Serum Beta-Defensin-2 is a biomarker for psoriasis but not subclinical atherosclerosis: Role of IL17a, PI-3 kinase and Rac1

Abstract

Background

Beta-defensins (BDs) are antimicrobial peptides secreted upon epithelial injury. Both chemotactic and antimicrobial properties of BDs function as initial steps in host defense and prime the adaptive immune system in the body. Psoriasis, a chronic immune-mediated inflammatory disease, has both visible cutaneous manifestations as well as known associations with higher incidence of cardiometabolic complications and vascular inflammation.

Objectives

We aimed to investigate the circulating expression of beta-defensin-2 (BD2) in psoriasis at baseline compared to control subjects, along with changes in BD2 levels following biologic treatment at one-year. The contribution of BD2 to subclinical atherosclerosis is also assessed. In addition, we have sought to unravel signaling mechanisms linking inflammation with BD2 expression.

Methods

Multimodality imaging as well inflammatory biomarker assays were performed in biologic naïve psoriasis (n=71) and non-psoriasis (n=53) subjects. A subset of psoriasis patients were followed for one-year after biological intervention (anti-Tumor Necrosis Factor-α (TNFα), n=30; anti-Interleukin17A (IL17A), n=21). Measurements of circulating BD2 were completed by Enzyme-Linked Immunosorbent Assay (ELISA). Using HaCaT transformed keratinocytes, expression of BD2 upon cytokine treatment was assessed by quantitative polymerase chain reaction (qPCR) and ELISA.

Results

Herein, we confirm that human circulating BD2 levels associate with psoriasis, which attenuate upon biologic interventions (anti-TNFα, anti-IL-17A). A link between circulating BD2 and sub-clinical atherosclerosis markers was not observed. Furthermore, we demonstrate that IL-17A-driven BD2 expression occurs in a Phosphatidylinositol 3-kinase (PI3-kinase) and Rac1 GTPase-dependent manner.

Conclusions

Our findings expand on the potential role of BD2 as a tractable biomarker in psoriasis patients and describes the role of an IL-17A-PI3-kinase/Rac signaling axis in regulating BD2 levels in keratinocytes.

Introduction

Psoriasis is a chronic-immune mediated inflammatory disease with clinical manifestations spanning both the skin and the inflammatory system [1]. While the cause of psoriasis remains under active investigation, studies point to both genetic and environmental components contributing to both its prevalence and severity [2, 3]. Common comorbidities include atherosclerotic cardiovascular disease (ASCVD), which remains a leading cause of death among psoriasis patients [1]. There are 5 psoriasis classifications, the most common is plaque-type psoriasis or psoriasis vulgaris, which is characterized by an itchy rash with scale-like patches commonly found on knees, elbows, scalp, and trunk [1]. The psoriasis and severity index score (PASI) is a metric used to quantify psoriasis severity with severe psoriasis encompassing >10% total body coverage [4].

Briefly, psoriasis begins with an initiation phase, characterized by local inflammation and keratinocyte proliferation [1, 5, 6]. This is followed by activation of inflammatory signaling pathways such as NF-kB and subsequently T-cell activation, both of which feed into a constitutive feedback loop [1, 7]. During T-cell activation, T-helper (Th) cells, in particular Th1 and Th17 differentiate and promote the stimulation of cytokines such as TNFα and IL-17A, respectively [7, 8]. As such, common biologic treatments for psoriasis target TNFα and IL17A signaling pathways, respectively [1, 9, 10]. During the psoriasis initiation phase, key mediators such as beta-defensin-2 (BD2), a 3.5–4.5kD antimicrobial peptide, are also released by keratinocytes which serve to primarily alert the adaptive immune system by chemotactically mobilizing human dendritic cells, monocytes and T-cells [11, 12]. As such, the presence of BDs during the initial response to epithelial injury has catapulted it study as a biomarker for psoriasis and other lesioned skin diseases such as atopic dermatitis [13, 14].

