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. 2023 Jan 26:14:1113883.
doi: 10.3389/fimmu.2023.1113883. eCollection 2023.

Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages

Affiliations

Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages

Charles Drummer 4th et al. Front Immunol. .

Abstract

Introduction: Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of 25% of the population and is a leading cause of cirrhosis and hepatocellular carcinoma. NAFLD ranges from simple steatosis (non-alcoholic fatty liver) to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs) and monocyte-derived macrophages, act as key players in the progression of NAFLD. Caspases are a family of endoproteases that provide critical connections to cell regulatory networks that sense disease risk factors, control inflammation, and mediate inflammatory cell death (pyroptosis). Caspase-11 can cleave gasdermin D (GSDMD) to induce pyroptosis and specifically defends against bacterial pathogens that invade the cytosol. However, it's still unknown whether high fat diet (HFD)-facilitated gut microbiota-generated cytoplasmic lipopolysaccharides (LPS) activate caspase-11 and promote NAFLD.

Methods: To examine this hypothesis, we performed liver pathological analysis, RNA-seq, FACS, Western blots, Seahorse mitochondrial stress analyses of macrophages and bone marrow transplantation on HFD-induced NAFLD in WT and Casp11-/- mice.

Results and discussion: Our results showed that 1) HFD increases body wight, liver wight, plasma cholesterol levels, liver fat deposition, and NAFLD activity score (NAS score) in wild-type (WT) mice; 2) HFD increases the expression of caspase-11, GSDMD, interleukin-1β, and guanylate-binding proteins in WT mice; 3) Caspase-11 deficiency decreases fat liver deposition and NAS score; 4) Caspase-11 deficiency decreases bone marrow monocyte-derived macrophage (MDM) pyroptosis (inflammatory cell death) and inflammatory monocyte (IM) surface GSDMD expression; 5) Caspase-11 deficiency re-programs liver transcriptomes and reduces HFD-induced NAFLD; 6) Caspase-11 deficiency decreases extracellular acidification rates (glycolysis) and oxidative phosphorylation (OXPHOS) in inflammatory fatty acid palmitic acid-stimulated macrophages, indicating that caspase-11 significantly contributes to maintain dual fuel bioenergetics-glycolysis and OXPHOS for promoting pyroptosis in macrophages. These results provide novel insights on the roles of the caspase-11-GSDMD pathway in promoting hepatic macrophage inflammation and pyroptosis and novel targets for future therapeutic interventions involving the transition of NAFLD to NASH, hyperlipidemia, type II diabetes, metabolic syndrome, metabolically healthy obesity, atherosclerotic cardiovascular diseases, autoimmune diseases, liver transplantation, and hepatic cancers.

