Background/Purpose: Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is an autosomal dominant autoinflammatory disorder caused by mutations in the PSTPIP1/CD2BP1 gene. PAPA syndrome is characterized by recurrent flares of sterile arthritis and severe skin inflammation in the absence of adaptive immune responses. Mutations in PSTPIP1 likely dysregulate the pyrin inflammasome leading to exuberant IL-1β production. PAPA patients respond to treatment with cytokine inhibitors, including IL-1 inhibitors. Neutrophils can undergo a form of cell death called NETosis where dying cells release a meshwork of decondensed DNA decorated with granule proteins called neutrophil extracellular traps (NETs). NETs have been shown to induce tissue damage in other systemic diseases. While previous studies have suggested that neutrophils may play critical roles in driving inflammation in PAPA syndrome, the mechanism by which it happens is still unclear. A subset of proinflammatory neutrophils called low-density granulocytes (LDGs) has been identified in other autoimmune/autoinflammatory conditions and these cells display an enhanced capacity to form NETs. Here, we aimed to elucidate the role of neutrophil subsets and NETs in the pathogenesis of PAPA syndrome.

Methods: Normal density neutrophils were isolated by dextran sedimentation from control and PAPA patients. LDGs were isolated by negative selection from the PBMC fraction. Immunofluorescence (IF) was used to assess the presence of NETs. To assess the effect of serum in NET formation, control neutrophils were incubated with 10% serum from control or PAPA patients in the presence or absence of the IL-1 receptor anatagonist anakinra for 2 h. Circulating NET remnants were quantified by ELISA against citrullinated histone H3 (citH3)-DNA complexes. A skin biopsy from a PAPA patient was analyzed by IF, quantitative PCR, and western blot analysis for the presence of NETs and proinflammatory cytokines.

Results: LDGs were detected in PAPA patients. Neutrophils and LDGs from PAPA patients displayed an enhanced capacity to spontaneously form NETs when compared to control neutrophils. Serum from PAPA patients induced NET formation in control neutrophils and this process was inhibited in the presence of IL-1 inhibitor, anakinra. NET products, as measured by cit-H3-DNA complexes in plasma from PAPA patients, were significantly higher when compared to control plasma. We detected NETs in a skin biopsy from a PAPA patient, in association with upregulation of tissue IL-1β and IL-8 when compared to healthy skin biopsies. Furthermore, an immature granulocyte signature was detected in the skin biopsy tissue suggesting the presence of LDGs.

Conclusion: Taken together, neutrophils and LDGs in PAPA patients display dysregulated NET formation as assessed by in vitro spontaneous NET formation and the presence of circulating NET remnants. Proinflammatory cytokines in serum and affected tissues of PAPA patients trigger the NET formation. Anakinra ameliorates serum-induced NETosis, suggesting a role of IL-1 signaling in NETosis. The identification of LDGs in skin tissue from PAPA patients point out a strong myeloid signature in this condition.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterization-of-neutrophil-subsets-and-neutrophil-extracellular-traps-in-pyogenic-arthritis-pyoderma-gangrenosum-and-acne-papa-syndrome/