Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD
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.
Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common form of chronic liver disease, with prevalence estimates ranging from 25–45% of the adult population and increasing in parallel with that of obesity and diabetes in Western world1. NAFLD was first described in 1980 and is divided into the histological categories of (1) Non-Alcoholic Fatty Liver, which includes patients with isolated hepatic steatosis and patients with steatosis and mild non-specific inflammation, and (2) Non-Alcoholic Steatohepatitis (NASH), which is distinguished from the former by the additional presence of features of hepatocellular injury with or without fibrosis2,3. Among patients with NAFLD, those with NASH are much more likely to progress to cirrhosis and hepatocellular carcinoma (HCC) than those with only hepatic steatosis, and these conditions are predicted to become the most common indication for liver transplantation1,4. NAFLD is associated with features of modern lifestyle, characterized by increased dietary caloric intake of saturated and trans-unsaturated fatty acids (FAs), sugar-sweetened beverages and sedentary lifestyle5,6,7. A significant association has been found between fructose intake and the prevalence of diabetes, obesity and NAFLD and the degree of fibrosis in NASH8.
In recent years, a key role of the gut microbiota in the pathogenesis of obesity and NAFLD has been identified. Preclinical studies have shown that transplantation of microbiota from obese to lean mice was associated with the occurrence of metabolic alterations in the recipients9. Many interactions of gut microbiota with food, bile components and intestinal epithelium have been demonstrated to contribute to NAFLD pathogenesis and progression. The predominant mechanisms are an increased energy harvesting and alterations of intestinal barrier function, which can lead to translocation of bacterial products into the portal circulation and activation of inflammatory processes9,10. High fat diet (HFD) has been demonstrated to strongly affect gut microbiota composition, by increasing the abundance of energy harvesting microorganisms, such as Firmicutes and Proteobacteria and by decreasing Bacteroidetes11,12,13,14. On this regard, inflammasomes are multiprotein complexes that orchestrate host defense mechanisms against infectious agents but their contribution to innate immunity might likely include the control of gut microbiota load and composition15. In agreement with this, genetic NLRP3-inflammasome deficiency-associated dysbiosis resulted in abnormal accumulation of bacterial products into the portal circulation and increased severity of liver injury during a methionine/choline-deficient diet (MCD)16. Moreover, NLRP3-inflammasome deficient mice develop exacerbated colitis in the dextran sulfate sodium (DSS) model17,18,19.
NLRP3 is also implicated in the pathogenesis of cardiovascular disease, obesity and type-2-diabetes, while controversial data exist on inflammasome activation in liver disease20,21,22,23. We have previously reported that during the development of liver injury, inflammasome components were upregulated in the liver and downregulated in the gut and this was associated with microbiota modifications and bacterial translocation24. On the other hand, an NLRP3 selective inhibitor improved NAFLD pathology and fibrosis in obese diabetic mice25,26,27. Thus, a complex balance exists between diet, gut microbiota, intestinal homeostasis and NLRP3 function, and controversial results have been provided concerning their respective role in the progression of liver injury. Thus, aim of this study was to provide an in depth evaluation of the relationship between innate immunity and Western-lifestyle diet in the progression of NAFLD.
Nlrp3−/−-HFHC gained more weight compared to WT-HFHC mice (Fig. 1a) despite a progressive reduction in the caloric intake/body weight ratio (Fig. 1b). The increased body weight in Nlrp3−/−-HFHC was associated with reduced total energy expenditure (TEE) (Fig. 1c). This higher average body weight correlated with an increased liver-to-body weight ratio in Nlrp3−/−-HFHC-fed mice (Fig. 1d). To assess whether the increased liver weight observed in Nlrp3−/−-HFHC-fed mice was associated with higher fat deposition in the liver, we evaluated hepatic fat content. First, liver sections were stained for Hematoxylin and Eosin (H&E). H&E staining revealed features of micro and macrovesicular steatosis in HFHC-fed mice, which were higher in Nlrp3−/− mice (Fig. 1e). To further confirm the increased lipid deposition in the liver of mice lacking NLRP3, we quantified triglyceride content, which was found significantly enhanced in these mice compared to WT animals (p < 0.01) (Fig. 1f). Lastly, to study whether the changes in body weight and composition could reflect different intestinal lipid absorption, quantification of triglycerides in the stool was performed. This analysis revealed that HFHC diet decreased faecal excretion of triglycerides compared to chow diet independently from the genotype, thus indicating that differences in lipid absorption cannot explain the higher hepatic triglyceride content in Nlrp3−/−-HFHC mice. (Fig. 1g).
