Single-cell transcriptomics identifies the differentiation trajectory from inflammatory monocytes to pro-resolving macrophages in a mouse skin allergy model

Both monocytes and macrophages are heterogeneous populations. It was traditionally understood that Ly6Chi classical (inflammatory) monocytes differentiate into pro-inflammatory Ly6Chi macrophages. Accumulating evidence has suggested that Ly6Chi classical monocytes can also differentiate into Ly6Clo pro-resolving macrophages under certain conditions, while their differentiation trajectory remains to be fully elucidated. The present study with scRNA-seq and flow cytometric analyses reveals that Ly6ChiPD-L2lo classical monocytes recruited to the allergic skin lesion sequentially differentiate into Ly6CloPD-L2hi pro-resolving macrophages, via intermediate Ly6ChiPD-L2hi macrophages but not Ly6Clo non-classical monocytes, in an IL-4 receptor-dependent manner. Along the differentiation, classical monocyte-derived macrophages display anti-inflammatory signatures followed by metabolic rewiring concordant with their ability to phagocytose apoptotic neutrophils and allergens, therefore contributing to the resolution of inflammation. The failure in the generation of these pro-resolving macrophages drives the IL-1α-mediated cycle of inflammation with abscess-like accumulation of necrotic neutrophils. Thus, we clarify the stepwise differentiation trajectory from Ly6Chi classical monocytes toward Ly6Clo pro-resolving macrophages that restrain neutrophilic aggravation of skin allergic inflammation.


Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Data exclusions
Replication parameters: -p 8 -N 1 -L 16 -very-sensitive-local -seed 656565 -nofw.Then, read counts of each gene in each samples were counted by using awk, sort, and uniq -c commands.Resulted gene-expression count data was summarized by same Gene Symbol and all of the expression table was full-outer joined by Gene Symbol by using dplyr-1.0.7 package.Normalization of count data and DE analyses were performed by utilizing the R software package TCC v.1.32.0 63.PCA visualization was conducted by ggbiplot.Differentially expressed genes (DEGs) for each Mo-Mac subpopulation was defined as follows: DEGs for Ly6C+PD-L2-Mo-Mac were defined as genes significantly upregulated in Ly6ChiPD-L2lo Mo-Mac compared to Ly6ChiPD-L2hi and Ly6CloPD-L2hi Mo-Mac; DEGs for Ly6ChiPD-L2hi Mo-Mac were defined as genes significantly upregulated in Ly6ChiPD-L2hi Mo-Mac compared to Ly6ChiPD-L2lo and Ly6CloPD-L2hi Mo-Mac; DEGs for Ly6CloPD-L2hi Mo-Mac were defined as genes significantly upregulated in Ly6CloPD-L2hi Mo-Mac compared to Ly6ChiPD-L2hi and Ly6CloPD-L2lo Mo-Mac; DEGs for Ly6CloPD-L2lo Mo-Mac were defined as genes significantly upregulated in Ly6CloPD-L2lo Mo-Mac compared to Ly6CloPD-L2hi and Ly6ChiPD-L2lo Mo-Mac.Module scores for each Mo-Mac subpopulation was calculated by AddModuleScore function in Seurat v4.0.4.

Antibodies
Antibodies used Animals used in this study were randomly assigned to their respective groups before the experiments were performed.
Blinding was not achieved in this study due to requirements for cage identification and labeling for treatment purposes.

nature portfolio | reporting summary
April 2023
2) APC-conjugated anti-CD200R3 (clone: Ba13, catalog#: 142208) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $0.25 µg per million cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.
3) APC-conjugated anti-CD64 (clone: X54-5/7.1, catalog #:139306) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $1.0 µg per million cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.4) APC-Cy7-conjugated anti-Ly6G (clone: 1A8, catalog#: 127624) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $0.25 µg per million cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.5) APC/Fire 750-conjugated anti-Ly6G (clone: 1A8, catalog#: 127652) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $0.5 µg per million cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.6) FITC-conjugated anti-CD45 (clone: 30-F11, catalog#: 103108)] Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $ 0.25 µg per 106 cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.7) PacificBlue-conjugated anti-c-Kit (clone: 2B8, catalog#: 105820) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.The suggested use of this reagent is $ 1.0 µg per 106 cells in 100 µl volume.It is highly recommended that the reagent be titrated for optimal performance for each application.8) PacificBlue-conjugated anti-CD45.1 (clone: A20, catalog#: 110722) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.The suggested use of this reagent is $ 1.0 µg per 106 cells in 100 µl volume.It is highly recommended that the reagent be titrated for optimal performance for each application.9) BV421-conjugated PD-L2 (clone: TY25, catalog#: 107219) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $ 0.25 µg per million cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.10) BV421-conjugated anti-CD11b (clone: M1/70, catalog#: 101251) Verified Reactivity: Mouse, Human, Cynomolgus, Rhesus, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining using the µg size, the suggested use of this reagent is $ 0.25 µg per million cells in 100 µL volume.For flow cytometric staining using the µL sizes, the suggested use of this reagent is 5 µL per million cells in 100 µL staining volume or 5 µL per 100 µL of whole blood.It is recommended that the reagent be titrated for optimal performance for each application.11) BV510-conjugated anti-CD45.1 (clone: A20, catalog#: 110741) Verified Reactivity: Mouse, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining, the suggested use of this reagent is $0.5 µg per million cells in 100 µl volume.It is recommended that the reagent be titrated for optimal performance for each application.12) BV605-conjugated anti-CD11b (clone: M1/70, catalog#: 101257) Verified Reactivity: Mouse, Human, Cynomolgus, Rhesus, Application: FC -Quality tested Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.For flow cytometric staining using the µg size, the suggested use of this reagent is $0.25 µg per million cells in 100 µl volume.For flow cytometric staining using the µl size, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood.It is recommended that the reagent be titrated for optimal performance for each application.For flow cytometric analyses, single cell suspensions were prepared from the ear skin by by treating excised ears with collagenase (125 U/mL, Wako) in in RPMI complete medium at at 37°C for 2 h, h, followed by by depletion of of red blood cells.After preincubation with TruStain FcX PLUS antibody (anti-CD16/32 antibody; BioLegend) and normal rat serum (Merck Millipore) on on ice for 10 10 min to to prevent the non-specific binding of of irrelevant Abs, cells were stained with indicated combination of of Abs, and analyzed with FACSLyric (BD Biosciences) or or sorted with FACSAriaIII (BD Biosciences).
Flow-cytometric analysis was perfoemed using BD BD FACS Lyric flow cytometer (BD Biosciences).Cell sorting was performed using BD BD FACS AriaIII (BD Biosciences).
Flow cytometry data were analyzed using FlowJo software (version 10.8.1, BD BD Biosciences) Purity for all cell populations were confirmed to to be be greater than 95%.
13) BV711-conjugated anti-F4/80 (clone: BM8, catalog#: 123147) to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.