Multi-Ingredient Supplement Supports Mitochondrial Health through Interleukin-15 Signaling in Older Adult Human Dermal Fibroblasts

: The macroscopic and microscopic deterioration of human skin with age is, in part, attributed to a functional decline in mitochondrial health. We previously demonstrated that exercise attenuated age-associated changes within the skin through enhanced mitochondrial health via IL-15 signaling, an exercise-induced cytokine whose presence increases in circulation following physical activity. The purpose of this investigation was to determine if these mitochondrial-enhancing effects could be mimicked with the provision of a novel multi-ingredient supplement (MIS). Cultured human ﬁbroblasts isolated from older, sedentary women were treated with control media (CON) or CON supplemented with the following active ingredients to create the MIS: coenzyme Q10, alpha lipoic acid, resveratrol, curcumin, zinc, lutein, astaxanthin, copper, biotin, and vitamins C, D, and E. Outcomes were determined following 24 or 72 h of treatment. MIS provision to dermal ﬁbroblasts signiﬁcantly increased the mRNA abundance of mitochondrial biogenesis activators and downstream IL-15 signaling pathways, and proteins for oxidative phosphorylation subunits and antioxidant defenses. These ﬁndings were co-temporal with lower cellular senescence and cytotoxicity following MIS treatment. In summary, MIS supplementation led to exercise-mimetic effects on human dermal ﬁbroblasts and their mitochondria by reproducing the molecular and biochemical effects downstream of IL-15 activation.


Introduction
It is widely appreciated by scientists and non-scientists alike that aging is associated with both visible, molecular, and microscopic alterations to skin health and function. This deterioration includes changes to both the outermost epidermis and the underlying dermis, and is attributed to attenuated cell proliferation (i.e., fibroblasts), dysregulation in extracellular matrix activities, and blunted connective tissue synthesis, among other cellular factors [1]. The molecular etiology of the aforementioned age-associated attributes is at least partially linked to mitochondrial capacity, whose functional decline is widely implicated in the aging process, including the skin [2][3][4][5][6]. This occurs via several independent mechanisms including oxidative damage due to electron transport chain generation of reactive oxygen species (ROS), age-associated damage to mitochondrial DNA (mtDNA), and a decline in energy metabolism [4,[7][8][9][10].
Corroborating these findings, our group has previously demonstrated that exercise increased mitochondrial content, improved mitochondrial metabolism, attenuated negative age-associated changes, and increased dermal collagen content within the skin in older 2 of 11 adults [5]. A major factor mediating these changes was IL-15, an exercise-stimulated cytokine (i.e., 'exerkine') whose secretion transiently increases following acute exercise [5,11]. In line with this finding, daily injections of recombinant IL-15 that mimicked an acute bout of exercise led to an increase in mitochondrial function and dermal collagen content that was comparable to the effect observed with exercise training for the same duration in older mice [5]. Despite these favorable outcomes, the use of recombinant IL-15 injections as a therapy for skin health would require extensive pharmacokinetic studies, and extensive safety and efficacy trials prior to approval. In contrast, nutritional compounds could potentially activate endogenous IL-15 expression or activate the downstream IL-15 signaling pathways independent of IL-15, and thus could be an alternative nutraceutical approach to benefit mitochondrial and dermal health.
Aging is associated with reduced oxidative respiration, mitochondrial gene expression, and biogenesis, concomitant with the buildup of ROS-induced cellular damage. Thus, a prominent anti-aging strategy should encompass the use of a combination of antioxidants and mitochondrial-enhancing ingredients to target the common pathways of cellular dysregulation, including mitochondrial dysfunction and oxidative stress. As evidence that a natural combinatorial therapy results in effective mitigation of a pathological phenotype, our group has shown that a multi-nutrient supplement including creatine monohydrate, alpha-lipoic acid (α-LA), coenzyme Q10 (CoQ10), and vitamin E led to improvements in mitochondrial function and reductions in oxidative stress in patients with genetic mitochondrial disease [12]. Supporting the concept of a multi-ingredient nutraceutical approach, CoQ10 in isolation did not produce antioxidant effects in a similar cohort of patients with mitochondrial disease [13]. Similarly, we have shown that three of these ingredients given as a multi-nutrient combination (α-LA, CoQ10, vitamin E) improved mitochondrial function and exercise performance in female mice [14], whereas others using these supplements in isolation found no ergogenic benefit [15][16][17].
Herein, we demonstrate that our novel MIS reproduces the biochemical effects of IL-15 activation, leading to the activation of gene expression patterns downstream of IL-15, and resultant protein adaptations in key mitochondrial and antioxidant pathways without increasing endogenous IL-15 expression. Furthermore, these adaptations are associated with less cellular senescence and improved survival in dermal fibroblasts, indicating that the benefits of the MIS extend beyond mitochondrial enhancement alone.

