Analysis of Intracellular Communication Reveals Consistent Gene Changes Associated with Early-Stage Acne Skin

A comprehensive understanding of the intricate cellular and molecular changes governing the complex interactions between cells within acne lesions is currently lacking. Herein, we analyzed early papules from six subjects with active acne vulgaris, utilizing single-cell and high-resolution spatial RNA sequencing. We observed significant changes in signaling pathways across seven different cell types when comparing lesional skin samples (LSS) to healthy skin samples (HSS). Using CellChat, we constructed an atlas of signaling pathways for the HSS, identifying key signal distributions and cell-specific genes within individual clusters. Further, our comparative analysis revealed changes in 49 signaling pathways across all cell clusters in the LSS– 4 exhibited decreased activity, whereas 45 were upregulated, suggesting that acne significantly alters cellular dynamics. We identified ten molecules, including GRN, IL-13RA1 and SDC1 that were consistently altered in all donors. Subsequently, we focused on the function of GRN and IL-13RA1 in TREM2 macrophages and keratinocytes as these cells participate in inflammation and hyperkeratinization in the early stages of acne development. We evaluated their function in TREM2 macrophages and the HaCaT cell line. We found that GRN increased the expression of proinflammatory cytokines and chemokines, including IL-18, CCL5, and CXCL2 in TREM2 macrophages. Additionally, the activation of IL-13RA1 by IL-13 in HaCaT cells promoted the dysregulation of genes associated with hyperkeratinization, including KRT17, KRT16, and FLG. These findings suggest that modulating the GRN-SORT1 and IL-13-IL-13RA1 signaling pathways could be a promising approach for developing new acne treatments.


Introduction
Acne vulgaris, the most common dermatological condition worldwide, presents as a chronic in ammatory and recurrent disease marked by a spectrum of lesions, including non-in amed (both open and closed comedones) and in amed (macules, papules, pustules, and nodules).Approximately 95% of boys and 85% of girls experience acne during adolescence, with nearly half of them experiencing it to adulthood 1 .The scarring and post-in ammatory hyperpigmentation caused by acne can severely affect an individual's quality of life, highlighting the importance of early and effective therapy.The development of acne is driven by four key processes within the pilosebaceous unit (PSU): in ammation, epithelial hyperkeratinization, hyperseborrhea accompanied by proin ammatory lipids, and colonization by Cutibacterium acnes (C.acnes) bacteria 1 .Despite signi cant progress in elucidating the pathophysiology and treatment mechanisms of acne, it is important to identify the shared dysregulated signaling pathways in individuals with acne.Targeting these shared signaling pathways can signi cantly improve the effectiveness of current acne treatments in affected patients.
Skin homeostasis relies on a sophisticated network of resident cells, each performing unique biological functions and engaging in complex signaling pathways mediated by intricate ligand-receptor interactions [2][3][4][5] .The epidermis hosts various cells such as keratinocytes, melanocytes, Langerhans cells and merkel cells.Among them, basal keratinocytes serve as epithelial stem cells, crucial for proliferation and differentiation, ensuring the daily renewal of the epidermis.In the dermis and hypodermis, a diverse array of cells including broblasts, immune cells, endothelial cells, nerves, and adipocytes form a harmonized network.Dysregulated signaling among these cells has been implicated in various skin disorders such as vitiligo, impaired wound healing, aging, psoriasis, and dermatitis [2][3][4][5] .In acne lesions, excessive squalene production by keratinocytes and sebocytes triggers TREM2 macrophage differentiation, enhancing immune cell migration and fueling the in ammatory cascade 6 .Moreover, sebocytes adjacent to the PSU in acne lesions release CXCL8, attracting neutrophils, monocytes, and T cells, in addition to secreting IL-6, TGF-β, and IL-1β, which drive the differentiation of T helper 17 cells (Th17 cells) 7 .Our previous studies demonstrated that C. acnes ribotypes differentially regulate the fate of Th17 responses in the skin 8,9 .However, the comprehensive and conserved changes in intercellular communication within acne-affected skin are yet to be thoroughly investigated.
