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. 2024 Aug;14(8):e1810.
doi: 10.1002/ctm2.1810.

Vitiligo auto-immune response upon oxidative stress-related mitochondrial DNA release opens up new therapeutic strategies

Ana C B Sant'Anna-Silva #  1 , Thomas Botton #  1 , Andrea Rossi  2 , Jochen Dobner  2 , Hanene Bzioueche  1 , Nguyen Thach  2 , Lauriane Blot  1 , Sophie Pagnotta  3 , Konrad Kleszczynski  4 , Kerstin Steinbrink  4 , Nathalie M Mazure  1 , Stéphane Rocchi  1 , Jean Krutmann  2   5 , Thierry Passeron #  1   6 , Meri K Tulic #  1
Affiliations

Vitiligo auto-immune response upon oxidative stress-related mitochondrial DNA release opens up new therapeutic strategies

Ana C B Sant'Anna-Silva et al. Clin Transl Med. 2024 Aug.
No abstract available

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Conflict of interest statement

Thierry Passeron is a consultant for AbbVie, Almirall, Amgen, Bristol Myers Squibb, Calypso, Galderma, Incyte Corporation, Janssen, Eli Lilly, Novartis, Pfizer, Roivant, UCB and VYNE Therapeutics; has received grants and/or honoraria from AbbVie, ACM Pharma, Almirall, Amgen, Astellas, Bristol Myers Squibb, Calypso, Celgene, Galderma, Genzyme/Sanofi, GlaxoSmithKline, Incyte Corporation, Janssen, LEO Pharma, Eli Lilly, Novartis, Pfizer, Roivant, Sun Pharmaceuticals, UCB and VYNE Therapeutics; is the cofounder of NIKAIA Therapeutics; and has patents on WNT agonists or GSK3b antagonist for re‐pigmentation of vitiligo and with Meri K. Tulic has a patent for the use of CXCR3B blockers in vitiligo. Meri K. Tulic is a consultant for Pfizer and ISIS Pharma. Jean Krutmann is a consultant for AbbVie and has received grants and/or honoraria from AbbVie.

