The Impact of Vitamin D on Skin Aging

The active metabolites of vitamin D3 (D3) and lumisterol (L3) exert a variety of antiaging and photoprotective effects on the skin. These are achieved through immunomodulation and include anti-inflammatory actions, regulation of keratinocytes proliferation, and differentiation programs to build the epidermal barrier necessary for maintaining skin homeostasis. In addition, they induce antioxidative responses, inhibit DNA damage and induce DNA repair mechanisms to attenuate premature skin aging and cancerogenesis. The mechanism of action would involve interaction with multiple nuclear receptors including VDR, AhR, LXR, reverse agonism on RORα and -γ, and nongenomic actions through 1,25D3-MARRS receptor and interaction with the nongenomic binding site of the VDR. Therefore, active forms of vitamin D3 including its canonical (1,25(OH)2D3) and noncanonical (CYP11A1-intitated) D3 derivatives as well as L3 derivatives are promising agents for the prevention, attenuation, or treatment of premature skin aging. They could be administrated orally and/or topically. Other forms of parenteral application of vitamin D3 precursor should be considered to avoid its predominant metabolism to 25(OH)D3 that is not recognized by CYP11A1 enzyme. The efficacy of topically applied vitamin D3 and L3 derivatives needs further clinical evaluation in future trials.


Skin Aging-Your Skin Can Reveal Stories
Skin aging is a complex process that is influenced by the total exposure of both intrinsic and extrinsic factors over the human lifespan (skin exposome), which is responsible for the progressive morphological and functional alterations of the aged skin [1,[67][68][69]. The main internal factors affecting the chronological (physiological) skin aging include a hormonal decline and changes in gene expression with advancing age [1]. In addition, the cutaneous regenerative potential decreases with the age due to the excessive senescence of keratinocytes, fibroblasts, and melanocytes over time, which contributes to skin aging [70][71][72]. The aged skin is characterized by fine wrinkles and atrophy with reduced elasticity. This chronological aging affects all skin areas but shows phenotypic differences among different anatomical regions, and it varies significantly within different populations [67,73]. The single nucleotide polymorphisms (SNPs) of the MC1R gene (main regulator of melanin pigmentation [74]) are significantly linked to a perceived facial age, providing a new molecular base of youthful looks [75].
Ultraviolet radiation (UVR) is the most harmful external factor contributing to the cutaneous photodamage on the sun-exposed areas. Photoaged skin is presented as a dry, deep-wrinkled skin with rough texture, dyschromia and senile lentigines, vascular complications, etc. [68,89]. UVR decreases the expression of filaggrin that contributes to epidermal hydration, so its downregulation from UVR could explain the skin xerosis in photoaging [90]. Both UVA (315-400 nm) and UVB (280-315 nm) have been shown to contribute to photoaging, either by imbalanced ROS production or by direct DNA damage [83]. However, UVA is considered to play a major role in the aging process. UVA represents more than 80% of total daily UV irradiation and can penetrate 5-10 times deeper into the reticular dermis, where it can damage the extracellular matrix (ECM) more significantly than UVB [91]. Moreover, UVA exposure increases the expression of matrix metalloproteinases (MMPs), especially the expression of the collagenolytic enzyme MMP-1 in dermal fibroblasts, which acts as an important regulator in photoaging [92,93]. Additionally, the chronic UVA irradiation inhibits hyaluronan synthesis, thus altering the composition of proteoglycans in the dermis [94]. A long-term exposure of UVA is related to photoaging and photocancer due to an overproduction of ROS and reactive nitrogen species (RNS), which can disrupt both the nuclear and mitochondrial DNA [95,96]. UVB can penetrate only through the epidermis but is biologically more active. It also induces the transformation of 7-DHC to vitamin D 3 [97,98]. UVB absorbed by DNA and RNA induces a formation of cyclobutane pyrimidine dimers (CPDs) and other photoproducts [99], thus inducing various solar signature mutations in specific genes, including the tumor suppressor gene p53 [100,101]. UVR induces an accumulation of p53 protein in the nucleus that in turn activates the transcription of genes responsible for cell cycle arrest allowing DNA repair, as well as causing an induction of apoptosis of the cells with unrepaired DNA damage [102,103]. Specific p53 mutations can be found in high rates, not only in actinic keratosis (precancerous state) and squamous cell carcinomas (60-90%) with typical UV signature, but also in the normal appearance of UV-exposed skin (about 75%), compared with a much lower rate of such mutations in healthy sun-protected skin (5% of all cases) [104].
