Upregulation of Anti-Oxidative Stress Response Improves Metabolic Changes in L-Selectin-Deficient Mice but Does Not Prevent NAFLD Progression or Fecal Microbiota Shifts

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  Figure 1. Lsel−/−Keap1Δhepa mice exhibit increased Nrf2 expression and an upregulation of Nrf2 target genes in the liver. (a) Representative immunofluorescence staining images indicate nuclear translocation of Nrf2 in hepatocytes of Lsel−/−Keap1Δhepa mice. Hepatocytes of untreated mice were labeled with anti-Nrf2 (orange) and DAPI (blue). Scale bar: 50 µm. (b) Representative flow cytometric analysis indicates increased Nrf2 in hepatocytes of Lsel−/−Keap1Δhepa mice. (c-e) Levels of the mRNAs indicated were measured in liver tissue of Lsel−/−Keap1flx/flx mice (red bars) and Lsel−/−Keap1Δhepa mice (blue bars) after 24 weeks of feeding with chow diet (CD) or Western diet (WD). mRNA levels are expressed as fold increase over the mean value obtained for healthy control liver tissue from Lsel−/−Keap1flx/flx mice. (Lsel−/−Keap1flx/flx CD-fed (n = 6), Lsel−/−Keap1flx/flx WD-fed (n = 12), Lsel−/−Keap1Δ hepa CD-fed (n = 4), Lsel−/−Keap1Δhepa WD-fed (n = 8). (c) Glrx (glutaredoxin), (d) Gstm-1 (glutathion S-transferase1) (e) Nqo-1 (NAD(P)H quinone dehydrogenase 1) Statistical significance was calculated by the one-way ANOVA (analysis of variance). Values are represented as mean ± SD. * p ≤ 0.05, ** p…

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  Figure 2. Lsel−/−Keap1Δhepa mice exhibit partial protection from metabolic dysfunction. Lsel−/−Keap1flx/flx mice (shown in red) and Lsel−/−Keap1Δhepa mice (shown in blue) were fed for 24 weeks with chow diet (CD) or Western diet (WD). (a) Weekly average WD consumption of Lsel−/−Keap1flx/flx mice (n = 4) and Lsel−/−Keap1Δhepa mice (n = 4). (b) Endpoint body weight, (c) endpoint glucose tolerance test (GTT), and (d) corresponding quantification as area under curve of Lsel−/−Keap1flx/flx CD-fed (n = 6), Lsel−/−Keap1flx/flx WD-fed (n = 12), Lsel−/−Keap1Δhepa CD-fed (n = 4), and Lsel−/−Keap1Δhepa WD-fed mice (n = 8). (e) Liver triglycerides and (f) liver free fatty acids (FFA) of Lsel−/−Keap1flx/flx CD-fed (n = 5), Lsel−/−Keap1flx/flx WD-fed (n = 5), Lsel−/−Keap1Δhepa CD-fed (n = 4), and Lsel−/−Keap1Δhepa WD-fed mice (n = 5). (g) Serum cholesterol and (h) LDL/HDL risk of Lsel−/−Keap1flx/flx CD-fed (n = 6), Lsel−/−Keap1flx/flx WD-fed (n = 12), Lsel−/−Keap1Δhepa CD-fed (n = 4), and Lsel−/−Keap1Δhepa WD-fed mice (n = 8). Statistical significance was calculated either by the one-way ANOVA (a,b) or unpaired t-test (d). Values are represented either as mean ± SD (a–c,e–h) or mean ± SEM (d). * p ≤…

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  Figure 3. Lsel−/−Keap1Δhepa mice are not additionally protected from WD-induced NASH progression. Lsel−/−Keap1flx/flx mice (shown in red) and Lsel−/−Keap1Δhepa mice (shown in blue) were fed for 24 weeks on chow diet (CD) or Western diet (WD). Shown are the endpoint values of Lsel−/−Keap1flx/flx CD-fed (n = 6), Lsel−/−Keap1flx/flx WD-fed (n = 12), Lsel−/−Keap1Δhepa CD-fed (n = 4), and Lsel−/−Keap1Δ hepa WD-fed mice (n = 8). (a) Liver/body weight ratio. (b) NAFLD activity score (NAS). (c) Representative images of H&E-stained liver sections of the indicated mice strains. Black arrows indicate the presence of inflammatory foci (original magnification X 20, scale bar = 100 µm). (d) mRNA levels of inflammatory mediators in whole liver tissue, expressed as fold increase over the mean value obtained for healthy control liver tissue from Lsel−/−Keap1flx/flx mice. (e) Quantification of serum aspartate aminotransferase (AST). (f) Quantification of serum alanine aminotransferase (ALT). (g) Quantification of alkaline phosphatase (ALP). (h) Comparative immune cell analysis of liver by flow cytometry. Representative FACS dot plots illustrating the gating strategy are shown in Supplementary Figure S1.…

