Background/Purpose: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by autoantibodies targeting RNA-associated antigens, anti-SSA/SSB. The IFN-signature is present in over half of pSS patients, and is associated with higher disease-activity and auto-antibody presence. Endosomal Toll-like receptors, TLR7 and TLR9, are crucial for both the generation of auto-antibodies by B-cells and immunecomplex-mediated IFN production by plasmacytoid Dendritic Cells (pDCs) in autoimmunity. Recently opposing effects were described for TLR7 and TLR9 in murine lupus-models, where TLR7-deletion limited autoimmunity and TLR9-deletion paradoxically exacerbated disease. Interestingly, we recently found the TLR7-pathway upregulated in IFNpositive pDCs of pSS patients, whereas TLR9 was not. Here we set out to further investigate this imbalanced endosomal TLR-signaling in IFN-driven pSS. 

Methods: Blood samples were obtained from 33 Healthy controls (HC) and 58 pSS patients, diagnosed according to the 2002 American-European criteria, and stratified according to their IFNsignature. Fluorescence-activated cell sorting was used to isolate CD123+BDCA4+ pDCs, CD14+ monocytes, CD3+ T-cells and CD19+ B-cells >98% purity, from peripheral blood mononuclear cells (PBMCs). Genome-wide Microarray analysis conducted on sorted pDCs and monocytes revealed increased expression of cytoplasmic and endosomal pattern recognition receptors: TLR7, retinoic acid inducible gene-I (RIG-I/DDX58), melanoma differentiation associated gene-5 (MDA-5/IFIH1), and further downstream MyD88-dependent signaling, confined to IFNpositive patients. mRNA expression of the resulting differentially expressed genes (DEGs), as assessed by Ingenuity pathway analysis (IPA), was validated in sorted cell-suspensions and whole blood (Paxgene) using real-time quantitative PCR. To further clarify the possible TLR7-driven activation of the IFN signature, PBMCs of HC were stimulated in vitrowith imiquimod, a TLR7 agonist, and inhibited with the TLR7 antagonist IRS661.

Results: Confirming our microarray results, we found an upregulation of TLR7 (p<0.05), but not TLR9, in IFNpositive pDCs, monocytes, B-cells and in whole blood (p<0.0001) as well as further downstream MyD88, RSAD2 and IRF7 (p<0.001). We also observed the upregulation of intracellular RNA-sensing receptors RIG-I and MDA-5 (p<0.01), recently described to collectively initiate effective IFN signaling. This widespread upregulation of TLR7 and its downstream signaling pathway is confined to IFNpositive pSS patients. In vitrostudies with HC-PBMCs reveal that triggering of the TLR7-pathway by Imiquimod causes further upregulation of the other RNA-sensing receptors RIG-I and MDA5, inflammatory cytokines, IFN-inducible genes and also BAFF. Specific TLR7-inhibition subsequently showed a dose-dependent decrease.

Conclusion: Taken together, these RNA-sensing receptors — TLR7, RIG-I and MDA5 — seem to collaborate in amplifying the pathogenic IFN-driven loop in pSS. A better understanding of this unrestrained and potentially autoreactive loop reveals novel targets for therapeutic interventions in pSS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/nucleic-acid-sensing-receptors-tlr7-rig-i-and-mda5-collaborate-in-driving-the-systemic-ifn-signature-and-amplify-the-pathogenic-loop-potential-new-targets-for-therapy-in-primary-sjogrens-syndrome/