Generalized pustular psoriasis (GPP) is an episodic, potentially life-threatening inflammatory disease that is considered a rare variant of psoriasis. However, differences in clinical presentation and distinct HLA alleles have been observed in patients with GPP compared with common forms of psoriasis, suggesting that disease classification may be misdiagnosed. In addition, patients with GPP do not respond to many therapies that have been successful for the treatment of plaque psoriasis. The genetic basis of GPP remains elusive as mutations in rare variants of IL36RN, CARD14 and AP1S3, genes which have been identified as susceptibility factors for GPP, are often non-mutated in many GPP patients. There is an urgent need for more treatments for GPP as many of the current first line therapies, acitretin, cyclosporine or methotrexate are ineffective.
To investigate GPP pathogenesis and the efficacy of drugs on molecular level, Wang et al performed RNA-sequencing on peripheral blood mononuclear cells (PBMCs) obtained from five adult GPP patients before and during treatment with the oral retinoid acitretin, the current “gold standard” for psoriasis.
PMBC samples were collected at T0 (prior to initiation of acitretin treatment), then 2 weeks after treatment (T1) and at 6 months following the treatment (T2). All five patients showed improvement of psoriatic inflammation symptoms at T1 and reached stable remission at T2.
RNA-seq was performed on all PMBC samples and approximately 80 million reads were generated per single sample. In total 2392 differentially expressed genes (DEGs) were identified when comparing T1 samples with T0. Of these, 901 were upregulated (38%) and 1491 (62%) were downregulated. When T2 samples were compared to T0, a total of 2296 genes were identified as differentially expressed, with approximately 40% (n=911) upregulated and 60% (n=1385) downregulated. 512 genes were common to both the T1 vs T0 and T2 vs T0.
Principal component analysis showed that pre-treatment gene cluster (T0) was more widely dispersed with less overlap then the two post-treatment clusters (T1 and T2).
The researchers used the Metascape analytical tool (http://metascape.org) to investigate gene ontology for GO Biological Processes, Reactome Gene Sets, KEGG Pathway genes and the Comprehensive Resource of Mammalian protein complexes (CORUM).
“Leukocyte activation involved in immune response” was the most significant ontological enrichment within differentially expressed genes, especially neutrophils, in the GPP patients' peripheral blood. Genes effecting nearly every aspect of neutrophil biology were identified, suggesting strongly that neutrophils are actively involved in the pathogenesis of GPP. Although is well-known that psoriasis is a T‑cell mediated immune disease, the function of neutrophils in immune disease pathology is much less well understood.
Other immune responses pathways were also overrepresented in the DEGs, including “cytokine signaling in immune system”, “cytokine production” and “positive regulation of immune response”.
The effect of acitretin treatment on cellular networks was investigated using Ingenuity Pathway Analysis (IPA) software. Using the Canonical pathway Analysis DEGs were enriched in 83 pathways at T1 and 74 pathways in T2. Using a z-score threshold of 2 reduced this to 9 and 13 pathways for T1 and T2 respectively. Most of the pathways affected were inhibited by acitretin treatment.
Many of the pathways effected were implicated in inhibition of innate immune responses with downregulation of genes such as TLR1, TLR5, TLR8, MYD88, NLRC4, NOD2, IRF7, IFNAR1 and STAT1.
Several cell cycle related pathways were also enriched, the mitotic genes of polo-like kinase as well as cyclins and cell cycle regulation pathways consistent with previous reports of dysregulated cell cycle signaling in GPP.
Cytokine pathways known to be implicated in psoriasis were also enriched including enrichment of the IL‑1, IL‑6 and IL‑8 signaling pathways. A more specific analysis for cytokine signaling using IPA demonstrated greater inhibition of cytokine signaling at T2 compared to T1. The “Role of IL‑17 in Psoriasis” pathway was enriched where genes such as CXCL3, IL‑8, S100A8, S100A9, IL17RA and CXCL1 had downregulated expression.
Regulatory networks analysis was also carried out to predict upstream molecules associated with cytokine pathways that may be responsible for gene expression changes. The list of top regulators included IL‑6, IL‑1B, IFN‑gamma, IL‑21, IL‑5, IL‑17A, TNF, IFN‑A2, IL‑15 and OSM and genes downstream of all these receptors were downregulated. Those genes have been reported previously to regulate multiple inflammatory diseases such as psoriasis, colitis and rheumatoid arthritis. Furthermore, biologic therapies targeting IL‑1, IL‑17A and TNF have previously been shown to hold promise in clinical setting.
In summary, RNA-seq technology permitted comprehensive dynamic gene expression profiling using liquid blood biopsies and identification of the molecular biomarker signature of GPP patients in response to drug treatment. Importantly it sheds light on mechanism of the disease and provided clues for potential new drug targets for much needed treatment of GPP and disease reclassification.