Inhibition of HSP90 and Activation of HSF1 Diminish Macrophage NLRP3 Inflammasome Activity in Alcohol-Associated Liver Injury

Abstract

Background: Activation of NLRP3 in liver macrophages contributes to alcohol-associated liver disease (ALD). Molecular chaperone heat shock protein (HSP) 90 facilitates NLRP3 inflammasome activity during infections and inflammatory diseases. We previously reported that HSP90 is induced in ALD and regulates proinflammatory cytokines, tumor necrosis factor alpha, and IL-6. Whether HSP90 affects IL-1β and IL-18 regulated by NLRP3 inflammasome in ALD is unknown. Here, we hypothesize that HSP90 modulated NLRP3 inflammasome activity and affects IL-1β and IL-18 secretion in ALD.

Methods: The expression of HSP90AA1 and NLRP3 inflammasome genes was evaluated in human alcoholic livers and in mouse model of ALD. The importance of HSP90 on NLRP3 inflammasome activation in ALD was evaluated by administering HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) to mice subjected to ALD, and in vitro to bone marrow-derived macrophages (BMDM) stimulated with LPS and ATP. The effect of activation of HSF1/HSPA1A axis during HSP90 inhibition or direct activation during heat shock of BMDMs on NLRP3 activity and secretion of downstream cytokines was evaluated.

Results: We found positive correlation between induction of HSP90 and NLRP3 inflammasome genes in human alcoholic cirrhotic livers. Administration of 17-DMAG in mouse model of ALD significantly down-regulated NLRP3 inflammasome-mediated caspase-1 (CASP-1) activity and cytokine secretion, with reduction in ALD. 17-DMAG-mediated decrease in NLRP3 was restricted to liver macrophages. Using BMDMs, we show that inhibition of HSP90 prevented CASP-1 activity, and Gasdermin D (GSDMD) cleavage, important in release of active IL-1β and IL-18. Interestingly, activation of the heat shock factor 1 (HSF1)/HSPA1A axis, either during HSP90 inhibition or by heat shock, decreased NLRP3 inflammasome activity and reduced secretion of cytokines.

Conclusion: Our studies indicate that inhibition of HSP90 and activation of HSF1/HSPA1A reduce IL-1β and IL-18 via decrease in NLRP3/CASP-1 and GSDMD activity in ALD.

Keywords: 17-DMAG; Caspase-1; Gasdermin D; HSPA1A; alcohol-associated liver disease.

Figure 1.. Induction and correlation between hepatic HSP90 and inflammasome components in patients with alcoholic hepatitis and alcoholic cirrhosis

A) Transcript levels of HSP90, NLRP3, pro-CASP-1, PYCARD, pro-IL-1β, and pro-IL-18 evaluated by RNA sequencing in patients with normal livers (n=10), early ASH (n=12), severe AH (n=11), and severe AH (explant, n=18). (B) mRNA expression of HSP90, NLRP3, pro-CASP-1, PYCARD, pro-IL-1β, and pro-IL-18 in patients with normal livers (controls; n=15) or alcoholic cirrhosis (n=18). (C) Correlation between the mRNA expression of HSP90 with pro-CASP-1, PYCARD, pro-IL-1β, and pro-IL-18 evaluated by the Pearsons test. Data are represented as mean ± SEM, ** p <0.01, ***p<0.001, **** p<0.0001. ASH, alcoholic steatohepatitis; AH, alcoholic hepatitis; TPM, transcripts per million.

Figure 2.. Therapeutic targeting of HSP90 alleviates NLRP3 activation and reduces IL-1β protein production in alcoholic livers.