Chronic sunlight exposure, together with the persistence of cellular senescence, can drive an impaired regenerative capacity of the skin, chronic inflammation, and photoaging, which correlates with enhanced cancer risk [77,[105][106][107]. Thereby, photoaging results in premature skin aging. Although some aging mechanisms share several similarities or overlapping, photoaged skin differs from physiologically aged skin in the ECM changes. Photoaged skin is characterized by degraded collagen and accumulated aberrant elastin fibers and glycosaminoglycans, whereas physiologically aged skin is presented by the atrophy of dermal structures [108].
The negative impact of ambient pollutants on human health and the human skin is of growing concern [109]. Ozone (O 3 ) from the smog and PM, primarily contacting with the skin, is capable to stimulate ROS production and generate oxidative stress, leading to phenotypic features of extrinsic aging [69]. It was found that chronic exposure to PM leads to pigment spots and deep nasolabial folds [110,111]. Moreover, ultrafine particles (<0.1 µm) can penetrate tissues and localize in the mitochondria, causing an aberrant mitochondrial function because of the oxidative processes [112]. Additionally, the photopollution exposure may aggravate UVR-mediated skin aging [113].
UVR, predominantly UVA, by the excessive amount of ROS activates the mitogenactivated protein kinases (MAPKs) and transcription factors such as nuclear factor erythroid 2-like (Nrf2), c-Jun-N-terminal kinase (JNK), and nuclear factor-κβ (NF-κB), and increases the transcription of MMPs [114]. Activated MMPs, together with the decreased expression of MMP inhibitors (TIMP), cause a dysregulation of the ECM homeostasis and a progressive damage of collagen and elastin [115]. Additionally, UVR impairs the endogenous antioxidant enzymes, leading to an increased oxidative damage of collagen. The destruction of ECM integrity is visualized as wrinkle appearance in photo-damaged skin [93]. The activation of redox-sensitive transcription factors, the activator protein-1 (AP-1) and NF-κβ, involved in wrinkle formation and inflammation, plays crucial roles in skin aging [88]. Both factors, NF-κβ and AP-1, are elevated within hours after cutaneous exposure to low-dose UVB. The upregulation of AP-1 suppresses the transforming growth factor β (TGF-β) receptors, which further blocks the synthesis of procollagen [116,117]. Additionally, activated AP-1 stimulates the degradation of collagen by MMPs and triggers the main activator of the inflammatory response NF-κβ [118]. NF-κβ signaling is a well-known regulator of tissue homeostasis, and its central role in skin aging was recently underlined [119]. The ROS-induced activation of NF-κβ drives an increase of proinflammatory cytokines and MMPs and decreases TGF-β and type I collagen synthesis [119]. The proinflammatory cytokines (interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α) stimulate inflammatory responses and enhance the activation of NF-κβ [93]. It was found that NF-κβ expression could increase in mitochondrial DNA (mtDNA)-depleter mice and after restoring the mitochondrial function, the NF-κβ expression could be reduced. These data confirm that NF-κβ signaling is a key mechanism contributing to the skin and hair follicle pathologies [120]. Due to the longer wavelength, UVA reaches the dermal fibroblasts in vivo with the activation of the Nrf2-mediated expression of antioxidant genes. Unlike UVA, UVB does not activate Nrf2 in skin cells or even appears to have an inhibitory effect [121]. However, vitamin D 3 derivatives, which are products of UVB action, do activate Nrf2 signaling [122]. The endogenous Nrf2 is essential for the protection of skin cells against oxidative insults and for regulating the redox balance during skin aging [123,124]. Many in vitro and in vivo studies confirmed the importance of the transcription factor Nrf2 and its downstream signaling in UV protection [125,126].
Indeed, the human skin aging is mainly driven by oxidative events. An extensive ROS production and insufficient scavenging activity or a mitochondrial dysfunction are crucial events in oxidative stress-induced skin aging. The high levels of ROS lead to oxidative damage of lipids, proteins, genomic, and mtDNA, and also can deplete and damage the antioxidant defense systems of the skin (both non-enzymatic and enzymatic one) [85,127].
