Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD - PubMed (original) (raw)

. 2017 Sep 22;7(1):12200.

doi: 10.1038/s41598-017-11744-6.

Chiara Rychlicki 1, Laura Agostinelli 1, Debora Maria Giordano 1, Melania Gaggini 2, Cristina Fraumene 3, Chiara Saponaro 2, Valeria Manghina 3 4, Loris Sartini 5, Eleonora Mingarelli 1, Claudio Pinto 1, Emma Buzzigoli 2, Luciano Trozzi 1, Antonio Giordano 5, Marco Marzioni 1, Samuele De Minicis 1, Sergio Uzzau 3 4, Saverio Cinti 5 6, Amalia Gastaldelli 2, Gianluca Svegliati-Baroni 7 8

Affiliations

Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD

Irene Pierantonelli et al. Sci Rep. 2017.

Erratum in

Abstract

Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A "multi-hit" theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3-/- and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3-/--HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3-/--HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Figure 1

Nlrp3−/−-HFHC mice showed increased weight gain and hepatic steatosis. Nlrp3−/−-HFHC mice showed increased weight gain (a), despite reduced calories intake/body weight ratio (b) and this was associated with reduced total energy expenditure (TEE) (c). Liver-to-body-weight ratio (d) correlated with hepatic lipid accumulation, evaluated by H&E staining (e) and triglyceride quantification in liver (f). These effects did not correlate with different intestinal lipid absorption (g). Mean ± SE: #p < 0.05 vs Nlrp3−/–-Chow diet; ##p < 0.01 vs Nlrp3−/−-Chow diet; ###p < 0.001 vs Nlrp3−/−-Chow diet; ####p < 0.0001 vs Nlrp3−/−-Chow diet; ç p < 0.05 vs Wt-Chow diet; çç p < 0.01 vs Wt-Chow diet; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Figure 2

Adipose tissue inflammation was higher in Nlrp3−/−-HFHC mice. Percentage of fat mass was increased in Nlrp3−/−-HFHC (a). MAC-2 immunohistochemistry (magnification, 40×) showed adipose tissue inflammation in Nlrp3−/−-HFHC mice (b). Gene expression of adipose tissue TNF-α (c) and MCP-1 (d) evaluated by qRT-PCR was also increased in Nlrp3−/−-HFHC mice. Mean ± SE: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Figure 3

Nlrp3−/−-HFHC showed higher hepatic lipid uptake and increased ROS production. HFHC increased PPAR γ1 in a genotype-independent manner, whereas Nlrp3−/−-HFHC mice showed higher expression of PPAR γ2 and its downstream effectors FABP4 and CD36 (a). Gene expression of ACC, FAS and SCD-1 (b). Measurements of plasma indexes of de novo lipogenesis and SCD-1 activity (c–d). Gene expression of PPAR α and of its downstream genes CPT1A and ACOX-1 and expression of the regulator of antioxidant response NRF2 (e). Representative images of liver sections stained with DHE (magnification, 20×) and its morphometric analysis (f). Mean ± SE: çp < 0.05 vs Wt-Chow diet; ççp < 0.01 vs Wt-Chow diet; çççp < 0.001 vs Wt-Chow diet; #p < 0.05 vs Nlrp3−/−-Chow diet; ##p < 0.01 vs Nlrp3−/−-Chow diet; ###p < 0.001 vs Nlrp3−/–Chow diet; ùp < 0.05 vs Wt-HFHC; ùùp < 0.01 vs Wt-HFHC; *p < 0.05; ***p < 0.001.

Figure 4

Figure 4

Nlrp3−/–HFHC had a more severe liver injury. qRT-PCR showed increased expression of TLR4, TLR5 and TLR9 in Nlrp3−/−-HFHC, whereas no differences were observed in TLR2 expression (a). F4/80 (b), MCP-1 (c) and Type I collagen (d) gene expression was higher in Nlrp3−/−-HFHC. The degree of liver injury was measured according to the Kleiner’s score (e). Mean ± SE: çp < 0.05 vs Wt-Chow diet; #p < 0.05 vs Nlrp3−/−-Chow diet; ###p < 0.001 vs Nlrp3−/−-Chow diet; ùp < 0.05 vs Wt-HFHC; ùùp < 0.01 vs Wt-HFHC; ùùùp < 0.001 vs Wt-HFHC; *p < 0.05; **p < 0.01.

