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Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly

Nature Immunology volume 21, pages 615–625 (2020)Cite this article

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Abstract

Increasing age alters innate immune–mediated responses; however, the mechanisms underpinning these changes in humans are not fully understood. Using a human dermal model of acute inflammation, we found that, although inflammatory onset is similar between young and elderly individuals, the resolution phase was substantially impaired in elderly individuals. This arose from a reduction in T cell immunoglobulin mucin receptor-4 (TIM-4), a phosphatidylserine receptor expressed on macrophages that enables the engulfment of apoptotic bodies, so-called efferocytosis. Reduced TIM-4 in elderly individuals was caused by an elevation in macrophage p38 mitogen-activated protein kinase (MAPK) activity. Administering an orally active p38 inhibitor to elderly individuals rescued TIM-4 expression, cleared apoptotic bodies and restored a macrophage resolution phenotype. Thus, inhibiting p38 in elderly individuals rejuvenated their resolution response to be more similar to that of younger people. This is the first resolution defect identified in humans that has been successfully reversed, thereby highlighting the tractability of targeting pro-resolution biology to treat diseases driven by chronic inflammation.

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Fig. 1: Reduced edema and protein in cantharidin blisters from aged as compared to young donors.
Fig. 2: Impaired PMN clearance in cantharidin blisters from elderly as compared to young donors.
Fig. 3: Apoptotic PMNs accumulate in cantharidin skin blisters of aged individuals.
Fig. 4: Efferocytosis is impaired in elderly individuals.
Fig. 5: Low TIM-4 results in failed resolution signaling in MPs from aged donors.
Fig. 6: Low TIM-4 expression coincides with elevated phospho-p38 in the MPs of elderly donors.
Fig. 7: p38 regulates TIM-4 expression through p300 in human MPs.
Fig. 8: Blocking p38-driven inflammaging in elderly individuals restores inflammatory resolution.

Data availability

The data supporting the findings of this study are available from the corresponding author upon request.

Change history

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Acknowledgements

We thank all the volunteers who participated in this study; M. Berkeley and M. Harries for assistance with recruitment; and V. Birault, I. Laws, R. Glaser, L. Sarov-Blat and R. Henderson from GSK for the provision of losmapimod (MRC Industry Collaboration Agreement)—specifically the losmapimod team at GSK for organizing the dispatch of the clinical supply of the drug in this investigator-led study. We thank our colleagues for invaluable discussion, especially E. Chambers. R.P.H.M. was funded by an AstraZeneca-MRC Industrial CASE PhD studentship (no. MR/J006610/1), jointly awarded to D.W.G. (UCL) and M.U. (AstraZeneca), and supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. R.C.M. was funded by the MRC Grand Challenge in Experimental Medicine grant no. MR/M003833/1 awarded to A.N.A. and D.W.G. D.R.G. was funded by the National Institutes of Health (grant nos. R01AG028082, R01HL127687 and R01HL127687). D.W.G. was further funded by the Wellcome Trust.

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Author notes

  1. These authors contributed equally: Roel P. H. De Maeyer, Rachel C. van de Merwe.

Authors and Affiliations

  1. Division of Medicine, University College London, London, UK

    Roel P. H. De Maeyer, Rachel C. van de Merwe, Rikah Louie, Olivia V. Bracken & Derek W. Gilroy

  2. Division of Infection and Immunity, University College London, London, UK

    Oliver P. Devine & Arne N. Akbar

  3. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    Daniel R. Goldstein

  4. Respiratory Global Medicines Development, AstraZeneca, Gothenburg, Sweden

    Mohib Uddin

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Contributions

D.W.G., A.N.A. and M.U. conceived the project. D.R.G. participated in the original experimental design. R.P.H.M., R.C.M. and D.W.G. designed, performed and analyzed all the experiments, except where otherwise stated, and wrote the manuscript. R.L. conceived the p300 experiments and designed, performed and analyzed the p300 chromatin immunoprecipitation experiments. O.V.B. and R.P.H.M. performed and analyzed the p300i experiments. O.P.D. performed the tissue sectioning, staining and analysis with R.C.M.

Corresponding author

Correspondence to Derek W. Gilroy.

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M.U. is an employee of AstraZeneca and holds shares in the company. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Extended cantharidin exudate data.

a, Scatter plot of total cantharidin blister cells and exudate volume at 24 hours. Pearson correlation data shown on the plot (n = 35 young, 33 aged). Quantification by multiplex ELISA of b, IL-6, c, IL-10 (n = 12 per group), d, CCL2, e, CCL3, f, CCL4, g, CCL7, h, CCL8, i, CXCL1, j, CXCL5, k, osteopontin (OPN), l, M-CSF, and m, platelet-derived growth factor (PDGF)-BB. Data from young are shown in black, aged in red. Each symbol represents a sample from a single participant. Data are shown on log scales with geometric means (n = 14 young, 15 aged unless otherwise stated). 2-way ANOVA on log-transformed data with Sidak’s multiple comparisons post-test. Where no * are shown, data were p > 0.05. * p < 0.05, *** p < 0.001.

