Enhancement of cutaneous immunity during aging by blocking p38 mitogen-activated protein (MAP) kinase-induced inflammation

Milica Vukmanovic-Stejic, Emma S Chambers, Mayte Suárez-Fariñas, Daisy Sandhu, Judilyn Fuentes-Duculan, Neil Patel, Elaine Agius, Katie E Lacy, Carolin T Turner, Anis Larbi, Veronique Birault, Mahdad Noursadeghi, Neil A Mabbott, Malcolm H A Rustin, James G Krueger, Arne N Akbar, Milica Vukmanovic-Stejic, Emma S Chambers, Mayte Suárez-Fariñas, Daisy Sandhu, Judilyn Fuentes-Duculan, Neil Patel, Elaine Agius, Katie E Lacy, Carolin T Turner, Anis Larbi, Veronique Birault, Mahdad Noursadeghi, Neil A Mabbott, Malcolm H A Rustin, James G Krueger, Arne N Akbar

Abstract

Background: Immunity decreases with age, which leads to reactivation of varicella zoster virus (VZV). In human subjects age-associated immune changes are usually measured in blood leukocytes; however, this might not reflect alterations in tissue-specific immunity.

Objectives: We used a VZV antigen challenge system in the skin to investigate changes in tissue-specific mechanisms involved in the decreased response to this virus during aging.

Methods: We assessed cutaneous immunity based on the extent of erythema and induration after intradermal VZV antigen injection. We also performed immune histology and transcriptomic analyses on skin biopsy specimens taken from the challenge site in young (<40 years) and old (>65 years) subjects.

Results: Old human subjects exhibited decreased erythema and induration, CD4+ and CD8+ T-cell infiltration, and attenuated global gene activation at the site of cutaneous VZV antigen challenge compared with young subjects. This was associated with increased sterile inflammation in the skin in the same subjects related to p38 mitogen-activated protein kinase-related proinflammatory cytokine production (P < .0007). We inhibited systemic inflammation in old subjects by means of pretreatment with an oral small-molecule p38 mitogen-activated protein kinase inhibitor (Losmapimod; GlaxoSmithKline, Brentford, United Kingdom), which reduced both serum C-reactive protein levels and peripheral blood monocyte secretion of IL-6 and TNF-α. In contrast, cutaneous responses to VZV antigen challenge were increased significantly in the same subjects (P < .0003).

Conclusion: Excessive inflammation in the skin early after antigen challenge retards antigen-specific immunity. However, this can be reversed by inhibition of inflammatory cytokine production that can be used to promote vaccine efficacy and the treatment of infections and malignancy during aging.

