Abstract

   Keloids are disfiguring, fibroproliferative growths and their
   pathogenesis remains unclear, inhibiting therapeutic development.
   Available treatment options have limited efficacy and harbor safety
   concerns. Thus, there is a great need to clarify keloid pathomechanisms
   that may lead to novel treatments. In this study, we aimed to elucidate
   the profile of lesional and non-lesional keloid skin compared to normal
   skin. We performed gene (RNAseq, qRT-PCR) and protein
   (immunohistochemistry) expression analyses on biopsy specimens obtained
   from lesional and non-lesional skin of African American (AA) keloid
   patients compared to healthy skin from AA controls. Fold-change≥2 and
   false-discovery rate (FDR)<0.05 was used to define significance. We
   found that lesional versus normal skin showed significant up-regulation
   of markers of T-cell activation/migration (ICOS, CCR7), Th2- (IL-4R,
   CCL11, TNFSF4/OX40L), Th1- (CXCL9/CXCL10/CXCL11), Th17/Th22- (CCL20,
   S100As) pathways, and JAK/STAT-signaling (JAK3) (false-discovery rate
   [FDR]<0.05). Non-lesional skin also exhibited similar trends. We
   observed increased cellular infiltrates in keloid tissues, including
   T-cells, dendritic cells, mast cells, as well as greater IL-4rα^+,
   CCR9^+, and periostin^+ immunostaining. In sum, comprehensive molecular
   profiling demonstrated that both lesional and non-lesional skin show
   significant immune alternations, and particularly Th2 and JAK3
   expression. This advocates for the investigation of novel treatments
   targeting the Th2 axis and/or JAK/STAT-signaling in keloid patients.

   Keywords: RNA-seq, keloids, immune, Th2, JAK3, inflammation

Introduction

   Keloids are common, benign fibroproliferative overgrowths that manifest
   as exophytic, hyperpigmented lesions, extending beyond the original
   wound and result from dysregulated wound-healing secondary to skin
   injury. Keloids may recur, are associated with pain and pruritus, and
   are disfiguring ([41]1). Numerous pathophysiologic processes have been
   investigated ([42]1–[43]3), including hypoxic ([44]4, [45]5),
   mechanical tension ([46]6, [47]7), and inflammatory etiologies
   ([48]8–[49]12). In addition, genetic studies have identified certain
   susceptibility loci, including inflammation-related genes, to be
   significantly associated with keloids ([50]13–[51]18). Yet, the
   pathogenesis still remains unclear, inhibiting therapeutic development.
   Moreover, current treatment options have limited efficacy and safety
   profiles and are associated with high recurrence rates of up to 100%
   ([52]2, [53]19). Thus, there is a large unmet need to understand the
   pathomechanisms of keloids and identify novel therapeutics.

   Recently, our group suggested a T-helper (Th)2-skewed pathogenesis of
   keloids by way of targeting Interleukin(IL)-4Rα with dupilumab, an
   FDA-approved biologic for moderate-to-severe atopic dermatitis (AD)
   ([54]20). In this report, treatment of severe AD in an African American
   patient resulted in both AD improvement and shrinkage of his
   concomitant keloids ([55]20). Furthermore, a limited analysis
   demonstrated increased mRNA expression of three Th2-related products
   ([56]20), suggesting a possible Th2 involvement in mechanisms
   underlying pathologic fibrosis ([57]21, [58]22). Clinical associations
   between keloids and other Th2-skewed diseases (e.g. AD ([59]23) and
   asthma ([60]24)) have also been reported. However, to our knowledge, a
   transcriptomic profiling of keloid skin lesions, that attempts to
   elucidate their immune alterations is lacking.

