Inflammasome Signaling and Impaired Vascular Health in Psoriasis

Abstract

Objective:

Psoriasis (PsO) is an inflammatory skin disease which heightens the risk of cardiovascular disease (CVD). This study directly investigated vascular endothelial health and systemically altered pathways in PsO and matched controls.

Approach and Results:

Twenty patients (mean age 40 years, 50% male) with active PsO and 10 age, sex-matched controls were recruited. To investigate systemically alerted pathways, a deep sequencing “omics” approach was applied including unbiased blood transcriptomic and targeted proteomic analysis. Vascular endothelial health was assessed by transcriptomic profiling of endothelial cells obtained from the brachial veins of recruited participants. Blood transcriptomic profiling identified inflammasome signaling as the highest differentially expressed canonical pathway (z-score 1.6, p=1×10⁻⁷) including upregulation of CASP5 and IL-1β. Proteomic panels revealed IL-6 as a top differentially expressed cytokine in PsO with pathway analysis highlighting IL-1β (z-score 3.7, p=1.02×10⁻²³) as an upstream activator of the observed upregulated proteins. Direct profiling of harvested brachial vein endothelial cells demonstrated inflammatory transcript (e.g. IL-1β, CXCL10, VCAM-1, IL-8, CXCL1, Lymphotoxin beta, ICAM-1, COX-2 and CCL3) upregulation between PsO versus controls. A linear relationship was seen between differentially expressed endothelial inflammatory transcripts and PsO disease severity. IL-6 levels correlated with inflammatory endothelial cell transcripts and whole blood inflammasome-associated transcripts including CASP5 and IL-1β.

Conclusion:

An unbiased sequencing approach demonstrated the inflammasome as the most differentially altered pathway in PsO versus controls. Inflammasome signaling correlated with PsO disease severity, circulating IL-6 and proinflammatory endothelial transcripts. These findings help better explain the heightened risk of CVD in PsO.

Introduction

Psoriasis (PsO) is a chronic inflammatory autoimmune skin disease affecting approximately 3% of the population in the United States.¹ Large population studies suggest PsO increases the risk of cardiovascular disease (CVD) upwards of 50%.^1–3 Young adults under 50 years of age with severe PsO are at even higher CVD risk, with risk of myocardial infarction 2 to 3 times that of age-matched controls.⁴ The dysfunction in PsO includes systemic inflammation involving cytokines such as interleukin (IL)-17, tumor necrosis factor (TNF)-α and interferon (IFN)-γ.⁵ Inflammation in active PsO has been associated with heightened CVD risk with a notable overlap between expressed cytokines seen in PsO and those associated with atherosclerosis.^5–9

Impaired vascular health, including endothelial inflammatory activation, is among the initiating factors of atherosclerosis.¹⁰ Increased expression of endothelial vascular adhesion molecules, a hallmark of endothelial activation, increases arterial wall translocation of leukocytes into the sub-endothelium, thus potentiating atherosclerosis.¹¹ Direct assessment of endothelial activation allows insights into the initiation of atherosclerosis in specific disease states.^12,13 Indirect assessment of the vasculature suggests that aortic inflammation as detected by [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) is present in PsO.¹⁴ However direct evidence of impaired vascular health and corresponding mechanisms are lacking in patients with PsO.

To gain insight into mechanisms of CVD risk in PsO, we used a deep sequencing “omics” approach to integrate circulating blood transcriptomic signatures and a targeted serum proteomic analysis among PsO patients and age-, sex-matched controls. Recruited participants then underwent analysis of harvested brachial vein endothelial cells to directly assess vascular health and the endothelial transcriptome ex vivo. We hypothesized that the endothelium in PsO is altered and proinflammatory and that those alterations correlate with PsO disease activity. The platform of integrating blood transcriptional signatures, targeted serum proteomics and ex vivo endothelial analyses would represent a provisional mechanistic pathway to explain impaired vascular health and hence CVD risk in patients with PsO.

Materials and Methods:

The authors declare that supporting data are available within the article and its online-only data supplement files. Additional data that support the findings of this study are available from the corresponding author upon reasonable request.

Participant recruitment:

Patients with PsO were recruited from New York University Langone Health outpatient clinics between July 2017 and April 2018 as part of an ongoing study (NCT03228017) investigating vascular health in PsO. Active psoriatic disease was confirmed and graded by a board-certified Dermatologist or board-certified Rheumatologist as appropriate (>1% body surface area of plaque PsO or >1 swollen/tender joint).

PsO participants were excluded if they were taking aspirin or lipid lowering therapy and/or had any major medical co-morbidity including CVD or autoimmune diseases aside from PsO or psoriatic arthritis. Control subjects from the community were recruited in a 2:1 (PsO to control) fashion. As PsO participation accrued, age and sex of the PsO participants was averaged and targeted recruitment used to find appropriate matched controls. The study protocol was approved by the New York University School of Medicine institutional review board (17–00692) in line with the Declaration of Helsinki. All subjects provided written informed consent prior to participation.

