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Vitamin A mediates conversion of monocyte-derived macrophages into tissue-resident macrophages during alternative activation

Abstract

It remains unclear whether activated inflammatory macrophages can adopt features of tissue-resident macrophages, or what mechanisms might mediate such a phenotypic conversion. Here we show that vitamin A is required for the phenotypic conversion of interleukin 4 (IL-4)-activated monocyte-derived F4/80intCD206+PD-L2+MHCII+ macrophages into macrophages with a tissue-resident F4/80hiCD206PD-L2MHCIIUCP1+ phenotype in the peritoneal cavity of mice and during the formation of liver granulomas in mice infected with Schistosoma mansoni. The phenotypic conversion of F4/80intCD206+ macrophages into F4/80hiCD206 macrophages was associated with almost complete remodeling of the chromatin landscape, as well as alteration of the transcriptional profiles. Vitamin A–deficient mice infected with S. mansoni had disrupted liver granuloma architecture and increased mortality, which indicates that failure to convert macrophages from the F4/80intCD206+ phenotype to F4/80hiCD206 may lead to dysregulated inflammation during helminth infection.

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Figure 1: Monocyte-derived inflammatory macrophages adopt a tissue-resident phenotype after long-term residency in the peritoneal cavity.
Figure 2: Fate-mapping of monocyte-derived macrophages that adopt a tissue-resident phenotype after long-term residency in the peritoneal cavity.
Figure 3: Transcriptional and chromatin landscape reprogramming during macrophage conversion.
Figure 4: Vitamin A deficiency disrupts tissue-resident macrophages.
Figure 5: Phenotypic conversion of inflammatory macrophages to a tissue-resident phenotype is disrupted in vitamin A–deficient mice.
Figure 6: Increased Ucp1 expression and proliferation in mature liver granulomas of S. mansoni–infected mice.
Figure 7: Fate-mapping of monocyte-derived macrophages in the liver granulomas of S. mansoni–infected mice.
Figure 8: Vitamin A–deficient mice show disruption of Ucp1 expression and proliferation in granulomas, and increased mortality of S. mansoni infection.

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Acknowledgements

We thank K. Cadwell and members of the K. Cadwell, J. Ernst and E. Fisher laboratories for their reading of this manuscript. We thank W. Gan and D. Littman (New York University Medical Center, Skirball Institute of Biomolecular Medicine, New York, New York, USA) for generously providing the Cx3cr1CreERT2-IRES-EYFP mice, and J. Collins (UT Southwestern, Dallas, Texas, USA) for providing S. mansoni eggs. We thank the NYUMC Genome Technology Core, NYUMC Flow Cytometry Core, NYUMC Microscopy Core and NYUMC Histopathology Core facilities for their assistance; these shared resources are partially supported by the Cancer Center (support grant P30CA016087) at the Laura and Isaac Perlmutter Cancer Center. This work was supported by the NIH (T32 microbiology training grant T32 AI007180 to U.M.G. and M.A.G.; T32 cardiology grant 104220 to U.M.G.), NIAID (grants AI093811 and AI094166 to P.L.), NIDDK (grant DK103788 to P.L.), a Ruth L. Kirschstein NRSA fellowship (F32AI102502 to N.M.G.), the Vilcek Foundation (U.M.G.) and AAI (M.S.T.). Biomphalaria glabrata snails were provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, Maryland, USA) through NIH–NIAID contract HHSN272201000005I for distribution through BEI Resources.

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U.M.G., N.M.G., M.A.G. and P.L. designed experiments, conducted research, analyzed data and contributed to writing of the paper. H.J.P.V.D.Z., M.S.T., J.-D.L., M.O., L.J.M. and J.P. carried out research and analyzed data. N.V., L.J.M. and J.P. conducted research. E.A.F. and K.J.M. provided necessary mice and materials, and contributed to writing of the paper.

Corresponding author

Correspondence to P'ng Loke.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Monocyte-derived inflammatory macrophages adopt a tissue-resident phenotype after long-term residency in the peritoneal cavity.

(a) Schematic of short-term transfer of monocyte-derived inflammatory macrophages from thioglycollate treated CD45.1 donor mice into CD45.2 recipient mice, rested for 24hrs and subsequently treated with IL-4c. (b) Quantification of mean fluorescence intensity values of PD-L2 and CD206 expression on donor and recipient macrophages. (c and d) Flow cytometric analysis of adoptive cell transfer of macrophages flow sorted from (c) thioglycollate-treated mice or (d) IL-4c treated mice and transferred into CD45.2 recipient mice and then treated with IL-4c 24hrs after transfer. Representative histograms display surface expression of F4/80, CD206, PD-L2 and MHCII in Donor CD45.1+ CD45.2 (Black line) and recipient CD45.1, CD45.2+ (Grey shaded) CD11b+, F4/80+ macrophages. (e) IL-4c induced tissue resident M2 macrophages rapidly disappear after transfer into an inflammatory environment. CD11b+, F4/80+ macrophages sorted from CD45.1 donor mice treated with IL-4c were adoptively transferred into CD45.2 recipient mice, 24hrs later the recipient mice were treated with thioglycollate + IL-4c prior to analysis.

Supplementary Figure 2 Fate-mapping of peritoneal macrophages after thioglycollate injection.

