Chronic overexpression of neuropeptide Y in the skin is sucient to induce inammation and epidermal and dermal pathology

Neuropeptide Y (NPY) is a pleiotropic peptide produced in the central nervous system and peripheral organs. Despite conjectures that NPY may have a role in skin physiology and pathology, the effects of NPY in this organ remain poorly understood. We reported that a knock-in mouse with entopic NPY overexpression exhibits signicantly elevated NPY in the skin, accompanied by premature and progressive hair graying secondary to depletion of melanocyte stem cells within hair follicles. However, the question remains as to whether NPY overexpression in the skin can induce non-melanocyte pathology. In this study, we employed this mouse to investigate the consequences of skin-specic overexpression of NPY. Our ndings show that chronic NPY overexpression in the skin induces dermal brosis and epidermal hyperkeratosis. Additionally, NPY overexpression induces signicant accumulation of macrophages and regulatory T cells in the dermis. RNA sequencing of whole skin from NPY-overexpressing mice further reveals NPY-mediated transcriptional changes consistent with inammatory processes and inammation-associated skin changes and highlights novel cell types involved in the NPY-mediated response in the skin. Together, these results provide long-awaited evidence of NPY’s involvement in skin pathology, providing a background for dening the precise role of NPY in the regulation of cutaneous homeostasis and disease.

Npy tet/tet skin. This model comes at a pivotal time in skin research because although NPY has long been implicated as a mediator of melanocyte and skin pathologies, there have yet to be any models with which to investigate these postulations. In the present study, we evaluated Npy tet/tet mice to assess whether NPY overexpression is indeed su cient to drive non-pigmentary pathologies within the skin.

Materials And Methods:
Animals: Animal care and experimental animal procedures were approved by the Institutional Animal Care and Use Committee associated with the University of Alabama at Birmingham under the animal protocol: UAB IACUC #20382 (MLH). The procedures were performed in accordance with the guidelines set forth by this protocol. Male and female B6;129S4-Npy tm2Rpa /J mice (RRID: IMSR_JAX:007585) were gifted from Lynn Dobrunz, PhD (UAB) and housed in standard cages separated by gender. Based on animal availability, skin from ve each of 22-week-old Npy +/+ and Npy tet/tet siblings was used for RNA sequencing, and a subset of these same mice (3 per genotype) was used for histopathological evaluations. Skin from three each of 35-week-old Npy +/+ and Npy tet/tet siblings was used for histopathological evaluations. Hair graying was determined subjectively by macroscopic identi cation of the obvious and signi cant appearance of depigmented or gray hairs. This study is reported in accordance with ARRIVE guidelines.
Histology and Immuno uorescence Skin harvested from the back of 22-week-and 35-week-old mice was immersed in 2% formaldehyde (Fisher Scienti c, Cat# PI28908) for 30 minutes on ice. Skins were cryoprotected in 10% sucrose for at least 24 hours, embedded in Tissue Plus TM O.C.T. Compound (Fisher Scienti c, Cat# 23-730-571), and frozen. Due to inadequate tissue morphology achieved by cryo-sectioning, reserved skins that were frozen in blocks were alternatively prepared for para n sectioning. Frozen skins were thawed in 4% formaldehyde for 1 hour and transferred to fresh 4% formaldehyde for further xation overnight. These skins were embedded in para n at the Pathology Core Research Lab (UAB) followed by sectioning with a microtome at 5µm thickness. Hematoxylin and eosin, as well as Toluidine blue, staining was performed on skin sections at the Pathology Core Research Lab (UAB).

RNA isolation and quantitative PCR
Skin for quantitative PCR (qPCR) and RNAseq analysis was harvested in TRIzol reagent (Thermo Fisher Scienti c, Cat# 15-596-018) and homogenized using a bead homogenizer (MP Biomedicals). Total RNA from skin was puri ed using the RNAEasy MiniKit (Qiagen) after phenol/chloroform separation.
For qPCR analysis, 1-1.5 µg of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scienti c, Cat# 4364103). qPCR was performed using the following TaqMan gene expression assays: Npy (Mm03048253_m1, Thermo Fisher Scienti c, Cat# 4331182) and the Mouse GAPD Endogenous Control (Thermo Fisher Scienti c, Cat# 4352339E). A standard curve was used to determine relative quantity based on C t values. Relative expression of the target gene was determined by dividing its relative quantity by the endogenous control gene relative quantity.

