Cd100 Regulating Keratinocytes Inflammation Is Implicated In The Pathogenesis Of Psoriasis

Background: Psoriasis is a common chronic inflammatory disease,and the treatment is difficult,which seriously affects the physical and mental health of patients.The pathogenesis of psoriasis is T cells abnormal activation to activate keratinocyte(KC)proliferation,differentiation disorder and abnormal immune activity phenotype.There are many cell growth microenvironment similarities between KC and tumor cells.In order to meet the need of rapid growth,tumor cells still exhibit active glucose uptake and glycolysis in aerobic conditions(Warburg effect).KC have also the same metabolic reprogramming process as tumor cells that convert oxidative phosphorylation into glycolysis,which is called glycolysis.This transformation helps to regulate the natural immune response function,and may participate in the regulation of disease development in some inflammatory diseases,which has become a new research hotspot in the field of metabolism and inflammation.Keratinocytes can also sense the damage associatedmolecular pattern molecules and/or pathogen associated molecular pattern molecules,which activate the inflammasome,produce IL-1β and IL-18,and elicit the inflammatory responses in psoriasis.Recent research shows that NLRP3 inflammasome and IL-1β were inplicated in the pathogenesis of psoriasis.Activation of NF-κB in keratinocytes has been reported in psoriasis lesions,which results in the production of multiple immune-related proteins such as CCL-20,CCR-6,CXCL-1,CXCL-9-11,and IL-36 and leads to further inflammatory responses.IL-1β has been identified as a key player in the pathogenesis of psoriasis and promotes the IL-23-triggered development of Th1 and Th17 cells and production of various cytokines.Furthermore,glycolysis metabolic pathways and lactate acid were elevated in psoriasis patients,and also the hypoxia inducing factor-1 alpha(HIF-1alpha)was highly expressed in psoriatic lesion,to regulate KC glycolytic metabolism and promote the formation of lactic acid,which provide favorable microenvironment for KC proliferation in psoriasis.However,the exact role of keratinocytes in psoriasis still remains to be elucidated.CD100 belongs to one of the family members of the Semaphorin family,and is the first brain-signaling protein that have immunomodulatory function,also known as Semaphorin 4D.CD100 is a membrane protein with a total length of 150 KD,membrane molecules(m CD100)which can be cut off from the form of membrane protein to functional soluble molecules(s CD100)by various proteolytic enzymes.s CD100 is elevated in serum or lesional skins of variety chronic inflammatory disease,and is closely correlated with various clinical indicators,such as the severity and prognosis of the disease.s CD100 stimulates tumor necrosis factor-a and IL-6 production by peripheral blood mononuclear cells in rheumatoid arthritis.Studies have also reported that semaphorin-plexin signaling activates the NF-k B pathway,NF-κB signaling regulates the transcription of NLRP3.According to the latest research,CD100 is the upstream regulatory gene of HIF-1α,and HIF-1α activate the essential enzyme for glycolysis,and thus CD100 promotes KC glycolytic metabolism.At the same time,the key restriction enzyme PKM2 and the end product of lactic acid regulate EIF2AK2 and NF-κB signaling to promote the activation of NLRP3 inflammasome and the secretion of IL-1β respectively,and participate in a variety of chronic inflammatory responses.Therefore,we propose the following hypothesis: CD100-Plxn B2 promotes the NF-κB signalling and NLRP3 inflammasome activation.Furthermore,CD100 influences the model of keratinocytes glucose metabolism and causes Warburg effect in KC to promote the transformation of glycolytic products to lactic acid.And then lactate regulates NF-κB signalling to increase IL-1β inflammatory cytokines release,on the other hand,PKM2 regulate EIF2AK2 signalling to promote NLRP3 activation and IL-1β、IL-18 secretion,which influence the KC immune activity,and participate in psoriasis immune inflammatory response.Aims: This study will illustrate the specific mechanism of CD100-Plxn B2 promoting the inflammatory cytokines expression and NLRP3 inflammasome activation in keratinocytes,and KC glycolysis on psoriasis inflammatory response,which may help to develop new therapeutic treatments for psoriasis.The specific research objective include: 1.To clarify the expression and distribution of CD100 and its receptors Plxn B2 in psoriasis lesions、imiquimod psoriasis-like dermatitis model and the correlation between serum CD100 levels and severity and activity in patients with psoriasis;2.To charify the signalling pathway that CD100-Plxn B2 promote the NLRP3 inflammasome and inflammatory cytokines expression;3.To clarify the signalling pathway that CD100-Plxn B2 regulate KC glycolysis and illustrate the specific mechanism of KC glycolysis on psoriasis inflammatory response.Methods: 1.Analysis of CD100 and its receptor expression in psoriasis and IMQ-induced psoriasis-like dermatitis model: We obtained serum samples from 25 patients with psoriasis、AD and 25 healthy individuals,and collected tissue samples from 10 patients with psoriasis、AD and 10 healthy individuals.CD100 and Plxn B2 expression were examined in serum and lesions of patients with psoriasis using immunohistochemistry staining,quantitative real-time PCR(q RT-PCR),western blots and enzyme-linked immunosorbent assay(ELISA).We also To collect 10 