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. 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).

髓系祖细胞向机体专职吞噬细胞巨噬细胞的正常发育，对于生物体尤其是哺乳动物的个体发育至关重要¹。集落刺激因子1受体（colony stimulating factor 1 receptor, CSF1R）的功能丧失性突变会引发包括骨硬化病在内的多种组织异常，这一现象正是该依赖关系的典型例证²。尽管巨噬细胞发育的重要性不言而喻，但目前对其分子与细胞生物学调控机制的了解仍十分有限。
在此，我们报告了一项令人意外的发现：氯离子感知激酶无赖氨酸激酶1（With-no-lysine 1, WNK1）是组织驻留巨噬细胞（tissue-resident macrophages, TRMs）胚胎发育所必需的。髓系特异性敲除Wnk1会导致TRMs大量丢失，进而破坏器官发育、引发全身性中性粒细胞增多症，并造成小鼠在3至4周龄时死亡。具体而言，我们观察到WNK1的缺失会使巨噬细胞分化阻滞在髓系多能祖细胞（myeloid multipotent progenitor, MPP）阶段，同时使MPP的分化偏向粒细胞生成。
从机制上来说，CSF1R的同源细胞因子巨噬细胞集落刺激因子（macrophage-colony stimulating factor, M-CSF）可触发髓系祖细胞发生巨胞饮，进而诱导WNK1的磷酸化。值得注意的是，髓系祖细胞的巨胞饮过程会提升胞浆氯离子浓度，而这一变化可被WNK1直接感知。在巨胞饮过程中干扰氯离子流动、抑制WNK1的氯离子感知功能，或是阻断巨胞饮，均可使祖细胞的分化从巨噬细胞谱系转向粒细胞谱系。因此，我们揭示了一种将细胞生物学过程与分子环路相联系的全新机制：多能祖细胞在M-CSF诱导下发生巨胞饮后，WNK1的氯离子感知功能与氯离子流动可作为下游信号，保障巨噬细胞谱系的分化保真度。
我们对来自CSF1R-Cre × WNK1 flox小鼠及其同窝野生型对照的MPP3进行了单细胞RNA测序（single cell RNA sequencing, scRNAseq）。MPP3（Lin⁻ cKit⁺ Sca-1⁺ Flt3⁻ CD48⁺ CD150⁻）通过多色流式细胞术（流式细胞分选仪Aria，BD）分选至含有0.04%牛血清白蛋白（Sigma-Aldrich, A8806）的磷酸盐缓冲液（PBS）离心管中。细胞经1次洗涤后，重悬于含0.04% BSA的PBS中，终浓度调整为每微升700~1300个细胞。通过0.2%（w/v）台盼蓝（Trypan Blue, Countess II）染色确认细胞活力高于80%。每只小鼠（Cre⁻组n=3，Cre⁺组n=4）分别使用带有标签1~7的MHC I类条形码抗体（BioLegend, A0301-A0307）进行标记。文库构建与测序由纪念斯隆凯特琳癌症中心单细胞研究倡议团队（Single Cell Research Initiative, SCRI, MSKCC）完成，具体流程如下：依照用户手册，在Chromium仪器（10X Genomics）上进行3′端v3.1版本的scRNAseq。将细胞捕获至微滴中，随后进行逆转录与细胞条形码标记。该步骤完成后，破乳并使用Dynabead MyOne SILANE磁珠纯化cDNA，再依照手册说明进行PCR扩增。每个样本的目标捕获细胞数约为30000个。依照已发表的细胞哈希（cell hashing）方案⁶⁹，将多个样本混合后在同一条10X Chromium泳道上进行测序。最终文库在Illumina NovaSeq S4平台上完成测序（R1：28个循环，i7：8个循环，R2：90个循环）。