Background/Purpose: Dysregulated neutrophil extracellular trap (NET) release has been proposed as a source of autoantigens in lupus.  Furthermore, it has recently been shown that the onset of lupus nephritis is predicted by a “neutrophil signature” in lupus blood.  Neutrophil elastase (NE) is a serine protease uniquely expressed by neutrophils, which is known to activate matrix metalloproteinases, inflammatory cytokines, and degrade extracellular matrix components.  NE is also required for many forms of NETosis, and has been reported to circulate at high levels in lupus patients.  Inhibition of NE has proven effective in models of cardiopulmonary disease and inflammatory arthritis.  Previously, we demonstrated the efficacy of a single NE inhibitor in mitigating organ damage (proteinuria, cardiac fibrosis) in lupus-prone NZW x BXSB F1 mice.  However, we have not previously characterized how specific immune-cell populations or autoantibody responses might be modulated by NE inhibition.

Methods: Lupus-prone MRL-lpr female mice were treated with two different selective NE inhibitors:  GW311616A (GW, 2.2 mg/kg by oral gavage three times per week) or Alvelestat (ALV, 10 mg/kg by oral gavage three times per week) from 8 to 16 weeks of age.  We measured NETosis efficiency and autoantibody levels using standard ELISAs.  Spleens, lymph nodes (mandibular, axillary, and inguinal), and kidneys were harvested and immune-cell profiling was performed with flow cytometry.

Results: Both GW- and the ALV-treated mice demonstrated reduced levels of autoantibodies against double-stranded DNA (both groups demonstrating ~50% reduction) and beta-2 glycoprotein I (~30% reduction).  Peripheral blood analysis revealed a significant reduction in the number of circulating neutrophils in inhibitor-treated mice (~20% reduction), but no difference in the number of cell-free NETs.  Intriguingly, elastase inhibition resulted in a marked reduction in CD45+ cells infiltrating kidneys (approximate 3-fold reduction for both inhibitor groups).  Regarding specific leukocyte populations, there was a 5-fold or greater reduction in renal-infiltrating CD19+ B cells, CD4+ T cells, and CD8+ T cells.  Analysis of lymph nodes demonstrated reduced expansion of activated CD4+ and CD8+ T cells (30% reduction) with NE inhibition, which was accompanied by reduced numbers of CD44+ CD62L+ central-memory T cells.  Furthermore, there was a 2-fold reduction in the percentage of germinal-center B cells, and a corresponding increase in immature B cells.  Experiments are underway to precisely define kidney histology and immune-complex deposition in NE inhibitor-treated mice as compared with vehicle-treated controls.

Conclusion: Treatment of MRL-lpr mice with two distinct orally-bioavailable NE inhibitors (GW311616A or Alvelestat) led to the reduction in autoantibody levels, decreased immune-cell infiltration into kidneys, and altered immune-cell profiles in lymph nodes.  These data provide further evidence that NE is a valid therapeutic target in lupus, and perhaps especially as a strategy for preventing lupus nephritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-neutrophil-elastase-reduces-autoantibody-levels-and-renal-inflammation-in-murine-lupus/