Study On The Pathogenesis Of Palmoplantar Pustulosis

Background and Objective:Pustular psoriasis can be divided into two types according to the different pathogenic sites:generalized pustular psoriasis?GPP?and palmoplantar pustulosis?PPP?.There are pustules all over the body of GPP patients who often have fever.IL36RN gene?Interleukin-36 receptor antagonist gene?mutation which triggers the abnormal inflammatory reaction pathway is the pathogenesis of some GPP patients.IL-36Ra,encoded by IL36RN,is a natural antagonist of IL-36y.It inhibits the inflammation mediated by IL-36y through binding to IL36R?IL-36 receptor?.The structural or functional abnormality of IL-36Ra caused by IL36RN mutation results in abnormal activation of IL-36y which acts via the mitogen-activated protein kinase?MAPK?and?nuclear transcription factor-KB?NF-?B pathways to stimulate keratinocyte secretion of IL-8.Interleukin?IL?-8 is a chemotaxin and activator of neutrophils,which can recruit neutrophils and is involved in the formation of aseptic pustules in generalized pustular psoriasis.Researches indicated IL-8,IL-36y serum level in GPP patients significantly elevated comparing with normal controls.Furthermore,IL-8 and IL-36y expressions in the skin lesion of GPP were higher than normal controls or other inflammatory skin disease such as lichen planus.But IL-36Ra expression levels decreased significantly.Palmoplantar pustulosis is a chronic,refractory and inflammatory skin disease which is characterized by erythema,pustule and scale restricted to palms and soles.It is considered as a localized form of pustular psoriasis.The epidemiological data of PPP is limited.It was reported that the prevalence rate in Japan was 0.12%,which occurs more common in women aged 45-65.The pathogenesis of PPP is complex.Heredity,immunity,sweat duct,type IV allergic reaction,infection and thyroid disease were reported to be related with PPP.Some researches indicated the abundant sweet gland and ducts on plams and soles with abnormalities such as sweat excretion disorder,structural damage are related with the inflammatory process of PPP.The secretion of sweat from skin lesions of PPP patients was lower than that of non-lesional lesions and healthy controls.Inflammatory cells such as histocytes and T cells were infiltrated around the sweet duct.However,studies of the pathogenesis in PPP are still lacking.There is no relevant research on the roles of IL-8,IL-36?,IL-36Ra in the pathogenesis of PPP.Also,the researches of IL36RN mutation in Chinese PPP patients are insufficient.In this study,we intend to explore the role of IL-8,IL-36y,IL-36Ra and sweat ducts in the pathogenesis of PPP,and explore whether PPP patients have IL36RN gene mutation like GPP patientsPatients:Blood samples and clinical materials were collected from all 51 PPP patients diagnosed according to their pathology or clinical features who consulted to the dermatological clinic of Peking Union Medical College Hospital from May,2014 to December,2016.Genomic DNA was extracted from peripheral blood leukocytes.All five IL36RN coding exons,as well as their flanking introns,were amplified by PCR using the extracted genomic DNA as template.The lesions of 17 patients with PPP were acquired for immunohistochemical staining,at the same time,14 PSV and 12 normal skin lesions were acquired as control groups.In addition,fresh tissue samples from 7 PPP patients were subjected to RNA extraction and reverse-transcription-quantitative real-time polymerase chain reactions?RT-PCR?.8 PSV patients and 6 healthy controls were included as controls.All PPP and PSV patients were in the active disease period and had not taken any topical or systemic therapy within 6 weeks before consulting at our hospital.The normal skin tissues were acquired from pigmented nevus patients.Methods:1.IL-8?IL-36y and IL-36Ra mRNA were evaluated by RT-PCR.The total skin tissue RNA was extracted by the Trizol method and then reverse-transcribed to make cDNA.Quantitative real-time PCR was performed using the Applied Biosystems 7500 Real-Time PCR System and FastStart Universal SYBR Green.The amplified PCR products were verified by agarose gel electrophoresis and observation under ultraviolet light.Gene expression was quantified using the relative quantity = 2-??CT method,normalized to an endogenous control?glyceraldehyde 3-phophate dehydrogenase?.2.Immunohistochemical staining for