Fetal hemogenic endothelium-derived multipotent progenitors are early responders to prenatal inflammation and shape postnatal myeloid immunity Free

Accumulating evidence suggests that prenatal factors, such as maternal infection, diet, and obesity, are linked to increased risk of immune dysfunction in offspring; yet, the mechanisms underlying these associations are unknown. During fetal development, hematopoietic stem and progenitor cells (HSPCs) emerge from intra-embryonic aortic hemogenic endothelium (HE). Recent evidence suggests that fetal hematopoietic stem cells (HSCs) and fetal multipotent progenitors (MPPs) arise separately from HE, and that fetal MPPs may contribute independently to postnatal blood production. However, the specific roles of these heterogeneous populations to fetal and postnatal hematopoiesis at homeostasis and under inflammation remain unclear. We hypothesize that fetal MPPs make distinct contributions to postnatal blood production and that prenatal inflammation reprograms the cellular output of distinct fetal HSPCs, shaping postnatal hematologic and immune function.

We used an inducible endothelial Cdh5-Cre-ERT2 fate-mapping approach to mark HE-labeled HSPCs at embryonic day (E)10.5 and track their contributions to hematopoiesis across time. HE-labeling was higher in myeloid-biased fetal MPP^GM and MPP^MK/E compared to fetal LT-HSCs suggesting that some fetal MPPs emerge independently from HE. This differential labeling allowed us to trace fetal HSC and fetal MPP contributions to hematopoiesis over time. By quantifying the cellularity of HE-labeled and unlabeled cells across the hematopoietic hierarchy and over the course of ontogeny, we found that fetal myeloid-biased MPPs contributed to myeloid-committed progenitors and mature cells during the first 2-3 weeks of life, while HSC contribution to peripheral blood increased by postnatal day (P)28 and was sustained into adulthood.

To test fetal HSPC responses to prenatal inflammation, we treated pregnant dams (from a Cdh5-Cre-ERT2 x Ai9 cross) with 20 ug Interferon gamma (IFNγ) at E14.5. Upon challenge with IFNγ, HE-labeling specifically increased in MPP^GM and MPP^MK/E which was propagated down to increased HE-labeling of myeloid and Mk/E committed progenitors at E17.5. This effect was due to an increase in cellularity of HE-labeled, but not unlabeled MPPs in response to IFNγ, whereas HE-derived labeling and cellularity were unchanged in fetal HSCs. This finding highlights distinct responses to IFNγ in fetal MPPs compared to HSCs and reveals functional differences in progenitors emerging from the HE at different developmental timepoints.

Prenatal IFNγ exposure reshaped the contributions of HE-labeled HSPCs to postnatal hematopoiesis. Specifically, HE-labeled myeloid-biased MPPs and progenitors remained expanded postnatally. Concurrently, IFNγ exposure led to expansion of unlabeled HSCs and MPPs by P14, implying that these unlabeled HSCs begin differentiating and contributing to downstream hematopoietic compartments. These findings support a model in which E10.5 HE-derived MPPs drive the first response to prenatal inflammation during gestation and unlabeled HSCs assume an earlier role in postnatal hematopoiesis compared to steady state.

To test the functional effects of prenatal IFNy on postnatal myeloid output, we generated bone marrow derived macrophages (BMDMs) from P14 pups exposed in utero to saline or IFNγ and challenged with Mycobacterium avium, a Type II IFN-inducing pathogen. BMDMs from IFNγ exposed pups showed enhanced phagocytosis and killing capacity at 4 and 24 hours post-infection, respectively, suggesting that prenatal priming with IFNγ confers a protective effect against postnatal Type II IFN-mediated infection. To assess if fetal HSPC function depends on timing of emergence, we generated BMDMs from sorted HE-labeled and unlabeled MPPs and HSCs and infected them with m. avium. HE-labeled BMDMs derived from MPPs, but not HSCs, exposed to prenatal IFNγ showed increased m. avium bacterial uptake at 4 hours and increased killing at 24 hours post infection compared to their unlabeled counterparts.

These data provide evidence that fetal HE-derived MPPs 1) give rise to functionally distinct immune progeny and 2) exhibit differential responses to IFNγ-induced prenatal inflammation.Together, our findings suggest that discrete fetal HSPCs have specific roles during development and across ontogeny, with MPPs being the first responders to inflammation. Importantly, the timing of emergence from the HE shapes the cell fate and functional output of fetal hematopoietic progenitors.