Increased adiposity and adipocyte dysfunction are known to contribute to metabolic diseases by altering adipose tissue-derived secretory factors, and adipose tissue inflammation represents a main mechanism in the pathogenesis of NAFLD5,6,7. Nlrp3−/−-HFHC mice had a greater increase in body fat mass (Fig. 2a) that was associated with adipose tissue inflammation. To assess the degree of fat inflammation, a morphological analysis by immunostaining of epididymal fat with the anti-MAC-2 antibody was performed. MAC-2 is a protein known to be expressed by activated macrophages that infiltrate hypertrophic, obese fat and surround death adipocytes, giving rise to distinctive morphological pictures called crown-like structures (CLS)28. Nlrp3−/−-HFHC mice exhibited a significant increase of the density of CLS when compared with WT-HFHC (Fig. 2b). Similarly, mRNA expression of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in adipose tissue was significantly increased in Nlrp3−/−-HFHC mice (Fig. 2c,d). Thus, the combination of immunohistochemical and gene expression data indicates a higher degree of adipose tissue inflammation in Nlrp3−/−-HFHC mice.
In addition to adipose tissue evaluation, we measured the expression of genes involved in hepatic lipid metabolism, in order to investigate the mechanisms behind the increased hepatic steatosis. HFHC increased PPAR γ1 independently from the genotype, whereas Nlrp3−/−-HFHC mice showed higher gene expression of PPAR γ2 and of its downstream effectors, such as the fatty acid binding protein-4 (FABP4) and CD36, which are involved in lipid uptake and storage (Fig. 3a)29. HFHC diet enhanced de novo lipogenesis (DNL)30, independently from the genotype, as shown by the expression of the acetyl Co-A carboxylase-1 (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) (Fig. 3b). An increased plasmatic ratio of palmitic/linoleic acid (16:0/18:2), as a marker of DNL (Fig. 3c), also confirmed mRNA results. Conversely, palmitoleic/palmitic acid (16:1/16:0) ratio, an index of SCD-1 activity, was increased in Nlrp3−/−-HFHC mice only (Fig. 3d). HFHC diet activated hepatic fatty acid oxidation, as shown by the expression of PPAR α and of its downstream genes palmitoyltransferase 1 A (CPT1A, a key enzyme in mitochondrial fatty acids β-oxidation) and Acyl CoA oxidase-1 (ACOX-1), a rate-limiting enzyme in peroxisomal fatty acids β-oxidation) (Fig. 3e)31. However, CPT1A expression was significantly increased in Nlrp3−/− compared to WT (p < 0.05), which was associated with lower mRNA levels of the “master regulator” of the antioxidant response NRF2 (Fig. 3e)32. Since excessive mitochondrial fatty acid β-oxidation is known to induce oxidative stress33,34, we measured the concentration of ROS by dihydroethidium (DHE) staining and found the highest concentration of anion superoxide in Nlrp3−/−-HFHC (Fig. 3f). These data indicate that increased steatosis in Nlrp3−/−-HFHC might be mediated by higher fatty acid uptake, which activates fatty acid catabolism and oxidative stress, also favored by decreased anti-oxidant response.
Oxidative stress due to ROS accumulation can contribute to either promotion or progression of chronic liver disease. Furthermore, hepatic injury can progress through intracellular pathways mediated by different Toll-like receptors (TLRs) via recruitment of various adaptor proteins9,10,35. Nlrp3−/−-HFHC mice showed higher expression of TLR4 (that recognizes LPS, a gram-negative bacteria wall component)36, TLR5 (known to recognize bacterial flagellin from invading mobile bacteria)37 and TLR9 (a specific receptor for double-stranded bacterial DNA)36 compared to WT-HFHC (Fig. 4a). No differences were observed in TLR2 expression, that mediates host response to gram-positive bacteria (Fig. 4a)36. HFHC diet increased mRNA expression of F4/80, a marker of macrophage infiltration, which was further enhanced in Nlrp3−/− mice (Fig. 4b). Differently, gene expression of the pro-inflammatory cytokine MCP-1 and of Type I collagen were increased only in Nlrp3−/−-HFHC-fed mice (Fig. 4c,d). Finally, hystological evaluation assessed by the NAS score38 showed the highest degree of liver injury in Nlrp3−/−-HFHC-fed mice (Fig. 4e).
