Examination On The Effects Of Neutrophils In The Application Of Local Hyperthermia Against Sporotrichosis

Introduction:Sporotrichosis is a common deep mycosis,primarily resulting from Sporothrix globosa.This condition is quite prevalent in Northeast China due to contact with reeds or cornstalks.It manifests in a variety of clinical forms,including disseminated cutaneous disease,fungemia and disseminated visceral disease,with fixed cutaneous sporotrichosis and lymphocutaneous sporotrichosis representing the most common types.While itraconazole is the preferred choice for treatment of sporotrichosis,due to the high cost of this treatment,local hyperthermia has also been applied for treating fixed cutaneous sporotrichosis and lymphocutaneous sporotrichosis.Local hyperthermia is also preferred for treating patients with fixed cutaneous sporotrichosis who cannot safely receive other regimens,such as pregnant and nursing women.Despite the recommendations for use,local hyperthermia for the treatment of fixed cutaneous sporotrichosis and lymphocutaneous sporotrichosis,no randomized placebo-controlled trials or comparisons with other treatments are available to enable any assessment of the validity and security of this procedure.Moreover,as there exist some case reports of spontaneous cures for this condition in the absence of any therapy,it is clear that more evidence is required to determine whether or not local hyperthermia is effective.In patients with sporotrichosis there is a mixed suppurative and granulomatous inflammatory reaction in the dermis and subcutaneous tissue,which is frequently accompanied by microabscess as revealed in histopathological examinations.Neutrophils represent one of the main infiltrating cells in this condition,but the role of neutrophils in the evolution of sporotrichosis is unclear.In addition,it has been reported that the formation of neutrophil extracellular traps(NETs)were observed in some sporotrichosis lesions.NETs are modified forms of chromatin that retain the periodicity of nucleosomal spacing;and,granular proteins bind in globular patterns to NETs.A number of fungi,including Aspergillus nidulans,Candida albicans,Candida dubliniensis,and Cryptococcus neoformans have all shown a susceptibility to NETs,but it remains unclear as to whether NETs exert antifungal effects against S.globosa.The purpose of this report was to evaluate the effect of hyperthermia in the mouse model of sporotrichosis,and assess whether neutrophils and NETs play a role in disease recovery of sporotrichosis after hyperthermia.Material and Methods:1.Experimental cells and miceHuman neutrophils were isolated from venous blood of 10 healthy volunteers.The protocol of collecting human blood was approved by Istitutional Review Board and was conducted in comform to the Declaration of Helsinki.One isolate strain of S.globosa was harvested from a patient with fixed cutaneous sporotrichosis and identified by ITS sequencing.The sporotrichosis models were all 6-8 weeks of age and 19-20g female BALB/c mice,and were injected subcutaneously with 0.1ml of 10~8/ml of yeast cell suspensions.2.Hyperthermia treatmentLocal hyperthermia in S.globosa infected mice:We employed a custom hyperthermia device and water polo.The target lesion was heated to 41°C by a custom hyperthermia device and water polo.The skin temperature of mice was monitored with a thermometer,and the temperature fluctuated between 40-41°C.Mice of hyperthermia group were anesthetized and subjected to a daily heating regimen for 30 minutes for 4 weeks with temperature ranging from 40-41°C.Mice within the control group were similarly anesthetized but received no heating treatments.Hyperthermia in cells:Cells cultured in 24-well plates were treated with the water bath at41°C or 37°C for 30 minutes,then put back to a cell incubator at 37°C atmosphere.3.Changes of rashes after hyperthermiaChanges in appearance of rashes were observed and recorded before treatment and weekly after treatment.The changes in rash widths between the two groups were compared.4.ImmunohistochemicalMice were euthanized at one week following treatment for skin removal.Tissue sections(3?m)were mounted on poly-l-lysine-coated glass slides.Immunohistochemical staining(SP method)was performed to observed the formation of NETs by detecting neutrophil elastase.5.Detectation of yeast cells in rashes by Periodic-Acid Schiff(PAS)stainingMice were euthanized for skin removal before treatment or after one or three weeks of treatment.For histology,tissue sections(3?m)were mounted on glass slides.The tissue was stained with Periodic-Acid Schiff(PAS)reagent,counterstained