The Effects And Mechanisms Of Local Hyperthermia On Macrophages For Sporotrichosis

Introduction:Sporotrichosis is a chronic granulomatous fungal disease caused by Sporothrix schenckii complex and the lesions usually locate in exposed areas such as the face,neck and limbs.Sporotrichosis is a worldwide disease and is very harmful.The clinical process usually begain with the invasion of sporothrix through the damaged skin and mucosa,then going along the lymphatic vessels resulting in subcutaneous nodules,suppurative,ulcerative,exudative or granulomatous lesions.And very few patients could be infected by inhaled spores.For individuals with the deficiency of immune system,sporothrix schencki may spread to the central nervous system and viscera which may cause systemic infection and life-threatening ending.At present,the treatment of the disease is limited,mainly potassium iodide therapy,antifungal drugs.Since most of the patients live in mountainous area with poor living conditions,so the treatment is always using in irregular way.Therefore,it is very important to find out the immune process of sporptrichosis and figure out a more effective treatment method for this disease.The immune system that human body against sporotricosis is mainly consist of innate immune system and adaptive immune system.Natural barriers,immune cells and immune factors constitute innate immune system.The intact skin and mucous membrane constitute a natural barrier,and the dense cuticle on the surface of the skin can effectively resist the invasion of the sporothrix.The innate immune cells against sporothrix are mainly macrophages,neutrophils and dendritic cells.Immune molecules include complement,cytokines and reactive oxygen species?ROS?.Innate immune cells are the main force against sporothrix.The typical pathological manifestation of sporotrichosis is the"three zone"phenomenon,a large number neutrophils infiltrated in the centre which showing that neutrophils are the earliest reaction cells of phagocytes in the initial stage of infection?24h?.But neutrophils have very limited ability of killing sporothrix.As phagocytes,dendritic cells have phagocytic function,but the main function of dendritic cells is antigen presentation.In the process of human body fighting aganist sporothrix,acquired immunity also plays an important role.The important evidence is that when a HIV infector with severe acquired immunodeficiency is infected by sporothrix,it is usually disseminated,and the patient always does not respond to treatment very well.Macrophages have strong phagocytosis and antigen-presenting functions.They are the bridge between innate and adaptive immunity,and the main force fighting against sporothrix in human body.But the sporothrix engulfed by macrophages may still survive even reproduct,and this may be the reason of sporothrix evasion from the immune system attacks.Therefore,to explore how to improve the ability of macrophages to kill sporothrix is very important.Material and Methods:1.Experimental cells and miceThe paraffin sections of sporotrichosis patients were collected from our department.The mouse macrophage cell line Ana-1 was selected as the research cells.The primary cells were derived from the bone marrow derived macrophages and Naive CD4⁺T cells in the spleen of BalB/C mice.The porotrichosis models mice were all 6-8 weeks old and20±0.1g female mice.2.Local hyperthermia treatmentThermotherapy instrument?Patent No.ZL200720185403.3?was used to provide a non-contact fixed-point heating method,and the temperature was kept in the specified temperature range by infrared thermometer.Local hyperthermia sporotrichosis mice model:The target lesion was subjected to local hyperthermia at 42?,once a day for 3 consecutive days,each treatment lasted 30 min.A week later,the mouse were received two more consecutive treatments for two days.Then the mouse were received treatment once a week.Local hyperthermia for patients with fixed type sporotrichosis:The target lesion was subjected to local hyperthermia at 44?,once a day for 3 consecutive days,each treatment lasted 30 min.A week later,the patient received two more consecutive treatments.Then the patient received treatment once a week.Hyperthermia treatment to cells:Cells were treated with the warm water bathing at 37?or 42?for 30 minutes,and then put back into the carbon dioxide cell incubator.3.ImmunohistochemicalThe paraffin section was 4?m,and the expression of macrophage M1 marker CD86 and macrophage M2 marker CD206 were detected by immunohistochemical staining?SP method?.Paraffin tissue sections were treated through dewaxing and antigen repair,and the primary and the second antibody were applied before chromogenic,double staining and photo taking.5 vision fields were randomly selected under objective lens?x20?,and the ratio of CD206 stained cells to CD86 stained cells in the field of vision was counted.The average value was calculated.4.The effect of hyperthermia on the expression of M2 macrophage and some cytokine genesThe expression M2 macrophage and some cytokine genes was detected by fluorescent real-time quantitative PCR.Total RNA was extracted by using RNeasy mini kit and