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Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation

Nature Immunology volume 22, pages 711–722 (2021)Cite this article

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Abstract

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

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Fig. 1: CTSG, ELANE and PRTN3 catalyze H3ΔN in monocytes.
Fig. 2: Morphologic and functional alterations in monocytic cells ablated of CTSG, ELANE and PRTN3.
Fig. 3: NSP and H3ΔN repression during monocyte-to-macrophage differentiation.
Fig. 4: Widespread H3ΔN genomic distribution and its permissive chromatin association in monocytic cells.
Fig. 5: Permissive chromatin and active transcription association of H3ΔN in primary monocytes.
Fig. 6: Global chromatin accessibility increase upon NSP and H3ΔNThr22 depletion.
Fig. 7: Chromatin priming mediated by NSP and H3ΔN repression facilitates gene expression reprogramming.
Fig. 8: Repressed H3ΔN in patients with sJIA.

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Data availability

ChIP–seq, ATAC-seq and RNA-seq datasets have been deposited in the Gene Expression Omnibus (GEO) with accession numbers GSE142661, GSE142660 and GSE142662, respectively. Other data generated and/or analyzed during the current study and all reagents, including cell lines and plasmid DNA, described in this work are available from the corresponding author on reasonable request. Figures associated with individual datasets are listed as follows: ATAC-seq, GSE142660 (Figs. 4e–g and 6c–f and Extended Data Figs. 4g, 6a,c,d,e and 7a–c); ChIP–seq, GSE142661 (Figs. 4a–c,e–h, 5a–c,e–g and 6d–f and Extended Data Figs. 4c,d,g, 5c–f and 6e); RNA-seq, GSE142662 (Fig. 6a and Extended Data Fig. 6a,b). Source data are provided with this paper.

Code availability

Custom code and mathematical algorithms to analyze ChIP–seq, ATAC-seq, RNA-seq and EpiTOF datasets are available from the corresponding author on reasonable request.

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Acknowledgements

We thank our colleagues at the Oklahoma Medical Research Foundation for helpful discussion. We also thank B. Nahal and the Division of Pediatric Rheumatology at the University of California San Francisco, led by E. von Scheven, for collection of several serum samples and associated clinical data. This work was supported in part by the Donald E. and Delia B. Baxter Foundation (to P.J.U.), Elizabeth F. Adler (to P.J.U.), the Henry Gustav Floren Trust (to P.J.U.), the Bill & Melinda Gates Foundation (grant no. OPP1113682 to P.J.U. and P.K.), EMD Serono (to P.J.U. and P.K.), the Department of Defense contracts no. W81XWH-18-1-0253 and no. W81XWH1910235 (to P.K.), the Ralph & Marian Falk Medical Research Trust (to P.K.), the sJIA Foundation (to E.D.M. and G.S.S.), the Lucile Packard Foundation for Children’s Health (to E.D.M.), the Fundación Bechara (to P.A.N.), the Arbuckle Family Foundation for Arthritis Research (to P.A.N.), Cincinnati Children’s Research Foundation ARC Grant (to G.S.S. and A.A.G.) and the NIH grants no. U19 AI110491 (Autoimmunity Center of Excellence) (to P.J.U.), no. R01 AI125197 (to P.K. and P.J.U.), no. U19 AI109662 (to P.K.), no. U19 AI057229 (to P.K.), no. R01 AR061297 (to E.D.M.), no. R35 GM139569 (to O.G.), no. P30 AR070253 (to P.A.N.), no. R01 AR073201 (to P.A.N.), no. K08 AR073339 (to L.A.H.), no. R01 AR059049 (to A.A.G.), no. P30 AR070549 (to G.S.S. and A.A.G.) and no. K08 AR072075 (to G.S.S.).

