.
O P E N A C C E S S S O U R C E : Cell Metabolism
Highlights
• CD4+ T cells from healthy older people preferentially produce a Th17 profile
• Autophagy, but not mitophagy, knockdown activates a Th17 profile in “young” cells
• Mitochondrial ROS is needed, but not sufficient, for a Th17 profile in “young” cells
• Metformin improves autophagy and mitochondria in parallel to decrease inflammaging
Summary
Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes.
Context and Significance
Inflammation increases naturally with age and contributes to many diseases that limit the amount of one’s life spent in good health, including type 2 diabetes, dementias, and many cancers. Researchers at the University of Kentucky and their colleagues identified a source of age-related inflammation that, if targeted by appropriate medications, holds great promise for promoting healthy aging. They manipulated immune cells from 30-year-old people to mimic inflammation in cells from 60-year-old people. This method identified specific breakdowns in the cellular machinery that cause age-related inflammation. The type 2 diabetes drug metformin repaired the broken machinery in cells treated outside the body to drastically lower inflammation, paving the way for clinical trials to test whether metformin lowers age-related inflammation to promote healthy aging.
Introduction
Aging-associated inflammation, or inflammaging, plays roles in increased risk of insulin resistance, type 2 diabetes (T2D), and cardiovascular diseases with age. Inflammaging thereby limits the length of one’s lifespan spent in good health (Mueller and Rose, 1996, Franceschi and Campisi, 2014, Franceschi et al., 2017, Bharath et al., 2017b). Aging-associated inflammation has been defined based on cytokines, such as TNFα and IL-6 (Bruunsgaard et al., 2000, Roubenoff et al., 1998, Ferrucci et al., 2005, Piber et al., 2019), without consideration of the relative importance of the many sources of what is collectively labeled “inflammation.” T cells are a major source of inflammatory cytokines in settings characterized by chronic low-level inflammation, and multiple lines of evidence showed that one particular T cell subset, Th17s, characterizes and mathematically predicts T2D (Ip et al., 2016). Th17s also promote arguably the most prevelant inflammatory disease in the world, periodontal disease, which fuels cardiovascular and other more deadly diseases (Abusleme and Moutsopoulos, 2017). Similarly defining age-associated T cell inflammation will significantly enhance our current appreciation of inflammaging toward the goal of improving health span.
Multiple mediators of cell maintenance are known to decline in aging, raising the possibility that age-associated changes in processes, such as macroautophagy, herein “autophagy,” and mitochondrial bioenergetics (Sun et al., 2016) both parallel and promote inflammaging. Autophagy has multiple immunomodulatory effects, including broad coordination of general immune cell responses, as evidenced by the importance of autophagy in development and function of anti-inflammatory regulatory T cells (Tregs) (Wei et al., 2016, Le Texier et al., 2016). Autophagy controls immune cell function in part by regulating mitochondrial bioenergetics, as shown by demonstrations that CD4+ T cell autophagy negatively regulates glucose metabolism in Tregs (Kabat et al., 2016). Given that ATP generation through mitochondrial oxidative phosphorylation (OXPHOS) versus non-mitochondrial glycolysis (deemed “glycolysis” herein) can determine T cell function (Kominsky et al., 2010, Priyadharshini et al., 2018), these findings suggest that autophagy enhancement may alter the mitochondrial response of T cells to stimulation, and thereby ameliorate inflammaging to prolong health span. Arguably, the top candidate drug for activating autophagy over the immediate term is metformin, a well-tolerated T2D drug that improves glycemic control and, in some studies, chronic inflammation (Cameron et al., 2016, Malínská et al., 2016). Putative effects of metformin on age-associated T cell inflammation justify targeted pre-clinical work to identify metformin-sensitive mechanisms that ameliorate a more sophisticated profile of inflammaging.
