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Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions

Abstract

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

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Figure 1: Heme impairs bacterial clearance in vivo.
Figure 2: Increased bacterial burden triggered by heme is independent of heme-iron acquisition.
Figure 3: Heme potently inhibits phagocytosis of bacteria.
Figure 4: Heme induces cell shape changes via actin cytoskeleton remodeling.
Figure 5: Heme interferes with actin cytoskeleton-dependent functions.
Figure 6: Identification of the mechanism of heme-induced cytoskeletal interference.
Figure 7: DOCK8 mediates heme-induced actin cytoskeleton changes and suppression of phagocytosis.
Figure 8: Quinine restores phagocytosis of bacteria in vitro and in vivo.

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Acknowledgements

Y. Fukui (Medical Institute of Bioregulation, Kyushu University) and J. Stein (Theodor Kocher Institute, University of Bern) are acknowledged for providing the DOCK8 deficient bone marrow. and H. Häcker (St. Judes Children's Research Hospital) for providing the ERHBD-HoxB8-encoding retroviral construct. pSpCas9(BB)-2a-Puro (PX459) was a gift from F. Zhang (Massachusetts Institute of Technology) (Addgene plasmid # 48139) and pGRG36 was a gift from N. Craig (Johns Hopkins University School of Medicine) (Addgene plasmid # 16666). LifeAct-GFP-encoding retrovirus was kindly provided by A. Leithner (Institute of Science and Technology Austria). pSIM8 and TKC E. coli were gifts from D.L. Court (Center for Cancer Research, National Cancer Institute). We acknowledge M. Gröger and S. Rauscher for excellent technical support (Core imaging facility, Medical University of Vienna). We thank D.P. Barlow and L.R. Cheever for critical reading of the manuscript. This work was supported by the Austrian Academy of Sciences, the Science Fund of the Austrian National Bank (14107) and the Austrian Science Fund FWF (I1620-B22) in the Infect-ERA framework (to S.Knapp).

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Authors and Affiliations

Authors

Contributions

R.M. performed and designed the in vivo, in vitro, pull-down and confocal microscopy experiments, analyzed data, and performed bioinformatics analysis. K.V.M.H. performed pull-down experiments. J.M. assisted with in vitro and in vivo experiments. A.-D.G. assisted with CRISPR/Cas9 knockout clone generation. O.S. performed efferocytosis assay. P.S., S.S., F.Q., R.G., K.L., A.H., A.K., B.R.-S., C.-H.L., K.V. and M.D. provided experimental support. T.D. designed and M.C.A. performed the L. monocytogenes experiments. M.B. performed the Cellix experiments. D.K., H.E. and S.C.E. provided reagents or mice. J.C. and K.L.B. analyzed the liquid chromatography mass spectrometry experiments. S. Kubicek supported and oversaw chemical screening instrumentation. M.S. and G.S.-F. provided technical and intellectual support. S. Knapp designed and supervised the research. R.M. and S. Knapp wrote the manuscript. All of the authors reviewed the manuscript.

Corresponding author

Correspondence to Sylvia Knapp.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Heme impairs bacterial clearance

(a,b) Plasma heme levels (a) of naïve WT mice (n=8) or WT mice pretreated with PBS (n=8) or heme (n=8) and respective bacterial counts (b) 6h post‑infection with E. coli. (c,d) Plasma heme levels (c) of naïve WT mice (n=8) or LysM-Cre−/−Hmox1fl/fl (n=8) and LysM-Cre+/−Hmox1fl/fl (n=8) mice and respective bacterial counts (d) 6h post-infection with E. coli. (e) Bacterial burden of WT mice pretreated with PBS or heme, 24h post-infection with L. monocytogenes (n=8 per group). (f) Survival of WT mice pretreated with PBS (n=5) or heme (n=6) and infected with L. monocytogenes. (g-i) Plasma cytokine (g) and PLF chemokine levels (h) of WT mice pretreated with PBS or heme 16h after control (PBS) or LPS treatment, and respective peritoneal lavage fluid absolute cell counts (i) total, macrophage and neutrophil; n=6-8 per group). (j) Growth curves for E. coli and ΔdppC::KanR E. coli in regular M63 minimal media or elemental iron-free M63 minimal media supplemented with 30μM heme. (k) Bacterial burden of WT mice pretreated with heme or equimolar elemental iron (Ferric ammonium citrate), 24h post-infection with L. monocytogenes (n=8 per condition). (l) Representative images of isogenic non-hemolytic E. coli and hemolytic E. coli hlyABCD::attTn7 colonies grown on Colombia sheep blood agar plates. Data in (a-d) are representative of 2 independent experiments. Data in (e) are pooled from 2 independent experiments. Data in (a-e, k) are presented as the mean; dots represent individual animals; Data in (g-i) are presented as mean ± S.E.M.. (a, c, g, h) one-way ANOVA with Tukey's multiple correction test, (b, d, e, k) Mann-Whitney test, (i) two-tailed t-test, (f) Mantel-Cox test; n.s. = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Supplementary Figure 2 Heme inhibits bacterial phagocytosis and the intact heme molecule is required to mediate its effect

