| Background:Pustular psoriasis（PP）is a special type of psoriasis.An epidemiological survey in china in 1984 showed that the prevalence of psoriasis was 0.123%,of which pustular psoriasis accounted for 0.69%of all psoriasis patients.The treatment of pustular psoriasis has always been a thorny problem for dermatologists,often encountering problems such as improper dose of systemic treatment,unreasonable application of systemic glucocorticoids and antibiotics,and lack of effective supportive treatment.In the context of the emerging application of psoriasis biological agents,in order to develop more rapid,effective and safe treatment programs,it is necessary to obtain a more precise understanding of the pathogenesis of psoriasis.Pustular psoriasis and psoriasis vulgaris（Ps V）share both commonalities and characteristics,and the uniqueness between these two types is reflected in the adaptive immune component（autoimmunity）and the innate immune component（autoinflammation）with different aspects of focus.In pustular psoriasis,the role of innate immunity cannot be ignored,with c-x-c motif chemokine ligand（CXCL）1,CXCL2,and CXCL8 recruiting neutrophils to the epidermis,forming Munro or Kogoj microabscesses and macroscopic pustular lakes,while innate immunity components such as interleukin（IL）-1,IL-6,and tumor necrosis factor（TNF）-αare released into the circulation,resulting in acute phase responses.As effector cells of innate immunity,neutrophils play a crucial role in the pathogenesis of pustular psoriasis,and they can connect the innate immune system and adaptive immune system,etc.,and participate in constituting a complex immune response network.Toll-like receptors（TLRs）act on the dendritic cell（DC）through the myeloid differentiation factor 88（My D88）signaling pathway to release a large number of chemotaxis factors that promote the further proliferation and abnormal differentiation of epidermal cells,migration and infiltration of dermal inflammatory cells in pustular psoriasis.TLR7 is a member of TLR9 subfamily and plays an important role in the induction of DC to regulate immune responses.CXCL16 is a novel chemokine mainly released by DC.The specific receptor chemokine receptor 6（CXCR6）of CXCL16exists on neutrophils.CXCL16 specifically binds to CXCR6,which may chemotactic neutrophils and further induce inflammation,but the specific mechanism is not yet clear.Through the research of this project,we will have a more comprehensive and in-depth understanding of the"innate immune response"of pustular psoriasis,which is expected to fundamentally block neutrophils and thereby inhibit the massive secretion of cytokines,so as to accurately target the disease chemotherapy provides a scientific theoretical basis.Objective:This project intends to clarify the following issues:（1）Through differential gene function enrichment analysis and transcription factor co-expression network construction,the genes and inflammatory factors related to pustular psoriasis were studied,and inflammatory signaling pathways for follow-up research were established.（2）To investigate the regulation of CXCL16 expression by TLR7 in pustular psoriasis and the effect of CXCL16 on inflammation-related factors dominated by neutrophil activation such as IL-8,IL-36α,IL-36β,IL-36γ,G-CSF,GM-CSF,etc.To further explore the correlation between the above inflammatory factors and the severity of the disease and the expression changes before and after targeted biological therapy.（3）Establishment of imiquimoid（IMQ）-induced wild-type（WT）,Cd11c-Cre Myd88 f/f and Mrp8-Cre Cxcr6 f/f knockout psoriasis-like mouse models were used as the research objects to explore the effect of activation/blocking of TLR7 on the expression of CXCL16 and neutrophil activation.（4）DC co-culture with neutrophils after in vitro activation/blocking of TLR7,and observed the chemotaxis to CXCL16,neutrophil activation,apoptosis and migration,as well as the influence of the expression of innate immune-related factors dominated by neutrophil activation.Methods:The study collected skin and blood samples from 25 patients with pustular psoriasis and their gender and age-matched 25 healthy controls who were treated in the dermatology ward of our hospital from December 2019 to September 2021.Healthy controls were obtained from outpatient volunteers.Among them,blood samples were obtained from 20 patients with pustular psoriasis and 20 healthy controls.Skin samples were obtained from 5 patients with pustular psoriasis and 5 healthy controls.We performed generalized pustular psoriasis area and severity index（GPPASI）,generalized pustular physician global assessment（GPPGA）,body surface area（BSA）and dermatology life quality index（DLQI）scores on the above patients with pustular psoriasis.The WT,Cd11c-Cre My D88 f/fand Mrp8-Cre Cxcr6 f/f gene knockout mouse models were selected,and the intervention experiments were carried out after IMQ modeling.In vitro cell experiments were performed at the level of DCs and neutrophils.