(a) Schematic of timecourse analysis of peritoneal macrophages 1 week (n=4), 4 weeks (n=4) or 8 weeks (n=4) after thioglycollate injection. Stacked bar graph showing the relative proportion of F4/80 and/or CD206 expression. (b) Representative FACS plots are shown displaying the frequency of CD45.1+ (blue) donor cells in CD45.2 (grey) recipient mice treated with or without IL-4c after 8 weeks of residency. Flow cytometry analysis of transferred cells shows acquisition of tissue resident phenotype by CD45.1 donor cells in the presence or absence of IL-4c treatment. IL-4c treatment increases the frequency of CD45.1+ F4/80+ cells. (c) Representative FACS plots showing the frequency of EdU+ cells in recipient and donor populations in response to IL-4c given after transfer and resting for 24hrs for short term residency or long term residency for 8 weeks. (d) Schematic of adoptive transfer of Thio-elicited monocyte-derived macrophages from Stat6−/− CD45.2 donor mice transferred into WT CD45.1 recipient mice rested for 24hrs and then treated with IL-4c. Histograms display expression of F4/80, CD206, PD-L2 and MHCII in donor CD45.2+ CD45.1 (Black) and recipient CD45.2, CD45.1+ (Grey shaded) CD11b+ cells. (e) Representative FACS plots showing the frequency of EdU+ cells in WT recipient and Stat6−/− donor CD11b+ F4/80+ macrophages in response to IL-4c given after resting for 24hrs.

Supplementary Figure 3 Transcriptional and chromatin landscape reprogramming during macrophage conversion.

(a) Pairwise Pearson’s correlation analysis and (b) PCA of transcriptional profiles in AAMmono, AAMconv and AAMres. (c) Heatmap visualizing the expression values of 6 specific genes across the different populations of macrophages. (d) Top 10 GO terms enriched in the 3966 genes upregulated in AAMmono when compared to AAMconv (top) and top 10 GO terms enriched in the 675 genes upregulated in AAMconv when compared to AAMres (bottom). (e) Pairwise Pearson’s correlation analysis and (f) PCA of accessible chromatin regions in AAMmono, AAMconv and AAMres. On PCA plots, red circles represent AAMmono, orange squares represent AAMconv and blue triangles represent AAMres in PCA plots.

Supplementary Figure 4 Baseline disruption of peritoneal tissue-resident macrophages in vitamin A–deficient mice.

(a) Total number of cells collected from the peritoneal cavity of vitamin A deficient (Vit-ADEF) or control (Vit-ACON) mice via peritoneal lavage. (b,c) Total number of F4/80hi CD206 (b) or F4/80int CD206+ (c) cells in the peritoneal cavity in Vit-ADEF or Vit-ACON mice. **P < 0.01. Unpaired Students T-test.

Supplementary Figure 5 Expression of UCP1 in S. mansoni infection associated with EdU localization in mature liver granulomas.

(a) Transcript expression of Ucp1 in whole liver from mice infected with S. mansoni at 9 weeks and 12 weeks post infection. (b) Representative immunofluorescence images of S. mansoni-infected liver granulomas at different timepoints post infection stained with DAPI (blue) and Click-it EdU (red) taken from mice pulsed with EdU 3 hours prior to sacrifice. Eggs are outlined in white. (c) Slide-scanned, immunofluorescence image of S. mansoni-infected liver granulomas taken at 8 weeks post infection and stained with DAPI (blue), anti-UCP1 (green) and Click-it EdU (red) in vitamin A control mice pulsed with EdU 3 hours prior to sacrifice. Scale bars represent 50 microns or 500 microns as indicated.

Supplementary Figure 6 STAT6 regulates phenotypic conversion of peritoneal AAMmono cells after S. mansoni egg injection.

(a) Schematic of S. mansoni egg injection in Cx3cr1creERT2-IRESYFP/+ Rosa26stopfl-tdTomato/+ vitamin A deficient (Vit-ADEF) or control (Vit-ACON) mice. Representative flow cytometry plots of fate-mapped monocyte-derived macrophages in the lung after S. mansoni egg challenge in the lung. (b) Schematic of S. mansoni egg injection in WT:WT (n=5) or Stat6−/−:WT (n=5) mixed bone marrow chimeric mice whereby mice were sensitized with eggs via i.p. injection then challenged i.v. with eggs after 2 weeks. PECs and lung macrophages were analyzed after 8 days of rest and pulsed with EdU 3hrs prior to sacrifice. Representative flow cytometry plots display macrophage phenotypes from the peritoneal cavity and lung in mixed bone marrow chimeric mice treated with or without eggs. (c) Schematic of S. mansoni infection in mixed bone marrow chimera Stat6−/−:WT mice. Cumulative body weight during the infection reveals loss of weight after 5 weeks post-infection. Kaplan-Meyer survival curve displays rapid mortality of Stat6−/−:WT chimeric mice at 7 weeks post-infection.

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Gundra, U., Girgis, N., Gonzalez, M. et al. Vitamin A mediates conversion of monocyte-derived macrophages into tissue-resident macrophages during alternative activation. Nat Immunol 18, 642–653 (2017). https://doi.org/10.1038/ni.3734

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