RNA sequencing
Sequencing of mRNA was performed on the Illumina NextSeq500 as described by the manufacturer (Illumina Inc., San Diego, CA). Brie y, RNA quality was assessed using the Agilent 2100 Bioanalyzer. RNA with a RNA Integrity Number (RIN) of ≥7.0 was used for sequencing library preparation. RNA passing quality control was converted to a sequencing ready library using the NEBNext Ultra II Directional RNA library kit as per the manufacturer's instructions (polyA mRNA work ow; NEB, Ipswich, MA). The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Cluster generation was performed according to the manufacturer's recommendations for onboard clustering (Illumina, San Diego, CA). We generated between 30-35 million paired end 75bp sequencing reads per sample for transcript level abundance.
Heatmaps were generated by applying the regularized log transformation (rlog) to the results data frame produced by the DESeq2 package. To generate gures, values for ltered genes of interest were loaded into the pheatmap (v1.0.12; RRID:SCR_016418) R package and scaled by row.
Clustered bar graphs were generated by using Gene Ontology Biological Process, Cell Type signature gene sets, and Human Phenotype Ontology analyses performed using the molecular signatures database (MSigDB v6.2, RRID:SCR_016863) through the Gene Set Enrichment Analysis website platform.
Visualization of data was performed using the ggplot2 (v3.3.3; RRID:SCR_014601) R-package. PCA plots were also generated using ggplot2 on the rlog le generated by DESeq2.
Statistical analysis: All data are presented as mean ± standard deviation and were analyzed using GraphPad Prism 9 (RRID: SCR_002798) statistical software. Unpaired t-tests were applied for statistical comparison of histological measurements and immune cell count data. p-values of < 0.05 were considered a statistically signi cant difference. Results:

Npy tet/tet skin is brotic with hyperkeratosis
To interrogate whether skin pathologies are present in Npy tet/tet mice, we performed histopathological evaluations on the dorsal skin of 22-week-old (i.e., pre-graying stage; [9]) and 35-week-old (i.e., postgraying stage; [9]) Npy +/+ and Npy tet/tet mice ( Figure 1). 22-week-old Npy tet/tet skin appears to exhibit epidermal thickening with hyperkeratosis, compared to their Npy +/+ littermates, as evidenced by hematoxylin and eosin (H&E) stained sections ( Figure 1A). Following this observation, we quanti ed the thickness of each layer of the skin (epidermis, dermis, and subcutaneous adipose tissue). Between Npy +/+ and Npy tet/tet mice, no difference was found in the thickness of the whole skin, dermis, or subcutaneous adipose tissue, however, the epidermis of 22-week-old Npy tet/tet skin is signi cantly thicker than that of Npy +/+ skin ( Figure 1B). At 35 weeks old, hyperkeratosis ( Figure 1C, D) in Npy tet/tet skin persists, and brosis, evidenced by an increased abundance of pink collagen bers, is now observed in the dermis. No differences between the genotypes are observed in the thickness of the dermis or subcutaneous adipose tissue at this timepoint. These ndings indicate that chronic overexpression of NPY in the skin is su cient to induce functional changes in epidermal keratinocytes and dermal broblasts that is pathological.

Npy tet/tet skin is in ltrated by macrophages and T cells
Following the identi cation of dermal brosis and epidermal thickening in Npy tet/tet skin, we questioned what mechanisms would contribute to these NPY-induced pathologies. Knowing that NPY has diverse functions on immune cells, including pro-in ammatory and anti-in ammatory effects [13, reviewed in (17), 30], we hypothesized that NPY can induce macrophage in ltration and can increase T cell proliferation in the skin [14,[31][32][33][34]. Accordingly, we assessed immunolabeled macrophages (F4/80) and T cells (CD3) to determine the average number of these immune cells within the skin of 22-and 35-weekold mice (Figures 2 and 3). Macrophages are signi cantly more abundant in the skin of 22-week-old Npy tet/tet mice when compared to their Npy +/+ littermates (Figure 2A,B). This accumulation of macrophages is observed in the dermis and subcutaneous adipose tissue of Npy tet/tet skin. At 35 weeks of age, chronic NPY overexpression induces an increase in macrophages in the dermis of Npy tet/tet skin, but this increase was only statistically signi cant at a less stringent p-value of 0.1 ( Figure 2C,D).