In this study, we examined global changes in intercellular communication by analyzing single cell RNA sequencing (scRNA-seq) and spatial transcriptomic data from six patients with papular acne.Our ndings reveal that acne triggers signi cant alterations in 49 signaling pathways across all skin cell clusters compared to nonlesional areas.We also identi ed 10 genes related to these signaling pathways that were consistently dysregulated in all donors.Our focus was primarily drawn to genes that were enriched and upregulated in keratinocytes and immune cells, particularly myeloid cells and lymphocytes, as they play key roles in in ammation and hyperkeratinization during the onset of acne.Among these genes, we observed signi cant upregulation of GRN and IL-13RA1 within TREM2-expressing macrophages and basal keratinocytes, respectively.Furthermore, we found that GRN and its receptor SORT1 were upregulated in IL-4-induced TREM2 macrophages, and treatment with GRN led to increased expression of proin ammatory cytokines and chemokines from these cells.Concurrently, treating human HaCaT cells with IL-13 to activate IL-13RA1 signaling resulted in dysregulation of KRT16, KRT17, and FLG expression, which are associated with hyperproliferation-associated phenotypes in acne.Our ndings suggest that GRN and IL-13RA1 are key players in the in ammation and hyperkeratinization process during acne development, highlighting their potential as novel therapeutic targets for acne treatment.

Overall cell-cell communications and signal distributions in normal skin
To investigate the cell-cell communication in acne-affected skin, we rst sought to display the overall signal distributions in normal skin samples as a reference point.Although several studies have explored the cell-cell interactions in aging, wound healing, psoriasis, and dermatitis in both mice and humans, there remains a gap in understanding the overall signal distribution in distinct cell types in normal human skin, which can be used as a baseline to compare with various skin diseases 2,3  As every signal pathway consists of numerous ligand-receptor pairs, we further narrowed down our scope and focused on identifying key ligand and receptor genes that were not only from the strongest pathways but also exhibited unique expression within speci c cell clusters.To achieve this, we analyzed all ligand-receptor pairs and their relative contribution within each cluster (Figure S2-S9).Through this comprehensive approach, we identi ed speci c ligand and receptor genes by compiling them from the top three outgoing and incoming signals (    21,22 .In endothelial cells, we identi ed ligands (FLT1 23 , CCL14 24 , CSF3 25 ) and receptors (ACKR1 26 , LIFR 14 , TGFBR2 Acne triggers signi cant signaling pathway changes across all cell clusters within the skin. To elucidate the changes of cell-cell interactions from nonlesional to lesional samples, we initially integrated the datasets of nonlesional and lesional samples (Fig. 2A), and as demonstrated by Tran et al.
we also observed signi cant changes in cellular compositions, especially in KC2 and broblasts 6 (Fig. 2B).Further analysis of each signaling pathways revealed an increase in both the strength and number of signaling pathways in lesional compared to nonlesional samples (Figure S18A-S18B).We identi ed changes in 49 signal distributions: (i) one signal was turned off (MSTN), (ii) three signals were decreased (CCL, FLT3, NT), (iii) ten signals were turned on (IL-17, CX3C, TAC, NPR1, TWEAK, PROS, ANGPTL, GALECTIN, MK and SPP1), and (iv) thirty-ve signals were increased (including BAFF, NGF, WNT) (Fig. 2C).We found that these signal changes involved all cell clusters, indicating the possibility that immune responses within acne skin trigger responses across every cell type (Fig. 2D-2E).Of the ten signaling pathways that were turned on, IL-17, NPR1, GALECTIN and SPP1 mainly derived from myeloid cells and targeted KC2, endothelial cells, lymphocytes, and broblasts, respectively.The presence of IL-17 signaling in acne, is consistent with our previous ndings 36 .PROS and TWEAK signals originated from melanocytes and can target both smooth muscle and melanocytes.A case-controlled study of 100 acne vulgaris patients reported that acne patients had signi cant elevation in TWEAK serum levels when compared to the control subjects, which is consistent with our ndings 37 .TAC and CX3C signals interact in an autocrine way in endothelial cells and KC2.MK signals mainly from broblasts target melanocytes, whereas ANGPTL signals from KC1 target broblasts.These changes occurred across all cell types were further supported by the observed signi cant increase in the expression level of ligands and receptors associated with the ten turn-on signaling pathways in lesional samples of acne (Fig. 2F-2G).
