Six-transmembrane epithelial antigens of the prostate comprise a novel inflammatory nexus in patients with pustular skin disorders

Abstract

Background: Pustular skin disorders are a category of difficult-to-treat and potentially life-threatening conditions that involve the appearance of neutrophil-rich pustules. The molecular basis of most pustular skin conditions has remained unknown.

Objective: We sought to investigate the molecular basis of 3 pustular skin disorders: generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), and acute generalized exanthematous pustulosis (AGEP).

Methods: Microarray analyses were performed to profile genome-wide gene expression of skin biopsy specimens obtained from patients with GPP, PPP, or AGEP and healthy control subjects. Functional enrichment, gene network, and k-means clustering analyses were used to identify molecular pathways dysregulated in patients with these disorders. Immunohistochemistry and immunofluorescence were used to determine protein localization. Quantitative RT-PCR and ELISA were used to determine transcript and secreted cytokine levels. Small interfering RNA was used to decrease transcript levels.

Results: Molecules and pathways related to neutrophil chemotaxis emerged as common alterations in patients with GPP, PPP, and AGEP, which is consistent with the pustular phenotypes. Expression of two 6-transmembrane epithelial antigens of the prostate (STEAP) proteins, STEAP1 and STEAP4, was increased in patients' skin and colocalized with IL-36γ around neutrophilic pustules. STEAP1/4 expression clustered with and positively correlated with that of IL-1, the IL-36 family proteins, and CXCL1/8. STEAP4 expression was activated by cytokines and suppressed by inhibition of mitogen-activated protein kinase kinase 1/2, whereas STEAP1 expression appeared less prone to such dynamic regulation. Importantly, STEAP1/4 knockdown resulted in impaired induction of a broad spectrum of proinflammatory cytokines, including IL-1, IL-36, and the neutrophil chemotaxins CXCL1 and CXCL8. STEAP1/4 knockdown also reduced the ability of keratinocytes to induce neutrophil chemotaxis.

Conclusion: Transcriptomic changes in 3 pustular skin disorders, GPP, PPP, and AGEP, converged on neutrophil chemotaxis and diapedesis and cytokines known to drive neutrophil-rich inflammatory processes, including IL-1 and members of the IL-36 family. STEAP1 and STEAP4 positively regulate the induction of proinflammatory neutrophil-activating cytokines.

Keywords: 6-transmembrane epithelial antigens of prostate; CXCL1; CXCL8; IL-1; IL-36; Pustular skin disorders; inflammation; neutrophils; transcriptomic profiling.

Figure 1. Transcriptional profiling of GPP, PPP, and AGEP

A) H&E staining of GPP, PPP, and AGEP skin lesions and normal skin (NN). B) Functional enrichment of signaling pathways in DEGs common and specific to GPP, PPP, and AGEP. C) Biological processes linked to genes commonly altered in GPP, PPP and AGEP.

Figure 2. Characterization of STEAPs in pustular psoriasis

A) qRT-PCR of STEAP1–4 mRNA levels in PP, NN, PPP, NNN, and GPP skin. Mann-Whitney test. *, p < 0.05. **, p < 0.01. B) Immunohistochemistry of STEAP1 and STEAP4 in GPP skin. C, D) Immunofluorescence co-staining of STEAP1 (C), or STEAP4 (D), and IL36γ in NN and GPP skin.

Figure 3. Cytokine profiling of pustular skin diseases and co-clustering with STEAP1 and STEAP4

Expression changes of cytokine and related genes were clustered by K-means clustering. The resultant groups of samples were shown on top and genes were shown on the right.

Figure 4. Correlation between STEAP and cytokine expression shown as table (A) or graphs (B)

Correlation analyses are shown between STEAPs (STEAP1 or STEAP4) with selected Group B cytokines (CXCL1, CXCL8, IL1A, IL1B, IL36A, and IL36G).

Figure 5. Regulation of STEAP1 and STEAP4 expression

A, B) Relative expression of STEAP1 (A) and STEAP4 (B) by qRT-PCR in untreated and cytokine treated keratinocytes. C–L) Relative expression of STEAP1 (C–G) and STEAP4 (H–L) by qRT-PCR under various conditions (no cytokines, TNF-α stimulation, IL-17A stimulation, IL-1β stimulation or IL-36α stimulation) with or without inhibitor co-treatment. * p<0.05, Student’s t-test.

Figure 6. Regulation of cytokine induction by STEAP1 and STEAP4

Relative expression of various cytokines under the indicated cytokine treatments upon STEAP1/4 RNAi (Ri) or scrambed RNAi (Scr Ri) by qRT-PCR. Expressions were shown for IL1A (A, D), IL1B (B, E), IL36G (C, F, H), CXCL1 (I, K, L) and CXCL8 (G, J). * p<0.05, (*) p<0.1, Student’s t-test.

Figure 7. Regulation of neutrophil chemotaxis by STEAP1 and STEAP4

Chemotaxis of neutrophils towards non-stimulated medium as negative control, and conditioned medium from IL-36α- or IL-1β-stimulated keratinocytes with scrambled RNAi (Scr Ri), STEAP1 RNAi (STEAP1 Ri) or STEAP4 RNAi (STEAP4 Ri). * p<0.05, Student’s t-test.