FOXP3+ macrophage represses acute ischemic stroke-induced neural inflammation

Abstract

Proper termination of cell-death-induced neural inflammation is the premise of tissue repair in acute ischemic stroke (AIS). Macrophages scavenge cell corpses/debris and produce inflammatory mediators that orchestrate immune responses. Here, we report that FOXP3, the key immune-repressive transcription factor of Tregs, is conditionally expressed in macrophages in stroke lesion. FOXP3 ablation in macrophages results in detrimental stroke outcomes, emphasizing the beneficial role of FOXP3+ macrophages. FOXP3+ macrophages are distinct from the M1 or M2 subsets and display superactive efferocytic capacity. With scRNAseq and analysis of FOXP3-bound-DNA isolated with CUT & RUN, we show that FOXP3 facilitates macrophage phagocytosis through enhancing cargo metabolism. FOXP3 expression is controlled by macroautophagic/autophagic protein degradation in resting macrophages, while initiation of LC3-associated phagocytosis (LAP) competitively occupies the autophagic machineries, and thus permits FOXP3 activation. Our data demonstrate a distinct set of FOXP3+ macrophages with enhanced scavenging capability, which could be a target in immunomodulatory therapy against AIS.Abbreviations: ADGRE1/F4/80: adhesion G protein-coupled receptor E1; AIF1/Iba1: allograft inflammatory factor 1; AIS: acute ischemic stroke; ARG1: arginase 1; ATP: adenosine triphosphate; BECN1/Beclin1: Beclin 1, autophagy related; BMDM: bone marrow-derived macrophages; CKO: conditional knockout; CSF1/M-CSF: colony stimulating factor 1 (macrophage); CSF2/GM-CSF: colony stimulating factor 2; CSF3/G-CSF: colony stimulating factor 3; CUT & RUN: cleavage under targets and release using nuclease; CyD: cytochalasin D; DAMP: danger/damage-associated molecular pattern; DIL: dioctadecyl-3,3,3,3-tetramethylin docarbocyanine; ELISA: enzyme linked immunosorbent assay; GO: Gene Ontology; FCGR3/CD16: Fc receptor, IgG, low affinity III; HMGB1: high mobility group box 1; IFNG/IFNγ: interferon gamma; IP: immunoprecipitation; KEGG: Kyoto Encyclopedia of Genes and Genomes; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11c: integrin subunit alpha X; LAP: LC3-associated phagocytosis; LC-MS: liquid chromatography-mass spectrometry; LPS: lipopolysaccharide; MRC1/CD206: mannose receptor, C type 1; O4: oligodendrocyte marker O4; PBMC: peripheral blood mononuclear cells; RBC: red blood cells; PTPRC/CD45: protein tyrosine phosphatase, receptor type, C; RBFOX3/NeuN: RNA binding protein, fox 1 homolog (C. elegans) 3; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; scRNAseq: single cell RNA sequencing; SQSTM1/p62 (sequestosome 1); TGFB/TGFβ: transforming growth factor, beta; tMCAO: transient middle cerebral artery occlusion; TNF/TNFα: tumor necrosis factor; Treg: regulatory T cell.

Keywords: FOXP3; inflammation; macrophage; phagocytosis; stroke.

Figure 1.