Activation of GRN and IL-13RA1-related signals.
To account for the diversity in signaling pathway alterations observed across different donors and the impact of outliers, we aimed to identify signi cant differences in gene expression within each matched pair of nonlesional and lesional samples from six donors.We performed a differential analysis on all 232 genes associated with the 49 altered signaling pathways, these genes exhibited seven distinct expression pro les (Fig. 3A).Among them, 26 genes (11.2%) displayed signi cant differences between nonlesional and lesional samples within each patient (Fig. 3B and Figure S19).Conversely, 52 genes (22.4%) showed no differences across all six individuals, while 27 (11.6%),24 (10.3%),27 (11.6%),37 (16%), and 39 (16.8%) genes exhibited signi cant differences in 1, 2, 3, 4, and 5 matched nonlesional and lesional sample pairs, respectively (Fig. 3A).Previous studies have indicated that papules can form in under 6 hours and exhibit a profound in ammatory response as evidenced by increased levels of CD4 T cells, neutrophils, and CD68 + macrophages in acne biopsies.However, KC did not exhibit abnormal proliferation compared to normal skin at that time point 38 .Given that our samples were collected from patients at approximately 24 hours into the disease course, later than the 6 hour-mark, we focused on genes linked to signaling pathways in lymphocytes, myeloid cells, and basal cells in KC.These genes may be associated with in ammation and hyperkeratinization during this period.In these three cell types, only 5 genes (GRN, IL13RA1, IL4R, FAS and SDC1) showed consistent expression patterns across all matched pairs in all patients (Fig. 3C).Among these, GRN, also known as the granulin precursor and a multifunctional growth factor, has been identi ed in macrophages across various organs, including the lung and brain 39,40 .GRN plays a dual function in regulating in ammation and is associated with processes such as tumorigenesis, neurodegeneration, wound healing, and early embryogenesis.In our dataset, GRN primarily originates from myeloid cells including TREM2 macrophages, M1 and M2 macrophages, CD1C dendritic cells (DCs), Langerhans and LAMP3 DCs (Figure S20A-S20B).Notably, we observed higher expression of GRN in TREM2 macrophages and M2-like macrophages in lesional skin compared to nonlesional skin (Fig. 3D).Given that TREM2 macrophages have been implicated in driving in ammation in acne 6 , our subsequent analyses focused on the function of GRN within these cells.
IL-13RA1 was also upregulated in lesional skin, primarily originating from myeloid cells (Fig. 3C).However, it was either downregulated or showed no signi cant difference in subsets of myeloid cells, implying that increased expression levels of IL-13RA1 came from other cell types in lesional skin (Fig. 3E).Therefore, our focus shifted to the second largest source of IL-13RA1, which was KC1.Speci cally, we focused on basal cells from KC1 given their critical role in skin self-renewal and their signi cant involvement in hyperkeratinization within the epidermis.We found that IL-13RA1 was markedly upregulated in lesional basal cells compared to nonlesional ones (Fig. 3F, Figure S20C-S20D).This observation aligns with a study suggesting that IL-13, produced by group 2 innate lymphoid cells in the crypt niche, interacts with IL-13RA1 on Lgr5 + intestinal stem cells 41 , suggesting potential involvement of IL-13RA1 in hyperkeratinization during acne development.Additionally, SDC1 was highly expressed in KC1, but showed no signi cant difference in basal cells between the two conditions (Fig. 3G).IL-4R and FAS were predominantly expressed in lymphocytes; however further analysis revealed that neither IL-4R nor FAS showed signi cant changes in lymphocytes subsets (Fig. 3H and Figure S20E-S20F).Consequently, we chose not to investigate these three genes further.