   While a few gene expression studies have been performed, the majority
   focused on specific genes or limited microarray studies
   ([61]25–[62]32). Furthermore, these studies did not entirely
   characterize the inflammatory profile of keloids. Earlier cDNA
   microarray analyses primarily associated keloids with alterations of
   the extracellular matrix, growth factors, apoptosis-related molecules,
   and/or chondrogenic or osteogenic tissue differentiation
   ([63]25–[64]28). A more recent microarray profiling study analyzed
   differences in lesional versus non-lesional skin and reported bone
   and/or cartilage abnormalities but did not evaluate inflammatory
   pathways ([65]29). Jumper et al. ([66]30) used a laser capture
   micro-dissection approach, describing dysregulation of a few immune
   markers in keloid tissues (e.g. IL-13Rα1, IL-1β). Onoufriadis et al.
   ([67]31) undertook an integrative mRNA and miRNA expression approach,
   focusing on pathway enrichment analysis and highlighting expression of
   mitogen-activated protein-kinase signaling pathway in keloid-prone
   individuals.

   Due to a greater prevalence of keloids in individuals of African
   descent ([68]19), we present a global RNA-seq profiling of skin
   biopsies obtained from both lesional and non-lesional skin of African
   American patients compared to healthy controls, complemented with
   additional validation using quantitative real-time PCR and
   immunohistochemistry. Our data show immune dysregulation, particularly
   Th2 and JAK3-skewing, along with Th1 and Th17/Th22 expression, in
   keloid lesions, extending to uninvolved skin, suggesting the potential
   for systemic therapies targeting these pathways in keloid patients.

Materials and Methods

Patient Characteristics and Sample Collection

   Three African American patients (three females, mean age 47.3 years),
   with history of severe chronic keloids and no concomitant atopy or
   other comorbidities ([69]29), and five healthy African American
   controls (two females, three males, mean age 39.8 years) were recruited
   under institutional review board-approved protocols. Biopsy specimens
   (6 mm) were collected from keloid lesions on the upper trunk.
   Non-lesional skin biopsies were obtained from a similar anatomic
   region, but at least 10 centimeters away from the keloid lesion. In one
   of the patients, a biopsy of an emerging keloid lesion was also
   obtained. Normal skin biopsy samples were collected from the trunk of
   healthy controls.

RNA-Sequencing

   RNA was extracted as previously described ([70]33, [71]34). Libraries
   were generated using TruSeq Stranded mRNA Library Prep kit (Illumina).
   Next generation sequencing was performed on Illumina NovaSeq6000
   (Illumina Inc., 100 cycles, single-read sequencing). Sample quality was
   assessed using FastQC. RNA-sequencing data was profiled by Illumina
   NovaSeq6000 to allow for more global analyses of genomic abnormalities.
   Data were pre-processed using standard pipeline incorporating quality
   control metrics, such as FastQC and MultiQC, sequence alignment based
   on STAR RNA-sequencing aligner, and sequencing reads assignment to
   genomic features by featureCounts and voom-transformed. A total of 4
   keloid lesions obtained from 3 different patients and 6 healthy
   controls were included in the RNA-sequencing analysis.

Quantitative Real-Time Polymerase Chain Reaction

   RNA was extracted for real-time polymerase chain reaction (RT-PCR)
   using the miRNAeasy Mini Kit (Qiagen, Hilden, Germany). Reverse
   transcription to complementary DNA (cDNA) from RNA was carried out
   using the High Capacity cDNA reverse transcription (Thermo Fisher).
   Pre-amplification was performed on all samples. Primers are listed in
   [72]Table S1 . Ten nanogram total RNA was used for PreAMP pool. Rplp0
   was used as endogenous control. Expression values were normalized
   to Rplp0.

Immunohistochemistry

   Immunohistochemistry was performed on frozen lesional and non-lesional
   skin sections from keloid patients and healthy skin from controls,
   using monoclonal antibodies as previously described ([73] Table S2 )
   ([74]35, [75]36). Cell counts were quantified from representative
   sections in both the epidermis and dermis using ImageJ V1.42 software
   (National Institutes of Health, Bethesda, Maryland).

