Hidradenitis Suppurativa and Comorbid Disorder Biomarkers, Druggable Genes, New Drugs and Drug Repurposing—A Molecular Meta-Analysis

Chronic inflammation and dysregulated epithelial differentiation, especially of hair follicle keratinocytes, have been suggested as the major pathogenetic pathways of hidradenitis suppurativa/acne inversa (HS). On the other hand, obesity and metabolic syndrome have additionally been considered as an important risk factor. With adalimumab, a drug has already been approved and numerous other compounds are in advanced-stage clinical studies. A systematic review was conducted to detect and corroborate HS pathogenetic mechanisms at the molecular level and identify HS molecular markers. The obtained data were used to confirm studied and off-label administered drugs and to identify additional compounds for drug repurposing. A robust, strongly associated group of HS biomarkers was detected. The triad of HS pathogenesis, namely upregulated inflammation, altered epithelial differentiation and dysregulated metabolism/hormone signaling was confirmed, the molecular association of HS with certain comorbid disorders, such as inflammatory bowel disease, arthritis, type I diabetes mellitus and lipids/atherosclerosis/adipogenesis was verified and common biomarkers were identified. The molecular suitability of compounds in clinical studies was confirmed and 31 potential HS repurposing drugs, among them 10 drugs already launched for other disorders, were detected. This systematic review provides evidence for the importance of molecular studies to advance the knowledge regarding pathogenesis, future treatment and biomarker-supported clinical course follow-up in HS.


Introduction
Hidradenitis suppurativa/acne inversa (HS) is a chronic, inflammatory, recurrent, debilitating skin disease of the hair follicle that usually presents after puberty with painful, deep-seated, inflamed lesions in the apocrine gland-bearing areas of the body, most commonly at the axillae, inguinal and anogenital regions [1]. A consistent finding, regardless of disease duration, is follicular hyperkeratosis, leading to follicular rupture, inflammation and possible secondary bacterial colonization. The deep part of the follicle appears to be involved. HS is further associated with an initial lymphohistiocytic inflammation, granulomatous reaction, sinus tract formation and scarring [2]. Current own transcriptome and proteome studies highlighted a panel of immunerelated drivers in HS, which induce an innate immunity response in epithelial skin cells in a targeted manner [3]. An inflammatory process coupled to impaired barrier function and bacterial activity were detected at the follicular and epidermal keratinocyte and at a minor

Drug Repurposing Sources
For drug repurposing, the detected overall HS molecular signature was compared with the drugs' molecular signatures of The Drug Repurposing Hub public domain, Eli and Edy L. Broad Institute, MIT and Harvard University (Cambridge, MA, USA) [24] and the Gene Cards, Weizmann Institute of Science (Rehovot, Israel) [25] public domains.

Study Selection Process
A total of 123 bibliographic records were identified after electronic database searches, 36 through other sources and six through bibliographic record citation search. Among them, 61 records were removed as duplicates, leaving 104 titles and abstracts to be screened. After careful screening and manual search, six records were excluded based on title and abstract and 49 records due to inappropriate design and two records due to overlapping data sets with another record, resulting in 47 studies that were included in the quantitative synthesis [3,4,11, (Figure 1).

Differentially Expressed Genes and Proteins in HS
The comparison of lesional skin vs. non-lesional skin as well as of blood of patients vs. controls at the mRNA and protein levels (cumulatively reported as "targets") without restrictions revealed 386 differentially expressed genes (DEGs) in HS (Table S1).

