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of the transcriptome and proteome.
This process is tightly regulated in different tissues, cell types, and stages of differentiation, but dysregulation of selective splicing can lead to the development of
tumors or immune-related diseases.
Clinically, atopic dermatitis (AD) and psoriasis are considered two chronic inflammatory skin diseases
that are not related to each other.
AD is driven by type 2 T helper cells (TH2 cells) and is associated with overproduction of interleukin-4 (IL-4) and IL-13, while psoriasis is mainly driven by TH17 cells and is associated with
IL-17 activation.
Although the abnormal response of keratinocytes to T cell-derived cytokines is an inherent feature of AD and psoriasis pathology, whether these two skin diseases have a common mechanism that modulates the inflammatory response of keratinocytes is unclear
.
Deep RNA-seq and high-throughput whole-gene transcriptome analysis of the skin of healthy controls, AD and psoriasis patients showed similar alternative splicing transcriptomes
for lesions in AD and psoriasis patients.
Analysis of splicing signatures revealed that multiple splicing factors, including HNRNPA1, U2AF1, and DDX5, may be involved in regulating alternative splicing
of RNA in AD and psoriasis lesions.
Among these splicing factors, DEAD (Asp-Glu-Ala-Asp)-box (DDX) RNA helicase DDX5 promotes splice factor binding to splicing sites by unlocking or disrupting the stem-loop structure of genes, thereby regulating physiological functions such as RNA splicing and being associated with
human disease 。 For example, the downregulation or deletion of DDX5 causes male infertility by altering the selective splicing pattern of multiple genes during sperm formation; In breast cancer, high expression of DDX5 drives tumor development
by regulating selective splicing of H-Ras or mH2A1.
DDX5 also promotes the transmission
of vesicular stomatitis virus by promoting RNA decay of antiviral transcripts in mouse embryonic fibroblasts and nuclear output of transcripts DHX58, p65, and IKKγ.
DDX5 is predicted to be a common biological factor that fights viral replication in reverse, erythroderma, and chronic plaque psoriasis, and that variations in the DDX5 gene locus predispose individuals to asthma, an atopic disease associated with AD, but whether dysregulation of DDX5 function or expression plays a role in skin inflammation is unknown
.
On October 21, 2022, Professor Lai Yuping's team from East China Normal University published a report entitled IL-17D-induced inhibition of DDX5 expression in keratinocytes amplifies in Nature Immunology Research results of
IL-36R-mediated skin inflammation.
This study found that RNA helicase DDX5 binds to the splicing factor SF2 to regulate the splicing of IL-36R in keratinocytes to produce IL-36R and sIL-36R, while the cytokine IL-17D in AD and psoriasis lesions activates CD93-p38 MAPK-AKT-SMAD2/3 signaling pathway to inhibit DDX5 in keratinocytes to promote IL-36R production.
However, it inhibits the expression
of sIL-36R.
Specific knockout of Ddx5 (Ddx5∆KC) in mouse keratinocytes made mice more susceptible to AD and psoriasis, while injecting Ddx5∆KC AD or psoriasis mice with sIL-36R or replacing sIL-36R in keratinocytes can inhibit skin inflammation and reduce disease symptoms
.
To demonstrate the role of DDX5 in skin inflammation, the researchers first analyzed RNA-Seq and single-cell sequencing (scRNA-Seq) results from the skin of healthy controls, AD and psoriasis patients in the GEO database and found DDX5 mRNA expression was significantly reduced
in skin lesions in AD and psoriasis patients.
Immunofluorescence staining further confirmed that DDX5 protein was mainly highly expressed in skin keratinocytes in healthy controls, while the expression was significantly reduced
in skin lesion keratinocytes in AD and psoriasis patients.
In addition, the expression of DDX5 mRNA and protein in skin lesion keratinocytes induced by OVA or MC903 (calcipotriol)-induced AD mice and imiquimod (IMQ)-induced psoriasis mice was also inhibited
.
To demonstrate the role of DDX5 in AD and psoriasis, they constructed conditionally hammered out Ddx5 (Ddx5∆KC) specifically in keratinocytes
.
Compared with Ddx5 fl/fl littermate mice, Ddx5∆KC mice were more sensitive to MC903 and IMQ, mainly manifested in: MC903-induced Ddx5∆KC AD mice had significantly thickened scales and more plaques on the skin, and the expression of Il4, Il13, Ccl11, Ccl17 and Ccl22, the key factors related to AD pathogenesis in the lesions, was significantly increased.
Infiltrated CD45+ immune cells in lesions (including CD11b+SiglecF+ eosinophils, CD49b+IgE+ basophils, and CD3+CD4+ T cells).
