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. 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.