FOXP3⁺ macrophage ameliorates post-stroke neural inflammation. (A) WT C57/Bl6 male mice were subjected to 60 min of tMCAO. Coronal brain sections of stroke mice were collected for immunostaining and confocal microscopy analysis. Representative images of sections labeled with FOXP3 (green), AIF1 (microglia/macrophage marker, red) and CD3 (T cell marker, blue) in peri-infarct region at 3 d after tMCAO were displayed. White dashed lines outline the stroke infarct along which AIF1⁺ cells gathered. Experiments were repeated for 3 times. (B) Foxp3^egfp mice with C57/Bl6 background were sacrificed at 1-14d after tMCAO. Coronal brain sections of stroke mice were collected for immunostaining and confocal microscopy analysis. Representative images of sections labeled with AIF1 (microglia/macrophage marker, red) in peri-infarct region are displayed. (C and D) WT C57/Bl6 male mice were sacrificed at 1-14d after tMCAO surgery or Sham (Sh) operation. Ipsilateral brains were isolated and subjected to flow cytometric analysis (C) and plasma was collected for ELISA experiments (D). (C) FOXP3 expression in ITGAM⁺ ADGRE1⁺ macrophage was accessed with flow cytometry. Fluorochrome minus one (FMO, without FOXP3 staining) staining was used as the negative control in gating of FOXP3⁺ macrophages. Percentages of FOXP3⁺ cells among ITGAM⁺ ADGRE1⁺ macrophages (mean ± standard deviation) were displayed on the right of the flow plot. Quantity of FOXP3⁺ macrophages and mean fluorescence intensity (MFI) of FOXP3 among ITGAM⁺ ADGRE1⁺ macrophages were calculated. **P < 0.01, ***P < 0.001 compared to 1 d after tMCAO; by one-way ANOVA (mean ± standard deviation). (D) Concentration of high mobility group protein B1 (HMGB1) in plasma of stroke mice at 1-14d after tMCAO was assessed with ELISA. N = 4 at 1 d, 3 d, 7 d and 14 d, N = 5 at 5 d. *P < 0.05, ***P < 0.001 compared with Sham operated mice (Sh); by one-way ANOVA (mean ± standard deviation). (E-G) Foxp3 CKO mice (Lyz2^cre-ERT2foxp3^fl/Y) and their WT littermates (Lyz2^cre-ERT2Foxp3 ^wt/Y) were subjected to 60 min of tMCAO at 5-7d after the last tamoxifen injection. Mice were sacrificed at 5 d after tMCAO. (E) Infarct volume was quantified with TTC or RBFOX3 staining. Dashed lines outlined the infarct area. N = 7 in both foxp3 CKO and WT group. *P < 0.05, compared with WT; by Student’s t test (mean ± standard deviation). Scale bar: 0.5 cm. (F) Left: Garcia score of mice were evaluated at 0–5 d after tMCAO. N = 7 in both foxp3 CKO and WT group. ***P < 0.001 compared to WT group at indicated time points; ###P < 0.001 compared to WT group in the whole experiment; by two-way ANOVA (mean ± standard deviation). Right: Neurological deficit score of mice were evaluated at 0–3 d after tMCAO. N = 7 in both foxp3 CKO and WT group. *P < 0.05, ***P < 0.001 compared to WT group at indicated time point; ###P < 0.001 compared to WT group in the whole experiment; by two-way ANOVA (mean ± standard deviation). In Fig. 1E-F, two-way ANOVA with Sidak’s multiple comparison tests was used in statistical analysis. (G) Leukocyte infiltration in the ipsilateral brains of foxp3 CKO and WT mice were analyzed with flow cytometry at 5 d after tMCAO. Cell counts of macrophage (PTPRC⁺ ADGRE1⁺), neutrophil (PTPRC⁺ LY6 G⁺), dendritic cell (PTPRC⁺ ITGAX⁺), T cell (PTPRC⁺ CD3⁺) and B cell (PTPRC⁺ CD19⁺) per hemisphere were recorded. N = 4 in both foxp3 CKO and WT group. ***P < 0.001, compared with WT group; by Student’s t test (mean ± standard deviation).

Figure 2.

