The Role Of Neutrophils In The Pathogenesis Of Psoriasis

Background:Psoriasis is a common chronic inflammatory skin disease with a complex pathogenesis of genetic,infection,immune disorder,and other factors.The typical clinical manifestations are recurrent scattered erythema with adherent silvery scales,severely affecting the physiological and psychological health of patients.In recent years,it is universally recognized that the T cell-dominant immune microenvironment disorder is the main cause of psoriasis.However,neutrophils migrate through the vasculature and infiltrate into the dermis,and then are further attracted to the epidermis to form Munro or Kogoj microabscess,which is one of the typical pathological manifestations of psoriasis in the early stage or pustular psoriasis.Several clinical trials have shown that selective depletion of elevated granulocytes and monocytes by adsorptive apheresis alleviates the symptoms and related disorders of generalized pustular psoriasis(GPP)and psoriasis arthritis,confirming that neutrophils contribute to the pathogenesis of psoriasis.However,the function of neutrophils in the development of psoriasis has remained unclear.Neutrophils are the most abundant immune cells in humans and are mainly known for the phagocytosis and killing of invading pathogens.They also play a vital role in a variety of diseases,including infections,autoimmune diseases,neoplastic diseases,and chronic inflammatory diseases.Neutrophils can promote the progression of inflammation by secreting important cytokines such as IL-1α,IL-6,IL-12,TNF-α,IFN-γ,and CXCL8.In addition,a variety of antimicrobial peptides(AMPs)released by neutrophils such as S100A8/S100A9,cathelicidin(LL-37)and lipocalin2(LCN2)can regulate and amplify the inflammatory responses.In recent years,studies have found that neutrophils can capture and kill pathogens by forming neutrophil extracellular traps(NETs).NETs are large,web-like structures composed of decondensed DNA,histones,and a variety of neutrophil granule proteins,including various granule enzymes and antimicrobial peptides.Although neutrophils are short-lived immune cells,NETs released by them can continue participating in the progression of various inflammation.Several studies have shown that NETs promote the development of a variety of autoimmune diseases,such as systemic lupus erythematosus(SLE),diabetes,and small vessel vasculitis(SVV).In addition,exosome derived from neutrophils is another way in which they exert immune functions.Exosomes are extracellular nano-size vesicles that transfer proteins,mRNA,and microRNAs,working as important mediators of intercellular communication and transport.Neutrophil exosomes are independent of the parent cells and participate in a series of inflammatory and cancer progression.However,it remains unclear whether the inflammatory response of psoriasis is mediated by neutrophil exosomes or not.According to our previous study,depleting neutrophils significantly alleviated the imiquimod(IMQ)-induced psoriasis-like inflammation in mice.The gene microarray analysis of neutrophils from psoriasis patients and normal controls showed that neutrophils were pre-activated in psoriasis patients.Based on these findings,we hypothesized that neutrophil in psoriasis patients,through releasing NETs or exosomes,activated keratinocyte to highly express and secrete inflammatory factors,thus participating in the development of psoriasis.Objectives:1.To explore the functions of neutrophils in the immune microenvironment disorder of psoriasis,and to identify the ways in which neutrophils play an immunomodulatory role;2.To identify the role of NETs in the development of psoriasis inflammation and the underlying mechanisms;3.To identify neutrophil exosomes and explore their immunomodulatory effects on the development of psoriasis.Methods:1.Analysis on the differences of neutrophils from psoriasis patients and healthy controlsNeutrophils were isolated using magnetic beads positive selection system from the peripheral blood of psoriasis patients and healthy controls,and then cultured in vitro for a certain time.Flow cytometry analysis was employed to compare the cell viability.Gene microarray analysis was used to screen and identify the highly expressed genes in neutrophils from psoriasis patients.The aim was to confirm how neutrophils participated in the immune microenvironment disorders of the psoriasis based on these results and previous literature.Quantitative real-time PCR(qRT-PCR),tissue immunofluorescence,and MPO-DNA complex ELISA were used to determine the level of NETs in the skin lesions and circulation of psoriasis patients.2.Analysis on the role of neutrophils in the psoriasis-like inflammation in IMQ-induced miceFirst,the ears of BALB/c mice were applied with IMQ for continuous 7 days to construct the psoriasis-like