M2-like dermis resident macrophages regulate type 1 2 circuitries with ILC2s and eosinophils to exacerbate cutaneous leishmaniasis

Tissue-resident macrophages (TRMs) are critical for tissue homeostasis/repair. We previously showed that dermal TRMs produce CCL24 which mediates their interaction with IL-4 producing eosinophils, required to maintain their M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we unveil another layer of TRM self-maintenance involving production of TSLP, an alarmin typically characterized as epithelial cell-derived. Both TSLP signaling and IL-5+ innate lymphoid cell 2 (ILC2s) were shown to maintain dermal TRM numbers and promote infection. Single cell RNA sequencing identified the dermal TRMs as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permitted specific labeling of dermal TRMs, as well as interstitial TRMs from other organs. Genetic ablation of TSLP from dermal TRMs reduced the number of dermal TRMs and ameliorated infection. Here, we show that by orchestrating localized type 2 circuitries with ILC2s and eosinophils, dermal TRMs are self-maintained as a replicative niche for L. major. Overall design: CD45+ hematopoietic and CD45- nonhematopoietic cells were FACs-sorted from the ears of either naïve or infected WT and eoCre : Il4/13f/f mice and mixed at 9:1 ratio (CD45+:CD45-), and then processed through the Chromium Single cell 3' v3.1 Library kit (10x Genomics) per the manufacturer's protocol. Samples were further stained with nucleotide sequence tagged antibodies for measuring surface protein expression and cell indexing: TotalSeqTM-B0182 anti-mouse CD3 (17A2, Biolegend), TotalSeqTM-B0001 anti-mouse CD4 (RM4-5, Biolegend); TotalSeqTM-B0014 anti-mouse CD11b (M1/70, Biolegend); TotalSeqTM-B0106 anti-mouse CD11c (N418, Biolegend); TotalSeqTM-B0093 anti-mouse CD19 (6D5, Biolegend); TotalSeqTM-B0118 anti-mouse NK1.1 (PK136, Biolegend); TotalSeqTM-B0015 anti-mouse Ly6G (1A8, Biolegend); TotalSeqTM-B0012 anti-mouse c-Kit (2B8, Biolegend); TotalSeqTM-B0847 anti-mouse ICOS (7E.17G9, Biolegend); TotalSeqTM-B0114 anti-mouse F4/80 (BM8, Biolegend); TotalSeqTM-B0157 anti-mouse CD45.2 (104, Biolegend); TotalSeqTM-B0115 anti-mouse FceR1a (MAR-1, Biolegend); TotalSeqTM-B0002 anti-mouse CD8a (53-6.7, Biolegend); TotalSeqTM-B0431 anti-mouse SiglecF (S17007L, Biolegend); TotalSeqTM-B0810 anti-mouse CD138 (281-2, Biolegend); TotalSeqTM-B0120 anti-mouse TCRb (H57-597, Biolegend); TotalSeqTM-B0211 anti-mouse TCRgd2 (UC3-10A6, Biolegend); TotalSeqTM-B0301 to 0304 anti-mouse Hashtag 1 to 4 (M1/42 and 30-F11, Biolegend). 8,300 sorted cells were loaded for GEM formation, targeting 5,000 single cells for analysis from each ear. The RNAs from lysed single cells in gel beads were reversed transcribed into cDNA with Barcodes. Library fragment-size distribution was assessed using the Bioanalyzer 2100 and the DNA high-sensitivity chip (Agilent Technologies), followed by amplification, fragmentation, adapter ligation, and size selection to generate both 3' gene expression libraries and cell surface protein libraries. Libraries were sequenced on an Illumina NovaSeq 6000 by Psomagen, Inc.

