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Safeguard function of PU.1 shapes the inflammatory epigenome of neutrophils

Abstract

Neutrophils are essential first-line defense cells against invading pathogens, yet when inappropriately activated, their strong immune response can cause collateral tissue damage and contributes to immunological diseases. However, whether neutrophils can intrinsically titrate their immune response remains unknown. Here we conditionally deleted the Spi1 gene, which encodes the myeloid transcription factor PU.1, from neutrophils of mice undergoing fungal infection and then performed comprehensive epigenomic profiling. We found that as well as providing the transcriptional prerequisite for eradicating pathogens, the predominant function of PU.1 was to restrain the neutrophil defense by broadly inhibiting the accessibility of enhancers via the recruitment of histone deacetylase 1. Such epigenetic modifications impeded the immunostimulatory AP-1 transcription factor JUNB from entering chromatin and activating its targets. Thus, neutrophils rely on a PU.1-installed inhibitor program to safeguard their epigenome from undergoing uncontrolled activation, protecting the host against an exorbitant innate immune response.

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Data availability

The data that support the findings of this study are available from the corresponding author on request. All sequencing data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE110865. Publicity available Hi-C data referenced in this study were extracted from GEO with the accession number GSE35156.

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Acknowledgements

We thank J. Rettkowski and J. Kamping for assistance with experiments, T. König for FACS sorting, J. Brands for help with figure preparations and C. Brennecka for linguistic support. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; RO2295/6-1) and the University of Muenster Medical Faculty (Ros2/007/15) to F.R. M.P. is supported by the DFG (SFB992, SFB1160, SFB/TRR167, Reinhart-Koselleck-Grant).

Author information

J.F., A.T., H.A., M.L., V.G., T.E. and I.D.J. conducted experiments and analyzed data, C.W., A.T. and M.D. performed computational analyses, T.V. and J.R. helped with the cell immortalization, G.L. provided important tools, and M.J.C.J. and M.P. conducted pathological analyses. J.F., I.D.J. and F.R. designed the study and wrote the manuscript. F.R. supervised the entire project and provided financial support.

Competing interests

The authors declare no competing interests.

Correspondence to Frank Rosenbauer.

Integrated supplementary information

Supplementary Figure 1 Validation of and gene expression in neutrophils of PU.1∆Neu mice.

(a) Scheme of the crossbreeding to generate a neutrophil-specific PU.1-knockout mouse strain. (b) Flow cytometry analysis of GFP expression in BM neutrophils (CD11b+Ly-6G+), splenic B cells (CD11b-CD19+), splenic Ly-6C- (CD11b+CD115+Ly-6C-) and Ly-6C+ (CD11b+CD115+Ly-6C+) monocytes as well as in the BM progenitor populations LSK (lin-c-kit+Sca-1+), common myeloid progenitors (CMP; lin-c-kit+Sca-1-CD34+FcγR-), granulocyte-monocyte progenitors (GMP; lin-c-kit+Sca-1-CD34+FcγR+) and megakaryocyte-erythrocyte progenitors (MEP, lin-c-kit+Sca-1-CD34-FcγR-) of wildtype (GFP-, black) and PU.1∆Neu mice (GFP+, blue). Data are representative of at least three independent experiments with similar results. (c) Flow cytometry analysis of YFP expression of neutrophils (Ly-6C+CD115-) and Ly-6C + monocytes (Ly-6C+CD115+) from the BM of PU.1∆Neu mice crossbred with loxSTOPlox-YFP reporter mice. Numbers in outlined areas indicate the percentages of cells within the gates. Data are representative of two independent experiments with similar results. (d) qPCR analysis of Spi1 expression in neutrophils (CD11b+Ly-6G+GFP+) isolated from the BM of PU.1WT (n = 5) and PU.1ΔNeu (n = 5) mice. Results are shown as fold over actin transcripts (mean±s.e.m.) and are from five independent experiments. ***P ≤ 0.0001 (unpaired t-test, two-tailed). (e) Immunoblot analysis to determine PU.1 protein levels in flow-sorted BM neutrophils (CD11b+Ly-6G+GFP+) from PU.1WT and PU.1∆Neu mice. Detection of valosin-containing protein (VCP) ensured comparable loading. Shown is one of three independent experiments with similar results. (f) qPCR analysis of Spi1 expression in monocytes (CD11b+CD115+) flow-sorted from PU.1WT and PU.1∆Neu mice (n = 2 each). Results are shown as mean fold over actin transcripts. (g,h) Flow cytometry analysis of peripheral blood B cells (CD11b-CD19+) and T cells (CD11b-CD3+) (g) and monocytes (CD11b+CD150+Ly-6C+ and CD11b+CD150+Ly-6C-) (h) in PU.1WT and PU.1ΔNeu mice (n = 8 each). Numbers in outlined areas indicate mean percent ± s.e.m. (i) White blood cell counts (WBC) of PU.1WT and PU.1∆Neu mice (n = 9 each). Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). *P = 0.0138 (unpaired t-test, two-tailed). (j,k) Gene set enrichment analysis differentially expressed between neutrophils (Gr-1+) isolated from the BM of PU.1WT (n = 3) and PU.1ΔNeu mice (n = 2). Enrichment score calculations are based on weighted Kolmogorov-Smirnov-like statistics, adjusted for multiple testing after Benjamini-Hochberg. Compared gene set sizes were n = 12 (granulocyte differentiation), n = 231 (inflammatory response) and n = 21 (Nf-kB). (l) Expression of downregulated genes in neutrophils from PU.1ΔNeu (n = 3) compared to PU.1WT (n = 2) mice. Results were obtained from microarrays and are given in mean intensity values.

