No abstract

The appropriate development of macrophages is essential for organismal development, especially in mammals. This dependence is exemplified by the observation that loss-of-function mutations in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities owing to an absence of macrophages. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 resulted in a dramatic loss of TRMs, disrupted organ development, systemic neutrophilia, and mortality between 3 and 4 weeks of age. Strikingly, we found that myeloid progenitors or precursors lacking WNK1 not only failed to differentiate into macrophages, but instead differentiated into neutrophils. Mechanistically, the cognate CSF1R cytokine macrophage-colony stimulating factor (M-CSF) stimulates macropinocytosis by both mouse and human myeloid progenitors and precursor cells. Macropinocytosis, in turn, induces chloride flux and WNK1 phosphorylation. Importantly, blocking macropinocytosis, perturbing chloride flux during macropinocytosis, and inhibiting WNK1 chloride-sensing activity each skewed myeloid progenitor differentiation from macrophages into neutrophils. Thus, we have elucidated a role for WNK1 during macropinocytosis and discovered a novel function of macropinocytosis in myeloid progenitors and precursor cells to ensure macrophage lineage fidelity.

The appropriate development of myeloid progenitors into macrophages, the body’s professional phagocyte, is essential for organismal development, especially in mammals1. This dependence is exemplified by the observation that loss-of-function mutation in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities including osteopetrosis2. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for embryonic development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 caused a dramatic loss of TRMs and subsequently disrupted organ development, induced systemic neutrophilia, and resulted in mortality between 3 and 4 weeks of age. Specifically, we observed that WNK1 absence stalled macrophage differentiation at the myeloid multipotent progenitor (MPP) stage, instead skewing MPP differentiation towards granulopoiesis. Mechanistically, the cognate CSF1R cytokine, macrophage-colony stimulating factor (M-CSF), triggers macropinocytosis in myeloid progenitors, which in turn induces phosphorylation of WNK1. Importantly, macropinocytosis by myeloid progenitors increases cytosolic chloride, which is directly sensed by WNK1. Perturbing chloride flux during macropinocytosis, inhibiting WNK1 chloride-sensing, and blocking macropinocytosis each skew progenitor differentiation from macrophage lineage to granulocyte lineage. Thus, we have uncovered a novel mechanism that links a cell biological process to a molecular circuit whereby WNK1 chloride-sensing and chloride flux act downstream of M-CSF-induced macropinocytosis by multipotent progenitors to ensure macrophage lineage fidelity.

Apoptotic cell clearance (efferocytosis), a process essential for organismal homeostasis, is performed by phagocytes that inhabit a wide range of environments, including physiologic hypoxia. Here, we find macrophages, the predominant tissue-resident phagocyte, display enhanced efferocytosis under prolonged (chronic) physiological hypoxia, characterized by increased internalization and accelerated degradation of apoptotic cells. Analysis of mRNA and protein programs revealed that chronic physiological hypoxia induces two distinct but complimentary states in macrophages. The first, primed state consists of concomitant induction of transcriptional and translational programs broadly associated with metabolism in apoptotic cell-naive macrophages that persist during efferocytosis. The second, poised state consists of transcription, but not translation, of phagocyte function programs in apoptotic cell-naive macrophages that are subsequently translated during efferocytosis. Importantly, we discovered that both states are necessary for enhanced continual efferocytosis. Mechanistically, we find that one such primed state consists of the efficient flux of glucose into a noncanonical pentose phosphate pathway (PPP) loop, whereby PPP-derived intermediates cycle back through the PPP to enhance production of NADPH. Furthermore, we found that PPP-derived NADPH directly supports enhanced continual efferocytosis under chronic physiological hypoxia via its role in phagolysosomal maturation and maintenance of cellular redox homeostasis. Thus, macrophages residing under chronic physiological hypoxia adopt states that both support cell fitness and ensure ability to perform essential homeostatic functions rapidly and safely.

The phagocytic clearance of dying cells, termed efferocytosis, is essential for both tissue homeostasis and tissue health during cell death-inducing treatments. Failure to efficiently clear dying cells augments the risk of pathological inflammation and has been linked to a myriad of autoimmune and inflammatory diseases. Although past studies have elucidated local molecular signals that regulate efferocytosis in a tissue, whether signals arising distally also regulate efferocytosis remains elusive. Interestingly, clinical evidence suggests that prolonged use of antibiotics is associated with an increased risk of autoimmune or inflammatory disease development. We therefore hypothesized that intestinal microbes produce molecular signals that regulate efferocytotic ability in peripheral tissue phagocytes. Here, we find that macrophages, the bodys professional phagocyte, display impaired efferocytosis in peripheral tissues in both antibiotic-treated and germ-free mice in vivo, which could be rescued by fecal microbiota transplantation. Mechanistically, the microbiota-derived short-chain fatty acid butyrate directly boosted efferocytosis efficiency and capacity in mouse and human macrophages, with both intestinal and local delivery of butyrate capable of rescuing antibiotic-induced peripheral efferocytosis defects. Bulk mRNA sequencing of primary macrophages treated with butyrate in vitro and single cell mRNA sequencing of macrophages isolated from antibiotic-treated and butyrate-rescued mice revealed specific regulation of phagocytosis-associated transcriptional programs, in particular the induction of programs involved in or supportive of efferocytosis. Surprisingly, the effect of butyrate on efferocytosis was not mediated through G protein-coupled receptor signaling, but instead acted by inhibition of histone deacetylase 3. Strikingly, peripheral efferocytosis was impaired well-beyond withdrawal of antibiotics and, importantly, antibiotic-treated mice exhibited a poorer response to a sterile efferocytosis-dependent inflammation model. Collectively, our results demonstrate that a process essential for tissue homeostasis, efferocytosis, relies on distal molecular signals, and suggest that a defect in peripheral efferocytosis may contribute to the clinically-observed link between broad-spectrum antibiotics use and inflammatory disease.