Statistical Analysis

   Analyses were performed using R-language (R-project.org) and
   Bioconductor Project packages ([76]www.bioconductor.org). Gene
   expression profiles were modeled by linear models using R’s lme
   function. limma framework was used for RNA-sequencing data, and
   p-values were adjusted for multiple hypotheses using the
   Benjamini-Hochberg procedure. Fold changes (FCHs)>2.0 and false
   discovery rates (FDR)<0.05 were considered differentially expressed. We
   also evaluated a curated immune gene-subset ([77]33, [78]34, [79]37)
   using P-values, due to the small sample size. Mean expressions are
   displayed in a heatmap, where unsupervised clustering was performed
   using Euclidean distance and average agglomeration criteria. PCR and
   immunohistochemistry data were log2 transformed and analyses were
   performed using Student’s t-test.

Results

   We performed RNA-seq global profiling ([80]GSE158395), followed by
   qRT-PCR and immunohistochemistry, on lesional and non-lesional keloid
   skin samples obtained from three African American patients (mean age
   47.3 years) with history of chronic (present for >10 years), large
   keloids, and no atopy or other comorbidities ([81]29), and healthy skin
   samples obtained from five African American controls (mean age 39.8
   years). In one of the patients, a biopsy of an emerging keloid lesion
   was also obtained. Patients did not have any treatments on their
   biopsied keloids. No significant differences in age or gender were
   detected between patients and controls.

RNA-Seq Molecular Profiling Shows Immune Dysregulation With Significant Th2,
Th1, Th17/Th22, and JAK3-Skewing in Keloid Lesions

   Using criteria of fold-change (FCH)>2 and false discovery rate
   (FDR)<0.05 to define differentially expressed genes (DEGs), we detected
   3,044 DEGs (1,437 up-regulated and 1,607 down-regulated) in lesional
   keloid versus normal skin, 2,929 DEGs between lesional and non-lesional
   skin, and few DEGs between non-lesional and normal skin, as depicted in
   a heatmap in [82]Figure S1 ([83] Table S3 ).

   The top 50 DEGs in lesional versus normal skin comparison included
   products related to fibrosis and cartilage/bone-differentiation
   (ADAM12, ASPN, COL10A1, COL11A1, FBN2), previously reported to be
   dysregulated in keloids ([84] Table S3 ) ([85]20, [86]29). Periostin,
   implicated both in fibrosis as well as in Th2 inflammation
   ([87]38–[88]40), was among the top DEGs in lesional versus normal skin
   (FDR<0.05 for all; [89]Figure 1 , [90]Table S3 ).

Figure 1.

   [91]Figure 1
   [92]Open in a new tab

   Summary heatmap of immune gene expression of multiple pathways in
   biopsied lesional and non-lesional keloid skin and normal skin from
   controls using RNA-seq. Table shows fold-changes in non-lesional versus
   normal (NL vs N), lesional versus normal (LS vs N), and lesional vs
   non-lesional (LS vs NL) skin. ***P < 0.001, **P < 0.01, *P < 0.05, ^+P
   < 0.1. Red color denotes higher mean expression levels and blue color
   denotes lower mean expression levels. LS, lesional; NL, non-lesional;
   N, normal.

   Among the significantly modulated genes in keloid lesions compared to
   normal skin also included markers related to various immune components,
   as depicted in a heatmap of a curated immune gene-subset ([93] Figure 1
   ) ([94]33, [95]34, [96]37). In keloid lesions, we observed significant
   increases in measures of T-cells (CD2, CD3, CD28),
   T-cells/NK-cells/T-cell activation and migration (ICOS, LCK, GZMA/B,
   CCR7, TNFRSF9), and cellular infiltrates, such as dendritic cell
   markers CD80 and CD86 (FDR<0.05; [97]Figure 1 , [98]Table S3 ).
   Measures of innate immunity (IL-6), and multiple immune pathways,
   including the Th2- (IL-4R, CCR5, CCL11, TNFSF4/OX40L), Th1-
   (CXCL9/CXCL10/CXCL11, OASL), and Th17/Th22- (CCL20, PI3, S100A7/8/9)
   pathways were also significantly up-regulated (FDR<0.05; [99]Figure 1 ,
   [100]Table S3 ).