Furthermore, given that psoriasis is a chronic-immune mediated disease, BDs have also been studied in context of autoimmune disorders such as type-1 diabetes, rheumatoid arthritis, and systemic lupus among others [15]. Prior reports have demonstrated associations of BD2 with psoriasis activity, potentially via Th1/Th17 cytokine upregulation, cell response to cytokine stimulation, or attributable to high defensin genomic copy number [14]. While there are reports implicating IL17A driving BD2 expression, the overarching signaling pathways remain elusive [16].

Our group and others have previously demonstrated that psoriasis patients have increased risk of ASCVD, stroke, and overall cardiovascular mortality [17, 18]. A role for alpha-defensin (AD) as a biomarker in cardiovascular disease has been proposed [19]. For example, AD mRNA and protein was elevated in seven patients with hyperlipidemia or coronary heart disease [19]. A role for BDs in cardiovascular disease states has not been reported. We therefore used similar methods to test BD2 expression in psoriasis and assess potential associations of BD2 with sub-clinical atherosclerosis markers, and cell-based assays to elucidate intracellular signaling pathways regulating BD expression under inflammatory conditions.

Materials and Methods

Study Participants and Human Serum Collection

Human serum samples were collected from patients enrolled in Psoriasis, Atherosclerosis, and Cardiometabolic Disease Initiative (PACI; NCT01778569) from January 2013 through August 2019 and healthy volunteers (ICKD; NCT01934660) from January 2013 to February 2020 at NIH/NHLBI, an ongoing longitudinal prospective study, in accordance with the principles of the Declaration of Helsinki. Detailed inclusion and exclusion criteria of the study and patient clinical evaluation has been previously reported [18]. Briefly, subject blood was collected and centrifuged within 2 hours of collection at 2400RPM for 30 minutes at room temperature (20–23°C) and serum supernatant was stored at −80°C until further use. Written informed consent was received from participants prior to inclusion in the study.

Clinical Measurements

The indicated lipoprotein subclass profiles along with GlycA, were measured using the LipoProfile-4 algorithm on the automated Vantera clinical NMR analyzer (Labcorp, NC, USA).

Measurement of Cytokines

Plasma levels of all indicated cytokines were measured using customizable human U-plex kits (Mesoscale Discovery, Gaithersburg, MD, USA) following manufacturer’s instructions.

Cholesterol Efflux Capacity

High density lipoprotein (HDL) cholesterol efflux capacity: Briefly, 3 × 10⁵ J774 cells/well were seeded in 24-well plate and radiolabeled with 2μCi of 3H-cholesterol/mL in RPMI-1640 media containing 1% FBS for 24-hours. Cells were incubated for 16-hours in RPMI media containing 2% BSA in the presence or absence of 0.3 mmol/L 8-(4-chlorophenylthio)-cAMP to upregulate ATP-binding cassette transporter A1 (ABCA1). This was followed by addition of 2.8% ApoB-depleted plasma to the efflux medium for 4 hours. A liquid scintillation counter was used to quantify the efflux of radioactive cholesterol from cells using the formula: (μCi of 3H-cholesterol in media containing 2.8% ApoB -depleted subject plasma -μCi of 3H-cholesterol in plasma-free media / μCi of 3H-cholesterol in media containing 2.8% ApoB-depleted pooled control plasma-μCi of 3H-cholesterol in pooled control plasma -free media). Pooled plasma was obtained from five healthy adult volunteers. All assays were performed in duplicate.

Coronary plaque measurements by coronary computed tomography angiography (CCTA)

Details of image acquisition and analysis have been recently described in Berg et al. (2022) [20].