Study visit:

Participants were asked to fast >4 hours prior to the visit. Clinical data including blood pressure, heart rate and anthropometric assessments were collected at the time of the visit by a licensed physician. Next, as previously described and published¹⁵ a 20-gauge angiocatheter was inserted into the brachial forearm. Three J – shaped vascular guidewires (Teleflex Inc., Reading Pa) were then sequentially advanced into the brachial vein up to 10cm and washed in dissociation buffer. Blood collection including serum, plasma and whole blood RNA was performed after vascular endothelial cell harvesting. Lipid profiles, high-sensitivity C-reactive protein (hs-CRP) and complete blood count were assessed using standard protocols.

Targeted proteomic panels:

As previously described and reported, aliquots of stored samples were analyzed using the OLINK Proseek multiplex assay Inflammation I, Cardiovascular II/III profiles.¹⁶ Briefly, oligonucleotide-labeled antibody probes with proximity extension assay technology bind to their designed target. These antibody pairs attach to their designed target and create a new DNA amplicon. The amplicons were quantified using a Fluidigm BioMark HD real-time PCR platform. Data is reported as Normalized Protein eXpression (NPX), a unit of measurement based on a Log2 scale.

Unbiased whole blood RNA transcriptomic sequencing:

Peripheral blood samples were collected in PAXgene Blood RNA tubes (PreAnalytiX, Qiagen/BD) with automated RNA extraction using a QIAsymphony PAXgene Blood RNA Kit (PreAnalytiX, Qiagen/BD). Prior to RNA sequencing, yield, quantity and quality of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA sequencing libraries were generated with the Illumina TruSeq (Sand Diego, CA) and 200ng total RNA used as starting input per sample. Samples underwent 12 cycles of amplification. Completed libraries were quantitated, normalized, and pooled. Pooled libraries were run on 2 lanes of single read 50 on the Illumina Hiseq 4000 sequencer.

Sequencing reads were mapped to the human reference genome (GRCh37/hg19) using the STAR aligner (v2.5.0c).¹⁷ Read count tables were generated using HTSeq (v0.6.0)¹⁸, normalized based on their library size factors using DESeq (v3.7)¹⁹, and differential expression analysis was performed. Pathway and gene set enrichment analysis was performed using ClusterProfiler R package (v3.6.0).²⁰ All downstream statistical analyses and generating plots were performed in R environment (v3.1.1) (http://www.r-project.org/). An exploratory p-value <0.05 was used to determine statistical significance. Ingenuity pathway analysis (Qiagen Bioinformatics, Redwood City, CA) was used to discover differentially expressed pathways.

In vitro human aortic endothelial methods and analysis:

Human aortic endothelial cells (HAECs) were cultured in Endothelial Cell Growth Medium MV 2 (Promocell® GmbH) at passage three. HAECs were stimulated with phosphate-buffered solution (PBS), IL-17 (200 ng/ml), IL-17+TNF-α (200 ng/ml +10ng/ml), IL-17+ IFN-γ (200 ng/ml + 20ng/ml) in duplicate. Cytokine combinations were chosen based on previous investigations by others evaluating in vitro inflammatory endothelial cell response (specifically IL-17+TNF-α) and known prominence (IL-17+ IFN-γ) in the pathogenesis of both PsO and atherosclerosis.^5,21–24 RNA extraction was performed using an RNeasy-MICRO kit (QIAGEN, Redwood City, CA). Gene expression analysis was performed using Affymetrix human transcriptome array 2.0 (Affymetrix, Santa Clara, California, USA). Data were analyzed through the expression console software 1.3.1 and Affymetrix’ transcriptome analysis console software (Affymetrix, Santa Clara, California, USA). Gene with a p-value <0.05 were considered statistically significant.

Brachial endothelial immunostaining:

As previously described¹³, dissociated endothelial cells were placed in RBC lysis buffer then fixed in 10% formaldehyde and dehydrated overnight. Endothelial cells were permeabilized with 0.1% Triton X-100 (Acros Organics), blocked with 4% fetal bovine serum and incubated 4 hours with CD144 (VE-cadherin) goat anti-human antibodies (1:20; R&D systems) along with 488-conjugated donkey anti-goat secondary antibodies (1:50; Jackson ImmunoResearch). Nuclear fluorescence of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) was identified by targeting the p65 subunit with rabbit anti-human antibodies (1:50; Millipore Sigma) and secondary staining with 594-conjugated donkey anti-rabbit antibodies (1:50; Jackson ImmunoResearch). Nuclei were identified though diaminophenylindole - DAPI staining. Staining was visualized with ultravoilet light under an AxioObserver. Z1 fluorescent microscope (Zeiss, Oberkochen, Germany). Negative controls were generated with their appropriate antibodies (supplement methods). Imaging processing and analysis for p65 NFκB nuclear translocation was performed using ImageJ (NIH). Endothelial cells with both cellular and nuclear integrity were assessed. The percentage of p65 NFκB/DAPI co-localization was calculated using the JACoP plug-in of ImageJ (NIH) after appropriate thresholds were established.²⁵

Brachial endothelial transcript analysis:

Brachial endothelial cell pellets were re-suspended in isolation buffer, incubated with biotinylated mouse anti-human monoclonal antibody directed against CD146 (1:200; Millipore Sigma) and isolated with streptavidin magnetic FlowComp Dynabeads (1:100). Endothelial cells underwent mRNA extraction using RNAqueous – micro RNA isolation kit (Invitrogen, Carlsbad CA). Messenger RNA was converted to cDNA (Quantbio, Beverly, MA) and amplified via PerfeCta PreAmp SuperMix (Quantabio). TaqMan (Life Technologies) primers were used on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA). To ensure reproducibility across analysis, results are represented as normalized to human acidic ribosomal protein (hARP) for each sample and gene.²⁶

Immunohistochemistry and immunofluorescence of human skin:

Frozen tissue sections of skin biopsies were blocked with 10% normal goat serum for 20 minutes and then incubated with Anti-NFκB p65 antibody (Millipore Sigman) overnight. Biotin-labeled goat anti-rabbit (Vector Laboratories) was used to detect the NFκB antibody. The staining signal was amplified with avidin-biotin complex (Vector Laboratories) and developed using chromogen 3-amino-9-ethylcarbazole (Millipore Sigma).

