Proprotein convertase subtilisin/kexin type 9 is a psoriasis-susceptibility locus that is negatively related to IL36G

Abstract

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a posttranslational regulator of the LDL receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-Seq-based variant discovery, we identified a possible psoriasis-susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C > T). This finding was verified in independently acquired genomic and RNA-Seq data sets. Single-cell RNA-Seq (scRNA-Seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-Seq and IHC demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression, including IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C > T was associated with lower PCSK9 expression and higher IL36G expression, when compared with heterozygous skin or cell lines. Together, these results support PCSK9 as a psoriasis-susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.

Keywords: Autoimmunity; Clinical practice; Dermatology; Skin.

Figure 1. RNA-Seq variant calling identified a psoriasis-associated SNP in 3′ UTR of the PCSK9 gene.

(A) SNP calling was performed on 2 separate psoriasis RNA-Seq data sets (12, 14), and ORs were calculated using the allele counting method. Fisher’s exact test was performed to calculate P values. Names of analyzed data sets are shown on the left with OR and P values on the right. The presence of SNP rs662145 C > T was associated with increased psoriasis risk. SNP rs662145-C constitutes the reference and minor allele, and SNP rs662145-T constitutes the alternative and major allele. (B) Meta-analysis of both RNA-Seq data sets using a random-effects model.

Figure 2. Genomic data confirmed a significant linkage between PCSK9 SNP rs662145 C > T and psoriasis.

(A) PCSK9 SNP linkage disequilibrium estimated from a psoriasis RNA-Seq data set (12) (see data set details in Supplemental Table 1). (B) Analysis of GWAS data from 2590 cases of psoriasis and 1720 controls (13) revealed a significant linkage between PCSK9 rs662145 C > T and psoriasis (P = 2.6 × 10^–4).

Figure 3. PCSK9 SNP rs662145 C > T is associated with altered expression of PCSK9 in cultured keratinocytes and nonlesional skin.

(A) In cultured keratinocyte cell lines and nonlesional skin, PCSK9 SNP rs662145 C > T HOMO phenotypes expressed lower levels of PCSK9 compared with PCSK9 SNP rs662145 C > T HET phenotypes. Box-and-whisker plots show normalized PCSK9 expression (log transformed reads on y axis). Each dot represents 1 keratinocyte line or skin sample. Differential gene expression was calculated using DESeq2. FDR-adjusted P values are displayed on each plot. (B) In cultured keratinocyte cell lines and nonlesional skin, there was no difference in the expression of PGK1, a housekeeping gene, between the reference allele and PCSK9 SNP rs662145 C > T HET and HOMO phenotypes. HOMO, homozygous; HET, heterozygous.

Figure 4. PCSK9 SNP rs662145 C > T is associated with altered expression of IL36 in cultured keratinocytes and nonlesional skin.

(A) In cultured keratinocyte cell lines and nonlesional skin, PCSK9 SNP rs662145 C > T HOMO phenotypes expressed higher levels of IL36B compared with PCSK9 SNP rs662145 C > T HET phenotypes. Box-and-whisker plots show normalized PCSK9 expression (log transformed reads on y axis). Each dot represents 1 keratinocyte line or skin sample. Differential gene expression was calculated using DESeq2. FDR-adjusted P values are displayed on each plot. (B) In cultured keratinocyte cell lines and nonlesional skin, PCSK9 SNP rs662145 C > T HOMO phenotypes expressed higher levels of IL36G compared with PCSK9 SNP rs662145 C > T HET phenotypes. HOMO, homozygous; HET, heterozygous.

Figure 5. PCSK9 expression negatively correlates with IL36B and IL36G expression in keratinocytes and skin.