Keywords: caspase-11; inflammation; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); pyroptosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
High-fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) and Caspase-11 deficiency decreases lipid droplet, steatosis score, and NAS score in HFD-induced NAFLD but does not change liver weight or gross anatomic fatty liver pathology. (A) Experimental diet timeline. 8-10 weeks old Wild-type (WT) male mice and Caspase-11 deficient (Casp11–/–) mice were fed HFD or normal chow diet (NCD) for 12 weeks. (B) Body weight (n = 15). (C) Liver weight (n = 15). (D) Plasma cholesterol levels (n = 3). (E) Representative images showed that HFD feeding changed liver color to light yellow color. (F) Representative 20X images of hematoxylin and eosin (H&E) staining showing hepatic steatosis in 12-week HFD-fed WT and Casp11–/– male mice compared to 12-week NCD. Scale bar 100 µm. (G). Description and grades of NAS scare. NAFLD activity score analysis indicated that lipids accumulation as judged by liver steatosis and ballooning is precedent to liver inflammation. In grade 1, when steatosis reaches 5-33%, and ballooning reaches mild, inflammation is minimal. Steatosis was in the graded section as: are 0, steatotic; 0% to 1, 5% greater than 5% to 33% of hepatocytes are steatotic; 2, greater than 33% to as: 66%; 0, absent; and 3, 1, greater mild (focal; than 66%. involving Ballooning fewer than 3 hepatocytes); 2, moderate (focal and involving 3 or more hepatocytes or multifocal); and 3, severe (multifocal, with more than 2 foci of 3 or more hepatocytes). (0 or 1 Inflammation focus per 20× was field); graded 2, as: mild 0, (2 absent; foci); 3, moderate (3 foci); and 4, severe (4 or more foci per 20× field). (H) Total NAS score for WT-NCD (n = 5), WT-HFD (n = 8), Casp11–/–NCD (n = 5) and Casp11–/–HFD (n = 8). (I) Hepatic steatosis score. (J) Hepatocyte ballooning. (K) Lobular inflammation. (L) Principal component analysis (PCA) demonstrating that WT-NCD, WT-HFD, Casp11–/–NCD, and Casp11–/–HFD mice are transcriptionally distinct (n = 3). (M) Volcano plot analysis showed the 3895 differentially expressed genes (DEGs) in the WT liver of 12-week HFD compared to12-week NCD control. Among 3895 DEGs, 2918 genes were significantly upregulated (red), and 977 genes downregulated (blue). (FC) > 1.5 and p < 0.05. Statistical Analysis: Bulk RNA-Seq analysis was performed using Qlucore Omics Explorer. PCA plot generated using significantly differentially regulated genes). Volcano plot generated using GraphPad Prism with significantly differentially regulated genes. Statistical Analysis: One-Way ANOVA. *p < 0.05, **p < 0.001, ***p < 0.0001 ****p < 0.0001. ns, Non-significant.
Figure 2
Figure 2
HFD upregulates the expressions of proinflammatory, NASH-related hepatic macrophage markers, GBPs, caspase-11, and increases N-terminal GSDMD cleavage. (A, B) The structure of hepatic acinus suggests that the liver is the first organ exposed to endotoxins-rich and Gram bacteria-rich blood in the body. Liver parenchymal and nonparenchymal cells are organized into structures called “acinus”. Hepatic acini form hexagonal structures with a central vein and portal triad at the vertices. The hepatic acinus can be histologically divided into three zones. Zone 1 represents the portal triad which includes a hepatic artery, a portal vein, and a bile duct. Zone 2 represents the parenchymal area, structurally consisting primarily of hepatocytes with a central vasculature composed of liver sinusoidal endothelial cells (LSECs). Mature immune cells including B cells, T cells, innate-like lymphoid cells (ILCs), natural killer cells (NK cells), and tissue-resident macrophages (Kupffer cells) reside in hepatic acinus zone 1 and 2. Zone 3 represents the central vein, the innermost hepatocytes, and infiltrating immune cells. The portal vein contains nutrient-rich, endotoxins-rich, and gram bacteria-rich blood. (C) Noncanonical pyroptosis is caspase-4 (human), caspase-11 (mouse) dependent. Guanylate binding proteins (GBPs) promote the outer membrane vesicles from gram-bacteria to activate caspase-11. Intracellular sources of inflammation (including LPS and oxidized phospholipids) directly bind to caspase-4/-11. Caspase-4/-11 cleaves gasdermin-D to initiate noncanonical pyroptosis. (D) The expressions of caspase-11 in the liver were in an upregulation trend in high-fat diet-fed wild-type mice. The microarray data were achieved from the NIH-NCBI-Geo-Profiles database (GDS4811). (E) Caspase-4 expression in 27 human tissues. The expression of caspase-4 in the liver is at a medium level among all 27 human tissues. Caspase-4 RNA-Seq data were analyzed from the NIH-NCBI-Gene database (https://www.ncbi.nlm.nih.gov/gene/837). (F) Caspase-4 expression in 45 hepatic cell types. Caspase-4 is expressed in all 40 immune cell types, the single-cell RNA-Seq data were analyzed from the MIT Broad Institute Single Cell RNA-Seq (scRNA-Seq) Porter database (https://singlecell.broadinstitute.org/single_cell/study/SCP1845/cross-tissue-immune-cell-analysis-reveals-tissue-specific-features-in-humans?genes=casp4%26tab=distribution#study-visualize). Among 45 immune cell types identified in scRNA-Seq, 19 immune cell types are significantly enriched in the human liver including dendritic cell 1 (DC1), DC2, classical monocytes, non-classical monocytes, erythrophagocytic macrophages, mononuclear phagocytes (MNP)/B doublets, age-associated B cells (ABCs), plasma cells, Plasmablasts, MNP/B doublets, T/B doublets, mucosal-associated invariant T (MAIT), T_CD4/CD8, T effector memory (Tem)/effector memory re-expressing CD45RA (emra)_CD8, T resident memory cell/effector memory cell (Trm/em)_CD8, gamma-delta T cell (Tgd)_CRTAM+, Cycling T cell & natural killer cell (NK), NK_CD16+, and NK-CD56bright_CD16-. (G) 12-week HFD promotes expression of HMΦ activation mediators in the WT liver. 