Spatial organization of pulmonary type 2 inflammation by a macrophage-derived cholesterol metabolite

Abstract

Effective pulmonary immunity requires the precise spatial organization of immune cells, yet the mechanisms guiding their intratissue positioning during inflammation remain unclear. Here, we identify a cholesterol-derived chemotactic axis that spatially organizes T helper 2 (T_H2) cells during fungal-induced pulmonary type 2 inflammation. Inflammation-expanded macrophages expressing cholesterol-25-hydroxylase (CH25H) produce 25-hydroxycholesterol, which is converted into the oxysterol 7α,25-dihydroxycholesterol to attract GPR183-expressing T_H2 cells into infectious lesions. This T_H2 positioning suppresses interferon-γ responsiveness in inflammatory Ly6C⁺ macrophages, promoting fungal persistence. Disruption of this axis via T_H2-specific GPR183 deletion restores type 1 macrophage activation and enhances fungal clearance. Our findings reveal a macrophage-driven, metabolite-based mechanism of immunosuppressive cell positioning in inflamed lung tissue.

Fig. 1.. T_H2 sensing of oxysterols antagonizes iNOS expression in Ly6C⁺ macrophages.

(A) Schematic of Cn intranasal infection model and intravenous (IV) CD45 labeling. (B) Representative histogram for Gpr183-GFP expression in IV⁺ and IV⁻ CD4⁺ T cells from Cn-infected mouse lung tissues at 10 dpi. (C) Geometric mean fluorescence intensity (gMFI) of Gpr183-GFP in IV⁺ versus IV⁻ CD4⁺ T cells from Cn-infected mouse lung tissues at 10 dpi. ^****P < 0.0001, paired two-tailed Student’s t-test. (D) Representative histogram of Gpr183-GFP expression across CD4⁺ T cell subsets from Cn-infected mouse lung tissues at 10 dpi. (E) Gpr183-GFP gMFI in CD4⁺ T cell subsets from Cn-infected mouse lung tissues at 10 dpi. ^**P < 0.01 and ^***P < 0.001 by one-way ANOVA. (F) Schematic of in vitro activation protocol for naïve T cells using anti-CD3/anti-CD28 beads with indicated cytokines and blocking antibodies. (G) Representative histogram of Gpr183-GFP expression in in vitro-generated T_H1 and T_H2 cells. (H) Quantification of Gpr183-GFP gMFI in in vitro-activated T_H1 and T_H2 cells. ^**P < 0.01, unpaired two-tailed Student’s t-test. (I) Schematic of trans-well migration assay comparing chemotactic responses of in vitro activated T_H1 and T_H2 cells to GPR183 ligand, 7α, 25-HC. (J) Quantification of T_H1 and T_H2 cell migration towards indicated concentrations of 7α, 25-HC and SDF-1. ^**P < 0.01 and ^***P < 0.001; ns, not significant; paired two-tailed Student’s t-test. (K) Schematic of mixed bone marrow chimera strategy for GPR183 specific depletion in T_H2 cells. (L) Quantification of tissue-resident eosinophils (Live/CD45⁺/IV⁻/CD90.2⁻/ B220⁻/SiglecF⁺/CD11b⁺/SSC-A^hi) in lungs from Cn-infected Gpr183^TH2Δ mice and their controls at 10 dpi. *P < 0.05, unpaired two-tailed Student’s t-test. (M) Quantification of lung resident AMs, Ly6C⁺ and Ly6C⁻ IMs at 0 and 10 dpi. (N) Representative flow cytometry plots showing iNOS expression in lung CD64⁺ macrophages from Cn-infected Gpr183^TH2Δ mice and their controls at 10 dpi. (O) Quantification of iNOS expression in lung CD64⁺ macrophages from Cn-infected Gpr183^TH2Δ mice and their controls at 10 dpi. *P < 0.05, ^**P < 0.01, unpaired two-tailed Student’s t-test.

Fig. 2.. Lung macrophages establish GPR183 chemotactic gradients via Ch25h induction.