Data herein show that a combinatorial Th17 cytokine profile differentiated CD4+ T cell inflammation in healthy sexagenarians compared to ∼30-year-old subjects. Physiologically achievable concentrations of metformin lowered overall T cell cytokine production ex vivo in samples from all subjects, but the Th17 profile was disproportionately susceptible in samples from older (O) subjects. By contrast, metformin reduced a Th2 profile in cells from younger (Y) subjects. Metformin increased autophagy in CD4+ T cells from older subjects and shifted measures of mitochondrial bioenergetics and T cell inflammation to values indistinguishable from young subjects’ cells. siRNA-mediated impairment of autophagy, but not mitophagy, in cells from younger subjects compromised mitochondrial function and activated a Th17 profile indistinguishable from T cell profiles produced by cells from older subjects. We conclude metformin-sensitive defects in immune cell autophagy (1) accompany natural aging in people, (2) shift mitochondrial bioenergetics, and (3) fuel a previously unappreciated Th17 inflammaging profile. Our findings highlight cause-and-effect relationships among defects in non-mitochondrial autophagy, mitochondrial function, and inflammaging to justify clinical trials to extend health span with metformin.
Results
A Th17 Profile Dominates CD4+ T Cell Function from Older Subjects through a Metformin-Sensitive Mechanism
To define an age-associated T cell cytokine profile, we quantified cytokines produced by αCD3/αCD28-stimulated CD4+ T cells from O and Y subjects (Table S1) by bioplex. O cells produced higher amounts of most classically defined Th17-associated/supportive cytokines (IL-6, IL-17A, IL-17F, IL-21, and IL-23) but similar amounts of cytokines typically produced by other CD4+ T cell subsets (Figures 1A and S1A–S1D). Age-associated shifts in CD4+ T cell subset distribution in our cohort was as previously published (Figures 1B and S1E), including CD57+ terminal effectors that were almost unique to samples from O subjects and fewer central memory T cells in Y samples. These results were consistent with recent work showing that Th17 frequency does not increase with age (Alpert et al., 2019). Age-associated changes in CD8+ T cell subsets were also as expected (Figure S1F). Partial least squares discriminant analysis (PLSDA) models, which combine all cytokines from one sample into a compendium multi-dimensional value for “inflammation,” showed that cytokine production differentiates O and Y samples (Figure 1C). Variable importance projection (VIP) calculations, which rank cytokines based on their overall importance for separating cytokine data clouds, showed almost all Th17 cytokines were disproportionately important for identifying higher overall inflammation produced by O-derived CD4+ T cells (VIP > 1.0, bracket in Figure 1D, red bars highlight classical Th17 cytokines). We conclude that a comprehensive Th17 profile defines and mathematically predicts age-related T cell inflammation.
Figure 1. Metformin Ameliorates an Age-Related Th17 Cytokine Profile
Cytokine production was assessed in T cells from BMI-matched normoglycemic Y and O subjects following 40 h αCD3/αCD28 stimulation ± 100 μm metformin (MET).
(A) Concentrations of IL-17A, IL-17F, IL-21, and IL-6 as indicated. Data are mean ± SEM. n = 10–14. For all panels, each n (i.e., each dot) represents T cells isolated from one subject. ∗p < 0.05 versus Y, #p < 0.05 versus O by ANOVA.
(B) Left: tSNE grouping of CD4+ T cell subsets based on markers shown in Figure S1E identified 5 subsets. Right: two representative analyses from subjects in age groups as indicated. Table shows frequencies (average and SD) of CD4+ T cell subsets in samples from Y or O subjects. ∗p < 0.05 by two-tailed t test.
(C, E, and G) PLSDA shows compendium measures of “inflammation” generated by combining all cytokines measured by © Y (blue) or O (green) CD4+ T cells, (E) CD4+ cells from O subjects stimulated in the presence (orange) or absence (green) of metformin (100 μM), or (G) CD4+ cells from Y subjects stimulated in the presence (purple) or absence (blue) of metformin (100 μM).
(D, F, and H) Bar graphs show VIP scores, which rank cytokines as most (leftmost) or least (rightmost) important for differentiating overall cytokine profiles between the groups indicated in key. A VIP score >1 (bracket) is considered important for differentiating inflammatory profiles between groups. All VIP cytokines indicated also differed in post hoc analyses (p < 0.05). n = 10–14.
See also Figure S1.