(A) C57BL/6 mice were fed control (pair-fed) or Lieber-DeCarli alcohol (ethanol-fed) diet and received a single dose of alcohol binge. Hepatic expression of Hsp90, Nlrp3, pro-Casp-1, Pycard, and pro-Il-1β was evaluated by RT-PCR. (B-F) Mice were subjected to pair-fed or Lieber-DeCarli diet with alcohol and administered with 50mg/kg 17-DMAG at the end of the feeding. mRNA expression of Hspa1a (B) and pro-Il-1β (C), protein levels of IL-1β (D) and IL-18 (E), and mRNA expression of Nlrp3 (F) was evaluated in the liver tissues (n=6–12). (G, H) Liver homogenates assessed for (G) Caspase-1 cleavage by western blot, and expression was quantitated and normalized to livers from pair-fed mice and (H) Caspase-1 activity was determined and normalized to pair-fed mice (n=8). Data are represented as mean ± SEM, * p <0.05, ** p <0.01, ***p<0.001, **** p<0.0001. EtOH, ethanol; DMAG, 17-Dimethylaminoethylamino-17-demethoxygeldanamycin.

Figure 3:. Inhibition of HSP90 reduces the expression of NLRP3 genes in alcoholic liver macrophages.

C57BL/6 mice were fed control (pair-fed) or Lieber-DeCarli alcohol (ethanol-fed) diet and received a single dose of alcohol binge. A group of mice were administered with 50mg/kg 17-DMAG at the end of the feeding. Hepatic macrophages isolated from these mice were assessed for the mRNA expression of pro-Il-1β (A), pro-Il-18 (B), Nlrp3 (C), pro-Casp-1 (D), Pycard (E), and Hspa1a (F) (n=4). Error bars represent mean ± SEM. *p<0.05. EtOH, ethanol; DMAG, 17-Dimethylaminoethylamino-17-demethoxygeldanamycin.

Figure 4.. Inhibition of HSP90 decreases NLRP3 inflammasome activity in LPS-induced BMDMs.

(A, B) BMDMs were treated with 1μM 17-DMAG for 6h. LPS (1μg/mL) was added to indicated group for the final 3h. RNA was assessed for the expression of (A) Nlrp3 and (B) pro-Il-1β. (C) For IL-18 induction, BMDMs were stimulated with 1μg/mL LPS for 8h. 1μM 17-DMAG was added for the final 6h and expression of pro-Il-18 was assessed. (D, E) BMDMs were stimulated with LPS (1μg/mL) for 4h (D) and 8h (E). 5mM ATP was added to the indicated group of cells. 1μM 17-DMAG was added with ATP for the final 1h and release of IL-1β (D) and IL-18 (E) was assessed in the culture supernatants by ELISA (n=6). (F, G) BMDMs were stimulated with LPS (1μg/mL) for 4h and 5mM ATP with or without 1μM 17-DMAG was added to the indicated group of cells for 1h. Proteolysis of pro-CASP-1 to CASP-1 p10 fragment (F) and GSDMD cleavage (G) was evaluated by western blotting (n=6). Data are represented as mean ± SEM, ***p<0.001, **** p<0.0001. LPS, lipopolysaccharide; 17-Dimethylaminoethylamino-17-demethoxygeldanamycin

Figure 5.. HSF1/HSPA1A axis contributes in decrease in LPS-mediated NLRP3 inflammasome signaling.

(A) WT and Hsf1^−/− BMDMs were heat shocked at 42°C for 1h and expression of Hspa1a was assessed (n=6). (B) WT and Hsf1^−/− BMDMs were stimulated with LPS (1μg/mL) for 4h and 5mM ATP was added to the indicated group of cells for 1h. 1μM 17-DMAG was added alongside ATP and release of IL-1β was evaluated by ELISA (n=6). (C, D) BMDMs were heat shocked at 42°C for 1h and stimulated with LPS (1μg/mL) for 2h. Real time PCR was carried out to evaluate the expression of C) pro-Il-1β and D) Nlrp3. (E, F) BMDMs were exposed to LPS (1μg/mL) for 4h before stimulation with 5mM ATP for 1h. Prior to ATP stimulation cells were heat shocked at 42°C for 1h and allowed to recover for 1h. (E) Culture supernatants were evaluated for secreted IL-1β. (F) Cell lysates were analyzed for Caspase-1 cleavage by western blotting. Data are represented as mean ± SEM, ***p<0.001, **** p<0.0001. WT, wild-type; LPS, lipopolysaccharide; HS, heat shock.

Figure 6:

Schematic representation demonstrating the effect of 17-DMAG in ameliorating NLRP3-mediated inflammasome activity.