(A) Schematic of trans-well migration assay used to assess GPR183-dependent chemotaxis in lung tissue extracts. (B) Quantification of migration activity of GPR183⁺ versus GPR183⁻ M12 cells in response to lung extracts collected at indicated time points, 1 nM 7α, 25-HC, or migration media alone. *P < 0.05, ^**P < 0.01; ns, not significant; paired two-tailed Student’s t-test. (C) Relative mRNA levels of Ch25h and Cyp7b1 in lung tissues over time following infection. Expression values were normalized to day 0 to show fold induction. ^**P < 0.01; ns, not significant; compared to the expression at day 0 by unpaired two-tailed Student’s t-test. (D) Schematic of Ch25h-tdTomato reporter experiments. (E) Representative flow cytometry plots of Ch25h-tdTomato expression across indicated immune cell populations from Cn-infected mouse lung tissues at 10 dpi. (F) Proportional contribution of each indicated population to total tdTomato⁺ (Ch25h-expressing) cells in Cn-infected mouse lung tissues at 10 dpi. (G) Relative mRNA levels of Ch25h to Rplp0 in lung tissues from Cn-infected CD11c^CrexCh25h^fl/fl mice or their controls at 10 dpi. ^**P < 0.01, unpaired two-tailed Student’s t-test. (H) Quantification of GPR183⁺ versus GPR183⁻ M12 cell migration towards lung extracts from CD11c^CrexCh25h^fl/fl mice and littermate controls at 10 dpi. ^**P < 0.01; ns, not significant; paired two-tailed Student’s t-test. (I) Relative mRNA levels of Ch25h to Rplp0 in lung tissues from Cn-infected STAT6-deficient mice or their controls at 10 dpi. (J) Relative mRNA levels of Ch25h to Rplp0 in lung tissues from Cn-infected T_H2-deficient mice or their controls at 10 dpi.

Fig. 3.. Single-cell RNA sequencing reveals distinct transcriptional programs of Ch25h-expressing macrophages

(A) Representative histogram of Ms4a3-tdTomato expression in Ly6C⁺ versus Ly6C⁻ IMs and AMs from Cn-infected mouse lungs at 10 dpi. (B) Ms4a3⁺ frequency of Ly6C⁺ versus Ly6C⁻ IMs and AMs from Cn-infected mouse lungs at 10 dpi. ^****P < 0.0001 by one-way ANOVA. (C) Representative flow cytometry plot of Ch25h-tdTomato expression in Ly6C⁺ versus Ly6C⁻ IMs and AMs from Cn-infected mouse lungs at 10 dpi. (D) Frequency of Ch25h⁺ within Ly6C⁺ versus Ly6C⁻ IMs and AMs from Cn-infected mouse lungs at 10 dpi. ^****P < 0.0001; paired two-tailed Student’s t-test. (E) UMAP visualization of annotated macrophage subsets from combined single-cell RNA sequencing replicates (21,069 cells from 15 mice; 3 mice per time point). (F) Violin plot showing Ch25h expression across indicated macrophage subsets. (G) Top differentially expressed genes within each macrophage population. (H) UMAP visualization of macrophage subset distribution across individual time points.

Fig. 4.. GPR183 guides intrapulmonary positioning of T_H2 cells to antagonize fungal clearance within granulomatous lesions.

(A) Representative full-projection confocal images of 500 μm thick lung sections from bone marrow chimeras overexpressing MSCV-Gfp or MSCV-Gpr183-Gfp from Cn gcs1Δ-infected at 35 dpi. (B) Representative full-projection confocal images of 500 μm thick lung sections from Cn gcs1Δ-infected Gpr183-knockout mixed bone marrow chimeras or their controls at 35 dpi. (C) Quantification of migration activity of GPR183⁺ versus GPR183⁻ M12 cells in response to lung extracts collected from CD11c^CrexCh25h^fl/fl mice or their controls at 35 dpi. *P < 0.05; ns, not significant; paired two-tailed Student’s t-test. (D) Representative flow cytometry plots of Ch25h-tdTomato expression in lung CD64⁺ macrophages at 35 dpi. (E) UMAP visualization of annotated myeloid cell subsets from merged scRNA-seq replicates (10,860 cells from 5 mice at 35 dpi). (F) Expression patterns of selected genes across indicated myeloid subsets. (G) Representative imaging of Ch25h transcripts detected by RNAscope, co-stained with CD11c by immunofluorescence on thin sections of Cn gcs1Δ-infected mouse lung tissues at 35 dpi. (H) Representative flow cytometry plots showing iNOS expression in CD64⁺ lung macrophages from Cn gcs1Δ-infected Gpr183^TH2Δ mice and their controls at 21 dpi. (I) Quantification of iNOS⁺ lung macrophages in Cn gcs1Δ-infected Gpr183^TH2Δ mice and their controls at 21 dpi. (J) Pulmonary fungal burden in Cn gcs1Δ-infected Gpr183^TH2Δ mice and their controls at 21 dpi. CFU, colony forming unit. (K) Schematic for the proposed model. During inflammation, Ly6C⁺ macrophages induce Ch25h expression, leading to the production of oxysterols that establish a GPR183-mediated chemotaxis. This guides T_H2 intrapulmonary positioning, which in turn antagonize local IFN-γ responses.