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Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity

Abstract

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state and expand during type 2 immunity. Whether shared mechanisms regulate the proliferation of macrophages in homeostasis and disease is unclear. Here we found that the transcription factor Bhlhe40 was required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages (LPMs), but not that of other tissue-resident macrophages. Bhlhe40 was necessary for the proliferation, but not the polarization, of LPMs in response to the cytokine IL-4. During infection with the helminth Heligmosomoides polygyrus bakeri, Bhlhe40 was required for cell cycling of LPMs. Bhlhe40 repressed the expression of genes encoding the transcription factors c-Maf and Mafb and directly promoted expression of transcripts encoding cell cycle-related proteins to enable the proliferation of LPMs. In LPMs, Bhlhe40 bound to genomic sites co-bound by the macrophage lineage-determining factor PU.1 and to unique sites, including Maf and loci encoding cell-cycle-related proteins. Our findings demonstrate a tissue-specific control mechanism that regulates the proliferation of resident macrophages in homeostasis and type 2 immunity.

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Fig. 1: Loss of Bhlhe40 dysregulates the cell cycle in LPMs.
Fig. 2: Bhlhe40 is cell-intrinsically required in LPMs to regulate the cell cycle.
Fig. 3: Bhlhe40 regulates a distinct set of genes related to alternative activation in LPMs.
Fig. 4: Bhlhe40 is required for normal accumulation of resident, but not recruited, macrophages in the peritoneum.
Fig. 5: Bhlhe40 is required for normal cycling, but not polarization, of peritoneal macrophages during type 2 immunity.
Fig. 6: Bhlhe40 is required for LPM proliferation in response to H. polygyrus.
Fig. 7: Bhlhe40 regulates gene expression to modulate proliferation, but not alternative activation, in LPMs during type 2 immunity.
Fig. 8: Bhlhe40 directly regulates gene expression in LPMs in an activation state-dependent manner.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The microarray and ChIP-sequencing data have been deposited in the GEO repository under accession code GSE125730.

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Acknowledgements

This work was supported by the National Institute of Allergy and Infectious Disease (NIAID) (AI113118 and AI132653) (B.T.E.) and a Burroughs Wellcome Fund Career Award for Medical Scientists (B.T.E.). N.N.J. was supported by grant 5T32AI007163 from the NIAID. C.-C.L. was supported by the McDonnell International Scholars Academy at Washington University. S.C.-C.H. was supported by the Case Comprehensive Cancer Center American Cancer Society IRG Award (IRG−16–186–21). M.E.C. was supported by the National Science Foundation Graduate Research Fellowship program (DGE-1745038). Research reported in this publication was supported by the Washington University Institute of Clinical and Translational Sciences grant UL1TR002345 from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. We thank E. Lantelme, A. Cullen and D. Brinja for help with fluorescence-activated cell sorting. We thank W. Beatty and L. Mwaghore for electron microscopy. We thank S. Van Dyken and C. Farnsworth for critical reading of the manuscript. We thank the members of the G. Randolph laboratory for helpful discussions about this project. We thank J. Bando, M. Robinette and T. Ai for help with flow cytometry of the gut.

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N.N.J designed, performed and analyzed experiments and wrote the manuscript. E.A.S., T.R.B., C.-C.L., M.E.C. and C.-W.L. performed experiments. S.C.-C.H. provided reagents, protocols and technical expertise. I.S. and M.N.A. analyzed ChIP-seq data. R.T. provided mice. T.S.S., G.J.R. and J.F.U. provided reagents and technical expertise. B.T.E. designed and analyzed experiments, supervised the studies and wrote the manuscript.

Corresponding author

Correspondence to Brian T. Edelson.