For immunofluorescence, skin samples were fixed with acetone and blocked in 10% normal chicken serum (Vector Laboratories) for 30 minutes. The skin tissue sections were incubated with NFκB p65 rabbit antibody (Millipore Sigma) and VE-cadherin polyclonal goat antibody (R&D systems) overnight at 4°C. The next day, the tissues were amplified with chicken anti-rabbit Alexa Fluor 594 (Invitrogen) and chicken anti-goat Alexa Fluor 488 (Invitrogen) for 30 minutes. Negative controls were generated with their appropriate antibodies (supplement methods).

Images were acquired using the appropriate filters of a Zeiss Axioplan 2 widefield fluorescence microscope with a Plan Neofluar 20 × 0.7 numerical aperture lens and a Hamamatsu Orca ERcooled charge-coupled device camera, controlled by METAVUE software (MDS Analytical Technologies, Downington, PA). Images in each figure are presented both as single color stains (green and red) located above the merged image, so that localization of two markers on similar or different cells can be appreciated. Cells that co-express the two markers in a similar location are yellow in color. A white line denotes the junction between the epidermis and the dermis

Statistical analysis comparing PsO to control participants:

Continuous data are presented as mean ± standard deviation (SD) or median [interquartile range, IQR] as appropriate. Categorical data are presented as total number (percentages). Normally distributed continuous variables were assessed through a students T-test while non-normally distributed continuous variables were assessed through Wilcoxon rank-sum test. Categorical variables were assessed through χ² analysis. Linear regression was used to evaluate the association between PsO severity and outcome transcripts with multivariable analysis to account for factors in the American Heart Association/American College of Cardiology 2013 pooled cohort CVD risk score. Regression analysis data is reported as beta coefficient (95% confidence interval) and p–value. A p-value <0.05 was considered statistically significant. All analyses were performed in STATA v.14 (College Station, TX: StataCorp LP). The authors had full access to all data in this study and take responsibility for its integrity and the data analysis.

Sample size:

Studies in inflammatory populations have noted 2 to 3 fold differences (disease versus control) in endothelial inflammatory activation.²⁷ Aortic vascular inflammation has been noted to be upwards of 13% higher in PsO compared to control.²⁸ Based on these previous studies and our own in vitro cytokine stimulated HAEC work we hypothesized to see at least a 2-fold increase between transcript expression in PsO compared to controls. A target analysis goal of n = 20 for psoriatic disease and n = 10 for controls gave us >80% power to detect a baseline endothelial difference of 2 times between groups with an alpha level of 0.05 (G*Power 3.1.9.2).

Results:

Clinical Characteristics of Recruited Subjects

This study sought to characterize vascular health in patients with PsO. Thirty participants were evaluated; 20 PsO and 10 matched controls. Demographics and clinical characteristics are presented in Table 1. Age, sex, race/ethnicity, body mass index, and blood pressure were similar between groups. Cardiovascular risk, measured using the American College of Cardiology/American Heart Association pooled cohort equation²⁹, was low and not different between groups (PsO - 4.0 ± 6.2% versus controls 4.1 ± 6.6%, p = 0.97).

Table 1.

Clinical Characteristics

Characteristics	Control (N=10)		Psoriasis (N=20)	p-value
Age, y, median (IQR)	42.5 (30 – 58)		39.5 (34.5 – 53)	0.71
Male sex, %	5 (50%)		10 (50%)	1
Caucasian, %	6 (60%)		10 (50%)	0.58
Body mass index, kg/m2	28 ± 3		28 ± 7	0.52
Hypertension, %	0 (0%)		2 (6%)	.
Systolic blood pressure, mm Hg	126 ± 16		138 ± 14	0.84
Diastolic blood pressure, mm Hg	73 ± 9		76 ± 10	0.54
Type 2 diabetes mellitus, %	0 (0%)		1 (3%)	.
Current tobacco use, %	0 (0%)		1 (3%)	.
ACC/AHA ASCVD Risk Score*, %	4.0 ± 6.2		4.1 ± 6.6	0.97
Psoriasis
Disease duration, y, median (IQR)			15 (10 – 22.5)	.
Psoriatic Arthritis, %			7 (35%)	.
BSA, %, median (IQR)			6 (3.5 – 10)	.
PASI score, median (IQR)			5.5 (3.8 – 14.1)	.
Biologic therapy, %			11 (55%)	.
Any other systemic therapy, %			6 (30%)	.
Light therapy, %			6 (30%)	.

Data are mean ± SD or N (%) unless otherwise stated.

