RELMa provides sex-specific protection from obesity through macrophages and eosinophils

The obesity incidence is increasing worldwide with the urgent need to identify new therapeutics. Sex differences in immune cell activation drive obesity-mediated pathologies where males are more susceptible to comorbidities with exacerbated inflammation, and females are protected. The focus of this study was to identify sex-specific immune effectors that regulate obesity pathogenesis, focusing on the M2 macrophage signature gene, Resistin-like molecule a (RELMa). Here we performed single cell RNA-sequencing (scRNA-seq) on visceral adipose tissue stromal vascular fraction on wildtype and RELMa knock-out animals, both males and females, on high fat diet. We utilized the 10x Genomics 3' CellPlex platform, to label cells with cell multiplexing oligos (CMOs) per sample in each group prior to pooling for downstream barcoding and library construction. This study revealed sex-specific and RELMa-dependent cellular targets and population differences of female and RELMa-mediated protection. Overall design: For multiplexing prior to generation of single-cell gel bead-in emulsions (GEMs), we took stromal vascular fractions for three animals per group (WT female, WT male, KO female, KO male) on high fat diet and labeled them with a lipid-containing CMO prior to pooling. Each group contained a pool of three animals to achieve a targeted cell recovery of 30,000 cells per group. After GEM creation, we sent the samples for sequencing, using the 10x Genomics 3' Gene expression and cell multiplexing library preparation method for each group. Per group in the gene expression library preparation, we sequenced an estimated number of 15,350 cells (WT females), 15,556 cells (WT males), 14,481 cells (KO females) and 16,642 cells (KO males). For the multiplexing library preparation, we sequenced the following of number of cells with positive CMO labeling animal per group: WT female (3,949, 4,676 and 4,360 cells), WT male (3,861, 3,190 and 3,105 cells), KO female (4,240, 4,342 and 3,710 cells) and KO male (4,025, 3,681 and 3,593 cells).