We previously reported no overt qualitative changes in the abundance of CD4 + and CD8 + T cells in Npy tet/tet skin [29], yet using the pan T cell marker and quantitative assessment we nd that T cells are signi cantly more abundant in Npy tet/tet skin compared to Npy +/+ littermates at 22 and 32 weeks old. This is attributable to T cell accumulation in the dermis and a trend for their accumulation in the adipose tissue ( Figure 3A,B). This pattern persists in 35-week-old Npy tet/tet skin ( Figure 3C,D). To further characterize the T cell pool, we assessed for the abundance of regulatory T cells (Tregs) via FOXP3 expression. In 22-week-old skin, Tregs are signi cantly more abundant in Npy tet/tet skin, which is attributed to their signi cant accumulation in the dermis ( Figure 4A,B). This persists in 35-week-old skin, along with a trend for their accumulation in the adipose tissue ( Figure 4C,D). Tregs are known to express FOXP3 as well as CD4 [35]. Although we previously observed no qualitative differences in CD4 + T cell abundance [29], the improved quality of the para n-embedded tissues used in the current study compared to the frozen tissue used in our previous study revealed quantitative differences in T cells expressing the FOXP3 marker for Tregs. These ndings suggest that chronic NPY overexpression in the skin can induce in ltration or proliferation of macrophages and T cells, speci cally Tregs, resulting in a chronically in amed environment within the skin.
NPY has also been shown to induce mast cell activation [36]. Accordingly, we assessed for NPY-mediated changes in mast cell numbers within 22-week-old Npy +/+ and Npy tet/tet skin and found that mast cells are similarly abundant in the skin of both genotypes (Supplementary Figure 5). Pathological phenotypes, by H&E staining and immunolabeling, are also observed in heterozygous Npy tet/+ mice and these phenotypes are generally similar to Npy tet/tet mice or are intermediate between those observed in Npy +/+ and Npy tet/tet mice ( Supplementary Figures 1-4).
3. Transcriptional changes in 22-week-old Npy tet/tet skin further supports in ammation as a major response to chronic NPY overexpression and a contributing factor to NPY-mediated skin pathologies To identify transcriptional changes in the skin that might elaborate the mechanisms by which NPY induces skin pathology and in ammation, we harvested whole skin from 22-week-old Npy +/+ and Npy tet/tet mice to be assessed by RNA sequencing (RNAseq) ( Figure 5A). Npy upregulation observed by RNAseq was independently validated in skin from a different anatomical region of the same mice via qPCR ( Figure 5B). From this RNAseq data we made several comparisons; all Npy tet/tet versus all Npy +/+ samples, Npy tet/tet versus Npy +/+ samples based on sex, an intra-genotype evaluation of Npy tet/tet samples ( Figure 5C). When comparing Npy tet/tet animals to Npy +/+ littermates, a short list of 32 differentially expressed genes (DEGs; -1 > log 2 FC < 1, padj < 0.05) was obtained. Six of these DEGs were upregulated. When comparing gene signatures speci c to a certain cell type and biological process were most apparent in the downregulated genes. Twenty-six of 32 DEGs were downregulated-11 of these DEGs were related to melanogenesis and pigmentation, such as Dct, Mc1r, Pmel, and Tyr, and 4 of these DEGs are usually expressed in anagen-stage mouse hair follicle keratinocytes, including Dlx2, Dlx3, Msx2, Mycn ( Figure 5D) [37,38].
By principal component analysis (PCA), we noticed that our RNAseq samples clustered somewhat by sex rather than by genotype (Supplementary Figure 6). Thus, repeating the differential expression analysis while only comparing Npy tet/tet to Npy +/+ animals of the same sex revealed some of the same DEGs as above along with additional DEGs not apparent when the samples of both sexes were evaluated together (see overlaps in Figure 5D). Interestingly, comparing male mice only (Npy tet/tet males (n=2) versus Npy +/+ males (n=3)) showed an upregulation of Retnla, a gene which participates in the negative regulation of Th2 responses and induction of brosis and is upregulated in response to IL-4 and IL-13 in macrophages [39][40][41]. Npy tet/tet males also downregulated Skint3 and Skint9. These genes are expressed by epidermal keratinocytes to signal dendritic epithelial T cells to promote proper skin wound healing and are downregulated during skin aging [42]. On the other hand, comparing Npy tet/tet and Npy +/+ females only [Npy tet/tet females (n=3) versus Npy +/+ females (n=2)], we discovered an abundance of new DEGs only signi cant in this sex (67 total). This included 9 upregulated genes, 4 of which are involved in adipogenesis and lipogenesis, Sult1e1, Thrsp, Fasn, Agpat2 [43][44][45], and one Pla2g2d, that is known as a pro-resolving lipid mediator that works to terminate in ammation [46]. The remainder of the DEGs downregulated by Npy tet/tet females are enriched in genes expressed by various layers of anagen-stage hair follicles, including Cryba4, Edn2, Fbp1, Fgf5, Foxe1, Krt28, Krt31, Krt33a, Krt35, Krt71, Krtap11-1, Krtap15, Padi3, S100a3, Sct, Scl39a8, and Tchh. It is unclear, however, whether this downregulation of hair follicle-related genes is re ective of an NPY-related pathology or differences in the hair stage of Npy tet/tet and Npy +/+ females at harvest as only a subset of these animals was evaluated for histopathology.
