Role of NLRP3 inflammasome in the immunopathogenesis of HEV infected AVH and ALF patients

Abstract

Background: Hepatitis E virus is one of the leading causes of acute viral hepatitis with worldwide distribution amounting to 3.3 million symptomatic cases and ∼60,000 deaths. 30–45% of acute liver failure (ALF) patients in India are positive for hepatitis E virus marker. High mortality (∼30%) in HEV infected pregnant patients has been reported. The pathogenesis of the disease is less understood, thus the present study focuses on the role of inflammasomes and pro-inflammatory cytokines in the immunopathogenesis of HEV. Methods and materials: 102 out of 1680 suspected cases were positive for HEV IgM and/or HEV RNA. 30 AVH and 15 ALF HEV positive sera were subjected for IL-1β, IL-18 and IL-1BPa cytokine and viral load estimation. PBMCs were isolated and processed for mRNA expressions of NLRs (NLRP1, LRP3, NLRC4); RLRs (RIG-1, MDA5, LGP2); cytokines (IL-1β, IL-18) and other necessary inflammasome pathway genes (CASP1, CASP3, PYCARD, NF-κβ). Mechanism of cell death in the PBMCs of HEV infected patients was studied by Annexin/PI apoptosis kit. Results: Lower limit of detection and quantification by HEV TaqMan Real-Time PCR was estimated as 9 and 30 viral copies/μl of sample respectively. High viral load was observed in ALF than AVH cases (1.55 × 105 ± 1.30 × 105 vs 8.18 × 103 viral copies/μl). mRNA expression of NLRP3 gene in AVH was positively correlated with IL-18 (r = 0.74, p ∼ 0.0001***) and IL-1β (r = 0.68, p ∼ 0.0001***). Significant levels of serum IL-1β and IL-18 were observed in AVH as compared to ALF patients (7.53 ± 3.92 pg/ml vs 3.39 ± 1.37 pg/ml; 1905 ± 751.9 pg/ml vs 1128 ± 705.2 pg/ml) respectively. Interleukin-18 binding protein a (IL-18BPa) levels were significantly high in AVH (24,960 ± 2351 pg/ml) in comparison to ALF (21,000 ± 2796 pg/ml) with the levels of 4960 ± 1639 pg/ml in healthy controls. Annexin/PI assay showed 36.82 ± 5.98% apoptotic cell death in AVH in comparison to healthy control. However no significant difference was observed between AVH and ALF patients. Conclusion: Significant upregulation of NLRP3 inflammasome leading to high production of IL-18 and IL-1β cytokines in sera of AVH patients indicated the role of TH1 response acting through NLRP3 pathway which might be helpful in recovery of AVH patients.