   Furthermore, keloid lesions displayed significant up-regulation of
   JAK/STAT signaling molecule, JAK3, as compared to normal skin
   (FDR<0.05; [101]Figure 1 , [102]Table S3 ). Markers related not only to
   fibrosis but also Tregs (TGFβ1 and TGFβ3) ([103]41, [104]42), were
   highly increased as well (FDR<0.05). Both CCR9, the homing receptor for
   the small intestine and lung ([105]43, [106]44), and its ligand CCL25,
   displayed increased expression in keloid tissues (FDR ≤ 0.1). The
   CCR9/CCL25 axis has been shown to induce cellular recruitment in early
   allergic asthma ([107]44). Negative immune regulators, IL-34 ([108]45)
   and IL-37 ([109]34), were significantly reduced in lesional keloid
   versus normal skin (FDR<0.05; [110]Figure 1 , [111]Table S3 ).

   Similar dysregulation of these immune axes was observed between
   non-lesional and normal skin comparisons, albeit these did not attain
   significance by FDR, however some achieved significance using criteria
   of P<0.05 (e.g. CCL11, CCR5, CXCL10/11, CCL20) ([112] Figure 1 ,
   [113]Table S3 ).

qRT-PCR Validates and Expands RNA-Seq Data

   qRT-PCR was performed to validate as well as evaluate key immune
   molecules that are often below detection limits on RNA-seq ([114]34,
   [115]46) ([116] Figure 2 ). Th2 (CCL11, TSLP, TNFSF4/OX40L,
   TNFRSF4/OX40) markers showed significant up-regulation in lesional or
   non-lesional versus normal skin (P<0.05; [117]Figure 2 ). Expression of
   CCR9 and its ligand CCL25 showed significant, or trending toward
   significant, up-regulation in lesional versus normal skin (P<0.05 for
   CCL25; [118]Figure 2 ). JAK3 was also highly expressed in keloid
   lesions compared to normal skin (P<0.01; [119]Figure 2 ).

Figure 2.

   [120]Figure 2
   [121]Open in a new tab

   Fold-changes of immune mediators in lesional and non-lesional skin of
   keloid patients, as well as normal skin, as measured by quantitative
   real-time PCR. Red bar represents mean. Black symbols: significance of
   comparison to normal skin; red symbols: significance of comparison
   between lesional and non-lesional skin. **P < 0.01, *P <0.05, ^+ P <
   0.1. PCR, polymerase chain reaction; LS, lesional; NL, non-lesional; N,
   normal.

   While the main Th1 cytokine, IFNγ, showed significant increases in
   keloid lesions (P<0.01), IL-17A, the Th17 cytokine, only showed a trend
   for increased expression in both lesional and non-lesional versus
   normal skin ([122] Figure 2 ). Treg markers, FOXP3 and TGFβ1, and
   T-cell activation measure ICOS, were up-regulated in lesional keloid
   versus normal skin (P<0.05 for TGFβ1; [123]Figure 2 ). We also
   evaluated an emerging keloid lesion and obtained similar gene
   expression results ([124] Figure S2 ).

Immunohistochemistry Shows Marked Cellular Infiltrates in Keloids

   Gradual increases were observed for multiple cellular infiltrate
   measures, such as T-cells (CD3^+, CD8^+) and dendritic cells (CD11c^+),
   including dendritic cell infiltrates that are characteristic to AD,
   such as markers typifying “atopic” dendritic cells (OX40L^+, FCεR1^+)
   ([125]33, [126]47, [127]48) (P<0.05 for CD3^+, FCεR1^+, OX40L^+ in
   lesional versus normal skin comparisons; [128]Figures 3A–T ). OX40L^+
   immunostaining was also significantly increased in non-lesional keloid
   versus normal skin (P<0.05; [129]Figures 3Q–T ).

