Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 19;118(42):e2102698118.
doi: 10.1073/pnas.2102698118.

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme

Elisa Jentho  1   2 , Cristian Ruiz-Moreno  3   4 , Boris Novakovic  4   5 , Ioannis Kourtzelis  6   7   8 , Wout L Megchelenbrink  3   9 , Rui Martins  2 , Triantafyllos Chavakis  6 , Miguel P Soares  2 , Lydia Kalafati  6   7 , Joel Guerra  1   10   11 , Franziska Roestel  1   11 , Peter Bohm  12 , Maren Godmann  12 , Tatyana Grinenko  6 , Anne Eugster  13 , Martina Beretta  1   14 , Leo A B Joosten  15 , Mihai G Netea  15   16 , Michael Bauer  1   10 , Hendrik G Stunnenberg  17   4 , Sebastian Weis  18   10
Affiliations

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme

Elisa Jentho et al. Proc Natl Acad Sci U S A. .

Abstract

Trained immunity defines long-lasting adaptations of innate immunity based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors [M. Divangahi et al., Nat. Immunol. 22, 2-6 (2021)]. Innate immune cells, however, do not exclusively differentiate between foreign and self but also react to host-derived molecules referred to as alarmins. Extracellular "labile" heme, released during infections, is a bona fide alarmin promoting myeloid cell activation [M. P. Soares, M. T. Bozza, Curr. Opin. Immunol. 38, 94-100 (2016)]. Here, we report that labile heme is a previously unrecognized inducer of trained immunity that confers long-term regulation of lineage specification of hematopoietic stem cells and progenitor cells. In contrast to previous reports on trained immunity, essentially mediated by pathogen-associated molecular patterns, heme training depends on spleen tyrosine kinase signal transduction pathway acting upstream of c-Jun N-terminal kinases. Heme training promotes resistance to sepsis, is associated with the expansion of self-renewing hematopoetic stem cells primed toward myelopoiesis and to the occurrence of a specific myeloid cell population. This is potentially evoked by sustained activity of Nfix, Runx1, and Nfe2l2 and dissociation of the transcriptional repressor Bach2. Previously reported trained immunity inducers are, however, infrequently present in the host, whereas heme abundantly occurs during noninfectious and infectious disease. This difference might explain the vanishing protection exerted by heme training in sepsis over time with sustained long-term myeloid adaptations. Hence, we propose that trained immunity is an integral component of innate immunity with distinct functional differences on infectious disease outcome depending on its induction by pathogenic or endogenous molecules.