Extended Data Fig. 2 Blister gating strategy.

Representative plots for flow cytometric gating are shown for a healthy aged participant at 24h. The same gating strategy (denoted by the arrows) was used for all volunteers at both time points for the characterization study. Particularly, HLA-DR+/CD14+ mononuclear phagocytes were gated out according to these criteria for more detailed analysis of this cell type. Briefly, doublets were excluded using a, Forward Scatter (FSC) and b, Side Scatter (SSC) height and width characteristics. c, Debris was excluded based on FSC vs SSC. d, Leukocytes were gated on CD45 and e, lineage (Lin, CD3/CD19/CD56) negative cells were taken forward. f, HLA-DR- populations contained g, CD16hiSSChi PMNs and i, CD16-SSChiSiglec-8+ eosinophils. h, The PMN population was examined for apoptosis using Annexin V. HLA-DR+ cells were j, gated on CD14 and CD16 to identify Dendritic Cells (DCs) and CD14hi monocytes/macrophages (MPs).

Extended Data Fig. 3 Extended PMN function and phenotype.

Paired flow cytometry analysis of PMNs in whole blood (WB) and 24 h cantharidin blisters (CB 24h) from young (black) and aged (red) participants of a, CD16 (p 0.5577 and 0.2230), b, CD11b (***p 0.0004 and 0.0003)¸ and c) CD66b (***p 0.0005) expression. Data are shown on a logarithmic scale (n = 6 young, 7 aged). Two-tailed paired Student’s t tests on log transformed data. Scatter plots showing TNF levels vs total PMNs in 24 h blisters of d, young and e, aged donors. Shown are linear regression with 95% confidence bands. Pearson r was calculated and is shown alongside p-values (n = 13 young, n = 12 aged). f, Fas-inducible neutrophil apoptosis measured by cell viability over a 24 h time course. Shown are means and s.d. (n = 3 per group). Multiple unpaired t tests with Holm-Sidak correction. Flow cytometric analysis of 24 h cantharidin blister PMN expression of g, TNFR1, h, TNFR2, and i, Fas. Data are shown on a logarithmic scale with geometric means (n = 5 per group). Two-tailed unpaired Student’s t tests on log transformed data ns: p > 0.05, *** p < 0.001, **** p < 0.0001.

Extended Data Fig. 4 Extended efferocytosis data.

a, Scatter plot of young (black, n =6) and aged (red, n = 7) PMN clearance (24 h – 48 h PMN numbers per blister) against 24 h MPs. Pearson correlation shown on the plot, alongside best-fit line. Representative cytospins from b, 24 h cantharidin blisters and c, ex vivo efferocytosis assay using cultured MPs. Arrows denote efferocytosis. Scale bars: 10 μm. Representative of three independent experiments with similar results. d, Cytochalasin B (2h, 10 μM) was used to block internalization. Data were paired and normalized to control, set to 1 (n = 4; *p 0.0231), means and s.d., paired two-tailed t test. e-i, Comparison between 24 h cantharidin blister (CB 24h) MPs, isolated blood MPs cultured for 24 h (MPs 24h), and isolated monocytes cultured for 7 days with M-CSF (20 ng/ml, monocyte-derived macrophages – MDMs). Flow cytometric analysis of e, CD14, f, TIM-4, g, MerTK, h, CD36, and i, CD51 (ITGAV) expression. e-g) Geometric means with geometric s.d. factor, h-i) means and s.d. One-way ANOVA with Sidak’s correction (n = 6 CB, n = 9 MPs, n = 4 MDMs, each dot represents a sample from a single donor). j, Representative ImageStreamX images from an ex vivo efferocytosis assay using 24 h cultured MPs (CD14, red) and autologous, blood-derived apoptotic ACs (green) at a ratio of 3:1 ACs:MPs. Images of a CFSE-stained apoptotic cell (AC), an AC-negative MP (MP), and MPs that have bound AC (<0) or that have ingested AC (>0). Representative image of six independent experiments with similar results. k, Summary data of MPs associated with ACs (internal and external) between MPs isolated from young (black) and aged (red) donors (n = 3 per group; **p 0.0024). Means with s.d., two-tailed unpaired Student’s t test * p < 0.05, ** p < 0.01, **** p < 0.0001.