Figure 5

Figure 5

Nlrp3−/−-HFHC-fed mice showed increased bacterial translocation and AMPs. HFHC induced bacterial translocation, expressed as turbidity of cultured mesenteric lymph nodes, that was further increased in Nlrp3−/−-HFHC-fed mice (a). Representative cropped Western blotting and densitometric analysis for caecal and ileal tight junction proteins (b-c) (full-length blots are presented in Supplementary Fig. S2). Regarding AMPs, in the caecum β-defensin 1 and 2 (BD1–2) expression was unchanged, whereas a lower expression of resistin-like molecule β (RELMβ) and angiogenin 4 (ANG4) has been observed after HFHC diet but independently from the genotype (d). In the ileum, HD4 and BD2 gene expression was not modified by neither diet or genotype, BD1 and RELMβ were reduced in HFHC-fed mice, whereas diet-induced reduction of ANG4 was more pronounced in NLRP3-deficient mice (e). Mean ± SE: *p < 0.05; **p < 0.01; ç p < 0.05 vs Wt-Chow diet; çç p < 0.01 vs Wt-Chow diet; ççç p < 0.001 vs Wt-Chow diet; #p < 0.05 vs Nlrp3−/−-Chow diet; ##p < 0.01 vs Nlrp3−/−-Chow diet; ###p < 0.001 vs Nlrp3−/−-Chow diet; ùù p < 0.01 vs Wt-HFHC.

Figure 6

Figure 6

Effect of diet and genotype on gut microbiota composition. Boxplots showing the most abundant microbial phyla. Features are ordered by decreasing median of the relative abundance among subjects. Boxplots are colored based on the relative phylum. Asterisks indicate statistical significance of phylum changes for each genotype when comparing the two diets (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 7

Figure 7

Effect of gut decontamination. Effect of antibiotic treatment on body weight (a), hepatic triglyceride deposition (b) and adipose tissue inflammation (c). Mesenteric lymph nodes colonization was decreased after antibiotics (d) and was associated with a significant reduction of hepatic TLR4, TLR5 and TLR9 gene expression (e). Antibiotics reduced hepatic gene expression of F4/80, MCP-1 and Type I collagen (f) and NAS Score (g) in Nlrp3−/−-HFHC mice. Mean ± SE: Mean ± SE: *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.05 vs Nlrp3−/−-Chow diet; ##p < 0.01 vs Nlrp3−/−-Chow diet; ###p < 0.001 vs Nlrp3−/−-Chow diet; ####p < 0.0001 vs Nlrp3−/−-Chow diet; çp < 0.05 vs Wt-Chow diet; ççp < 0.01 vs Wt-Chow diet; ùp < 0.05 vs Wt-HFHC; ùùp < 0.01 vs Wt-HFHC; ùùùp < 0.001 vs Wt-HFHC; §p < 0.05 vs Nlrp3−/−-HFHC; §§p < 0.01 vs Nlrp3−/−-HFHC; §§§p < 0.001 vs Nlrp3−/–HFHC.

Figure 8

Figure 8

Representative scheme of the mechanisms linking lack of NLRP3 and diet in adipose tissue inflammation and progression of liver injury. NLRP3 deficiency associated with a Western-lifestyle diet drive to dysbiosis and alteration of the intestinal barrier which increase bacterial translocation. Bacterial translocation induces adipose tissue dysfunction, known to increase fatty acids efflux to the liver. Hepatic PPAR γ2 expression, responsible of fatty acids uptake, is significantly higher and is associated with increased β-oxidation, which in turn leads to ROS synthesis. In addition, circulating gram-negative bacteria trigger liver injury via specific TLRs.

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