(A) In cultured keratinocyte cell lines, PCSK9 expression negatively correlated with IL36B and IL36G expression. PCSK9 expression did not correlate with PGK1 expression, a housekeeping gene. In these plots, each dot represents an in vitro cultured keratinocyte cell line under a different culture condition (control, IL-4, IL-13, IL-17A, IFN-α, IFN-γ, TNF-α, IL-4 and IL-13, IL-17A and IFN-γ, IL-17A, and TNF-α). Normalized log₂ transformed reads for each gene are plotted on the x axis and y axis. Pearson’s correlation coefficients and P values are displayed on each plot. (B) Epidermal expression of PCSK9 and IL36G. Representative pictures of an IHC staining for PCSK9 and IL36G as well as the matching isotype controls in lesional skin of a patient with psoriasis (upper row) and healthy control nonlesional skin (lower row). The single arrow points to the basal layer. Double arrows point to the granular layer. Scale bar: 100 μm.

Figure 6. PCSK9 expression is negatively and directly related to IL36B and IL36G expression.

(A) Single-cell sequencing of psoriatic nonlesional and lesional skin (n = 9). The UMAP method was used to create 2-dimensional representation of the resulting data. Keratinocyte populations were identified by the expression levels of established keratinocyte markers (red, basal layer keratinocytes, DST high; green, spinous layer keratinocytes, KRT5 low and KLK7 low; and blue, granular layer keratinocytes, KLK7 high). PCSK9- and IL36G-expressing cells are depicted in maroon. (B) Expression of IL36G in individual basal, spinous, and granular layer keratinocytes shown as box-and-whisker plots of log₂ transformed gene expression. P values were calculated for each data set using 1-way ANOVA. (C) PCSK9-positive keratinocytes were parsed into 2 groups, PCSK9-high and PCSK9-low (x axis). Box-and-whisker plots of indicated intracellularly expressed genes are plotted on the y axis (log₂ reads). P values were calculated using Student’s t test. (D) Box plots showing the effects of in vitro siRNA knockdown of PCSK9 in keratinocyte cell lines on IL36B and IL36G. PCSK9 (positive control) and PGK1 (negative control) expression is also shown for scrambled siRNA transfected and PCSK9 siRNA transfected cultures. Each dot represents an independently cultured and independently transfected HaCaT keratinocyte cell line (n = 3). P values were calculated with Student’s t test.

Figure 7. PCSK9 expression clusters with inflammatory mediators of psoriasis.

(A) A 2-dimensional plot of the keratinocyte transcriptome was constructed using a nonlinear dimensionality reduction strategy, the t-SNE method. Each point represents a gene and the distance between the points is inversely related to how well the genes correlate with one another. Within this plot, PCSK9 clusters with various genes of interest, shown on the right of the plot. (B) PCSK9 coexpression network with each circle representing a different gene and the lines connecting each circle representing the strength (thickness of line) and direction (red = positive, blue = negative) of each correlation.

Figure 8. PCSK9 expression directly correlates with inflammatory mediators of psoriasis.

(A) Individual scatter plots showing correlations between PCSK9 and IL1RL1, IL27RA, ISG20, STX3, and STX11 in cultured keratinocytes. In these plots, each dot represents an in vitro cultured keratinocyte cell line under a different culture condition (control, IL-4, IL-13, IL-17A, IFN-α, IFN-γ, TNF-α, IL-4 and IL-13, IL-17A and IFN-γ, IL-17A, and TNF-α). PCSK9 expression is shown on the x axis with each plot depicting a different gene on the y axis. (B) Box-and-whisker plots showing expression of genes of interest in cultured keratinocytes displaying the REF allele or rs662145 C > T variant PCSK9 allele. Differential gene expression was calculated using DESeq2. REF, reference; HET, heterozygous; HOMO, homozygous. (C) Box plots showing effects of in vitro siRNA knockdown of PCSK9 in keratinocyte cell lines on expression of genes of interest. Each dot represents an independently cultured and independently transfected HaCaT keratinocyte cell line (n = 3). The y axis depicts log₂ transformed normalized reads and the x axis compares control keratinocyte cell lines with PCSK9 knockdown cell lines, with P values calculated via Student’s t test.