HS Biomarkers
DEGs and differentially expressed proteins in blood and involved skin of HS patients in comparison to controls in at least two relevant articles or two targets were defined as HS biomarkers. Among the 109 detected genes/proteins out of the 386 genes/proteins detected without restrictions, which fulfilled this requirement, 43 DEGs (including the coding genes of detected differentially expressed proteins) have been described in 2/4 targets in two articles, seven in 3/4 targets (CXCL10, IL6, IL17A, IL36A, IL36G, S100A8, S100A9) and none in all four targets (Table 1). Additional 10 DEGs have been described in 2/4 targets, however, in a diversified direction (upregulated/downregulated). Among the 109 HS biomarkers, 65 are druggable. S100A9) and none in all four targets (Table 1). Additional 10 DEGs have been described in 2/4 targets, however, in a diversified direction (upregulated/downregulated). Among the 109 HS biomarkers, 65 are druggable. Figure 1. Preferred reporting items for systematic reviews and meta-analyses (PRISMA 2020 [15]) flow diagram. Table 1. HS biomarkers resulting from the DEGs after transcriptomic profiling and protein expression studies between lesional HS and non-lesional skin biopsies and blood samples from HS patients and healthy controls, respectively and reported in at least two relevant articles. Bold letters indicate druggable genes. Background: white = similar results reported in one target (biological material) in at least two independent studies; orange = similar results reported in two targets in at least two independent studies; yellow = similar results reported in three targets in at least two independent studies. Gray = diversified result reported in at least two independent studies; + = upregulation; -= downregulation; +/-= diversified dysregulation in different studies; () = lower level of evidence.   Table 1. HS biomarkers resulting from the DEGs after transcriptomic profiling and protein expression studies between lesional HS and non-lesional skin biopsies and blood samples from HS patients and healthy controls, respectively and reported in at least two relevant articles. Bold letters indicate druggable genes. Background: white = similar results reported in one target (biological material) in at least two independent studies; orange = similar results reported in two targets in at least two independent studies; yellow = similar results reported in three targets in at least two independent studies. Gray = diversified result reported in at least two independent studies; + = upregulation; − = downregulation; +/− = diversified dysregulation in different studies; () = lower level of evidence.

Enrichment Analysis of HS-Associated Genes
The 386 detected HS-associated DEGs and the 109 HS biomarkers were enriched into relevant signaling pathways, which were assessed according to the g:Profiler [19], the KEGG GO, [20], the REAC [21], the WP [22] and the HP [23] public domains in order to identify the major organismal and signal transduction pathways involved in HS. Gene clustering in chromosome 2 and 4 was detected.
Among the 386 HS-associated DEGs, 101 genes were enriched in the cytokine-cytokine (C-C) receptor interaction pathway (−log 10         Epithelial differentiation signaling dysregulation in HS was represented by the epidermal growth factor receptor (EGFR), IL-1, IL-1 receptor, formation of the cornified envelope, TLRs and antimicrobial peptides ( Figure 5).
Metabolic/obesity-associated dysregulation in HS was detected through type I diabetes mellitus signaling, lipid and atherosclerosis, C-type leptin receptor signaling, estrogendependent nuclear events and extranuclear signaling, adipogenesis and resistin signaling ( Figure 5).
Interestingly, infection-indicating signaling pathways did not exhibit any major involvement in our study ( Figure 5).
At last, the REAC evaluation of globally involved pathways [70] revealed the innate immune system, the cytokine signaling in immune system (major pathways: regulation of IFNG signaling), signal transduction (nuclear receptor, GPCR and leptin pathways) and developmental biology (formation of the cornified envelope pathway) pathways as the mainly HS-associated ones ( Figure S2).
The protein-based connectivity map occurring from an assumed gene biomarker translation (103 proteins our of 109 genes) resulted in 2465 interactions compared with the expected 531 interactions (4.64-fold; p < 0.0001), a result that indicates a robust strong protein-protein association in HS ( Figure 6). On the other hand, the protein-based connectivity map occurring from the 386 HS-associated DEGs (372 proteins out of 386 genes) resulted in 19,823 interactions compared with the expected 6502 interactions (3.05-fold; p < 0.0001), indicating that the biomarker selection procedure increased the HS/protein association. Epithelial differentiation signaling dysregulation in HS was represented by the epidermal growth factor receptor (EGFR), IL-1, IL-1 receptor, formation of the cornified envelope, TLRs and antimicrobial peptides ( Figure 5).
Metabolic/obesity-associated dysregulation in HS was detected through type I diabetes mellitus signaling, lipid and atherosclerosis, C-type leptin receptor signaling, estrogen-dependent nuclear events and extranuclear signaling, adipogenesis and resistin signaling ( Figure 5).
Interestingly, infection-indicating signaling pathways did not exhibit any major involvement in our study ( Figure 5).
At last, the REAC evaluation of globally involved pathways [70] revealed the innate immune system, the cytokine signaling in immune system (major pathways: regulation of IFNG signaling), signal transduction (nuclear receptor, GPCR and leptin pathways) and developmental biology (formation of the cornified envelope pathway) pathways as the mainly HS-associated ones ( Figure S2).
The protein-based connectivity map occurring from an assumed gene biomarker translation (103 proteins our of 109 genes) resulted in 2465 interactions compared with the expected 531 interactions (4.64-fold; p < 0.0001), a result that indicates a robust strong protein-protein association in HS ( Figure 6). On the other hand, the protein-based connectivity map occurring from the 386 HS-associated DEGs (372 proteins out of 386 genes) resulted in 19823 interactions compared with the expected 6502 interactions (3.05fold; p < 0.0001), indicating that the biomarker selection procedure increased the HS/protein association.

