Background/Purpose: Blau syndrome is a pediatric rheumatic disease characterized by dermatitis, arthritis, and uveitis. Blau syndrome is caused by inborn or de novo mutations in NOD2, the gene encoding the microbial sensing molecule NOD2. However, the cellular mechanism by which NOD2 causes sterile inflammation in Blau syndrome remains unknown. Recent studies have challenged the established dogma that Blau syndrome is caused by NOD2 gain-of-function mutations to show Blau NOD2 mutations result in a loss-of-function in NOD2 activation downstream of bacterial ligand stimulation. We previously demonstrated a novel T cell-intrinsic role for murine Nod2 in blocking pathogenic Th17/IL-17 responses and protecting against experimental autoimmune uveitis and arthritis in mice. Here we sought to determine how NOD2 mutations in human patients with Blau syndrome alter T cell composition and function including Th17 responses.

Methods: Peripheral blood mononuclear cells (PBMCs) from patients with Blau representing 6 unique NOD2 mutations, ages 2-64 (n=8) or controls (n=10) were analyzed before and after activation by anti-CD3 in combination with co-stimulatory agent, anti-CD28, or phorbol myristate acetate (PMA) and ionomycin (io). Cellular composition, T cell transcription factors, and cytokine expression were quantified by flow cytometry.

Results: Patients with Blau had comparable frequencies of peripheral blood CD4+ and CD8+ T cells to controls. However, these patients had a higher fraction of memory CD4+ T cells that expressed the Th17 lineage-defining transcription factor Rorgt, suggesting patients with Blau have enhanced baseline Th17 responses. Following T cell receptor (TCR)-independent stimulation (PMA/io) we found that cells from Blau patients had increased percentages of CD4+ T cells that produced IL-17A+ or IL-17F+ compared to controls, yet no difference in percent of TNF+, IFNg+, or IL-2+ CD4 T cells. These data indicate that Blau patient T cells, regardless of individual NOD2 mutations or age, have a predisposition to increased peripheral blood Th17 cells. To mimic antigen/cognate MHC-II interactions, T cells were then activated with a TCR-dependent stimulus (anti-CD3/CD28) over 5 days. On day 3 post-stimulation, PBMCs from Blau patients had increased IL-17F+, TNF+ and IL-2+ CD4 T cells with IL-17F+ CD4 T cells persistently increased through day 5 compared to controls. Cumulatively these data suggest that patients with Blau have primed Th17 immunity due to underlying NOD2 mutations that enhance antigen-independent and -dependent T cell production of IL-17.

Conclusion: To our knowledge we are the first group to implicate T cells and Th17 immunity in Blau syndrome pathogenesis. These data provide foundational evidence for consideration of T cells and more specifically IL-17A and IL-17F as future targets for therapeutic intervention.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/nod2-mutations-mediate-il-17-predisposition-in-patients-with-blau-syndrome/