Previously we reported that the onset of macroscopic hair graying in the Npy tet/tet mice initiates at 25-27 weeks [29], yet this timeframe is variable as graying was visible in a subset of this cohort of mice as early as 22 weeks ( Figure 5A). Interested in interrogating transcriptomic signatures that might explain the accelerated onset of graying in the "gray" subset of Npy tet/tet mice, we reanalyzed our RNAseq data from the 22-week-old skin to compare the "gray" and "non-gray" Npy tet/tet mice. Interestingly, the gray subset is comprised of male Npy tet/tet mice, while the non-gray subset contains female Npy tet/tet mice. By PCA analysis, Npy tet/tet animals clustered independently based on both graying and sex with 71% variance across principal component 1 (PC1) ( Figure 6A). Npy +/+ animals also cluster independently across PC1 based on sex, yet with only 57% variance. This suggests that the additional variance seen between Npy tet/tet animals across PC1 may be explained by DEGs that are speci c to graying. It is important to note that although the male Npy tet/tet mice of this particular 22-week-old cohort exhibited hair graying before their female counterparts, this male-rst presentation of the phenotype is not consistent across multiple litters (data not shown).
To identify the genes that contribute to graying onset within the gray cohort of Npy tet/tet mice, we rst removed any DEGs that could be considered sex-associated and may be confounding. First, differential expression analysis between the sexes was performed for each genotype and a list containing both upregulated and downregulated genes for each contrast were generated (Npy tet/tet males versus Npy tet/tet females = 918 DEGS; and Npy +/+ males versus Npy +/+ females = 303 DEGs). Next, we overlapped these two DEG lists and removed the 138 genes which were present in both lists and which we deemed to be common, sex-associated genes ( Figure 6B). The remaining 780 DEGs within the Npy tet/tet group were evaluated via gene set enrichment analysis (GSEA) to investigate the transcriptomic differences between gray (i.e., male) and non-gray (i.e., female) Npy tet/tet skin.
GSEA to identify the biological processes upregulated in non-gray Npy tet/tet skin indicates that immune system development is the second-most enriched process. Speci cally, the processes of lymphocyte activation, interleukin-4 (IL-4) production, and cell proliferation are among the most enriched and match expectations based on known NPY signaling. Additionally, processes such as thermogenesis and neurogenesis are enriched in non-gray Npy tet/tet skin (Supplementary File). Conversely, GSEA shows that epidermal cell differentiation is the third-most enriched process in gray Npy tet/tet skin. Additionally, myeloid leukocyte (i.e., macrophage) activation and response to cytokines are enriched in the skin of gray Npy tet/tet mice ( Figure 6C). Despite the gray Npy tet/tet mice clearly exhibiting a more progressed graying phenotype, the expression of pigment genes is not consistently up-or down-regulated in either subset of the Npy tet/tet group. Interestingly, genes related to T cells and B cells are more enriched in the non-gray Npy tet/tet skin, while genes related to macrophages are enriched in the gray Npy tet/tet skin ( Figure 6D). This observation is further supported by GSEA for the cell types that are enriched in the skin of each subset of the Npy tet/tet genotype. The transcriptome of non-gray Npy tet/tet skin shows high enrichment for cells of neural origin (Supplementary File), as well as enrichment for broblasts and immune cells, more speci cally B cells ( Figure 6E). This contrasts with gray Npy tet/tet skin, in which dendritic cells, proliferating macrophages, and proliferating basal cells are within the 10 most enriched cell types ( Figure  6E). These changes in transcriptomic signatures from non-gray to gray may re ect a progression in cellular pathology that ultimately drives the more severe hypopigmentation observed in 35-week-old mice.