Supplementary Figure 2 The role of PU.1 during distinct neutrophil defense steps.

(a,b) Experimental setups of systemic Candida albicans (C. albicans) infections of PU.1WT and PU.1ΔNeu mice with a lethal (5x105 colony forming units, cfu) (a) or sublethal (2x105 cfu) (b) fungal dose to obtain survival curves (a) or perform time point analyses (b). (c,d) Fungal burden of brains from PU.1WT (c: n = 5, d: n = 6) and PU.1ΔNeu (c,d: n = 6) mice one (c) and three (d) days after C. albicans-infection (2x105 cfu). Data are from two independent experiments. Small horizontal lines indicate the mean ( + s.e.m.) *P = 0.0173 (unpaired t-test, two-tailed). (e) Flow cytometry analysis of CD62L expression on the surface of peripheral blood neutrophils of PU.1WT and PU.1ΔNeu mice one (d1) and three (d3) days post C. albicans-infection (2x105 cfu). (f) Frequency of CD62Llo expressing neutrophils in the PB of unstimulated PU.1WT (n = 3) and PU.1∆Neu (n = 4) mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). The experiment was independently performed twice with similar results. (g) Mean fluorescence intensity (MFI) of CD11b on PB neutrophils in C. albicans-challenged (2x105 cfu) PU.1WT (n = 3) and PU.1ΔNeu (n = 3) mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). (h) Ratio of neutrophil numbers between kidneys and the peripheral blood (number of kidney neutrophils divided by the number of blood neutrophils) in PU.1WT and PU.1ΔNeu mice (n = 3 each) three days post C. albicans-infection (2x105 cfu). Data are the mean ± s.e.m. **P = 0.0036 (unpaired t-test, two-tailed). (i,j) Expression of NADPH oxidase subunit genes Cybb, Cyba, Ncf, Ncf2 and Ncf4 (i) and genes coding for immune response receptors important for C. albicans sensing: Clec7a, Clec4e and Clec4n (j) in flow-sorted neutrophils (Gr-1+) isolated from the BM of PU.1WT (n = 2) and PU.1ΔNeu mice (n = 3). Results were obtained from microarrays and are given in normalized intensity values. Small horizontal lines indicate the mean. All expression differences of the indicated genes were independently reproduced by qRT-PCR (data not shown).

Supplementary Figure 3 PU.1 inhibits neutrophil activation.