The glycemic control drug metformin variably impacts inflammation and inflammatory comorbidities like T2D in part through undefined age-associated mechanisms (Chakraborty et al., 2011, Smith et al., 2010, Fidan et al., 2011). We tested the effect of physiologically achievable metformin (100 μM) (Madiraju et al., 2019) added coincidence with T cell-targeted stimuli on the newly defined age-related inflammation profile. Metformin specifically decreased production of Th17 cytokines but failed to decrease most Th2 cytokines (IL-4, IL-5, and IL-10) by O cells (O + met), as indicated by single cytokine (Figures 1A and S1A–S1D) or PLSDA (Figures 1E and 1F) analysis. In contrast, single cytokine analyses showed metformin did not change cytokine production by Y-derived T cells (Y + met; Figures 1A and S1A–S1D), while PLSDA showed metformin ameliorated a Th2/type 2 immune profile produced by Y cells (IL-13, IL-33, IL-31, IL-10, and IL-5; Figures 1G and 1H). Cytokine profiles from O + met cultures were indistinguishable from profile produced in Y cultures as indicated by statistically similar profiles (p > 0.05), no VIP cytokines with values >1.0, and a non-predictive value in “leave-one-out” analysis of p = 0.15 (data not shown). We conclude that metformin restores age-related T cell inflammation to profiles generated by Y cells.
Mitochondria Dysfunction in CD4+ T Cells Is Regulated by an Age-Related, Metformin-Sensitive Mechanism
To identify metformin-sensitive mechanisms that control Th17 inflammaging, we quantified indicators of mitochondrial function that promote pro-inflammatory T cells (De Rosa et al., 2015, Hong et al., 2013, Bharath et al., 2017a) using a mito stress test in extracellular flux (XF, Seahorse). αCD3/αCD28-stimulated CD4+ T cells from O subjects had higher OXPHOS (OXPHOS/oxygen consumption rate [OCR]; baseline and maximal), extracellular acidification rate ratio (OCR:ECAR), and proton leak. Spare respiratory capacity was similar between O and Y cells. CD4+ T cells from O compared Y subjects produced less lactate and had lower ECAR (Figures 2A–2D and S2A–S2D). Mitochondrial membrane potential (MMP) was lower in O cells, as measured by tetramethylrhodamine, ethyl ester (TMRE) (Figure 2E), perhaps in part due to intrinsically lower membrane potential differences in O compared with Y cells (Figure 2F). Addition of metformin (100 μM) concomitant with stimulation decreased basal and maximal OCR, OCR:ECAR ratio, and proton leak of CD4+ T cells from O subjects (Figures 2A, 2B, 2D, S2A, and S2B). Metformin increased lactate production and ECAR (Figures 2C and S2C) and supported a trend toward increase in MMP in CD4+ T cells from O subjects (Figure 2E; p = 0.055). Metformin action on mitochondrial bioenergetics was independent of AMPK, as indicated by similar outcomes from cells treated with AMPK-specific or scrambled siRNA prior to stimulation and extracellular flux (XF) analysis (Figures S2E and S2F). Metformin had no effect on the mitochondrial function of T cells from Y subjects (Figures 2A–2E and S2A–S2D). We conclude that higher OXPHOS corresponds with lower glycolysis and Th17 inflammation in T cells from O compared with Y subjects, and that metformin shifts O cells to recapitulate characteristics of Y cells.
Figure 2. Metformin Ameliorates OXPHOS and Promotes Non-mitochondrial Glycolysis in CD4+ T Cells from O Subjects
(A) OCR in a mito stress test assayed by XF of CD4+ T cells following 40 h αCD3/αCD28 stimulation ± 100 μm MET as indicated.
(B) OCR:ECAR ratio calculated by profiles in (A) and Figure S2C.
© Relative lactate production after 40-h stimulation per (A).
(D) Proton leak calculated from (A) data.
(E) MMP measured with TMRE after stimulation per (A). #p = 0.055 versus O.
(F) MMP measured following addition of the mitochondrial uncoupler fluoro-carbonyl cyanide phenylhydrazone (FCCP) to unstimulated CD4+ T cells from Y or O subjects.
n = 8–10 (A–E) and 12–13 (F).
