Lactation-associated macrophages exist in murine mammary tissue and human milk.

Macrophages are involved in immune defense, organogenesis and tissue homeostasis. Macrophages contribute to the different phases of mammary gland remodeling during development, pregnancy and involution postlactation. Less is known about the dynamics of mammary gland macrophages in the lactation stage. Here, we describe a macrophage population present during lactation in mice. By multiparameter flow cytometry and single-cell RNA sequencing, we identified a lactation-induced CD11c+CX3CR1+Dectin-1+ macrophage population (liMac) that was distinct from the two resident F4/80hi and F4/80lo macrophage subsets present pregestationally. LiMacs were predominantly monocyte-derived and expanded by proliferation in situ concomitant with nursing. LiMacs developed independently of IL-34, but required CSF-1 signaling and were partly microbiota-dependent. Locally, they resided adjacent to the basal cells of the alveoli and extravasated into the milk. We found several macrophage subsets in human milk that resembled liMacs. Collectively, these findings reveal the emergence of unique macrophages in the mammary gland and milk during lactation.

Single-cell profiling of immune system alterations in lymphoid, barrier and solid tissues in aged mice.

Aging exerts profound and paradoxical effects on the immune system, at once impairing proliferation, cytotoxicity and phagocytosis, and inducing chronic inflammation. Previous studies have focused on individual tissues or cell types, while a comprehensive multisystem study of tissue-resident and circulating immune populations during aging is lacking. Here we reveal an atlas of age-related changes in the abundance and phenotype of immune cell populations across 12 mouse tissues. Using cytometry-based high parametric analysis of 37 mass-cytometry and 55 spectral flow-cytometry parameters, mapping samples from young and aged animals revealed conserved and tissue-type-specific patterns of both immune atrophy and expansion. We uncovered clear phenotypic changes in both lymphoid and myeloid lineages in aged mice, and in particular a contraction in natural killer cells and plasmacytoid dendritic cells. These changes correlated with a skewing towards myelopoiesis at the expense of early lymphocyte genesis in aged mice. Taken together, this atlas represents a comprehensive, systematic and thorough resource of the age-dependent alterations of the mammalian immune system in lymphoid, barrier and solid tissues.

Cold exposure protects from neuroinflammation through immunologic reprogramming.

Autoimmunity is energetically costly, but the impact of a metabolically active state on immunity and immune-mediated diseases is unclear. Ly6Chi monocytes are key effectors in CNS autoimmunity with an elusive role in priming naive autoreactive T cells. Here, we provide unbiased analysis of the immune changes in various compartments during cold exposure and show that this energetically costly stimulus markedly ameliorates active experimental autoimmune encephalomyelitis (EAE). Cold exposure decreases MHCII on monocytes at steady state and in various inflammatory mouse models and suppresses T cell priming and pathogenicity through the modulation of monocytes. Genetic or antibody-mediated monocyte depletion or adoptive transfer of Th1- or Th17-polarized cells for EAE abolishes the cold-induced effects on T cells or EAE, respectively. These findings provide a mechanistic link between environmental temperature and neuroinflammation and suggest competition between cold-induced metabolic adaptations and autoimmunity as energetic trade-off beneficial for the immune-mediated diseases.

Monocytes promote UV-induced epidermal carcinogenesis.

Mononuclear phagocytes consisting of monocytes, macrophages, and DCs play a complex role in tumor development by either promoting or restricting tumor growth. Cutaneous squamous cell carcinoma (cSCC) is the second most common nonmelanoma skin cancer arising from transformed epidermal keratinocytes. While present at high numbers, the role of tumor-infiltrating and resident myeloid cells in the formation of cSCC is largely unknown. Using transgenic mice and depleting antibodies to eliminate specific myeloid cell types in the skin, we investigated the involvement of mononuclear phagocytes in the development of UV-induced cSCC in K14-HPV8-E6 transgenic mice. Although resident Langerhans cells were enriched in the tumor, their contribution to tumor formation was negligible. Equally, dermal macrophages were dispensable for the development of cSCC. In contrast, mice lacking circulating monocytes were completely resistant to UV-induced cSCC, indicating that monocytes promote tumor development. Collectively, these results demonstrate a critical role for classical monocytes in the initiation of skin cancer.

Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis.

Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.

Emerging roles of IL-34 in health and disease.

Macrophages are part of the innate immune system and are present in every organ of the body. They fulfill critical roles in tissue homeostasis and development and are involved in various pathologies. An essential factor for the development, homeostasis, and function of mononuclear phagocytes is the colony stimulating factor-1 receptor (CSF-1R), which has two known ligands: CSF-1 and interleukin-34 (IL-34). While CSF-1 has been extensively studied, the biology and functions of IL-34 are only now beginning to be uncovered. In this review, we discuss recent advances of IL-34 biology in health and disease with a specific focus on mononuclear phagocytes.

Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal and identity.

Tissues in multicellular organisms are populated by resident macrophages, which perform both generic and tissue-specific functions. The latter are induced by signals from the microenvironment and rely on unique tissue-specific molecular programs requiring the combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs)-a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41 deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity.

The Cytokine TGF-ß Promotes the Development and Homeostasis of Alveolar Macrophages.

Alveolar macrophages (AMs) derive from fetal liver monocytes, which colonize the lung during embryonic development and give rise to fully mature AMs perinatally. AM differentiation requires granulocyte macrophage colony-stimulating factor (GM-CSF), but whether additional factors are involved in AM regulation is not known. Here we report that AMs, in contrast to most other tissue macrophages, were also dependent on transforming growth factor-ß receptor (TGF-ßR) signaling. Conditional deletion of TGF-ßR in mice at different time points halted the development and differentiation of AMs. In adult mice, TGF-ß was also critical for AM homeostasis. The source of TGF-ß was AMs themselves, indicative of an autocrine loop that promotes AM self-maintenance. Mechanistically, TGF-ßR signaling resulted in upregulation of PPAR-γ, a signature transcription factor essential for the development of AMs. These findings reveal an additional layer of complexity regarding the guidance cues, which govern the genesis, maturation, and survival of AMs.

TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.

Large-scale identification and analysis of suppressive drug interactions.

One drug may suppress the effects of another. Although knowledge of drug suppression is vital to avoid efficacy-reducing drug interactions or discover countermeasures for chemical toxins, drug-drug suppression relationships have not been systematically mapped. Here, we analyze the growth response of Saccharomyces cerevisiae to anti-fungal compound ("drug") pairs. Among 440 ordered drug pairs, we identified 94 suppressive drug interactions. Using only pairs not selected on the basis of their suppression behavior, we provide an estimate of the prevalence of suppressive interactions between anti-fungal compounds as 17%. Analysis of the drug suppression network suggested that Bromopyruvate is a frequently suppressive drug and Staurosporine is a frequently suppressed drug. We investigated potential explanations for suppressive drug interactions, including chemogenomic analysis, coaggregation, and pH effects, allowing us to explain the interaction tendencies of Bromopyruvate.