Cell Culture and Treatment
Cultured human fibroblasts (derived from skin samples collected from human inner forearm skin biopsies) were collected at McMaster University (Hamilton Integrated Research Ethics Board no. . Fibroblasts were isolated from sedentary females with an average age of 61.6 years. Fibroblasts (passage 3-6; same passage used for all treatment groups for each method of analysis) were cultured in Fibroblast culture media (MEM) supplemented with 10% fetal bovine serum, L-glutamine, and HEPES (all Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained at 37 • C with 5% CO 2 , and culture medium was changed every two days. The following active ingredients were dissolved via gentle stirring into cell culture media (as above) to create the MIS: CoQ10, α-LA, vitamin E, vitamin C (10 µM; Sigma Aldrich, St. Louis, MO, USA), resveratrol and curcumin (5 µM; Sigma Aldrich), zinc (4 µM; Thermo Fisher Scientific), lutein and astaxanthin (3 µM; lutein, Cayman Chemical Company, Ann Arbor, MI, USA; astaxanthin, Sigma Aldrich), vitamin D and copper (0.5 µM; Sigma Aldrich), and biotin (0.25 µM; Sigma Aldrich). Control treatment (CON) was composed solely of cell culture media, as above.
Each treatment group consisted of a minimum of three replicates of dermal fibroblast cultures per donor. Cell harvests occurred following either 24 or 72 h of either CON or MIS treatment. As such, four experimental groups were created: CON-24, MIS-24, CON-72, MIS-72.

RNA Isolation and cDNA Synthesis
RNA was isolated from 1-2 million cells via needle aspiration for membrane lysis followed by a standardized Trizol extraction protocol. RNA was resuspended in DNase/RNase-free water and purity was validated using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using SuperScript IV VILO Master Mix (Thermo Fisher Scientific; 11,756,500) as per the manufacturer's protocol. Reaction mixtures were incubated in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) with thermocycling conditions of 25 • C for ten minutes (annealing), 50 • C for ten minutes (elongation), and 85 • C for five minutes (enzyme inactivation).
Fold-increase in mRNA was calculated via the 2 −∆CT method [50]. Content levels were normalized to the reference gene beta-2-microglobulin (B2M) and subsequently to the respective timepoint CON group dataset. B2M content was unchanged between individually collected samples and was not affected by MIS versus CON treatment.

Cytotoxicity and Cellular Senescence
Cytotoxicity was quantified using cell culture media within the LDH Assay Kit (Abcam; ab65393) using standard manufacturer instructions. Briefly, following treatment as above, ten microliters of media were extracted from each well of cells on a 96-well plate that was plated at 3000 cells per cm 2 . All assays were conducted in triplicate.
Cellular senescence was quantified using the Beta-galactosidase Senescence Detection Kit (ab65351; Abcam). Briefly, cells were plated at 3000/cm 2 within a 12-well dish and treated as above. The detection kit was then carried out as per manufacturer instructions.

Statistics
All statistical analyses were performed using Prism 9 (GraphPad Software, La Jolla, CA, USA). All data herein are presented as mean ± standard error of the mean (SEM). A * within a figure indicates statistical significance, set at p < 0.05, via unpaired Student's t-test with the respective timepoint control (i.e., CON-24 or CON-72).

MIS Treatment Increases mRNA Content of Mitochondrial Biogenesis Activators in Human Fibroblasts via Downstream Targets of IL15
Compared to the respective timepoint control groups, mRNA content of IL15 was not significantly altered by 24 or 72 h of MIS treatment (p = 0.26 and 0.15, respectively; Figure 1A). Downstream effectors of IL15 gene activation, namely STAT5A and PPARG, did show significantly higher mRNA content at 24 h of MIS supplementation (STAT5A 1.68-fold, PPARG 2.21-fold increases when comparing CON-24 and MIS-24; Figure 1B,C). These early elevations in pathway intermediates were associated with a 1.81-fold increase in TFAM mRNA content following 72 h of MIS treatment ( Figure 1D).
Cellular senescence was quantified using the Beta-galactosidase Senescence Detection Kit (ab65351; Abcam). Briefly, cells were plated at 3000/cm 2 within a 12-well dish and treated as above. The detection kit was then carried out as per manufacturer instructions.

