Leukocyte cytokine responses in adult patients with mitochondrial DNA defects

Abstract

Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n = 21) and patients (n = 12) with either the m.3243A > G mutation or single, large-scale mtDNA deletions, we examined (i) cytokine responses (IL-6, TNF-α, IL-1β) in response to acute lipopolysaccharide (LPS) exposure and (ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC₅₀), relative to controls, leukocytes from patients with mtDNA deletions showed 74-79% lower responses for IL-6 and IL-1β (p_IL-6 = 0.031, p_IL-1β = 0.009). Moreover, whole blood from patients with mtDNA deletions (p_IL-6 = 0.006), but not patients with the m.3243A > G mutation, showed greater sensitivity to the immunosuppressive effects of dexamethasone. Together, these ex vivo data provide preliminary evidence that some systemic OxPhos defects may compromise immune cytokine responses and increase the sensitivity to immune cytokine suppression by glucocorticoids. Further work in larger cohorts is needed to define the nature of immune dysregulation in patients with mitochondrial disease, and their potential implications for disease phenotypes. KEY MESSAGES: Little is known about leukocyte cytokine responses in patients with mitochondrial diseases. Leukocytes of patients with mtDNA deletions show blunted LPS sensitivity and cytokine responses. Leukocytes of patients with mtDNA deletions are more sensitive to glucocorticoid-mediated IL-6 suppression. Work in larger cohorts is needed to delineate potential immune alterations in mitochondrial diseases.

Keywords: 3243A > G; Cytokine; Glucocorticoid; Inflammation; Inflammation Suppression; Interleukin; Mitochondrial disease; mtDNA deletion.

Fig. 1

Inter-individual differences in stimulated cytokine responses in patients with mitochondrial disease. a Schematic of study groups included in whole blood LPS experiments. b Pro-inflammatory cytokine (IL-6, TNF-α, and IL-1β) levels from healthy controls, patients with 3243A > G mutation and single deletions after 6 h of exposure to increasing concentrations of LPS. The grey boxes indicate missing/undetermined data. c Dose-dependent cytokine responses and fitted models used to determine the maximal (ECmax) and half maximal concentration of LPS (EC₅₀) in controls (grey) and in patients with mitochondrial disease (green = mutation, orange = deletion). d Inter-individual differences in cytokine sensitivity among patients with mitochondrial diseases and healthy controls. e Cytokine sensitivity bi-plots based on LPS EC₅₀ values. The dotted line crosses the origin and average of the control group. f Standardized effect sizes (Hedge’s g) of cytokine response (EC₅₀, left; max response, right) in mitochondrial disease groups relative to healthy controls. g > 0.5 is a medium effect size, and g > 0.8 is a large effect size. Non-linear regression analysis was used in dose–response curves presented in c, and one-way ANOVA with Dunnett’s multiple comparison post hoc analysis was used in d. *p < 0.05; **p < 0.01; ns not significant. Assays were performed once in technical duplicates. Error bars indicate SEM in all panels. Controls n = 21, Mutation n = 7, Deletion n = 5

Fig. 2

Glucocorticoid sensitivity to LPS-stimulated cytokine responses in mitochondrial diseases. a Overview of the dexamethasone (Dex) sensitivity assay in whole blood. b LPS dose–response curves for IL-6, TNF-α, and IL-1β without Dex (top traces, same as in Fig. 1) and with Dex (bottom traces). The dotted lines mark the maximal cytokine responses (EC_max) in healthy participants (grey) and patients with the m.3243A > G mutation (green) and single, large-scale mtDNA deletions (orange). c Effect of Dex on EC₅₀ LPS sensitivity; note that higher EC50 values represent lower sensitivity (greater dose required to elicit half of the maximal cytokine response). d Summary of effect sizes (Hedge’g) for deletion and mutation groups relative to control. g > 0.5 is a medium effect size, g > 0.8 is a large effect size. One-way ANOVA with Dunnett’s multiple comparison test was used in c to test for group differences. Pre- to post-Dex differences were tested with paired t-tests. *p < 0.05. Assays were performed once in technical duplicates. Error bars indicate SEM. Controls n = 21, mutation n = 7, deletion n = 5