^*American College of Cardiology/American Heart Association Atherosclerotic Cardiovascular Risk Score. BSA, body surface area of psoriasis; IQR, interquartile range; PASI, psoriasis area severity index.

Participants with PsO had on average 15 years of disease duration with a median body surface area of 6% (IQR 3.5 – 10) PsO plaque and median PASI (PsO area severity index) score of 5.5 (3.8 – 14.1) consistent with a cohort of mild to moderate active PsO at the time of enrollment. Seven patients (35%) had concomitant psoriatic arthritis (Table 1). Hs-CRP trended higher in PsO versus controls (1.8 mg/L IQR; 0.5 – 1.4 versus 0.80 mg/L IQR; 0.7 – 4.1, respectively p=0.15, Table 2).

Table 2.

Laboratory Characteristics

Laboratory parameters	Control (N=10)		Psoriasis (N=20)	p-value
Hematologic studies
WBC, × 103 cells/mm3	5.9 ± 1		7.5 ± 3	0.34
Hematocrit, %	40.0 ± 4		38.8 ± 5	0.44
Platelets, cells/L	256 ± 75		258 ± 83	1.0
Mean platelet volume, fL	9.2 ± 2		9.3 ± 1	0.52
Absolute neutrophils, × 103 cells/mm3	3.7 ± 1.3		4.8 ± 2.8	0.57
Absolute monocytes, × 103 cells/mm3	0.36 ± 0.2		0.52 ± 0.2	0.17
Neutrophil:lymphocyte ratio	2.2 ± 1		2.5 ± 1	0.78
Serum laboratory measurements
hs-CRP, mg/L, median (IQR)	0.80 (0.5 – 1.4)		1.8 (0.7 – 4.1)	0.15
Total cholesterol, mg/dl	154 ± 24		175 ± 48	0.09
Triglycerides, mg/dl, median (IQR)	62 (56 – 83)		83 (55 – 131)	0.30
LDL Cholesterol, mg/dl	90 ± 22		105 ± 41	0.49
HDL Cholesterol, mg/dl	49 ± 9		52 ± 16	0.64
Interleukin - 17A, median (IQR)*	0.9 (0.4 – 1.8)		2.1 (1.0 – 5.0)	<0.01
Interleukin – 6, median (IQR)*	3.9 (3.5 – 4.6)		4.5 (3.9 – 5.5)	0.03

Data are mean ± SD or N (%) unless otherwise stated.

^*Data reported as NPX (normalized protein eXpression - log2 scale, entire cohort assessed n = 15 controls, n = 22 psoriasis). HDL, high-density lipoprotein; hs-CRP, high sensitivity C-reactive protein, IQR, interquartile range; LDL, low-density lipoprotein WBC, white blood cell.

Transcriptome Profiling of Blood Using RNA Sequencing Reveals an Inflammatory Signature in PsO

High throughput sequencing technologies are a valuable platform to investigate complex and dynamic disease processes. To further our understanding of a heightened inflammatory state in PsO, transcriptomics through unbiased whole blood RNA sequencing was performed. Overall, 758 transcripts were differentially expressed (p<0.05 – 430 upregulated and 328 downregulated) between 10 PsO and 10 age- and sex-matched controls (Figure 1A and 1B). Ingenuity pathway analysis demonstrated inflammasome signaling to be the highest differentially expressed canonical pathway (z-score 1.6, p = 1×10⁻⁷; supplement Figure I). There was significant overexpression of CASP5, SOCS3, TNF and IL-1β (Figure 1B and 1C). Predicted top regulators of differentially expressed transcripts were signaling proteins and transcription factors known to be elevated in PsO including IFN-γ, IL-6, IL-4, NFκB and IL-12 (supplement Table I).⁵ Biological process analysis revealed substantial pathway activation in inflammatory response, cell-to-cell signaling and interaction, along with lipid metabolism and cardiovascular system development (atherosclerosis and immune function related pathways, Figure 1D). To better understand the clinical relevance of the transcripts identified from blood RNA sequencing, we investigated their association with PsO severity and noted a positive correlation between the top 20 upregulated blood transcripts and PsO disease severity (supplement Table II). Collectively, these data demonstrate the inflammasome as the most differentially altered pathway in PsO (versus controls) which correlates with PsO disease activity.

Figure 1. Enrichment in inflammatory and atherosclerosis-associated pathways in the blood transcriptome of psoriasis patients.

(A) Heat map of 758 differentially expressed transcripts between 10 psoriasis and 10 age- sex-matched controls as determined by blood RNA sequencing (p<0.05). (B) Volcano plots of transcripts in whole blood from participants with psoriasis versus controls. The y-axis corresponds to the p value (-log₁₀), and the x-axis displays the log2 fold change. The red dots represent the significantly upregulated transcripts, and blue dots significantly downregulated transcripts (p<0.05). (C) Differential expression of IL-1β, TNF-α, and the top two upregulated transcripts (CASP5, SOCS3) in psoriasis patients compared to controls. (D) Pathway analysis (IPA) of differentially regulated transcripts highlights the inflammatory, immune response and cardiovascular disease biological process which are upregulated in psoriasis over control subjects p<0.05*, p<0.01**, p<0.001***. FPKM, fragments per kilobase of transcript per million mapped reads; IL, Interleukin; IPA, ingenuity pathway analysis; TNF, tumor necrosis factor.