组织驻留巨噬细胞（Tissue-resident macrophages, TRMs）在组织稳态维持与损伤修复过程中发挥关键作用。本课题组前期研究证实，皮肤驻留TRMs可分泌趋化因子配体24（CC chemokine ligand 24, CCL24），介导其与分泌IL-4的嗜酸性粒细胞产生相互作用，该过程是维持利什曼原虫（Leishmania major）感染皮肤的TH1型免疫环境中TRMs的M2样表型所必需的。本研究进一步揭示了TRMs自我维持的另一调控机制：TRMs可分泌胸腺基质淋巴细胞生成素（Thymic stromal lymphopoietin, TSLP）——这类警报蛋白通常被认为仅由上皮细胞分泌。研究发现，TSLP信号通路与表达IL-5的2型固有淋巴细胞（Innate Lymphoid Cell 2, ILC2s）均可维持皮肤TRMs的数量，并促进利什曼原虫感染。单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）结果显示，皮肤TRMs是TSLP与CCL24的唯一来源。我们构建的Ccl24-cre基因工程小鼠可特异性标记皮肤TRMs，同时也能标记其他器官中的间质TRMs。对皮肤TRMs的TSLP进行基因敲除后，皮肤TRMs的数量显著减少，且小鼠的感染症状得到缓解。本研究证实，皮肤TRMs可通过与ILC2s及嗜酸性粒细胞协同构建局部2型免疫环路，实现自我维持，从而成为利什曼原虫的复制微环境。实验整体设计：从未致敏（naïve）或利什曼原虫感染的野生型与eoCre:Il4/13f/f小鼠的耳部组织中，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分选出CD45+造血细胞与CD45-非造血细胞，按9:1的比例（CD45+:CD45-）混合后，依照制造商说明书使用Chromium Single Cell 3' v3.1文库制备试剂盒（10x Genomics）进行文库构建。后续采用核苷酸序列标记抗体进行细胞表面蛋白表达检测与细胞索引，所用抗体包括：TotalSeq™-B0182抗小鼠CD3（克隆号17A2，Biolegend）、TotalSeq™-B0001抗小鼠CD4（克隆号RM4-5，Biolegend）、TotalSeq™-B0014抗小鼠CD11b（克隆号M1/70，Biolegend）、TotalSeq™-B0106抗小鼠CD11c（克隆号N41，Biolegend）、TotalSeq™-B0093抗小鼠CD19（克隆号6D5，Biolegend）、TotalSeq™-B0118抗小鼠NK1.1（克隆号PK136，Biolegend）、TotalSeq™-B0015抗小鼠Ly6G（克隆号1A8，Biolegend）、TotalSeq™-B0012抗小鼠c-Kit（克隆号2B8，Biolegend）、TotalSeq™-B0847抗小鼠ICOS（克隆号7E.17G9，Biolegend）、TotalSeq™-B0114抗小鼠F4/80（克隆号BM8，Biolegend）、TotalSeq™-B0157抗小鼠CD45.2（克隆号104，Biolegend）、TotalSeq™-B0115抗小鼠FceR1a（克隆号MAR-1，Biolegend）、TotalSeq™-B0002抗小鼠CD8a（克隆号53-6.7，Biolegend）、TotalSeq™-B0431抗小鼠SiglecF（克隆号S17007L，Biolegend）、TotalSeq™-B0810抗小鼠CD138（克隆号281-2，Biolegend）、TotalSeq™-B0120抗小鼠TCRβ（克隆号H57-597，Biolegend）、TotalSeq™-B0211抗小鼠TCRγδ2（克隆号UC3-10A6，Biolegend）以及TotalSeq™-B0301至0304抗小鼠Hashtag 1至4（克隆号M1/42与30-F11，Biolegend）。将8300个分选细胞用于凝胶微滴（Gel Bead-in-Emulsion, GEM）形成，每只耳部样本目标捕获5000个单细胞用于后续分析。凝胶微滴中裂解的单细胞RNA经逆转录生成带有条形码的cDNA。采用Agilent 2100生物分析仪与DNA高灵敏度芯片对文库片段长度分布进行质检，随后依次完成扩增、片段化、接头连接与尺寸筛选，分别构建3'基因表达文库与细胞表面蛋白表达文库。最终由Psomagen公司在Illumina NovaSeq 6000测序平台上完成文库测序。