cases peripheral blood from patients of psoriasis and healthy individuals,then separate PBMC,flow cytometry detected the expression level of m CD100 in peripheral blood PBMC in different immune cells.We also constructed IMQ-induced psoriasis-like dermatitis model and MC903-induced AD mouse model,flow cytometry and q RT-PCR detected the expression of Plxn B2 in epidermis of these animal model.2.The role and mechanism of CD100 regulated Rho GTPase signal to promote NLRP3 inflammasome activation: We treated Ha Ca T and primary keratinocytes with recombinant CD100 protein and trasfected with Plxn B2-si RNA,western blots、ELISAs and GST pull down revealed the activation of NLRP3 inflammasome、proinflammatory cytokines and Rho GTPase.We also treated primary keratinocytes with recombinant CD100 protein and Rho GTPase inhibitors,western blots detected the formation of NLRP3 inflammasome and the production of proinflammatory chemokines/cytokines.3.Analysis of key restriction enzyme of glycolysis expression in psoriasis: We collected tissue samples from 10 patients with psoriasis and 10 healthy individuals,immunohistochemistry staining,q RT-PCR and western blots detected the expression of key restriction enzyme of glycolysis in psoriatic lesions.4.The role and mechanism of CD100 regulated Rho GTPase signalling to promote KC glycolysis: We treated primary keratinocytes with recombinant CD100 protein and trasfected with Plxn B2-si RNA,q RT-PCR and western blots detected the expression of key restriction enzyme of glycolysis;Spectrophotometer detected the consumption of glucose、lactate production、LDH activity and p H.q RT-PCR and western blots detected the expression of PKM2、PFK1 and HK2 in CD100 KO and wild type mice.We also treated primary keratinocytes with recombinant CD100 protein and Rho GTPase inhibitors,q RT-PCR and western blots detected the expression of key restriction enzyme of glycolysis.5.The role and mechanism of KC glycolysis involved in prosiasis inflammation: We treated primary keratinocytes with lactic acid and NF-κB inhibitors,western blots and ELISA detected the NF-κB and IL-1β expression and secretion;and treated primary keratinocytes with recombinant CD100 protein and blocking PKM2,western blots detected the expression of EIF2AK2 and NLRP3 inflammasome.Results: 1.CD100 and its receptor Plxn B2 were increased in psoriasis and IMQ-induced psoriasis-like dermatitis: ELISA assay results showed that s CD100 levels in serum from patients with psoriasis were elevated compared with AD and healthy controls,and positively correlated with disease severity and activity.IHC,q RT-PCR and western blots showed that CD100 and its receptor Plxn B2 were up-regulated in psoriasis lesions compared with AD and healthy controls.In a mouse model of IMQ-induced psoriasis-like dermatitis,the expression level of Plxn B2 was increased as indicated by flow cytometry and q RT-PCR.The expression of m CD100 on T cells,monocytes,and platelets,but not B cells,from blood mononuclear cells of psoriasis patients was lower than that of healthy control subjects,as evidenced by flow cytometry.2.CD100-Plxn B2 regulated Rho GTPase signalling to promote NLRP3 inflammasome activation: q RT-PCR,ELISA,and western blots results showed that CD100-Plxn B2 promoted the production of proinflammatory chemokines/cytokines and formation of the NLRP3 inflammasome in keratinocytes.q RT-PCR、western blots and GST pull down results showed that s CD100 activated the small GTPase-NF-k B signaling pathway in keratinocytes through Plxn B2.3.KC glycolysis involved in the process of psoriasis: IHC,q RT-PCR and western blots showed that the expression of PKM2、HK2 and PFK1 in psoriasis lesions were significantly upregulated.4.CD100-Plxn B2 regulated Rho GTPase signalling to promote KC glycolysis: q RT-PCR and western blots results showed that the expression of PKM2、HK2 and PFK1 in CD100 KO mice were significantly downregulated.q RT-PCR、western blots and Spectrophotometer results showed that CD100-Plxn B2 promoted the expression of PKM2、PFK1、HK2、HIF-1α and also the consumption of glucose、lactate production and LDH activity.q RT-PCR and western blots results showed that CD100-Plxn B2 regulated Rho GTPase signalling to promote keratinocyte glycolysis.5.Lactate-NF-κB and PKM2-EIF2AK2 signalling participated in psoriasis inflammation: Western blots and ELISA results showed that CD100 regulated keratinocyte glycolysis to promote the formation of NLRP3 inflammasome and the production of pro-inflammatory chemokine/cytokine secretion via lactate-NF-κB and PKM2-EIF2AK2 signalling.Conclusion: We found that the increased expression of CD100 through Plxn B2-Rho GTPase signaling to promote the NLRP3 inflammasome activation and also CD100-Plxn B2 through Rho GTPase-HIF1α signaling to influence the model of keratinocytes glucose metabolism to cause the Warburg effect and then promoted the glycolysis to produce lactate.Lactate regulates NF-κB signalling to increase IL-1β proinflammatory cytokines release;on the other hand,PKM2 regulate EIF2AK2 signalling to promote NLRP3 activation and IL-1β cytokines secretion,which influence the keratinocyte immune activity,and participate in psoriasis immune inflammatory response.Our research confirmed that CD100-Plxn B2-keratinocytes glycolysis-NLRP3 inflammasome axis is involved in inflammatory response of psoriasis,and will illustrate the specific mechanism of keratinocyte glycolysis on psoriasis inflammatory response,which may help to develop new therapeutic treatments for psoriasis.