Path of Differentiation Distinctly Influences Macrophage Identity
Authors: Frenis Katie , Badalamenti Brianna , Lummertz Da Rocha Edroaldo , Weng Chen , Wang Dahai , Fierstein Sara , Rowe R. Grant , Sankaran Vijay G. 
Published: December 2024

The impact of human hematopoietic ontogeny on macrophage heterogeneity is incompletely understood. Studies have compellingly shown that microenvironmental signals shape macrophage identity, but this only partially explain differences in macrophage subsets. Improved understanding of the macrophage specification has the potential to transform management of macrophage-driven inflammatory diseases.
To understand the influence of ontogeny on macrophage specification, we performed single cell RNA sequencing (scRNA-seq) of human fetal liver and adult bone marrow HSPCs, monoblasts, monocytes, and macrophages. Analysis of this dataset identified populations of monocyte-derived macrophages (mMacs) expressing canonical monocyte markers (S100A8, ANXA1, VCAN) with clear differentiation trajectories from HSPCs via monoblasts and monocytes. We also identified a fetal-specific population of tissue-resident-like macrophages (TRMs) that did not express monocyte markers and clustered independently, suggestive of an alternative monocyte-independent mechanism of specification.
We then performed macrophage colony stimulating factor-driven differentiation assays of human fetal or adult Lineage-CD34+CD38- HSPCs. Fetal macrophages arose within 4-5 days with no apparent monocyte stage, whereas adult HSPCs required 10-14 days to generate mature macrophages. Flow cytometry, gene expression, and phagocytosis assays showed that the rapidly emerging fetal macrophages adopted a less inflammatory phenotype than the adult mMacs. We observed analogous results upon xenotransplantation of HSPCs into immunodeficient mice, where fetal macrophages adopted a TRM profile. These results posit a fetal-specific alternative pathway of monocyte-independent TRM specification from definitive HSPCs.
Since ability to self-renew is a hallmark of TRMs, we speculated that rapid fetal differentiation could result in retention of HSPC-like self-renewal programs. In our scRNA-seq dataset, the proliferating macrophage population resembled the TRM population and clustered near hematopoietic progenitors. Compared to other macrophage populations, proliferating TRMs downregulated anti-self-renewal transcription factors (TFs) MAF and MAFB. We confirmed that fetal macrophages showed sustained self-renewal in culture compared to adult mMacs, suggesting that self-renewal was restricted by acquisition of monocyte identity.
We sought to identify TFs potentially impacting macrophage self-renewal. Expression of transcription factor AHR was prominent in both the fetal and adult monocyte and mMac clusters but was low in the fetal TRM cluster. AHR inhibition is a strategy for expanding HSPCs ex vivo, so we hypothesized that AHR could play a similar role regulating self-renewal of TRMs given their close ontologic relationship to HSPCs. We therefore differentiated and cultured fetal TRMs and adult mMacs in the presence of the AHR antagonists StemRegenin1 (SR1) or Ch223191. Fetal-derived TRMs exhibited amplified expansion in response to AHR, an effect not observed in adult mMacs.
Since mMac accumulation is present in many inflammatory disorders, we hypothesized that selective expansion of long-lived TRMs of fetal origin could be leveraged therapeutically. We therefore employed two independent mouse models of chemical-induced atopic dermatitis (AD). We found that application of SR1 at sites of AD induction could abrogate local inflammation and adjacent lymphadenopathy with relative expansion of TRMs compared to mMacs.
Overall, we find that the cell of origin and pathway of differentiation specify the functionality and self-renewal potential of macrophages. We show that selective induction of TRM self-renewal is a plausible approach in the treatment of inflammatory diseases.