Preliminary Study On Immune Response And Molecular Mechanism Of Human Keratinocytes Infected With Sporothrix Globosa

Sporotrichosis is a type of subcutaneous mycosis caused by the pathogenic clade of the genus Sporothrix(sporothrix spp),which course is chronic and prolonged.Sporothrix globosa(S.globosa)infection has a high incidence in northeast China.S.globosa are dimorphic fungi,which grows in the form of mycelium in the natural environment,and then invades the host epidermis to reach the deep tissue to transform into the form of yeast-like cells and cause infection.Currently,it is unclear how S.globosa infects.Previous studies have suggested that trauma could cause Sporothrix spp.invading the host’s epidermal tissue.However,reports from Asian countries have shown that most of the patients with sporotrichosis have no clear history of trauma,and the proportion of trauma reported in the literature is only 26.4-29.1%,which suggested that sporotrichosis may still occur in the case of intact or micro-damaged epidermis.Keratinocytes,as the main cells in the epidermis,become the first immune defense against sporothrix spp.invasion.However,the mechanism of keratinocytes resisting S.globosa remains unclear.Therefore,in this study,we use conidia and yeast-like cells of S.globosa inducing human epidermal keratinocytes(nHEKs)respectively,to explore the barrier function,immune response and regulation of TLRs/NF-κB signaling pathway of keratinocytes in S.globosa infection.The first part of this study was to investigate the immune barrier role of nHEKs against S.globosa.Firstly,the killing effect of nHEKs on conidia and yeastlike cells of S.globosa was detected.The results showed that the inhibition rate of nHEKs against S.globosa was time and MOI dependent.The inhibition rate against conidia was highest in MOI=5 and co-incubation for 8 hours,and for yeast-like cells at12 hours.In order to elucidate whether n HKEs has intracellular killing effect on S.globosa,we used LIVE/DEAD fluorescent dye Fun1 to label the activity of S.globosa and to trace nHEKs with Cell Trace to observe with confocal microscopy.Most conidia and yeast-like cells were found to accumulate in the interstitial space of nHEKs;only a few of them adhered to nHEKs surface;and no conidia or yeast-like cells were found to be swallowed by nHEKs.It was speculated that nHEKs did not kill S.globosa by intracellular.Therefore,the extracellular killing of nHEKs against S.globosa was further analyzed.LIVE/DEAD fluorescent dye Fun1 was used to label conidia and yeast-like cells in the supernatant,and the dead/alive fluorescence ratio of conidia and yeast-like cells was detected by enzyme labeling.The results showed that the dead/live ratio of conidia and yeast-like cells was higher than that of control group after 4 hours of co-incubation(P<0.001).It can be detected that nHEKs have an extracellular killing effect on S.globosa.Then,the levels of antimicrobial peptides in the supernatant of coincubated were detected by ELISA.The expressions of h BD1,h BD2 and h BD3 were significantly increased in the conidia group during 4-8 hours of co-incubation(P<0.001),while the expressions of h BD1,h BD2 and h BD3 were significantly increased in the yeast cell group in 4 hours of co-incubation(P<0.01).The level of LL-37 did not change significantly.Finally,the ROS levels produced by S.globosa induced nHEKs were detected by fluorescent probes.The results showed that a large amount of ROS produced by conidia induced nHEKs in 4-12 hours(4h,P<0.01;8h,P<0.01;12h,P<0.001),while ROS levels produced significantly by the yeast-like cells higher than the control group during 12-24 hours(12h,p<0.05;24h,P<0.01).In summary,keratinocytes can kill S.globosa by extracellular secreting defensins and producing ROS.In the second part of this study was to determine the immune response of keratinocytes in S.globosa infection.Firstly,the expression levels of inflammatory factors and chemokines were detected by q PCR,ELISA and multifactor flow cytometry.The results showed that conidia can induce nHEKs to express the levels of IL-1βcontinued to increase,comparing with the control group,and the expression levels of G-CSF increased at 8h and 12 h.Yeast-like cells can induce nHEKs to express the levels of G-CSF significantly increased at 12 h,and the expression levels of IL-1β