We assessed tissue macrophage gene expression in different mouse organs. Diversity in gene expression among different populations of macrophages was remarkable. Only a few hundred mRNA transcripts stood out as selectively expressed by macrophages over DCs and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of novel transcripts were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBPα, BACH1, and CREG-1 were among the top transcriptional regulators predicted to regulate these core macrophage-associated genes. Other transcription factor mRNAs were strongly associated with single macrophage populations. We further illustrate how these transcripts and the proteins they encode facilitate distinguishing macrophage versus DC identity of less characterized populations of mononuclear phagocytes.

Summary Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. Using parabiosis and fate mapping approaches, we confirmed that monocytes do not show significant contribution to tissue macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurred by stochastic cellular proliferation in situ in an macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage (GM)-CSF-dependent manner but independently of interleukin-4 (IL-4). We also found that after bone marrow transplantation, host macrophages retained the capacity to expand when the development of donor macrophages was compromised. Expansion of host macrophages was functional and prevented the development of alveolar proteinosis in mice transplanted with GM-CSF receptor-deficient progenitors. Collectively, these results indicate that tissue resident macrophages and circulating monocytes should be classified as mononuclear phagocyte lineages that are independently maintained in the steady state.

Summary To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single cell analysis of the tumor, non-involved lung and blood cells together with multiplex tissue imaging to assess spatial cell distribution, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies.

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

Although, much progress has been made in our understanding of DC ontogeny and function, the transcriptional regulation of DC lineage commitment and functional specialization in vivo is poorly understood. We performed a comprehensive comparative analysis of CD8+, CD103+, CD11b+, and plasmacytoid DC subsets and the recently identified Macrophage DC precursors and Common DC precursors across the entire immune system. Here we characterize candidate transcriptional activators involved in myeloid progenitor commitment to the DC lineage and predicted regulators of DC functional diversity in tissues. We identify a molecular signature that distinguishes tissue DC from macrophages. We also identify a transcriptional program expressed specifically during steady-state tissue DC migration to the draining lymph nodes that may control tolerance to self-tissue antigens.

SUMMARY The multistep sequence leading to leukocyte migration is thought to be locally regulated at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves, regulate rhythmic recruitment of leukocytes in tissues. Constitutive leukocyte adhesion and migration in murine bone marrow (BM) and skeletal muscle microvasculature fluctuated with circadian peak values at night. Migratory oscillations, altered by experimental jetlag, were implemented by perivascular SNS fibers, acting on β-adrenoreceptors expressed on non-hematopoietic cells, and leading to tissue-specific, differential circadian oscillations in the expression of endothelial cell adhesion molecules and chemokines. We showed that these rhythms have physiological consequences by alteration of hematopoietic cell recruitment and overall survival in models of septic shock, sickle cell vaso-occlusion and BM transplantation. These data provide unique insight in the leukocyte adhesion cascade and the potential for time-based therapeutics for transplantation and inflammatory diseases.

Summary The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential ofspecific myeloid cellprogenitors of both yolk sac and bone marrow origin. Homeostaticinnate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral antibiotic treated mice display increased pathogen burden and acute death. Re-colonization of germ-free mice with a complex microbiota restores defects inmyelopoiesis and resistance to Listeria. Thesefindingsreveal that gut bacteriadirect innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota.

The role of macrophages in erythropoiesis was suggested several decades ago with the description of “erythroblastic islands” in the bone marrow (BM) composed of a central macrophage surrounded by developing erythroblasts. However, the in vivo role of macrophages in erythropoiesis under homeostasis or disease remains unclear. Specific depletion of CD169+ macrophages markedly reduced erythroblasts in the BM but did not result in overt anemia under homeostasis likely due to concomitant alterations in RBC clearance. However, CD169+ macrophage depletion significantly impaired erythropoietic recovery from hemolytic anemia, acute blood loss and myeloablation. Furthermore, macrophage depletion normalized the erythroid compartment in a JAK2V617F-driven murine model of polycythemia vera (PV), suggesting that erythropoiesis in PV, unexpectedly, remains under the control of macrophages in the BM and splenic microenvironments. These data indicate that CD169+ macrophages promote late erythroid maturation and that modulation of the macrophage compartment represents a novel strategy to treat erythropoietic disorders.