Proteomics in PsO Reveal an Inflammatory Signature

Protein panels revealed plasma levels of IL-17A and IL-6 as the top two differentially expressed cytokines in PsO compared to controls (Table 2). Consistent with findings in other PsO cohorts, serum levels of inflammatory proteins including IL-6, but not IL-17A correlated with PsO disease activity¹⁶ (supplement Table III). Ingenuity pathway analysis revealed the top three predicted upstream regulators of protein expression were TNF (z-score 4.2, p=2.9×10⁻²⁴), IL-1B (z-score 3.7, p=1.02×10⁻²³) and IFN-γ (z-score 2.8, p=2.4×10⁻¹⁹). Finally, the top differentially expressed canonical pathway was high mobility group box 1 (HMGB1, z-score 2.6, p=1.2×10⁻⁸, data not shown), a known regulator of inflammatory gene expression and a pathway induced by inflammasome activation.^30,31

Inflammatory PsO Profiles Associate with Activated Endothelial Cells

Systemic inflammation in PsO associates with heightened CVD risk.⁶ However, the mechanisms underlying this risk are not entirely understood. Endothelial cells act as a “first responder” to inflammatory cytokines and are involved in the early pathogenesis of atherosclerosis.³² To model how PsO inflammatory processes relates to vascular health, endothelial cells were assessed in vitro in response to implicated cytokines from our proteomic studies, each of which are also involved in the pathogenesis of PsO (IL-17, TNF-α, IFN-γ).^21–23 Unbiased microarray analysis of HAECs stimulated with IL-17+IFN-γ compared to PBS revealed 3,904 differentially expressed genes (p<0.05, 1,657 upregulated, 2,247 downregulated) and IL-17+TNF-α compared to PBS revealed 4,687 differentially expressed genes (p<0.05, 2,155 upregulated, 2,532 downregulated, data not shown). Pathway analysis revealed NFκB signaling as a key regulator of the cytokine stimulated HAEC inflammatory response (supplement Figure II). Additionally, in canonical pathway analysis, inflammasome signaling was highly upregulated after IL-17+IFN-γ stimulation (z-score 2.3, p=1.1×10⁻⁵). Finally, biological pathway analysis of the PsO blood transcriptome along with in vitro transcriptomics of cytokine stimulated HAECs revealed overlapping biological processes related to atherosclerosis and inflammation (supplement Figure III). In summary, the blood transcriptome, proteome and in vitro endothelial transcriptome all suggested a connection between PsO disease activity, inflammasome signaling and markers of endothelial inflammation and activation.

Inflammatory Transcriptomic Signature is Present in Endothelial Cells Harvested from PsO Patients

To directly investigate the endothelium in PsO, we harvested primary brachial venous endothelial cells from PsO and controls. Candidate transcripts were evaluated based on cytokine stimulation HAEC studies (Figure 2A) and through existing literature describing endothelial inflammatory activation (VCAM-1, ICAM-1, MCP-1, CCL3, COX-2, vWF, VEGFA)^33,34 as well as transcripts observed in the pathophysiology of PsO (CXCL10, CX3CL1, IL-8, CXCL1, Lymphotoxin beta, IL-1β.^35–37 Transcriptome profiling of harvested brachial vein endothelial cells were positive for the endothelium-specific marker VE-cadherin and revealed no difference between groups in VE-cadherin or housekeeping genes hARP or β-actin (supplement Table IV). Comparing PsO to controls, we found significant elevations in IL-1β (3 – fold), IL-8 (10 – fold), CCL3 (8-fold), COX-2 (3 – fold), and Lymphotoxin beta (3.5 – fold; p<0.05, Figure 2B).

Figure 2. Endothelial cells from psoriasis patients reveal inflammatory activation.

(A) Differential transcript expression following in vitro human aortic endothelial cells stimulation with IL-17 versus PBS, IL-17+TNF-α versus PBS, IL-17+IFN-γ versus PBS (p<0.05 for all changes). (B) Direct analysis of venous endothelial cells harvested from psoriasis patients compared to controls show transcript upregulation in intracellular adhesion molecules (VCAM-1, ICAM-1), inflammation (COX-2) as well as chemokines (CXCL10, CXCL1, CX3CL1, CCL3, MCP-1) interleukins and tumor necrosis factors (IL-1β, IL-8, Lymphotoxin beta). (C) Differential endothelial transcript expression in psoriasis over controls stratified by psoriasis severity (control, PASI: ≤ 10, > 10). (D) Endothelial inflammatory activation in psoriatic patients, as assessed by nuclear p65 NFκB localization in the vascular endothelium of brachial vein endothelial cells. Direct immunofluorescence staining of harvested venous endothelial cells (VE-cadherin, green) of psoriasis patients compared to healthy controls show increased p65 NFκB (red) nuclear (DAPI - blue) co-localization (n=6). p<0.05*, p<0.01**. DAPI, 4’,6-diamidino-2-phenylinodle; IL, interleukin; NFκB, Nuclear factor kappa-light-chain-enhancer of activated B cells; PASI, psoriasis area and severity index; PSB, phosphate buffered-saline; TNF, tumor necrosis factor; VEGFA, vascular endothelial growth factor A; vWF, von willebrand factor.

