In a Dish, Male and Female Macrophages Wage Inflammation Differently

Men and women do not experience inflammation in the same way, and clinicians have known this for decades. Women mount stronger immune responses to infection and vaccination, yet they shoulder the burden of roughly 80 percent of all autoimmune diseases. Men, by contrast, suffer disproportionately from severe sepsis and show higher mortality after hemorrhagic shock. The macrophage, a versatile white blood cell that devours pathogens, presents antigens to T cells, and coordinates tissue repair, sits at the heart of many of these divergent outcomes. But disentangling why male and female macrophages behave differently has been maddeningly difficult, because cells taken from living donors carry a lifetime of confounding influences: hormones, age, diet, pollution, illness. Now a research team led by Pamela Graney and Gordana Vunjak-Novakovic has sidestepped that problem with an elegant trick, growing macrophages from induced pluripotent stem cells so that the only meaningful difference between two batches of cells is the biological sex of the donor they came from.

The study, published in the journal iScience, used induced pluripotent stem cell, or iPSC, lines derived from three healthy male donors aged 27 to 36 and three healthy female donors aged 20 to 49. The researchers guided these stem cells through a developmental journey in a dish: first into mesoderm, then into hemogenic endothelium that produces hematopoietic stem and progenitor cells, and finally into monocytes nurtured with interleukin-3 and macrophage colony-stimulating factor. The monocytes were collected over multiple weeks and cultured further until they matured into macrophages. Crucially, the entire process took place in serum-free medium, which limits exposure to circulating hormones. Because the cells never lived inside a body, any differences that emerged between male-derived and female-derived macrophages could be attributed largely to inherent cellular genetics, namely the chromosomal complement, rather than to years of hormonal and environmental conditioning.

Before any experiments on inflammation, the team verified that their lab-grown macrophages were the real thing. Flow cytometry confirmed expression of the surface markers CD11b, CD14, HLA-DR, and SIRP-alpha, the fingerprint of cells in the myelomonocyte lineage, and immunostaining revealed the pan-macrophage marker CD68. Functional testing showed that the cells could phagocytose fluorescently tagged latex beads, a proxy for their ability to engulf dead cells and debris, and here the sexes performed identically. The same held true when the researchers compared their iPSC-derived macrophages with primary macrophages grown from monocytes isolated from male and female blood donors, supporting the validity of the stem cell model. Migration toward the chemoattractant CXCL12 in a Boyden chamber assay also showed no statistically significant difference between the sexes, although female macrophages trended toward slightly greater movement.

The transcriptome told a subtler story. Bulk RNA sequencing of the resting macrophages, analyzed by principal component analysis and hierarchical clustering, showed that the male lines clustered tightly together while the female lines spread across a much wider range, hinting at hidden variables such as unknown menopausal status among the female donors. Although the first two principal components explained only about 30 percent of the variance, the top 1,000 genes still sorted samples by biological sex rather than by donor, suggesting that sex, not individual identity, was the dominant driver of the transcriptional landscape. When the differentially expressed genes were mapped to their chromosomes, more than half landed on the X and Y chromosomes, a striking signal that sex-linked gene expression underlies much of the baseline dimorphism.

Those baseline genetic differences translated into at least one measurable functional gap. Resting female macrophages secreted significantly more monocyte chemoattractant protein-1, or MCP-1, than their male counterparts. MCP-1 is no bit player; it is a major chemotactic and activating signal that recruits monocytes and macrophages to sites of inflammation, so elevated production by female cells could shape how an inflammatory response unfolds. Female cells also produced slightly higher levels of interleukin-1 beta, IL-6, IL-10, and interferon-alpha 2, while male cells produced somewhat more IL-8, though these differences did not reach statistical significance. Enrichment analysis of the differentially expressed genes flagged pathways including ferroptosis, complement and coagulation cascades, HIF-1 signaling, the cell cycle, and regulation of the actin cytoskeleton, along with gene ontology terms tied to macrophage activation and cytokine production.