(G) LDH quantification on western blots. Top: representative blot and bottom averages n = 4–6 of group indicated beneath. ∗p < 0.05 versus Y, #p < 0.05 versus O. Data shown are mean ± SEM.
(H) VIP scores, which rank cytokines as most (leftmost) or least (rightmost) important for differentiating overall cytokine profiles between CD4+ T cells from young subjects stimulated ± the LDH inhibitor OA in an orthagonalized model. A VIP score >1 (bracket) is considered important for differentiating inflammatory profiles between groups. All VIP cytokines indicated also differed in post hoc analyses (p < 0.05). Fold change is compared to Y or Y + FCCP.
See also Figure S2.
Although inflammation is traditionally fueled by glycolysis, our data showing association between mitochondrial respiration and inflammation in CD4+ T cells from O subjects raise the possibility that O cells ineffectively shift to glycolysis to fuel inflammation. To begin testing this possibility, we quantified protein levels of glycolytic pathway enzymes. CD4+ T cells from O subjects had lower expression of lactate dehydrogenase A (LDH), which catalyzes pyruvate ↔ lactate (Figure 2G), providing a mechanistic explanation for low lactate in O cells (Figure 2C). Expression of enzymes that regulate pyruvate production from glucose, including hexokinase and pyruvate kinase M2 (PKM2), was higher or equivalent, respectively, in CD4+ T cells from O compared with Y subjects (Figures S2G and S2H), suggesting age did not change pyruvate production to limit lactate. In contrast to lower glycolysis, pyruvate hydrolysis through the citric acid cycle was not likely compromised by age-dependent changes in citric acid cycle enzymes, as suggested by protein levels of isocitrate dehydrogenase (IDH2) and oxoglutarate dehydrogenase (OGDH) (Figures S2I and S2J). NADH:ubiquinone oxidoreductase (mitochondrial respiratory complex 1) was quantitatively equal in CD4+ T cells from O and Y subjects, although western blots suggested an age-related post-translational modification (Figure S2K). Metformin increased protein levels of LDH (Figure 2G) and OGDH (Figure S2J) in CD4+ T cells from O subjects but had no effect on the other enzymes measured. We conclude age-related decreases in LDH, which are sensitive to metformin, mechanistically explain lower glycolysis and may thereby promote compensatory OXPHOS in CD4+ T cells from O subjects.
To test causal relationships between low LDH expression in O cells and the Th17 inflammaging profile, we pharmacologically inhibited LDH activity in CD4+ T cells from Y subjects with 20 mM oxamic acid (OA; Figure S2L). Some Th17-associated cytokines, including IL-21 and the Th17 supporters IL-6 and IL-23, were activated by OA (Figure S2M). However, PLSDA showed that the majority of Th17 signature cytokines (IL-17A, IL-17F IL-6, and IL-21; Figure 1F) were not important for distinguishing compendium cytokine profiles from LDH inhibitor-treated Y cells. Exceptions were IL-22 and IL-23, highlighted as important by this method (Figure 2H). We conclude that changes in glycolytic machinery do not play critical roles in Th17 inflammaging.
Mitochondrial Dysfunction Disrupts Redox Balance to Support Th17 Cytokine Production by CD4+ T Cells from O Subjects and Is Corrected by Metformin
Higher OXPHOS in the absence of parallel increases in anti-oxidants can generate excess oxidative stress, as measured by reactive oxygen species (ROS), which in turn can support Th17 number and function (Zhi et al., 2012, Ungvari et al., 2009, Murphy, 2009, Liu et al., 2002). CD4+ T cells from O subjects had more ROS than Y counterparts, as measured by DCFDA (Figure 3A), and consistent with higher ATP-linked respiration (Figure 3B). Lower glutathione (GSH) and more nicotinamide nucleotide transhydrogenase (NNT) in O compared with Y cells indicated that less antioxidant, perhaps in response to “reverse” NNT function (Nickel et al., 2015), contributed to higher oxidative stress (Figures 3C and 3D). Lower expression of mitochondrial manganese superoxide dismutase (MnSOD/SOD2) in T cells from O subjects (Figure 3E) was also consistent with higher ROS, despite age-independent expression of the anti-oxidants SOD1 and PRDX2 (Figures S3A and S3B), all of which associated with lower MMP (Figure 3F). Metformin increased the expression of GSH and SOD1 (Figures 3C and S3A), decreased ROS and NNT (Figures 3A and 3D), and showed a trend toward increased MnSOD (Figure 3E; p = 0.062) in cells from O subjects.