FOXP3 signaling defines a distinct subset ofmacrophage with superactive efferocytic capacity in stroke lesion. (A-C) a total of 6 Foxp3^egfp mice were sacrificed at 5 d aftertMCAO. PTPRC+ITGAM+CD3⁻ADGRE1+macrophages from the ipsilateral brains were isolated with flow cytometry andsubjected to 10x single cell RNA sequencing (scRnaseq). A total of 22,908 cellswere scanned and 1474 macrophages that expressed Foxp3 mRNA wereidentified. (A) T-distributed stochastic neighbor embedding (tSNE) plotof Foxp3 expression in the isolated macrophages. (B) Gene expression profiles of macrophages with (+) or without (-) Foxp3 mRNA expression werecompared. Left: Volcano plot showing the up- and down-regulated genes in Foxp3expressing macrophages compared with macrophages without Foxp3mRNAexpression. Part of phagocytosis related genes are specified. Right:Quantification of indicated phagocytosis related genes. **P <0.01,***P <0.001, compared with Foxp3⁻ cells; byStudent’s t test (median and quartiles were displayed with violinplots). (C) GO-BP enrichment of the up-regulated genes in Foxp3expressingmacrophages. Phagocytosis related GO terms were specified. (D-F) Scavenging activity analysis of FOXP3⁺ macrophages. (D) Wildtype C57/Bl6 mice were sacrificed at 5 d after tMCAO and subjected to flowcytometric analysis. RBFOX3+, O4⁺ and LY6 G cells in PTPRC+ITGAM+CD3⁻ADGRE1⁺ macrophages wereidentified as macrophages that had engulfed neuronal corpse, myelin debris andneutrophil corpse, respectively. N = 7. *P <0.05, ***P <0.001 compared with FOXP3⁻ macrophages; by Student’s t test(mean ± standard deviation). (E andF) Foxp3^egfpmice were sacrificed at 5 d after tMCAO. Coronal brain sections were subjectedto immunol labeling of AIF1 (red) and RBFOX3 (blue, in E) or MBP (blue,in F). White arrows emphasized AIF1⁺ cells engulfed neuron (E) or myelin debris (F) expressing FOXP3-EGFP. White arrowheads showed AIF1+cells without FOXP3-EGFP expression nor phagocytosis of neuronal corpse (E) or myelin debris (F). (G) Foxp3 CKO mice and the WTlittermates were subjected to tMCAO at 5-7d after the last tamoxifen injectionand sacrificed at 5 d after stroke. Brain cells were analyzed with flowcytometry. RBFOX3+, O4⁺ and LY6 G cells in PTPRC+ADGRE1⁺ macrophages were identified as macrophages that hadengulfed neuronal corpse, myelin debris or neutrophil corpse,respectively. N = 4 in each group. *P <0.05, ***P <0.001, compared with WT; by Student’s t test (mean ± standarddeviation). (H) Coronal brain sections of foxp3 CKO and WT micewere collected at 5 d after tMCAO and subjected to immunol labeling of AIF1(red) and MBP (green). Representative images are displayed. (I-J) BMDM from foxp3 CKO mice and the WT littermates were cultured at 5-7dafter the last tamoxifen injection. Myelin labeled with DIL was treated to BMDM(100 ug/ml) for indicated time period. Phagocytic efficiency of BMDM atindicated time points was assessed with immunol staining (I) and flowcytometric analysis (J). Experiments were repeated for 4 times. *P <0.05, **P <0.01, ***P <0.001; by two-way ANOVA (mean± standard deviation).

Figure 3.

M1/m2markers expression in FOXP3⁺ macrophage after stroke. (Aand B)Wild type C57/Bl6 mice were sacrificed at 5 d after tMCAO. Expression of M1 (A)and M2 (B) markers in FOXP3⁺ macrophages was assessed withflow cytometry. N = 5. *P <0.05, **P <0.01, ***P <0.001, compared with FOXP3⁻ cells; by Student’s ttest (mean ± standard deviation). (C) foxp3 CKO mice and the WTlittermates were subjected to tMCAO at 5-7d after the last tamoxifen injectionand sacrificed at 5 d after stroke. Expression of the indicated markers wereanalyzed with flow cytometry. N = 4 in each group. *P <0.05,**P <0.01, ***P <0.001 compared with WT group; byStudent’s t test (mean ± standard deviation). (D) Coronal brainsections of foxp3 CKO and WT mice were collected at 5 d after tMCAO andsubjected to immunol labeling of AIF1 (red) and IL10 or TGFB (green).Representative images are displayed. (E) Expression of the indicated M2signature genes in macrophages with (+) or without (-) Foxp3 expressionas revealed by scRnaseq. ***P <0.001, compared with Foxp3⁻macrophages; by Student’s t test (median and quartiles were displayedwith violin plots).

