Mice fed with a high-fat and choline-deficient (HFCD) diet develop Metabolically Dysfunctional-Associated Fatty Liver Disease (MAFLD) and are more susceptible to endotoxemia.

(A) Schematic illustration of the experimental design. (B) Survival curves of mice with MAFLD (n=5) and Chow diet (controls) (n=5) which were injected intraperitoneally with LPS (10mg/kg) and the survival rates were determined daily for 7 days (C and D) Serum ALT and Urea levels from mice with MAFLD 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 MAFLD 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 IFN-γ and TNF-α is increased during the development of MAFLD in mice and humans.

(A) Volcano plots illustrating the fold change and P-value for gene expression comparisons between livers of MAFLD 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 the 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, IFN-γ, and TNF-α genes within hepatic immune cell populations.

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

(A) IFN-γ and (B) TNF-α secretion in the tissue livers of Chow and MAFLD mice injected with LPS (10mg/kg) (n = 5). (C) Serum ALT levels from mice with MAFLD 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 MAFLD and Chow and/or knockout of IFNγ-/- and TNF-α (TNFR1R2-/-). (F) Survival curves of mice with MAFLD (n=5) and Chow (n=5) and/or knockout of IFN-γ (IFN-γ-/-) and (G) TNF-α (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 MAFLD to endotoxemia.

(A) CD4 IFN-γ+ and (B) CD8 IFN-γ+ T cell frequency in animals with MAFLD and Chow 6h after LPS inoculation. (C) displays survival curves of mice with MAFLD (n=5) and Chow (n=5) and T-cell knockout (RAg-/-). (D) Serum ALT levels from mice with MAFLD in knockout of Rag -/-, 6 hours after 10 mg/kg LPS (n=5). (E) and (F) represent the number of cells and frequency of NK IFNγ+ or IFN-γ- in animals with MAFLD and Chow 6h after LPS inoculation (G) The survival curves of mice with MAFLD (n=5) and Chow (n=5) and/or depleted NK cells (Anti NK 1.1), 6 days after intraperitoneal inoculation of LPS (10mg/kg) (H) presents the serum ALT levels from mice with MAFLD and Chow of mice with MAFLD (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). (I) and (J) depict IFN-γ and TNF-α secretion in the livers of Chow and MAFLD mice injected with LPS (10mg/kg) (n=5), respectively. (K) shows TNF-α secretion in the livers of Chow and MAFLD and/or knockout of IFN-γ (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 MAFLD 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 MAFLD and Chow after LPS inoculation. (D) Effect of LPS inoculation on liver histopathology of mice with MAFLD and Chow treated with a CXCR2 inhibitor (CXCR2i) and neutrophil-depleted animals (anti-Ly6G). (E) Serum ALT levels of mice with MAFLD 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 MAFLD mice injected with LPS (10mg/kg) (n = 5). (G) Survival curves of mice with MAFLD (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 MAFLD.

(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 MAFLD 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 MAFLD 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-γ, or LPS+ IFN-γ 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 MAFLD to endotoxemia.

(A) Representative flow cytometry plot showing TNF-α expression in PD-L1+ neutrophils in the liver of animals with MAFLD or fed with Chow diet 6h after LPS inoculation. (B) Number of TNF-α+ neutrophils. (C) Number of TNF-α+PD-L1-neutrophils. (D) number of TNF-α+PD-L1+ neutrophils. (E) Representative flow cytometry plot showing TNF-α expression in PD-L1+TNF+ neutrophils in the liver of animals with MAFLD 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-α+ 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 MAFLD during endotoxemia.

(A) Schematic illustration of experimental design. (B) Serum ALT levels of mice with MAFLD 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-γ and TNF-α secretion in the liver of Chow and MAFLD mice injected with LPS (10mg/kg) treated with anti-PDL1 (n = 5). (D) Survival curves of mice with MAFLD (n=5) and Chow (n=5) treated or not with anti-PD-L1. (E) Representative flow cytometry graph showing TNF-α expression in PD-L1+ neutrophils in the liver of animals with MAFLD and Chow and/or treated with anti-PD-L1 6h after LPS inoculation. (F) Effect of LPS inoculation in liver histopathology of mice with MAFLD 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 MAFLD 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.

Validation of the HFCD-induced MAFLD experimental model based on liver weight, blood glucose levels, and histological analysis (H&E and Picro Sirius staining).

(A) Liver height, (B) plasma glucose, and (C) representative liver histological sections of lipid accumulation, steatotic morphology and collagen deposition (Picro Sirius Red staining) of mice fed a high-fat, choline-deficient (HFCD) or control chow diet for two weeks and subsequently challenged or not with LPS (10 mg/kg, i.p.)(n=5) (Data 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 as *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated at least twice to ensure reproducibility of the findings

The IFNγ-deficient mice display LPS responses comparable to controls.

(A–B) Splenocytes isolated from C57BL/6 and IFNγ⁻/⁻ mice fed with Chow or HFCD for two weeks were stimulated in vitro with LPS (100 ng/mL) for 24 hours. Cytokine levels of (A) IL-6 and (B) TNF-α were quantified in the culture supernatants. Data are expressed as mean ± standard deviation (SD) from one representative experiment (n = 5 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated twice to ensure reproducibility.

