Background/Purpose: Leukocyte immunoglobulin-like receptor A3 (LILRA3) is a secreted protein belongs to LILR family. Our research group previously reported that the functional LILRA3 is a novel genetic risk for multiple autoimmune diseases including systemic lupus erythematosus (SLE). However, the function of LILRA3 in development of lupus is unclear. Dysfunctional neutrophils play a pathogenic role in SLE, particularly through the release of neutrophil extracellular traps (NETs). The aim of this study was to functionally characterize the role of LILRA3 in NET formation in the TLR7-induced lupus-like model.

Methods: To functionally study the role of LILRA3 in autoimmune diseases, we constructed the LILRA3 knock-in (KI) mice. The lupus-like disease was induced by the epicutaneous application of the TLR7 agonist imiquimod (IMQ) three times a week. The C57BL/6 wild-type and LILRA3-KI mice were sacrificed on day 14, 28, and 56 after treatment of IMQ. Flow cytometry was used to evaluate the proportions of neutrophils in peripheral blood (PB), spleen, and bone marrow (BW), respectively. To assess NETosis (NETs release), complementary approaches were utilized. The total extracellular DNA was quantified as relative fluorescence units upon the addition of Sytox Green by using the SYTOX Green assay. Immunofluorescence staining was applied to label the neutrophil elastase and citrullinated Histone H3(Cit-H3). Enzyme-linked immunosorbent assay was applied to detect antibodies in serum.

Results: he proportions of CD11b⁺ly6G⁺ neutrophils were significantly increased in PB, spleen and BM from IMQ-treated LILRA3-KI (KI-IMQ) mice, compared to IMQ-treated WT (WT-IMQ) mice and WT control mice on day 14 and 28 (P < 0.01). After 56 days of IMQ treatment, there was no significant difference in the proportion of PB and BM CD11b⁺ly6G⁺ neutrophils across the 3 groups, but the splenic CD11b⁺ly6G⁺ neutrophils were remarkably increased from KI-IMQ mice, compared to WT-IMQ and WT control mice (P < 0.01). Furthermore, BM neutrophils from KI-IMQ mice displayed an enhanced release of NETs as compared to that from WT-IMQ and WT control mice at three time points (P < 0.01). After phorbol myristate acetate (PMA) or Calcimycin (A23187) stimulation, a similar phenomenon was also observed for BM neutrophils from KI-IMQ mice. Concentration of serum anti-dsDNA IgG was significantly elevated in KI-IMQ mice as compared to WT-IMQ and WT control mice (P < 0.001).

Conclusion: Our data suggest that LILRA3 may promote lupus-like disease through excessive neutrophil activation and NET formation in TLR7-induced lupus mice.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/leukocyte-immunoglobulin-like-receptor-a3-facilitates-neutrophil-extracellular-trap-formation-in-tlr7-induced-lupus-mice/