(a) Automated quantification of phagocytosis of bacteria by control- (DMSO) or heme-treated RAW264.7 macrophages as assessed by confocal microscopy (representative images in Fig. 3b) and displayed as bacteria per cell (n=4-5 images per condition). (b-e) Phagocytosis of heat-killed E. coli by RAW264.7 macrophages pretreated with (b) control (DMSO), heme or hemoglobin (n=4 per condition), (c) control (DMSO), heme or elemental iron (iron sulfate; n=4 per condition), (d) control (DMSO), heme or SnPPIX (n=4 per condition) and (e) control (DMSO), heme or PPIX (n=4 per condition). Hemoglobin concentration was calculated as heme content-equivalent concentration. Phagocytosis was quantified by flow cytometry. (f) Binding of FITC-labeled E. coli to RAW264.7 macrophages pretreated with control (DMSO) or heme (n=5 images per condition) at room temperature and incubated at 4°C for 1h as assessed by confocal microscopy. (g) Bacterial killing time course by RAW264.7 macrophages after treatment with control (DMSO) or heme (n=3 per condition). (h, i) Representative images (h) and quantification of phagocytosis (i) of FITC-labelled heat-killed E. coli by BMDM pretreated with control (DMSO) or heme (n=510-1009 cells per condition). Scale bar = 25 μm. (j) Phagocytosis of heat-killed E. coli, P. aeruginosa and S. pneumoniae by WT BMDM pretreated with control (DMSO) or heme (n=4 per condition) as assessed by flow cytometry. (k) Phagocytosis of live L. monocytogenes by WT BMDM pretreated with control (DMSO) or heme (n=4 per condition). (l) Phagocytosis of heat-killed FITC-labeled E. coli by human monocyte-derived macrophages pretreated with control (DMSO) or heme, shown as the percentage of FITC+ macrophages; representative histograms shown (n=3 per condition; related to Fig. 3e). (m) Efferocytosis of apoptotic thymocytes by RAW264.7 macrophages treated with control (DMSO) or heme (n=4 per condition). (n) Scheme showing the workflow for human whole blood phagocytosis assays. (o) Phagocytosis of bacteria by human blood neutrophils (CD11b+, SSChigh) and monocytes (CD11b+, SSClow) from control, heme-treated (30μM) and phenylhydrazine (PHZ)-hemolytic whole blood as quantified by flow cytometry. Background signal from bound, non-internalized bacteria was calculated from Cytochalasin D (10μM) treated control samples. (p) Percentage of cell death of RAW264.7 macrophages upon incubation with control (DMSO) or heme in the presence of heat-killed E. coli, as measured by LDH release (n=4 per condition). Data in (a, e, f-i, k, l, p) are representative of 2 independent experiments. Data in (d) are pooled from 2 independent experiments. Data in (a-g, i-k, m, o, p) are presented as mean ± S.E.M.. (a-f, i, m, o) one-way ANOVA with Tukey's multiple correction test, (g, p) two-way ANOVA with Tukey's multiple correction test, (j, k) two-tailed t-test; n.s. = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Supplementary Figure 3 Heme induces cell shape changes due to effects on the actin cytoskeleton

(a) Live cell imaging of RAW264.7 macrophages expressing LifeAct-GFP treated with control (DMSO), heme or PPIX (see Supplementary Video 3). Scale bar = 10 μm. (b,c) Cell shape analysis and automatic quantification of cell perimeter of RAW264.7 (b; n=177-282 cells) and human monocyte-derived macrophages (c; n=6 images per condition) treated with control (DMSO) or heme for 15 min (related to Fig. 4d and 4e, respectively). (d) Cell shape analysis and automatic quantification of cell area, perimeter and form factor of BMDM incubated with control (DMSO) or heme for 15min (n=169-182 cells). Data in (b, d) are representative of 2 independent experiments. Data in (b, c, d) are presented as mean ± S.E.M., one-way ANOVA with Tukey's multiple correction test; ** p ≤ 0.01, **** p ≤ 0.0001.

