The Role And Mechanism Of IL-6-induced LMO4 In Psoriasis And Its Co-morbid Inflammatory Bowel Diseases

Background Psoriasis is a chronic inflammatory skin disease that cannot be completely cured,and its most common histopathologic features are erythema,epidermal hyperplasia,and numerous scaling.The pathogenesis of psoriasis is complex.In people with a genetic background of psoriasis,trauma or environmental factors induce innate and adaptive immune interactions and secrete a variety of types of inflammatory cytokines,such as interleukin(IL)-23,IL-1β,IL-6,and tumor necrosis factor(TNF)-alpha,that stimulate the differentiation and growth of helper T cells.Activated T cells secrete cytokines such as IL-17,IL-22,and TNF-α,which act on epidermal keratinocytes and affect cell proliferation and differentiation.IL-23 mediates the activation of T-helper 17,the central cells in psoriasis pathogenesis.In addition,IL-23 has been shown to affect keratinocyte proliferation and differentiation.Hyperplasia and abnormal differentiation of keratinocytes are classic features of psoriasis.In patients with psoriasis,the abnormally activated immune system secretes large quantities of inflammatory cytokines that act on keratinocytes and activates intracellular signaling,resulting in abnormal cell proliferation and differentiation,leading to hyperplasia of the skin epidermis and mass scaling.The inflammatory milieu induces the proliferation and differentiation of keratinocytes,depending on various intracellular transcription factors such as STAT1,STAT3,NF-κB,and LMO4.LMO4(LIM only 4)is a member of the LIM protein family,an intranuclear transcriptional regulator that binds to other transcriptional factors to form a polyprotein complex,regulates gene transcription,and plays an important role in cellular decision,cell growth,differentiation,tissue formation,and organ development.During embryonic development,LMO4 is involved in the regulation of epithelial cell proliferation and differentiation.LMO4-deficient mice exhibited severe neural tube defects,eyelid insufficiency,and impaired formation of skin protective barriers.In normal skin,LMO4 is expressed primarily in keratinocytes in the basal layer of the epidermis.LMO4 was highly expressed in the proliferating intestinal epithelium.Thus,LMO4 may play an important role in epithelial cell proliferation and differentiation.Our previously study demonstrates that LMO4 regulates keratinocytes proliferation and differentiation.IL-23 binds to IL-23 R,activates JAK2/(AKT)/STAT3 signaling pathway,upregulates LMO4 expression,and promotes cell proliferation and differentiation.Keratinocytes play an important role in the pathogenesis of psoriasis,and their function is regulated by a variety of factors,and IL-23 is not the only cytokine influencing keratinocyte proliferation and differentiation.IL-6 is a member of the pro-inflammatory cytokines network in the psoriasis pathogenesis and is expressed at high levels in the serum and skin lesions of patients with psoriasis.IL-6 is generated by different cell types and acts in a variety of biologically active forms to regulate different cell types.It has been reported that IL-6is derived from inflammatory cells and skin cells in psoriatic lesions.IL-6 contributes to the differentiation of Th17 cell and inhibits regulatory T cell(Treg)differentiation,and promotes keratinocuyes proliferation and differentiation.Ravipati,et al.reported that the presence of s IL-6R in Ha Ca T cell cultures enhanced IL-6-induced Stat3 activation,leading to an abnormality in keratinocyte proliferation and differentiation.However,it is unclear whether high levels of LMO4 expression in psoriatic keratinocytes require IL-6 and its downstream signaling pathways.Psoriasis is a systemic inflammatory disease with an increased risk of many complications such as psoriatic arthritis,inflammatory bowel disease,malignancy,obesity,and cardiovascular disease.Inflammatory bowel disease is a chronic relapsing inflammatory disease in