Study Drugs and Drug Repurposing for HS
The majority of registered, studied or off-label administered drugs modify HS-associated DEGs. On the other hand, the evaluation of the detected 105 HS-associated druggable genes proposed 452 potentially therapeutic compounds, among them 120 launched drugs, 178 compounds in clinical studies and 154 in preclinical evaluation (Table S2). Among these potentially therapeutic compounds, the 31 drugs, which regulate three or more genes with all of them being HS-associated DEGs or at least four genes with 60% of them been DEGs were classified as probable repurposing drugs for HS (Table 2).

HS Pathogenesis
Inflammation doubtlessly plays a major role in the pathogenesis of HS [3,7,8]. Proteome studies provide evidence that the innate immunity system and both IL-1 and IL-17 signaling pathways are activated in HS lesions and circulating neutrophils [27,40,45,[71][72][73], findings that have been confirmed in our systematic review. In addition, Th17 differentiation of CD4+ lymphocytes is activated in HS [57]. Among others, Kelly et al. [38] provided evidence that CD45+CD4+ T cells are responsible for IL-17 production and CD11c+CD1a-CD14+ dendritic cells are the main producers of IL-1β in lesional HS skin. The IL-17 cytokine family has been linked to the pathogenesis of diverse autoimmune and inflammatory diseases and also plays an essential role in host defense against extracellular microorganisms [2,74]. IL-17 has been shown to increase the expression of skin antimicrobial peptides, including human β-defensin 2, psoriasin (S100A7) and calprotectin (S100A8/9) in keratinocytes and of a number of cytokines attracting neutrophils [75]. Thus, IL-17 may contribute to inflammation by increasing the influx of neutrophils, dendritic cells and memory T cells into the lesions. On the other hand, the involvement of IL-1 signaling pathway is also prominent in HS with upregulation of molecules causing immune cell infiltration and extracellular matrix degradation and could be reversed by application of IL-1 receptor antagonist [40,76]. IL1B signaling pathway-associated genes, such as IL1R1, IL1RN,  IFNG, IL6, IL18, IL18R1, IL32, IL33, IL36A, IL36B, IL36G, IL36RN, IL37, TLR2, TLR3, TLR4, S100A7, S100A7A, S100A8, S100A9 and S100A12 were HS-associated DEGs, as detected in our systemic review.
The inflammatory process in HS seems to be coupled with impaired barrier function, altered epidermal cell differentiation, formation of the cornified envelope, TLRs and antimicrobial peptides [3], the latter not being associated with any infection, as clearly shown in the present study. These events have been observed at the follicular and epidermal keratinocytes and at a minor grade at the skin glands [3]. Moreover, we could confirm a dysregulated expression pattern of serpins, small proline-rich proteins and certain keratins, which further support the involvement of the follicular infundibulum in the initiation of the lesions, especially at the anatomic area of communication with the apocrine gland duct and the ductus seboglandularis [3].
Although HS has well-documented associations with the metabolic syndrome, which is characterized by systemic inflammation identified at a molecular level [77], the role of adipose tissue in HS has barely been investigated. Obesity is currently shown to represent the primary risk factor in HS at the molecular level [4,28]. A chronic low-grade subclinical inflammatory response is strongly implicated in the pathogenesis of insulin resistance and metabolic syndrome. The clinically relevant peroxisome proliferator-activated receptor (PPAR) pathway was down-regulated in adipocytes of HS lesions [4]. In agreement with these data, reduced serum levels of adiponectin were currently found in non-diabetic patients with HS [28]. Since adiponectin inhibits the production of TNF-α, IL-6 and chemokines of human macrophages the upregulation of ADIPOQ and PLIN1, shown in this systematic review, might be beneficial in HS treatment. Indeed, thiazolidine derivatives act as PPARγ agonists and effectively increase the adiponectin concentration and adipogenic gene expression [28,78]. Unsaturated fatty acids, eicosanoids and non-steroidal anti-inflammatory drugs function in a similar manner [79]. Further metabolic pathways, e.g., the IGF transport and uptake of IGF-binding proteins pathway, type I diabetes mellitus signaling, lipid and atherosclerosis, C-type leptin receptor signaling, estrogen-dependent nuclear events and extranuclear signaling and RETN signaling, encoding resistin, are dysregulated in HS, as shown in the present review.
In conclusion, inflammatory signaling, mainly innate immunity signaling pathways, mostly that of IL-1 and IL-17, epithelial differentiation signaling pathways, primarily of follicular keratinocytes and skin gland duct cells and metabolic signaling pathways, especially that of obesity/adipogenesis, represent pathogenetic HS cascades, whose activity may be targeted by future therapeutic means.