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Author notes

  1. These authors contributed equally: Peggie Cheung, Steven Schaffert, Sarah E. Chang.

Authors and Affiliations

  1. Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA

    Peggie Cheung, Steven Schaffert, Sarah E. Chang, Mai Dvorak, Michele Donato, Mariko H. Foecke, Purvesh Khatri, Paul J. Utz & Alex J. Kuo

  2. Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Peggie Cheung, Sarah E. Chang, Mai Dvorak, Mariko H. Foecke, Paul J. Utz & Alex J. Kuo

  3. Center for Biomedical Informatics Research, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Steven Schaffert, Michele Donato & Purvesh Khatri

  4. Program in Immunology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Claudia Macaubas & Elizabeth D. Mellins

  5. Department of Biology, Stanford University, Stanford, CA, USA

    Tie-Mei Li, John P. Coan & Or Gozani

  6. Chan Zuckerberg Biohub, Stanford, CA, USA

    Lichao Zhang & Joshua E. Elias

  7. Division of Rheumatology, Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Grant S. Schulert & Alexei A. Grom

  8. Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Lauren A. Henderson & Peter A. Nigrovic

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Contributions

P.C., S.S. and A.J.K. conceived the molecular biology, cell biology and biochemistry experiments. P.C., S.E.C. and M. Dvorak performed experiments with assistance from M.H.F. S.S. performed computational analyses of EpiTOF, ChIP–seq, ATAC-seq and RNA-seq data with assistance from M. Donato. P.C. and A.J.K. interpreted the data with help from S.S. C.M. coordinated sJIA clinical sample selection and compiled clinical information. T.-M.L. performed mass spectrometry analysis to identify proteolytic cleavage sites with assistance from L.Z., J.P.C. and J.E.E. under the supervision of O.G. G.S.S., A.A.G., L.A.H. and P.A.N. collected sJIA samples and associated clinical data. E.D.M. provided input on experimental strategies, monocyte biology and sJIA pathophysiology and sJIA samples with associated clinical data. P.K. supervised the computational analyses. P.J.U. supervised the work conducted in the experimental laboratory. P.C. and A.J.K. wrote the manuscript with contributions from all coauthors. All authors discussed and commented on the manuscript.

Corresponding authors

Correspondence to Elizabeth D. Mellins, Purvesh Khatri, Paul J. Utz or Alex J. Kuo.

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Competing interests

sJIA-related consultation or research support: E.D.M., Novartis; P.A.N., Novartis; A.A.G., Juno, Novartis, NovImmune and AB2Bio; G.S.S, Novartis. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Jamie D. K. Wilson and Ioana Visan were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 CTSG, ELANE, and PRTN3-Mediated H3ΔN in Monocytes.

a, H3ΔNThr22 enrichment in monocytes is not associated with cell death. Viability of the cells shown in Fig. 1a measured by cisplatin staining. Data represent mean ± S.E.M. (N = 20). b, CD14+CD16− classical monocyte-specific H3ΔNThr22 enrichment. EpiTOF analysis of the indicated monocyte subsets. Y-axis, normalized H3ΔNThr22 level; center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Statistical significance is determined by two-tailed Welch’s t-test with P values depicted. c, Inverse relationship between class II MHC expression and H3ΔNThr22 in monocytes. Single-cell analysis of EpiTOF data as in (a). Each dot represents a single monocyte. X-axis, HLA-DR; y-axis, H3ΔNThr22 (top) or bulk H3 levels (bottom). d, Chromatin localization of H3ΔN in monocytic cells. Western blot analysis of THP-1 cells biochemically separated into cytoplasmic and insoluble chromatin fractions. e, H3ΔN in monocytes is not catalyzed by cathepsin L. Western blot analysis of WCE from THP-1 cells expressing CRISPR-Cas9 and two independent sgRNAs targeting CTSL. Control cells express CRISPR-Cas9 but lack sgRNA. f, H3ΔN in monocytes is not catalyzed by matrix metalloproteases (MMPs). Western blot analysis of WCE from U937 (top) or THP-1 (bottom) cells treated with MMP9-specific or broad-spectrum MMP inhibitors. Control, DMSO treated. g, H3ΔN in monocytes is not catalyzed by cysteine proteases. Western blot analysis of WCE from U937 (left) or THP-1 (right) cells cultured in the presence of a cell-permeable broad-spectrum cysteine protease inhibitor E-64d. Control, DMSO treated. h, Serine proteases generate H3ΔN in monocytic THP-1 cells. Western blot analysis of WCE from THP-1 cells treated with nonselective serine protease inhibitor AEBSF. Control, PBS treated. i, Chromatin localization of CTSG, ELANE, and PRTN3. Western blot analysis of the cytoplasmic and chromatin-enriched fractions purified from THP-1 cells. j, Release of chromatin-bound NSPs in high-salt solution. Chromatin pellet as in Fig. 1d is washed extensively with a buffer containing physiological ionic strength and is subsequently treated with a high-salt solution to solubilize chromatin proteins. Supernatant (left) and pellet (right) fractions are subject to immunoblotting analysis. HP1α serves as a chromatin protein control. k, Controlled proteolytic activities of CTSG, ELANE, and PRTN3 on nucleosomal H3 in vitro. Tandem mass spectrometry analysis of protease assays as in Fig. 1h using individual NSPs and recombinant nucleosomes. Primary cleavage sites accounting for greater than 20% of proteolytic products are labeled. CTSG (red); ELANE (green); PRTN3 (blue). l,m, Distinct histone modification profiles between FL- and truncated H3. Immunoblotting analysis of WCE from U937 cells (l) or primary monocytes (m) using the indicated antibodies. Three biological replicates are shown. These results are used for the quantitative analyses shown in Figs. 1i and 1j.