（1）Transcriptome data analysis of skin lesions in pustular psoriasisWe mainly used Fastqc to evaluate the quality of the raw data of the dataset sequencing.Gene annotation was performed on the matched data with the genome database,and the expression of each gene was quantified with Feature Counts software to obtain the original expression matrix of each sample.Principal component analysis（PCA）and hierarchical clustering analysis（HCA）clustering analysis was performed using R language.For the differentially expressed genes found in pustular psoriasis skin lesions and healthy control skin tissues,the kyoto encyclopedia of genes and genomes（KEGG）database was used to analyze the enrichment results of signaling pathways.The functions of genes were analyzed at three gene ontology（GO）levels of process,molecular function and cellular composition.The differential transcription factor co-expression network was constructed to study inflammatory factors associated with pustular psoriasis.To investigate changes in the immune microenvironment（IME）during the progression of pustular psoriasis,we inferred the composition of immune cells and stromal cells from ribonucleic acid-sequencing（RNA-seq）data and analyzed for the proportions of cell subsets.（2）To investigate the effect of TLR7-regulated CXCL16 expression on neutrophil activation in pustular psoriasisDCs and neutrophils were isolated and purified,and the relative expression level of DC-released CXCL16 in pustular psoriasis and healthy controls was detected by ELISA before and after TLR7 stimulation,as well as the inflammation-related factors dominated by neutrophil activation such as IL-8,IL-36α,IL-36β,IL-36γ,G-CSF,GM-CSF,etc.before and after rh CXCL16 stimulation.The gene expression levels of inflammatory factors dominated by neutrophil activation in the skin of pustular psoriasis and healthy controls were detected by real-time quantitative polymerase chain reaction（RT-PCR）.The factors with the most significant expression were selected,and their expression was detected by skin immunofluorescence and immunohistochemistry.The correlation between the severity of pustular psoriasis and the expression levels of inflammatory factors dominated by neutrophil activation was statistically analyzed by GPPASI,GPPGA,BSA,and DLQI scores.Through the phase II clinical study of IL-36R antagonists,the GPPASI,GPPGA,BSA and DLQI scores,and the changes in the expression levels of therapeutic targets and neutrophil activation-related factors such as TLR7,CXCL16,IL-36α,IL-36βand IL-36γin patients with representative follow-up nodes were observed.The efficacy of adalimumab,secukinumab,ixekizumab,ustekinumab and guselkumab before and after treatment was evaluated,and the response rates of GPPASI 50,GPPASI 75,and GPPASI 90 were observed,as well as GPPGA,BSA and DLQI scores improvement were evaluated.The expression levels of therapeutic target and neutrophil activation-related factors such as TLR7,CXCL16,TNF-α,IL-17,IL-12,IL-23,IL-36α,IL-36βand IL-36γwere further detected before and after treatment.（3）In vivo verification in animal models of TLR7-regulated CXCL16 expression on neutrophil activationThe WT,Cd11c-Cre Myd88 f/fand Mrp8-Cre Cxcr6 f/fgene knockout mouse models were established with imiquimod,and the successful modeling was determined by epidermal thickness detection,PASI score and skin pathology.H&E staining was used to observe the changes in the severity of skin lesions in each mouse model before and after TLR7 interference,and immunohistochemistry was used to detect its regulatory effect on CXCL16.Furthermore,immunofluorescence detection was used to observe the expression of neutrophil surface activation marker in each mouse model before and after TLR7 interference.RT-PCT was used to investigate the gene expression of inflammatory factors dominated by neutrophil activation in each mouse model before and after TLR7 intervention.（4）In vitro verification of TLR7-regulated CXCL16 expression on neutrophil activation,apoptosis and migrationWe used TLR7 agonist and TLR7 block to to act on DCs,and then co-cultured them with neutrophils,respectively,and used RT-PCR,western blot（WB）,Transwell,apoptosis detection and other methods to investigate the activation,apoptosis and migration of neutrophils before and after TLR7 interference in vitro,as well as the secretion of innate immune-related factors dominated by neutrophil activation.Result:（1）The results of transcriptome data analysis of skin lesions in pustular psoriasisThrough transcriptome analysis of skin lesions in pustular psoriasis,228 up-regulated genes and 242 down-regulated genes were obtained from differentially expressed genes analysis of pustular psoriasis skin lesions and healthy control skin tissues co-founded in the dataset.In the differential gene volcano plot,genes such as IL-36RN,TLR7,SERPINB4,CARD14,DEFB4A,DEFB4B,SPRR2A,SPRR2B,SPRR2D and SPRR2F were highly expressed in the pustular psoriasis group;Genes such as THRSP,GPD1,FADS2,ANGPTL7,SEC14L6,GAL,ADIPOQ,PPP1R1A,AWAT2 and PM20D1 were lowly expressed in the pustular psoriasis group.Transcription factor co-expression network construction results established a positive correlation between TLR7-CXCL16 inflammation positively correlation pathway.The results of IME detection and analysis indicated that there was an immune microenvironment with significantly increased proportion of neutrophils in patients with pustular psoriasis.