Statistics
All statistical analyses were performed using Prism 9 (GraphPad Software, La Jolla, CA, USA). All data herein are presented as mean ± standard error of the mean (SEM). A * within a figure indicates statistical significance, set at p < 0.05, via unpaired Student's ttest with the respective timepoint control (i.e., CON-24 or CON-72).

MIS Treatment Increases mRNA Content of Mitochondrial Biogenesis Activators in Human Fibroblasts via Downstream Targets of IL15
Compared to the respective timepoint control groups, mRNA content of IL15 was not significantly altered by 24 or 72 h of MIS treatment (p = 0.26 and 0.15, respectively; Figure  1A). Downstream effectors of IL15 gene activation, namely STAT5A and PPARG, did show significantly higher mRNA content at 24 h of MIS supplementation (STAT5A 1.68-fold, PPARG 2.21-fold increases when comparing CON-24 and MIS-24; Figure 1B,C). These early elevations in pathway intermediates were associated with a 1.81-fold increase in TFAM mRNA content following 72 h of MIS treatment ( Figure 1D).

MIS Treatment Increases Production of Key mRNA and Proteins Responsible for Mitochondrial Function and Oxidative Phosphorylation in Human Fibroblasts
Following 72 h of MIS supplementation, fibroblasts showed significant increases in MTCO1 (1.67-fold), COX4I1 (1.67-fold), and ACO2 (2.25-fold) mRNA content when compared to CON-72 fibroblasts (Figure 2A-C). These elevations were also reflected by positive changes in oxidative phosphorylation protein content. When compared to their respective timepoint controls, both MIS-24 and MIS-72 increased Complex I (76% and 161%), Complex II (23% and 65%), and Complex III (41% and 169%) protein content (Figure 2D-F). Increases in Complex IV content with MIS treatment were only significant

MIS Treatment Increases Production of Key mRNA and Proteins Responsible for Mitochondrial Function and Oxidative Phosphorylation in Human Fibroblasts
Following 72 h of MIS supplementation, fibroblasts showed significant increases in MTCO1 (1.67-fold), COX4I1 (1.67-fold), and ACO2 (2.25-fold) mRNA content when compared to CON-72 fibroblasts (Figure 2A-C). These elevations were also reflected by positive changes in oxidative phosphorylation protein content. When compared to their respective timepoint controls, both MIS-24 and MIS-72 increased Complex I (76% and 161%), Complex II (23% and 65%), and Complex III (41% and 169%) protein content ( Figure 2D-F). Increases in Complex IV content with MIS treatment were only significant following 24 h of supplementation (73% increase at 24-h, p = 0.11 at 72-h; Figure 2G). Although trending towards significance, MIS-24 and MIS-72 did not cause significant changes in Complex V protein content (p = 0.08 and 0.06, respectively; Figure 2H). following 24 h of supplementation (73% increase at 24-h, p = 0.11 at 72-h; Figure 2G). Although trending towards significance, MIS-24 and MIS-72 did not cause significant changes in Complex V protein content (p = 0.08 and 0.06, respectively; Figure 2H).

Seventy-Two Hours of MIS Supplementation Modifies mRNA and Protein Content of Key Cellular Antioxidants in Human Fibroblasts
MIS-72 increased mRNA abundance relative to CON-72 for GPX1 (1.52-fold; Figure  3A), CAT (1.81-fold; Figure 3B), and SOD1 (1.54-fold; Figure 3C). When compared to CON-72, SOD2 mRNA content was lower following 72 h of MIS supplementation ( Figure  3D). The aforementioned elevation in GPX1 mRNA abundance was reflected by an 84% increase in GPX1 protein content with MIS-72 treatment ( Figure 3E). Although significant increases in CAT and SOD1 mRNA content were seen as a result of MIS-72, these increases did not translate to an increase in catalase or SOD1 protein content following 72 h of MIS supplementation (p = 0.79 and 0.73, respectively; Figure 3F,G). Similar to GPX1, MIS-72 was associated with a significant 117% elevation in SOD2 protein content compared to CON-72 ( Figure 3H).