with hematoxylin and then examined for pathological changes and fungal burden within the skin.6.NETs visualization by immunofluorescenceImmunofluorescence was performed to detect the formation of NETs,when neutrophils and yeast cells were co-cultured at 41°C or 37°C.The number of NETs and neutrophils were counted and the percentage of NETs formed was calculated.7.S.globosa viability assaySpore cultures were plated on petri dishes after different treatment.Colony forming units(CFU)were counted after fourteen days and the number of CFU was compared among the groups.8.Statistical analysisData were analyzed with use of SPSS 22 and plotted by GraphPad Prism 6.0.The two tables were generated with use of Office 2016 and data were presented as means±SD.Results of differences in rash widths during treatment were analyzed using a repeated measures analysis of variance.Fisher's exact test was used to evaluate differences in cure rates between the two groups.The number of yeast cells/per pixel area of the two groups,as determined at the end of the third week,failed to show a normal distribution and was therefore analyzed using the nonparametric Mann-Whitney test,with data presented as median.The number of yeast cells/per pixel area of two groups in the first week was normally distributed and analyzed with use of independent-sample t test,but data were also presented as median.Independent-sample t test was used to evaluate the viability of S.globosa after different treatment in vitro.A P<0.05 was required for results to be considered as statistically significant.Results1.Changes of lesion size after hyperthermiaWhen calculating the value of differences in the lesion width(Difference=lesion width before treatment-lesion width during treatment)these differences were found to significantly increase over this 4-week period of the experiment within both groups(P<0.05).In addition,these differences in the value of lesion width were significantly greater at each time point during the treatment period within the hyperthermia versus and control group(P<0.05).Cure rates in hyperthermia group were higher than control group at the end of the second or third week after treatment cessation(P<0.05).2.Number of fungi from infection sitesYeast cells were limited to the abscess cavity in all mice of both groups.Yeast cell count/per pixel area in the abscess cavity decreased over the duration of the experiment,but no statistically significant differences were present between the control and hyperthermia groups after one(P>0.05)or three weeks(P>0.05)of treatment.3.Formation of neutrophil extracellular trap nets(NETs)The formation of NETs was observed within abscess cavities of some mice in both the hyperthermia and control groups.After co-culture with yeast cells at 41°C or 37°C,the NETs observed were similar to those induced by PMA,but the percentage of NETs formed was not significantly different after co-culture with yeast cells at 41°C or 37°C4.Effect of neutrophils and temperature on S.globosa viabilityWhen yeast cells were co-cultured with neutrophils at 41°C versus 37°C for 30 minutes,followed by transfer to 37°C and continued in culture for 6 hours,no differences in CFU were found between the two groups.Nor were significant differences present in CFU between the two groups,after yeast cells were cultured at 41°C versus 37°C for 6.5 hours.Yeast cells were co-cultured with or without neutrophils at 41°C for 30 minutes,then transferred to 37°C and continued in culture for 6 hours.No statistically significant differences in CFU were obtained between the two groups.We also compared the number of yeast cells cultured at 37°C for 6.5 hour in the presence or absence of neutrophils,and found no significant differences between these two groups.When examining the effects of neutrophils or NETs induced by PMA on yeast cell viability,no significant differences were obtained between two groups.Finally,we investigated the effects of PMA on yeast cell viability in the presence or absence of neutrophils and found no significant differences.Conclusions:1.The rash in mice infected with S.globosa can be cured spontaneously,but hyperthermia promotes an earlier onset and more complete recovery of this condition.2.Although hyperthermia serves as an effective therapy for mice with sporotrichosis,antifungal effects in sporotrichosis were not improved by the hyperthermia treatment administered,and this ability does not involve any fungicidal effects of neutrophils.3.Yeast cells of S.globosa can induce the formation of NETs.4.Hyperthermia is effective to eliminate mouse model of sporotrichosis,independent of NETs formation.