(a) Mixed BM chimeras were generated by intravenous injection of 7.5x105 BM cells from SJL.Bl6 mice (CD45.1+) mixed with 7.5x105 BM cells from either PU.1WT or PU.1ΔNeu (both CD45.2+) mice into lethally irradiated (9 Gy) SJLxBl6 (CD45.1+CD45.2+) or SJL.Bl6 mice (CD45.1+). After 2 to 3 month of engraftment, chimera were intravenously infected with a sublethal (2x105 cfu) dose of C. albicans and time point studies were performed. (b) Weight loss of C. albicans-infected (2x105 cfu) mixed BM chimeras (n = 5) three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.).(c) Kidney weights of C. albicans-infected (2x105 cfu) mixed BM chimeras (n = 5) three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). (d) Fungal burden of kidneys from mixed BM-reconstituted chimeras (n = 4) one and three days post C. albicans-infection (2x105 cfu). Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). (e) Frequency of CD62Llo expressing neutrophils in the peripheral blood (PB) of C. albicans-infected (2x105 cfu) mixed BM chimeras (n = 5) three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). *P = 0.0191. (f) Mean fluorescence intensity (MFI) of the degranulation marker CD63 on the surface of neutrophils (CD11b+Ly-6G+) from the BM of unstimulated PU.1WT (n = 5) and PU.1ΔNeu (n = 4) mice as determined by flow cytometry. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). ***P ≤ 0.0001. (g) qPCR analysis of the proinflammatory cytokine-encoding genes Il1b and Tnfa in BM derived macrophages (BMM) coincubated with in vitro derived PU.1WT and PU.1ΔNeu neutrophils (n = 3 each). Results are shown as fold difference relative to untreated BMM control (mean + s.e.m). Left to right: *P = 0.0443, *P = 0.0154. (h) Weight loss of C. albicans NRG1 mutant-infected (1x106 cfu) PU.1WT (n = 6), PU.1ΔNeu (n = 4) and ΔCYBB (n = 4) mice one and three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: ***P = 0.0008, *P = 0.0494. (i) Kidney weights of C. albicans NRG1 mutant-infected (1x106 cfu) PU.1WT (n = 6), PU.1ΔNeu (n = 4) and ΔCYBB (n = 4) mice one to three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). *P = 0.0302. (j) Fungal burden of kidneys from C. albicans NRG1 mutant-infected (1x106 cfu) PU.1WT (n = 6), PU.1ΔNeu (n = 4) and ΔCYBB (n = 2) mice two and three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: ***P ≤ 0.0001, **P = 0.0012. (k) Absolute number (by flow cytometry) of total macrophages (CD11b+F4/80+) in the kidneys of PU.1WT (n = 6), PU.1ΔNeu (n = 4) and ΔCYBB (n = 4) mice that were challenged with the NRG1 overexpression mutant of C. albicans (1x106 cfu) for one to three days. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: **P = 0.0085, **P = 0.0032 (l) Percentage of CD62Llo expressing neutrophils in the BM of C. albicans NRG1 mutant-challenged (1x106 cfu) PU.1WT (n = 6), PU.1ΔNeu (n = 4) and ΔCYBB (n = 4) mice one to three days post infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). Left to right: ***P ≤ 0.0001, ***P ≤ 0.0001. All P-values in this figure: unpaired t-test, two-tailed.

Supplementary Figure 4 Generation and validation of immortalized PU.1∆Neu neutrophil progenitor lines.

(a) Experimental procedure of the generation of immortalized neutrophil progenitor cell lines from PU.1WT and PU.1ΔNeu mice. BM cells were isolated and enriched for progenitor cells by gradient centrifugation. Progenitor cells were transfected with the ER-Hoxb8 (HoxER) retrovirus and were cultured with supplementation of β-estradiol and stem cell factor (SCF). For neutrophil differentiation, progenitor cells were cultured without β-estradiol with supplementation of granulocyte colony-stimulating factor (G-CSF) for three to four days. To stimulate neutrophils, they were exposed to heat inactivated C. albicans cells for two hours. (b) PCR analysis of the PU.1 excision in progenitors (d0) and neutrophils (d4) of HoxER-PU.1WT (WT) and HoxER-PU.1ΔNeu (ΔNeu) lines. The experiment was independently performed more than five times with similar results. (c) qPCR analysis of Spi1 expression in progenitors (d0, n = 3 both genotypes) and neutrophils (d4, n = 4 both genotypes) of HoxER-PU.1WT (WT) and HoxER-PU.1ΔNeu (ΔNeu) lines. Results are shown as fold over actin transcripts (mean±s.e.m.) of three (d0) and four (d4) independent experiments. ***P = 0.0003 (unpaired t-test, two-tailed). (d) Immunoblot analysis of PU.1 expression in HoxER-PU.1WT and HoxER-PU.1ΔNeu progenitors (d0) and neutrophils (d4). Detection of valosin-containing protein (VCP) served as loading control. The experiment was independently performed twice with similar results. (e,f) Flow cytometry analysis of HoxER-PU.1WT and HoxER-PU.1ΔNeu progenitors (e) and neutrophils (f). The experiment was independently performed more than five times with similar results. (g) Morphological analysis of cytospins stained with Giemsa of HoxER-PU.1WT and HoxER-PU.1ΔNeu neutrophils. Scale bar resembles 10 µm. The experiment was independently performed twice with similar results. (h,i) Gene set enrichment analysis of gene signatures generated from the top 500 upregulated genes in HoxER-PU.1WT (h; Top 500 HoxER-PU.1WT) or HoxER-PU.1ΔNeu (i; Top 500 HoxER-PU.1ΔNeu) neutrophils obtained from mRNA-seq in flow-sorted (Gr-1+) neutrophils from the BM of PU.1WT (n = 2) and PU.1ΔNeu (n = 3) mice. Enrichment score calculations (for n = 500 genes each) are based on weighted Kolmogorov-Smirnov-like statistics, adjusted for multiple testing after Benjamini-Hochberg. (j) ROS production in HoxER-PU.1WT (PU.1WT; n = 3) and HoxER-PU.1ΔNeu (PU.1ΔNeu; n = 3) neutrophils stimulated with heat inactivated C. albicans cells. Data were obtained from three independent experiments. Each symbol represents and individual sample, small horizontal lines indicate the mean ( + s.e.m.). ***P = 0.0003, (unpaired t-test, two-tailed). (k) Distribution of expression of cluster genes (I-IV) visualized as the log2 of the reads per kilobase per million (RPKM) values.