Next, to spatially localize GRN and IL-13RA1 expression in acne skin, we used the Seq-Scope sequencing dataset obtained from acne lesions and segmented the histological area using 10 µm-sided square grids 6 .The analyzed specimen featured a hair follicle surrounded by an in ammatory in ltrate.Each grid detected an average of 145 genes across 3558 grids, enabling the identi cation of eight distinct cell populations including KRT5 and KRT16 keratinocytes, broblasts, endothelial cells, TREM2 macrophages, B cells, other macrophages, and various other cell types.(Fig. 4A).Our initial focus on GRN expression revealed its prominence in TREM2 macrophage where it co-localized with TREM2 macrophage marker, APOE, both in scRNA-seq and Seq-Scope dataset (Fig. 4B-4C).To identify the receptor for GRN, we analyzed the contribution of all ligand-receptor (L-R) pairs in GRN signaling.Our ndings revealed that only one receptor, Sortilin (SORT1), was detected and signi cantly upregulated in TREM2 macrophages.Furthermore, SORT1 was found to colocalize with GRN in acne lesions (Fig. 4D-4F).This L-R binding was rst identi ed in the brain, underscores SORT1's role in mediating rapid endocytosis and lysosomal localization of GRN, central in the development of inherited frontotemporal lobar degeneration 42 .Other studies have also shown that both GRN and SORT1 are key regulators of in ammation 43,44 .Our ndings showing GRN + cells also expressing SORT1 in lesions, suggest that the GRN-SORT1 axis functions in an autocrine manner within TREM2 macrophages.Additionally, IL-13RA1 was co-localized with basal KCs, marked by KRT14 and KRT5 45,46 , consistent with the scRNA-seq data (Fig. 4G-4H).Subsequent analysis of the relative contribution of each L-R pair revealed that both IL-4 and IL-13 ligands can interact with IL-13RA1 (Fig. 4I).Subsequently, IL-13RA1 may be regulated by IL-4 and IL-13 in the basal KCs of the skin.
Activation of GRN and IL-13RA1 exacerbates in ammation and hyperkeratinization both of which are critical in acne progression.
Next, to explore the function of GRN in TREM2 macrophages, we induced TREM2 macrophage differentiation in vitro using macrophage colony-stimulating factor (M-CSF) and IL-4 as previously described (Fig. 5A) 6, 47 .We observed that the combination of M-CSF/IL-4 induced higher TREM2 expression compared to M-CSF alone (Fig. 5B).Further analysis revealed that both GRN and SORT1 were upregulated in MCSF/IL-4-induced TREM2 macrophages, suggesting that GRN may play a signi cant role in TREM2 macrophages activation through its interaction with SORT1 (Fig. 5C).Tran et al. reported that TREM2 macrophages elicit a proin ammatory response by increasing the expression of proin ammatory cytokines and chemokines, such as IL-18, CCL5, and CXCL2 6 .To investigate the involvement of GRN in the proin ammatory activity of TREM2 macrophages, we treated these cells with recombinant GRN protein, which induced SORT1 expression (Fig. 5D).Our results demonstrated that treatment with 10 ng/ml of GRN activated the upregulation of SORT1.Intriguingly, higher concentrations of GRN did not enhance the SORT1 response, prompting the selection of 10 ng/ml of GRN as the optimal concentration for further studies (Fig. 5E).This treatment also elevated levels of proin ammatory cytokines (IL-18, CCL5, and CXCL2) known to activate the canonical in ammatory NF-kB pathway, recruiting T cells, mast cells, and natural killer cells, as well as promoting neutrophil in ltration 48-50 (Fig. 5F).These observations were corroborated by the colocalization of GRN + cells with IL-18, CCL5, and CXCL2expressing cells in acne lesions (Fig. 5G-5H).Additionally, we observed that GRN promotes the expression of proin ammatory cytokines (TNFA, IL-1B, and IL-6) in TREM2 macrophages (Fig. 5I), and the coexpression of TNFA and IL-1B can be found within GRN + cells (Figure S21A-S21B).Altogether, these data suggest that GRN ampli es the in ammatory response in TREM2 macrophages.