Accumulating evidence support a strong link between the mitochondrial dysfunction and the aging process [126]. Many studies demonstrate a decrease in mtDNA content and mitochondrial number during aging. It is thought that mitochondrial dysfunction plays a role in accelerated cellular senescence, seen in advancing age [128][129][130]. Furthermore, mitochondria are believed to contribute to 90% of generated ROS in the cells [95]. mtDNA, as an important target for ROS, is highly vulnerable to the oxidative damage and possesses inefficient DNA repair mechanisms [96,131]. The functional decline of the mitochondria leads to vicious cycle effect contributing to further enhancement of ROS production [127,132].

Impact Paths on the Skin
Excessive exposure to solar UVR accelerates skin aging and could trigger cutaneous cancerogenesis [133]. However, UVR plays a beneficial role in the regulation of many skin functions [56,77,134]. The same UVB, responsible for the increase of non-melanoma skin cancer, is required for vitamin D 3 production in the skin that supplies more than 90% of the vitamin D 3 body's requirement [44,55,135]. In the skin, vitamin D 3 is essential for the formation of the epidermal barrier and hair follicles, and its deficiency has been linked to many proliferative and inflammatory cutaneous disorders [20,29,44,136].
Upon the absorption of UVB, 7-DHC is transformed to vitamin D 3 in the skin, a process accelerated by thermal energy. Prolonged UVB exposure can also generate tachysterol (T 3 ) and lumisterol (L 3 ) [24,97]. These reactions are non-enzymatic and dependent on the UVB dose and the temperature. Vitamin D 3 can be activated through canonical and noncanonical pathways with similar activation of L 3 to biologically active metabolites (Figure 1). In the classical pathway, vitamin D 3 is hydroxylated to 25-hydroxyvitamin D 3 (25(OH)D 3 ) by CYP2R1 and/or CYP27A1 in the liver with further hydroxylation by CYP27B1 in the kidney, skin, and other tissues to its biologically active metabolite 1,25(OH) 2 D 3 [20,21,137].
The main genomic effects and biological responses of vitamin D metabolites in the skin are mediated through their binding to the nuclear VDR [32,61,[154][155][156]. Notably, VDR has been reported to regulate about 3% of mammalian genome due to its broad expression in all tissues [4,34,157,158]. The skin also expresses the VDR and serves not only as a source but also as a site for the action of vitamin D 3 [28,39]. Additionally, the VDR activated by classical 1,25(OH) 2 D 3 can induce rapid response signaling through a non-genomic, membrane-associated mechanism based on an alternative ligand-binding site [159] or through action on 1,25D 3 -MARRS receptor [156,160,161]. Similar non-genomic activities for CYP11A1-derived hydroxyderivatives are still not established. SNPs can affect VDR activity favoring a development of melanoma and non-melanoma skin tumors [162,163]. VDR functions as a tumor suppressor [164] and a decrease in its expression is associated with progression of cutaneous melanoma [165,166]. On the opposite, the nuclear VDR expression has been found significantly elevated (moderate to strong) in squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) compared to in normal skin [167,168]. Thus, targeting VDR with vitamin D secosteroids (especially low calcemic ones) would be rational in skin cancer prevention, attenuation, or therapy [62,64,169].