Source data

Extended Data Fig. 2 Reintroduction of H3ΔN into ΔNSPs Cells Reverses the Morphologic and Functional Alterations Associated with NSP Depletion.

a, b, c, Depletion of individual NSPs does not affect global H3ΔN level in monocytes. Western blot analysis of WCE from U937 cells depleted of CTSG (a), ELANE (b), or PRTN3 (c). Control cells express CRISPR-Cas9 but lack sgRNA. d, Highly specific anti-H3ΔNThr22 antibody used in this study. Dot blot analysis of the anti-H3ΔNThr22 antibody using the indicated synthetic peptides. This affinity reagent is employed throughout the entire study. e, f, g, Clonal selection by FACS does not alter H3ΔN level and pattern, cell morphology, or activation markers CD11b and CD11c expression. Western analysis of WCE from control cells as in Fig. 2a with (six samples on the right) or without (three samples on the left) clonal selection by FACS e, Representative light microscopy images of the control cells as in (e) (f). Mass cytometry analysis of the cells as in (e) (g). Y-axis, mean signal intensities of CD11b (left) or CD11c (right) from the indicated cells relative to the signals from PMA-activated U937 cells as a positive control. Data represent mean ± S.E.M. (three pool controls (left) or six clonal controls (right)). Clonal controls are randomly selected from approximately 400 sorted clones. Statistical significance is determined by two-tailed Student’s t-test. h, Simultaneous NSP depletion does not affect cell viability. Mass cytometry analysis of ΔNSPs and control cells. The percentage of cells negative for cisplatin staining for each cell line is shown. Data represent mean ± S.E.M. (three technical replicates). i, Strategy to reintroduce H3ΔN into ΔNSPs cells. j, Expression of epitope-tagged exogenous H3 (H3ENLYFQS-FLAG) in ΔNSPs cells. Western blot analysis of WCE from ΔNSPs cells transduced with H3ENLYFQS-FLAG (right lane) or empty vector (left lane). The relative abundance of H3ENLYFQS-FLAG to endogenous H3 determined by ImageJ software is shown. k, Chromatin localization of exogenous H3. Western blot analysis of cytoplasmic and chromatin fractions from ΔNSPs cells expressing H3ENLYFQS-FLAG. l, Doxycycline treatment to induce TEV protease expression and H3ENLYFQS-FLAG cleavage does not affect cell viability. Viability of the indicated cells is determined by trypan blue staining and an automatic cell counter. Data represent mean ± S.E.M. (three technical replicates). m, H3ENLYFQS-FLAG cleavage in response to doxycycline-induced TEV protease expression. Western analysis of WCE from the indicated cells. The abundance of cleavage product relative to total H3ENLYFQS-FLAG is determined by ImageJ software. n, o, p, Reintroduction of H3ΔN into ΔNSPs cells reverses morphological and functional alterations associated with NSP depletion. Light microscopy (n), transwell cell migration (o), and phagocytosis (p) analyses of the cells as in (m). Data represent mean ± S.E.M. (three technical replicates). q, Reintroduction of H3ΔN into ΔNSPs cells alters global chromatin architecture. MNase sensitivity analysis of the cells as in (m).