Keywords: heme; myelopoiesis; sepsis; single-nuclei analysis; trained innate immunity.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Heme imposes trained immunity and evokes a specific transcriptional signature. (A) Experimental setup. (B) Cytokine release from vehicle- or heme-trained (50 μM) Mφ after LPS restimulation (10 ng/mL) at day 7, measured with LegendPlex. IL-8 measurement by enzyme-linked immunosorbent assay. n = 16 independent donors. (C) TNF release from vehicle- or heme-pretreated Mφ second hit at day 7 with Pam3CSK4. n = 6 independent donors. (D) t-distributed stochastic neighbor embedding (t-SNE) plot of dynamic acetylation peaks in monocytes and vehicle, heme, or β-glucan Mφ 24 h after stimulation. Dim1: differentiation processes. Dim2: variation because of treatment. (E) Genes with highest number of differential H3K27ac peaks within 1 Mb of their TSS. (F) Representative gene loci. (G–I) Transcriptional analysis by RNA-seq. (G) Heatmap of regulated genes at baseline (t0) and at indicated time points after heme or β-glucan exposure. GO terms are listed next to each group of genes. (H) Most abundant pathways based on gene expression analysis 24 h after treatment. White numbers indicate the number of affected genes. (I) Time-resolved expression patterns of genes found with increased H3K27ac. All analyses were performed from n = 5 individual donors. All data mean ± SD unless otherwise stated. Student’s t test, *P ≤ 0.05; **P ≤ 0.01. β-Glu, β-glucan; Ctrl. control; d, days; h, hours; H3K27ac, histone 3 lysine 27 acetylation; HMOX1, heme oxygenase 1; P3C, Pam3CSK4; t0, baseline before stimulation; dim, dimension.
Fig. 2.
Fig. 2.
Heme training relies on Syk/JNK activation in human monocytes. (A) Western blot of p-S6 24 h after heme ± rapamycin stimulation. (B) TNF production after restimulation with LPS at day 7 of vehicle- or heme-trained Mφ ± rapamycin. The representative Western blots of at least three independent donors are pictured. (C) Western blot of Syk phosphorylation after heme treatment of human monocytes at indicated time points. (D) Syk phosphorylation 20 min after heme stimulation ± the Syk inhibitor R406. (E) TNF production after restimulation with LPS at day 7 of vehicle- or heme-trained Mφ ± R406. (F) Western blot of total H3K27ac 24 h after heme treatment ± R406. (G) ChIP-qPCR analysis of H3K27ac at the IL8 promoter (n = 3 independent donors). (H) Western blot of JNK phosphorylation after heme treatment of human monocytes at indicated time points. (I) Western blot of JNK phosphorylation 20 min after heme stimulation ± R406. (J) Western blot and densitometry of JNK activation 20 min after heme stimulation ± the JNK inhibitor SP600125. (K) TNF production after restimulation with LPS at day 7 of vehicle- or heme-trained Mφ ± SP600125. All enzyme-linked immunosorbent assay results derived ≥3 independent experiment à 2 to 3 donors; Two-way ANOVA with Fisher’s LSD; mean ± SEM, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H3, histone 3; H3K27ac, histone 3 lysine 27 acetylation; JNK-I, JNK inhibitor SP600125.
Fig. 3.
Fig. 3.
Persistent heme effects in vivo. (A) Cytokine release of bone marrow–derived macrophages (BMDM), bone marrow (BM) isolated at day 7 after control (Ctrl; n = 8) or heme (n = 8) injection and 24 h after LPS (10 ng/mL) stimulation. Pool of two independent experiments. (B) Phagocytic capacity of BMDM, BM isolated at day 7 after Ctrl (n = 4) or heme (n = 4) injection and 2 h after pHrodo Red E. coli stimulation. Pool of two independent experiments. (C) Gating of myeloid cells. Neutrophils are CD11b+Ly6G+ and monocytes are CD11b+Ly6G‒ and further distinguished as Ly6Chigh (inflammatory monocytes) and Ly6Clow (patrolling monocytes). Peritoneal Mφ were gated as CD11bhighLy6G‒F4/80+. (D) Cell count and flow cytometry analysis of peritoneal lavage fluid of vehicle- or heme-pretreated mice at day 7 and 6 h after endotoxemia. (E) Experimental setup. (F) Survival of polymicrobial sepsis induced 7 d after vehicle (n = 10) or heme (n = 10) treatment in mice. Pool of two independent experiments. Survival: Fisher’s exact test. (G) Pathogen load and (H) serology of vehicle- (n = 8) or heme- (n = 7) pretreated mice 24 h after sepsis induction. Pool of two independent experiments. Mann–Whitney U test; mean, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (I) Experimental setup. (J) Flow cytometry analysis of indicated HSPC cellular subsets of vehicle- or heme-pretreated mice at day 7. n = 5 to 6 animals per group derived from two independent experiments. Student’s t test; mean ± SD, *P ≤ 0.05; **P ≤ 0.01. Ae, aerob; ALT, alanin aminotransferase; An, anaerob; h, hours; hi, high; IFN, interferon; IL, interleukin; LDH, lactate dehydrogenase; LSK, Lin−Sca1+cKit+; Neutrop, neurophils; n.s., non-significant; PCI, peritoneal contamination and infection; p.i., post injection; PLF, peritoneal lavage fluid.
Fig. 4.
Fig. 4.
Single-cell epigenetic landscape of heme-trained HSPCs in mice. (A) UMAP projection of 4,842 (Control), 5,562 (Heme 7d), and 6,052 (Heme 28d) single-nuclei (sn) ATAC profiles from sorted bone marrow–derived LSK cells. Each dot represents an individual cell, and colors represent the identity of the cluster. An annotation of the clusters was performed according to the overlay of epigenetic signatures (ImmGen Database) and known marker genes per cell type. (B) Enrichment of epigenomic ImmGen Database signatures (area under the curve [AUC] scores) of the HSPCs onto UMAP projection of LSK cells. (C) Pseudotime trajectory of pGN differentiation and heme-trained cells from ST-HSCs in control LSK cells and after 28 d of heme training, respectively. (D) TF footprint analysis in the indicated snATAC-seq clusters of non- and heme-treated LSK cells subtracted for Tn5 insertion bias shown below. (E) TF motif scores from bone marrow cells projected onto the UMAP. (F) UMAP projection colored by log-normalized gene scores calculated on the individual LSK cell types. (G) Enhancer–promoter connections (coaccessibility) of the Nfix locus in control- and heme-trained cells. The height of connections indicates the coaccessibility score between the connected peaks. Ctrl, control; pGN, precursor of granulocytes; pDC, precursor of dendritic cells; pNK, precursor of NK cells; pLB, precursor of B lymphocytes; NC, normalized count.
See this image and copyright information in PMC