mouse model.The anti-Ly6G mAb or rat IgG2a isotype control antibody was intraperitoneally(i.p.)injected 24h prior the first IMQ induction and every 48 h thereafter.In some groups,murine NETs were subcutaneously injected back to the ears.The inflammation degree of skin lesions was observed for each group,and H&E staining was done for observing the histopathological features.Tissue immunofluorescence was employed to compare the number of infiltrated neutrophils and CD4/CD8~+T cells in skin lesions of these mice.In addition,the mRNA levels of various inflammatory factors in the lesions of each group were detected by qRT-PCR.3.Analysis on the role of NETs and its downstream signaling pathways in IMQ-induced psoriasis-like mouse modelFor the IMQ-induced psoriasis-like mouse model,NETs inhibitors or small molecules that clear NETs structure were subcutaneously injected to reduce NETs in mice;or siRNA targeting receptors that receive NETs stimulus were mixed with emulsion matrix and applied to the ears to downregulate its expression level.Then H&E staining,tissue immunohistochemistry and fluorescence,Western Blot,and qRT-PCR were employed to analyze and compare the psoriasis-like inflammation in each group.4.Analysis on the effects of NETs on keratinocytes(KCs)and the underlying mechanismsNETs were isolated from neutrophils of psoriasis patients and healthy controls,and then used to stimulate primary KCs for indicated time.Western Blot,qRT-PCR,and ELISA were used to detect the expression and secretion of proinflammatory factors such as LCN2 in KCs.The protein components of NETs from psoriasis patients and healthy controls were compared and analyzed by mass spectrometry.The siRNA targeting multiple signaling pathways were transfected into KCs to validate the activation of receptors and the downstream signaling pathways triggered by NETs.5.Analysis on the role of LCN2 in psoriasis-like mouse modelLCN2 neutralizing antibody and isotype control antibody were i.p.injected to reduce LCN2 protein levels in IMQ-induced or K14-VEGF knockout psoriasis-like mouse model.H&E section staining,tissue immunohistochemistry and fluorescence,and qRT-PCR were used to compare the psoriatic-like inflammation in mice of each group.6.Analysis on the underlying mechanism of LCN2 regulating neutrophil chemotaxis and activationTranswell experiment was used to study the effect of LCN2 on neutrophil chemotaxis.Cell fluorescence,qRT-PCR,Western Blot,and ELISA were used to detect the activation of neutrophils induced by LCN2.7.Analysis on the functions of GPP neutrophils on KCsNeutrophils were isolated from peripheral blood of GPP patients and healthy controls using magnetic beads positive sorting system,and then co-cultured with KCs.In some conditions,the exosome release inhibitor,GW4869,was used to pretreat neutrophils to block the release of exosomes.CCK-8 and qRT-PCR were used to detect the proliferation and cytokine expression of KCs.8.Analysis on the functions of neutrophil exosomes in the autoinflammation of GPPExosomes were isolated and collected using ultracentrifugation method from the supernatant of neutrophils that isolated from GPP patients and healthy controls.Transmission electron microscopy(TEM),Western Blot,and nanoparticle size analysis were used to identify exosomes.Then Dil labeled exosomes were co-cultured with KCs,and cell immunofluorescence was used to observe the uptake of exosomes by KCs.Western Blot,qRT-PCR,cell immunofluorescence,and ELISA were used to detect the activation of KCs by neutrophil exosomes.The protein components and functional molecules of exosomes were analyzed by mass spectrometry.Results:1.Psoriatic neutrophils are pre-activated and form NETsFlow cytometry results showed that psoriasis neutrophils underwent spontaneous death at a higher rate than healthy neutrophils in vitro.The microarray gene expression data showed that a large number of genes were upregulated.Using Gene ontology analysis,the differentially expressed genes in psoriasis vs.healthy neutrophils could be assigned to various inflammatory functions,including“chemotaxis”,“response to stimulus”,and“leukocyte activation”,indicating that circulating neutrophils are pre-activated in psoriasis patients.And these increased genes found in psoriasis neutrophils were genes encoding for enzymes,proteases,and cathelicidin such as NE,MPO,LCN2,etc.,which have been shown to be associated with NETs formation.2.Neutrophils participate in IMQ-induced psoriasis-like inflammation by releasing NETsWe depleted neutrophils first and then injected back NETs subcutaneously into the ears,followed by IMQ induction for 7 days in vivo.Strikingly,we observed less erythematous and scaly plaques based on H&E staining,thinner epidermis,and decreased influx of dermal immunocytes in the lesions in neutrophil-depleted group.In