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Extended Data Fig. 3 NSP and H3ΔN Repression as Primary Monocytes Mature into Macrophages.

a, H3ΔNThr22 repression during monocyte-to-macrophage differentiation. Mass cytometry analysis of monocytes and monocyte-derived macrophage. Cells collected at days one (red), three (green), and five (blue) in culture for differentiation are analyzed. Independent biological replicate of Fig. 3a from a different donor. b, H3ΔNThr22 repression during monocyte-to-macrophage differentiation is not associated with cell death. Viability of the cells as in Fig. 3a (donor 1) and (a) (donor 2) assessed by cisplatin staining and mass cytometry. c, Repression of all species of H3ΔN as monocytes differentiate into macrophages. Western blot analysis of WCE from primary monocytes and monocyte-derived macrophages. Macrophages, cells collected at day seven in culture. Molecular weight markers, see Source Data. Independent biological replicate of Fig. 3b from a different donor. d, Macrophages depleted of H3ΔNThr22 show robust phagocytosis capability. Mass cytometry analysis of mature macrophages as in (a) (five-day differentiation in vitro) incubated with osmium-labelled E. coli to measure phagocytosis capability. Control, no E coli particle. Cytochalasin D is used to demonstrate the specificity of phagocytosis measurement. H3ΔNThr22 (x-axis) and osmium (y-axis) levels measured by mass cytometry are shown. Independent biological replicate of Fig. 3c from a different donor. e, H3ΔN repression is maintained in polarized macrophages. Western blot analysis of freshly isolated monocytes, naïve, classically activated, and alternatively activated macrophages from a healthy volunteer. Independent biological replicate of Fig. 3d. f, CTSG, ELANE, and PRTN3 repression during monocyte-to-macrophage differentiation. Western blot analysis of the samples as in (c) using the indicated antibodies. Molecular weight markers, see Source Data. Independent biological replicate of Fig. 3e from a different donor. g, Transcriptional repression of CTSG, ELANE, and PRTN3 in U937 cells treated with PMA. Differential gene expression analysis of CTSG, ELANE, and PRTN3 (x-axis) in undifferentiated (green) and differentiated (purple) U937 cells using the publicly available dataset GSE107566. Each dot represents an independent sample. Y-axis, reads per kilobase per million mapped reads (RPKM). h, Repression of CTSG, ELANE, and PRTN3 proteins in U937 cells treated with PMA. Western blot analysis of WCE from U937 cells treated with or without PMA using the indicated antibodies. i,j, Pharmacological inhibition of NSPs accelerates macrophage development. Western blot analysis of WCE from peripheral blood monocytes cultured in the absence or presence of ELANE inhibitor GW311616 in combination with CTSG inhibitor CAS 429676−93-7 (i). Phagocytosis analysis of the cells as in (i) using mass cytometry and osmium-labeled E. coli (j). Independent biological replicate of Fig. 3g,h from a different donor. k, Exogenous NSP expression is unaffected by NSP repression during cellular differentiation. Western blot analysis of WCE from U937 cells stably expressing exogenous CTSG (left), ELANE (middle), or PRTN3 (right) under the control of a cytomegalovirus promoter treated with or without PMA to induce differentiation. Control, cells transduced with lentiviral vector only without a transgene. (l) Constitutive NSP expression suppresses cellular differentiation. Ectopic overexpression of the indicated NSPs (x-axis) in U937 cells treated with PMA. CD11c expression is determined by mass cytometry. Data represent mean ± S.E.M. (three technical replicates).

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Extended Data Fig. 4 Widespread H3ΔN Genomic Enrichment in Monocytic Cells is Associated with Permissive Chromatin and Active Transcription.