NK cells produce high levels of IFN-γ during endotoxemia in MAFLD

(A) Representative flow cytometry dot plot showing CD4⁺IFN-γ⁺ T cells from the liver of mice fed a high-fat, choline-deficient (HFCD) or chow diet for two weeks and subsequently challenged or not with LPS. (B) Representative dot plot of hepatic CD8⁺IFN-γ⁺ T cells under the same experimental conditions. (C) Representative dot plot showing NK cells (NK1.1⁺CD3⁻) from the liver of HFCD and chow-fed mice challenged or not with LPS. (D) Representative dot plot of IFN-γ expression by NK cells (NK1.1⁺CD3⁻) from the same groups.

Anti-NK1.1 treatment depletes NK cells in animals with MAFLD during endotoxemia

(A) Representative flow cytometry dot plot showing depletion of NK cells (NK1.1⁺CD3⁻) in the liver of mice fed a high-fat, choline-deficient (HFCD) or chow diet for two weeks and challenged with LPS, following treatment with anti-NK1.1 antibody.

Monocytes do not participate in the susceptibility of mice with MAFLD to endotoxemia

(A) Representative flow cytometry dot plot showing neutrophils (CD45⁺CD11b⁺Ly6G⁺) and monocytes (CD45⁺CD11b⁺Ly6G⁻) in the liver of C57BL/6 and CCR2⁻/⁻ mice fed a high-fat, choline-deficient (HFCD) or chow diet for two weeks and challenged with LPS. (B) Survival curves of C57BL/6 and CCR2⁻/⁻ mice under the same experimental conditions. (C) Representative dot plot of total leukocytes (CD45⁺) in the liver of C57BL/6 and CCR2⁻/⁻ mice after LPS challenge. (D) Frequency of leukocytes (CD45⁺) in the liver of C57BL/6 and CCR2⁻/⁻ mice fed HFCD or chow diet and challenged with LPS (n=5). Data are representative of one experiment reproduced independently twice with similar results. 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. Results are expressed as the mean ± standard deviation (SD).

Neutrophils are the main PD-L1–expressing subset among CD45⁺CD11b⁺ leukocytes in MAFLD.

(A and B) Representative flow cytometry histogram showing PD-L1 expression on neutrophils (CD45⁺CD11b⁺Ly6C⁺Ly6G⁺) and monocytes (CD45⁺CD11b⁺Ly6C⁺Ly6G⁻) isolated from the liver of mice fed a high-fat, choline-deficient (HFCD) or control chow diet for two weeks and challenged with LPS (10 mg/kg, i.p.) (n=5) (C) Representative dot plot showing PD-L1 expression within total hepatic CD45⁺CD11b⁺ leukocytes from chow- and HFCD-fed animals for two weeks, either challenged or not with LPS. (D) Representative dot plot depicting the relative proportions of neutrophils(CD45⁺CD11b⁺Ly6C⁺Ly6G⁺) and monocytes (CD45⁺CD11b⁺Ly6C⁺Ly6G⁻) within total CD45⁺CD11b⁺ leukocytes in the liver of chow- and HFCD-fed mice after LPS challenge (n=5). Data 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 as *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was independently repeated at least twice to ensure reproducibility of the findings.

CXCR2i treatment reduces IFN-γ in the liver tissue of animals with MAFLD during endotoxemia

(A) IFN-γ levels in the liver of mice fed a high-fat, choline-deficient (HFCD) or chow diet for two weeks, challenged with LPS, and treated with either anti-Ly6G antibody or a CXCR2 inhibitor (CXCR2i) (n=5).. (B) IL-10 levels in the liver of the same experimental groups (n=5). The data are representative of one experiment reproduced independently twice with similar results. 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. Results are expressed as the mean ± standard deviation (SD).

TNFR1/R2⁻/⁻ animals do not show reduced neutrophil infiltration in the hepatic tissue of mice with MAFLD during endotoxemia.

(A) Representative flow cytometry dot plot showing neutrophils (CD45⁺CD11b⁺Ly6G⁺) in the liver of TNFR1R2⁺/⁺ and TNFR1R2⁻/⁻ mice fed a high-fat, choline-deficient (HFCD) or chow diet for two weeks and challenged with LPS. (B) Representative dot plot of PD-L1⁺ neutrophils (CD45⁺CD11b⁺Ly6G⁺PD-L1⁺) under the same experimental conditions. (C) Quantification of neutrophils (CD45⁺CD11b⁺Ly6G⁺) in the liver of TNFR1R2⁺/⁺ and TNFR1R2⁻/⁻ mice after LPS challenge. (D) Quantification of PD-L1⁺ neutrophils (CD45⁺CD11b⁺Ly6G⁺PD-L1⁺) in the liver(n=5). (E) IL-10 levels in the liver of TNFR1R2⁺/⁺ and TNFR1R2⁻/⁻ mice fed HFCD or chow and challenged with LPS(n=5). (F) IFN-γ levels in the liver of TNFR1R2⁺/⁺ and TNFR1R2⁻/⁻ mice under the same conditions. (G) Representative histogram showing TNF-α expression in the liver of C57BL/6 mice fed HFCD or chow for two weeks, challenged or not with LPS. The data are representative of one experiment reproduced independently twice with similar results. 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. Results are expressed as the mean ± standard deviation (SD).

PD-L1 expression in neutrophils occurs in the liver and not in the lung in animals with MAFLD during endotoxemia

Gating strategy for identification of NK, CD4 and CD8 T cells

Gating strategy for identification of PD-L1+ neutrophils