Figure 3

Cellular infiltration and pro-inflammatory macrophage activity are abrogated in neutrophil depleted mice during NASH development. IHC for detection of monocyte derived macrophages and other CD11b⁺ infiltrating myeloid cells (a), hepatic resident F4/80⁺ Kupffer cells (b) and pro-inflammatory Ly6C^hi monocytes/macrophages was performed and quantified for comparison among the control diet receiving group and the mice receiving either IgG or anti-Ly6G antibodies during ongoing FFC diet . mRNA expression was analyzed via RT-qPCR and fold-change was compared between the mentioned groups regarding the main monocyte chemotactic pathway CCL2/CCR2 (d) and pro-inflammatory macrophage activity markers such as components of the NLRP3 inflammasome pathway, chitinase-3 like 1 protein (CHI3L1) and TNF-α (e). Data was compared by using ANOVA analysis followed by the Turkey post hoc test. Western Blots targeting components of the NLRP3 downstream cascade were performed to compare the mentioned groups in a protein expression level (f). α-tubulin (band imported from Supplementary Fig. 1b) was used as housekeeping protein in both assessments. Full-length blottings of pro-caspase 1 and pro-IL-1β are shown in Supplementary Fig. 1b and 2, respectively. Ccl2 C–C motif chemokine ligand 2, ccr2 C–C motif chemokine receptor 2, NLRP3 NLR family pyrin domain containing 3, CHI3L1 chitinase 3-like 1, TNF-α/tnfa tumor necrosis factor alpha.