Cell Culture

The immortalized keratinocyte cell line (HaCaT) (#T002001) was purchased from Addexbio and cultured in Dulbecco’s Modified Eagle Medium with high glucose and L-glutamine (DMEM; ATCC, CA# 30–2002) and supplemented with 10% fetal bovine serum (FBS, ATCC, CA# 30–2020), 10,000U/mL penicillin (GIBCO, CA# 15140122),10,000ug/mL streptomycin (GIBCO, CA# 15140122). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Enzyme-Linked Immunosorbent Assay

BD2 levels from cell supernatants and serum were measured by ELISA (Phoenix Pharmaceuticals; CA# EK-072–37) according to manufacturer’s protocol. Briefly, undiluted samples were loaded onto capture antibody-coated plate, incubated for 2 hours at room temperature (20–23°C) on a plate shaker (300–400 rpm) and washed 4 times with assay buffer. Samples were then incubated with anti-BD2 capture antibody incubated for 2 hours at room temperature (20–23°C) on a plate shaker (300–400 rpm), washed 4 times, incubated 30 minutes at room temperature (20–23°C) on a plate shaker (300–400 rpm) with streptavidin-horseradish peroxidase (SA-HRP) and briefly washed 4 times. In darkness, TMB substrate solution was added and allowed to incubate for 10–20 minutes at room temperature (20–23°C) on a plate shaker (300–400 rpm) before hydrochloric acid stop solution was added. Absorbance values were read at 450nm using Biotek Synergy HT Microplate Reader.

Cell treatments and protein isolation

HaCaT cells were maintained to confluence and then split 1:2 overnight at 37°C and 5% CO2, and then treated for 24-hours with combinations of the following: 100ng/mL IL-17a, 2μM BAY 11–7082 (Invivogen, San Diego, CA, USA), 10μM ZINC 69391 (Aobious Inc, MA, USA), 10μM IA-116 (Aobious Inc, MA, USA), 50μM LY294002 (Cayman chemical, Ann Arbor, MI, USA), and 10μM U0126 (Cayman chemical, Ann Arbor, MI, USA). At this point, conditioned cell media was collected and used for subsequent ELISA assays.

Quantitative Polymerase Chain Reaction (qPCR)

HACAT cells were treated as indicated above. RNA was isolated using Direct-zol RNA miniprep plus kit (#R2072) (Zymo Research, Irvine, CA) following manufacturer’s instructions. Reverse transcription was performed using RT2 first strand kit (Qiagen, Germantown, MD) as recommended using 500ng RNA. qPCR was performed using SYBR green (#330503) (Qiagen, Germantown, MD) using default cycles in a Roche Lightcycler 96, using the following primers: BD2-forward, 5’-ATCTCCTCTTCTCGTTCCTC-3’, BD2-reverse, 5’-ACCTTCTAGGGCAAAAGACT-3’, GAPDH-forward 5’- GGCAAATTCAACGGCACAGT-3’, GAPDH-reverse 5’- CGCTCCTGGAAGATGGTGAT. Samples were run in triplicate, and fold changes were determined using the comparative DC_T method.

Statistical Analysis

Data were reported as mean (standard deviation) for parametric variables, median (interquartile range) for nonparametric variables, and N (percentage) for categorical variables. The comparison between psoriasis and healthy controls was conducted and statistical significance was assessed by a student’s t-test when comparing two groups for parametric variables, Wilcoxon rank-sum test for nonparametric variables, Pearson’s χ2 test for categorical variables. To assess the effect of therapeutic treatment, the changes of BD2 and PASI score between baseline and one year were tested based on Wilcoxon signed-rank test. All statistical analyses were performed using and R Statistical Software (version 4.1.3; R Foundation for Statistical Computing, Vienna, Austria). Two-tailed p-values ≤ 0.05 was deemed statistically significant (bolded values).

Results

Serum BD2 levels are upregulated in psoriasis patients.

