In-Depth Analysis of the Hidradenitis Suppurativa Serum Proteome Identifies Distinct Inflammatory Subtypes

Abstract

Hidradenitis suppurativa is a chronic inflammatory dermatosis with presentations ranging from painful nodules and abscesses to draining tunnels. Using an unbiased proteomics approach, we assessed cardiovascular-, cardiometabolic-, and inflammation-related biomarkers in the serum of patients with moderate-to-severe hidradenitis suppurativa. The serum of patients with hidradenitis suppurativa clustered separately from that of healthy controls and had an upregulation of neutrophil-related markers (Cathepsin D, IL-17A, CXCL1). Patients with histologically diagnosed dermal tunnels had higher serum lipocalin-2 levels compared with those without tunnels. Consistent with this, patients with tunnels had a more neutrophilic-rich serum signature, marked by Cathepsin D, IL-17A, and IL-17D alterations. There was a significant serum‒skin correlation between proteins in the serum and the corresponding mRNA expression in skin biopsies, with healthy-appearing perilesional skin demonstrating a significant correlation with neutrophil-related proteins in the serum. CSF3 mRNA levels in lesional skin significantly correlated with neutrophil-related proteins in the serum, suggesting that CFS3 in the skin may be a driver of neutrophilic inflammation. Clinical significantly correlated with the levels of lipocalin-2 and IL-17A in the serum. Using an unbiased, large-scale proteomic approach, we demonstrate that hidradenitis suppurativa is a systemic neutrophilic dermatosis, with a specific molecular signature associated with the presence of dermal tunnels.

Figure 1:. HS serum is molecularly distinct from healthy controls and other systemic skin diseases

a) Principal Component Analysis and b) unsupervised hierarchical clustering of all differentially expressed proteins (abs(FCH) ≥1.2, and p≤0.05) between HS and healthy volunteer (HV) controls. Fold changes relative to healthy controls are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. c-Kit-ligand is found in both inflammation and cardiovascular panel. c) Gene Ontology (GO) biological process pathway enrichment analysis of differentially expressed proteins in HS relative to HV using the exploring Genomic Relations (XGR) tool. Vertical line is FDR=0.05. d) PANTHER Statistical overrepresentation test for GO cellular component for differentially expressed proteins in HS relative to HV e) Venn diagram of differentially expressed proteins between HS, psoriasis and atopic dermatitis (AD).

Figure 2:. Serum LCN2 levels differentiate HS into two subgroups

a) Unsupervised hierarchical clustering of all proteins in HS and healthy volunteers (HV) control serum arranged by increasing levels of LCN2 in the serum. Red indicates upregulated and blue indicates downregulated protein expression levels. Blue circle demonstrates node of interest on the dendrogram with black box identifying a cluster of proteins that are directly proportional with LCN2 levels b) Magnification of the LCN2-related cluster of proteins identifies a clear demarcation of two HS sub-groups based on protein level of LCN2 in serum c) Pearson’s correlation between LCN2 protein levels and levels of other neutrophil-related proteins in serum is shown. r is Pearson correlation. d) High LCN2 sub-group consists of patients with histologically diagnosed tunnels, which cluster separately and away from patients without tunnels and low LCN2 levels.

Figure 3:. HS patients with tunnels have a different serum proteome profile compared to patients without tunnels

a) Heatmap of all differentially expressed proteins (abs(FCH) ≥1.2, and p≤0.05) between healthy control volunteers (HV) and HS patients without tunnels, HV and HS patients with tunnels, or HS patients with and without tunnels. Fold changes relative to HV are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. b) Enriched biological processes in serum of HS patients with tunnels by canonical/KEGG/REACTOME/biocarta pathways using the exploring Genomic Relations (XGR) tool. Vertical line is FDR=0.05 c) Venn Diagram of differentially expressed proteins in HS patients with and without tunnels relative to HV d) Olink expression of serum protein levels, shown in Log2(Expression) for neutrophil and cardiovascular-risk related proteins in serum of HS and HV, HS patients with and without tunnels, and HS patients with draining and non-draining tunnels. Each dot represents an individual sample.

Figure 4:. There is a significant serum-skin correlation in HS

a) Lesional (LS) skin was biopsied at an edge of an active inflammatory lesion. Perilesional (PL) and nonlesional (NL) skin biopsies were taken from healthy-appearing skin 2cm and 10cm from the edge of the active inflammatory lesion, respectively, and biopsied on the same anatomical area as the LS biopsy. b) Correlation plots of IL-6 protein serum levels and the IL-6 mRNA levels in LS, PL and NL skin; scatterplots are shown with estimated linear regression and 95% confidence interval; r Pearson Correlation. c) Serum-skin correlation of serum protein levels (Log2 Olink expression) with their corresponding mRNA levels in PL skin (Log2 mRNA expression) d) Serum-skin correlation between mRNA levels of CSF3 in LS skin and levels of neutrophil-related proteins in the serum.

Figure 5:. Serum protein correlations with clinical markers and skin disease severity

Correlation of White Blood Cell Count (WBC), Absolute Neutrophil Count (Abs. Neutrophil), Absolute Nodule Count (defined as the sum of nodules and abscesses), Draining Tunnel Count, Pain Score, and IHS4 scores with A) LCN2 and B) IL-17A protein levels in the serum. Scatterplots are shown with estimated linear regression and 95% confidence interval; r Pearson Correlation. Blue dots represent healthy control samples and red dots are HS samples.