IL-8.IL-36?,IL-36Ra and EMA:Specimens were embedded in paraffin blocks and 4 ?m sections were prepared for immunohistochemistry.Envidion two steps methods was applied for staining and DAB was used for chromogenic reaction.Phosphate-buffered saline was substituted for the primary antibody as a negative control.Each digital slice was evaluated by two investigators,independently and blind.The staining intensity of IL-8,IL-36? and IL-36Ra in the epidermis was evaluated using semi-quantitative grading system.Staining intensity scores were represented by numbers as follows:0 = no staining,1=weak staining/light brown-yellow,2 = moderate staining/brown-yellow,3 = strong staining/chocolate brown?range of scores:0-3?.3.HaCaT cells were cultured and stimulated with IL-36??80,100 and 120 ng/ml?.HaCaT cells treated with each concentration of IL-36? were seeded into 6-well culture plates.After 48 hours,total RNA was isolated and reverse-transcribed to make cDNA for quantitative real-time PCR to evaluate the IL-8 mRNA.In addition,cell supernatants were collected and IL-8 cytokine levels were measured by enzyme-linked immunosorbent assay.4.The genomic DNA was extracted from blood leukocytes of all 51 PPP patients using the AxyPrep Blood Genomic DNA Miniprep kit?Axygen Biosciences?and amplified by polymerase chain reaction?PCR?to include each IL36RN coding exon?NM₀12275?and flanking introns.The PCR products were sequenced using the Sanger method with ABI3730XL.SIFT/PolyPhen software was used to predict the function of mutant Proteins.Results:1.IL-8 mRNA was expressed at significantly higher levels in PPP lesions compared with PSV or healthy control skin?P?0.01 for both?.The expression of IL-36y mRNA was significantly greater in PPP lesions than in control skin?p=0.01?,whereas no significant difference was seen between the PSV and HC groups?P = 0.15?.IL-36Ra mRNA was significantly over-expressed in PPP lesions and PSV compared with control skin?P ? 0.001&P ? 0.01?.2.The IL-8 immunoreactive intensity of PPP specimens was markedly higher than PSV patients and controls?P<0.01&P=0.04?.The expression of IL-36y was significantly higher in PPP lesions than in controls?p=0.01?.Compared with PSV,the intensity of IL-36y in PPP was not significantly different?p=0.65?.No significant difference was seen in IL-36Ra protein expression between the PPP,PSV and healthy control groups?p?0.10?.3.IL-8 was abundantly expressed in neutrophils in the pustules of PPP lesions.Apart from Munro's abscesses,PSV tissues were negative for IL-8.IL-36Ra was positive in PPP,PSV and controls,with the cytoplasm of keratinocytes being stained.IL-36y and EMA staining was positive in the cells lining and keratinocytes surrounding PPP pustules.The localization of IL-36? and EMA staining overlapped.Moreover,the sweat duct cells in dermis stained positively for IL-36y.4.Interleukin-8 mRNA transcription level and IL-8 secretion in cell supernatant were noth higher in HaCaT cells treated with different concentration of IL-36y?80ng/ml,100ng/ml,120ng/ml?compared with blank control group.When IL-36y was cultured at a concentration of 100 ng/ml,the IL-8 increased most significantly,and there was a statistically significant difference from the control group?P=0.03&P=0.01?.5.Among the 51 PPP patients,four different single-base substitutions were identified in nine patients.The mutations were c.140A>G/p.Asn47Ser in five patients,c.258G>A/p.Met86IIe in two patients,and c.115+6T>C and c.169G>A/p.Va157IIe in one patient each.All mutations were heterozygous.Comparison with the human genome database and reported literature suggested that these variants may not be pathogenic mutations causing PPP.Furthermore,there was no difference in disease severity,onset age or disease duration between patients with these heterozygous IL36RN variants and those without?all P>0.1?.Conclusion:1.IL-8 and IL-36? were overexpressed in PPP lesions and IL-36Ra expression was normal.IL-8 and IL-36y play key roles in the pathogenesis of PPP,whereas IL-36Ra may not be a pathogenic factor for PPP.2.The pustules may form at acrosyringia associating with the overexpression of IL-36y and IL-8.Sweet duct cells can secrect IL-36y.3.IL-36? can stimulate HaCaT cells to abnormally secrete IL-8.4.No pathogenic mutation of IL36RN was found in 51 Chinese PPP patients.The four variants of IL36RN identified did not appear to be associated with the generation of PPP.