Figure 3.

   [130]Figure 3
   [131]Open in a new tab

   Representative immunohistochemistry images and corresponding cell count
   quantification of CD3^+ T-cells (A–D), CD8^+ T-Cells (E–H), CD11c^+
   dendritic cells (I–L), FCεR1^+ dendritic cells (M–P) and OX40L^+
   dendritic cells (Q–T) in normal skin from controls, and lesional and
   non-lesional skin of keloid patients, viewed at 10x magnification. Red
   bar represents mean. Black symbols: significance of comparison to
   normal; red symbols: significance of comparison between lesional and
   non-lesional skin. **P < 0.01, *P < 0.05, ^+ P < 0.1. LS, lesional; NL,
   non-lesional; N, normal.

   Supporting our mRNA expression data, we noted increased IL-4Rα^+
   immunostaining in both lesional and non-lesional keloid skin as
   compared to normal skin (P<0.05 for lesional versus non-lesional skin
   comparison; [132]Figures 4A–D ). Mast cells (tryptase^+), associated
   with atopy ([133]49, [134]50), were increased in keloid lesions (P<0.1
   for lesional versus non-lesional comparison; [135]Figures 4E–H ).
   Trending toward significantly greater CCR9^+ immunostaining was noted
   in lesional compared to normal skin (P<0.1 [136]Figures 4I–L ).
   Finally, we observed significantly greater periostin immunostaining,
   concentrating in the lower dermis of keloid lesions, as compared to
   non-lesional and normal skin (P<0.05; [137]Figures 4M–P ). We also
   evaluated an emerging keloid lesion and obtained similar cellular
   infiltrates ([138] Figure S3 ).

Figure 4.

   [139]Figure 4
   [140]Open in a new tab

   Representative immunohistochemistry images and corresponding cell count
   quantification of IL-4Rα^+ cells (A–D), tryptase^+ mast cells (E–H),
   CCR9^+ cells (I–L) and periostin^+ cells (M–P) in normal skin from
   controls, and lesional and non-lesional skin of keloid patients, viewed
   at 10x magnification. Red bar represents mean. Black symbols:
   significance of comparison to normal; red symbols: significance of
   comparison between lesional and non-lesional skin. **P < 0.01, *P <
   0.05, ^+ P < 0.1. LS, lesional; NL, non-lesional; N, normal.

Discussion

   Our study combines gene and protein expression analyses, broadly
   characterizing the molecular and cellular phenotype of lesional and
   non-lesional keloid skin, highlighting the possible contribution of
   immune abnormalities to its pathogenesis. While various etiologies for
   keloid formation have been investigated, the pathogenesis remains to be
   clearly elucidated, hindering the development of novel therapeutics.
   Global RNA-seq profiling, as successfully performed in other
   inflammatory skin diseases, such as AD and psoriasis ([141]34,
   [142]51), may uncover further insights into disease mechanisms, and
   possibly lead to the development of newer, immune-based treatments.

   In light of the role of inflammation in normal wound healing, a
   dysregulated immune component can contribute to an abnormal wound
   repair process ([143]8, [144]9, [145]52). However, to our knowledge,
   the role of inflammation in the pathogenesis of keloids has not been
   adequately investigated. Published studies investigating an immune
   component have largely focused on specific keloidal cell cultures or
   specific genes. One report identified up-regulation of the
   proinflammatory cytokines, IL-1α, IL-1β, IL-6, and TNFα, in keloid
   fibroblasts ([146]10). Our group recently examined a few Th2 markers in
   keloids ([147]20), while another study found increases for the
   IL-17/IL-22-induced products, S100A7 and hBD2 ([148]53). Additionally,
   dysregulation of the IL-17/IL-6 axis ([149]11) and/or
   JAK/STAT-signaling ([150]54, [151]55) have also been investigated. The
   present study expands on prior investigations and provides a
   comprehensive molecular profiling of keloids, identifying a significant
   immune component in both lesional and non-lesional skin, as well as
   linking keloids with fibrosis and cartilage/bone-differentiation.

