Fetal liver macrophages contribute to the hematopoietic stem cell niche by controlling granulopoiesis.

During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche.

Fate-Mapping of Yolk Sac-Derived Macrophages.

To better understand the distinct functions of yolk-sac-derived tissue-resident macrophages (TRMs) and bone-marrow-derived macrophages in homeostasis and disease, it is important to trace the ontogeny of these cells. The majority of TRMs originate from erythro-myeloid progenitors (EMPs). EMPs develop into pre-macrophages (pMacs), which can be detected starting at embryonic developmental day (E)9.0, and which give rise to all TRM during early development. pMacs start expressing the gene Cx3cr1, allowing us to genetically target the early yolk-sac wave of pMacs and their progeny. Here, we describe the protocol for the identification of yolk sac-derived TRMs utilizing in utero labelling of the inducible fate mapping Cx3cr1CreERT; Rosa26LSL-eYFP mouse model.

Segmented filamentous bacteria-induced epithelial MHCII regulates cognate CD4+ IELs and epithelial turnover.

Intestinal epithelial cells have the capacity to upregulate MHCII molecules in response to certain epithelial-adhesive microbes, such as segmented filamentous bacteria (SFB). However, the mechanism regulating MHCII expression as well as the impact of epithelial MHCII-mediated antigen presentation on T cell responses targeting those microbes remains elusive. Here, we identify the cellular network that regulates MHCII expression on the intestinal epithelium in response to SFB. Since MHCII on the intestinal epithelium is dispensable for SFB-induced Th17 response, we explored other CD4+ T cell-based responses induced by SFB. We found that SFB drive the conversion of cognate CD4+ T cells to granzyme+ CD8α+ intraepithelial lymphocytes. These cells accumulate in small intestinal intraepithelial space in response to SFB. Yet, their accumulation is abrogated by the ablation of MHCII on the intestinal epithelium. Finally, we show that this mechanism is indispensable for the SFB-driven increase in the turnover of epithelial cells in the ileum. This study identifies a previously uncharacterized immune response to SFB, which is dependent on the epithelial MHCII function.

Deletion of TLR2+ erythro-myeloid progenitors leads to embryonic lethality in mice.

Early embryonic hematopoiesis in mammals is defined by three successive waves of hematopoietic progenitors which exhibit a distinct hematopoietic potential and provide continuous support for the development of the embryo and adult organism. Although the functional importance of each of these waves has been analyzed, their spatio-temporal overlap and the lack of wave-specific markers hinders the accurate separation and assessment of their functional roles during early embryogenesis. We have recently shown that TLR2, in combination with c-kit, represents the earliest signature of emerging precursors of the second hematopoietic wave, erythro-myeloid precursors (EMPs). Since the onset of Tlr2 expression distinguishes EMPs from primitive progenitors which coexist in the yolk sac from E7.5, we generated a novel transgenic "knock in" mouse model, Tlr2Dtr , suitable for inducible targeted depletion of TLR2+ EMPs. In this model, the red fluorescent protein and diphtheria toxin receptor sequences are linked via a P2A sequence and inserted into the Tlr2 locus before its stop codon. We show that a timely controlled deletion of TLR2+ EMPs in Tlr2Dtr embryos results in a marked decrease in both erythroid as well as myeloid lineages and, consequently, in embryonic lethality peaking before E13.5. These findings validate the importance of EMPs in embryonic development.

Toll-like receptor signaling in thymic epithelium controls monocyte-derived dendritic cell recruitment and Treg generation.

The development of thymic regulatory T cells (Treg) is mediated by Aire-regulated self-antigen presentation on medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), but the cooperation between these cells is still poorly understood. Here we show that signaling through Toll-like receptors (TLR) expressed on mTECs regulates the production of specific chemokines and other genes associated with post-Aire mTEC development. Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirpα+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines. Consistently, the cellularity of CD14+moDC is diminished in mice with MyD88-deficient TECs, in which the frequency and functionality of thymic CD25+Foxp3+ Tregs are decreased, leading to aggravated mouse experimental colitis. Thus, our findings describe a TLR-dependent function of mTECs for the recruitment of CD14+moDC, the generation of Tregs, and thereby the establishment of central tolerance.

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors.

Hematopoiesis in mammalian embryos proceeds through three successive waves of hematopoietic progenitors. Since their emergence spatially and temporally overlap and phenotypic markers are often shared, the specifics regarding their origin, development, lineage restriction and mutual relationships have not been fully determined. The identification of wave-specific markers would aid to resolve these uncertainties. Here, we show that toll-like receptors (TLRs) are expressed during early mouse embryogenesis. We provide phenotypic and functional evidence that the expression of TLR2 on E7.5 c-kit+ cells marks the emergence of precursors of erythro-myeloid progenitors (EMPs) and provides resolution for separate tracking of EMPs from primitive progenitors. Using in vivo fate mapping, we show that at E8.5 the Tlr2 locus is already active in emerging EMPs and in progenitors of adult hematopoietic stem cells (HSC). Together, this data demonstrates that the activation of the Tlr2 locus tracks the earliest events in the process of EMP and HSC specification.