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Integrated supplementary information

Supplementary Fig. 1 Bhlhe40 is specifically required in peritoneal and pleural macrophages.

a, Flow cytometry of Bhlhe40GFP transgene reporter expression in pleural macrophages from Bhlhe40GFP+ and Bhlhe40GFP mice (representative of 2 experiments, n = 3 Bhlhe40GFP+, 2 Bhlhe40GFP-). b-e, Flow cytometry for AMs (b), red pulp macrophages (c), kidney macrophages (d), and Kupffer cells (e) from Bhlhe40+/+ and Bhlhe40-/- mice as in Fig. 1c. f, Flow cytometry for Tim4 expression on LPMs from Bhlhe40+/+ and Bhlhe40-/- mice (representative of 11 experiments, n = 40/group). g, Numbers of Tim4+ LPMs as in f. h, Flow cytometry for CD226 expression on SPMs from Bhlhe40+/+ and Bhlhe40-/- mice (representative of 8 experiments, n = 27/group). i, Numbers of CD226+ SPMs as in h. j, Numbers of peritoneal B cells from Bhlhe40+/+ and Bhlhe40-/- mice (pooled from 12 experiments, n = 35/group). k, Numbers of large pleural macrophages from Bhlhe40+/+ and Bhlhe40-/- mice (pooled from 6 experiments, n = 16 Bhlhe40+/+, 17 Bhlhe40-/-). l, Frequency of Ki67+ large pleural macrophages from Bhlhe40+/+ and Bhlhe40-/- mice (pooled from 3 experiments, n = 7 Bhlhe40+/+, 8 Bhlhe40-/-). m, frequency of 7-AAD+ LPMs from Bhlhe40+/+ and Bhlhe40-/- mice (pooled from 8 experiments, n = 19 Bhlhe40+/+, 18 Bhlhe40-/-). Data in g,i-m are mean ± s.e.m; each symbol represents an individual mouse (g,i-m). ***P < 0.001; ****P < 0.0001, unpaired two-sided Student’s t-test.

Supplementary Fig. 2 Bhlhe40 is cell-intrinsically required in LPMs.

a, Ratio of CD45.1 to CD45.2 cells for small pleural macrophages and pleural B cells from mixed bone marrow chimeras as in Fig. 2d. b, Flow cytometry for the discrimination of donor and recipient LPMs and peritoneal B cells from CD45.1/CD45.2 recipients of transferred peritoneal cells as in Fig. 2k, l. c,d, Gene expression microarray data from Bhlhe40+/+ and Bhlhe40-/- LPMs (in this study) and LysM-Cre- Gata6fl/fl, and LysM-Cre+ Gata6fl/fl LPMs (reanalyzed from 36) were analyzed for shared and unique Bhlhe40 and/or Gata6-dependent genes ( ≥ 2-fold differentially expressed, depicted as a Venn diagram) (c) and differentially expressed genes dependent on both Bhlhe40 and Gata6 (d). e, Gene expression microarray data were analyzed for expression of an LPM gene signature in LPMs and AMs from Bhlhe40+/+ and Bhlhe40-/- mice. Microarray data from LPMs (n = 3/group) and AMs (n = 2/group) are from a single experiment. Data in a are mean ± s.e.m; each symbol represents an individual mouse (a). Significance calculated with an unpaired two-sided Student’s t-test.

Supplementary Fig. 3 Further analysis of responses to IL-4c in Bhlhe40-deficient mice.