GSEA to identify human phenotypes with similar gene enrichment pro les indicates that abnormal function in the human gastrointestinal tract and personality abnormalities, including depressivity, are among the only 5 human phenotypes with similar gene enrichment as the non-gray Npy tet/tet skin, the former of which could be due to the upregulation of immune cell activation in non-gray skin ( Figure 6F).
The same GSEA shows that gray Npy tet/tet skin gene enrichment is similar to that of multiple hair abnormalities, with alopecia being the most similar human phenotype (Supplementary File). Notably, gray Npy tet/tet skin is highly enriched for abnormal hair growth, thickened skin, and hyperkeratosis ( Figure 6F), which match our histopathological ndings (Figure 1).

Discussion:
Skin neuroendocrinology literature has long suggested the involvement of NPY in skin pathology [47]. In support of this hypothesis, clinical studies have shown that genetic mutations in NPY are associated with increased susceptibility for vitiligo [48][49]. Studies have also shown that NPY is more highly expressed in the circulation and affected skin from patients suffering from atopic dermatitis and vitiligo [20][21][22][23][24][25]27]. Despite these long-standing theories and correlative data from the clinic, there has yet to be de nitive data to show whether and how NPY is involved in skin pathology.
We previously reported that chronic, entopic overexpression of NPY induces McSC depletion from murine hair follicles to result in premature and progressive hair graying [29]. This McSC loss and hair graying are associated with elevated expression of Npy in the skin, with NPY protein being elevated in multiple skin cell types. Our previous results were reminiscent of what has been found in some vitiligo patients, where NPY is elevated in the depigmented skin [27], leading us to further inquire into whether this skin-speci c elevation of NPY in the Npy tet/tet mouse may also be relevant to other skin pathologies.
In the present study, we show that chronic overexpression of NPY in Npy tet/tet skin induces epidermal hyperkeratosis by 22 weeks of age, which persists, along with the presentation of dermal brosis, at 35 weeks of age ( Figure 1). Epidermal thickening in Npy tet/tet skin is in line with similar histopathology that is seen in mouse models of atopic dermatitis [50]. Additionally, we show that in ammation plays a role in these skin pathologies, indicated by increased macrophage in ltration and increased Treg abundance in the dermis at 22 weeks old, the latter of which persisted at 35 weeks (Figures 2 and 3). Likewise, increased abundance of macrophages and T cells have been shown in the skin of mouse models of atopic dermatitis [50]. RNAseq data from whole skin of 22-week-old Npy +/+ and Npy tet/tet mice reveal that genes involved in pigmentation, proper wound healing, and the hair follicle are downregulated in response to chronic NPY overexpression ( Figure 5), while genes that promote brosis, adipocyte functions, and in ammatory responses are upregulated. Our RNAseq ndings also suggest mechanisms that may contribute to the differential progression of hair graying in response to NPY in the skin. In 22-week-old Npy tet/tet skin with advanced presentation of hair graying, Npy is more highly expressed, and there is gene enrichment for macrophage activation and keratinocyte differentiation. In contrast, non-gray Npy tet/tet skin has lower Npy expression and is enriched for lymphocyte activation and cell proliferation ( Figure 6). These differences in the enrichment for different immune cell populations, biological processes, and cell types in gray versus non-gray Npy tet/tet skin suggest that the level of Npy expression may in uence the progression of skin in ammation and pathology. Additionally, these differences could explain the wide variances in the pathological characteristics that we have shown within the skin of Npy tet/tet mice (Figures 1-4). As a note, the accelerated graying observed in this cohort of mice was also associated with the male gender, but this male-rst presentation was not consistent across Npy tet/tet mice in other litters. While NPY has been linked to sex-speci c biological changes, we hesitate to make similar conclusions here without further evaluation.
Using the Crowd function from the online Enrichr database, we further compared our gene datasets to previously published datasets [51][52][53]. Interestingly, DEGs identi ed from the skins of both gray and nongray Npy tet/tet mice are similar to that of human and mouse samples of psoriasis and atopic dermatitis. These similarities are largely due to increased expression of Gjb2, Krt27, Krt33a, Psors1c2, and Fabp5 in gray Npy tet/tet skin, which have been shown to be signi cantly upregulated in skin from mouse models of psoriasis and in human psoriatic skin [54][55][56]. Likewise, transcripts for Ecm2 and Plscr4 are reduced in non-gray Npy tet/tet skin, and the downregulation of these genes has been associated with human atopic dermatitis and psoriasis, respectively [55,57]. The dysregulation of a number of genes in Npy tet/tet skin that are similarly dysregulated in samples of psoriasis and atopic dermatitis suggests that overexpression of NPY may in uence the skin's transcriptional landscape that mirrors aspects of these diseases.