Our study used two cohorts of participants (psoriasis (n=71) and non-psoriasis (n=53), both recruited at the National Institutes of Health Clinical Center (Bethesda, MD, USA). The psoriasis study cohort at baseline were biologic treatment naïve, had a mean age of 48 (SD 12.7), composed primarily of male participants (67%) with mean body mass index of 29.6 (Table 1). These participants had moderate to severe skin disease as measured by the Psoriasis Area Severity Index (PASI) score (median 9.0; IQR 6.4–15.0) and mean psoriasis disease duration of 20 years (SD 14) (Table 1). As expected, the psoriasis cohort had elevated high-sensitivity C-Reactive Protein (hsCRP) compared to non-psoriasis participants (median 2.0, IQR 0.8–4.2 mg/dL vs median 1.0, IQR 0.5–1.9 mg/dL; P= 0.005), and GlycA (median 398, IQR 354–477 μmol/L vs median 323 IQR 304–362 μmol/L; P< 0.001). Insulin resistance, assessed by homeostatic model assessment for insulin resistance was higher in psoriasis participants (median 2.9 IQR 1.5–4.4 vs 2.2 IQR 1.0–3.3; P= 0.016). Framingham risk score was also greater in psoriasis than in non-psoriasis (median 1.9, IQR 0.3–5.2 vs median 0.8, IQR 0.1–2.1; P=0.017).

Table 1:

Baseline Characteristics of Patients with Psoriasis compared with Control Subjects.

Parameter	Non-psoriasis	Psoriasis	P Value
Clinical Characteristics	N=53	N= 71
Age, years	40.2 (16.2)	48.1(12.7)	0.006
Sex, male, (n)	32 (70)	41 (67)	0.80
BMI	26.5 (5.5)	29.6 (6.2)	0.010
Current smoker, (n)	2 (5)	6 (8)	0.54
Hypertension, n	2 (5)	17 (24)	0.014
Diabetes, n	1 (3)	4 (6)	0.48
Hyperlipidemia, n	11 (29)	26 (37)	0.42
Lipid lowering medication, n	7 (18)	13 (18)	0.99
Hypertension treatment, n	4 (11)	14 (20)	0.22
Diabetes treatment, n	1 (3)	3(4)	0.67
Psoriasis Characteristics
PASI score	-	9.6 (6.4–15)
Disease duration, years		20.8 (14.8)
Clinical and Lab Values
Systolic blood pressure, mm Hg	114.5(110.5–128)	121 (110–131)	0.14
Diastolic blood pressure, mm Hg	71 (65–76.5)	73 (66–78)	0.092
hsC-reactive protein, mg/dL	1 (0.5–1.86)	2 (0.8–4.22)	0.005
GlycA, μmol/L	323.5 (304.5–362)	398 (354–477)	<0.001
HOMA-IR	2.18 (1.0–3.4)	2.9 (1.5–4.4)	0.016
Framingham risk score	0.75 (0.1–2.1)	1.9 (0.32–5.25)	0.017
Beta defensin ng/mL	10960.5 (2463.8–5)	60026.23 (20361–162436.3)	<0.001
Natural log Beta defensin	9.02 (2.7)	10.9 (1.6)	<0.001
Cytokine Characterization
IL-1b	0.05 (0.02–0.1)	0.14 (.09–0.2)	<0.001
IL-17a	0.83 (0.4–1.51)	2.1 (1.09–4.2)	<0.001
IL-8	3.1 (2.0–3.9)	3.96 (2.9–6.2)	<0.001
TNFa	1.1 (0.8–1.6)	1.2 (0.8–2.8)	0.62
S100A7	0.83 (0.4–2.6)	1.6 (0.5–4.6)	0.078
S100A8	28.6 (18.5–46.8)	51.1 (39.1–80.5)	0.003
S100A9	2.7 (1.7–3.6)	2.7 (1.9–3.7)	0.53
S100A12	21.7 (15.03–35.3)	36.4 (23.8–64.5)	0.004
S100A8/A9	236.6 (150.1–362.1)	447.2 (325.6–601.4)	<0.001
Lipid and Lipoprotein Profile
Triglycerides, mg/dL	78 (63.5–122.5)	101 (72–150)	0.027
Total cholesterol, mg/dL	177 (47.1)	185.01 (38.04)	0.32
HDL cholesterol, mg/dL	51.5 (44–76)	48 (42–59)	0.099
LDL cholesterol, mg/dL	98.7 (40.98)	111.4 (31.7)	0.070
Cholesterol efflux capacity	1.02 (0.88–1.12)	0.9 (0.85–1.07)	0.14
Imaging Characteristics
Total plaque burden, mm2 (x100)	1.048 (0.3)	1.3 (0.6)	0.004
Non-calcified plaque burden, mm2 (x100)	1.0 (0.32)	1.3 (0.5)	0.005
Global aortic vascular inflammation, TBR	1.6 (0.1)	1.74 (0.31)	0.006
Visceral Adipose Tissue	10107.9 (8619.9)	16288.09 (9339.72)	0.023