The real drama began when the researchers dosed the cells with lipopolysaccharide, or LPS, a component of the outer membrane of Gram-negative bacteria that triggers acute inflammation through toll-like receptor 4 signaling. In the sequencing data, LPS clearly separated stimulated cells from resting controls along one principal axis, and male from female cells along another. The separation between sexes was far more pronounced under inflammation than at rest, indicating that the dimorphism is not merely present but actively amplified by an inflammatory challenge. When the team searched for genes that changed in opposite directions between the sexes, or changed significantly in one sex while remaining flat in the other, the chemokine signaling pathway emerged as the most significantly perturbed, alongside metabolic pathways, cytokine-cytokine receptor interactions, Th1 and Th2 cell differentiation, apoptosis, and extracellular matrix-receptor interactions.

Within the chemokine signaling circuit, the analysis pointed to upregulation of CCL15 and CCL20 and of the signaling intermediates SOS1, SOS2, and BRAF, with downregulation of MAPK3, perturbations expected to cascade through Jak-STAT and MAPK pathways and drive changes in cytokine output, migration, and cell death. Protein-level measurements bore this out in a sex-specific pattern. Both sexes ramped up cytokine production in response to LPS, but the profiles diverged sharply: male macrophages produced significantly more IL-1 beta, TNF-alpha, IL-6, IFN-alpha 2, IL-10, and IL-23, whereas female macrophages produced significantly more IFN-gamma, IL-8, and IL-17A. Notably, these patterns echo published reports from primary human cells, in which men show stronger monocyte-derived cytokine responses to endotoxin, lending physiological credibility to the stem cell model.

Perhaps the most intriguing finding was chromosomal geography. At rest, most differentially expressed genes sat on the sex chromosomes; after LPS, that flipped. Of the 304 genes differentially expressed between male and female macrophages under inflammatory conditions, only 18.1 percent mapped to sex chromosomes, and once baseline differences were accounted for, the sex-linked fraction fell below 3 percent, with the largest share of genes on chromosome 1. The sex-linked genes that did change included X-linked players such as CSTF2, UXT, MBTPS2, HUWE1, and MECP2, upregulated in males, and NOX1, SLC6A8, and TSPYL2, downregulated in males, plus Y-linked shifts in DDX3Y and RPS4Y1. Several of these genes govern NF-kappaB signaling, epigenetic regulation, and reactive oxygen species production, processes with obvious inflammatory stakes. One candidate, the NADPH oxidase NOX1, was confirmed at the protein level: male macrophages produced modestly but significantly more NOX1 protein during inflammation, consistent with prior reports that oxidative stress runs higher in males across species.

The authors are careful about the limits of their work. Six iPSC lines cannot capture the full breadth of human population diversity, and differences between iPSC-derived macrophages and their blood-derived counterparts remain. The single LPS time point misses the dynamic arc of inflammation, and the female donors’ menopausal status, unknown here, may have inflated variability. Still, the model scales naturally through large iPSC banks holding lines from hundreds of donors, and future iterations could embed these macrophages into organ-on-a-chip systems to restore cell-cell interactions lost in isolation. The implications reach into some of medicine’s most stubborn sex disparities, from COVID-19 severity, where cytokine differences between the sexes are well documented, to autoimmune disease, obesity, asthma, cardiovascular disease, and aging. If most inflammation-driven dimorphism is written on the autosomes, steered by epigenetic echoes of the sex chromosomes, then drug developers may need to rethink a long-standing habit: dosing men and women identically on the assumption that their immune cells are interchangeable.

Subject of Research: Sex-based differences in human iPSC-derived macrophage inflammatory responses

Article Title: LPS stimulation reveals male and female dimorphism in human iPSC-derived macrophages

Article References: Graney, P. L., Tavakol, D. N., Lock, R. I., Chen, C., Samaritano, M., Sanchez, E., Hachmann, N. P., Rosales, M. P., Friedman, R., & Vunjak-Novakovic, G. (2026). LPS stimulation reveals male and female dimorphism in human iPSC-derived macrophages. iScience, 29(10), Article 117565. https://doi.org/10.1016/j.isci.2026.117565

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117565

Keywords: macrophages, iPSC, sexual dimorphism, LPS, inflammation, cytokines, sex chromosomes, NOX1, chemokine signaling, immunology, transcriptomics, TLR4