H HUTTON1,2, J OOI1, S HOLDSWORTH1,2, AR KITCHING1,2
1Centre for Inﬂammatory Diseases, Monash University, Clayton, Victoria; 2Department of Nephrology, Monash Health, Clayton, Victoria
Aim: To determine the therapeutic potential of inﬂammasome inhibition in ANCA-associated vasculitis (AAV).
Background: The NLRP3 inﬂammasome innate immune system molecular complex responds to cellular stress by producing the pro-inﬂammatory cytokines IL-1β and IL-18. MCC950 is a speciﬁc NLRP3 inﬂammasome inhibitor.
Methods: To assess anti-MPO autoimmunity, mice were injected with 20mg rMPO/FCA and studied at 10 days. For anti-MPO GN, mice were immunised with 20mg rMPO/FCA day 0 and 20mg rMPO/FIA on day 7. On day 16, a low (3mg) dose of sheep anti-mouse anti-GBM globulin was administered to recruit neutrophils to glomeruli. Experiments ended on day 19. Experiments used Nlrp3-/- mice, and WT mice treated with MCC950 (20mg/kg) daily). Renal and immunological (MPO-ANCA ELISA, ELISPOT or flow cytometry) endpoints were studied. Neutrophil recruitment was assessed after 4 hours by administering 10μg LPS, then 1mg of anti-MPO monoclonal antibody.
Results: After immunization, Nlrp3-/- mice had fewer activated (CD62LloCD44hi)CD4+T cells (9±1 vs 15±1 %CD4+ cells, p=0.001) and decreased Th1 responses (0.65±0.05 vs 0.83±0.06 %CD4+ cells, p=0.05) Compared to WT mice, Nlrp3-/- mice developed less 24-hour albuminuria (92±21 vs 199±15mg p=0.001) and histological injury (64±6 vs 13±1 glomeruli abnormal; p<0.0001). MCC950 treated mice also had significantly reduced 24-hour albuminuria (54±7 vs 153±29 mg/24hr, p=0.0036) and histological injury (25±2 vs 47±4% glomeruli abnormal; p=0.0006) compared to saline treated mice. MCC950 reduced MPO-stimulated IFN-g production (ELISPOT) and T cell activation, with fewer CD62LloCD44hi cells (51±2 vs 36±2% CD4+ cells p=0.001). Acute anti-MPO antibody induced neutrophil recruitment was similar in MCC950 treated and control mice.
Conclusions: NLRP3 inﬂammasome inhibition attenuates T cell responses and disease in experimental anti-MPO GN, and may be a target for treatment in AAV.