HS Comorbid Disorders
HS has been associated with a variety of comorbid disorders, such as inflammatory bowel diseases, especially Crohn's disease, axial spondylarthritis without or with follicular occlusion, triad signs, genetic keratin disorders associated with follicular occlusion, such as pachyonychia congenita, steatocystoma multiplex, Dowling-Degos disease without and with arthritis, as well as other genetic disorders, such as keratitis-ichthyosis-deafness syndrome and Down syndrome [80]. Moreover, HS has been associated with reduced quality of life, metabolic syndrome, sexual dysfunction, working disability, depression and anxiety. Like in psoriasis, HS patients have higher prevalence of cardiovascular disease risk factors and suicide risk [81]. At last, the development of epithelial tumors on chronic HS lesions at the anogenital region may be considered as the consequence of chronic severe inflammatory skin disease. The current work has provided molecular evidence of HS association with inflammatory bowel disease pathway, rheumatoid arthritis pathway, type I diabetes mellitus signaling, lipid and atherosclerosis and adipogenesis signaling.

Conclusions
The current review provides robust molecular evidence on the pathogenetic triads of HS, namely upregulated inflammation, dysregulated epithelial cell differentiation and obesity signaling/hormone involvement. In addition, evidence of the negligible role of infectious agents is included. Moreover, HS biomarkers with strong protein-protein connectivity in HS are presented. While adalimumab, the only currently registered drug in HS, and the majority of studied and off-label administered drugs regulate DEGs and their proteins in HS, numerous compounds are eligible for HS repurposing due to their molecular signaling. Among them, 31 compounds are designated probable, following our classification, with 10 of them already being launched for other indications.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics14010044/s1: Figure S1: Enrichment of HS-associated DEGs in signaling pathways; Figure S2: Global REAC evaluation of possibly involved signaling pathways in HS; Figure  S3: Enrichment of druggable HS-associated genes in signaling pathways; Table S1: DEGs resulting from the comparison of transcriptomic profiles and protein expression studies between lesional HS and non-lesional skin biopsies and blood samples from HS patients and healthy controls, respectively; Table S2. Drugs regulating HS-associated DEGs.
Funding: This research received no external funding.
Institutional Review Board Statement: Ethical review and approval were waived because the article reviews ethically approved published studies involving humans.