Significant associations were also noted between PsO disease severity and many of the upregulated endothelial transcripts (Figure 2C, Figure 3, Table 3, supplement Figure IV). After adjustment for traditional cardiovascular risk factors (age, sex, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, diabetes, smoking) and systemic PsO biologic therapy, the associations between PsO disease severity and inflammatory endothelial transcripts remained significant. Analysis of PsO disease severity using body surface area yielded similar findings (supplement Table V/supplement Figure V).

Figure 3. Representative regression plot showing a linear relationship between psoriasis disease severity (psoriasis area and severity index) and inflammatory transcript expression.

VCAM-1, r=0.81, p<0.001; CXCL10, r=0.89, p<0.001.

Table 3.

Association between Psoriatic Disease Severity and Inflammatory Endothelial Transcripts by PASI Score

Gene Transcript	Univariable†	Multivariable‡
	Beta (95%, CI)	Beta (95%, CI)	p-value
Lymphotoxin Beta	1.22 (0.47 – 1.96)**	1.19 (0.47 – 1.92)	<0.01
ICAM-1	0.48 (0.22 – 0.75)**	0.46 (0.26 – 0.66)	<0.001
VCAM-1	1.42 (0.90 – 1.93)***	1.44 (0.91 – 1.96)	<0.001
MCP-1	0.21 (0.09 – 0.32)**	0.20 (0.02 – 0.38)	0.03
CCL3	0.08 (−0.10 – 0.19)	.
CX3CL1	0.08 (−0.39 – 0.56)	.
CXCL1	0.08 (−0.002 – 0.17)	.
CXCL10	1.89 (1.41 – 2.37)***	1.83 (1.15 – 2.52)	<0.001
COX-2	0.32 (−0.12 – 0.77)	.
IL-8	−0.06 (−0.77 – 0.64)	.
IL-1β	1.01 (0.34 – 1.68)**	0.93 (0.28 – 1.58)	0.01

^†Data on table represented as regression coefficients.

^‡Multivariable model adjusted for age, sex, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, diabetes, smoking and biologic therapy. Psoriatic disease severity assessed by PASI, Psoriasis area severity index.

^**p<0.01,

^***p<0.001. IL, interleukin.

NFκB is Activated in PsO Endothelial Cells

To investigate the clinical significance of our blood transcriptomic (supplement Table II) and in vitro HAEC experiments (supplement Figure II), we performed direct ex vivo p65 NFκB staining of the subcutaneous vascular endothelium in skin biopsies and harvested brachial vein endothelial cells. Subcutaneous PsO lesional skin (psoriatic plaque) and non-lesional skin biopsies from PsO patients, compared to skin biopsies from healthy controls demonstrated increased endothelial cell staining of p65 NFκB (Figures 4A, 4B, 4C). Consistent with data in the subcutaneous tissue and cytokine stimulated HAEC pathway analysis (supplement Figure II), nuclear translocation of p65 NFκB in the harvested vascular endothelial cells was greater in PsO than in control (Figure 2D).

Figure 4. Skin biopsies reveal vascular subcutaneous endothelial inflammation is present in psoriatic patients.

(A) Immunohistochemistry of p65 NFκB in lesional skin, non-lesional skin from a psoriasis patient and normal skin from a healthy control. (B) Immunofluorescence staining of skin biopsies show increased p65 NFκB (red) expression in the vascular endothelium (VE-cadherin, green) in both lesional skin and non-lesional skin of a psoriatic patient compared to normal healthy skin. Co-localization of p65 NFκB and VE-cadherin (yellow). (C) Increased magnification of images in B. NFκB, Nuclear factor kappa-light-chain-enhancer of activated B cells.

Inflammasome Signaling May be Linked to the Endothelial Inflammatory Transcriptome

Finally, we explored associations between the blood transcriptome, serum proteome signatures and the vascular endothelium. As noted above, pathway analysis demonstrated inflammasome signaling as the top upregulated canonical pathway (supplement Figure I). The NFκB signaling pathway (regulator in PsO; supplement Table I) regulates the transcription of genes encoding components of the inflammasome and mediates the induction of inflammatory cytokines, such as IL-1β leading to downstream IL-6 production, thereby contributing to the initiation and development of inflammatory diseases.^38,39 In our proteomic data, IL-17A and IL-6 were the two highest differentially expressed cytokines (Table 2). Serum levels of both IL-17A and IL-6 correlated with many of the top differentially expressed blood RNA sequencing transcripts. However, serum IL-17A only correlated with the ex vivo endothelial transcript CXCL10 (supplemental Table VI). In contrast, serum IL-6 levels correlated with many differentially expressed ex vivo endothelial cell transcripts directly harvested from the brachial vein (Table 4). Altogether, these data suggest an important link between inflammasome (IL-1β/IL-6) signaling and endothelial activation, a major precursor to atherosclerosis.⁴⁰

Table 4.