   Our results associate keloid tissues with an inflammatory milieu
   representing multiple T-helper pathways, including the Th2 (e.g. IL-4R,
   CCL11, TSLP, TNFSF4/OX40L, TNFRSF4/OX40), Th1 (e.g. IFNγ, CXCL10/11),
   Th17/Th22 (e.g. PI3, CCL20, S100As) axes, as well as the JAK/STAT
   signaling molecule JAK3. The up-regulation of Th17 markers expands upon
   prior reports demonstrating a role of Th17 mediators in keloids
   ([152]11, [153]55). Additionally, our results suggest that, similar to
   other fibrotic diseases ([154]56), the Th1 axis is also involved to
   keloid pathogenesis. Of note, IL-6 which is associated with innate
   immunity ([155]57), is highly up-regulated in our study and has also
   been shown to drive a chronic pro-fibrotic state via a Th1-mediated
   response ([156]58).

   In the present study, we observed a significant Th2 signature in
   keloids as well as increased cellular infiltrates associated with the
   Th2 microenvironment, such as tryptase^+ mast cells ([157]12, [158]49,
   [159]50), markers of dendritic cells characteristic to atopic
   dermatitis (AD) (OX40L^+ and FCεR1^+) ([160]33, [161]47, [162]48),
   IL-4Rα^+ cells, and periostin^+ cells. In human fibroblasts, it has
   been found that IL-4 and IL-13 increased TGF-β signaling and enhanced
   fibrosis via periostin ([163]59). Periostin is a matricellular protein
   that plays important physiologic and pathogenic roles in skin fibrosis
   ([164]60, [165]61) and serves as a biomarker for several known
   Th2-associated diseases (e.g. AD, asthma, nasal polyps, systemic
   sclerosis) ([166]38, [167]39, [168]62, [169]63). Its significantly
   increased upregulation in keloids may represent an important link
   between immune responses and fibrosis. In fact, one study found
   periostin to be expressed by a novel subpopulation of Th2-associated
   fibroblasts in AD ([170]64). These data implicate a cross-talk between
   Th2 immune mechanisms and fibrosis that is also likely relevant in
   keloid pathogenesis.

   Further reinforcing the concept of immune dysregulation in driving
   fibrotic processes in keloids is the increased T-cell expression,
   including Th2-related markers, in several fibrotic conditions, such as
   frontal fibrosing alopecia and scleroderma ([171]65, [172]66). In vitro
   studies have also demonstrated the profibrotic effects of the type 2
   cytokines IL-4 and IL-13 ([173]22), including increased collagen
   production in keloid fibroblasts after IL-13 stimulation ([174]67).
   These studies strengthen our hypothesis that immune dysregulation,
   possibly Th2 driven, plays a significant role in keloid formation.
   Additionally, we found increased expression of CCR9 and its receptor
   CCL25, which are involved in inflammatory cell recruitment in early
   allergic asthma ([175]44). Taken together, our results highlight
   commonalities between keloids and other atopic disorders such as AD and
   asthma ([176]68), as well as those that exhibit exophytic growth (e.g.
   nasal polyps) ([177]69, [178]70). Keloids also display commonalities
   with connective tissue diseases associated with excessive extracellular
   matrix deposition, such as scleroderma ([179]65).

   The up-regulation of T-cell related genes in non-lesional skin compared
   to normal skin (e.g. CCL11, CCR5) highlight that the seemingly normal
   skin of keloid patients is in fact abnormal and predisposed to
   formation of keloids. These data are also supported by the clinical
   observation that individuals who develop keloids are prone to future
   keloid formation after skin wounding ([180]19). These incipient
   findings in non-lesional skin are important as it might mean that
   patients with significant keloids may necessitate systemic treatment to
   target not only lesional but also non-lesional skin to prevent future
   recurrence of keloids.

