| Objective: Sporothrix is a two-state fungus that can cause Sporothrix disease.It is composed of a mixture of Sporothrix brasiliensis,Sporothrix schenckii,Sporothrix globosa,Sporothrix Luri,Sporothrix Mexico and Sporothrix pallidus.It exists in the form of mycelium at a temperature below 37 ° C and budding yeast at a temperature of 37 ° C or higher.This characteristic form is important for determining the transformation from mycelial to yeast.Sporotrichosis occurs by inoculating soil,plants and organic matter contaminated by fungi.Sporotrichosis is usually characterized by papules or pustules,forming ulcerative nodules involving local lymphatic vessels.Sporotrichosis can be divided into skin type,lung type and disseminated type,of which skin type is the most common form.When Sporothrix infects the body,innate immunity is the initial defense line.Some studies have shown that sporotrichosis mouse model involves a variety of immune reactions.Sporothrix infection can cause type 2 T cell immunity,and the antigen components of Sporothrix schenckii cell wall inhibit type 1 T cell immunity in the experimental model of sporotrichosis.Macrophages,as important immune cells in natural immunity,are type1 T cell responses,which can kill invading pathogens through phagocytosis and oxygen burst.Disabled homolog 2(DAB2)was originally identified in CSF-1stimulated macrophages as the low-density lipoprotein receptor(LDHR)and type II growth factor β receptor(TGFβR)And integrin β 1.DAB2 is involved in multiple immunity signaling pathways,such as Ras /MAPK and TGF-β pathway.Studies have shown that in adipose tissue,DAB2 inhibition significantly activates dendritic cells and their immune response function.However,the expression of DAB2 in macrophages and its immunomodulatory effect in sporotrichosis are not clear.c-JUN amino terminal kinase(JNK)signaling pathway is involved in many important cellular processes such as cell differentiation,transformation and survival.Studies have shown that the activation of JNK pathway is a switch for macrophages to transform from anti-inflammatory phenotype to pro-inflammatory phenotype,and is necessary for macrophage aggregation in inflammatory tissues and insulin resistance induced by high-fat diet.The role of JNK pathway in sporotrichosis and whether it participates in the regulation of DAB2 to affect macrophage inflammatory response are unknown.It has been reported that hyperthermia therapy can effectively treat sporotrichosis,but it is not clear whether hyperthermia therapy plays an immunomodulatory role through DAB2.Therefore,we aimed to explore the immune response caused by Sporothrix schenckii infection of macrophages and its mechanism,as well as the regulation of 42 o C hyperthermia on the immune function of macrophages.Methods: 1.In this study,firstly,the expression of DAB2 gene in the skin lesions of sporotrichosis mice and the skin of normal mice was detected by immunoblotting and fluorescence quantitative PCR.To further detect whether the expression of DAB2 in macrophages in sporotrichosis lesions was increased.In order to further clarify whether the DAB2 gene expression of macrophages in sporotrichosis has been changed,we extracted bone marrow primary macrophages from mouse femoral shaft for in vitro study,stimulated bone marrow primary macrophages according to the ratio of bacteria to cells reported in the previous literature,and detected the expression of DAB2 gene.2.Because the effect of DAB2 gene on some inflammatory factors in macrophages has been reported,we used fluorescence quantitative PCR to detect the m RNA levels of Il6,Tnfα,Il1β,Arg1,Il10,and Mgl1 in primary macrophages stimulated by Sporothrix schenckii.Then we knocked down the DAB2 gene of the primary macrophages to detect m RNA levels of Il6,Tnfα,Il1β,Arg1,Il10,and Mgl1.3.In order to detect the possible regulatory mode of Sporothrix on DAB2 gene in macrophages,we found that c-JUN may be a possible transcription factor of DAB2 gene through http://jaspardev.genereg.net.The expression of c-JUN and the JNK pathway in macrophages stimulated by Sporothrix schenckii were detected by Western Blot.After activating the JNK activity of macrophages with the activator,then the macrophages were stimulated by Sporothrix schenckii,the protein levels of DAB2,c-Jun and JNK phosphorylation were detected by immunoblotting,so as to explain the regulation of JNK pathway on c-JUN and DAB2 in the macrophages stimulated by Sporothrix schenckii.4.We knocked down the c-Jun protein of bone marrow primary macrophages and added Sporothrix