Background/Purpose: Up to 80% of patients with pediatric systemic lupus erythematosus (pSLE) can present with renal abnormalities. Treatment of pSLE is often difficult and includes broad immunosuppression, which can lead to further damage, necessitating the discovery of new targets for therapy. In adult SLE (aSLE), neutrophil extracellular traps (NETs) have been shown to play a role in the development of lupus nephritis (LN). Further, loss-of-function mutations in the genes encoding for DNASE1 and DNASE1L3 are associated with aSLE and DNASE1L3 mutations have been implicated in early onset monogenic lupus. Anti-NET protective antibodies have been also shown to be present in aSLE serum, thus protecting the NETs from DNASE digestion. In a cohort of treatment naïve pSLE patients with and without LN, we detected a significant increase in both blood and urinary NETs. The purpose of this study was to determine the mechanisms driving spontaneous NETosis in these patients, pre- and post-treatment, and evaluate whether NET levels may be used as an indicator of treatment response.

Methods: Plasma was obtained from 21 pSLE treatment-naïve patients, 13 post-treatment patients and 12 age- and sex-matched healthy children (pHC). Lupus disease activity was measured using SELENA-SLEDAI scoring, dsDNA antibody levels, and urine protein/creatine ratios. ELISA and smear assay were used to detect NETs in plasma and urine samples. DNase1L3 concentration was measured by ELISA, DNase enzymatic activity was determined via targeted assay (Dnase1 versus DNase1L3), NET degradation was determined with patient plasma, and blocking assays were used to assess autoantibody functionality. Targeted and whole exome sequencing was performed to identify genetic mutations. Immunofluorescence in renal biopsies was performed to evaluate NETs at end organ.

Results: Accumulation of NETs was observed with significant inhibition of DNase1 and DNase1L3 activities pre-treatment. Notably, NET levels remained high post-treatment. No known loss-of-function variants in DNASE1L3 or DNASE1 were found. Finally, we found higher anti-DNASE1 and anti-DNASE1L3 antibodies selectively playing role in inhibition of DNASE1L3 activity. Moreover, higher anti-NET protective antibodies were found in pLN plasma pre- and post-treatment.

Conclusion: Our data show that defective functional DNase1/1L3 activity and increased anti-NET protective antibodies drive delayed clearance and accumulation of NETs in pLN. Taken together, our results suggest NETs and the mechanisms driving spontaneous NETosis as novel non-invasive biomarkers of pLN and potential novel targets for therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/trapped-in-the-net-impaired-dnase-function-and-targeted-antibodies-in-the-pathogenesis-of-pediatric-lupus-nephritis/