（2）The results of TLR7 regulation of CXCL16 expression on neutrophil activation in pustular psoriasisCompared with healthy controls,patients with pustular psoriasis had higher expression of inflammation-related factors dominated by neutrophil activation such as CXCL16,TNF-α,IL-1,IL-6,IL-8,IL-12,IFN-α,IFN-β,IFN-γ,G-CSF,GM-CSF,IL-36α,IL-36βand IL-36γ.The expression of CXCL16 increased after stimulation by TLR7,and the expression of the expressions of various neutrophil inflammation-related factors were significantly increased after rh CXCL16 stimulated.The severity of pustular psoriasis scores was positively correlated with inflammation-related factors dominated by neutrophil activation.The results of phase II clinical studies suggested that IL-36R antagonists could reduce the GPPASI,GPPGA,BSA and DLQI scores of patients,inhibited the continuous chemotaxis of CXCL16 and the continuous activation of neutrophils,and reduced the expression levels of neutrophil activation-related factors such as IL-36α,IL-36βand IL-36γ.The response rates of GPPASI 50,GPPASI 75,and GPPASI 90 in 19 patients with pustular psoriasis treated with adalimumab,secukinumab,ixekizumab,ustekinumab,and guselkumab all increased steadily,and the GPPGA,BSA and DLQI scores all showed a continuous downward trend.The expression levels of target factors and neutrophil activation-related factors such as CXCL16,TNF-α,IL-17,IL-12,IL-23,IL-36α,IL-36βand IL-36γall showed a downward trend compared with those before treatment.（3）The results of in vivo verification in animal models of TLR7-regulated CXCL16 expression on neutrophil activationThe skin appearance,epidermal thickness detection,PASI scores and pathological results indicated that the IMQ-induced psoriasis-like animal model was successfully established.TLR7 stimulation could aggravate IMQ-induced WT mouse model psoriasis-like mouse model skin lesions,promote CXCL16 expression,and stimulate neutrophil activation,innate immune activation-based related inflammatory factors such as TNF-α,IL-1,IL-6,IL-12,IFN-α,IFN-β,IFN-γ,G-CSF,GM-CSF,IL-36α,IL-36βand IL-36γgene expression increased;TLR7 block showed the opposite.The Cd11c-Cre Myd88 f/f gene knockout psoriasis-like mouse model did not change the skin lesions through TLR7 interference,the expression of CXCL16 and the neutrophil chemotaxis and activation were restricted.The gene expression of each related inflammatory factors was also disturbed.The Mrp8-Cre Cxcr6 f/fgene knockout psoriasis-like mouse model did not change the skin lesions through TLR7 interference.The expression of CXCL16 was increased by stimulation of TLR7,and decreased by inhibition of TLR7.The neutrophil chemotaxis and activation were still restricted.The gene expression of each related inflammatory factors was also disturbed.Therefore,Cd11c-Cre Myd88 f/f and Mrp8-Cre Cxcr6 f/f gene knockout psoriasis-like mouse models confirmed that TLR7 is required to induce DC release of CXCL16 by activating the My D88 signaling pathway to act on neutrophils,thereby promoting the development of psoriatic skin inflammation.development occurs.（4）The results of in vitro verification of TLR7-regulated CXCL16 expression on neutrophil activation,apoptosis and migrationIn vitro experiments,TLR7 agonist could slow down neutrophil apoptosis,enabled neutrophil activation and migration,as well as the increased expression levels of innate immune-related inflammatory factors such as CXCL16,TNF-α,IL-1,IL-6,IL-12,IL-36α,IL-36βand IL-36γ.TLR7 block could accelerate neutrophil apoptosis,weaken neutrophil activation and migration ability,and reduce the expression of the above inflammatory factors.Conclusion:（1）Genes on pustular psoriasis such as IL-36RN,TLR7,SERPINB4 and CARD14were up-regulated,the TLR7-CXCL16 was a inflammation positively correlation pathway and the IME was a significantly increased proportion of neutrophils.（2）The inflammation-related factors dominated by neutrophil activation were highly expressed in pustular psoriasis,and were positively correlated with the severity of the disease.（3）TLR7 can regulate the expression of CXCL16,which can chemotactically activate neutrophils,make high expression of related inflammatory factors and induce innate immune responses.（4）IL-36R antagonists could inhibit the continuous chemotaxis of CXCL16 and the continuous activation of neutrophils.Adalimumab,secukinumab,ixekizumab,ustekinumab,and gusekinumab were effective in the treatment of pustular psoriasis,and the expression levels of neutrophil activation-related factors declined with initial improvement of the inflammatory microenvironment.（5）Animal models confirmed in vivo that TLR7 might affect the release of CXCL16 from DCs by interfering with the My D88 signaling pathway,and the specific binding of CXCL16 to CXCR6 was blocked,thereby affecting neutrophils to play a pro-inflammatory role.（6）It was confirmed that TLR7 could affect the release of CXCL16 from DCs through the My D88 signaling pathway,and then act on the specific binding of CXCL16 to CXCR6,affecting neutrophils to play a pro-inflammatory role.From this,we speculated that TLR7-DC-CXCL16-neutrophils played an important role in pustular psoriasis and were expected to control disease progression by blocking this pathogenic axis. |
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