Figure 4.

GHNI+N Danger/damage-associated molecular patternsactivate FOXP3 signaling in macrophages, enhancing the anti-inflammatoryproperty.Danger/damage-associated molecular patterns (DAMPs) extractedfrom brain was utilized as simulation of the in-situ environment instroke brain. (AandB) FOXP3 expression in BMDM after 24 h ofDamps treatment (20% in culture medium) and the indicated anti-inflammatorymarkers expression in FOXP3⁺ and FOXP3⁻ BMDM was assessedwith flow cytometry. Experiments were repeated for 4 times. **P <0.01, ***P <0.001; by Student’s t test (mean ± standarddeviation). (C) BMDM were stimulated with brain derived DAMPs (20% inculture medium), IL4 (20 ng/ml, M2 inducer) or LPS (100 ng/ml, M1 inducer) for 1 h. The level of FOXP3 and other M1/M2 relevant transcriptional factors wasanalyzed with western blot. Experiments were repeated for 3 times. (D)BMDM from foxp3 CKO mice and the WT littermates at 5-7d after the lasttamoxifen injection was stimulated with DAMPs (20% in culture medium) for 24 h.Expression of ARG1, IL10, IL4 or TGFB was analyzed with flow cytometry.Experiments were repeated for 4 times. **P <0.01, ***P <0.001 compared with WT BMDM without DAMPs stimulation; by one-way ANOVA. (EandF) BMDM from Foxp3^egfp mice were treated with brainderived DAMPs for 24 h to activate FOXP3 signaling. The FOXP3-EGFP+cells were then isolated with flow cytometry and further subjected to M1 (LPS,100 ng/ml) or M2 (IL4, 20 ng/ml) induction for another 24 h (E). Thelevel of FOXP3-EGFP and M1/M2 markers, including IL10, MRC1, ARG1 and IFNG, wasfurther accessed with flow cytometry (F). Experiments were repeated for4 times. *P <0.05, **P <0.01, ***P <0.001; byone-way ANOVA (mean ± standard deviation). () WT BMDM weretreated with DAMPs or 20 μg/ml of Aβ40 for 1 h. FOXP3 was co-labeled with thenuclei stain DAPI. The number of cells with FOXP3 translocation into thenucleus was quantified. Experiments were repeated for 3 times. **P <0.01,***P <0.001; by one-way ANOVA (mean ± standard deviation). () Plasma of Sham-operatedand stroke mice (3 d after tMCAO) was collected and Aβ40 concentration wasassessed with ELISA. = 3 in each group.**P <0.01; by Student’s t test (mean ± standard deviation). () Aβ40 was injected to miceafter ischemic stroke (10 mg/kg, i.P., one dose per day during the first 3 daysafter AIS), the percentage of FOXP3 cells among infiltratedmacrophages in ipsilateral brain was assessed with flow cytometry at 3 d aftertMCAO. = 4 in each group. *P <0.05;by Student’s t test (mean ± standard deviation).

Figure 5.

