Mice fed with a high-fat and choline-deficient (HFCD) diet develop non-alcoholic fatty liver disease (NAFLD) and are more susceptible to endotoxemia.

(A) Schematic illustration of the experimental design. (B) Survival curves of mice with NAFLD (n=5) and Chow diet (controls) (n=5) which were injected intraperitoneally with LPS (10mg/kg) and the survival rates were determined daily for 6 days (C and D) Serum ALT and Urea levels from mice with NAFLD and Chow, 6 hours after LPS (10 mg/kg) and PBS administration (n=5). (E) Effect of LPS inoculation on histopathology (H&E stain) of liver from mice with NAFLD or controls (Chow diet). The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

The expression of genes from the inflammatory pathways of IFNγ and TNFα are increased during the development of NAFLD in mice and humans.

(A) Volcano plots illustrating the fold change and P-value for gene expression comparisons between livers of NAFLD and healthy patients. Genes of interest are highlighted on the volcano charts. (B) Gene Ontology (GO) functional analysis of differentially expressed genes (DEGs). GO enrichment analysis of DEGs was conducted using DAVID, presenting the 20 most significantly (P < 0.05) enriched GO terms in biological process, molecular function, and cellular component categories. All adjusted statistically significant values are presented as negative log-transformed base 10 values. (C) UMAP plots depicting immune cells from livers of mice subjected to 30 weeks of HFCD diet compared to Chow diet. (D) Frequency distribution of immune cell populations. (E) Expression levels of CD14, CD274, IFNγ, and TNF genes within hepatic immune cell populations.

TNF-α and IFN-γ participate in susceptibility in mice with NAFLD to endotoxemia.

(A) IFNγ and (B) TNF-α secretion in the tissue livers of Chow and NAFLD mice injected with LPS (10mg/kg) (n = 5). (C) Serum ALT levels from mice with NAFLD and Chow and/or knockout of IFNγ (IFNγ-/-) and (D) TNFα (TNFR1R2-/-), 6 hours after 10 mg/kg LPS and PBS administration (n=5). (E) Effect of LPS inoculation on liver histopathology from mice with NAFLD and Chow and/or knockout of IFNγ-/- and TNF (TNFR1R2-/-). (F) Survival curves of mice with NAFLD (n=5) and Chow (n=5) and/or knockout of IFN (IFNγ-/-) and (G) TNF (TNFR1R2-/-), after intraperitoneal inoculation of LPS (10mg/kg). The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

IFN-γ secreted by NK cells increases susceptibility of animals with NAFLD to endotoxemia.

(A) CD4 IFN+ and (B) CD8 IFN+ T cell frequency in animals with NAFLD and Chow 6h after LPS inoculation. (C) displays survival curves of mice with NAFLD (n=5) and Chow (n=5) and T-cell knockout (RAg-/-). (D) and (E) represent the number of cells and frequency of NK IFN+ or IFN- in animals with NAFLD and Chow 6h after LPS inoculation (F) The survival curves of mice with NAFLD (n=5) and Chow (n=5) and/or depleted NK cells (Anti NK 1.1), 6 days after intraperitoneal inoculation of LPS (10mg/kg) (G) presents the serum ALT levels from mice with NAFLD and Chow of mice with NAFLD (n=5) and Chow (n=5) and/or depleted NK cells (Anti NK 1.1), 6 hours after 10 mg/kg LPS or PBS administration (n=5). (H) and (I) depict IFN and TNF-α secretion in the livers of Chow and NAFLD mice injected with LPS (10mg/kg) (n=5), respectively. (J) shows TNF-α secretion in the livers of Chow and NAFLD and/or knockout of IFNγ (IFNγ-/-) mice injected with LPS (10mg/kg) (n=5). The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

Neutrophils but not monocytes mediate susceptibility of animals with NAFLD to endotoxemia