Correlation between Serum IL-6 Levels and Blood and Endothelial Transcripts

Transcript		Correlation		p-value
Blood transcripts
IL-1β*†		0.60		<0.001
NFKB1*†		0.68		<0.001
CASP5*		0.77		<0.001
MYD88*		0.73		<0.001
NAIP*		0.63		<0.01
SOCS3†		0.77		<0.001
CD14†		0.77		<0.001
MAPK11†		0.59		<0.01
IRS1†		0.47		<0.05
IL-1RN†		0.65		<0.01
TNF†		0.46		0.07
SRF†		0.57		0.01
Endothelial ex vivo transcripts
Lymphotoxin Beta		0.34		<0.05
ICAM-1		0.38		<0.05
VCAM-1		0.66		<0.001
MCP-1		0.51		<0.01
CCL3		0.32		0.08
CX3CL1		0.00		0.99
CXCL1		0.20		0.26
CXCL10		0.80		<0.001
COX-2		0.02		0.89
IL-8		−0.04		0.81
IL-1β		0.24		0.18

^*Inflammasome and

^†IL-6 signaling determined by ingenuity pathway analysis. Correlation: Pearson’s correlation coefficient r assessing the correlation between serum IL-6 levels and inflammatory related transcripts. Whole blood transcriptome evaluated as FPKM values. Endothelial transcripts were obtained from freshly harvested endothelial cells obtained from the Brachial vein. FPKM, fragments per kilobase of transcript per million mapped reads; IL, interleukin.

Discussion

In this study, unbiased whole blood transcriptomic and targeted proteome analysis pointed to systemic inflammasome signaling as the top differentially expressed pathway. Direct ex vivo analysis of harvested endothelial cells revealed differentially expressed inflammatory endothelial transcripts between PsO and control with strong associations observed between PsO disease severity, systemic inflammasome signaling and endothelial inflammatory activation. Finally, NFκB, a known transcription factor implicated in inflammasome signaling⁴¹ was enhanced in PsO subcutaneous skin and brachial venous endothelial cells. Altogether, we directly characterized impaired vascular health using ex vivo and in vitro techniques and described altered pathways in PsO that contribute to early CVD.^6,7,11,23

Endothelial inflammatory activation is among the early vascular abnormalities in the development of atherosclerosis.^10,42 The vascular endothelium is the key regulator of vascular system homeostasis including maintenance of vasomotor tone, regulation of cellular trafficking and adhesion, and thromboresistance.¹⁰ In a disease state, activated endothelium express pro-inflammatory cytokines and adhesion molecules which attract further inflammatory cells, contributing to and perpetuating the development of atherosclerosis.^42,43 Among patients with PsO and low CVD risk (as noted by a mean cardiac risk score of 4% - Table 1), we demonstrated impaired endothelial health both in vitro and ex vivo and identified upregulated inflammatory transcripts in PsO, each of which have previously been implicated in the pathogenesis of atherosclerosis.^42,44,45

Prior studies have shown that PsO immune mediated inflammation is associated to aortic inflammation as assessed by FDG-PET.^28,46 However, no study has directly investigated the vascular endothelium and systemically altered pathways in an unbiased manner to explore how PsO relates to CVD. In our analysis, the majority of harvested endothelial transcripts assessed ex vivo correlated with PsO disease severity. The findings described in our study suggest that expression of the cytokines MCP-1, CXCL10, IL-1β, the tumor necrosis factor Lymphotoxin beta and vascular adhesion molecules, ICAM-1 and VCAM-1 may, in part, explain FDG-PET imaging studies describing the association between PsO disease severity and large vessel vascular inflammation.^{14,28,36,37,46–49} However, this concept would need to be investigated in future studies.

Despite the known connection between PsO disease severity and vascular inflammation^14,49,50, we observed that the transcripts IL-8, COX-2 and CCL3, were upregulated in PsO compared to controls, yet not associated with PsO disease activity. Epidemiologic studies support the concept that while PsO disease severity influences CVD risk, a residual risk of CVD may remain even in those with mild disease.⁵¹ Through direct analysis of harvested endothelial cells, our study suggests potential pathways through which CVD risk exists independent of PsO disease activity. Finally, the findings that cytokines upregulated in PsO lesional skin biopsies^21,35,52,53 are also present in large vessel endothelium highlights the systemic inflammatory nature of PsO affecting organs beyond the skin.

To expand upon the significance of PsO endothelial inflammatory activation, we used whole blood transcriptomics and targeted serum proteome analysis to perform a deep sequencing “omics” approach to inflammation in PsO and correlate this with impaired vascular health. In our study, unbiased analysis revealed inflammasome signaling as the top activated canonical pathway in PsO which correlated with IL-6 protein levels. The inflammasome is a multimeric protein complex involved in the immune response whose end products including IL-1β and IL-18 along with downstream production of IL-6.^40,54 CASP5, our top upregulated whole blood transcript, encodes caspase 5, which is present within the inflammasome and activates caspase 1, the enzyme required to cleave pro-IL-1β and pro-IL-18 into active forms.⁵⁵ Others have noted caspase 5 and inflammasome pathway upregulation in PsO plaque keratinocytes.^56,57 Therefore, our findings of a circulating inflammasome transcriptomic signature in PsO are not surprising and suggest that mechanisms driving skin pathology in PsO also drive systemic pathology in PsO such as vascular endothelial inflammation.^56,58

In our analysis, serum IL-17A, a known key pathogenic cytokine in PsO⁵, correlated only with the ex vivo endothelial transcript CXCL10 and not others (supplement Table VI). CXCL10 is a known chemoattractant and found at all stages of atherosclerotic development in addition to lesional PsO skin.^37,59 Notably, this cytokine was also highly differentially expressed after in vitro HAEC analysis and strongly correlated to PsO disease severity. Given the controversial role of IL-17A in promoting atherosclerosis,⁶⁰ our data suggests that CXCL10, IL-17A and their relationship to CVD risk in PsO deserves future investigation.^37,61 However ultimately, the question of inhibiting IL-17A to improve vascular health in PsO will need to be tested such as in the ongoing VIP-U trial (NCT02690701).

