Figure 3. GRIP1 deficiency in macrophages promotes HFD-induced inflammation in WAT in vivo.
(a) FA uptake is impaired in GRIP1-deficient M(IL4). FA uptake was measured 6 min post-FA addition and expressed in relative fluorescence units (RFU). n>5 per group; error bars are s.d.; Student's t-test was used to determine significance. (b) WT and GRIP1 cKO gain similar amount of body weight. Shown are averages of 9 mice per group for chow-fed mice and 7 WT and 5 cKO mice per group for HFD-fed mice. (c) eWAT weight in WT and cKO after 20 weeks of chow or HFD. eWAT weight is expressed as percentage of total body weight for each mouse; n>10 per group. Significance was determined using ANOVA followed by Tukey's HSD test. The F-statistic and the P value for the significant main effect are shown (Methods section, Supplementary Fig. 4a and Supplementary Table 1 for detailed statistics). (d) Haematopoietic cell infiltration in cKO eWAT following HFD. eWAT was fixed in 4% PFA, paraffin-embedded and subjected to H&E staining or F4/80 immunohistochemistry. Magnification is × 40. (e) Augmented expression of inflammatory mediators in eWAT of the HFD-fed GRIP1 cKO. Gene expression was assessed by RT-qPCR with Hprt for normalization. n>5 per group; error bars are s.d. The Mann–Whitney test was used to determine significance. (f) SVF cells from HFD-fed GRIP1 cKO show increased expression of macrophage and inflammatory markers. Gene expression in WT and cKO SVF was assessed as in e. n=3/group; error bars are s.d. (g) GRIP1 cKO SVF shows increased macrophage abundance. The percentage of CD45+F4/80+CD11b+ macrophages in the SVF of WT and cKO mice and frequencies of the sorted CD11c+ (CD45+F4/80+CD11b+CD206-) and CD206+ (CD45+F4/80+CD11b+CD11c-) macrophages were quantified by FACS using FlowJo software. n=5/group; values are mean±s.e.m. The Mann–Whitney test was used to determine significance. (h) GRIP1 cKO ATMΦ display an exaggerated inflammatory profile. The expression of indicated genes in CD11c+ and CD206+ populations from g was assessed as in e. n>4 per group; error bars are s.d. Student's t-test was used to determine significance. (i) Efficient deletion of Ncoa2 (GRIP1) in CD11c+ macrophages of cKO mice. RNAseq shows read distribution across Ncoa2 exons 8–11 and a complete deletion of Ex11. (j) GSEA in WAT-derived CD11c+ macrophages shows upregulation in cKO versus WT of multiple gene signatures related to inflammatory response and interferon signalling. Shown are top gene enrichment profiles with corresponding FDR q.