Tetrahydroxystilbene Glucoside Recruit Neutrophil Extracellular Traps To Inhibit IL-36γ Inflammatory Loop In Psoriasis

OBJECTIVE:Psoriasis is one of the most common inflammatory disease worldwide characterized by increased IL-36 cytokines,which is indicated to play a crucial pathogenic role in psoriasis.Regulating IL-36 subfamily of cytokines has led to new pharmacologic strategies to reverse psoriasiform dermatitis.Psoriatic lesions are rich in neutrophils,which are activated to release DNA scaffold decorated with granule-derived proteins named neutrophil extracellular traps（NET）.This experiment investigated the pharmacological effects of tetrahydroxy stilbene glucoside（2,3,5,4-Tetrahydroxy stilbene-2-O-β-D-glucoside,2354Glu）from Polygonum multiflorum on psoriasis and the mechanism of regulating IL-36 signaling,the characteristics of NET,and its role in the inflammatory response of psoriasis.Methods:1)Psoriasiform dermatitis was induced by Imiquimod.8-12-week-old wildtype C57BL/6 mice were randomly divided into 5 groups（n=5）:（1）the normal group,（2）Aldara group,Aldara plus 2354Glu（（3）100 or（4）25 mg/kg body weight）.and（5）Aldara plus DXMS group.Psoriasiform dermatitis were induced by apply 62.5 mg Aldara cream once daily on the shaved back skin for 4d,except the normal group.2354Glu were intraperitoneally given as the pretreated regimen for psoriasis administered at 8:00 am once daily for 8d,and together with Aldara from the 4^th d.Western blotting,immunohistochemistry,quantitative real-time PCR and multiple immunofluorescence staining were used to detect IL-36 associated inflammatory cytokines,chemotaxis,keratosis and NETosis of skin tissue.2)Human epidermal keratinocyte line Ha Ca T cells and mouse primary keratinocytes were pretreated with various inhibitors and 2354Glu before administering imiquimod（2.5μg/ml）,Poly（I:C）（25μg/ml）or IL-36γrecombinant protein（100 ng/ml）.Poly（I:C）was used to stimulate P2X7R-si RNA,IL-36G-si RNA or Con-si RNA transfected Ha Ca T cells to investigate the regulatory effect of P2X7R in the process of TLR3 activation induced IL-36γproduction.Detect the production and secretion of IL-36γ,IL-1βand caspase-1 in keratinocytes.3)Use Poly（I:C）（TLR3 ligand）,IMQ（TLR7 ligand）or IL-36γto treat the skin tissue of newborn mice to establish an ex vivo model of psoriasis with high expression of IL-36γ.The effects of 2354Glu on the activation of IL-36γ,IL-1βand caspase-1 in these three models were tested.Ac-YVAD-CHO（caspase-1 inhibitor）,A438079（P2X7R antagonist）,IL-36Ra（IL-36R antagonist）and TLR3/ds RNA Complex Inhibitor（TLR3 inhibitor）were used to detect the effect of 2354Glu on the mechanism of regulating IL-36γsignal in the local skin.4)Stimulating Primary dermal fibroblasts with IL-36γat different times points for0,2,4,6,8,16 and 24 hours,and detect the secret levels of IL-36γand IL-1βby Western blotting.A438079,Ac-YVAD-CHO,NF-κB inhibitor,TLR3/ds RNA Complex Inhibitor,CLI-095 or IL-36Ra were pretreated to detect and the effect of various inhibitors on IL-36γself-amplification was determined.5)Neutrophils were treated with Poly（I:C）,Poly（I:C）/ATP,IMQ,IMQ/ATP or PMA for 4 hours,or pretreated with A438079,Ac-YVAD-CHO,IL-36Ra,TLR3/ds RNA Complex Inhibitor,CLI-095,cl-amidine or GSK484 1h prior to the stimulation.NE-MPO co-localization of extracellular DNA and ds DNA-MPO were labeled by double stained immunofluorescence staining to determine the NET formation.Western blotting was used to detect the release of IL-1βand IL-36γduring NET formation.6)The crosstalk model between NETing neutrophils and keratinocytes was established by giving the keratinocytes conditioned medium from NETing neutrophils.Neutrophils were stimulated with Poly（I:C）for 4 hours combined with ATP for 30 minutes to induce NET formation,or pretreated with PAD4 inhbitor to prevent NET formation.Remove intact（no NET）neutrophils,and the neutrophil culture medium that containing NET was