Figure 3. ROS Amelioration Prevents Th17 Profile Production by CD4+ T Cells from O Subjects
(A–E) ROS production (A), ATP-linked respiration (B), GSH ©, NNT (D), or MnSOD expression (E) by CD4+ cells stimulated for 40 h with αCD3/αCD28 ± 100 μm MET. #p = 0.062 versus O.
(F and G) Outcomes following stimulation the ROS scavenger Tempol ± 100 μM MET as indicated.
(H) Production of Th17 cytokines by CD4+ cells stimulated for 40 h with αCD3/αCD28 ± tempol.
n = 7–10 (A–D, F, and G), 4–10 (H), and 4–7 (E); ∗p < 0.05 versus Y, #p < 0.05 versus O. Data are represented as mean ± SEM. Fold change is compared with Y. See also Figure S3.
To explore the possibility that metformin regulates the Th17 inflammaging profile through effects on ROS, we tested the ability of the ROS-specific scavenger Tempol to recapitulate metformin effects on CD4+ T cells from O subjects. Tempol reduced ROS and trended toward increased MMP (TMRE, p = 0.058) in CD4+ T cells from O subjects (Figures 3F and 3G), but perhaps more importantly, Tempol uniformly decreased Th17 profile cytokines in O cells with no effect on Y cells, though effects on other cytokines somewhat differed from effects of metformin (Figures 3H and S3C–S3F). Tempol and metformin together increased TMRE signal more than either alone in O cells (Figure 3F), further indicating overlapping but non-identical effects of ROS scavenging and metformin, but consistent with previous demonstrations that ROS dissipation is a mechanism of metformin action (Madiraju et al., 2019). We conclude mitochondrial OXPHOS in O cells coincides with lower antioxidant to cause oxidative stress and a ROS-downstream Th17 profile. Metformin-sensitive pathways that are partially redundant with mitochondrial ROS-scavenging re-establish redox balance to ameliorate T cell inflammaging.
Autophagy Defects in CD4+ T Cells from O Subjects Are Corrected by Metformin
Accumulation of defective mitochondria stemming from general age-related declines in autophagy may, in part, explain excessive OXPHOS and thus redox imbalance in CD4+ T cells from O subjects. We quantified mitochondrial accumulation in CD4+ T cells with Mitotracker green fluorescence and flow cytometry. Cells from O subjects had more mitochondrial mass and mitochondrial matrix proteins, such as m-aconitase, consistent with mitochondrial accumulation. Metformin decreased mitochondrial accumulation in O, but not Y cells (Figures 4A–4D). Metformin action was redundant with Tempol-mediated decrease in mitochondrial mass (Figure 4D), raising the possibility that metformin corrects age-related changes in autophagy that impact redox balance that in turn drives T cell inflammaging.
Figure 4. Metformin Promotes Mitochondrial Turnover and Mitophagy in CD4+ T Cells from O Subjects
(A and B) MitoTracker green fluorescence in CD4+ T cells from O and Y subjects assessed by flow cytometry. n = 6.
© Mitochondrial matrix protein m-aconitase in CD4+ T cells from O and Y subjects as measured on western blots. n = 7–10.
(D) Mitochondrial mass assessed via Mitotracker green fluorescence in the presence of Tempol (TEMP) ± MET as indicated. n = 7–8.
(E and F) Expression of the autophagy proteins LC3II (E) or p62 (F) in cells, measured on western blots. n = 8–10.
(G and H) Autophagosome formation as indicated by puncta and quantitated by confocal microscopy. 3-MA is an inhibitor or autophagy thus serves as a negative control. n = 3.
(I) LC3II expression in CD4+ T cells as indicated, following treatment with metformin + BAF A1 as a positive control for autophagy. n = 5.
(J and K) Localization in representative CD4+ cells (J), and quantitation of co-localization of the mitochondrial protein TOM20 and the lysosomal protein (LAMP1) ± metformin as indicated (K). n = 4 with multiple dots from some N’s shown.
