Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism

Abstract

Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.

Keywords: innate immunity; interferons; neutrophils; sex differences.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Adult female neutrophils up-regulate ISGs. (A) Volcano plot of differentially expressed genes comparing healthy young adult female and male neutrophils (cohort 1; n = 7/group). The dataset was filtered to include the 10,000 genes with highest average expression. Genes in red have higher expression in female neutrophils and genes in blue have higher expression in male neutrophils. (B) Pathway enrichment analysis of differentially expressed genes between female and male neutrophils (cohort 1; n = 7/group). Statistical analysis was performed with the clusterProfiler package in RStudio. (C) GSEA plots of ISGs and neutrophil activation genes in female vs. male neutrophils (cohort 1; n = 7/group). Differentially expressed genes were ordered using a calculated rank metric = −log10(P value) * log2(fold change). Adjusted P values for each gene set are shown. (D) Heatmap and hierarchal clustering of ISG expression in female and male neutrophils (cohort 1; n = 7/group). Data are represented as z scores as compared to average of the male samples. (E) Sex differences in type I IFN score between female and male neutrophils (cohort 1; n = 7/group). IFN score was calculated for each individual using 21-gene ISGs from RNA-sequencing data. P values were calculated with Mann–Whitney U test; **P ≤ 0.005. (F) Heatmap and hierarchal clustering of ISG expression in female (n = 10) and male (n = 8) neutrophils in cohort 2. Data are represented as z scores as compared to average of the male samples. (G) Sex differences in IFN score between female (n = 10) and male (n = 8) neutrophils in cohort 2. IFN score was calculated for each individual using 21-gene ISGs from RNA-sequencing data. P values were calculated with Mann–Whitney U test; **P ≤ 0.005.

Fig. 2.

Enhanced type I IFN response in female whole blood leukocytes is restricted to neutrophils. (A) Uniform manifold approximation and projection (UMAP) clustering of whole blood human leukocytes from seven healthy human controls (n = 40,724 cells, females = 4, males = 3). (B) UMAP representation of female (red, n = 24,083 cells; 4 donors) and male (blue, n = 16,641 cells; 3 donors) whole blood leukocytes. (C) Log2 fold change difference between main male and female white blood cell clusters with differentially expressed ISGs highlighted in red (female = 4, male = 3). (D) Up-regulated GO pathways in female neutrophils relative to male neutrophils (female = 4, male = 3). (E) Heatmap representation of the 21 ISGs in each cell cluster split by sex (female = 4, male = 3). (F) IFN score in T cells and monocytes from healthy postpubertal young males and females from a publicly available database (GSE103147). IFN score was calculated for each individual using 21 ISGs from RNA-sequencing data publicly available performed on sorted T cells (female = 40, male = 17) and monocytes (female = 25, male = 17). Mann–Whitney U test was used to determine differences in the score between males and females.

Fig. 3.

The IFN signature in neutrophils is driven by a distinct subset and ISGs in this subset are elevated in females. (A) UMAP clustering of neutrophils from human whole blood leukocytes from females (n = 4) and males (n = 3). (B) UMAP feature map showing expression of various neutrophil genes in different neutrophil subsets from all healthy controls (n = 7, female = 4, male = 3). Colony-stimulating factor 3 receptor (CSF3R), Fc fragment of IgG receptor IIIb (FCGR3B), S100 calcium-binding protein A9 (S100A9), ferritin heavy chain 1 (FTH1), MX dynamin-like GTPase 1 (MX1), signal transducer and activator of transcription 1 (STAT1), C-X-C motif chemokine ligand 8 (CXCL8, IL8), and prostaglandin-endoperoxide synthase 2 (PTGS2, COX2). (C) The top defining genes of the four neutrophil clusters from females (F, n = 4) and males (M, n = 3) based on gene expression, with cluster 2 showing high expression of ISGs. (D) Expression of 21 ISGs in different neutrophil subsets display increased expression of ISGs in subset 2 in females (n = 4) and males (n = 3), and higher ISG expression in females compared to males.

Fig. 4.

Increased type I IFN gene signature in female neutrophils is driven by enhanced response to these cytokines. (A) Serum type I IFN activity in healthy young adult females and males. ISGs were evaluated by RT-PCR in HeLa cells after culturing them with male or female sera (n = 5/group) or recombinant IFNα (1 ng/mL or 10 ng/mL) for 6 h. Each subpanel represents an individual ISG. Results represent mean + SEM. (B) Immunofluorescence analysis of intracellular localization of IFN-related transcription factors (IRF9 in red, highlighted by white arrows; p65 in red, highlighted by white arrows; and pIRF3 in red, highlighted by white arrows) was performed on permeabilized neutrophils isolated from male and female subjects. Nuclei were counterstained with Hoechst (blue). Original magnification, 400×. Images shown are from one representative dataset of three independent experiments. (C) Transcription factor analysis of RNA-sequencing data from cohort 1 showed increased expression of STAT1 and its regulated genes in female compared to male neutrophils (n = 7/group). (D) Cell membrane expression of IFNAR was quantified by flow cytometry in neutrophils from healthy males and females (n = 4/group). Mann–Whitney U test was used to calculate significance. P value was nonsignificant.

Fig. 5.

(A) Priming of neutrophils with type I IFNs enhances responses to TLR agonists: TNF and IL6 mRNA were quantified after exposing male neutrophils (n = 8) to IFNα (10 ng/mL) or media for 15 min, followed by incubation in the presence or absence of R484 for 3 h. Data were analyzed using Mann–Whitney U test; *P < 0.05, ns, not significant. Results represent mean + SEM. (B–D) Female and male neutrophils differ in their bioenergetic capacity. Mitochondrial and glycolysis stress tests analyses of peripheral blood neutrophils. Graphs display (B) OCR, to assess mitochondrial respiration, and (C) ECAR to measure glycolysis (females = 6; males = 6). (D) Bar graph shows ratio of basal OCR vs. basal ECAR. (E–G) Male neutrophils have increased mitochondrial content. (E) Quantification of mitochondrial DNA by qPCR in neutrophils from males (n = 6) and females (n = 6). (F) Quantification of MitoTracker by flow cytometry in neutrophils from males (n = 6) and females (n = 6). (G) Representative example of a male and female neutrophil stained with MitoTracker green dye. (H–K) Estradiol stimulation decreases mitochondrial metabolism in male neutrophils. Effect of estradiol (200 pg/mL) stimulation on male neutrophils (n = 6) on (H) OCR and (I) ECAR. Also shown, male neutrophils’ (J) basal respiration and (K) maximal respiration after incubation with estradiol. Oligo, oligomycin (inhibitor of ATP-synthase); FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (mitochondrial uncoupler); Rot + AA, rotenone + antimycin (complex I and complex III inhibitors, respectively). Data (D, E, F, J, and K) were analyzed using Mann–Whitney U test. Data (B and H) were analyzed using multiple t tests; *P < 0.05, **P < 0.01, ***P < 0.001. Results represent mean + SEM.

Fig. 6.

The enhanced response to type I IFNs and maturation status in female neutrophils is not explained by X chromosome gene dosage. ISGs were quantified by RT-PCR in (A) neutrophils from young adult males (n = 10), and females (n = 15) and males with Klinefelter syndrome (47, XXY) (n = 17); (B) and in neutrophils from healthy prepubertal males (n = 8) and females (n = 4). Data were analyzed with Mann–Whitney U test; *P < 0.05, **P < 0.01. Results represent mean + SEM. (C) Primary granule genes were quantified by PCR in neutrophils from prepubertal healthy males (n = 8) and females (n = 5). Results represent mean + SEM.