We also found a significant elevation in triglycerides in psoriasis vs control (median 101, IQR 72–150 vs median 78 IQR 64–123) and cholesterol function, as measured by efflux capacity was to be diminished in psoriasis as expected and previously described [21] (Table 1). As anticipated, serum concentrations of cytokines IL1β, IL17A, IFNγ and IL-8 along with members of the S100 family of proteins were all significantly elevated in psoriasis (Table 1). As we have previously reported in psoriasis, total plaque burden was higher (mean 1.3 mm2, SD 0.06 vs mean 1.048 mm², SD 0.3; P=0.004), predominantly comprised of non-calcified plaque burden (mean 1.3 mm^2, SD 0.5 vs mean 1.0 mm^2, SD 0.3; P=0.005) [22].

Next, we assessed the levels of BD2 in the serum of our two cohorts (Table 1). Circulating BD2 was significantly more elevated in psoriasis patients than non-psoriasis subjects (median 60026, IQR 20361–162436 ng/mL vs median 10961, IQR 2465 ng/mL; P<0.001) (Table 1). On comparing BD2 levels with psoriasis disease severity, we found a positive trend between BD2 and median PASI score, but this did not reach significance (beta=0.18, P=0.13).

Serum BD2 does not associate with subclinical atherosclerosis.

Given that our diseased cohort exhibits elevated coronary plaque burden and that several of our psoriasis biomarkers have previously been associated with subclinical atherosclerosis, we hypothesized that BD2 levels in psoriasis might reveal similar findings [20, 23]. However, an unadjusted analysis between groups in the present study revealed a slight positive association between BD2 in psoriasis with both aortic vascular inflammation and non-calcified burden, but both were not significant (β=0.10, p=0.77) and (β=0.18, p=0.85) respectively.

BD2 levels are reduced in psoriasis subjects undergoing anti-IL17A and anti-TNFα treatments.

Of our original 71 psoriasis patients, 70 were followed for one-year, comprising those treated with anti-TNFα (n=30) or anti-IL17A (n=21), while 19 patients remained free of biologics (topicals (n=9), systemics including methotrexate (n=5), untreated (n=5)) (Table 2). After one-year all three groups had an improvement in psoriasis severity which was significant (anti-TNFα; 8.9 (6.0–14.7) to 2.7 (1.4–4.1), anti-IL17A; 12.3 (8.5–16.8) to 1.2 (0.3–3.5), non-biologic; 8.4 (3.0–14.4) to 3.9 (3.0–9.3).

Table 2:

Changes in circulating BD2 levels in PSO patients with the indicated treatments.

Parameter	Baseline	1 Year	P Value
Entire Cohort	N=70	N=70
Beta defensin 2	62578.4 (20361.2–162436.3)	19753 (4415.2–111263.2)	<0.001
PASI score	9 (6.2–15.2)	2.9 (1.2–5.2)	<0.001
Anti TNF Treated	N=30	N=30
Beta defensin 2	28742.1(18636.1–117827.5)	13499.3 (6586.214–45868.8)	0.0062
PASI score	8.9 (6.0–14.7)	2.7 (1.4–4.1)	<0.001
Anti-IL-17 Treated	N=21	N=21
Beta defensin 2	72866.9 (54261.7–322891.7)	39503 (1850.8–143324.6)	0.0046
PASI score	12.3 (8.5–16.8)	1.2 (0.3–3.5)	<0.001
No biologic	N=19	N=19
Beta defensin 2	87281.1(30393.9–150083.5)	76068.66(12367.2–149628.2)	0.1262
PASI score	8.4 (3.0–14.4)	3.9 (3–9.3)	0.0249