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  Figure 4. Epidydimal white adipose tissue of WD-fed Lsel−/−Keap∆hepa mice exhibit decreased immune cell infiltration. Lsel−/−Keap1flx/flx mice (shown in red) and Lsel−/−Keap1Δhepa mice (shown in blue) were fed for 24 weeks on chow diet (CD) or Western diet (WD). Depicted are the endpoint values of Lsel−/−Keap1flx/flx CD-fed (n = 6), Lsel−/−Keap1flx/flx WD-fed (n = 12), Lsel−/−Keap1∆hepa CD-fed (n = 4), and Lsel−/−Keap1Δhepa WD-fed mice (n = 8). (a) Epidydimal white adipose tissue (eWAT)/body weight ratio. (b) Representative images of H&E stained eWAT sections of the indicated mice strains (original magnification X 20, scale bar = 100 µm. Areas with immune cell infiltration are labeled with black arrows. (c,d) Comparative immune cell analysis of eWAT by flow cytometry. Representative FACS dot plots illustrating the gating strategy are shown in Supplementary Figure S2. Depicted are the percentages of CD45+ cells of (c) CD4+ T cells (CD45+CD3+CD8-CD4+), CD8+ T cells (CD45+CD3+CD4-CD8+), B cells (CD45+CD3-CD19+), and (d) NK cells (CD45+NK1.1+), neutrophils (CD45+CD11b+Ly6G+), and monocytes/macrophages (CD45+Ly6G-CD11b+F4/80+). Statistical significance was calculated by the one-way ANOVA.…

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  Figure 5. Keap1 inactivation in L-selectin-deficient mice has only minor effects on WD-induced gut changes. Lsel−/−Keap1flx/flx mice (shown in red) and Lsel−/−Keap1Δhepa mice (shown in blue) were fed for 24 weeks on chow diet (CD) or Western diet (WD). Depicted are the endpoint values of Lsel−/−Keap1flx/flx CD-fed (n = 6), Lsel−/−Keap1flx/flx WD-fed, n = 12; Lsel−/−Keap∆hepa CD-fed, n = 4, and Lsel−/−Keap∆hepa WD-fed mice n = 12 (a) Colon length. (b,d) Comparative immune cell analysis of colon lamina propria by flow cytometry and (c,e) corresponding representative FACS dot plots. Representative FACS dot plots illustrating the gating strategy are shown in Supplementary Figure S1. Depicted are the percentages of CD45+ cells of (b) B cells (CD45+CD3-CD19+) and (d) NK cells (CD45+NK1.1+). Statistical significance was calculated by the one-way ANOVA. Values are represented as either as mean ± SD (a,b,d). * p ≤ 0.05, *** p ≤ 0.001.

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  Figure 6. WD feeding causes robust shifts in gut microbiota composition. Lsel−/−Keap1flx/flx mice (shown in red) and Lsel−/−Keap∆hepa mice (shown in blue) were fed for 24 weeks with Western diet (WD). (a) Schematic diagram of the sampling strategy. Fecal samples were collected of Lsel−/−Keap1flx/flx and Lsel−/−Keap∆hepa littermates at the indicated time points. The number of samples (n) has been demarcated under each point. (b) NMDS plot of microbiota profiles based on generalized UniFrac distances from Lsel−/−Keap1flx/flx and Lsel−/−Keap∆hepa mice at different time points. Individual time points that were considered for calculations have been marked within their respective circles. (c) Diversity and composition of the microbiota from different mouse groups. Shown is the Shannon effective count in samples based on time (treatment duration). The diversity of operational taxonomic units (OTUs) within a given sample (alpha-diversity) was calculated in Rhea. (d) Taxonomic binning of fecal microbiota of Lsel−/−Keapflx/flx and Lsel−/−Keap∆hepa mice at the phylum level. (e) Relative abundance of various genera in feces of Lsel−/−Keapflx/flx and Lsel−/−Keap∆hepa mice. In all graphs shown,…