Extended Data Fig. 5 Annexin V binding and extended phagocytosis data.

a, Flow cytometric analysis of Annexin V binding to 24 h blister PMNs gated as live (CD16hiAnnexin V-), CD16hi early apoptotic (CD16hiAnnexin V+) and CD16lo late apoptotic (CD16loAnnexin V+). Means and two-way ANOVA with Tukey’s correction, no significant differences detected (n = 5 young [black], n = 7 aged [red]). b, Representative ImageStreamX images from ex vivo phagocytosis assays using 24 h cultured MPs (stained with CD14, red). Shown are images of unchallenged control MPs (Ctrl), MPs challenged with CFSE-labelled ACs at a 3:1 AC:MP ratio (AC), MPs challenged with 10:1 fluorescently labelled latex beads that were either opsonised in serum (OLB) or left in their native state (LB). Representative of 9 independent experiments with similar results.

Extended Data Fig. 6 Extended data on MP phenotype and signaling.

Flow cytometric analysis on 24 h cantharidin blister MPs between young (black) and aged (red) for expression of a, CD14, b, CD16, c, HLA-DR, d, CD163, e CD206 (mannose receptor), f, CD36, g, CD51 (ITGAV, Integrin αV), and h, CD11b (a-c n = 20 young, 18 old; d-g n = 7 young, 11 old; h n = 6 young, 7 old). Geometric means and two-tailed unpaired Student’s t tests on log-transformed data, except CD36 and CD51 where means and Mann-Whitney tests were used. All tests showed no significant change. Flow cytometric analysis of i, TIM-4 (n = 8 donors per group; **p 0.0026) and j) MerTK expression on 24 h cultured MPs isolated from young (n = 8) and aged (n = 5) donors (ns p 0.2371). Geometric means with geometric s.d. factor and two-tailed unpaired Student’s t tests on log-transformed data. k, Isolated blood monocytes were cultured for 24 h before stimulation with vehicle (Ctrl), 1 ng/ml LPS (LPS), 3:1 AC:MP CFSE-labelled autologous ACs (AC), or simultaneous addition of LPS and ACs (LPS + AC). MPs were stained with CD14-AF647 (red), DAPI (blue), phospho-STAT3 (P-STAT3, green), and NFκB subunit p65 (p65, yellow). Overlays show CD14, DAPI and P-STAT3, and CD14, DAPI and NFκB respectively. Scale bar denotes 7 μ μm. Representative of six independent experiments with similar results. ** p < 0.01.

Extended Data Fig. 7 p300 blockade using A-485.

a, Flow cytometry was used to confirm TIM-4 expression in MPs cultured with 10 µg/ml LPS with or without 3 µM losmapimod and the indicated dose of A-485 (in nM). The experiment was performed five times with five different donors and summary data of all experiments are shown (means and s.d.; *p 0.0324, 0.0459 respectively, ns 0.5596). One-way repeated-measured ANOVA with Dunnett’s post-test correction, ns p > 0.05, * p < 0.05. b, Normalised TIM-4 expression of the same experiments is shown alongside CD14 expression on the same cells (means and s.e.m, n = 5). One-way repeated-measured ANOVA with Dunnett’s post-test correction. *p 0.0160 with respect to LPS alone, #p 0.0275 compared to LPS + losmapimod. c, ChIP to confirm Histone3 Lysine 27 acetylation in MPs treated with 3 µM losmapimod with either C646 and SGC-CBP30 (2.5 μM and 5 μM respectively) or A-485 (200 nM). Results are normalised by setting losmapimod samples to 1 (n = 2 pooled donors).

Extended Data Fig. 8 Extended data on losmapimod-treated individuals.

a, The effects of losmapimod (15mg BID/PO for 4 days) on inflammation induced by topical application of cantharidin (0.1% w/v) to the skin were investigated in healthy old subjects (>65 years old, n=11, 6 female, 5 male). Dose-dependent relationship between 24 h ex vivo LPS stimulation and b, TNF and c, IL-6 (measured by cytometric bead array) in blood pre- and post-losmapimod (n = 11, means and s.d., multiple paired t tests per row). d, Representative photographs of 24 h and 72 h cantharidin blisters on an untreated (Aged) and losmapimod-treated (Losmapimod) individual. Arrows denote 72 h blisters. e, Total blister exudate volume and f, total cell-free exudate protein levels were measured (means, two-way ANOVA with Tukey’s correction). ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

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De Maeyer, R.P.H., van de Merwe, R.C., Louie, R. et al. Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly. Nat Immunol 21, 615–625 (2020). https://doi.org/10.1038/s41590-020-0646-0

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