Hyperkeratinization, a key initial event in microcomedone formation, can be caused by anomalies in the differentiation, adhesion, and proliferation within the follicular infundibulum.Molecular markers such as KRT6, KRT16 and KRT17 are upregulated, whereas laggrin (FLG), a marker for keratinocyte differentiation, is downregulated in established microcomedones [51][52][53] .To investigate the role of IL-13RA1 in hyperkeratinization, we activated IL-13RA1 in the keratinocyte cell line (HaCaT) with its ligands IL-13 and IL-4.Our ndings revealed that compared to the control group, IL-13 treatment signi cantly upregulated the expression of both IL-13RA1 and IL-4R in HaCaT cells (Fig. 5J-5K).In contrast, IL-4 treatment either did not alter or downregulate IL-13RA1 and IL-4R expression (Figure S21C), indicating that only IL-13 activate IL-13RA1 in keratinocytes, which is consistent with the ndings in intestinal epithelial cells and bone marrow-derived macrophage 41 54 .Subsequent analysis showed that IL-13 treatment led to increased expression of KRT16 and KRT17, accompanied by reduced FLG expression, these gene expression patterns were consistent with our scRNA-seq data (Fig. 5L-5M).However, no signi cant change was observed in KRT6A expression (Figure S21D).Collectively, these data suggest that IL-13RA1 signaling may play a signi cant role in driving the dysregulation of genes related to hyperkeratinization, contributing to the development of acne in human skin.

Discussion
In our study, we rst investigated the distribution of signaling pathways within different cell types in both normal and acne skin.Through detailed analysis, we identi ed 49 signaling pathways that were altered in acne, along with genes showing consistent expression changes across all donors.Our subsequent focus centered on examining the roles of GRN in TREM2 macrophages and IL-13RA1 in keratinocyte basal cells given their consistent alterations across donors and potential importance in acne development.Using spatial-seq datasets, we con rmed the expression and colocalization of these genes with their respective cell types in acne samples.Further exploration of their functional roles in vitro revealed that GRN may exacerbate acne progression by enhancing in ammation in TREM2 macrophages, as demonstrated by its induction of in ammatory cytokines and chemokine expression.Conversely, the upregulation of IL-13RA1 in basal cells suggests its potential involvement in hyperkeratinization.We activated IL-13RA1 by IL-13 in the HaCaT cell line, which resulted in the dysregulation of genes associated with hyperkeratinization, further implicating its role in acne development.Together, our ndings shed light on the complex interplay between in ammation and hyperkeratinization in acne pathogenesis, while also highlighting GRN and IL-13RA1 as promising therapeutic targets for acne (Fig. 6).
The initial phase of acne is characterized by the presence of microcomedones, which progresses into papules, pustules, nodules, and cysts as the severity worsens.Studies have indicated the involvement of various innate and adaptive immune cells, including Th1 55 , Th17 56 , Foxp3 + , CD1 + , CD83 + DCs 57 , CD68 + macrophages, and activated mast cells in the early events, along with the secretion of proin ammatory cytokines and chemokines.Limited studies have comprehensively explored dysregulated signaling pathways in different skin cell clusters.In our study, we detected 49 altered signaling pathways encompassing 232 genes in lesional samples compared to nonlesional samples across all cell clusters.Subsequent analysis revealed that not all these genes exhibit signi cant changes in all donors.However, we identi ed 10 genes that were consistently dysregulated in all donors and speci cally expressed in lymphocytes, myeloid cells, keratinocytes, broblasts, and smooth muscle.Among these, Dahl et al. observed C3 presence at the dermo-epidermal junction in the majority of in ammatory acne lesions, contrasting with non-in ammatory samples 58 .This observation was further supported by Scott et al., who associated early complement activation with acne in ammation 59 , and these ndings also align with our results that broblast-derived C3 is upregulated in acne.The A > G polymorphism in the IL-4R gene has been associated with heightened allergic and immune-mediated disorders 60 .In a study by Robaee et al., a comparison of genetic polymorphisms in IL-4R between 95 acne patients and 87 unrelated healthy controls revealed a signi cant difference in IL-4R (Q551R A/G) genotypes between the two groups 61 , yet its role in acne remains unknown.In our data, IL-4R was mainly expressed in lymphocytes, but no signi cant difference was found in lymphocyte subsets, so further investigation is needed to understand its function in other cell types.Moreover, the function of the other genes, including FAS, SDC1, ANGPTL2, IL-15RA, INHBA, and OSMR in lymphocytes, keratinocytes, broblasts, and smooth muscle, are yet to be explored, suggesting that acne involves not only the skin's surface but also a wider systemic dysregulation.Future studies should focus on these genes to advance our understanding of acne pathogenesis.