The main genomic effects and biological responses of vitamin D metabolites in the skin are mediated through their binding to the nuclear VDR [32,61,[154][155][156]. Notably, VDR has been reported to regulate about 3% of mammalian genome due to its broad expression in all tissues [4,34,157,158]. The skin also expresses the VDR and serves not only as a source but also as a site for the action of vitamin D3 [28,39]. Additionally, the VDR activated by classical 1,25(OH)2D3 can induce rapid response signaling through a nongenomic, membrane-associated mechanism based on an alternative ligand-binding site [159] or through action on 1,25D3-MARRS receptor [156,160,161]. Similar non-genomic activities for CYP11A1-derived hydroxyderivatives are still not established. SNPs can affect VDR activity favoring a development of melanoma and non-melanoma skin tumors [162,163]. VDR functions as a tumor suppressor [164] and a decrease in its expression is associated with progression of cutaneous melanoma [165,166]. On the opposite, the nuclear VDR expression has been found significantly elevated (moderate to strong) in squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) compared to in normal skin [167,168]. Thus, targeting VDR with vitamin D secosteroids (especially low calcemic ones) would be rational in skin cancer prevention, attenuation, or therapy [62,64,169].   [61] with the permission from Springer). D 3 , L 3 , and 7-DHC are substrates for CYP11A1 that produces the corresponding hydroxyderivatives. In the case of L 3 and 7-DHC, the side chain can be cleaved by CYP11A1 to produce 7DHP or pL that can be further metabolized by steroidogenic enzymes (ES). In the skin, UVB acting on 5,7-dienes can lead to the production of D 3 , L 3 , and T 3 derivatives with a full-length side chain and pD, pL, and pT derivatives with a shortened side chain. While the cut-off for UVC/UVB is 280 nm, we show the range of 290-315 nm because wavelengths below 290 nm are filtered by the ozone layer and no additional pre-D 3 is produced above 315 nm [170]. 7DHC, 7-dehydrocholesterol; 7DHP, 7-dehydropregnenolone; pD, preganacalciferol; pL, preganalumisterol; D 3 , vitamin D 3 ; L 3 , lumisterol; T 3 , tachysterol; OH, hydroxyl group; number before OH, carbon number with OH; number in subscripts after (OH), number of hydroxyl groups. CYP11A1-derived hydroxyderivatives can regulate some skin functions through other nuclear receptor such as retinoic acid-related orphan receptors (ROR) α and γ, which expressed in the skin [171]. The endogenously produced nonclassical vitamin D 3 hydroxyderivatives, 20(OH)D 3 and 20,23(OH) 2 D 3 can act as weak RORα and RORγ inverse agonists [155,171,172]. Moreover, these hydroxyderivatives could exert anti-inflammatory effect and could inhibit tumor progression in the skin via RORγ-mediated mechanism [173].
Alternative, the classical 1,25(OH) 2 D 3 and CYP11A1-initiated vitamin D 3 derivatives can act as agonists on aryl hydrocarbon receptor (AhR) [174] and liver X receptors (LXR) [175]. Moreover, the activation of AhR is the top canonical pathway for 20,23(OH) 2 D 3 [174]. This receptor regulates cellular proliferation, inflammation, and melanogenesis in the skin [176]. Although many different ligands can target AhR, some functional studies and molecular modeling can predict that secosteroidal signal transduction further leads to the downregulation of proinflammatory responses [177], detoxification, and antioxidative action [61,174].
Summarizing, the biologically active classical and novel vitamin D 3 metabolites exert different affinities to multiple receptors in the skin and through their modulation they can influence different cutaneous pathologies. In addition to act on the VDR, the active forms of vitamin D can act on alternative nuclear receptors including RORs, AhR, LXR, and 1,25D 3 -MARRS receptor. The active forms of vitamin D 3 have various functions, which partially overlap in their anti-inflammatory, antimicrobial, antiproliferative, prodifferentiation, antifibrotic, and antioxidative effects on the skin [20,38,63,141,145,178]. Along with the best characterized 1,25(OH) 2 D 3 , CYP11A1-derived products of vitamin D 3 and L 3 exhibit photoprotective properties against UVR-induced skin damage ( Figure 2) [37,61,[179][180][181][182][183].