Seventy-Two Hours of MIS Supplementation Modifies mRNA and Protein Content of Key Cellular Antioxidants in Human Fibroblasts
MIS-72 increased mRNA abundance relative to CON-72 for GPX1 (1.52-fold; Figure 3A), CAT (1.81-fold; Figure 3B), and SOD1 (1.54-fold; Figure 3C). When compared to CON-72, SOD2 mRNA content was lower following 72 h of MIS supplementation ( Figure 3D). The aforementioned elevation in GPX1 mRNA abundance was reflected by an 84% increase in GPX1 protein content with MIS-72 treatment ( Figure 3E). Although significant increases in CAT and SOD1 mRNA content were seen as a result of MIS-72, these increases did not translate to an increase in catalase or SOD1 protein content following 72 h of MIS supplementation (p = 0.79 and 0.73, respectively; Figure 3F,G). Similar to GPX1, MIS-72 was associated with a significant 117% elevation in SOD2 protein content compared to CON-72 ( Figure 3H).

MIS Treatment Supports Whole-Cell Health in Human Dermal Fibroblasts
After 24 h of MIS treatment, there was a 514% lower relative cellular cytotoxicity in cultured dermal fibroblasts than observed in CON-24 (p < 0.0001, Figure 4A). Furthermore, when compared to CON-24, cellular senescence was 29% lower in fibroblasts after 24 h of MIS supplementation (p = 0.01, Figure 4B).

Discussion
The interconnection between poor mitochondrial health, the physiological decline in skin health, and the restoration of these impairments via exercise is an intriguing target for anti-aging strategies [2][3][4][5][6]. Previously, we found IL-15 to be an important regulator of

MIS Treatment Supports Whole-Cell Health in Human Dermal Fibroblasts
After 24 h of MIS treatment, there was a 514% lower relative cellular cytotoxicity in cultured dermal fibroblasts than observed in CON-24 (p < 0.0001, Figure 4A). Furthermore, when compared to CON-24, cellular senescence was 29% lower in fibroblasts after 24 h of MIS supplementation (p = 0.01, Figure 4B).

MIS Treatment Supports Whole-Cell Health in Human Dermal Fibroblasts
After 24 h of MIS treatment, there was a 514% lower relative cellular cytotoxicity in cultured dermal fibroblasts than observed in CON-24 (p < 0.0001, Figure 4A). Furthermore, when compared to CON-24, cellular senescence was 29% lower in fibroblasts after 24 h of MIS supplementation (p = 0.01, Figure 4B).

Discussion
The interconnection between poor mitochondrial health, the physiological decline in skin health, and the restoration of these impairments via exercise is an intriguing target for anti-aging strategies [2][3][4][5][6]. Previously, we found IL-15 to be an important regulator of

