WNK1 enforces 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. Overall design: Single cell RNA sequencing (scRNAseq) of MPP3s from CSF1R-Cre x WNK1 flox mice or littermate controls. MPP3s (Lin- cKit+ Sca-1+ Flt3- CD48+ CD150-) were sorted using multi-color flow cytometry (FACS Aria, BD) into fresh tubes containing PBS with 0.04% BSA (Sigma-Aldrich, A8806). Cells were washed 1x and resuspended in PBS containing 0.04% BSA at a final concentration of 700–1,300 cells per µl. Cell viability was confirmed to be above 80% via staining with 0.2% (w/v) Trypan Blue (Countess II). Each mouse (n=3 Cre-, n=4 Cre+) was labeled separately by staining with MHC Class I- barcoded antibodies bearing hashtags 1-7 (BioLegend, A0301-A0307). Library prep and sequencing were performed by the Single Cell Research Initiative (SCRI) at MSKCC as follows: scRNAseq was performed on a Chromium instrument (10X Genomics) following the user guide manual for 3' v3.1. Cells were captured in droplets, then subjected to reverse transcription and cell barcoding. Following this step, emulsions were broken, and cDNA purified using Dynabead MyOne SILANE, followed by PCR amplification per manual instruction. Approximately 30,000 cells were targeted for each sample. Samples were multiplexed together on one lane of a 10X Chromium following a published cell hashing protocol69. Final libraries were sequenced on the Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).