Effects on the Cutaneous Immune Function
Vitamin D3 and its analogues and precursors play an important complex role in t regulation of both the innate and adaptive immune systems, including in the sk [8,184,185]. There is a clear connection between vitamin D deficiency and incidences

Effects on the Cutaneous Immune Function
Vitamin D 3 and its analogues and precursors play an important complex role in the regulation of both the innate and adaptive immune systems, including in the skin [8,184,185]. There is a clear connection between vitamin D deficiency and incidences of infections and immune-mediated skin diseases [31,[186][187][188]. The expression of the VDR has been found in almost all immune cells including T-and B-lymphocytes (Lym), macrophages, mast cells, natural killer (NK) cells, and regulatory T cells (Tregs), but it is differently regulated [189]. Monocytes, for example, lose VDR expression levels during differentiation towards macrophages and dendritic cells (DCs) [190,191]. Immunomodulatory actions of active vitamin D 3 metabolites include the induction of Tregs [192] and Thelper-2 (Th2)-Lym, together with the downregulation of proinflammatory Th1/Th17/Th9-Lym [193]. 1,25(OH) 2 D 3 can have both direct and indirect effects on T-Lym [194]. The indirect effect is based on DC-derived cytokines, which modulate Th-Lym response [30]. Directly, 1,25(OH) 2 D 3 can suppress the immune cell production of inflammatory cytokines such as interferon gamma (IFN-γ), TNF-α, and IL-2 (Th1 cytokines), IL-17/21 (Th17 cytokines), and Th9 cytokines [193,195,196], while it enhances either the levels of anti-inflammatory IL-10 from Tregs [197] or Th2-derived IL-4 [198]. As a result, vitamin D shifts Th1 inflammatory response towards more tolerogenic Th2 response with an increase of CD4 + CD25 + Tregs reflected to a change in cytokine profile in the skin [199]. Additionally, 1,25(OH) 2 D 3 influences the activated B-Lym by inducing apoptosis [200], suppressing immunoglobulin E (IgE)-dependent mast cell activation [201,202], and upregulating IL-10 production [203]. Enhanced IL-10 synthesis contributes to a suppressed mast cell-mediated inflammation and IgE-related allergic reactions [201]. 1,25(OH) 2 D 3 and its analogues directly regulate antimicrobial peptide (AMP) gene expression in innate immune cells [204,205]. Moreover, it has been found that vitamin D is able to induce essential for antimicrobial defense, production of cathelicidin (LL37) [204] and to modulate the phagocytic activity of macrophages and NK cells [193]. Additionally, vitamin D exerts immunosuppressive effects through the modulation of epidermal Langerhans cells [206] and the proliferation of the Tregs number [184,185,197].
CYP11A1 is expressed also in immune cells [207], where vitamin D can be further metabolized to biologically active hydroxyderivatives [31,39]. Via the activation of VDR or through the inhibition of RORγ-mediated activation, 20(OH)D 3 and 20,23(OH) 2 D 3 , like 1,25(OH) 2 D 3 , can attenuate Th17 differentiation, as well as the formation and activity of inflammatory cytokine IL-17 by immune cells [155,208,209]. Thus, Th17-related cutaneous inflammation could be successfully modulated via RORγ inverse agonists such as CYP11A1-derived D 3 -hydroxyderivatives, causing the regulation of the immune system and a resistance against autoimmunity [210,211]. The most recently inhibition of collageninduced autoimmune arthritis by CYP11A1-derived 20(OH)D 3 was reported [212].

Impact on Skin Aging
The normal vitamin D 3 status is important for a general prevention of premature aging maintaining a healthful skin aging [213,214]. Vitamin D 3 metabolites including its classical (1,25(OH) 2 D 3 ) and novel (CYP11A1-intitated) D 3 hydroxyderivatives exert many beneficial protective effects on the skin, which could influence the process of premature aging via many different mechanisms, leading to a delay or attenuation of both chronological skin aging and photoaging. Skin-resident cells (keratinocytes, fibroblasts, and sebocytes) are capable of locally activating vitamin D 3 [23,36,215] and exhibiting a diverse biological effect such as photoprotection and immunosuppression, similar to the UVR-induced one [179,216].
The process of chronological aging is associated with immunological alteration and the imbalance between inflammatory and anti-inflammatory mechanisms, leading to a chronic low-grade inflammation, known as "inflammaging" state [217,218]. The "inflammaging" phenotype of the skin and hair follicles is a result of both chronic antigen stimulation and continued exposure to oxidative stress caused by ROS and RNS [219,220]. With advancing age, skin is affected by the profound remodeling of the immune system, leading to a decline in its adaptive capacity [221,222]. Th1-and Th17-related markers, together with the number of epidermal DCs are increased as a function of age [223][224][225]. DCs during aging appear to be functionally impaired, which contributes to an initiation of inflammatory and autoimmune skin disorders and a loss of their protective role against cutaneous infections. The active forms of vitamin D 3 are able to decrease the proliferation and cytotoxicity of T-Lym, as well as to suppress the differentiation of B-Lym and the maturation of DCs [193]. Therefore, vitamin D 3 hydroxyderivatives exert potent anti-inflammatory activities including the inhibition of TNF-α, INF-γ, and IL-1/6/9/17 production [4,38,185], suggesting their implication in the modulation of skin inflammation. Moreover, the noncalcemic and nontoxic 20(OH)D 3 has shown a similar anti-inflammatory property in vivo to 1,25(OH) 2 D 3 (hypercalcemic in high doses) through the suppression of the immune responses by T-and B-lym [155,212].
