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. 2021 Aug 17;2(8):100370. doi: 10.1016/j.xcrm.2021.100370

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© 2021 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway

(A) Circulating mtDNA was quantified in plasma by qPCR and inflammatory molecules were measured in sera by flow cytometry, from 12 healthy donors (6 for mtDNA) and 6 patients (Mann-Whitney U test). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs.

(B) IFN signature in peripheral blood mononuclear cells was identified by qPCR in 4 controls and 4 patients. Dot plots (mean of 3 technical replicates/dot) show the mean ± SD of the CT value of a given gene normalized to the CT value of BACT (Mann-Whitney U test).

(C) DC maturation reflected by CD83 expression (mean effect of interaction F(4,70) = 2.114, p = 0.0881, of stimulus F(4,70) = 36.27, p < 0.0001, of subjects F(1,70) = 14.21, p = 0.0003) and interleukin-6 (IL-6) production (mean effect of interaction F(3,64) = 2.847, p = 0.0444, of stimulus F(3,64) = 18.20, p < 0.0001, of subjects F(1,64) = 2.807, p = 0.0987) were evaluated by flow cytometry and ELISA, respectively, in 10 controls and 6 patients, after exposure to Poly:IC (PIC), lipopolysaccharide (LPS), imiquimod (Im), and CpG-A. Dot plots (mean of 2 [CD83] or 4 [IL-6] technical replicates/dot) show the means ± SDs.

(D) CpG-A-induced IFN responses in DCs from 3 controls and patients were evaluated by qPCR by quantifying SIGLEC1 (mean effect of interaction F(1,8) = 4.436, p = 0.0683, of stimulus F(1,8) = 9.255, p = 0.0160, of subjects F(1,8) = 20.78, p = 0.0019), ISG15 (mean effect of interaction F(1,8) = 0.3191, p = 0.5876, of stimulus F(1,8) = 21.75, p = 0.0016, of subjects F(1,8) = 41.33, p = 0.0002), and IFI27 (mean effect of interaction F(1,8) = 21.99, p = 0.0016, of stimulus F(1,8) = 25.45, p = 0.0010, of subjects F(1,8) = 91.20, p < 0.0001) expression. Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of CT value calculated as in (B).

(E) Production of IL-6 (ELISA) by myoblasts from 8 controls and 6 patients (mean effect of interaction F(2,36) = 3.882, p = 0.0297, of stimulus F(2,36) = 45.49, p < 0.0001, of subjects F(1,36) = 15.28, p = 0.0004), IFNA (mean effect of interaction F(3,24) = 23.44, p < 0.0001, of stimulus F(3,24) = 74.13, p < 0.0001, of subjects F(1,24) = 42.33, p < 0.0001), and IFNB (mean effect of interaction F(3,24) = 2.207, p = 0.1133, of stimulus F(3,24) = 10.19, p = 0.0002, of subjects F(1,24) = 13.60, p = 0.0012).

(F) As in (E), but using sh-Sc or sh-LPIN1-transduced immortalized myoblasts from 1 healthy donor (1 different cell vial from the same single healthy donor/dot) and primary myoblasts from 4 patients. Dot plots (mean of 4 technical replicates/dot) show the means ± SDs of the IL-6 concentration (mean effect of interaction F(2,14) = 6.234, p = 0.0116, of stimulus F(1,14) = 0.0874, p = 0.3653, of subjects F(2,14) = 4.397, p = 0.0330).

(G) As in (F), but primary myoblasts from 4 controls and patients were transduced with a lentivirus expressing a plasmid encoding for a myc-DDK-tagged lipin1 protein (+LPIN1) or an empty vector (+vector) before being challenged (mean effect of interaction F(2,18) = 0.2196, p = 0.8050, of stimulus F(1,18) = 0.2906, p = 0.5965, of subjects F(2,18) = 14.80, p = 0.0002).

(H) Myoblasts from 6 controls and patients were exposed to a vehicle or ethidium bromide (Et. B.). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of the ratio of the CT value for 12S mitochondrial DNA normalized to the CT value for BACT (mean effect of interaction F(1,12) = 3.186, p = 0.0996, of stimulus F(1,12) = 101.6, p < 0.0001, of subjects F(1,12) = 5.268, p = 0.0405).

(I) Myoblasts from 6 controls and patients pre-treated with Et. B. for 5 days were exposed to EBSS and challenged or not with CpG-A for 16 h. Dot plots (mean of 4 technical replicates/dot) show the mean ± SD of IL-6 concentration in culture supernatants at the end of the experiment (mean effect of interaction F(3,24) = 4.989, p = 0.0079, of stimulus F(3,24) = 8.218, p = 0.0006, of subjects F(1,24) = 15.14, p = 0.0007).

(J) Myoblasts were transfected with a plasmid encoding HA-tagged TLR9 and exposed to GM or EBSS and with vehicle or CpG-A before refeeding (Refed) cells exposed to EBSS and CpG-A with GM. Cells were then stained for HA-TLR9 (anti-HA antibody, red) and 8OHDG (green). Box whisker plots (25 images/condition) show the percentage of proximity of TLR9 with 8OHDG (mean effect of interaction F(4,240) = 1.712, p = 0.1481, of stimulus F(4,240) = 25.07, p < 0.0001, of subjects F(1,240) = 68.55, p < 0.0001) in myoblasts from 1 of 3 controls and patients.

(K) As in (J) but myoblasts were immunostained for HA-TLR9 (green) and LC3 (red). Mean effect of interaction F(4,240) = 18.56, p < 0.0001, of stimulus F(4,240) = 125.8, p < 0.0001, of subjects F(1,240) = 61.39, p < 0.0001.

IL-6 concentration is presented after background (which corresponds to values obtained with a control vehicle) subtraction (C, E, F, H, and I). Scale bars, 10 μm (J and K). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001: adjusted p values after between-subjects 2-way ANOVA (B–K) and post hoc Sidak’s correction for multiple comparisons. Data are representative of 1 of at least 3 independent experiments.

See also Figure S5.