Discussion
The interconnection between poor mitochondrial health, the physiological decline in skin health, and the restoration of these impairments via exercise is an intriguing target for anti-aging strategies [2][3][4][5][6]. Previously, we found IL-15 to be an important regulator of the benefits of exercise on mitochondrial and overall skin health via AMPK-and PGC1α-dependent pathways of mitochondrial biogenesis and an increase in antioxidant defenses [5].
There lies a challenge in translating the science of IL-15 and its benefits on skin health into a meaningful commercial product, because IL-15 cannot be ingested, as it will be degraded in the stomach during digestion. Furthermore, creating a topical IL-15 cosmetic or cream would not likely produce favorable results, as IL-15 would be unstable in a cream/gel matrix, and would not likely penetrate to the dermis to produce visible anti-aging effects. Another challenge to the intravenous or subcutaneous pharmacological administration of IL-15 would lie in selecting an appropriate dose to mimic the exercise "pulse" in vivo, as both positive and negative clinical effects have been observed with different dosages of IL-15 [52]. As such, the most logical approach for recreating the benefits of IL-15 on the skin in humans is via an ingestible MIS that increases endogenous IL-15 expression and/or mimics the downstream effects of IL-15-receptor-mediated signaling. Furthermore, having an MIS in an ingestible format allows for endogenous bathing of the dermis and epidermis upon ingestion in a manner similar to the pulsatile, transient increases in IL-15 release observed following exercise.
Within the current investigation, we indeed mimic the desired exercise-induced effect on the dermal fibroblasts of older adults via provision of a novel MIS containing ingredients known to facilitate mitochondrial or skin health without leading to an increase in endogenous IL-15 expression. Given that IL-15 is not known to be produced in fibroblasts, the cell type used within this body of work, it is not surprising that we did not see an increase in IL-15 mRNA in response to our MIS. Instead, we observed robust activation of the downstream canonical pathways induced by IL-15 receptor binding with MIS supplementation, namely STAT5A, PPARG, and TFAM genes. These downstream pathways are major targets for the pharmaceutical treatment of a variety of mitochondrial-associated disorders and have also been shown to be upregulated by some of our individual MIS ingredients in a variety of cell types [53][54][55][56][57][58][59][60][61].
A potential concern for the usage of a multi-target combinatorial antioxidant supplement is the ability for exogenous antioxidants to have complex and unpredictable interactions [62]. For example, previous studies have reported that combined vitamin C and E supplementation attenuates mRNA responses in mitochondrial proteins and antioxidant enzymes in skeletal muscle, blunting the adaptive response to endurance training [63,64]. Others, however, have not been able to reproduce these findings [65,66], and our group has not found any deleterious metabolic effects of combined vitamin E, CoQ10, and α-LA therapy in humans or rodents [12,14]. Similarly, our recent development of a combination multi-ingredient antioxidant supplement targeting mitochondrial dysfunction in obese mice found no deleterious health effects, and in fact an enhanced benefit of exercise [67]. Similarly, positive effects of MIS supplementation on fibroblast health were observed in the current investigation.
During aging, mitochondrial aconitase, an integral component of the TCA cycle, is deleteriously affected by oxidative damage, resulting in the loss of catalytic function [68]. Within the current investigation, 72 h of MIS supplementation caused an increase in aconitase gene content, potentially indicating the onset of reversal of this aging effect. As accumulation of mtDNA mutations have been associated with phenotypic aging of the skin [69][70][71], the significance of this result is of extended importance due to the additional role of aconitase in associating with and stabilizing mtDNA [72,73]. Furthermore, as mtDNA mutations have also been identified to propel metabolic reprogramming and subsequent cellular senescence [74,75], this may be a key component causing the decrease in cellular senescence observed with MIS supplementation.
Mitochondria accumulate damage with time, resulting in cellular senescence, a process in which cells cease division, develop apoptotic resistance, exhibit altered gene expression patterns, and develop morphological changes [76]. There is clear evidence to suggest that fibroblast senescence is a major driver of age-associated pathology [77,78], making our finding of attenuated senescence with MIS one of clinical relevance to the aging population. In addition to the aforementioned association of mtDNA mutations with cellular senescence, overt mitochondrial dysfunction and oxidative stress also contribute to this pathological state [76,79,80]. As such, additional factors contributing to our decrease in fibroblast senescence likely include the metabolic restoration of mitochondrial health by way of the observed MIS-associated upregulation of mitochondrial respiratory chain complex proteins and antioxidant defenses.
Another highlight of the current investigation is the supra-mitochondrial effect of our MIS in mitigating cytotoxicity. Similar to our findings, resveratrol, a polyphenol within our MIS, has recently been identified to concomitantly decrease both cellular senescence and cytotoxicity on its own in human fibroblasts [81]. Evidently, the benefits of the MIS and its ingredients on mitochondrial health extend beyond this organelle, facilitating whole-cell health and rejuvenation.
In conclusion, the provision of our MIS to aged dermal fibroblasts mimicked exercise through activation of signaling pathways known to be downstream of IL-15 receptor binding. This activation led to elevations in key factors of mitochondrial health, including those involved in mitochondrial biogenesis, oxidative phosphorylation, and antioxidant defense systems. Furthermore, these mitochondrial benefits were associated with improvements in overall fibroblast health. Given these results, future studies will evaluate the benefits of the current MIS in other preclinical disease models associated with aging and mitochondrial dysfunction.  Informed Consent Statement: Written informed consent has been obtained from the patients to publish this paper. Data Availability Statement: Not applicable.

Conflicts of Interest:
Exerkine Corporation is a biotechnology company that develops and commercializes therapies based on nutritional supplements, exercise-derived factors ('exerkines'), and extracellular vesicles to treat and diagnose genetic disorders, chronic diseases, and aging. M.A.T. is the founder, C.E.O., and C.S.O. of Exerkine Corporation, which provided support in the form of a salary for I.A.R. Exerkine Corporation owns shares in HERB + FLORA, a clean beauty corporation, who has marketed a commercial product that contains the same ingredients as the MIS embodied within this research. Exerkine has patents for the use of IL-15 in skin and muscle health (Patent Number CA 2917944).