Western diet-induced NAFLD can be associated with dysbiosis, gut barrier dysfunction and increased intestinal permeability leading to bacterial translocation, that drives the progression toward NASH9. We then looked at mesenteric lymph nodes cultures, as a measure of bacterial translocation24, and found the highest increase in bacterial growth in Nlrp3−/−-HFHC (Fig. 5a), indicating a combined role of Western-lifestyle diet and NLRP3 deficiency in inducing bacterial translocation. The maintenance of a proper gut barrier against bacterial translocation is mediated by several mechanisms, including the secretion of enzymes/proteins and mechanical barrier such as the presence of tight junctions, which exert a key role in the protection against intraluminal microorganisms39. To study the eventual alterations of intestinal barrier associated to bacterial translocation, Western blot for tight junction proteins was performed both in the caecum and in the ileum. Caecal Zonula Occludens-1 (ZO-1), an intestinal protein which binds different transmembrane proteins and modulates intestinal permeability by disassembling the intercellular tight junctions40, was decreased in HFHC-fed mice and in chow-fed Nlrp3−/− (Fig. 5b). Differently, protein expression of caecal Occludin, a transmembrane protein which forms the core of the tight junctions and controls ion selectivity and permeability of the paracellular pathway between adhering cells41, did not change (Fig. 5b). To also evaluate eventual alterations of the gut barrier in the upper part of the intestine, we analyzed ZO-1 and Occludin protein expression in the ileum and found a trend similar to what was observed in the caecum (Fig. 5c).
Thus, we measured the expression of antimicrobial peptides (AMPs) both in large and small intestine (Fig. 5d,e). At colon level, expression of β-defensin 1 and 2 (BD1-2), antimicrobial peptides produced by epithelial cells, did not change among the experimental groups (Fig. 5d)39. The expression of the inducible BD2 was also not modified in the ileum, where we could observe a diet-dependent decrease of the constitutive BD1 (Fig. 5e). α-defensin 4 (HD4) expression, the most abundant peptide produced by ileal Paneth cells, was unaffected by both diet and genetic background (Fig. 5e). A lower expression of Resistin-like molecule β (RELMβ), an antimicrobial peptide secreted by goblet cells, which stabilizes mucin polymer and regulates intestinal mucin secretion42, was found decreased in both WT and Nlrp3−/− mice after HFHC, either in the caecum or in the ileum (Fig. 5d,e). Finally, Angiogenin-4 (ANG4), an antimicrobial peptide which belongs to a family of RNases and possesses a well-known antibacterial and antiviral function42, was significantly lower in HFHC-fed mice, independently from the genotype in the caecum, whereas was significantly (p < 0.01) reduced in the ileum of Nlrp3−/−-HFHC mice compared to WT-HFHC (Fig. 5d,e). Taken together these results indicate that HFHC alters the physical and chemical mechanisms involved in the maintenance of gut barrier functions, by impairing tight junctions expression and AMPs secretion. In addition, NLRP3 deficiency further affects intestinal barrier by decreasing the expression of ANG4. Thus, our data suggest that the combination of a Western-lifestyle diet with immunological dysregulation due to NLRP3 deficiency alters intestinal homeostasis leading to increased bacterial translocation.
The Western-lifestyle diet can induce gut dysbiosis that, in turn, has been implicated in bacterial translocation and worsening of liver injury in NAFLD9. Further, lack of NLRP3 affects, per se, the composition of gut microbiota16. However, no data exist regarding the effect of a Western-lifestyle diet on Nlrp3−/− host gut microbiota. Thus, we evaluated the different gut microbial composition that possibly occurs in WT and Nrlp3−/− mice with either HFHC or chow diet (Supplementary Fig. S1). The microbial communities associated to each group were compared according to their α-diversity, richness and β-diversity. A dramatic reduction in α-diversity and richness values was induced by HFHC independently from the genotype. Analysis of β-diversity clearly illustrates group-level differences in the taxonomic composition, with the highest variation according to diet treatment and the variation due to genetic background being more evident in HFHC-fed mice (Supplementary Fig. S1).
