-7)/IFN axis. METHODS: Primary cultured SGECs from control subjects and patients with primary SS were stimulated with poly(I-C), IFNα, or IFNγ. T cells were sorted from blood and stimulated with IL-7. CD25 expression was assessed by flow cytometry. SG explants were cultured for 4 days with anti-IL-7 receptor (IL-7R) antagonist antibody (OSE-127), and transcriptomic analysis was performed using the NanoString platform. RESULTS: Serum IL-7 level was increased in patients with primary SS compared to controls and was associated with B cell biomarkers. IL7R expression was decreased in T cells from patients with primary SS compared to controls. SGECs stimulated with poly(I-C), IFNα, or IFNγ secreted IL-7. IL-7 stimulation increased the activation of T cells, as well as IFNγ secretion. Transcriptomic analysis of SG explants showed a correlation between IL7 and IFN expression. Finally, explants cultured with anti-IL-7R antibody showed decreased IFN-stimulated gene expression. CONCLUSION: These results suggest the presence of an IL-7/IFNγ amplification loop involving SGECs and T cells in primary SS. IL-7 was secreted by SGECs stimulated with type I or type II IFN and, in turn, activated T cells that secrete type II IFN. An anti-IL-7R antibody decreased the IFN signature in T cells in primary SS and could be of therapeutic interest.

Serum IL-33 level is associated with auto-antibodies but not with clinical response to biologic agents in rheumatoid arthritis.

TRIAL REGISTRATION: Rotation or Change of Biotherapy After First Anti-TNF Treatment Failure for Rheumatoid Arthritis (ROC), registered 22 October 2009, NCT01000441.

Clinicopathological features of multiple mononeuropathy associated with systemic lupus erythematosus: a multicenter study.

Multiple mononeuropathy (MM) occurs rarely during systemic lupus erythematosus (SLE) but may lead to major disability. The aim of this study was to investigate the clinic-pathological presentations of MM during SLE, as well as long-term outcomes. We conducted a multicentric retrospective study that included patients receiving a diagnosis of MM during SLE. Ten patients were included (8 women and 2 men, median age at MM diagnosis: 40.4 years). SLE was diagnosed before MM in 9/10 patients (median time 8.2 years). When MM occurred, the SLEDAI score was ≥6 for 6/9 patients. Presenting symptoms consisted of sensory deficits (n = 10), neuropathic pain (n = 9), and/or motor deficits (n = 9), sometimes symmetrical, affecting the lower limbs (10/10) and occasionally the upper limbs (5/10). All patients presented with uni- or bilateral damage of the common fibular nerve, with less frequent involvement of the tibial nerve. Serum cryoglobulinemia was positive in 5/9 patients. Electrophysiological studies confirmed the non-symmetrical involvement of multiple nerve trunks in all patients. Neuromuscular biopsy (performed in five patients) showed histological signs of vasculitis in two patients and perivascular lymphocytic inflammatory infiltrates in two others. All patients were treated with glucocorticosteroids combined with cyclophosphamide (n = 6), rituximab (n = 3), or mycophénolate-mofétil (n = 1). The median follow-up was 5 years. Two patients relapsed during follow-up. All patients had motor and/or sensory sequelae upon follow-up. MM associated with SLE is frequently caused by a vasculitis mechanism. Patients improve with steroids and immunosuppressive drugs. Long-term outcomes include frequent clinical sequelae and possible relapses.

Takayasu Arteritis and Spondyloarthritis: Coincidence or Association? A Study of 14 Cases.

OBJECTIVE: Spondyloarthritis (SpA) and Takayasu arteritis (TA) are 2 chronic inflammatory diseases; their coexistence in a single patient is uncommon. The aims of our study were to describe clinical features of patients having SpA associated with TA and to identify some characteristics of the types of patients with SpA associated with TA. We also analyzed treatments used in this context. METHODS: This French multicenter retrospective survey called for observations on behalf of the Club Rhumatismes et Inflammations, with a standardized questionnaire established by the investigators. RESULTS: We included 14 patients (women: 10/14; median age at SpA diagnosis: 43.5 yrs, ranging from 19 to 63). Subtypes of SpA were ankylosing spondylitis (n = 11), psoriatic arthritis (n = 2), and synovitis, acne, pustulosis, hyperostosis, and osteitis syndrome (n = 1). HLA-B27 was positive in 3 cases, negative in 9, and unknown in 2. SpA was diagnosed before TA in 13 cases. Imaging findings compatible with the diagnosis of TA were found with computed tomography (11/14) and/or Doppler ultrasound (10/14). Laboratory tests showed increased acute-phase reactants in all cases (C-reactive protein ≥ 25 mg/l in 71% of the cases). All patients except 1 received corticosteroids and 7 were treated with anti-tumor necrosis factor (anti-TNF). CONCLUSION: Association of SpA and TA is rare but probably not coincidental. Peripheral pulse palpation and vascular auscultation should be systematic and are the first indicators of TA in patients with SpA. Moreover, increased acute-phase reactants during SpA followup should lead to search for TA. Finally, there are therapeutic implications because anti-TNF are efficient in SpA and might be efficient in TA.

Serum IL-33, a new marker predicting response to rituximab in rheumatoid arthritis.

BACKGROUND: Recent works have suggested a possible link between interleukin (IL)-33 and B-cell biology. We aimed to study the possible association between serum IL-33 detection and response to rituximab (RTX) in rheumatoid arthritis (RA) patients in different cohorts with an accurate enzyme-linked immunosorbent assay (ELISA). METHODS: Serum IL-33, rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and high serum immunoglobulin (Ig)G levels were assessed in 111 RA patients receiving a first course of 2 g RTX (cohort 1) in an observational study and in 74 RA patients treated with the same schedule in routine care (cohort 2). Univariate and multivariate analyses identified factors associated with a European League Against Rheumatism (EULAR) response at 24 weeks. RESULTS: At week 24, 84/111 (76%) and 54/74 (73%) patients reached EULAR response in cohorts 1 and 2, respectively. Serum IL-33 was detectable in only 33.5% of the patients. In the combined cohorts, the presence of RF or anti-CCP (odds ratio (OR) 3.27, 95% confidence interval (CI) 1.13-9.46; p = 0.03), high serum IgG (OR 2.32, 95% CI 1.01-5.33; p = 0.048), and detectable serum IL-33 (OR 2.40, 95% CI 1.01-5.72; p = 0.047) were all associated with RTX response in multivariate analysis. The combination of these three factors increased the likelihood of response to RTX. When serum IL-33 detection was added to seropositivity and serum IgG level, 100% of the patients with the three risk factors (corresponding to 9% of the population) responded to RTX (OR versus patients with none of the three risk factors 29.61, 95% CI 1.30-674.79; p = 0.034). CONCLUSION: Detectable serum IL-33 may predict clinical response to RTX independently of, and synergistically with, auto-antibodies and serum IgG level. TRIAL REGISTRATION: NCT01126541 ; 18 May 2010.