which Crohn’s disease and ulcerative colitis are the two major forms.Studies have shown that psoriasis and inflammatory bowel disease share common clinical course,genetic susceptibility,and immunologic features.Shared susceptibility loci between psoriasis and Crohn’s disease have been identified,including IL23 R,IL12B,REL,TYK2,JAK2,SOCS1,and STAT3,but relatively few studies have examined the genetic associations between psoriasis and ulcerative colitis.On the immunologic side,the IL-23/Th17 axis is the common immune pathway in psoriasis and inflammatory bowel diseases,and the two main cytokines involved in the IL-23/Th17 pathway are IL-23 and IL-17.When stimulated by IL-23,naive T cells differentiate into a Th17 phenotype and produce cytokine IL-17,which induces inflammatory responses within the intestine and skin.Th17 cells secrete a variety of cytokines,including IL-6,TNF,IL-21,and IL-22,in addition to IL-17.There is functional redundancy and reciprocal regulation between IL-17 and IL-22,and the IL-17 cytokines may consequently drive mucosal inflammation while aiding in the restoration and repair of intestinal mucosa after inflammation has subsided.TNF-α is one of the most important proinflammatory cytokines directly affecting the intestinal epithelium,and IL-6 is a key mediator of the development of inflammatory bowel disease.Cytokines necessary for immune and mesenchymal intestinal homeostasis,such as IL-6,IL-10,IL-2,or IL-22,and those described as mediators of pathological responses in inflammatory bowel disease(IFN-γ,IL-12,IL-23,or IL-9),are all depend on JAK-STAT-mediated signaling.Variants of JAK2,TYK2,STAT1,STAT3,and STAT4 have been associated with both ulcerative colitis and Crohn’s disease.In addition,overexpression or inappropriate activation of NF-κB protein,which promotes the expression of more than 150 genes,including inflammation modulation,has been demonstrated in human inflammatory bowel diseases.Transcription factors play an important role in inflammatory bowel disease.Transcription factor LMO4 regulates both epithelial cell proliferation and differentiation and immune cell differentiation.Whether LMO4 plays a role in inflammatory bowel diseases and modulates the biological activities of intestinal epithelial cells or immune cells is unknown.PART Ⅰ.Overexpression of LMO4 in Psoriatic Keratinocytes Requires Proinflammatory Cytokine IL-6 via Activation AKT/STAT3 Signaling PathwayObjective To clarify overexpression of LMO4 in psoriatic keratinocytes requires the pro-inflammatory cytokine IL-6 that activate AKT/STAT3 signaling pathway.Methods1.Plasma samples from healthy volunteers and patients with psoriasis were collected and IL-6 levels in plasma were determined using chemiluminescence immunoassay system.2.Collect prepuce tissues that discard from circumcision,surrounding tissues and lesions of patients with psoriasis.Animal models of psoriasis were generated using 5% imiquimod cream in BALB/C mice.Hematoxylin and eosin(H&E)staining was performed to visualize histological structures and detect IL-6 and LMO4 levels using immunohistochemistry in human and animal tissues,as well as activation of AKT and STAT3.3.After stimulation of Ha Ca T cells with recombinant human interleukin-6,LMO4 expression and downstream signaling pathways of IL-6 were examined using western blotting.Ha Ca T cells were treated with IL-6 plus inhibitors,detection signaling pathway and LMO4 expression using immunoblotting.4.Ha Ca T cells were induced with IL-6 to observed changes in cell morphology,and cell proliferation was analyzed using Ed U incorporation assay.Furthermore,both immunoblotting and immunofluorescence were used to detect molecular markers of differentiated epidermal cells.And then,we used IL-6 and inhibitors to stimulate Ha Ca T cells,Ed U incorporation assay was used to analyze cell growth,and immunoblotting was used to detect molecular markers of differentiated epidermal cells.Results1.Chemiluminescence immunoassay results showed that plasma IL-6 