Supplementary Figure 5 Cluster enhancer determination and allocation.

(a) Spearman correlations for individual ATAC-seq samples (n = 2 each) based on the combined peaks of unstimulated (WT u) and 1-hour C. albicans-stimulated (WT Ca) HoxER-PU.1WT neutrophils. (b) Expression of AP-1 transcription factors in the indicated neutrophils. All samples were n = 3 except for HoxER-PU.1ΔNeu which was n = 2. Expression is shown as in reads per kilobase per million (RPKM) obtained from mRNA-seq. Bars represent mean ± s.e.m. expect for HoxER-PU.1ΔNeu which is only shown as mean. (c) Scheme of the used enhancer determination strategy and their allocation to genes. Enhancers were determined as promoter distal regions marked with H3K4me1. Enhancers with differential H3K27ac occupancy in one condition were allocated to the next differential expressed gene within one TAD. Box indicates the active enhancer, which was allocated to the differentially expressed gene. (d) PU.1 occupancy on gene regulatory elements (promoters and enhancers) of the cluster genes. (e) Number of genes dedicated to clusters I-IV, number of promoters of cluster genes with PU.1 occupancy, number of enhancers which could be allocated to the cluster genes, the number of these enhancers with PU.1 occupancy and the number of expressed genes that could be allocated to the PU.1 bound enhancers. (f) Reporter activities of putative enhancers measured by luciferase assay in transiently transfected THP-1 cells. Data represent the mean of two (E3–5, negative regions), three (E2) or four (E1) independent experiments. (g,h) Integrative genomics viewer images showing tracks of normalized tag counts of H3K4me1 (K4me1), H3K27ac (K27ac) and PU.1 ChIP-seq as well as ATAC-seq in HoxER-PU.1WT and HoxER-PU.1ΔNeu neutrophils. Shown are the PU.1 activated gene loci of the cluster I gene Cd34 and the cluster II gene Il1b (g), and PU.1 inhibited loci of the cluster III gene Spp1 and the cluster IV gene Cd63 (h). Grey boxes indicate relevant enhancer positions. Sown are representative tracks of two independent samples each.

Supplementary Figure 6 JUNB in PU.1-deleted neutrophils.

(a) Homer motif analysis in PU.1-bound cluster I and II or III and IV enhancers. Data were derived from two independent samples each. Motif enrichment was calculated with HOMER software, applying cumulative hypergeometric distribution adjusted for multiple testing with the Benjamini-Hochberg method. (b) Average and normalized (events per million) cutting events based on the ATAC-seq data over cluster enhancers containing a CEBP motif. (c) Genomic distribution of JUNB and PU.1 cobound regions in HoxER-PU.1WT neutrophils. (d) qPCR analysis of Junb expression in C. albicans stimulated HoxER-PU.1ΔNeu neutrophils that were infected with retroviruses carrying a scramble control or one of three independent shRNAs against Junb. Expression is shown as percent of the control value. Dashed line represents the mean expression value of the controls.

