NLRP3 inflammasome activation is required for fibrosis development in NAFLD - PubMed (original) (raw)

NLRP3 inflammasome activation is required for fibrosis development in NAFLD

Alexander Wree et al. J Mol Med (Berl). 2014 Oct.

Abstract

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3(-/-) mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3(-/-) mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease. Key message: Mice with NLRP3 inflammasome loss of function are protected from diet-induced steatohepatitis. NLRP3 inflammasome gain of function leads to early and severe onset of diet-induced steatohepatitis in mice. Patients with severe NAFLD exhibit increased levels of NLRP3 inflammasome components and levels of pro-IL1β mRNA correlate with the expression of COL1A1.

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Conflict of interest statement

Conflict of interest The authors state that they have nothing to disclose.

Figures

Fig. 1

Fig. 1

Nlrp3−/− are protected from CDAA-induced hepatomegaly and liver damage. Nlrp3−/− and WT mice weaned after 21 days were placed on normal chow (NC) diet for 4 weeks and thereafter placed either on CDAA or CSAA diet for an additional 16 weeks (a); all groups gained weight throughout the investigational period (b). Notably, Nlrp3−/− mice were protected from CDAA-induced hepatomegaly (b) and liver damage—assessed by ALT serum levels (c). Analysis of liver histology revealed marked liver steatosis in all groups, with mice fed with CDAA showing larger droplets and higher variability in droplet size (d, e). WT mice fed with CDAA diet showed higher grades of inflammation when compared to WT on CSAA diet, and Nlrp3−/− mice on CDAA diet showed a trend toward lower grades when compared to diet-matched WT mice (e). TUNEL analysis showed a significant increase in WT mice fed with CDAA diet when compared to WT mice on CSAA diet; this increase was not found in Nlrp3 knockout mice (d, f)

Fig. 2

Fig. 2

Nlrp3 loss of function alleviates CDAA-diet-induced inflammation. Myeloperoxidase (MPO) expressing cells are significantly increased in mice fed with CDAA diet compared to mice on CSAA diet in both WT mice and Nlrp3−/− mice (a, b). Similarly, mRNA levels of CXCL2 and MCP1 were increased in WT mice on CDAA diet when compared to WT mice on CSAA diet and were significantly reduced in Nlrp3−/− when compared to WT mice (e, f).Expression of F4/80 was increased in WT mice when compared to _Nlrp3−/−_regardless of diet fed (a, c). mRNA levels of Ly6c (a, d), iNOS (g), and TNF-α (h) (markers for inflammatory or M1-polarized macrophages) were significantly increased in WT mice fed with CDAA when compared to WT mice fed with CSAA, while iNOS and TNF-α expression were significantly lower in Nlrp3−/− mice fed CDAA than WT mice fed with CDAA. Protein levels of pro-Casp1 were increased in Nlrp3−/− and WT mice fed with CDAA, while only WT mice showed the corresponding increase in Casp1 subunits P10 and P20 (d). The protein levels of pro-IL-1β were elevated in mice fed with CDAA diet compared to mice fed with CSAA diet and Nlrp3−/− mice on CDAA exhibited significantly lower IL-1β protein levels when compared to WT mice on CDAA (d)

Fig. 3

Fig. 3

Nlrp3−/− mice are protected from CDAA-induced liver fibrosis. Quantitative image analysis of liver sections stained with Sirius Red (a) reveals that CDAA diet significantly increases collagen deposition in WT mice when compared to WT mice fed with CSAA diet (b). Notably, Nlrp3−/− mice on CDAA diet showed a significantly lower percentage of Sirius Red stained liver area when compared to diet-matched WT mice (a, b). In line with this histological finding, mRNA levels of α-SMA, COL1A1, and MMP2 were significantly higher in liver samples of WT mice fed with CDAA diet when compared to WT mice fed with CSAA diet. Moreover, aforementioned mRNA levels were significantly reduced in Nlrp3−/− mice on CDAA diet when compared to diet-matched WT mice (c). Expression levels of TIMP1 were significantly increased in Nlrp3−/− and WT mice on CDAA when compared to mice fed with CSAA, while its expression was reduced in Nlrp3−/− mice when compared to WT mice on CDAA diet (c). Protein levels of α-SMA were increased in WT mice fed with CDAA diet when compared to WT mice fed with CSAA diet, and those levels were significantly reduced in Nlrp3−/− mice fed with CDAA when compared to WT mice on CDAA diet for 16 weeks (d)

Fig. 4

Fig. 4

NLRP3 activation leads to severe liver inflammation. WT and Nlrp3 mutant mice were weaned after 21 days and placed on a normal chow for 4 weeks prior to induction of the knock-in mutation (in Nlrp3 mutants) by subcutaneous injections of tamoxifen. Analysis of liver histology revealed that Nlrp3 mutant mice fed with normal chow develop significantly more liver inflammation when compared to WT mice (a, b). Nlrp3 mutant mice also showed a significant increase in myeloid cells (assessed via immunohistochemistry for MPO) when compared to WT mice (a, d). In line with this finding, mRNA levels of Casp1, pro-IL-1β (c), CXCL2, and ICAM1 (e) were significantly elevated (p<0.05) in Nlrp3 mutant mice. Total macrophages (a, f) (assessed via immunohistochemistry and mRNA levels for F4/80) and Ly6c–positive macrophages (a, h) were increased in Nlrp3 mutant mice when compared to WT mice. Also, macrophages from Nlrp3 mutant mice showed aninflammatory M1 profile as evidenced by increased iNOS (g) and unchanged Arg1 (i) mRNA expression when compared to WT mice

Fig. 5

Fig. 5

Short-term CDAA feeding induces liver fibrogenesis in Nlrp3 mutant mice. After receiving normal chow for 4 weeks, Nlrp3 mutant and WT mice were placed on either CDAA or NC diet and injected with tamoxifen for 4 weeks (a). Quantitative analysis of Sirius Red staining revealed significantly increased areas in Nlrp3 mutant mice fed with CDAA diet (b). Expression analysis of pro-fibrogenic genes (α-SMA and COL1A1) and genes associated with hepatic stellate cell activation (TIMP1 and CTGF) showed significant increases in Nlrp3 mutants fed with CDAA diet when compared to Nlrp3 mutants fed with NC diet (with the exception of TIMP1) and also WT mice fed with CDAA (c). Protein analysis showed significantly increased levels of desmin and α-SMA in mice fed with CDAA diet when compared to mice on NC with protein levels being further elevated in Nlrp3 mutants on CDAA diet (d)

Fig. 6

Fig. 6

NLRP3 inflammasome-associated proteins are increased in livers of patients with NASH and correlate with collagen deposition. The mRNA levels of NLRP3, pro-IL-1β, and pro-IL-18 were analyzed in the liver samples of a cohort with NAFLD and NASH. Table 1 describes the characteristics of the patient population. The mRNA levels of NLRP3 (a), pro-IL-18 (b), and pro-IL-1β (c) were significantly increased in patients with NASH when compared to patients with non-NASH NAFLD. Moreover, mRNA levels of pro-IL-1β were significantly correlated with levels of COL1A1 (d) (mRNA values are graphed at log10 scale)

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