FOXP3 regulatesphagocytosis and inflammatory resolution-associatedgenes in macrophages.(A) FOXP3 expression time course in wholeBMDM and in nucleus under stimulation of DAMPs (20% in culture medium) wasassessed with western blot. In analysis of the whole cell, experiments wererepeated for 4 times. In analysis of the nuclear protein, experiments wererepeated for 5 times. *P <0.05, **P <0.01; compared with0 h, by one-way ANOVA (mean ± standard deviation). We observed that the proteinlevel of FOXP3 peaked at 2–4 h after DAMPs stimulation. (BandC)BMDM was stimulated with brain derived DAMPs (20% in culture medium) for 2 h.Foxp3 binding DNA was isolated with CUT & RUN (Cleavage under targets andrelease using nuclease) using anti-FOXP3 antibodies (Invitrogen, FJK-16s, 2ugper 10⁷ cells) and the following sequencing. (B) Schematicplot of the experimental process. (C) Left: Top 10 pathways enrichedamong FOXP3 binding genes by KEGG (Kyoto Encyclopedia of Genes and Genomes)analysis. Middle: GO-BP (Gene Ontology-biological process) analysis of FOXP3binding genes in BMDM. Right: FOXP3 binding genes in the GO term of Regulationof phagocytosis (GO: 0050764). (Dand E) BMDM from foxp3CKO mice and the WT littermates at 5-7d after the last tamoxifen injection wasstimulated with DAMPs (20% in culture medium) for 2 h. (D) Expression ofphagocytosis and inflammatory resolution-associated genes in foxp3 CKOBMDM was evaluated with RT-PCR. Data were interpreted as Log₂ (foldchange) compared with WT group after normalization with Gapdh mRnalevel. Experiments were repeated for 4 times. *P <0.05, **P <0.01, ***P <0.001, compared with WT BMDM; by Student’s ttest (mean ± standard deviation). (F) FOXP3 binding peaks in Trem2and Il10.

Figure 6.

FOXP3 is undercontrol of autophagic protein degradation system.(A) BMDM was stimulated with DAMPs(20% in culture medium) forindicated time period and Foxp3 mRnalevel was assessed with RT-PCR. Experiments were repeated for 4 times. *P <0.05; by one-way ANOVA (mean ± standard deviation). (B) BMDM was stimulated with DAMPs (20% in culture medium)for 1 h. Cycloheximide (CHX, 10 ug/ml) was applied to inhibit proteinsynthesis. FOXP3 expression was assessed with western blot. Experiments were repeated for 4 times. **P <0.01; by one-way ANOVA (mean ± standard deviation). (C) FOXP3 binding protein was isolated with immunoprecipitation(ip), then subjected to liquid chromatography-mass spectrometry (LC-MS)analysis and the subsequent GO-biological process (BP) projection. Theubiquitin recognizing protein SQSTM1 was identified and could be enriched in GOterms emphasized with red characters. Coverage of the detected peptide in theprotein and the best peptide-spectrum match of SQSTM1 were displayed in Fig. S7 H. We thus inferred that FOXP3interacted with autophagic machineries in macrophages. (D) Co-IP and thefollowing immunol blot (IB) validation of the interactions between FOXP3 andthe autophagy-associated molecule SQSTM1 or LC3A/B with or without DAMPsstimulation (20% in culture medium,1 h). Experiments were repeated for 3 times. (E) Immunofluorescence staining of BMDM with LC3A/B (red) and FOXP3(green) showing colocalization of the two proteins. White arrows emphasizeddisassembly of FOXP3 and LC3A/B after 1 h of DAMPs stimulation (20% in culture medium). (F) BMDM was treated with the autophagyenhancer Rapamycin (50 nM), or the autophagy inhibitor Chloroquine (25 μM), 3 MA(0.5 mM), or Vacuolin1 (0.5 μM) respectively for 1 h. FOXP3 expression wasassessed with western blot. The treatment of DAMPs (20% in culture medium, 1 h) was set as positive control. Experiments were repeated for 3 times. *P <0.05, **P <0.01; by one-way ANOVA (mean ± standard deviation).(Gand H) BMDM were treated with the autophagy inhibitor 3 MA (0.5 mm) for 1 h. Expression of FOXP3 (G) and the indicated anti-inflammatorymarkers (H) was analyzed with flow cytometry. Experiments were repeated for 4 times. **P <0.01, ***P <0.001; by Student’s t test (mean ± standard deviation).

Figure 7.