supplemented to keratinocytes for 24 hours.WB,IF and q PCR were used to detect the protein expression changes of IL-36γ,PCNA and keratins in keratinocytes.Results:1)IMQ-induced psoriasis-like skin inflammation is manifested as epidermal hyperplasia,and the mRNA expression of IL-36Ra,IL-36α,IL-36βand the pro-inflammatory factor IL-1βare significantly increased.Both 100 mg/kg and 25 mg/kg of2354Glu can effectively reduce the expression of keratin 17,improve epidermal hyperkeratosis,and inhibit the expression of inflammatory factors related to IL-36signaling.Among them,100 mg/kg showed the best effect which was better than Dexamethasone.2354Glu can down-regulate the mRNA levels of CXCL1 and CXCL2.A large amount of NET was accumulated in the psoriasis-like skin lesions induced by IMQ.The high-dose（100 mg/kg）2354Glu significantly reduced the formation of NET,and the low-dose 2354Glu（25 mg/kg）recruited neutrophils to produce NET.2)2354Glu inhibits the production and release of IL-36γand IL-1βin primary keratinocytes and Ha Ca T cells by regulating TLR3-P2X7R-caspase-1,and this process is positively correlated with caspase-1 activity.P2X7R gene-deficient Ha Ca T cells do not respond to produce or secrete IL-36γand IL-1βby Poly（I:C）stimulation.IL-36G-si RNA blocked the production and release of IL-36γand IL-1βin Ha Ca T cells,and the corresponding caspase-1 activity was also reduced.Psoriasis-like ex vivo showed significant secretion of IL-36γ,and treatment with 2354Glu（100 m M,25 m M and 6.25m M）showed similar inhibitory effect with Ac-YVAD-CHO,A438079,IL-36Ra,TLR3/ds RNA Complex Inhibitor.The effect of 2354Glu is dose-dependent,and 100 m M showed the best effect.3)Direct stimulation of IMQ and Poly（I:C）cannot induce fibroblasts produce IL-36γ,however,fibroblasts showed high sensitivity to IL-36γ,and IL-36γsignal can be significantly amplified in only 2 hours.TLR3,P2X7R and caspase-1 inhibitors have antagonistic effects on the signal amplified by IL-36γin dermal fibroblasts,while PDTC,CLI-095 and IL-36Ra have no obvious effects.4)Stimulating mouse bone marrow-derived neutrophils with Poly（I:C）,IMQ alone or in combination with ATP to develop NETosis,which is similar with PMA stimulation,and NE and MPO co-expression with decondensed DNA was detected in extracellular.Poly（I:C）/ATP stimulated neutrophils within 24 hours with the increase of stimulation time,the denser the formation of NET,the higher the NE level.A large amount of IL-36γand IL-1βare produced during NETosis.A438079,Ac-YVAD-CHO,IL-36Ra,TLR3/ds RNA Complex Inhibitor or CLI-095 are all related to the inhibition of neutrophil activation and NE release,but only TLR4,P2X7R and IL-36R are related to the inhibition of IL-36γand IL-1β,and the inhibition of TLR3 can promote the production of IL-36γ.The Poly（I:C）/ATP induced NET formation is driven by autophagy;NET structure is decorated with IL-1βrather than IL-36γprotein.A large amount of IL-36β,IL-36γ,and IL-36Ra are produced,as well as IL-1β,HMGB1,NE and Histone-3 are produced.5)The culture medium of Poly（I:C）/ATP stimulated neutrophils that containing NET structure was applied to keratinocytes to establish the crosstalk model.The culture medium inhibited the expression of IL-36γprotein in keratinocytes.The higher the NET concentration,the longer the co-cultivation time,the more effective it is to inhibit IL-36γprotein production.The conditioned medium consumed IL-36Ra during the process of inhibiting IL-36γproduction in keratinocytes,but promotes the mRNA expression of IL-36αand IL-36β.Conclusion:Poly（I:C）/ATP-induced NET inhibited keratinocyte proliferation and IL-36γproduction;2354Glu improves epidermal hypokeratosis and inflammatory infiltration by inhibiting IL-36 cytokines in keratinocytes and recruiting NET to psoriatic lesions;NET showed a potential therapeutic target for psoriasis,and 2354Glu may be a potential drug candidate for the treatment of psoriasis.