Growth Differentiation Factor 15 As A Targeted Therapeutic Strategy For Psoriasis By Regulating Keratinocytes And Neutrophils

Background:Psoriasis is a chronic inflammatory skin disease characterized by erythematous scaly skin lesions accompanied by systemic manifestations,affecting over 60 million adults and children worldwide.The main pathogenic driver of psoriasis is generally considered the intricate interplay between infiltrated inflammatory cells and activated keratinocytes.Keratinocytes are the target cells and play a pivotal role in psoriasis development.Keratinocyte-derived proteins,such as S100A8/9,lipocalin-2（LCN2）,IL-1 family,IL-6,TNF-α,CXCL1,CXCL9,10,11,and CCL-20 accumulate and lead recruit and activate innate immune cells such as neutrophils,macrophages,and dendritic cells.Infiltrated immune cells,in return,stimulate keratinocytes to produce more chemokines,cytokines,and antimicrobial peptides to magnify the immune circuits in psoriasis.Among the infiltrating immune cells,neutrophils that aggregate in Munro’s microabscesses and scatter in the dermis play a critical pathogenic role by releasing cytokines,enzymes,and NETs.NETs further release IL-17A and IL-36,and interact directly with keratinocytes by activating TLR4/IL-36R crosstalk,leading to increasing expressions of multiple proinflammatory cytokines and chemokines.Therefore,the communication of keratinocytes and neutrophils may play a crucial role in the pathogenesis of psoriasis.With an depth understanding of the pathogenesis of psoriasis,biological agents,which targeted tumor necrosis factorα（TNF-α）,interleukin-23（IL-23）,and IL-17A,have achieved faster and better efficacy in the treatment of psoriasis.Further exploring the pathogenesis of psoriasis and finding more specific therapeutic targets are the trend and hope of psoriasis research in the future.Growth differentiation factor 15（GDF15）,also known as macrophage inhibitory cytokine 1（MIC-1）,placental transforming growth factorβ（PTGFB）,belongs to the transforming growth factor-beta（TGF-β）superfamily.Recently,studies have deeply elucidated the functions of GDF15 in cachexia,metabolic control,and cancer invasion.Still,its expression and role in inflammatory diseases have not been clarified,and its immunomodulatory function remains controversial.Our previous studies showed that GDF15 has a vital immunosuppressive part,which can promote the differentiation and function of Treg cells,inhibit the maturation and activation of macrophages,and inhibit the killing function of NK cells.Did recombinant GDF15 be used to treat psoriasis,and the expression and function of GDF15 in patients with psoriasis are still unknown.In conculusion,our project intends to focus on the role and mechanism of GDF15 in psoriasis and explore the possibility of GDF15 in the treatment of psoriasis.Objectives:1.To identify the expression of GDF15 in psoriasis and explore the possible causes of GDF15 downregulation in psoriasis.2.To identify the role of GDF15 in the development of psoriasis inflammation and the underlying mechanisms.3.To explore the therapeutic role of GDF15 in psoriasis.Methods:1.Analysis of the expression of GDF15 in psoriasis.We collected tissue samples from 15 patients with psoriasis and five healthy individuals.We also obtained serum samples from 49 patients with psoriasis and ten healthy individuals.Enzyme-linked immunosorbent assay（ELISA）,immunohistochemistry staining,quantitative real-time PCR（q RT-PCR）,and Western blots were used to examine the GDF15 expression in the patients with psoriasis.2.Investigate the effect of GDF15 in psoriasis.We generate IMQ-induced psoriasis mice in GDF15 knockout mice(Gdf15^-/-)to investigate the effect of GDF15 on psoriatic.IMQ cream was applied daily onto the shaved backs of indicated mice for six days（62.5 mg:3.125 mg/mouse/day）.We observed the inflammation degree of skin lesions for each group.Mice were sacrificed and collected skin samples were for subsequent experiments on the seventh day.H&E staining was employed to observe the histopathological features.Tissue immunofluorescence staining and tissue flow cytometry were done for compared the number of