reverse-transcribed with PrimeScript^TMRT reagent kit.The cDNA obtained was used as a template for subsequent PCR amplification and relative quantitative result was analyzed with double standard curve method.5.The effect of hyperthermia on the proliferation of cellsThe effect of hyperthermia on the proliferation of macrophages was detected by MTS method.The cells were inoculated in 96 and were tested on zeroth days,1 days,2 days,3days and 4 days respectively.The absorbance value was observed at 490nm.6.Fluorescent Ca²⁺measurementThe Ca²⁺-sensitive fluorescent single wavelength dye Fluo-4 AM was used to measure changes in[Ca²⁺]_i at 1 mins,5 mins and 120 mins post hyperthermia.In Brief,cells were loaded with 5?M of Fluo-4 AM?Invitrogen?and then treated with hyperthermia for30 mins.The fluorescent density of Fluo-4 was then measured by fluorescence microplate reader?PerkinElmer?at 37?.Fluorescence was evoked by 488nm excitation wavelength and collected at 510 nm.7.Flow cytometry to detect the effect of hyperthermia on the apoptosis of cellsFor apoptosis analysis,cells were stained with FITC-conjugated Annexin V apoptosis detection kit?BD Biosciences,USA?or APC-conjugated Annexin V?BD Biosciences,USA?,respectively,in accordance with the manufacturer's instructions.In brief,the cells were trypsinized and washed with cold PBS,stained with APC-conjugated Annexin V?5ul?and PI?5ul?,or FITC-conjugated Annexin V?5ul?and PI?5ul?for 15 minutes at room temperature in the dark,and then analyzed by BD LSRFortessa?BD Bioscience?within 1 hour.8.Statistical analysisStudent's t test and one-way analysis of variance?ANOVA?were performed by GraphPad Prism software?Version 5.0?for statistical analysis,and a p value<0.05 was considered to be statistically significant.RESULTS:1.The polarization of macrophages and the differentiation of Th1 and Th2 in the lesions of patients with sporotrichosis.In the lesions of patients with sporotrichosis,the relative expression of Arginase-1 RNA was higher than that of iNOS,the relative expression of CD206 higher than normal skin.The relative expression of IL-2 RNA in patients with sporotrichosis is significantly lower than that in normal people?p<0.05?.The relative expression of IL-4 in patients with sporotrichosis is significantly higher than that in normal people?p<0.05?.The expression of IFN-?and IL-10 in lesions have no difference from that in normal people.By immunohistochemical staining,we observed that CD86 was slightly expressed in the lesions,and a strong expression of CD206 was observed in the lesions.2.BTP2 treated cells were resistant to hyperthermia induced apoptosis except those harboring HPV cervical cell linesBy using CRAC channel selective inhibitor BTP2,we tried to test if hyperthermia induces cellular apoptosis via regulating CRAC channel.For each cell line,cells were divided into four groups:negative control group?37°C?,BTP2 treated group?37°C?,hyperthermia treated group?45°C for all the cell lines?,and BTP2 and hyperthermia treated group?45°C for all the cell lines?.There was no difference of numbers of apoptosis and necrosis cells between negative control group?37°C?and BTP2 treated group?37°C?.Hyperthermia significantly increased the apoptosis levels in most cell lines,but failed to induce intense apoptosis in BTP2 pre-treated cells suggesting blockade of the CRAC channel by BTP2 inhibited hyperthermia induced apoptosis.However,for the HPV positive cervical cell lines of Caski,SiHa and HeLa,pre-treatment with BTP2 did not alter the percentage of apoptosis induced by hyperthermia,suggesting that hyperthermia related calcium overload may not be the main mechanism of apoptosis in HPV positive cervical cell lines,or there are rescue pathways in the cell lines.3.The effect of 42?hyperthermia on the antigen presentation ability of macrophage and the killing of intracellular sporothrix.42?hyperthermia increased the antigen presentation ability of macrophages through CRAC calcium channel.42?hyperthermia increased the ability of killing intracellular sporothrix of macrophages,however not through the CRAC calcium channel.In addition,42?hyperthermia increased the ability of macrophages killing intracellular sporothrix after co-culture with CD4⁺T cells through CRAC calcium channel.4.42?hyperthermia treatment could promote the healing of murine sporotrichosis42?hyperthermia treatment could promote the healing of murine sporotrichosis.No obvious promoting in the healing of sporotrichosis was observed in mouseintracutaneous injected with CRAC channel selective inhibitor BTP2 before application of hyperthermia.CONCLUSIONS:1.The macrophages in the lesions of the sporotrichosis patients are mainly polarized to the M2 direction that inhibit the inflammatory response,and the T helper lymphocytes mainly differentiate to Th2 type.2.The antigen-presenting ability of macrophages and the ability of killing intracellular sporothrix are improved after treated with 42?hypethermia,and the ability of antigen presentation of macrophages also improved after treated with 42?hypethermia through CRAC channel.3.Hyperthermia may treats sporotrichosis model mice mainly through activating CRAC calcium channel.