Altogether, our ndings provide the long-awaited evidence that NPY can indeed have a pathomechanistic role in the skin. In Npy tet/tet skin, NPY overexpression is su cient to induce epidermal thickening, hyperkeratosis, dermal brosis, and macrophage and Treg accumulation in the dermis. These histopathologies are reminiscent of pathological hallmarks that are seen in human and mouse skin affected by atopic dermatitis or psoriasis. For example, skin affected by atopic dermatitis has been shown to have a T-helper 2 (Th2) immune pro le that is accompanied by increased numbers of Tregs [58,59], both of which are present in our ndings from RNAseq and histological evaluations, respectively. Furthermore, it has been shown that psoriatic skin exhibits epidermal thickening and macrophage in ltration (reviewed in [60]), two pathological ndings identi ed in our study.
Gene enrichment within Npy tet/tet skin supports the pathological ndings of NPY-induced in ammation and epidermal thickening, along with reduction in pigment genes. The current study, along with the ndings from our previous study, suggests that the B6;129S4-Npy tm2Rpa /J mouse line is a novel model that can be used to further evaluate NPY-mediated in ammatory skin pathologies. For instance, RNAseq analysis indicated that gene signatures for B cells and NK cells are enriched in non-gray Npy tet/tet skin, and signatures for dendritic cells are enriched in gray Npy tet/tet skin. Thus, future studies to evaluate the prevalence of these immune cells in the skin of Npy tet/tet mice will determine whether these cells are also involved in the skin in ammation that is induced by NPY overexpression.
Although vitiligo is the in ammatory skin disease whose primary phenotype is a loss of skin color, or hypopigmentation, other in ammatory skin diseases have also been shown to induce hypopigmentation. There have been severe cases of psoriasis and atopic dermatitis that have been reported to induce skin hypopigmentation in a process termed post-in ammatory hypopigmentation [61][62][63]. By mechanisms that remain unclear, post-in ammatory hypopigmentation occurs when chronic cutaneous in ammation induces epidermal melanocyte dysfunction or loss, resulting in temporary or permanent loss of skin color, respectively (reviewed in [64]). Our ndings that in ammation in Npy tet/tet skin precedes hair graying, and that in ammation persists following hair graying, suggests that overexpression of NPY in the skin may be a contributory mechanism to post-in ammatory hypopigmentation. Future studies with this knock-in mouse line should investigate the order by which in ammation and other skin tissue changes (e.g., hyperkeratosis) occur in Npy tet/tet skin. This will elucidate the primary pathological effect of NPY in the skin and identify a potential pathway that can be targeted to mitigate early detrimental effects of NPY. Additional mechanistic studies to investigate speci c cellular responses to NPY overexpression (e.g., using in vitro approaches and/or conditional knock-out of Y receptors in speci c cell types) will be important to identify the critical cell mediators of the NPY response in the skin. Finally, the Npy tet/tet mouse now allows for novel evaluations into NPY's contribution to skin pathology in the context of other models of skin disease.       Non-gray Npytet/tet skin is enriched for in ammation, and gray Npytet/tet skin is enriched for skin pathology. A) PCA plots comparing samples within Npy+/+ or Npytet/tet groups show that graying and/or sex in uences the variations within genotypes. (B) A Venn diagram representing the number of DEGs when comparing between sexes of Npy+/+ (303 DEGs) or Npytet/tet (918 DEGs) groups. The 138

Declarations
DEGs that were common, sex-associated genes were removed from downstream analyses. (C) Clustered bar graph depicting the Gene Ontology: Biological Processes that are upregulated in non-gray Npytet/tet skin (green) compared to gray Npytet/tet skin (blue). The legend (box) indicates that for panels C, E, and F, non-gray Npytet/tet samples are shown in green, and gray Npytet/tet samples are shown in blue. (D) A heatmap of some of the top DEGs in non-gray vs gray Npytet/tet skin shows an enrichment for T and B cells in non-gray skin in contrast to an enrichment for macrophages in gray skin. (E) Clustered bar graph depicting the Cell Types that are enriched in non-gray Npytet/tet skin (green) compared to gray Npytet/tet skin (blue). (F) Clustered bar graph depicting the Human Phenotype Ontologies that are enriched in nongray Npytet/tet skin (green) compared to gray Npytet/tet skin (blue).