at 24 h was increased compared with control group.Conidia can induce nHEKs to express the high levels of the chemokine IL-8(4h,P<0.05;8h,P<0.05;12h,P<0.01)and CXCL-1(12h,P<0.01);Yeast-like cells induced nHEKs with the high expression of IL-8(4h,P<0.01),CXCL-1 was highly expressed in m RNA level(12h,P<0.01).The results showed that both conidia and yeast-like cells can induce keratinocytes to overexpress IL-1β,IL-8,and CXCL-1.Subsequently,we using the supernatant to chemotaxis neutrophils and THP-1macrophages.The results showed that the supernatant of coculturing conidia with nHEKs could chemotaxis neutrophils,while the yeast cells themselves could chemotaxis neutrophils.Compared with the control group,the supernatant of co-culturing conidia or yeast-like cells with nHEKs showed chemotaxis macrophage obviously.For further verification,we used immunofluorescence double labeling to label keratinocytes in frozen sections of sporotrichosis patients’ tissues and to observe the expression of IL-8 and CXCL-1.Similarly,the neutrophils in frozen sections were labeled to see if they expressed receptors for IL-8 and CXCL-1.The results showed that keratinocytes expressed IL-8 and CXCL-1,and neutrophils expressed the receptors of IL-8RA and IL-8RB,respectively.Finally,to confirm the polarization of macrophages induced by cytokines in infection supernatant.The supernatant of co-culturing conidia or yeast-like cells was incubated with THP-1 cells,and the M1 and M2 polarization of macrophages were detected by immunofluorescence double labeling and Flow cytometry assay.The results showed that the ratio of M1 and M2 polarization of THP-1 macrophages was the same in conidia group,and the cytokines secreted by yeast-like cells induced keratinocytes were more inclined to cause M1 polarization of THP-1 macrophages.The third part of this study is to further demonstrate the regulatory role of TLRs and downstream signaling pathways in keratinocytes infected with S.globosa.First,we extracted RNAs for RNA-seq sequencing.The results showed that compared with yeast-like cells group,there were 18 different gene expressions in conidia group,including IL1β,NLRP3,TICAM2 and other genes of the downstream signaling molecule of TLR receptor.The signaling pathways involved included toll-like receptor signaling pathway,C-type lectin receptor signaling pathway and NOD-like receptor signaling pathway.These results suggestted that keratinocytes may regulate immune response through different TLRs in S.globosa infection.Then,Western blotting was used to detect the expression of TLR2,TLR4,My D88,TRIF,and downstream p65,PP65,IKB-α,and P-IKB-α.The results showed that the conidia could induce nHEKs to overexpress TLR2,and the expression of TRIF and My D88 was increased,and the expression of My D88 was significantly increased;while the yeast-like cells group could overexpress TLR4.Both conidia and yeast-like cells could induce nHEKs to express the high levels of p65 and IKB-α,and activate the NF-κB signaling pathway to up-regulate the expression of phosphorylated p65 and IKB-α.Finally,TLR2 and TLR4 of nHEKs were knocked down by siRNA technology,and then the expression of IL-8and CXCL-1 of TLR2 siRNA/TLR4 siRNA nHEKs which were induced by S.globosa was detected.The results showed that TLR2-siRNA /TLR4-siRNA nHEKs induced by S.globosa could reduce the expression of IL-8 and CXCL-1.In summary,conidia can induce keratinocytes to participate in immune response,through TLR2 and My D88 activation of NF-κB signaling pathway,while yeast-like cells may induce keratinocytes to participate in immune response by activating NF-κB signaling pathway through TLR4.The completion of this study has proved that keratinocytes could not only resist S.globosa by extracellular secreting defensins and producing ROS,but also play a role in immune regulation by secreting inflammatory factors,chemokines and neutrophils and macrophages.Keratinocytes overexpress TLR2 and TLR4 and rely on My D88 to activate the NF-k B signaling pathway to complete the immune response.The TLR2/TLR4 /My D88/NF-k B signaling pathway play an important role in the immune response of keratinocytes against S.globosa infection.The above research results provide a new scientific basis for revealing the pathogenic mechanism of S.globosa and the role of keratinocytes in S.globosa infection.