a, Validation of affinity reagents for ChIP-seq. Immunoprecipitation under the stringent ChIP condition using formaldehyde-crosslinked chromatin and the indicated antibodies. ChIP samples are subject to immunoblotting analysis using an antibody raised against H3. Control, bare magnetic beads. b, Mono-nucleosome-enriched sheared chromatin for ChIP-seq analysis. Gel electrophoresis analysis of chromatin input for ChIP-seq analysis. DNA ladder in base pairs is shown. c, Widespread H3ΔNThr22 distribution across the genome. Percentages of H3ΔNThr22 peaks at the indicated 22 pairs of autosomes. d,e, Widespread H3ΔNThr22 enrichment and ELANE occupancy across the genome. Representative genomic tracks of H3ΔNThr22 (top) and bulk H3 (bottom) ChIP-seq data. Genomic regions with high (red) or low (green) H3ΔNThr22 ChIP-seq signals are tested (d). qPCR analysis of H3ΔNThr22 (top) or ELANE (bottom) ChIP DNA from control (gray) or ΔNSPs (white) cells using the indicated primer pairs. Y-axes, fold enrichment over bulk H3. Data represent mean ± S.E.M. (three biological replicates (three control cell lines or thee ΔNSPs clones) and three technical replicates (N = 9)) (e). f, Validation of affinity reagents for ChIP analysis of NSP occupancy. Immunoprecipitation as in (a) using antibodies recognizing the indicated NSPs. Immunoprecipitation samples are subsequently analyzed by immunoblotting using an antibody recognizing bulk H3. Control, bare magnetic beads. g, Peak association between H3ΔNThr22 ChIP-seq and ATAC-seq datasets. Representative genomic tracks of H3ΔNThr22 ChIP-seq data from wild-type U937 cells (top) and ATAC-seq data from control cells (bottom). h, Positive correlation between transcriptional activity and chromatin accessibility. TSS-proximal ATAC-seq signals for the three groups of genes with high (red), medium (gray), or low (blue) gene expression activity (GSE107566).

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Extended Data Fig. 5 Widespread H3ΔN Genomic Enrichment in Primary Monocytes is Associated with Permissive Chromatin and Active Transcription.

Purity of primary monocytes used for ChIP-seq analysis. Representative mass cytometry data showing the percentages of CD14+ monocytes in bulk PBMC (blue) or purified monocyte samples (red) used for ChIP-seq analysis. X-axis, CD14 level; y-axis, percentage of the maximal count. b, Fully differentiated macrophages used for ChIP-seq analysis. Representative mass cytometry data showing H3ΔNThr22 (x-axis) level and CD68 expression (y-axis) in macrophages (blue) relative to those in monocytes (red) for the cells used for ChIP-seq analysis. c, Widespread H3ΔNThr22 distribution across the genome in primary monocytes. Percentages of H3ΔNThr22 peaks at the indicated 22 pairs of autosomes. Data represent mean ± S.E.M. (three biological replicates). d,e, H3ΔNThr22 enrichment and ELANE occupancy at the p65 subunit (RELA) of NF-κB locus in primary monocytes. Representative genomic tracks of H3ΔNThr22 ChIP-seq data from primary monocyte (top) or the matching monocyte-derived macrophages (bottom). Genomic regions with high (red) or low (green) H3ΔNThr22 are tested (d). qPCR analysis of H3ΔNThr22 enrichment (top) or ELANE occupancy (bottom) in primary monocytes (gray) or the matching macrophages (white) using the indicated primer pairs. Y-axes, fold enrichment over bulk H3. Data represent mean ± S.E.M. (three technical replicates) (e). f, H3ΔNThr22 is associated with permissive chromatin in primary monocytes. Representative genomic tracks of H3ΔNThr22 ChIP-seq (top) and ATAC-seq (GSE87218) (bottom) datasets from primary monocytes.

Extended Data Fig. 6 NSP and H3ΔN Depletion Is Associated with Increased Chromatin Accessibility.

a, Differential gene expression in ΔNSPs cells is associated with chromatin accessibility changes. Integrative analysis of ATAC-seq and RNA-seq datasets focusing on differentially expressed genes in Fig. 6a. Y-axis, relative chromatin accessibility determined by ATAC-seq; left, genes downregulated in ΔNSPs cells relative to controls (N = 220); right, genes upregulated in ΔNSPs cells relative to controls (N = 170). Statistical significance is determined by two-tailed Welch’s t test with P value depicted. Differential gene expression in U937 cells depleted of NSPs affects selective biological processes. Gene ontology analysis of the differentially expressed genes in Fig. 6a. Gene ontology terms in which differential genes are enriched are ranked by FDR. b, Increased chromatin accessibility across the genome in U937 cells depleted of NSPs and H3ΔN. The distribution of differentially accessible peaks across 22 pairs of autosomes (x-axis) in ΔNSPs cells. Color, three independent ΔNSPs clones; y-axis, percentages of peaks at specific chromosomes. c, Genic region enrichment of differentially accessible peaks in ΔNSPs cells. X-axis, the presence of differentially accessible peaks in ΔNSPs cells in the indicated genomic regions over their relative proportions in the genome. Center line, genomic distribution with no enrichment. Data represent mean ± S.D. (three biological replicates). d, Further increase in chromatin accessibility upon NSP and H3ΔNThr22 depletion. Representative genomic tracks of the H3ΔNThr22 ChIP-seq dataset from wild-type U937 cells (top) and the ATAC-seq datasets from control (middle) or ΔNSPs (bottom) cells at the STAT5A-STAT3 locus.