Overall, studies of therapies to improve CVD risk in PsO have primarily used PsO disease specific medications^5,16,62,63 and our findings suggest that inflammatory mediators which may not necessarily drive the maintenance phase of PsO lesional skin pathogenesis,⁵ strongly correlate with impaired vascular health. The inflammasome IL-1β/IL-6 axis correlated more strongly than IL-17A to ex vivo endothelial upregulated transcripts, a finding which may allow for improved development and evaluation of therapies to reduce CVD risk. PsO preferentially increases the relative risk of CVD in the young, an area where the evidence for primary cardiac prevention therapies are lacking.⁶⁴ Furthermore, randomized clinical studies assessing biologic therapy in PsO (TNF-α inhibitors) to reduce vascular inflammation using in vivo imaging techniques have yielded discrepant results.^62,63 Studies have shown that progression of carotid plaque and aortic inflammation are reduced following TNF-α inhibitor therapy.⁶² However, a recent randomized trial of TNF-α inhibitor therapy in PsO improved skin disease and circulating markers of systemic inflammation but failed to improve aortic vascular inflammation assessed by FDG-PET.⁶³

By characterizing the vascular endothelium and linking this to differentially expressed systemic pathways in PsO, we have identified promising surrogate endpoints to evaluate the impact of PsO specific treatment on CVD risk as the endothelium may be more responsive to changes in circulating cytokines than FDG-PET vascular imaging. Additionally, the inflammasome (IL-1β/IL-6 axis) has been implicated in the pathogenesis of CVD and a recent randomized clinical trial investigating IL-1β inhibition has demonstrated a reduction in CVD events.^40,65 Thus, a logical hypothesis emerges that targeting inflammasome signaling in PsO is a more precise approach to reduce CVD risk in PsO patients.

Limitations

The primary goal of this study was to directly investigate mechanisms of impaired vascular endothelial health in PsO. To do this, because of practical and logistical reasons, we studied freshly harvested venous as opposed to arterial endothelial cells. Direct harvesting of venous endothelial cells to describe molecular mechanisms of endothelial activation is a well validated innovative technique.^12,13,15,66 While venous and arterial endothelium exhibit different gene expression characteristics,^67,68 our findings and approach to studying endothelial activation was supported by in vitro HAEC analysis. We also confirmed that endothelial inflammation (as assessed by p65 NFκB) is present both in the venous (Figure 2D) and arterial (Figure 4) endothelium. Finally, the use of whole blood PAXgene to assess the systemic inflammatory response may limit complete mechanistic insight as this technique does not allow identification of the specific cell types which are responsible for the observed systemic inflammatory changes. However, in support of our findings, the whole blood transcriptomic and serum proteomic findings are supported by an extensive literature investigating inflammatory mediators present in PsO plaque.^5,16,35–37.

There are several other limitations to this current study. The cross-sectional study design does not permit causal inference based on the observed associations of endothelial inflammation, activation and severity of PsO, and by itself does not inform on the potential impact on future CVD risk. Additionally, this study may actually underrepresent systemic and endothelial inflammation in PsO as the median PASI score was only 5.5, signifying mild to moderate disease but similar to other studies investigating CVD risk in PsO.⁶⁹ Finally, half of PsO patients were receiving immune modulating therapies which has been shown to reduce CVD risk associated serum proteins.¹⁶

Conclusion

In conclusion, in PsO patients considered to be at low cardiovascular risk we show direct evidence of impaired vascular health. Using an “omics” approach, both blood transcriptomic and targeted proteomic analysis implicating inflammasome signaling as the major circulating inflammatory signature in PsO patients. PsO disease severity and inflammasome signaling each correlated with the observed upregulated inflammatory endothelial transcripts suggesting that the inflammasome signaling pathway may mediate the observed impaired vascular endothelial health and increased CVD risk in PsO. These findings have future implications for mechanistic studies assessing CVD risk in PsO and highlight several therapeutic pathways which may be a target in future trials to reduce CVD risk in PsO.

Highlights:

• Psoriasis is associated with endothelial inflammatory activation

• Inflammasome signaling is highly upregulated in patients with psoriasis

• Altered inflammasome signaling is associated with impaired vascular health in psoriasis patients.

Abbreviations list:

CVD

Cardiovascular Disease

HAEC

Human aortic endothelial cells

hARP

human acidic ribosomal protein

hs-CRP

High sensitivity C-reactive protein

FDG-PET

[18F]-fluorodeoxyglucose positron emission tomography

IFN

Interferon

IL

Interleukin

NFκB

Nuclear factor kappa-light-chain-enhancer of activated B cells

PASI

Psoriasis area severity index

PBS

phosphate-buffered solution

PsO

Psoriasis

TNF

Tumor necrosis factor