Next, we tested whether changes in serum BD2 were concomitant with a reduction of psoriasis severity (Table 2). We found that patients undergoing anti-TNFα biologic treatment over one-year exhibited a 51% reduction in serum BD2 (27842 to 13499.3 ng/ml, p=0.0062) and those treated with anti-IL17A, a 45.8% reduction (72866.9 to 39503 ng/ml, p=0.0046). However, a significant reduction (12.9%) in serum BD2 was not observed in patients lacking biological therapy (87281.1 to 76068.66 ng/ml, p=0.127, though psoriasis disease severity was diminished (p=0.0249). Of these 19 patients, 13 used topical treatments for psoriasis, 3 used phototherapy and 3 had no treatment.

IL-17A signaling increases BD2 expression via Rac and PI-3 kinase.

Given that anti-IL17A biologic treatment of human psoriatics reduces circulating BD2, we hypothesized that IL17A signaling would regulate BD2 expression, which has previously been reported in [24]. While Kao et al. demonstrated that JAK and NF-κB pathways were involved in this BD2 expression [24], we hypothesized that additional signaling pathways would be involved. To this end, we focused on pathways involving the PI3-kinase, MAP kinase and the Rac1 GTPase given that both PI3-kinase and MAP kinase have been described to be downstream of IL17 receptor activation [25–27]. In addition, elevated Rac1 activity has been observed in the epidermis of human psoriasis [28].

As a first step, we treated HaCaT, a normal human keratinocyte cell line, with 100ng/mL IL17A to confirm that BD2 gene expression is elevated (Figure 1a). Secondly, conditioned media treated with IL17A increased BD2 levels approximately 5-fold in comparison to vehicle controls, as assessed by ELISA (Figure 1b). Furthermore, pretreatment with the PI3-kinase inhibitor, LY294002, but not the MEK1/2 inhibitor, U0126, blocked the IL-17A-dependent secretion of BD2 from HaCaT cells (Figure 1b). To test the involvement of Rac1 GTPase in this process, we employed two Rac1 inhibitors, IA-116 and ZINC 69391, of which function by blocking the interaction between Rac1 and associated GEF protein, such as Tiam-1 or P-Rex-1 [29]. Treatment of HaCaT cells with both inhibitors blocked both passive and IL-17A-induced secretion of BD2 (Figure 1c).

Figure 1: IL17a regulates BD2 expression in a PI3kinase- and Rac1-dependent manner.

(a) Bar graphs representing qPCR data assessing mRNA expression of BD2 in HaCaT keratinocytes treated with 100ng/mL IL17a for 24 h. BD2 mRNA expression was normalized using GAPDH as an internal control. (b, c) HaCaT keratinocytes were pre-incubated with the indicated pharmacological inhibitors for one-hour prior to IL17A (100ng/ml) for 24 h. Cell supernatants were collected and the BD2 content measured by ELISA (n=5).

Discussion

Skin is the first barrier protecting the human body from environmental pathogens. Herein the production of antimicrobial peptides, such as the family of β-defensins serve as a defense mechanism. Psoriasis is a systemic T-cell mediated inflammatory skin disease where a subset of T-helper cells along with other cells primarily release IL17A as its signature cytokine. This elevated IL17a acts on the IL17 receptor in keratinocytes to increase cell proliferation, produce further cytokines and chemokines which serve as a chemoattractant for T and Dendritic cells [1]. Two key components of both antimicrobial defense and psoriasis pathogenesis have been thoroughly described with IL17A increasing BD2 expression [16, 24].

Given the inflamed psoriatic skin is likely to contribute to the large increases in systemic BD2 protein, we were able to recapitulate these in vivo findings and probe further signaling mechanisms in HaCaT keratinocyte cells. A key orchestrator of both psoriatic disease and BD2 expression is the transcription factor NFκB. Lesioned psoriatic skin has elevated activated and phosphorylated NFκB and promotes cell proliferation [30] and an NFκB-like binding sequence has been found to be present in the promoter region of the BD2 gene [24]. Hence, it is not surprising that previous reports have demonstrated that NFκB plays a key role in IL17A-dependent increase in BD2 expression [24].