According to NLRP3-inflammasome deficiency and diet, significant differences between microbial taxa were found (Fig. 6). In WT mice, as expected, Firmicutes/Bacteroidetes ratio was higher in HFHC-fed animals due to a reduction of Bacteroidetes abundance (Fig. 6b). HFHC also promoted an increased abundance of gram-negative Proteobacteria and a reduction on Verrucomicrobia (Fig. 6b). Akkermansia was the most prevalent genus of Verrucomicrobia and accounted for 4.6 ± 4.1% of the microbial community in WT-chow (Fig. 6a) and was almost undetectable (0.04 ± 0.05%) in WT-HFHC (Fig. 6b). The increased abundance of Proteobacteria concerned mostly OTUs classified at the family level as Desulfovibrionaceae, including Desulfovibrio and Bilophila pathobiont genera, representing 56.5% and 42.2% of this family group respectively. In Nlrp3−/− mice, HFHC diet induced similar changes in gut microbial composition but to a higher extent (Fig. 6d). Energy harvesting Firmicutes were significantly higher in HFHC diet compared to Nlrp3−/− mice fed chow, and Proteobateria were more consistently increased (Fig. 6; p < 0.001). Noteworthy, Verrucomicrobia showed great variation in its relative abundance when mice were challenged by diet (HFHC) and by innate immunity impairment (Nlrp3−/−) (Fig. 6d). In Nlrp3−/−mice fed with chow (Fig. 6c), Akkermansia abundance was similar to that observed in WT mice on the same diet (Fig. 6a), while its relative abundance showed an impressive increase at 14.62 ± 5.82% solely in the Nlrp3−/−mice fed with HFHC diet (Fig. 6d). Taken together, these data suggest that lack of NLRP3 does not lead to a derangement of gut microbial composition in chow-fed mice. However, the impact of HFHC in mice lacking NLRP3-inflammasome functions was more dramatic than in WT mice, suggesting that this pivotal player of the innate immunity might contribute to control dysbiosis induced by Western-lifestyle diet.
To confirm that intestinal dysbiosis and permeability contribute to NAFLD severity in Nlrp3−/−-HFHC, antibiotic treatment was performed24. We first analyzed whether antibiotics could modify microbiome composition. Antibiotic treatment, as expected16,24, had a dramatic effect on bacterial composition (Fig. 6e–f and Supplementary Fig. S1). Gram-negative bacteria (i.e., Bacteroidetes and Proteobacteria) were substantially depleted by antibiotics, while, not surprisingly, cell wall-free Tenericutes were the most prevalent taxa adapted to the antibiotic pressure. Similarly, Euryarchaeota was among the most abundant phyla represented after antibiotic treatment, as it comprises a large number of genera either cell-wall free or with a pseudo-peptidoglycan wall (i.e., Methanobacteria) (Fig. 6e,f).
This modified microbiota was associated with a different phenotype in Nlrp3−/−-HFHC mice. Antibiotic treatment was associated with reduced weight gain in Nlrp3−/−-HFHC compared to WT-HFHC (Fig. 7a). In Nlrp3−/−-HFHC, antibiotics decreased hepatic triglyceride accumulation (Fig. 7b) and adipose tissue inflammation (Fig. 7c). Furthermore, following antibiotic treatment, bacterial growth in cultured lymph nodes was lowered (Fig. 7d) and this was associated with reduced TLR4, TLR5 and TLR9 expression in the liver of Nlrp3−/−-HFHC mice (Fig. 7e), indicating a role of bacterial products translocation in these processes. Reduced bacterial translocation in Nlrp3−/−-HFHC mice was associated with decreased gene expression of pro-inflammatory markers, such as F4/80 and MCP-1 and of Type I collagen (Fig. 7f) together with a significant reduction in NAS score (Fig. 7g).
An exciting hypothesis in the pathogenesis of NAFLD/NASH is that multiple steps of interaction between intestinal microbiota and the host might be the cause of derangement in either glucose and/or lipid metabolism leading to metabolic diseases9. In this scenario, diet-induced intestinal dysbiosis provides pathogen-associated molecular patterns (PAMPs), that cross gut epithelial barrier and activate the innate immune response (by mean of TLRs and “the inflammasomes”) with the release of inducible antimicrobial peptides16,43,44,45. While the lack of inflammasome has been reported to protect from the development of metabolic syndrome and associated diseases35,46, this possibility in NAFLD is still debated16,25. Activation of inflammasome components has been shown in patients with chronic hepatic injury, particularly NASH, and in experimental models of NAFLD21,23. However, lack of NLRP3 was associated with reduced steatosis in some studies but it had no effects in others21,46,47,48. In the choline-deficient L-amino acid-defined (CDAA) model of liver injury, although not associated with features of the metabolic syndrome, inflammasome activation occurred during steatohepatitis development, whereas Nlrp3−/− mice were protected during CDAA treatment22,23. More recently, an NLRP3 selective inhibitor improved NAFLD pathology either in the appetite-defective foz/foz mice overnutrition model fed an atherogenic diet or in the MCD model25. These controversial results might be explained by the different methods used and by the complex role of inflammasome components in regulating cellular homeostasis5.