   While studies in blood can clarify the presence of systemic activation
   in keloid patients, our results, which demonstrate immune dysregulation
   extending beyond skin lesions to uninvolved skin, advocate for the
   investigation of systemic treatments for keloids, such as those
   targeting the Th2 axis. The potential clinical utility of anti-IL-4Rα
   antagonism ([181]20) is encouraged by the significant overlap observed
   between dupilumab-regulated markers in AD and those that are
   dysregulated in keloids, including markers of the Th2 axis, as well as
   cellular infiltrates (CD3^+, CD11c^+, FCεR1^+), T-cell
   activation/migration (GZMB, ICOS, CCR7), and the Th17/Th22 (PI3,
   S100As) pathway ([182]37, [183]71). Antagonism of the OX40-TSLP axis,
   which is known to perpetuate Th2 activation in both AD and asthma
   ([184]72–[185]74), could also be explored for keloids. GBR830 and
   KHK4083 are anti-OX40 monoclonal antibodies in clinical trials
   ([186]75, [187]76). In one analysis ([188]75), GBR830 significantly
   decreased measures that are also upregulated in keloid tissues in the
   present study, including OX40^+ T-cells, OX40L^+ dendritic cells, and
   Th2- (CCL11), Th1- (IFNγ, CXCL10), and Th17/Th22- (S100A9/12) related
   markers. Given the significantly increased expression of TSLP in
   non-lesional keloid skin, exploration of tezepelumab, an anti-TSLP
   monoclonal antibody ([189]77), may also be warranted.

   Furthermore, biologics targeting IL-6 signaling, such as tocilizumab,
   sarilumab, and siltuximab, could be explored ([190]78). Indeed, our
   data showing significant IL-6 up-regulation in keloids expands on
   previous studies that demonstrated elevated IL-6 expression in keloidal
   fibroblasts ([191]79, [192]80). Ghazizadeh et al. ([193]80) further
   showed that inhibition of IL-6 or IL-6Rα in keloid fibroblasts resulted
   in a reduction of collagen synthesis, underscoring the potential
   utility of IL-6 antagonism for this disease.

   In contrast to the aforementioned biologics, Janus kinase (JAK)
   inhibitors comprise a class of broad-acting small molecules. The
   JAK/STAT signaling and spleen tyrosine kinase (SYK) pathways are
   implicated in numerous autoimmune and inflammatory diseases (e.g. AD,
   psoriasis, alopecia areata) ([194]81), modulating a range of immune
   responses, including the Th2 (IL-4, IL-13, CCL18), Th1 (IFNγ), and
   Th17/Th22 (CCL20, S100As) pathways ([195]82). Preliminary studies
   evaluating inhibition of JAK/STAT signaling in human keloid fibroblasts
   ([196]54, [197]55), and in a humanized keloid animal model ([198]83),
   have demonstrated promising results. There are several JAK inhibitors
   in clinical development showing efficacy for various dermatologic
   disorders ([199]81, [200]82, [201]84) and could be a therapeutic
   strategy worth investigating in keloids.

   This is a preliminary study with a few limitations, including a small
   sample size. While future studies need to evaluate larger cohorts, we
   were still able to obtain large differences in lesional and
   non-lesional skin versus normal skin, as well as a significant signal
   after adjusting for multiple hypothesis testing by false-discovery rate
   using a stringent cut-off for significance. Our study group only
   included African American patients, representing a greater prevalence
   of keloids in this ethnicity ([202]1, [203]19). Additional studies
   should evaluate keloids in other races and ethnicities, such as in
   Hispanics, Asians, and Caucasians ([204]1, [205]19). Finally, as our
   study raises the hypothesis of possible immune-based treatments for
   keloids, this needs to be proven in future proof of concept studies and
   clinical trials.