Keywords: Aging; inflammation; p38 mitogen-activated protein kinase; varicella zoster virus.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Fig 1
Fig 1
Cutaneous response to VZV antigen is reduced in old subjects. Healthy young and old volunteers were injected with VZV skin antigen. Circles, Female subjects; diamonds, male subjects. Clinical score at day 3 in response to VZV was calculated based on induration, palpability, and redness. A, Clinical score versus participant's age. B, Hematoxylin and eosin staining (×10 magnification). PV, Perivascular infiltrates. Five-millimeter punch biopsies were performed on days 0, 1, 3, or 7 after injection (with 4-7 volunteers per time point). C, Representative skin sections stained for CD4 (green; original magnification ×400). D, Collated data of T-cell numbers at different times after VZV injection in young and old volunteers. Each symbol represents the average number of CD4+ T cells within perivascular infiltrates for each subject (n = 4-7 per time point; Mann-Whitney test; horizontal bar represents the mean). E, Clinical score at 48 hours (peak clinical response) correlated with the number of CD4+ T cells in the perivascular infiltrate at the peak of cellular response on day 7 (n = 10 young [solid circles] and 22 old [open circles] subjects). *P < .05, **P < .01, and ***P < .001. ns, Not significant.
Fig 2
Fig 2
Perivascular cluster formation is reduced in the skin of old subjects. Five-millimeter punch biopsies were performed on days 0, 1, 3, or 7 after VZV injection (with 3-6 volunteers per time point). A, Representative staining of skin sections immunostained for CD11c (original magnification ×10). B, Cumulative data showing mean CD11c+ cell numbers per field (solid bars, young subjects; open bars, old subjects). Data are shown as means ± SEMs. *P < .05 and **P < .01. C, Representative staining showing CD11c+ DCs (red) and CD4+ T cells (green) in a perivascular cluster (representative young donor on day 3 after VZV injection, ×400 magnification). Dapi, 4′-6-Diamidino-2-phenylindole dihydrochloride.
Fig 3
Fig 3
Transcriptomic analysis of skin from young and old subjects after VZV antigen challenge. Three-millimeter punch biopsy specimens were collected from old (n = 10) and young (n = 6) subjects at 6 and 72 hours after VZV injection. Normal skin punch biopsy specimens were collected from an additional group of young (n = 9) and old (n = 6) subjects. Total skin RNA was isolated, amplified, and hybridized to Affymetrix Human Genome U133 2.0 plus arrays. A, Heat map showing relative expression of DEGs between VZV-injected and normal skin from young (left) and old (right) subjects at a fold change (FCH) of greater than 2 and a false discovery rate of greater than 0.05 in normal/unmanipulated skin and skin 6 and 72 hours after VZV challenge in each group. For each gene, only the probe set with the largest average expression is shown. Unsupervised clustering was carried out by using Pearson correlation distance with the McQuitty agglomeration scheme of DEGs at 6 hours after VZV. B, The table shows the top 30 upregulated genes at 72 hours in young and old subjects compared with normal skin in each group.
Fig 4
Fig 4
Comparison of global gene expression between normal, saline-injected, and VZV antigen–injected skin. A, Schematic representation of biopsy collection for transcriptional analysis. B, Heat map showing relative expression of DEGs (fold change [FCH] > 2 and false discovery rate > 0.05) between normal skin and saline-injected skin at 6 hours after treatment in young (left) and old (right) subjects. C, The table shows the top 20 upregulated genes at 6 hours in saline-injected skin from old and young subjects compared with normal skin. Genes not reaching statistical significance are indicated in blue. Asterisks indicate genes related to p38 MAP kinase signaling. D, Bubble plot shows expression of pathways in saline-injected skin versus normal skin. Kyoto Encyclopedia of Genes and Genomes (KEGG) and GO collection, as well as curated skin-related collection, were interrogated, and the most relevant pathways among them with an false discovery rate of less than 0.05 are presented. E, Inflammatory index was calculated for each subject (see the Methods section) and plotted against VZV clinical scores at 72 hours (young subjects, n = 6; old subjects, n = 10).
Fig 5
Fig 5
Identification of a monocyte/macrophage-related gene expression signature in saline-injected aged skin. A, Transcriptomic analysis using the tool BioLayout Express3D of genes upregulated in the skin of elderly human subjects 6 hours after saline treatment, which clusters together in a large network of monocyte/macrophage-related genes (C_, cluster no.; nodes represent individual genes, and edges represent Pearson correlations > 0.7). B and C, Representative images of CD163-stained saline-injected skin from skin from young and old subjects (CD163 is pink, Dapi blue; Fig 5, B) and cumulative data of CD163+ cells in paired analysis from normal and saline-injected skin at 6 hours (Fig 5, C; n = 4-5 per age group). *P < .05. D, Frequency of mononuclear phagocytes determined as being CD45+ Lineage− (CD3−, CD19−, CD20−, and CD56−) and HLA-DR+ and either CD14+ and/or CD16+ expressed as a percentage of CD45+ Lineage− cells in young (open symbols) and old (solid symbols) subjects before and after saline, as assessed by using flow cytometry. Data are assessed by using the paired t test.
Fig 6
Fig 6
Effects of Losmapimod treatment on VZV response in the skin. A, Responses to VZV skin challenge were investigated in old subjects (n = 18, 8 male and 10 female subjects) before and after Losmapimod treatment (15 mg administered twice daily for 4 days). B, Serum CRP levels before and after Losmapimod treatment (n = 18; P = .04, Wilcoxon paired test). C, Whole-blood LPS stimulation was performed before and after Losmapimod treatment, and TNF-α production was measured by using CBA (LPS: P < .0001 and Losmapimod: P < .0001, 2-way ANOVA [n = 18]). D, Clinical score was measured at 48 hours after VZV antigen challenge before and after Losmapimod (P = .0006, Wilcoxon paired test; red symbols indicate the mean). E, Correlation between change in serum CRP level and change in clinical score after Losmapimod treatment (Pearson correlation). F, Representative images of skin sections collected 7 days after VZV injection stained for CD4 (red) and CD11c (pale blue) before and after Losmapimod treatment in one of the subjects with an increased clinical score in response to VZV improved after Losmapimod treatment (top and bottom right panels) and one of the subjects whose clinical score remained low after Losmapimod treatment (top and bottom left panels). White arrows indicate a DC interacting with surrounding T cells.
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