Supplementary Figure 7 PU.1-inhibits immune gene expression by HDAC1.

(a) Table summarizing expression (with a minimal RPKM value of 1) of HDACs in HoxER neutrophils. Expression was obtained from mRNA-seq. (b) qPCR analysis of expression of the indicated cluster III genes in HoxER-PU.1WT and HoxER-PU.1ΔNeu neutrophils (n = 3 each) treated for 6 h with DMSO, 100 nM TSA, 5 µM Entinostat, 5 µM Mocetinostat or 1 µM TMP195. Relative expression values of three independent experiments are shown as mean ± s.e.m. and were calculated as fold difference over the expression values of DMSO treated HoxER neutrophils. (c) qPCR analysis of expression of the indicated cluster III genes in HoxER-PU.1WT and HoxER-PU.1ΔNeu neutrophils (n = 3 each) treated for 6 h with DMSO or 1 µM TMP269. Relative expression values of two independent experiments are shown as mean ± s.e.m. and were calculated as fold difference over the expression values of DMSO treated HoxER neutrophils. (d) qPCR analysis of expression of the indicated cluster II genes in HoxER-PU.1WT neutrophils (n = 3) treated for 6 h with DMSO or 5 µM Entinostat. Relative expression values of three independent experiments are shown as mean ± s.e.m. and were calculated as fold difference over the expression values of DMSO treated HoxER-PU.1WT neutrophils. b-d: ns (not significant) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (unpaired t-test, two-tailed). (unpaired t-test, two-tailed). (e) Immunoblot analysis (IB) of the immunoprecipitation (IP) of HDAC1, HDAC2 and HDAC3 from nuclear extracts of HoxER-PU.1WT neutrophils. This ensured that HDAC1–3 were precipitated from the nuclear extracts. IgG precipitated nuclear extract from the same cells was used as control. Data are presentative of three independent experiments for HDAC1 and HDAC2, and two for HDAC3. (f) qPCR analysis of HDAC1 ChIP at cluster III/IV gene enhancers (indicated below) of chromatin extracted from HoxER-PU.1WT and HoxER-PU.1ΔNeu neutrophils. As negative control (NC), a region 7 kb downstream of the enhancer allocated to Gm14005 was used. Results are shown as mean ± s.e.m. and were calculated as percent of input divided through the IgG value. Each symbol represents a biological replicate (n = 3) from two independent experiments. (g) Immunoblot analysis of HDAC1 protein expression in tetracycline induced (72 h) shRNA HDAC1 (shHDAC1) and scramble (SC4) control THP-1 cells. Two different shRNAs against HDAC1 were used. Detection of valosin-containing protein (VCP) ensured comparable loading. The blot is representative of four experiments with similar results. (h) qPCR analysis of expression of the indicated cluster III genes in doxycycline induced (72 h) shRNA HDAC1 (bp642 or pb1719) and scramble (SC4) control THP-1 cells. Expressions of two independent experiments are shown and were calculated as fold difference over the expression values of SC4 control. Doted lines represent the expression values of the controls.

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Supplementary Figures 1–7 and Supplementary Tables 1–13.

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Fig. 1: Neutrophil homeostasis in PU.1∆Neu mice.
Fig. 2: PU.1ΔNeu mice are deficient in clearing C. albicans infections.
Fig. 3: Functional hyperactivity of PU.1∆Neu neutrophils.
Fig. 4: PU.1 simultaneously activates or inhibits gene expression in neutrophils.
Fig. 5: Chromatin structure alterations following C. albicans stimulation and PU.1 deletion.
Fig. 6: PU.1 limits JUNB access to enhancers.
Fig. 7: PU.1 recruits HDAC1 to inhibit accessibility of immune-gene enhancers.
Supplementary Figure 1: Validation of and gene expression in neutrophils of PU.1∆Neu mice.
Supplementary Figure 2: The role of PU.1 during distinct neutrophil defense steps.
Supplementary Figure 3: PU.1 inhibits neutrophil activation.
Supplementary Figure 4: Generation and validation of immortalized PU.1∆Neu neutrophil progenitor lines.
Supplementary Figure 5: Cluster enhancer determination and allocation.
Supplementary Figure 6: JUNB in PU.1-deleted neutrophils.
Supplementary Figure 7: PU.1-inhibits immune gene expression by HDAC1.