   In sum, the current genomic and cellular profiling study of keloids
   provides a comprehensive molecular fingerprint of the immune
   alterations in lesional and non-lesional keloid skin compared to normal
   skin. Our results reveal broad immune dysregulation, including
   up-regulation of markers of the Th1 and Th17/Th22 axes as well as
   significant Th2 and JAK3 expression in keloid lesions, extending to
   uninvolved skin, suggesting the potential use of systemic therapeutics.
   Future clinical trials for keloid patients may include those targeting
   the Th2 axis, inhibitors of the TSLP-OX40 axis, antagonists of
   JAK/STAT-signaling, or IL-6 inhibition.

Data Availability Statement

   The original contributions presented in the study are publicly
   available. This data can be found here:
   [206]https://www.ncbi.nlm.nih.gov/geo with accession number
   [207]GSE158395.

Ethics Statement

   The studies involving human participants were reviewed and approved by
   The Rockefeller University IRB. The patients/participants provided
   their written informed consent to participate in this study.

Author Contributions

   JW contributed to the study design, data analysis, data interpretation,
   and writing the manuscript. ED, ME, and AG contributed to the histology
   stainings, data interpretation, and writing the manuscript. IC, NZ, and
   YE contributed to the experiments. AP contributed to the study design,
   data analysis, data interpretation and writing the manuscript. JK
   edited and improved the manuscript. EG-Y contributed to the study and
   experimental design, data interpretation, and writing the manuscript.
   All authors contributed to the article and approved the submitted
   version.

Conflict of Interest

   EG-Y is an employee of Mount Sinai and has received research funds
   (grants paid to the institution) from: Abbvie, Celgene, Eli Lilly,
   Janssen, Medimmune/Astra Zeneca, Novartis, Pfizer, Regeneron, Vitae,
   Glenmark, Galderma, Asana, Innovaderm, Dermira, UCB. EG-Y is also a
   consultant for Sanofi Aventis, Regeneron, Stiefel/GlaxoSmithKline,
   MedImmune, Celgene, Anacor, AnaptysBio, Dermira, Galderma, Glenmark,
   Novartis, Pfizer, Vitae, Leo Pharma, Abbvie, Eli Lilly, Kyowa,
   Mitsubishi Tanabe, Asana Biosciences, and Promius. JK has received
   research support (grants paid to his institution) and/or personal fees
   from Pfizer, Amgen, Janssen, Lilly, Merck, Novartis, Kadmon, Dermira,
   Boehringer, Innovaderm, Kyowa, BMS, Serono, BiogenIdec, Delenex,
   AbbVie, Sanofi, Baxter, Paraxel, Xenoport, and Kineta. AP, NZ, and ED
   are employees of Mount Sinai.

   The remaining authors declare that the research was conducted in the
   absence of any commercial or financial relationships that could be
   construed as a potential conflict of interest.

Supplementary Material

   The Supplementary Material for this article can be found online at:
   [208]https://www.frontiersin.org/articles/10.3389/fimmu.2020.597741/ful
   l#supplementary-material
   [209]Click here for additional data file.^ (3MB, csv)
   [210]Click here for additional data file.^ (21.8KB, docx)
   [211]Click here for additional data file.^ (14.6KB, docx)
   [212]Click here for additional data file.^ (13.2KB, docx)
   [213]Click here for additional data file.^ (722.7KB, jpeg)
   [214]Click here for additional data file.^ (10.2MB, jpeg)
   [215]Click here for additional data file.^ (10.3MB, jpeg)

Abbreviations

   AD, atopic dermatitis; DC, dendritic cell; FCH, fold-changes; FDR,
   false discovery rate; IL, interleukin; qRT-PCR, quantitative real time
   polymerase chain reaction; RNA-seq, RNA-sequencing; Th, T-helper; Treg,
   T-regulatory cell.

References