LC3-Associatedphagocytosis (LAP) facilitates FOXP3 activation in macrophages.(A) Expression of autophagy relatedproteins RUBCN, SQSTM1, BECN1 and LC3A/B in BMDM after 1 h of DAMPs (20% in culture medium) stimulation wasassessed with western blot. Experiments were repeated for at least 3 times. (BandC) Wild typeC57/bl6 mice were subjected to 60 min of tMCAO and sacrificed at 5d. (B) Protein in the contralateral brain(cl) and ipsilateral hemisphere (Ip) was isolated and subjected to western blotto analyze the expression of RUBCN. N = 3 in Cl and Ip groups. *P <0.05; by Student’s t test (mean ±standard deviation). (C) Coronalbrain sections were collected and labeled with RUBCN (red, LAP marker), BECN1(green, autophagy machinery) and AIF1 (red, microglia/macrophage marker).Representative images of confocal microscopy revealed that the process of LAPwas activated in AIF1⁺ microglia/macrophages in peri-infarct area asrevealed by colocalization of RUBCN and BECN1. (D)Representative image of confocal microscopy with brain slice of tMCAO mice (5 dafter stroke) labeled with RUBCN (gray), BECN1 (red), FOXP3 (green) and (ADGRE1+,blue). Macrophages with ongoing LAP (with RUBCN andBECN1 colocalization) were emphasized with yellow arrows, those without wereemphasized with yellow arrow heads. (E)BMDM was stimulated with DAMPs (20%) for 1 h. Protein was extracted andsubjected to immunoprecipitation (IP) experiments with anti-RUBCN or anti-BECN1antibodies. IP products were analyzed with immunol blot (IB) to examine theinteraction of RUBCN and BECN1. Experiments were repeated for 3 times. (F) BMDM were stimulated with DAMPs (20%in culture medium) for 1 h and subjected to immunofluorescence labeling ofBECN1 (green), RUBCN (red) and FOXP3 (cyan). Nucleus was stained with DAPI(blue). (G-N) the impact of inhibiting or enhancing LAP on FOXP3expression in macrophages. (G) Experimental design. (H) BMDM wastreated with DAMPs (20% in culture medium) for 1 h. Cytochalasin D (CyD, 10 μM)was applied to inhibit phagocytosis. FOXP3 expression was measured with westernblot. Experiments were repeated for 3 times. *P <0.05; by one-wayANOVA (mean ± standard deviation). (I) BMDM were stimulated with DAMPs(20% in culture medium) for 1 h. The specific inhibitor of NADPH oxidase (NOXi)GSK2795039 (25 μM) was applied to interrupt LAP in BMDM during DAMPsstimulation. Expression of FOXP3 in the whole BMDM and the nucleus was assessedwith western blot. Experiments were repeated for 3 times. *P <0.05;by one-way ANOVA (mean ± standard deviation). (J andK) WT male mice were subjected to tMCAO. NOXi was treated to miceright after reperfusion (10 mg/kg, i.P., single dose). Mice were sacrificed at 3d after tMCAO. (J) Representativeconfocal microscopy images showing decreased FOXP3 (green) expression ininfiltrated macrophages (ADGRE1+, red) in the ipsilateral brain. (K)Percentage of FOXP3 expressing cells in macrophages and FOXP3 MFI ininfiltrated macrophages in the ipsilateral brain were analyzed with flowcytometry. N = 4 in each group. **P <0.01; by Student’s t test (mean ± standard deviation). (L-N) BMDM was infected with lentiviralvectors carrying RubcncDNA (RubcnOE) for 48 h. (L)Expression of RUBCN and FOXP3 was assessed with western blot. Experiments wererepeated for 3 times. (M) Expression of phagocytosis receptor TREM2, aswell as the M2 markers MRC1, ARG1, IL10 and IL4 in RubcnOE BMDM wasassessed with flow cytometry. Experiments were repeated for 3 times. **P <0.01, ***P <0.001; by Student’s t test (mean ± standarddeviation). (N) Myelin (100 ug/ml) was treated to RubcnOE BMDMfor indicated time period. Myelin engulfment capacity of BMDM was assessed withflow cytometry and was calculated as O4⁺ cells. Experiments wererepeated for 4 times. **P <0.01, ***P <0.001; by two-wayANOVA (mean ± standard deviation).