8-10 weeks old male WT mice were fed with HFD for 12 weeks. Heatmap of significant, differentially regulated macrophage (HMΦ) genes. (H) Bulk RNA-seq expression (mean transcripts per kilobase million, TPM) of macrophage mediators. (I) Schematic representing caspase-4/11 pathway genes. (J) Bulk RNA-seq expression (mean TPM) of noncanonical pyroptosis-associated mediators. (K) Western blot analysis showed that HFD feeding increased caspase-11 and N-terminal GSDMD cleavage. Statistical Analysis: Bulk RNAseq analysis was performed using Qlucore Omics Explorer. Heatmap was generated using significantly differentially regulated genes (p.adj < 0.01). Differential gene expression presented as Z-score calculated from log2 transformed TPM. *P < 0.05, **p < 0.01.
Figure 3
Figure 3
High-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) drives the increase of NASH-related F4/80+ hepatic macrophages (HMΦs) in WT mice, which are caspase-11 activation-independent. 8-10 weeks old male WT and Casp11–/– mice were fed HFD for 12 weeks. (A) Bulk RNA-seq expression (TPM) of NASH-associated activated HMΦ genes. (B) Representative flow cytometry gating of HMΦ. Gated on CD45+ > CD11b+ Ly6G- > Ly6Clow MHCIIhigh. (C) F4/80+ mean fluorescence intensity (MFI) for HMΦ populations. Statistical Analysis: Bulk RNAseq analysis was performed using Qlucore Omics Explorer. PCA generated using significantly differentially regulated genes (p.adj < 0.01). Included genes were significant (p < 0.05) in multi-variant analysis (Two-Way ANOVA). Marked significance (*) determined by One-Way ANOVA. *p < 0.05, **p < 0.001, ***p < 0.0001 ****p < 0.0001. Flow cytometry data was analyzed with FlowJo, and statistical analysis was performed using Prism. One-Way ANOVA.
Figure 4
Figure 4
Hepatic inflammatory monocyte (IM) and monocyte-derived macrophage (MDM) caspase-11 deficiency protective against pyroptosis, WT bone marrow transplantation to Casp11–/– mice restored IM and MDM pyroptosis. 8-10 weeks old male WT and Casp11–/– mice were fed HFD for 12 weeks. (A) Western blot for noncanonical pyroptosis mediators. (B) Liver IL-1β concentrations. (C) Experimental design for bone marrow transplantation (BMT). (D) Representative flow cytometry gating of hepatic macrophages (HMΦ, Green, CD45+ > CD11b+ Ly6G- > Ly6Clow MHCIIhigh) inflammatory monocytes (IM, Red, CD45+ > CD11b+ Ly6G- > Ly6Chigh MHCIIlow). (E) Gating strategy for designing pyroptosis populations. HMΦs and IMs gated on GSDMD MFI vs. Casp11-Inhibitor MFI. RED: GSDMD MFI for IM (CD45+ > CD11b+ Ly6G- > Ly6Chigh MHCIIlow). GREEN: Percentage of the parent for HMΦ pyroptosis gating CD45+ > CD11b+ Ly6G- > Ly6Chigh MHCIIlow > Casp11 Activity vs GSDMD). (F) Schematic diagram showed that WT mice had more monocyte migration and more hepatic pyroptosis, however, Casp11–/– had less monocyte migration and hepatic pyroptosis resulting in an unchanged total number of hepatic macrophages. Statistical Analysis: Flow cytometry data was analyzed with FlowJo, and statistical analysis was performed using Prism. One-Way ANOVA. One-Way ANOVA. *p < 0.05, **p < 0.001, ***p < 0.0001 ****p < 0.0001. ns, Non-significant.
Figure 5
Figure 5
Caspase-11 significantly contributes to maintaining dual fuel bioenergetics- glycolysis and OXPHOS in macrophages potentially for cholesterol synthesis and trained immunity. Bone marrow macrophages were isolated from 3 WT and 3 Casp11–/– mice and treated with palmitic acid (500 µM) for 8 hours then pulled for seahorse analysis. (A) Seahorse XF96 Extracellular Flux Analyzer to measure extracellular acidification rate (glycolysis) of Casp11–/– vs WT BMDMs in palmitic acid supplemented medium. (B) Seahorse mitochondrial function assay of Casp11–/– vs WT BMDMs in palmitic acid supplemented medium.*P < 0.0, ****p < 0.0001.
Figure 6
Figure 6
The working model showed that HFD increased hepatic lipid accumulation (steatosis). HFD promotes increased gut microbiota Gram-negative bacteria-generated endotoxin LPS leading to elevations in circulating LPS and metabolic endotoxemia and increased LPS endocytosis and intracellular LPS. Gram-negative bacteria promoted by HFD enter the bloodstream and enter cells which are mediated by GBPs to increase intracellular bacteria and LPS to activate caspase-11. Caspase-11 activation is triggered by its interaction with LPS from Gram-negative bacteria. Being an initiator caspase, activated caspase-11 functions primarily through its cleavage of key substrates. GSDMD is the primary substrate of caspase-11, and the GSDMD cleavage fragment generated (GSDMD-NT) leads to the formation of pores in the plasma membrane and secretion of caspase-1 produced IL-1B into the extracellular space to promote liver inflammation (NASH) and subsequently increased hepatic pyroptosis and promotes NAFLD. Thus, caspase-11 functions as an intracellular sensor for LPS and an immune effector. Palmitic acid produced by lipolysis in HFD-fed mice caspase-11 activation and GSDMD cleavage. Furthermore, LPS-induced caspase-11 activation and GSDMD cleavage also maintain dual fuel bioenergetics-glycolysis and OXPHOS and. Casp11–/– decreased GSMDM cleavage and IL-1β secretion, reduced liver inflammation, and hepatic pyroptosis.

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