In the present study, we planned a set of experiments to elucidate the role of inflammasome in the development of NAFLD. Given the pivotal role of food in the development of dysbiosis, metabolic disorders and NAFLD, we used a well-defined model of Western diet associated with increased body weight, fat mass, fasting glucose and insulin-resistance, with development of minimal fibrosis after 12 weeks49. In our model, Nlrp3−/− mice showed increased liver steatosis, macrophage infiltration and liver injury (NAS score). Furthermore, Nlrp3−/−-HFHC mice increased fat mass and adipose tissue inflammation (MAC-2 positive staining and gene expression of TNF-α and MCP-1 in adipose tissue), indicating the occurrence of “inflamed” adipose tissue and the development of adipose tissue insulin-resistance, that is associated with fatty acids overflow and hepatic fat accumulation as a consequence50,51. This study is in agreement with and extends previous observations showing that Nlrp3−/− mice fed MCD diet, a model associated with steatohepatitis but with a cachectic phenotype, had worsened liver injury, and this was attributed to a pathogenetic microbiota16. However, no detailed mechanisms on the effect of NLRP3 deficiency were obtained in a Western-lifestyle diet. Studies evaluating the effect of a Western-lifestyle diet on microbiota composition in the presence of innate immunity defects are lacking.
The worsen degree of adipose tissue inflammation and liver injury in NLRP3 deficient mice was associated with specific modifications of gut microbiota composition. Specifically, in the caecal content of Nlrp3−/−-HFHC mice we observed: a) an increased abundance of Proteobacteria, the main pathobiont bacteria expressing endotoxins associated with the highest degree of liver injury in a model of HFD and fibrosis24, b) a significant increase of mucus degrading bacteria such as Akkermansia muciniphila (phylum Verrucomicrobia) and Desulfovibrio (phylum Proteobacteria). Akkermansia muciniphila is a gut commensal bacteria that resides in the mucus layer and exerts mucin-degrading function with a controversial role in basal metabolism homeostasis and immune tolerance toward commensal bacteria52. To this end, preparations of Akkermansia muciniphila as therapeutic options to target human obesity and associated disorders have been proposed53,54. Indeed, although Akkermansia can be considered as a regulator of the thickness of gut barrier, exaggerate mucus degradation has been shown to contribute to intestinal and systemic inflammation by increased layer crossing of luminal antigens13,55. More recently, in vivo isotope labeling combined with metaproteomics showed that the active microbiome in HFD-fed mice increased bacterial taxa as Verrucomicrobia and Desulfovibrionaceae, and this active microbiome affected metabolic pathways such as energy production and carbohydrate metabolism56. Again HFD was associated in mice with the expansion of Firmicutes (appearance of Erysipelotrichi), Proteobacteria (Desulfovibrionales) and Verrucomicrobia, a decrease in AMPs expression, increased intestinal permeability and finally a decrease in ileal secretion of chloride, likely responsible for massive alteration in mucus phenotype13. As suggested in this last study, a collapse of the mucus barrier colonized by a dysbiotic microbiota might promote the emergence of mucus degrading bacteria (Akkermansia and Desulfovibrio) that may further participate in the alteration of the mucus barrier.
Several mechanisms, in addition to the mucus layer, act simultaneously in order to prevent bacterial translocation, such as the efficiency of the immune function, the production of AMPs, and the presence of active and functional tight junctions39. No data are available on the effects of a Western-lifestyle diet on these mechanisms in the presence of innate immunity defects. In our hands, both the Western diet and the genetic background were independently associated with intestinal barrier alterations. While no differences in Occludin were observed, protein expression of ZO-1, which is involved in the assembly of tight junctions57 was reduced in WT-HFHC, Nlrp3−/− and Nlrp3−/−-HFHC. On the other hand, bacterial translocation was observed in HFHC mice, more prominently in Nlrp3−/−, indicating a major role of the modified microbiota on this process.
In our study, no co-housing experiments were performed. Henao-Mejia & coll.16 have already shown that in this condition a transmissible microbiota present in inflamm