Figure 7. Scavenging of mtROS in Treg cells attenuates the DDR response, reverses Treg cell death and ameliorates Th1 and Th17 autoimmune responses.

For A-D CD4⁺Foxp3⁺ Treg cells were isolated from dLNs of naïve or pre-diseased Foxp3gfp.KI mice treated with or without MT.

(A) Caspase-3 and pH2AX (***P < 0.0001) puncta/cell measured by immunofluorescence confocal microscopy, in Treg cells isolated from dLNs of untreated (n = 4) or MT treated (n = 4) pre-diseased mice. One representative experiment of three.

(B) Lamp-1 (*P = 0.0223, ***P < 0.0001), p62 (**P = 0.0029, ***P < 0.0001), Rab7 (**P = 0.0017, ***P = 0.0005, ***P < 0.0001) and CathD (***P < 0.0001) puncta/cell measured by IF are depicted (n = 4 mice per group). One representative experiment of three.

(C) Mean clinical score (*P = 0.0152, *P = 0.0160) and EAE severity (*P = 0.0160) from diseased mice treated with (n = 6) or without MT (n = 4). Representative H&E sections and score from spinal cords of control diseased (clinical score 4) and diseased mice treated with mitotempo (clinical score 1.5) at 14d post immunization (p.i.) (n = 8 mice per group, ***P = 0.0004). One representative experiment of two is depicted.

(D) Flow cytometric analysis and frequencies of CD4⁺IL-17⁺ Th17 cells (*P = 0.0456) and CD4⁺IFNγ⁺ Th1 cells (*P = 0.0151) in spinal cords of diseased (clinical score 4) and diseased mice treated with MT (clinical score 1.5) at 14d post-immunization (p.i.). One representative experiment on two is depicted.

(E) dLN cells were isolated from (7-9 weeks old) Mog35-55/CFA immunized Atg5^AFoxp3 (denoted as pre-diseased Atg5^AFoxp3) mice treated or not with MT, 9d p.i. (n = 8 mice per group) and cultured in the presence or absence of Mog35-55 peptide for 48hrs. IFNγ (**P = 0.036) and IL-17 (*P = 0.0101) measured by ELISA in the supernatants.

For F-I CD4⁺Foxp3⁺ Treg cells were isolated from dLNs of Mog35-55/CFA immunized mCAT^Foxp3 mice, over-expressing the antioxidant enzyme mCAT in their Treg cell compartment, 9d p.i. (n = 4 mice per group). One representative experiment of two is depicted.

(F) Immunofluorescence confocal microscopy for 8-OHDG (red), TOM20 (green) and DAPI (blue). Representative fields (10 μm scale bar). Pearson correlation analysis for colocalization efficiency of 8-OHDG and TOM20 (****p < 0.0001).

(G) Immunofluorescence confocal microscopy for pH2Ax (red), caspase 3 (green) and DAPI (blue). pH2AX (***P < 0.0001) puncta/cell.

(H) Frequency of 7AAD⁺CD4⁺Foxp3⁺ Treg cells (*P = 0.0452).

(I) dLN cells were isolated and cultured in the presence or absence of Mog35-55 peptide for 48hrs. IFNγ (**P = 0.0041) and IL-17 (*P = 0.0163) measured by ELISA in the supernatants (n = 6 mice per group).

Results are presented as mean ± SEM. Statistical significance was obtained by One-way ANOVA or unpaired Student’s t-test. See also Figure S6.