Active vitamin D 3 metabolites can protect skin against the hazardous effects of skin aging-triggering agents, including UVR, pollution, and microbial infections [179,[226][227][228][229][230]. It has been shown that the oral administration of high-dose vitamin D 3 shortly after UVB exposure could reverse rapidly the photo-induced cutaneous damage by decreasing the inflammation and induction of the repair mechanisms of the epidermal barrier [38]. There is strong experimental evidence that active vitamin D 3 and L 3 hydroxyderivatives can induce, in a dose-dependent manner, antioxidative responses and reverse the UVB-mediated ROS production in keratinocytes by the activation of Nrf2 that works for cytoprotection and detoxification, thus attenuating photoaging [122]. Therefore, they serve as protective agents against UVB-induced oxidative stress in cells, pre-treated with each of these active metabolites for 24 h prior to UVB irradiation (50 mJ/cm 2 ) [122]. These hydroxyderivatives stimulate the expression of antioxidant-response genes downstream of Nrf2 (GR, HO-1, CAT, SOD-1, and SOD-2) as well as the expression of HO-1, CAT, and MnSOD at the protein level [122]. The transcription factor Nrf2 plays an important role in the detection of excessive ROS and RNS and in the induction of mechanisms counteracting the oxidative damage and skin pigmentations produced by UVA [121,125,231].
Chronic UVR irradiation, mainly UVB [232] and UVA [233], induces DNA damage and the formation of CPDs that potentially lead to premature skin aging and carcinogenesis. CYP11A1-derived D 3 and L 3 hydroxyderivatives, along with 1,25(OH) 2 D 3 , demonstrate photoprotective and reparative properties by increasing the expression and phosphorylation of p53 with its translocation to the nucleus [61,229,234,235]. The P53 gene family, in particular its isoform p63, might be an important molecular target for vitamin D action in premature aging and cancer [236], which are promoted by similar mechanisms [237].
Additionally, 1,25(OH) 2 D 3 can induce rapid and dose-dependent reduction in skin cell apoptosis, and it can increase CPDs repair and decrease the oxidative DNA damage through non-genomic energy-conserving autophagy and mitophagy [227], thus contributing to the intrinsic skin photoprotection mechanism [242].

Conclusions and Future Perspectives
Vitamin D 3 and its active metabolites exert a variety of antiaging and (photo) protective effects on the skin. These are achieved through immunomodulation that include anti-inflammatory actions and regulation of keratinocytes proliferation and differentiation program to build the epidermal barrier necessary to maintain skin homeostasis. In addition, they induce antioxidative responses, inhibit DNA damage and induce DNA repair mechanisms to attenuate premature skin aging and cancerogenesis. Similar actions can be assigned to lumisterol metabolites. Therefore, active forms of vitamin D 3 including its canonical (1,25(OH) 2 D 3 ) and noncanonical (CYP11A1-intitated) D 3 -hydroxyderivatives as well as L 3 -derivatives are promising agents for the prevention, attenuation, or treatment of premature skin aging, when applied topically. It is expected that they will attenuate photoaging and perhaps repair the existing damage induced by external stressors. The mechanism of action would involve interaction with nuclear receptors including VDR, AhR, LXR, reverse agonism on RORα and RORγ, and nongenomic actions through 1,25D 3 -MARRS receptor and interaction with the nongenomic binding site of the VDR. The regulatory mechanism affected by D 3 and L 3 derivatives would include the activation of Nrf2 and p53 and the downregulation of NFκβ signaling pathways or the regulation of mitochondrial functions. To prevent skin aging, vitamin D 3 and lumisterol or their derivatives could be administrated orally and/or topically. Other forms of parenteral application of the vitamin D 3 precursor should be considered to avoid channeling its metabolism to 25(OH)D 3 , which is not recognized by CYP11A1 enzyme [243]. The efficacy of topically applied vitamin D 3 and L 3 derivatives needs further clinical evaluation in future trials.

Funding:
We acknowledge the support of NIH grants (1R01AR073004 and R01AR071189), a VA merit grant 1I01BX004293 to A.T.S., a grant R21 AI152047 to A.T.S. and C.R., and a grant R21 MD015319 to C.R.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.