Busulfan and cyclosphamide induce liver inflammation through NLRP3 activation in mice after hematopoietic stem cell transplantation

Abstract

The aim of this study was to evaluate the role of NLRP3 inflammasome on BU/CY-induced liver inflammation in mice after HSCT. HSCT mice model was established through infusion of 5 × 10(6) bone marrow mononuclear cells after conditioned with BU/CY. On day 7, 14, 21 and 28 after HSCT, mice were sacrificed for analysis of liver inflammation, cytokine secretion, NLRP3 expression and caspase-1 activation as well as release of ATP and high-mobility group protein B1 (HMGB1). Furthermore, NLRP3 selective inhibitor (BAY 11-7082) was administrated into mice after HSCT to evaluate its effects on liver inflammation. Severe liver inflammation and damage with elevated secretion of IL-1β and IL-18 were found in mice after HSCT. Meanwhile, elevated expressions of NLRP3 and caspase-1 activation in liver were found. In addition, increased release of ATP and HMGB1 were observed. Selective inhibition of NLRP3 decreased caspase-1 activation and secretion of IL-1β and IL-18. Furthermore, NLRP3 inhibition also reduced infiltration of macrophages and neutrophils and improved liver function. In conclusion, NLRP3 was involved in BU/CY-induced liver inflammation after HSCT and selectively inhibited it ameliorated liver inflammation and improved liver function, suggesting targeting NLRP3 might be a new approach in the prophylaxis of liver inflammation after HSCT.

Figure 1. Liver pathological changes and numbers of mononuclear cells in liver post HSCT.

At indicated time points after HSCT, mice were sacrificed and liver was isolated and fixed with formaldehyde, dehydrated, waxed and sliced into 4 μm thickness by RM2126 microtome for H&E staining (A). For immunohistochemical staining, the slices were incubated with 3% H₂O₂ at room temperature and blocked with 5–10% goat serum. Then the slices were incubated with pan-endothelial cell monoclonal antibody (MECA-32) at 37 °C for 1–2 h followed by the incubation with secondary antibody. Color was developed with 3,3′-diaminobenzidine. Mason staining was performed using a commercial kit. A representative staining graph selected from 6 independent stainings in each group was shown. Meanwhile, numbers of infiltrated mononuclear cells in liver were also counted (B).

Figure 2. Expression of IL-1β and IL-18 in liver post HSCT.

On d7, d14, d21 and d28 post HSCT, RNA was extracted from liver for analysis of mRNA expression of IL-1β (A) and IL-18 (B) by quantitative real-time PCR. Meanwhile, protein expressions of IL-1β (C) and IL-18 (D) on d7 and d14 post HSCT were also measured by western blot.

Figure 3. Expression of caspase-1 and NLRP3 in liver after HSCT.

At indicated time points after HSCT, RNA and protein were isolated from liver for the measurement of mRNA and protein expression of caspase-1 and NLRP3 by quantitative real-time PCR and western blot respectively. Protein expressions of p20 and NPRP3 were quantified as a ratio to β-actin.

Figure 4. Level of ATP and HMGB1 in plasma.

Plasma was isolated from mice at indicated time points after HSCT for analysis of release of ATP and HMGB1 by ELISA. Mice without any treatments were served as normal.

Figure 5. Secretion of IL-1β and IL-18, expression of NLRP3 and caspase-1, inflammatory cell infiltration and liver function in mice after treatment with BAY 11-7082.

Mice receiving NLRP3 inhibitor (BAY) or vehicle were sacrificed on day 14 post HSCT and liver was obtained for analysis of the level of IL-1β and IL-18 in liver by quantitative real-time PCR (A). Expression of NLRP3 and caspase-1, inflammatory cells infiltration, such neutrophils and macrophages, were evaluated by western blot (B). In addition, immunohistochemical staining of CD11b and Ly6G expression in liver (C), H&E staining of liver (D) and liver function (AST and ALT level) measurement by automatic biochemistry analyzer (D) was also performed. A representative graph selected from 6 independent experiments at different time points was shown. ^**P < 0.01, ^*P < 0.05.