Participant characteristics
Consistent with the eligibility criteria, study participants had chronic low-grade inflammation as indicated by a median hs-CRP concentration greater than 3 mg/mL (Table 1). Participant characteristics and measures of inflammation were similar between females and males, with the exception of diastolic blood pressure which was lower in females than in males (two-sided t test p < 0.02). Similarly, monocytes, as a percent of total leukocytes in peripheral blood, were similar between the sexes.
Monocyte-specific sex signature genes
Monocyte transcriptional sex dimorphism was clearly observed, as shown in the separation of the samples based on sex in the PCA plot (Fig. 1A). Of the 41 sexually differentially expressed genes identified, the expression of approximately half (22 genes, 54%) was higher in females than males. Of the 22 genes, 7 were autosomal and 15 genes were X-linked. These genes are involved in immune cell activation (CEACAM1, FCGR2B, and SLAMF7) and antigen-recognition or presentation (AIM2, CD1E, and UBA1), suggesting enhanced activation of innate and adaptive immune responses in monocytes from females compared to males. Expression of 19 genes (46%) was higher in males than females. These included three autosomal genes (SERPINB2, BNIP3, and EBPL) and 16 Y-linked genes. The overall median absolute log2 fold difference in females versus males was 1.38 (Fig. 1B, Supplementary Table S1).
Increased activation of immune responses in monocytes
To gain further insight into the biological pathways that are associated with the transcriptional sexual dimorphism in monocytes, we selected genes that were significant at p < 0.03 for the sexual difference (360 female-biased and 205 male-biased genes; Fig. 1C, Supplementary Table S2) and mapped them to the canonical pathways of the IPA database. Out of the 11 canonical pathways that were significantly upregulated in females (z-score ³ 1, -log(p) > 1.30 by Fisher’s exact test), most pathways (8/11, 73%) were involved in immune or inflammatory responses (Table 2). IFN signaling was the top (z-score = 2.45, -log(p) = 3.88) among the 11 female-biased pathways, consistent with its documented association with female-biased autoimmunity (23,32). Genes involved in IFN signaling (IFN-induced protein with tetratricopeptide repeats [IFIT] 1-3, IFN-induced transmembrane protein 1 [IFITM1], Janus kinase 2 [JAK2], signal transducer and activator of transcription 1 [STAT1]), antigen-presentation (CD1 molecules [CD1B, CD1C], major histocompatibility complex, class II, DO alpha [HLA-DOA], major histocompatibility complex class II, DR beta 5 [HLA-DRB5]), and immune cell activation (C-C motif chemokine receptor 5 [CCR5], CD274 molecule [CD274], CD40 molecule [CD40], Fc fragment of IgG receptor IIb [FCGR2B]) were principally implicated in the other significant pathways (i.e., T helper type 1 (Th1) pathway, dendritic cell maturation, crosstalk between dendritic cells and NK cells, and SLE in B cell signaling pathway) as well. In addition to the 11 significant female-biased pathways, we identified two male-biased pathways with borderline significance: pathways related to programmed cell death protein 1 (PD-1), programmed cell death-ligand1 (PD-L1) cancer immunotherapy (z-score = -0.82, -log(p) = 2.01) and p53 signaling (z-score = -0.45, -log(p) = 1.64).
Comparison of IFN signature and regulatory pathways
Immune transcriptional sex dimorphism has been documented in unstimulated mouse macrophages, showing higher IFN responsiveness in females (19). Based on this evidence and our pathway analysis results, we sought to further examine how the IFN pathways differ between males and females in human monocyte transcriptome. To this end, we first compared the monocyte sex-biased genes with p < 0.03 to a set of genes that were recently demonstrated to be upregulated by IFN in mouse macrophages (i.e., MF-ISGs, 601 genes). Of the 498 out of 601 MF-ISGs that have human orthologs, 485 genes were expressed in our monocyte samples. Out of the 360 female-biased genes in monocytes, 48 were also MF-ISGs (13.3%). In contrast, only six of the 205 (2.9%) male-biased genes were MF-ISGs (Fig. 2, Supplementary Table S2). Next, we assessed the expression of the entire set of MF-ISG human orthologs in our monocytes. The log2 fold change distribution of MF-ISGs was skewed toward females (mean = 0.09) and was significantly different from the symmetrical distribution of all other genes (20,602 genes, mean = 0.003) (two-sided t test p = 2.9 x 10-4; Fig. 3). Consistent with the pathway analysis, these results suggest that upregulation of IFN-response genes is more frequently observed in female than male monocytes under unstimulated conditions.
Despite conserved transcriptomic signatures of immune cell lineages between human and mouse, specific differences exist due to divergent evolutionary paths (33). Moreover, monocytes have limited contribution to tissue-resident macrophages (34). Thus, we applied a larger set of genes (623 genes), instead of MF-ISGs, that had been validated in various immune cells both from mouse and human and that cover more complicated IFN responses (29). These genes are from an IFN transcriptional network consisting of 92 predicted regulators and 628 ISGs that was built based on co-expression in human and mouse responses (29). Similar to the comparison with MF-ISGs, we assessed the expression of target ISGs in our monocytes. We identified expression of 623 genes out of the 628 ISGs in unstimulated monocytes and their log2 fold change distribution was significantly skewed to the female side (mean = 0.11), compared to that of all other genes (20,464 genes, mean = 0.002) (two-sided t test p = 2.2 x 10-16; Supplementary Fig. S1). We further examined sexual bias in expression of those ISGs based on the five clusters (C1–C5) that were parsed within the network and characterized by distinct functionalities; C1 and C2 enriched for RNA processing, C3 for antiviral effectors, C4 for metabolic regulation, and C5 for inflammation mediators or regulators (29). While most of the clusters, except for C1, displayed significantly higher expression in females, the antiviral cluster C3 showed the strongest positive log2 fold change (median = 0.28, one sample two-sided t test Bonferroni-adjusted p < 2.2 x 10-26; Fig. 4), followed by the C5 cluster of inflammation (median = 0.13, Bonferroni-adjusted p = 7.6 x 10-6; Fig. 4). These results further confirm the higher baseline expression of IFN-responsive genes observed in female monocytes, particularly for the genes involved in antiviral and inflammatory responses, which may contribute to the phenotypic sex differences in autoimmunity.