a, Flow cytometry of peritoneal macrophage subsets from Bhlhe40+/+, Bhlhe40-/-, and Bhlhe40-/- Il10-/- mice treated with PBS or IL-4c (representative of 2 experiments, n = 3 PBS-treated Bhlhe40+/+, 2 PBS-treated Bhlhe40-/-, 3 PBS-treated Bhlhe40-/- Il10-/-, 5 IL-4c-treated Bhlhe40+/+, 7 IL-4c-treated Bhlhe40-/-, 4 IL-4c-treated Bhlhe40-/- Il10-/-). b, Numbers of LPMs as in a. c, Immunoblotting of cyclins D1-3, cyclin-dependent kinase (CDK) 2, CDK4, CDK6, E2F2, and beta actin in lysates of LPMs from Bhlhe40+/+ and Bhlhe40-/- mice unstimulated or treated with IL-4c (representative of 2 experiments, n = 2/group). d, Frequency of BrdU+ Bhlhe40+/+ (CD45.1), Bhlhe40+/+ (CD45.2), or Bhlhe40-/- (CD45.2) LPMs from mixed bone marrow chimera mice (generated as in Fig. 2a–f) treated with PBS or IL-4c, with LPMs from each donor recovered from the same recipient connected by a line (pooled from 2 experiments, n = 2 PBS-treated [Bhlhe40+/+ (CD45.1) +Bhlhe40+/+ (CD45.2)] and [Bhlhe40+/+ (CD45.1) +Bhlhe40-/- (CD45.2)]; 4 IL-4c-treated [Bhlhe40+/+ (CD45.1) +Bhlhe40+/+ (CD45.2)]; 5 [Bhlhe40+/+ (CD45.1) +Bhlhe40-/- (CD45.2)])). Data in b,d are mean ± s.e.m; (b) each symbol or (d) paired symbols represent an individual mouse. ***P < 0.001, (b) unpaired or (d) paired two-sided Student’s t-test.

Supplementary Fig. 4 Loss of Bhlhe40 causes morphological changes in in vivo IL-4c-stimulated peritoneal macrophages.

a,b, Transmission electron microscopy images of LPMs from naïve (a) and IL-4c-treated (b) Bhlhe40+/+ and Bhlhe40-/- mice (representative of 2 experiments, n = 2 mice (45–50 images)/group). Scale bar, 2 μm. c-h, Transmission electron microscopy for cellular cross-sectional area (c), endoplasmic reticulum (ER) cross-sectional extent (d), ER luminal width (e), vesicle cross-sectional area (f), nucleoli/cross-section (g), mitochondria/cross-section (h) of LPMs from Bhlhe40+/+ and Bhlhe40-/- mice unstimulated or treated with IL-4c (pooled from 2 experiments, n = 2 mice/group [27–60 cells analyzed, except for 17–20 cells analyzed in (d)]). Data in c-h are mean ± s.e.m; each symbol represents an individual cell (c-h). *P ≤ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, unpaired two-sided Student’s t -test.

Supplementary Fig. 5 Bhlhe40 expression is tightly regulated in resident macrophages.

a, Flow cytometry of Bhlhe40GFP transgene reporter expression in LPMs, SPMs, red pulp macrophages, Kupffer cells, kidney macrophages, and AMs from Bhlhe40GFP+ and Bhlhe40GFP- mice after PBS or IL-4c treatment (representative of 1–2 experiments, n = 3 PBS-treated Bhlhe40GFP+, 1 PBS-treated Bhlhe40GFP-, 5 IL-4c-treated Bhlhe40GFP+, 3 IL-4c-treated Bhlhe40GFP-). b, Numbers of LPMs, red pulp macrophages, Kupffer cells, and AMs from Bhlhe40+/+ and Bhlhe40-/- mice treated with PBS or IL-4c (pooled from 2 experiments, n = 4 PBS-treated Bhlhe40+/+, 3 PBS-treated Bhlhe40-/-, 8 IL-4c-treated Bhlhe40+/+, 7 IL-4c-treated Bhlhe40-/-). c-e, Flow cytometry of BrdU incorporation by red pulp macrophages (c), Kupffer cells (d), and AMs (e) from Bhlhe40+/+ and Bhlhe40-/- mice treated with PBS or IL-4c as in Fig. 5g. Data in b are mean ± s.e.m; each symbol represents an individual mouse (b). *P ≤ 0.05, unpaired two-sided Student’s t-test.