infiltrated immune cells.q RT-PCR was used to confirm the various inflammatory factors in the lesions of each group.3.Identify the possible causes of GDF15 downregulation in psoriasisWe treated primary keratinocytes with a panel of cytokines and single cytokines.q RT-PCR and western blot were employed to detect the expression of GDF15.Then,we used the JASPAR database to predict the-2000 to+100 region of the GDF15promoter and evaluated whether GATA2 could regulate GDF15 promoter activity in Ha Ca T cells using a dual-luciferase reporter assay.We also performed a Chromatin immunoprecipitation assay（Ch IP）to test whether GATA2 directly interacted with the GDF15 promoter.4.Explored the role of GDF15 in keratinocytesWe treated primary keratinocytes with a panel of cytokines to promote psoriatic inflammation in keratinocytes.Based on this cell model,we evaluated if GDF15exerted immunosuppressive effects on keratinocytes.q RT-PCR and ELISA were employed to detect psoriasis-related inflammatory mediators’expression in keratinocytes.To elucidate the underlying mechanisms of GDF15 inhibited the inflammatory responses in keratinocytes,we performed RNA-sequencing using rh GDF15-treated or untreated psoriasis-like Ha Ca T cells.The receptor inhibitor and c DNA plasma targeting signaling pathways were transfected into keratinocytes to validate the receptors and downstream signaling pathways triggered by GDF15.5.Explored the role of GDF15 in neutrophils.Neutrophils were isolated using CD15 magnetic beads from the peripheral blood of psoriasis patients and healthy controls.We employed trans-well migration assays to examine if GDF15 could directly inhibit neutrophil migration.We also used neutrophils and endothelial cells（HUVECs）co-culture systems to test if GDF15 could directly affect the adhesion of neutrophils.Finally,we used receptor inhibitor and GTP-pull down assay to clarify the potential mechanisms.6.Investigate the therapeutic role of GDF15.We subcutaneously injected recombinant murine GDF15（rm GDF15）in the IMQ-induced psoriasis-like model for six consecutive days.We observed the inflammation degree of skin lesions for each group.Mice were sacrificed,and collected skin samples were for subsequent experiments on the seventh day.H&E staining was employed to observe the histopathological features.Tissue immunofluorescence staining and tissue flow cytometry were done for compared the number of infiltrated immune cells.q RT-PCR was used to confirm the various inflammatory factors in the lesions of each group.Results:1.GDF15 expression is downregulated in skin lesions of psoriasis patients and IMQ-induced psoriasiform miceBy q RT-PCR,we found the m RNA levels of GDF15 were markedly decreased in psoriatic lesions compared to normal controls.Consistently,protein levels of GDF15were decreased in psoriatic epidermis compared with normal skin.In addition,tissue immunohistochemical and immunofluorescence staining showed that GDF15 was expressed in the epidermis of normal skin,whereas barely detected in psoriatic lesions.However,by ELISA,we found no significant difference in the expression of serum GDF15 between psoriasis patients and healthy controls.2.GDF15 knockout mice develop more severe skin inflammation in an IMQ-induced psoriasis modelIMQ-treatment drastically reduced GDF15 expression in the affected mouse skin,as confirmed by q RT-PCR,western blot,and tissue immunohistochemical and immunofluorescence staining in lesions of mouse skin.We generated IMQ-induced psoriasis in GDF15 knockout mice(Gdf15^-/-).Clearly,Gdf15^-/-mice developed more severe scaly erythematous skin lesions on day 6 after IMQ treatment compared to wild-type mice（WT）.H&E staining revealed that Gdf15^-/-mice exhibited remarkable epidermal hyperplasia and pronounced microabscesses on the skin surface.Immunofluorescence staining and flow cytometry analysis confirmed the vigorous accumulation of Ly6G⁺neutrophils in the skin lesions in Gdf15^-/-mice.Furthermore,CXCL1,CXCL10,LCN2,S100A8,S100A9,IL-1β,TNF-α,and IL-6were significantly increased in keratinocytes from Gdf15^-/-mice.Intriguingly,the differences in the distribution patterns of CD4⁺T cells in