Increase; IMQ-induced Ddx5∆KC psoriasis mice had increased plaque on the skin, thickened the spinous layer of the epidermis, and significantly increased the infiltration of key cytokines, chemokines and immune cells related to psoriasis in the lesions, proving that the loss of DDX5 in keratinocytes induced skin inflammation
.
Subsequently, by screening cytokines that are highly expressed in AD and psoriasis, the researchers found that IL-17D is a key cytokine
that inhibits DDX5 expression in keratinocytes.
It inhibits DDX5 expression
by activating the CD93-p38 MAPK-AKT-SMAD2/3 signaling pathway.
The deletion of IL-17D and its receptor, CD93, made mice resistant to MC903 and IMQ-induced skin inflammation
.
In addition, the researchers demonstrated through in vitro cell experiments that the deletion of DDX5 can selectively enhance the response
of keratinocytes to IL-36.
To look for the molecular mechanism by which DDX5–/– keratinocytes selectively enhance IL-36 response, the researchers evaluated WT vs DDX5–/– keratinocytes, Ddx5 fl/fl vs Ddx5∆ KCAD mouse lesions, and Ddx5 fl/fl vs Ddx5 ∆ KCs After comprehensive analysis of three RNA-Seq data, DDX5 mainly regulates the expression of genes related to leukocyte migration, positive regulation of cytokine production and cell chemotaxis, which are the key factors
leading to AD and psoriasis.
In addition to upregulating the expression of these factors, DDX5 deletion can also upregulate the expression
of IL-36R in keratinocytes, Ddx5∆ KCAD and psoriasis lesions.
Moreover, knocking down IL-36R in DDX5–/– keratinocytes can significantly inhibit DDX5–/– keratinocyte response to IL-36, proving that DDX5 deletion promotes keratinocyte response
to IL-36 by upregulating IL-36R expression.
Next, the researchers set out to explore the mechanism
by which DDX5 negatively regulates IL-36R signaling to promote the inflammatory response of keratinocytes.
Pre-mRNA splicing is a post-transcriptional regulation of gene expressionIn quantifying changes in splicing events in RNA-Seq data, they found that the absence of DDX5 altered multiple splicing events, especially exon jumps
in alternative splicing.
Therefore, they speculate that DDX5 may regulate IL-36R expression
by regulating the alternative splicing of IL-36R pre-mRNA 。 The use of DNA-PAGE to detect the splicing of endogenous IL-36R and the IL-36R miniGene reporting system also confirmed that DDX5 can indeed regulate the jump of exon 3 of human IL-36R pre-mRNA or exon 6 of murine IL-36R pre-mRNA, which can terminate the translation of IL-36R pre-mRNA early and produce soluble protein containing IL-36R extracellular segment （sIL-36R）
。
Since DDX5 cannot directly bind exonic splicing enhancers (ESEs) to regulate the splicing of IL-36R, the researchers further looked for DDX5-bound splicing factors through immunoprecipitation and mass spectrometry
。 Of the six DDX5-bound splicing factors identified, only SF2 (also known as SRSF1) binds to exon 3 of the IL-36R pre-mRNA and flank exons
2 and 4.
Furthermore, RNA immunoprecipitation experiments have demonstrated that SF2 binds to flank exon 4 significantly more than it binds to exon 3, resulting in a jump in exon 3 to generate sIL-36R
.
When DDX5 is knocked out, the expression of SF2 is reduced, and the ability to bind to exon 4 is weakened, so that it is impossible to mediate the jump of exon 3 to generate IL-36R
.
After determining that DDX5 combined with SF2 co-modulates IL-36R splicing to produce sIL-36R, the researchers demonstrated through competitive immunoprecipitation and FACS that sIL-36R competes with IL-36R to bind to their ligand IL-36 to inhibit the IL-36R signaling
pathway 。 Expression of sIL-36R in keratinocytes inhibits the immune response of keratinocytes to IL-36, and injection of recombinant sIL-36R in AD and psoriasis mice inhibits skin inflammation
in mice.
Finally, they confirmed that injecting sIL-36R into Ddx5∆KC AD or psoriasis mice or supplementing sIL-36R in keratinocytes can inhibit skin inflammation and reduce disease symptoms, proving that the reduction of sIL-36R in AD and psoriasis lesions is the cause
of aggravated skin inflammation in Ddx5∆KC mice.
In summary, the study revealed that IL-17D-CD93 inhibits the expression of DDX5 in keratinocytes and aggravates skin inflammation, suggesting that IL-17D and DDX5 can be used as therapeutic targets for inflammatory skin diseases
.
In addition, the identification of sIL-36R provides insight into how RNA splicing abnormalities are involved in the occurrence of skin inflammation, and also provides alternative drugs
for the treatment of AD and psoriasis.
Dr.
Ni Xinhui, Xu Yi and Wang Wang from the Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, are the co-first authors of this paper, and Lai Yuping is the corresponding author
.
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