Recent research has extensively investigated the speci c expression of GRN and TREM2 on microglia, the brain-resident macrophages, revealing their links to neurodegenerative disorders such as frontotemporal lobar degeneration and Alzheimer's disease 62,63 .However, Götzl et al. discovered that microglia isolated from GRN −/− mice exhibited a hyperactivated state of the neurodegenerative phenotype molecular signature and suppression of genes characteristic of homeostatic microglia.
Conversely, loss of TREM2 enhanced the expression of genes associated with a homeostatic state but reduced glucose metabolism in both conditions.This suggests that opposite microglial phenotypes lead to similar widespread brain dysfunction 64 .Our in vivo data initially revealed GRN expression predominantly in myeloid cells, with a signi cantly higher expression in TREM2 macrophages in lesional compared to nonlesional samples.Additionally, colocalization of GRN and TREM2 macrophages was observed in spatial-seq data.Further investigation detected higher expression of GRN in IL-4-induced TREM2 macrophages compared to non-IL-4-treated cells, indicating a strong correlation between GRN and TREM2 macrophages.
The role of GRN in in ammation is diverse, showing variability across different disease conditions, tissues, and even cell types.A wealth of evidence from in vitro and animal models suggests that GRN possesses anti-in ammatory properties.GRN competitively binds with TNFR1/2 to disrupt TNF-α function, which in turn leads to increased IL-10 production in T regulatory cells in conditions such as rheumatoid arthritis and in ammatory bowel disease 65,66 .Additionally, GRN can selectively inhibit the release of TNF-α and IFN-γ-induced CXCL9 and CXCL10 in CD4 + T cells 67,68 .However, the interaction between GRN and TNFR1/2 appears to be complex, with some studies suggesting that GRN does not bind to TNF receptors, thus not directly in uencing TNF signaling in various cell lines [69][70][71] .On the contrary, GRN can exhibit a pro-in ammatory effect by promoting the expression of proin ammatory cytokines such as IL-6 and IL-8 in different diseases such as psoriasis, obesity, and systemic lupus erythematosus [72][73][74][75][76] .These contradictory ndings suggest that GRN possesses characteristics of a double-edged sword in in ammation, acting both as a protector and provocateur depending on the condition.Our studies on the effect of recombinant GRN on TREM2 macrophages indicate that GRN activates its receptor SORT1 and promotes the expression of proin ammatory cytokines and chemokines from TREM2 macrophages, thereby activating downstream NF-kB signaling pathways [48][49][50] .
These ndings suggest that the proin ammatory function of TREM2 macrophages can be driven through the GRN-SORT1 axis.
