CD36 deficiency affects depressive-like behaviors possibly by modifying gut microbiota and the inflammasome pathway in mice

Abstract

Both inflammatory processes and gut microbiota have been implicated in the pathophysiology of depressive disorders. The class B scavenger receptor CD36 is involved in the cytotoxicity associated with inflammation. However, its role in depression has not yet been examined. In this study, we investigated whether CD36 affects depression by modulating the microbiota-gut-inflammasome-brain axis. We used CD36^-/- (knockout) mice subjected to chronic social defeat stress, and measured the expression of CD36 in these depressed mice and in patients with depression. The hippocampus of CD36^-/- mice was used to investigate changes in the NLRP3 inflammasome signaling pathway. The 16S rRNA gene sequence-based approach was used to compare the cecal microbial communities in CD36^-/- and WT mice. The CD36 deficiency in CD36^-/- mice alleviated chronic stress-induced depression-like behaviors. CD36 was upregulated in depressed mice as well as in depressed patients. Furthermore, the NLRP3 inflammasome signaling pathway was downregulated in the hippocampus of CD36^-/- mice. The Simpson Diversity Index revealed increased cecal bacterial alpha-diversity in the CD36^-/- mice. Among genera, Bacteroides, Rikenella, and Alloprevotella were significantly more abundant in the CD36^-/- mice, whereas Allobaculum was less abundant, consistent with the attenuated inflammation in the hippocampus of CD36^-/- mice. Our findings suggest that CD36 deficiency changes the gut microbiota composition, which in turn may impact depressive-like behaviors by affecting the inflammasome pathway.

Fig. 1. The relationship between CD36 and depression.

Upregulated expression of CD36 in susceptible mice and depressed patients (a–g). a SI ratios for control and CSDS-exposed mice. b CSDS-exposed mice displayed anhedonia as measured by a reduction in sucrose preference (n = 15–17 mice/group, P < 0.05). c–e Open-field test. Compared with control mice, the distance (c), proportion of time (d), and proportion of entries (e) into the center were significantly decreased in the CSDS-exposed mice. f Representative western blot quantification of CD36 protein expression in the hippocampus, PFC and cerebellum after CSDS (n = 5–6 mice/group, P < 0.05). g CD36 mRNA level and representative western blotting of PBMCs in depressed patients (n = 16 mice/group for qPCR; n = 5–6 mice/group for western blotting; P < 0.05). Genetic ablation of CD36 in mice prevents chronic social defeat stress-induced depression-like behaviors (h–k). h Social interaction (SI) ratios for WT, WT-CSDS, CD36^−/− and CD36^−/−-CSDS mice. CD36^−/− mice showed a significant increase in social interaction after CSDS. [^#significant main effect of CSDS^: F (1, 46) = 6.419, P < 0.05; *P < 0.05 (post hoc Tukey test)]. i CD36^−/− mice did not exhibit anhedonia, as shown by an increase of 1% in sucrose preference after CSDS. [⁺significant main effect of interaction: F (1, 40) = 5.523, P < 0.05; *P < 0.05 (post hoc Tukey test)]. j WT mice showed decreased entrance into the open arms of the EPM. [^#significant main effect of CSDS: F (1, 46) = 10.13, P = 0.093; *P < 0.05 (post hoc Tukey test)]. k WT mice showed decreased entry into the center of the OFT. [^#significant main effect of CSDS: F (1, 46) = 5.114, P = 0.093; *P < 0.05 (post hoc Tukey test)]. WT wild-type mice, CD36^−/− CD36 knockout mice, CSDS chronic social defeat stress, SI ratio social interaction ratio, PFC prefrontal cortex, PBMC peripheral blood mononuclear cell.

Fig. 2. CD36 knockout affects the gut microbiome compared with WT mice.

a A Venn diagram of OTUs detected in WT and CD36^−/− mice. b The majority of rarefaction curves tended to approach the saturation plateau. c Alpha-diversity analysis showed that CD36^−/− mice were characterized by lower microbial richness (Chao, n = 8–9 mice/group, *P < 0.05) and higher microbial diversity (Simpson, n = 8–9 mice/group, *P < 0.05) relative to WT mice. d At the phylum level, partial least-squares discriminant analysis (PLS-DA) showed that gut microbiota composition in CD36^−/− mice was greatly different from that in WT animals. e LEfSe identified the most differentially abundant taxons between WT and CD36^−/− mice—(red) WT taxa; (blue) taxa enriched in CD36^−/− mice. The brightness of each dot is proportional to its effect size. f Taxa enriched in CD36^−/− mice are indicated with a positive LDA score (green), and taxa enriched in WT mice have a negative score (red). Only taxa meeting an LDA significance threshold >2 are shown.

Fig. 3. Taxonomic differences in cecal microbiota between WT and CD36^−/− mice.

a The differences in taxonomic abundance between the different groups. b Classification and abundance of cecal contents at the phylum level. c Classification and abundance of cecal contents at the family level. d Classification and abundance of cecal contents at the genus level (n = 8–9 mice/group, *P < 0.05).

Fig. 4. The most differentially abundant taxa between the two groups.

a Heatmap of the 119 discriminative OTU abundances between WT and CD36^−/− mice (LDA > 2.0). OTUs (raw) were sorted by taxa and enrichment. The intensity of the color (green to red) indicates the score normalized abundance for each OTU. b Associations of gut microbial family with behavioral phenotype. Heat map of Spearman’s rank correlation. Red squares indicate positive associations between microbial family and behavioral phenotype; blue squares indicate negative associations. The statistical significance level is indicated in the squares (n = 8–9 mice/group, *P < 0.05). SPT sucrose preference test, EIOA entrance in open arms, EICZ entrance in center zone.

Fig. 5. Genetic ablation of CD36 in mice alters the gut microbiota functional and the NLRP3/caspase-1 inflammatory pathway.

a Function prediction analysis by COG functional classification. b KEGG pathway enrichment of gut microbiota between the two groups (n = 8–9 mice/group, *P < 0.05). Genetic ablation of CD36 in mice alters the NLRP3/caspase-1 inflammatory pathway (c, d). c Hippocampal mRNA levels of NLRP3, ASC, pro-caspase-1 and caspase-1 in CD36^−/− mice (n = 6–7 mice/group, P < 0.05). d Representative western blotting for hippocampal NLRP3, ASC, IL-1β, Cleaved-IL-1β, NF-кB, p-NF-кB, caspase-1 and pro-caspase-1 in CD36^−/− mice (n = 5–7 mice/group, P < 0.05).