levels were significantly higher in patients with psoriasis than in healthy controls.2.The hematoxylin and eosin staining demonstrates the typical pathologic features of psoriasis in lesions.Pathological changes in the skin of psoriasis-like mice were similar to those seen in patients with psoriasis.Immunohistochemical analysis revealed lower levels of IL-6 expression in healthy epidermis,whereas the expression of IL-6 was significantly increased in the lesions of patients with psoriasis.LMO4 was highly expressed in each layer of psoriatic epidermis,consistent with IL-6 expression in prelesional tissues and lesions,whereas LMO4 was only expressed in the basal cell layer of epidermis of healthy control.In vitro studies showed that IL-6 induced LMO4 expression in a time-dependent manner in Ha Ca T cells.3.Immunohistochemical staining showed abnormal activation of AKT and STAT3 in human psoriatic lesions and mice psoriasis-like skin.After treated with IL-6,AKT/STAT3 pathway was activated and LMO4 expression was elevated in Ha Ca T cells.And then Ha Ca T cells were treated with inhibitor plus IL-6 to block the signaling pathway,we found that LMO4 expression was markedly decreased,consistent with the inhibition of AKT and STAT3 activities.4.Ha Ca T cells were cultured in D-KSFM for 5 days to maintain the primary keratinocyte state and then treated with IL-6.Compared with PBS treatment,IL-6-treated Ha Ca T cells formed tight junctions and presented a cobblestone-like morphology.The results of the Ed U incorporation assay showed that the cells grew faster after IL-6 treatment.Immunoblotting and immunofluorescence staining analysis showed that the expression levels of involucrin,keratin 5,and keratin 1 were significantly increased in Ha Ca T cells exposed to IL-6,consistent with up-regulation of LMO4 expression.Jak2,AKT and STAT3 inhibitor r Ed Uced cell growth compared with IL-6-induced cells.And molecular markers of differentiated epidermal cells were significantly r Ed Uced after inhibitor exposure.Conclusion LMO4 expression is regulated by the inflammatory cytokine IL-6,mediated by the AKT/STAT3 signaling pathway,which regulates keratinocyte proliferation and differentiation.PART Ⅱ.IL-6 Activates MEK/ERK/NF κB Signaling Pathway to Modulate LMO4 Expression in Psoriatic Keratinocytes Objective To demonstrate that cytokine IL-6 can activate the MEK/ERK/NF κB signaling pathway to regulate LMO4 expression in psoriatic keratinocytes.Methods1.The expression levels of IL-6 and LMO4 in human and mouse skin were determined by immunohistochemistry.2.LMO4 levels in Ha Ca T cells treated with IL-6 at different concentrations or different time points of the same concentration were performed using immunoblotting analysis.Cell proliferation was analyzed using Ed U incorporation assay and cell differentiation was detected using WB.3.Ha Ca T cells were treated with IL-6 alone or IL-6 plus inhibitors,immunoblotting was performed to detect MEK/ERK/NF κB/LMO4 signaling pathway.The promoter of human LMO4 gene was cloned to constructed a luciferase reporter vector,and the binding site of P65 were mutated.Dual-luciferase reporter assay was performed to determine promoter activity.4.Cell growth was determined using Ed U incorporation assay after Ha Ca T cells were treated with IL-6 alone or IL-6 plus inhibitors.And cell differentiation was detected using immunoblotting analysis.5.Activation of MEK/ERK/NF κB signaling pathway was performed using immunohistochemical analysis at the histologic level.Results1.Immunohistochemical studies showed that IL-6 expression was low in healthy skin and LMO4 was only expressed in the epidermal basal cell layer.IL-6expression levels were significantly increased in psoriatic lesions.Consistent with the high expression of IL-6,LMO4 was overexpressed in all cell layers of the epidermis in psoriatic lesions.2.Immunoblotting analysis revealed that LMO4 expression gradually increased in cells exposed to IL-6 in a dose-and time-dependent manner.Changes in