IL-13RA1 serves as the receptor or coreceptor for IL-13 and IL-4, playing a critical role in type 2 immunity, which encompasses both host-protective and pathogenic functions 77 .Our data revealed that IL-13RA1 levels were either downregulated or remained unchanged in certain myeloid subsets, a trend contrary to that observed in whole-sample analyses.Therefore, we redirected our focus towards its role in keratinocytes and observed that IL-13RA1 was notably upregulated in basal cells.Previously, studies have demonstrated that IL-13 promotes the self-renewal of intestinal stem cells solely through IL-13RA1 but not IL-4R, underscoring the proliferative function of the IL13-IL13RA1 axis 41 .In the skin, IL-13 activation of IL-13RA1 disrupts the skin's barrier function and facilitates terminal differentiation by downregulating the expression levels of epidermal barrier proteins such as FLG, loricrin (LOR), and involucrin in primary human epidermal keratinocytes [78][79][80] .Our ndings align with these observations, as we discovered that IL-13 activation of IL-13RA1 resulted in the downregulation of FLG expression and upregulation of hyperproliferation-associated keratins KRT16 and KRT17 81,82 .Thus, our data suggests that the IL-13-IL-13RA1 axis signi cantly in uences keratinocyte proliferation and plays a key role in acne pathogenesis.Interestingly, TREM2 macrophages-recruited mast cell, NKT cell, T cell and neutrophils, all capable of secreting IL-13 83,84 , this connection bridges in ammation and hyperkeratinization processes in acne, indicating that in ammation precedes and triggers hyperkeratinization.Our data collectively suggest that in ammation and hyperkeratinization, driven by common dysregulated GRN and IL13RA1 may be pivotal in acne development.Targeting these pathways holds promise for more effective acne treatments.

PBMC and monocyte isolation
PBMCs were obtained from the blood of healthy donors after signed written informed consent as approved by the Institutional Review Board at UCLA following the Helsinki Guidelines.PBMCs were then isolated using Ficoll-Paque density gradients (GE Healthcare) as previously described 36 .Monocytes were isolated from PBMC by positive selection with CD14 MicroBead (Miltenyi Biotec, Cat#130-050-201), then seeded at 800,000 cells per well in 12-well plates.
RNA isolation, cDNA synthesis, and real-time PCR Total RNA was isolated using Trizol reagent (Thermo Fisher, Cat#15596018) following manufacturer's protocol.RNA samples were reverse transcribed to cDNA using Script Reverse Transcription Supermix (Bio-Rad, Cat#1708841).Reactions were performed at 25°C for 5 min, 46°C for 20 min and 95°C for 1 min.Real-time PCR was applied using SensiFAST SYBR & Fluorescein Kit (Thomas Scienti c, Cat#C755H99).40 cycles were carried out at 95°C for 5 min, then 95°C for 10 sec, 60°C for 12 sec, 72°C for 12 sec.GAPDH was used as a control.The gene expression level was quanti ed by the comparative method 2 −ΔΔCT .The primers used for gene assessment are summarized in Supplementary Table 2.

Data and code availability
For the scRNA-seq data, the sample processing and analysis for this dataset were described in a previous study 6 , and downstream analysis (Data visualization, clustering, cell type mapping, subsetting) was performed according to the Seurat tutorial series (https://satijalab.org/seurat/articles/visualization_vignette),and cell-cell interaction and comparison analysis according to the CellChat tutorial series (https://github.com/jinworks/CellChat).The sample processing and analysis for the Seq-Scope spatial dataset were conducted as previously described 6 85 .Brie y, this dataset included a 6 mm punch biopsy from a back acne papule.This sample was frozen in OCT medium and stored at -80°C until sectioning.For the Seq-Scope array, HISEQ2500 ow cells were used instead of the usual MISEQ ow cells.The distinctions between these two types of ow cells can be found in 6 .Published seq-scope datasets, step-by-step protocol, and data processing tools of Seq-ScopeMISEQ and Seq-ScopeHISEQ will be available at http://www.seq-scope.com and updated regularly.

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 9.0, with P values ≤ 0.05 were assigned as signi cant.For comparisons between two groups, an unpaired Student's t test with twotailed p-value analysis was performed, unless otherwise stated in the gure legend.

Study approval
This study was conducted according to the principles expressed in the Declaration of Helsinki.The study was approved by the UCLA IRB (no.22-000400).

Figure 1 Overall
Figure 1

Figure 6 A
Figure 6

Table
27 ), that are either known cell markers for endothelial cells or are involved in processes such as angiogenesis, migration, immune cell recruitment,