the skin between Gdf15^-/-and WT mice were not appreciable.3.TNF-αreduces the expression of GDF15 in keratinocytesq RT-PCR showed that cytokine cocktails or TNF-αalone significantly reduced GDF15 expression in keratinocytes,and western blot validated cytokine cocktails or TNF-αalone suppressed the expression of GDF15.q RT-PCR and western blot revealed that GATA2 was markedly reduced under cytokine mix or TNF-αtreatment.Using a dual-luciferase reporter assay and Chromatin immunoprecipitation assay,we proved the TNF-αnotably inhibited the activation of the GDF15 promoter and the expression of GDF15 protein via the transcript factor GATA2.4.GDF15 blocked pro-inflammatory mediators in keratinocytesA panel of cytokines,IL-17A,IL-22,IFN-λ,and TNF-α,are known to promote psoriatic inflammation in keratinocytes.Treatment with these four cytokine cocktails dramatically increased the expression of psoriasis-related inflammatory mediators in keratinocytes,including neutrophil chemoattractants（CXCL1,CXCL8,CXCL10）,antimicrobial peptides（LCN2,S100A8,S100A9）,and proinflammatory cytokines（L-6,IL-1β,TNF-α）.The addition of 25 ng/m L,50 ng/m L,or 100 ng/m L of rh GDF15 into cytokine cocktails when treating keratinocytes significantly decreased the m RNA levels of CXCL1,CXCL10,LCN2,IL-1β,IL-6 and TNF-αin a dose-dependent manner.In line with m RNA expression,rh GDF15 treatment significantly reduced the cytokine cocktails-induced high protein levels of CXCL1,CXCL10,IL-1β,IL-6,and TNF-αin the culture medium of keratinocytes,as determined by ELISA.The m RNA sequencing analysis and Gene Set Enrichment Analysis（GSEA）revealed that the markedly differential genes in GDF15-treated vs.GDF15-untreated cells could enrich the"NF-κB signaling pathway."Immunofluorescence staining,western blotting,and TAK1transfection confirmed that TAK1/IKK/IκB/NF-ΚB is an essential axis via which GDF15 exhibits anti-inflammatory function in keratinocytes.5.GDF15 directly inhibits neutrophil migration and adhesionTrans-well migration assays indicated that treatment of human peripheral neutrophils with rh GDF15 inhibited their migration through the trans-well filters along an f MLP gradient.The cell co-culture system showed that treating human peripheral neutrophils with rh GDF15 inhibited their adhesion to TNF-α-activated HUVECs cells and ICAM-1.GDF15 treatment reduced CXCL1-triggered Rap1 activation,as assessed by measuring the level of GTP-bound Rap1.6.Topical supplement of recombinant GDF15 alleviates psoriasis-like inflammation in vivorm GDF15 efficiently ameliorated psoriasis phenotype,including reducing silvery scales and alleviating erythema.H&E-staining revealed that rm GDF15 treatment remarkably reduced the epidermal hyperplasia and pronounced microabscesses on the skin surface.In addition,the number of infiltrated neutrophils was significantly decreased in rm GDF15 treated IMQ mice,but there was no observable difference in CD4⁺T cells infiltration.Flow cytometry analysis confirmed the reduced Ly6G⁺neutrophils in the skin of rm GDF15 treated mice.Moreover,q RT-PCR of mouse skin lesions showed the amounts of psoriasis-related inflammatory cytokines,and chemokines（CXCL1,CXCL10,IL-1β,IL-6,IFN-γ,and LCN2）were significantly decreased in rm GDF15 treated mice,compared with IMQ treated mice.Conclusion:This study found that GDF15 was significantly decreased in the epidermis in lesional skin in psoriasis patients.The specific deficiency of GDF15 is due to locally high concentrated TNF-αin psoriatic lesions.More importantly,the lack of GDF15 can drive psoriasis-like skin inflammation.Topical supplement of recombinant GDF15suppressed psoriatic inflammation induced by imiquimod in C57/BJ mice.We then dissected the underlying mechanism and established that GDF15 blocked proinflammatory mediators produced by suppressing the TAK1/NF-κB activation in keratinocytes,which partly limited neutrophil migration.In addition,GDF15 also directly inhibited neutrophil adhesion and migration by inhibiting the activation of the chemokine-related protein Rap1 in neutrophils.These findings indicate that increasing GDF15 may be an appropriate therapeutic approach in psoriasis.