For both confocal analyses (G, H, J, and K), 3 cells/field and 3 fields/slide were imaged using 63× oil immersion in Zeiss microscope. The average fluorescence/field is reported.
(L) Expression of mitochondrial fission protein Drp 1 on western blots. n = 5.
(M–O) Indicators of autophagy (M) LC3II, (N) m-aconitase, or (O) GRP78 quantified in CD4+ T cells from pre-diabetes subjects sampled before or after 3 months’ administration of metformin (1,000 mg/day; n = 4). ∗p < 0.05 versus Y or pre-met, #p < 0.05 versus O.
Data are represented as mean ± SEM. Fold change is compared to either Y, Y + MET, or PRE-MET.
To more broadly test the possibility that age-related autophagy defects impact CD4+ T cells and thereby age-related inflammation, we quantified autophagy indicators in CD4+ T cells from O subjects. O cells had less robust autophagy than Y counterparts, as shown by lower LC3II, p62 accumulation, and fewer LC3II-labeled puncta. Metformin improved all measures of autophagy in O cells (Figures 4E–4H). More LC3II in cells treated with metformin + bafilomycin A1 (BAF A1, autophagy inhibitor) indicated that metformin enhanced autophagosome flux rather than stalled cargo degradation (Figure 4I). In addition, O cells had less co-localization of the mitochondrial protein translocase of outer membrane 20 protein (TOM20) and the lysosomal protein lysosome associated membrane protein 1 (LAMP1) than Y cells had, definitively confirming defective mitochondrial turnover was restored by metformin (Figures 4J and 4K). Metformin similarly increased expression of the mitochondrial fission protein dynamin related protein 1 (Drp1), a mitochondrial indicator of improved health span in Drosophila (Rana et al., 2017) in cells from O subjects (Figure 4L). Metformin did not affect general indicators of autophagy, nor specific mitophagy indicators in Y cells (Figures 4E–4H and 4J–4L), and also failed to impact age-related decreases in humanin and prohibitin (Figures S4A and S4B), two mitokines that regulate inflammation in some circumstances (Conte et al., 2019, Zapała et al., 2010, Kathiria et al., 2012). We conclude that mitochondrial turnover is defective in CD4+ T cells from older people, and that this defect, but not age-related declines in mitokines, is neutralized by metformin.
To test the clinical significance of our in vitro demonstration that metformin improves CD4+ T cell autophagy, we collected cells from obese, pre-diabetes subjects (Table S2) before and 3 months after clinically indicated metformin (1,000 mg/day). Metformin activated autophagy in purified CD4+ T cells from these subjects, after cells were stimulated (without additional metformin) ex vivo, as indicated by more LC3II and multiple indicators of organelle clearance (less m-aconitase and GRP78, indicating mitochondrial and endoplasmic reticulum [ER] clearance; Figures 4M–4O). These data indicate that metformin intervention in older subjects is likely to improve autophagy and autophagy-downstream effects of aging on CD4+ T cells.
To begin testing the possibility that metformin-sensitive defects in autophagy, alone or in combination with fundamental changes in mitochondria, fuel Th17 inflammaging, we activated CD4+ T cells from Y subjects in the presence of the autophagy activator trehalose, or the fatty acid oxidation inhibitor trimetazidine (to induce partial mitochondrial dysfunction), alone or in combination. Trimetazidine increased production of the Th17 cytokines IL-17A/F, IL-21, and IL-23 as previously reported in peripheral blood mononuclear cells (PBMCs) (Nicholas et al., 2019), although, in contrast to PBMCs, trimetazidine also activated Th1 cytokine production by CD4+ T cells (Figures 5A and S4C–S4E). Trehalose did not affect cytokine production, as expected from the inability of metformin or other manipulations to alter Y cells. However, trehalose partially blocked trimetazidine-activated cytokine production (Figures 5A and S4C–S4E), supporting the conclusion that autophagy improvement cannot entirely prevent inflammatory cytokine production by cells with compromised mitochondrial function.
.../...
.
Edited by Engadin, 14 May 2020 - 09:23 PM.