Supplementary Figure 4 Heme impairs functions dependent on cytoskeleton rearrangement

(a) Adhesion (left panel) and rolling (right panel) of human monocytes pretreated with control (DMSO; n=7) or 10μM heme (n=6) on activated human endothelium monolayer, and (b) representative images shown with detected cell tracks overlaid on the endothelium monolayer; scale bar = 100 μm, (see Supplementary Video 5) (c) Absolute cell numbers (total, macrophage and neutrophil) in the peritoneal cavity of WT mice pretreated with PBS or heme (n=8) 6h post-infection with E. coli (n=8). (d) Absolute cell numbers (total, macrophage and neutrophil) in the peritoneal cavity of LysM-Cre−/−Hmox1fl/fl and LysM-Cre+/−Hmox1fl/fl mice 6h post-infection with E. coli (n=8). (e) Chemokine levels in the peritoneal cavity of mice presented in c. (f) Chemokine levels in the peritoneal cavity of mice presented in d. (g) Representative flow cytometry plots displaying the percentage of neutrophils (viable CD45+, Ly6G+, CD11b+, CD3, CD19 cells; left panels), quantification of the absolute number of peritoneal neutrophils (right panel), and (h) the percentage of viable peritoneal cells 6h post-injection of thioglycollate broth in WT mice pretreated with PBS or heme (n=6 per condition). (i) Absolute peritoneal cell numbers (total, macrophage and neutrophil) 6h post-injection of thioglycollate broth to WT mice pretreated with PBS or heme, as assessed by cytospin preparations (n=6 per condition). Data in (a) are pooled from 2 independent experiments. Data in (c-f) are representative of 2 independent experiments. Data in (c, d, g-i) are presented as mean ± S.E.M.; Data in (a, e, f) are presented as mean; dots represent technical replicates (a) or individual animals (e, f). Data in (c, e) are related to Supplementary Fig. 1a and 1b, and (d, f) are related to Supplementary Fig. 1c and 1d. (a, c-i) two-tailed t-test; n.s. = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Supplementary Figure 5 Heme-induced cell spreading is independent of calcium influx signaling and ROS

(a) Representative images and (b) automatic quantification of cell area and form factor of RAW264.7 macrophages treated with control (DMSO) or heme in the presence or absence of calcium in the medium (n=4 images per condition). (c) Representative images and (d) automatic quantification of cell area and form factor of RAW264.7 macrophages treated with control (DMSO) or heme in the presence or absence of the ROS scavenger N-acetyl-L-cysteine (NAC; n=4 images per condition). Data in (b, d) are presented as mean ± S.E.M., one-way ANOVA with Tukey's multiple correction test; * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bars = 20 μm.

Supplementary Figure 6 Heme further disrupts actin cytoskeleton organization through ARP2/3

(a) Chemical proteomic workflow to determine specific heme-binding proteins in RAW264.7 macrophage lysates. (b) Western blot showing the protein level of Cdc42 (molecular weight = 21kDa) from whole-cell lysates of RAW264.7 macrophage treated with control (DMSO) or heme (10μM) for the indicated time points. Beta actin (ACTB) is shown as loading control. (c) Scheme showing the inhibitors used to assess the pathways involved in heme-mediated cell shape changes, and their respective targets. (d) Automatic quantification of cell area of RAW264.7 macrophages pretreated with control (DMSO), ML141 (Cdc42 inhibitor) or ML141 treatment and subsequent washing to remove ML141, and treated with control (DMSO) or heme for 15min. (n=4 random fields per condition; see representative images and automatic quantification of form factor in Fig. 6e and 6f). (e,f) Representative images (e) and automatic quantification of form factor and cell area (f) of RAW264.7 macrophages pretreated with control (DMSO) or CK666 (ARP2/3 complex inhibitor), and treated with control (DMSO) or heme (n=4-8 images per condition). (g) Western blots showing LGALS3 or MPP1 protein expression in wild-type and CRISPR/Cas9-deleted RAW264.7 macrophage clones, and (h) representative images of these cells upon treatment with control (DMSO) or heme. Data in (d-f) are representative of 2 independent experiments. Data in (d, f) are presented as mean ± S.E.M., one-way ANOVA with Tukey's multiple correction test; n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bars = 20 μm.