Similar articles

  • Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity.
    Mitroulis I, Ruppova K, Wang B, Chen LS, Grzybek M, Grinenko T, Eugster A, Troullinaki M, Palladini A, Kourtzelis I, Chatzigeorgiou A, Schlitzer A, Beyer M, Joosten LAB, Isermann B, Lesche M, Petzold A, Simons K, Henry I, Dahl A, Schultze JL, Wielockx B, Zamboni N, Mirtschink P, Coskun Ü, Hajishengallis G, Netea MG, Chavakis T. Mitroulis I, et al. Cell. 2018 Jan 11;172(1-2):147-161.e12. doi: 10.1016/j.cell.2017.11.034. Cell. 2018. PMID: 29328910 Free PMC article.
  • Trained Immunity and Cardiometabolic Disease: The Role of Bone Marrow.
    Mitroulis I, Hajishengallis G, Chavakis T. Mitroulis I, et al. Arterioscler Thromb Vasc Biol. 2021 Jan;41(1):48-54. doi: 10.1161/ATVBAHA.120.314215. Epub 2020 Nov 19. Arterioscler Thromb Vasc Biol. 2021. PMID: 33207931 Free PMC article. Review.
  • DAMPs and Innate Immune Training.
    Jentho E, Weis S. Jentho E, et al. Front Immunol. 2021 Oct 22;12:699563. doi: 10.3389/fimmu.2021.699563. eCollection 2021. Front Immunol. 2021. PMID: 34745089 Free PMC article. Review.
  • Maladaptive innate immune training of myelopoiesis links inflammatory comorbidities.
    Li X, Wang H, Yu X, Saha G, Kalafati L, Ioannidis C, Mitroulis I, Netea MG, Chavakis T, Hajishengallis G. Li X, et al. Cell. 2022 May 12;185(10):1709-1727.e18. doi: 10.1016/j.cell.2022.03.043. Epub 2022 Apr 27. Cell. 2022. PMID: 35483374 Free PMC article.
  • C/EBPβ-Dependent Epigenetic Memory Induces Trained Immunity in Hematopoietic Stem Cells.
    de Laval B, Maurizio J, Kandalla PK, Brisou G, Simonnet L, Huber C, Gimenez G, Matcovitch-Natan O, Reinhardt S, David E, Mildner A, Leutz A, Nadel B, Bordi C, Amit I, Sarrazin S, Sieweke MH. de Laval B, et al. Cell Stem Cell. 2020 May 7;26(5):657-674.e8. doi: 10.1016/j.stem.2020.01.017. Epub 2020 Mar 12. Cell Stem Cell. 2020. PMID: 32169166
See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bozza M. T., Jeney V., Pro-inflammatory actions of heme and other hemoglobin-derived DAMPs. Front. Immunol. 11, 1323 (2020). - PMC - PubMed
    1. Gouveia Z., et al. ., Characterization of plasma labile heme in hemolytic conditions. FEBS J. 284, 3278–3301 (2017). - PMC - PubMed
    1. Englert F. A., et al. ., Labile heme impairs hepatic microcirculation and promotes hepatic injury. Arch. Biochem. Biophys. 672, 108075 (2019). - PubMed
    1. Ferreira A., Balla J., Jeney V., Balla G., Soares M. P., A central role for free heme in the pathogenesis of severe malaria: The missing link? J. Mol. Med. (Berl.) 86, 1097–1111 (2008). - PubMed
    1. Larsen R., et al. ., A central role for free heme in the pathogenesis of severe sepsis. Sci. Transl. Med. 2, 51ra71 (2010). - PubMed

Publication types

LinkOut - more resources