Extended Data Fig. 7 NSP and H3ΔN Repression Primes the Chromatin to Facilitate Transcription Reprogramming During Cellular Differentiation.

a-c, Increased chromatin accessibility at genes with important immune regulatory functions in ΔNSPs cells. ATAC-seq sequencing tracks of the loci encoding proinflammatory cytokines, immune regulators (a), differentiation markers (b), or differential genes upon differentiation (c) in ΔNSPs (blue) or control (purple) cells. X-axis, relative position to TSS in base pairs; y-axis, normalized read count in ΔNSPs (above zero) or control (below zero) cells; gray block, exon. H3ΔNThr22 genomic track in wild-type U937 cells is shown.

Extended Data Fig. 8 IL-6 and Ferritin Contribute to H3ΔN Repression in Monocytes from sJIA patients.

a, Robust epigenetic alterations in sJIA patients captured by independent replicates. EpiTOF analysis of the global levels of 40 histone marks in 16 major immune cell subtypes from sJIA patients and healthy volunteers. Effect size comparisons of 560 data points are computed using the biological replicates 1 (x-axis) or 2 (y-axis) datasets. The trendline and Pearson’s correlation coefficient are shown. Statistical significance is determined by Student’s t-test for Pearson correlation with P value depicted. b, Minimal batch effect between the two biological replicates. PCA of the EpiTOF data as in (a) where each dot represents a single subject from biological replicate 1 (red) or 2 (blue) using the variance of 40 histone marks in 16 major immune cell subtypes (560 data points). The proportion of the variance explained by each principal component is shown. c, Separation of sJIA patients from healthy volunteers by epigenetic landscape. PCA as in (b). Orange, healthy donors; green, sJIA patients with quiescent disease; blue, sJIA patients with active disease. d, Repressed H3ΔNThr22 in sJIA patients with the exception of those on IL-6-blocking therapy. Box plot representation of the H3ΔNThr22 levels in monocytes from healthy volunteers, sJIA patients on IL-1 (blue) or IL-6 targeting therapies (green), or on no or other biologic treatments (purple). Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Statistical significance is determined by two tailed Student’s t-test with P value depicted. e, Reduced H3ΔNThr22 in monocytes from sJIA patients. EpiTOF analysis of PBMCs from 14 sJIA patients and 4 healthy volunteers focusing on H3ΔNThr22 in monocytes. An independent validation cohort in addition to the one described in Fig. 8a. Each dot represents the H3ΔNThr22 level in monocytes from a subject. sJIA patients are colored based on treatments as in (d). Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Statistical significance is determined by two tailed Student’s t-test with P value depicted. f, Sera from sJIA patients with MAS induce H3ΔNThr22 repression in monocytes from healthy volunteers. Mass cytometry analysis of monocytes from two healthy volunteers (top and bottom) cultured in vitro with sera from a healthy donor (left) or a sJIA patient with MAS (right) for 24 hours. Biological replicate of the experiment described in Fig. 8f using independent serum sample from an sJIA patient with MAS and independent PBMCs from two healthy volunteers. g,h, Clinically important cytokines IL-1β and IL-18 do not induce H3ΔNThr22 repression in monocytes. Mass cytometry analysis of PBMCs from two healthy volunteers (top and bottom) treated with (right) or without (left) IL-1β (g) or IL-18 (h) for 24 hours. X-axis, bulk H3; y-axis, H3ΔNThr22 level; each dot represents a single monocyte. i, Purity of recombinant ferritin for ex vivo monocyte stimulation. Coomassie staining of ferritin complex purified from E. coli ectopically co-expressing the heavy and light subunits of ferritin. Molecular weight marker is shown.

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Cheung, P., Schaffert, S., Chang, S.E. et al. Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation. Nat Immunol 22, 711–722 (2021). https://doi.org/10.1038/s41590-021-00928-y

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