(A) Representative flow cytometry plot showing monocytes (CD45+CD11b+MHC-Ly6C+Ly6G-) and neutrophils (CD45+CD11b+MHC-Ly6C+Ly6G+) and a number of monocytes (B) and neutrophils (C) in animals with NAFLD and Chow after LPS inoculation. (D) Effect of LPS inoculation on liver histopathology of mice with NAFLD and Chow treated with a CXCR2 inhibitor (CXCR2i) and neutrophil-depleted animals (anti-Ly6G). (E) Serum ALT levels of mice with NAFLD and Chow and/or CXCR2i and anti-Ly6G, 6 hours after administration of 10 mg/kg of LPS and PBS (n=5). (F) TNF-α secretion in the liver of Chow and NAFLD mice injected with LPS (10mg/kg) (n = 5). (G) Survival curves of mice with NAFLD (n=5) and Chow (n=5) treated with anti-Ly6G or (H) CXCR2i, and the survival rates were analysed daily for 6 days after intraperitoneal administration of LPS (10mg/kg). The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

IFN-γ induces PD-L1 expression and reduces apoptosis in mouse neutrophils with NAFLD.

(A) Representative flow cytometry plot showing the expression of PD-L1 in gated neutrophils and (B) the quantification of PD-L1+ neutrophils in the liver of mice with NAFLD or that received Chow diet, 6 hours after LPS inoculation (n=5 per group). (C) Representative flow cytometry plot showing the expression of PD-L1 in gated neutrophils and (D) the quantification of PD-L1+ neutrophils in the liver of mice with NAFLD and Chow and/or IFNγ knockout (IFNγ-/-) 6 hours after LPS inoculation (n=5 per group). (E) Representative flow cytometry plot showing the expression of PD-L1 and apoptosis (Annexin V+ 7AAD+) in gated neutrophils and (F) the frequency of apoptosis in neutrophils cultured in medium, LPS, IFN, or LPS+IFN for 24 hours. The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

IFN-γ-dependent secretion of TNF-α by PD-L1+ neutrophils induces susceptibility of animals with NAFLD to endotoxemia.

(A) Representative flow cytometry plot showing TNF expression in PD-L1+ neutrophils in the liver of animals with NAFLD or fed with Chow diet 6h after LPS inoculation. (B) Number of TNF+ neutrophils. (C) Number of TNF+PD-L1-neutrophils. (D) number of TNF+PD-L1+ neutrophils. (E) Representative flow cytometry plot showing TNF expression in PD-L1+TNF+ neutrophils in the liver of animals with NAFLD and Chow and/or depleted NK cells (anti-NK 1.1) 6h after LPS inoculation. (F) Number of PD-L1+ neutrophils. (G) Frequency of TNF+ neutrophils. The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

Anti PD-L1 treatment reduces PD-L1 neutrophils in animals with NAFLD during endotoxemia.

(A) Schematic illustration of experimental design. (B) Serum ALT levels of mice with NAFLD or feded with Chow and/or treated with anti-PDL1, 6 hours after administration of 10 mg/kg of LPS or PBS (n=5). (C) IFN and TNF-α secretion in the liver of Chow and NAFLD mice injected with LPS (10mg/kg) treated with anti-PDL1 (n = 5). (D) Survival curves of mice with NAFLD (n=5) and Chow (n=5) treated or not with anti-PD-L1. (E) Representative flow cytometry graph showing TNF expression in PD-L1+ neutrophils in the liver of animals with NAFLD and Chow and/or treated with anti-PD-L1 6h after LPS inoculation. (F) Effect of LPS inoculation in liver histopathology of mice with NAFLD and Chow treated or not with anti-PD-L1. (G and J) Frequency of PDL1 neutrophils, (H and K) Number of neutrophils, (I and L) Number of NK cells from animals with NAFLD and Chow and/or treated with anti-PDL1 after 6h of LPS inoculation. The results are expressed as the mean ± standard deviation (SD) from one representative experiment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined with the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated 2 times to ensure reproducibility of the findings.

Antibodies Cytometry