addition,the expression of inflammatory factors such as IL-1βand LCN2 in the epidermis of mice in neutrophil-depleted group was significantly downregulated.However,injecting back murine NETs subcutaneously would increase the above-mentioned indicators of inflammation.3.Targeting NETs in vivo attenuates the psoriasis-like inflammation in IMQ miceIn the IMQ-induced mouse model,subcutaneous injection of NETs inhibitor CI-amidine or Dnase I that disrupts NETs structures over a 7-day period resulted in lighter scaling,thinner epidermis,and fewer inflammatory infiltrates,as observed by visual inspection and H&E staining.The qRT-PCR analysis of inflamed back skin from mice revealed statistically lower expressions of inflammatory mediators in the group with CI-amidine or Dnase I treatment.Accordingly,the number of infiltrated immune cells in the dermis were dramatically decreased because of CI-amidine or Dnase I disposition.4.NETs activate inflammatory responses in KCs through TLR4 receptor and its downstream MyD88-NF-κB p65 pathwayFirst,the qRT-PCR results showed that NETs could promote the expression and secretion of various inflammatory molecules in KCs,such as cytokine IL-1βand IL-36γ,chemokines CXCL1/2/8 and CCL20,and LCN2,among which LCN2 expression was most up-regulated.Then,qRT-PCR,Western Blot,and tissue immunofluorescence detected that NETs induced the expression of TLR4 in KCs,which was also highly expressed in psoriatic lesions.Silencing TLR4 and its downstream signaling molecules such as MyD88 or NF-κB p65 using siRNA in KCs would inhibit the regulatory effects of NETs on the gene and protein level of proinflammatory mediators.5.Targeting NETs downstream signaling TLR4-LCN2 attenuated IMQ-induced psoriatic-like dermatitisThe qRT-PCR,Western Blot,tissue immunohistochemistry and fluorescence resulted illustrated that the siRNA or inhibitor targeting TLR4 in IMQ mice,or neutralizing LCN2 in IMQ or K14-VEGF mice reduced the psoriasis-like inflammation,the expression of various inflammatory factors,and especially the infiltration of neutrophils in skin lesions.6.LCN2 modulates the chemotaxis and activation of neutrophilsTranswell experiments and qRT-PCR results showed that LCN2 could induce neutrophil chemotaxis and further activate them to produce a variety of pro-inflammatory and chemokines.Pre-incubating neutrophils with signaling inhibitors revealed that ERK1/2 and P38 MAPK signaling pathways were the main signaling pathways involved in LCN2-induced chemotaxis and activation in neutrophils.7.GPP neutrophils and their exosomes upregulate the expressions of GPP-associated genes in keratinocytesThe CCK-8 result showed that neither GPP neutrophils or healthy neutrophils could promote the proliferation of primary KCs.However,as the qRT-PCR results showed,GPP neutrophils induced higher expression of a variety of inflammatory mediators in keratinocytes than healthy neutrophils did,which could promote more neutrophil influx and autoinflammation.However,neutrophils pre-treated with exosome inhibitors could not significantly upregulate the inflammatory genes in KCs.The cell immunofluorescence staining showed that these Dil-labeled neutrophil exosomes were already internalized by keratinocytes at 3 h,and then could further activate the innate immune functions of KCs.8.Neutrophil exosomes carry a variety of immune proteins to promote the autoinflammatory responses of GPPMass spectrometry analysis of neutrophil exosomes from GPP patients and healthy controls showed that GPP neutrophil exosomes carried a variety of granule proteins and inflammation-related molecules such as OLFM4.The qRT-PCR,Western Blot,and ELISA experiments confirmed that OLFM4 recombinant protein could activate KCs to secrete the above-mentioned proinflammatory mediators and chemokines,thereby inducing more neutrophil chemotaxis and aggregation in the skin lesions to form sterile pustules,aggravating the phenotype of GPP.Conclusion:In this study,we reveal that psoriatic neutrophils are pre-activated and form NETs,which can activate the TLR4 and its downstream signaling pathway in KCs,thus inducing the expression and secretion of various proinflammatory mediators.The highest expressed protein in NETs-treated KCs,LCN2,induces the chemotaxis and activation of neutrophils,forming a positive inflammatory loop that aggravates the local inflammation of the psoriatic lesions.In addition,exosomes derived from GPP neutrophils activate keratinocytes to express and secrete proinflammatory factors and neutrophil chemokines,promoting the formation of pustules and aggravating the autoinflammation of GPP.Our research clarifies the way in which neutrophils participate in the development of psoriasis,enriches the understanding of the pathogenesis of psoriasis,and provides new ideas for the clinical treatment of psoriasis in the future.