Multicenter Systems Analysis of Human Blood Reveals Immature Neutrophils in Males and During Pregnancy

Abstract

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20-30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women.

FIGURE 1.

ILNS in males compared with females. (A) Differences in GM77 gene expression between males and females. Mean expression of module 77 was computed and compared between young women and young men or between older women and older men. Differences were estimated by the Student t test. (B) Heat map of gene expression of GM77 in young (20–30-y-old) individuals (14 females and 16 males), shown for different genes individually, as well as averaged expression for the whole module (module profile). (C) Venn diagram depicting overlaps between GM77 and immature neutrophil (IN)-specific genes. The 13 common genes are listed. Validation of the ILNS in two additional data sets of 22 males and 22 females (D) and of 23 males and 33 females from public domain data (GEO accession numbers GSE29615 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29615] and GSE29617 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29617]) (23) (E), by QuSAGE, expressed as a summarized module mean expression fold change of males versus females.

FIGURE 2.

Cell surface markers and functional analysis of males’ and females’ neutrophils. Analysis of cell surface marker expression in males and females was conducted using a CyTOF instrument. (A) Multiple regression analysis with permutations for 34 molecules, with cut-off line at 5% FDR. Box plots showing standardized CD11c (B) and CD16 (C) expression in males and females. (D–F) Neutrophil responses to GM-CSF stimulation. (D) Multiple regression analysis with permutations for 244 features, with cut-off line at 5% FDR. Box plots showing the expression of standardized phosphorylated NF-κB (E) and phosphorylated S6 ribosomal protein (F) in males and females. (G and H) NET formation in vitro. (G) Fluorescent microscopy of NETs in neutrophils from healthy young males and females, in unstimulated conditions and after stimulation with bacterial LPS. (H) Box plots showing normalized percentage of NETs in healthy males and females in unstimulated conditions, after stimulation with LPS, and after stimulation with sera from five SLE patients. Significance of difference between males and females was calculated by combined p values (31) for two unstimulated conditions and five SLE sera–stimulated conditions.

FIGURE 3.

Neutrophil phenotype in pregnancy. Cell surface markers CD10 and CD15, measured by CyTOF, differentially expressed in four stages of pregnancy (first trimester, second trimester, third trimester, and postpartum [PP]), expressed as viSNE plots (A) and as standardized expression levels (B). Neutrophils, highlighted in the top right plot by arrow and dotted circle, were defined by expression of the following markers: CD19⁻, CD3⁻, CD56⁻, CD66⁺, and CD33⁺. For gating strategy, see Supplemental Fig. 2. The level of expression is depicted by the color scale, as shown on the right side of each plot. (C) Differential RNA expression of three representative ILNS genes (defensin α-4, CEACAM6, and CEACAM8) in whole blood of pregnant women (red lines) and healthy controls (black lines) during pregnancy. Solid lines represent median expression; dotted lines indicate the boundaries in which >95% of the values for the group are contained.