Supplementary Figure 7 Heme interferes with actin cytoskeleton dynamics via DOCK8

(a) Cellular morphology of WT and Dock8−/− HoxB8 macrophages treated with control (DMSO), heme or the CDC42 activator bradykinin (10μM) for 15min. Scale bar = 20 μm. (b, c) Representative images (b) and automated quantification of phagocytosis of bacteria (c) by WT and Dock8−/− BMDM treated with control (DMSO) or heme, as assessed by confocal microscopy and displayed as bacteria per cell (n=6 images per condition). (d) Immune cell composition in the bone marrow, blood and peritoneal cavity of chimeric mice, 6 weeks after bone marrow transplant, displayed as percentage of donor (CD45.1+) and recipient (CD45.2+) cells. (e) Scheme depicting the working model proposed. Data in (b, c) are pooled from 2 independent experiments and are representative of 3 independent experiments. Data in (c) are presented as mean ± S.E.M., one-way ANOVA with Tukey's multiple correction test; n.s. = not significant, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bars = 20 μm (a) and 50 μm (b).

Supplementary Figure 8 Quinine restores host bacterial resistance

(a) Chemical screening workflow. (b) Compound screen scoring. (c) Phagocytosis of FITC-labeled heat-killed bacteria by RAW264.7 macrophages pretreated with control (DMSO) or quinine, and treated with control (DMSO) or heme. Data in (c) represent the original phagocytosis values for the top scoring compound from the compound screen (see Fig. 8a and Supplementary Fig. 8b). (d) Phagocytosis of FITC-E. coli by RAW264.7 macrophages (n=4 images per condition) pretreated with control (saline) or indicated amounts of quinine, and treated with control (DMSO) or heme as assessed by confocal microscopy. (e, f) Cellular morphology (e) and automatic quantification of cell area (f) of RAW264.7 macrophages pretreated with control (saline) or quinine and treated with control (DMSO) or heme (n=172-368 cells per condition). Scale bar = 20μm. (g, h) Plasma heme levels (g) of naïve WT mice (n=8) or WT mice pretreated with mock (PBS) or quinine and treated with PBS or heme, and (h) respective bacterial counts 6h post-infection with E. coli (n=7 per group of infected mice). (i) Growth curves and (j) respective area under the curve (AUC) for E. coli grown on LB medium supplemented with control (saline) or quinine (n=3 per condition). Data in (d) is representative of 2 independent experiments. Data in (c, d, f, i, j) is presented as mean ± S.E.M. Data in (g, h) is presented as mean, and dots represent individual animals. Statistical comparisons in (d, f) are calculated versus control conditions (no heme) at the same dose of quinine. (a) C-score analysis – see Online Methods, (c, d, f-h, j) one-way ANOVA with Tukey's multiple correction test, (i) two-way ANOVA with Tukey's multiple correction test; n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–3 (PDF 3491 kb)

Heme induces changes in 3D shape of macrophages

3D reconstruction from Z-stacks of RAW264.7 macrophages treated with control (DMSO), or 3μM heme. Z-stack thickness was 17.88 μm with 0.33 μm between slices. Volume rendering performed using Huygens Professional (SFP renderer module). Videos presented are representative of 2 independent experiments. (AVI 4685 kb)

Fast heme-induced changes on macrophage cell shape – time lapse

Live cell imaging of RAW264.7 macrophages expressing LifeAct-GFP treated with control (DMSO), or 3μM heme. Time-lapse images taken every 25s for 35min. Timestamp presented as the time post-stimulation. Videos presented are representative of 2 independent experiments. (MP4 3099 kb)

PPIX cannot reproduce heme-induced effects on macrophage cell shape– time lapse

Live cell imaging of RAW264.7 macrophages expressing LifeAct-GFP treated with control (DMSO), 3μM heme, or PPIX. Time lapse images taken every 25s. Timestamp presented as minutes post-stimulation. (MP4 3007 kb)

Heme inhibits DC migration in a 3D collagen gel

Migration of control (DMSO) or heme treated DC on a 3D collagen gel in response to CCL19. Time lapse images taken every 2min for 6h. Timestamp presented as minutes post-stimulation with CCL19. Videos presented are representative of 3 independent experiments. (MP4 4441 kb)

Heme inhibits human monocyte adhesion and rolling

Adhesion and rolling of peripheral blood human monocytes treated with control (DMSO) or 3μM heme onto activated human endothelium. Time-lapse images taken every second for 2min. Timestamp presented as minutes after flow start. Videos presented are representative of 2 independent experiments. (MP4 6044 kb)

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Martins, R., Maier, J., Gorki, AD. et al. Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions. Nat Immunol 17, 1361–1372 (2016). https://doi.org/10.1038/ni.3590

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