Supplementary Fig. 6 Bhlhe40 is required for normal proliferation of thioglycollate-elicited macrophages during type 2 immunity.

a, Flow cytometry of BrdU incorporation by LPMs and Thio-elicited macrophages from Bhlhe40+/+ and Bhlhe40-/- mice treated with PBS, IL-4c, thioglycollate (Thio), or Thio and IL-4c as in Fig. 5j. b,c, Frequency of pHH3+ LPMs and Thio-elicited macrophages (b) (pooled from 2 experiments, n = 3 PBS-treated Bhlhe40+/+; 4 PBS-treated Bhlhe40-/-; 4 IL-4c-treated and Thio-treated Bhlhe40+/+ and Bhlhe40-/-; 6 Thio and IL-4c-treated Bhlhe40+/+ and Bhlhe40-/-) and RELMα+ LPMs and Thio-elicited macrophages (c) (pooled as in b) from Bhlhe40+/+ and Bhlhe40-/- mice treated with PBS, IL-4c, thioglycollate (Thio), or Thio and IL-4c. Data in b,c are mean ± s.e.m; each symbol represents an individual mouse (b,c). *P ≤ 0.05; ***P < 0.001, unpaired two-sided Student’s t-test.

Supplementary Fig. 7 Bhlhe40 directly regulates gene transcription in LPMs.

a, Gene expression microarray data were analyzed for expression of genes encoding selective regulators of LPM proliferation (Myo18a and C1q) in LPMs from Bhlhe40+/+, Bhlhe40-/-, and LysM-Cre+ Bhlhe40fl/fl mice unstimulated or treated with IL-4c. b-d, Tracings of Bhlhe40 binding, PU.1 binding, and vertebrate conservation at the Il10 (b), Ccl2 (c), and Plac8 (d) loci. e, Bhlhe40-bound, Bhlhe40-dependent genes ( ≥ 2-fold differentially expressed in Bhlhe40+/+ and Bhlhe40-/- LPMs) in LPMs from naïve mice and Bhlhe40-bound, Bhlhe40-dependent genes ( ≥ 2-fold differentially expressed in Bhlhe40+/+ and Bhlhe40-/- LPMs) in LPMs from IL-4c-treated mice, as in Fig. 8i. Underlined genes are highlighted elsewhere in this study. f-h, Tracings of Bhlhe40 binding, PU.1 binding, and vertebrate conservation at the Maf (distal) (f), Mafb (g), and Mafb (distal) (h) loci. LPM Bhlhe40 ChIP-seq data (n = 1/group), microarray data from naïve LPMs (n = 3/group), and microarray data from IL-4c-stimulated LPMs (n = 2/group) are from single separate experiments. LPM PU.1 ChIP-seq data reanalyzed from 35.

Supplementary Fig. 8 Bhlhe40 directly binds to cell cycle-related loci and is required to sustain normal gene expression.

a-d, The proportion of Bhlhe40-bound members of gene sets enriched in Bhlhe40+/+ compared to Bhlhe40-/- LPMs from IL-4c-treated mice. Tracings of Bhlhe40 binding, PU.1 binding, and vertebrate conservation for a representative member of the core enrichment signature for each gene set is presented. Hallmark E2F Targets (a), Hallmark Myc Targets (v1) (b), C5 Chromosome Organization (c), and C5 Cell Cycle Process (d). e-h, GSEA of expression of Bhlhe40-bound genes from gene expression microarray data from LPMs from Bhlhe40+/+ and Bhlhe40-/- mice treated with IL-4c for Hallmark E2F Targets (e), Hallmark Myc Targets v1 (f), C5 Chromosome Organization (g), and C5 Cell Cycle Process (h). C5 Cell Cycle Process is also presented in Fig. 8i. NES, normalized enrichment score. FWER, family-wise error rate. LPM Bhlhe40 ChIP-seq data (n = 1/group) and microarray data (n = 2/group) are from single separate experiments. LPM PU.1 ChIP-seq data reanalyzed from 35. (e-h) NES and FWER.

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Jarjour, N.N., Schwarzkopf, E.A., Bradstreet, T.R. et al. Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity. Nat Immunol 20, 687–700 (2019). https://doi.org/10.1038/s41590-019-0382-5

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