schenckii stimulation to detect the transcription of DAB2 gene.At the same time,chromatin immunoprecipitation and luciferase gene reporting were used to detect whether c-Jun participated in DAB2 gene transcription as a transcription factor.5.Finally,proteins were extracted from the skin lesions of mice with sporotrichosis and the skin of normal mice without sporotrichosis.The expressions of phosphorylated proteins of c-JUN and JNK pathways were detected by Western Blot to help verify the results of cell experiment.Results: 1.Western Blot showed that the expression of DAB2 protein in the skin lesions of sporotrichosis mice was significantly higher than that of normal mice.Immunofluorescence showed that the number of macrophages expressing DAB2 protein increased in the same field of view with the same magnification,and the difference was statistically significant.According to the ratio of Sporothrix schenckii:cell = 5:1,when the corresponding number of Sporothrix schenckii stimulated bone marrow primary macrophages,the m RNA level of DAB2 first increased and then decreased within 24 hours,and the difference was statistically significant.The expression of DAB2 protein in macrophages was stimulated by Sporothrix schenckii and increased,and maintained a high level within 24 hours.The difference was statistically significant.2.Inflammatory factor Tnfα in macrophages stimulated by Sporothrix schenckii、 The m RNA levels of Il10 and Mgl1 increased,Il6 and Il1 increased and Arg1 m RNA levels decreased,the difference was statistically significant.Compared with negative control cells,macrophages with DAB2 gene knockdown had lower levels of Il6 and Tnfα after stimulation by Sporothrix schenckii.The m RNA levels of Il10 and Mgl1 decreased and Il1 and Arg-1m RNA have no difference.3.In Sporothrix schenckii stimulated macrophages,the protein expression of c-jun increased at 1 hour,decreased continuously at 3 ～ 6 hours,increased at 12 hours and decreased again at 24 hours.Moreover,the stimulation of Sporothrix schenckii reduced the phosphorylation level of JNK.When macrophages were stimulated with JNK activator Juglanin,there was no significant change in the expression of DAB2 protein,the expression of c-JUN protein and the level of JNK phosphorylation.4.In c-JUN protein knockdown macrophages,the m RNA level of DAB2 decreased,and the difference was statistically significant.Compared with negative control cells,the m RNA level of DAB2 decreased after c-JUN protein knockdown macrophages were stimulated by Sporothrix schenckii,and the difference was statistically significant.Chromatin immunocoprecipitation showed that c-JUN antibody could precipitate more DAB2 gene promoter fragments than negative control antibody.The results of luciferase reporter gene showed that the plasmid constructed with-2100 base length upstream of the promoter region emitted high-energy fluorescence.The fluorescence intensity of the plasmid constructed with-630 base length upstream of the promoter region decreased significantly.5.Compared with normal skin tissue,JNK phosphorylation level and c-JUN protein expression level in skin lesions of sporotrichosis mice were lower,and the differences were statistically significant.6.Compared with 37 o C treatment group,42 o C hyperthermia decreased the expression of c-JUN and DAB2,and the difference was statistically significant.7.The number of promoter regions of Dab2 that c-JUN and c-FOS combined in the 42 o C group was reduced compared with that in the 37 o C group in macrophages.8.c-JUN and c-FOS were located in the nucleus when cells were treated with 37 o C.c-JUN of macrophages treated with 42 o C were located in the nucleus and c-FOS was in the cytoplasm.Conclusion: 1.The expression of DAB2 protein in the rash of Sporothrix schenckii mice increased,and the expression of DAB2 protein in dermal macrophages increased.The transcription and translation levels of DAB2 in primary bone marrow macrophages in vitro were induced by Sporothrix schenckii,and Sporothrix schenckii regulated the m RNA levels of inflammatory factor Tnfα,Il1 β,Arg-1,Il10 and Mgl1 through DAB2 gene.2.Sporothrix schenckii inhibited JNK phosphorylation and induced c-JUN expression.3.c-JUN can play a regulatory role as a transcription factor of DAB2 gene.4.JNK pathway was inhibited and c-JUN protein expression was increased in sporotrichosis mice.5.42 o C hyperthermia inhibited DAB2 transcription by inhibiting the expression of c-JUN and promoting c-JUN / c-FOS dimer separation. |
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