cell morphology suggested that IL-6 can induce cell differentiation compared to PBS.Consistent with the up-regulation of LMO4 expression,immunofluorescence staining and western blotting analysis showed a significant increase in the expression of IVL,K5,and K1 upon stimulation with IL-6.Results of Ed U assay showed that IL-6 significantly accelerated the growth of keratinocytes.3.Immunoblotting analysis showed that IL-6 activated MEK/ERK/NF κB signaling and upregulated LMO4 expression in keratinocytes.Dual-luciferase assays confirmed that IL-6 enhanced LMO4 promoter activity,but luciferase activity was significantly reduced after deletion the binding site of P65 on the LMO4 promoter.4.IVL expression was significantly reduced in Ha Ca T cells treated with IL-6 and ERK inhibitors,whereas changes in K1 and K5 were not significant.But,IVL,K5,and K1 were significantly reduced after treated with IL-6 plus NF κB inhibitors.Ed U assay showed that both ERK and NF κB inhibitor significantly inhibited cell growth.5.Immunohistochemical analysis showed abnormal activation of MEK/ERK/NF κB signaling pathway both in human and mouse psoriatic lesions.Conclusion Cytokine IL-6 regulates LMO4 expression via activation the MEK/ERK/NF κB signaling pathway,and regulates the proliferation and differentiation of psoriatic keratinocytes.PART Ⅲ.IL-6 signaling pathway regulates LMO4 expression and contributes to the development of inflammatory bowel disease.ObjectiveTo investigate the role of IL-6 signaling pathway and LMO4 in psoriatic-associated inflammatory bowel disease.Methods(1)Establishment of a psoriasiform animal model and acquisition of mouse colon tissues.Hematoxylin and eosin(H&E)staining of sections of mouse colon tissue was performed to observe structural changes in intestinal tissue.(2)Expression of IL-6,LMO4,p-STAT3,p-ERK1/2,and p-NF-κB(p65)in the intestine of mouse models of psoriasis using immunohistochemistry.(3)Collection of bowel biopsy specimens from patients with inflammatory bowel disease and normal bowel tissue,fixation,dehydration,and embedding.Hematoxylin and eosin staining was used to investigate the histopathological changes of intestinal tissue in patients with inflammatory bowel disease.(4)Expression of IL-6,LMO4,p-STAT3,p-ERK1/2,and p-NF-κB(p65)in the human intestine by immunohistochemistry.(5)Animal models of ulcerative colitis were created using trinitrosylbenzoic acid(TNBS)to obtain colon tissue.(6)Structural changes in ulcerative colitis were observed using hematoxylin and eosin staining.(7)Expression of IL-6,LMO4,p-STAT3,p-ERK1/2,and p-NF-κB(p65)in animal models of ulcerative colitis were detected using immunohistochemical staining.Results(1)Animal models of psoriasis were successfully established,and H&E staining showed inflammation in the mouse colorectal tissue.(2)Immunohistochemical staining showed elevated levels of IL-6 and LMO4 expression and increased p-STAT3,p-ERK1/2,and p-NF-κB(p65)activity in the pancreatic lesions of animal models of psoriasis.(3)Expression of IL-6 and LMO4,p-STAT3,p-ERK1/2,and p-NF-κB(p65)activity is increased in normal human gut,ulcerative colitis,and Crohn’s disease.(4)Successful establishment of an animal model of ulcerative colitis with trinitrosylbenzoic acid(TNBS)by H&E staining revealed intact colonic mucosa,normal crypt number and morphology,normal goblet cell number,well-organized glands,and no inflammatory cell infiltration in the control mice.In the model group,mice had reduced goblet cells in the colon,a disordered glandular arrangement,and marked inflammatory cell infiltration.(5)In an animal model of ulcerative colitis,IHC assays revealed elevated levels of IL-6 and LMO4 expression and increased p-STAT3,p-ERK1/2 and p-NF-κB(p65)activity.Conclusion In psoriasis-associated inflammatory bowel disease,IL-6 and LMO4 expression levels were elevated,and p-STAT3,p-ERK1/2 and p-NF-κB(p65)activity were increased.IL-6 may regulate LMO4 expression and contribute to the pathogenesis of inflammatory bowel disease.