While there are numerous mouse models of psoriasis, one model of particular interest in our group is the K14-Rac1 model, where an activated form of Rac1 (V12) is expressed in the epidermal layer [28, 31]. This model exhibits hallmarks of psoriasis including elevated cytokines and enhanced NFκB activity [28]. This knowledge prompted the hypothesis that Rac1 may be involved in the NFκB-mediated induction of BD2 by IL17A. Using two-different pharmacological inhibitors for Rac1, we demonstrate that both passive and IL17A-mediated BD2 secretion in HaCaT cells is significantly reduced.

We also questioned the role of PI3kinase in this pathway. PI3-kinase and Rac1 are closely intertwined in numerous cellular processes. For example, in response to extracellular cues, such as by growth factors, the levels of activated Rac are increased and shown to be dependent on PI3-kinase [32]. While endogenous BD2 expression may be produced by gene copy number, our observation that a PI-3 kinase inhibitor will block the induction of BD2 by IL17A lends weight to an IL17RA-PI3kinase-Rac-NFκB pathway being essential for the inflammation-induced expression of this protein. As described previously, JAKs have also been implicated in the upregulation of BD2 in response to inflammatory stimuli, and like several steps in the above pathway are targets for therapeutic intervention in psoriasis [33]. Though the currently clinical available treatments for PSO are safe and effective (reviewed in [34]), the data presented adds further weight that targeting PI3kinase, NFκB and particularly Rac1 may be beneficial as anti-PSO therapeutics. Previous reports have demonstrated the suppression of PSO keratinocyte hyperproliferation by PI3kinase inhibition, and there are numerous small molecule inhibitors of NFκB under clinical testing [35] [36]. However, we are not aware of any anti-Rac1 therapeutic agents in clinical trial, possibly due to predicted side-effects given the plethora of Rac1-dependent cellular pathways, but it is plausible that the two Rac1 pharmacological inhibitors used in this study, IA-116 and ZINC 69391 may serve as a topical agent for the treatment of PSO.

Numerous studies have demonstrated the benefit of increasing BD2 secretion in the innate immune response to invading pathogens (reviewed in [37]). During this study, we also surprisingly noted that baseline BD2 expression in our psoriasis cohort inversely and significantly associated with absolute lymphocyte count (Rho=−0.271; p=0.0227) and the absolute eosinophil count (Rho=−2627; p=0.0305). Given that eosinophils are known to provide proinflammatory signals that accelerate the pathogenesis of psoriasis our data further supports the idea that secretion of BD2 will diminish this response [38]. We hope to investigate the link between BD2, eosinophils and psoriasis in future studies.

Finally, a common symptom of psoriasis is pruritis, or itch. A recent study has suggested a role for BD proteins in this disease manifestation [39]. In a genetic screen on psoriatic and atopic dermatitis skin, BD3 and BD2 genes were the two most upregulated genes in both diseases [39]. In the same article, the mechanism by which BDs evoke their itch is by stimulating Mrgpra3 neurons [39]. So, perhaps an additional and untested benefit of anti-IL17A therapeutic agents in psoriasis is the reduction of psoriatic itch.

While our data offers insight on the role of BD2 as tractable biomarker for psoriasis and the mechanisms underpinning BD2 expression, there is much to be learned about the functional role of BD2 on psoriatic skin or in the circulation. Further studies should focus on direct targets of BD2 and whether these targets are responsible for chemotactic and inflammatory response BDs orchestrate upon epithelial injury. We conclude by stating that here we add to the growing literature that BD2 is a reliable quantitative marker for PSO severity but not cardiometabolic comorbidities.

Data Availability Statement

The data used to generate the results in this study can be obtained by e-mail request to the corresponding author, Martin Playford at playfordmp@nhlbi.nih.gov