Figures

FIGURE 1
FIGURE 1
Some vitiligo melanocytes present elevated frequency of mitochondrial DNA (mtDNA) variants associated with mitochondrial nucleic acids DNA release. (A) Number of mtDNA variants in melanocyte cultures from healthy donors (n = 14) and non‐active vitiligo patients (n = 16); (B) Measurement of double‐stranded DNA (dsDNA) in the culture supernatant of melanocytes from healthy (n = 6), Vitiligo low variants (LVs; n = 7) and Vitiligo high variants (HV; n = 5); (C) Representative images of melanocytes from healthy donors, Vitiligo LV or Vitiligo HV were immunostained for DNA (green) and the outer membrane mitochondrial marker TOM20 (red; n = 3 for each condition). (D) Relative abundance of MT‐ND1 or MT‐CO1 transcript in melanocyte culture supernatant (n = 6 healthy, 6 LV and 5 HV measured in at least three technical triplicates). (E) Correlation between the number of mtDNA variants present melanocytes and the amount of MT‐ND1 or MT‐CO1 transcripts detected in their culture supernatant. (F) Number of mtDNA variants found in melanocytes or PBMCs of four LV and five HV vitiligo melanocytes cultures. All data represent mean  ±  SD. One‐way analysis of variance (ANOVA) compared to healthy controls. *p < .05; **p < .01; ***p < .001.
FIGURE 2
FIGURE 2
Vitiligo high variant (HV) melanocytes display increased mitochondrial function, elevated reactive oxygen species (ROS) production and are associated with decreased peripheral catalase activity. (A) Representative transmission electron microscopy (TEM) images from three independent cultures of healthy, Vitiligo low variant (LV) and Vitiligo high variant (HV) melanocytes. Scale bar represents 2 µm (1×) and zoomed image is 4× the original picture; (B) Quantification of mitochondrial mass (n = 30 healthy, 11 LV and 5 HV melanocyte cultures) and scoring of mitochondrial branching (n = 20 healthy, 9 LV and 5 HV) per cell; (C) Correlation between mitochondrial mass and the number of mitochondrial DNA (mtDNA) variants in melanocytes; (D) Total ATP production; (E) Left panel: oxygen consumption rate. Cells were treated with oligomycin (1 µM), carbonyl cyanide p‐trifluoro methoxy phenylhydrazone (FCCP; 20 nM titrations), rotenone (1 µM) and antimycin A (1 µM). Right panel: quantification of basal respiration. (F) ATP‐coupled respiration; (G) Measurement of ATP contribution from glycolysis and mitochondria; (H) ROS measurement at baseline and (I) in response to oxidative stress induced by 50 µM menadione; (J) Catalase activity assay in matched PBMCs (n = 5 healthy, 6 LV and 4 HV). Experiments from panels (D) to (I) were performed on three independent melanocyte cultures for each condition in at least three technical triplicates. All data represent mean  ±  SD. One‐way analysis of variance (ANOVA) compared to healthy controls. *p < .05; **p < .01; ***p < .001; ****p < .0001.
FIGURE 3
FIGURE 3
Vitiligo high variant (HV) triggers inflammatory response via the cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) pathway. (A) Melanocytes from healthy donors, Vitiligo low variant (LV) or high variant (HV) were immunostained for phosphorylated TANK‐binding kinase 1 (p‐TBK1) and the percentage of cells with p‐TBK1 foci was quantified (n = 3 melanocyte cultures per condition and at least 45 cells per datapoint). (B) Immunoblot analysis of melanocyte lysates for STING, TBK1, p‐TBK1, NF‐kB p65, NF‐kB p100/p52, interferon regulatory factor (IRF)3 and IRF7 (n = 3 melanocyte cultures per group). Vinculin was used as a loading control. (C) ELISA assay from supernatants measuring interleukin (IL)‐1β, IL‐18, interferon (IFN)α, IFNβ, CXCL9 and CXCL10 (n = 3 melanocyte cultures per condition measured in technical triplicates). (D) Chemotaxis assay of CD8+ cells from healthy donors or vitiligo patients using culture supernatants from healthy melanocytes, Vitiligo LV or HV melanocytes (n = 3 melanocyte cultures per condition measured in technical triplicates). All data represent mean  ±  SD. One‐way analysis of variance (ANOVA) compared to healthy controls. *p < .05; **p < .01; ***p < .001; ****p < .0001.
FIGURE 4
FIGURE 4
Preventing mitochondrial DNA (mtDNA) release or sensing blunts inflammatory response responsible from immune cells recruitment. (A) Measurement of double‐stranded DNA (dsDNA) and a panel of cytokines (B–E) and chemokines (F–H) in the supernatant of melanocytes treated with nuclear factor erythroid 2‐related factor 2 (NRF2) activators dimethyl fumarate (DMF; 50 µM) or NK‐252 (100 µM), recombinant mitochondrial antioxidant superoxide dismutase 2 (SOD2; 50 µg/mL), VDAC‐1 inhibitor VBIT‐4 (20 µM) or TANK‐binding kinase 1 (TBK1) inhibitor GSK8612 (10 µM); (I, J) Chemotaxis assay of CD8+ cells from healthy or vitiligo subjects using culture supernatants form healthy melanocytes, Vitiligo low variant (LV) or high variant (HV) melanocytes previously treated (or not) with SOD2 (50 µg/mL) (I) or TBK1 inhibitor GSK8612 (10 µM) (J). All data represent mean  ±  SD. n = 3 melanocyte cultures per condition measured in at least three technical triplicates. Two‐way analysis of variance (ANOVA) compared to healthy controls. *p < .05; **p < .01; ***p < .001; ****p < .0001. (K) Schematic summary of our findings illustrating the induction of vitiligo auto‐immune response upon oxidative stress‐related mitochondrial DNA release. A subset of vitiligo subjects harbouring polymorphisms in their CAT gene have decreased catalase activity and elevated level of reactive oxygen species (ROS), which was associated with supraphysiological levels of mtDNA variants in their melanocytes. In this context, release of mtDNA triggers the activation of TBK1 downstream of the cyclic GMP‐AMP synthase‐stimulator of interferon genes (cGAS‐STING) pathway and the induction of pro‐inflammatory cytokines and chemokines responsible for the attraction of CD8+ cells and the initiation of an auto‐immune response against melanocytes. This process can be blocked at various levels using mitochondrial antioxidant SOD2, NRF2 activators or TBK1 inhibitors. Created with BioRender.com.
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