| BackgroundFungal keratitis(FK)is an infectious corneal disease caused by pathogenic fungal infection,which has a high rate of blindness.In China,aspergillus flavus(A flavus)and fusarium solani(F.solani)are the main pathogenic bacteria of FK.Clinically,we also found that the most serious fungal corneal ulcer infected strains were fusarium solanum and aspergillus flavus(there were statistically significant differences between the two strains during excision and penetrating corneal transplantation)by statistical analysis of the data of patients hospitalized with fungal corneal ulcer between January 2013 and October 2015 in Shandong province hospital.The incidence of fungal keratitis has increased in recent years,due to the misuse of antibiotics and hormones and the prevalence of contact lens wearing.At present,limited understanding of the pathogenesis of FK has greatly restricted its clinical treatment.Therefore,it is crucially important to explore the pathogenesis of FK for the prevention and treatment of keratitis.At present,the early diagnosis of FK in clinic is slow and there is no effective surgical treatment method,resulting in a very difficult treatment process,and the surgical treatment has the possibility of postoperative complications.Based on a variety of basic and clinical researches,the corneal infection of pathogenic fungi in the process of occurrence and development was caused by immune response and inflammatory response,while clearing fungi also can lead to corneal tissue damage,and the final outcomes of FK(corneal perforation,eyeball lost or healing)which mainly depend on the body’s ability to remove pathogenic fungi and pathogenic virulence,followed by corneal inflammatory response caused by the violation and repair the balance between the related factors of healing.The infiltration of neutrophils and mononuclear macrophages is the main process of FK.Macrophages are essential components of the inflammatory response,which rapidly recruit into the corneal matrix to produce various cytokines.Macrophages remove fungi including phagocytosis,lysosomal degranulation,and sterilization.In this process,oxidative metabolites are produced and trigger respiratory burst reactions.Macrophages phagocytosis and sterilization simultaneously produce metalloproteinase,elastase collagenase,acidic hydrolysis enzyme,such as lysosomal enzymes and oxygen free radicals which can decompose phagocytic pathogens but also dissolve corneal matrix to form necrotic abscess.Therefore,proper regulation of inflammatory response is essential for fungal clearance.If the inflammatory response is out of control,persistent and deep inflammatory cell infiltration may lead to corneal structure destruction.The occurrence of infiltration,early apoptosis and effective elimination of apoptotic cells reduced the side effects of inflammatory response and tissue injury.It is well known that innate immune response is the host’s first line of defense against invasion of foreign pathogens.When the body is attacked by pathogenic fungi,one or more pathogen-associated molecular patterns(PAMPs)were non-specifically identified by pattern-recognition receptors(PRRs),which in turn activated the downstream signal transduction pathway to trigger the cytophagocytosis and inflammatory reaction,thereby producing adaptive immune response,and completely eliminating the pathogenic bacteria.In addition to being a natural immune barrier,corneal epithelial cells can also express a variety of PRRs,that can effectively identify pathogenic fungi,secrete inflammatory cytokines and participate in the innate immune response of FK.Currently,the identified PRRs mainly include cytomembrane-located Toll-like receptors(TLRs)and C-type lectin receptors(CLRs),cytoplasm-located NOD like receptors(NLRs)as well as RIG-I like receptors(RLRs).The role of NLRs in the immunity against fungal infection has been confirmed.NOD-like receptor protein 3(NLRP3),an important member of the NLRs family,is a multiprotein complex in existence of cytoplasms of macrophages and dendritic cells(DCs).Under pathological conditions,NLRP3 inflammasome can be activated by PAMP of bacteria,fungi,viruses and DAMP of high concentrations of extracellular ATP and uric acid crystals to trigger the assembly of the NLRP3/apoptosis-associatedspeck-like protein containing a CARD(ASC)/pro-cysteinyl aspartate-specific protease-1(pro-caspase-1)inflammasome complex.Active caspase-1 can lead to the activation of IL-1β and IL-18,which then participate in the innate immune response of the host.However,the role and mechanism of NLRP3 and its downstream cytokines in corneal antifungal infection remained unclear.Part Ⅰ The expression and significance of NLRP3 inflammasome in Corneal specimens of patients with FKObjective:To explore the expression and clinical significance of NLRP3 inflammasome in Corneal specimens of patients with FK.Methods:Fifteen cases of patients with FK at Anhui Provincial Hospital between June 2017 and June 2018 and fifteen controls were included.The mycelial content in the lesions were determined by PAS staining and the expression of NLRP3 inflammasome was detected by immunohistochemistry.Results:In the pathologic corneal tissue of the clinically diagnosed fungal keratitis patients,obvious positive staining of NLRP3 inflammasome was observed.The staining intensity and range of the inflammatory infiltration of NLRP3 inflammasome were significantly increased in the lesions,while no obvious staining of NLRP3 inflammasome was observed in the control group.Conclusion:Fungal corneal ulcer can cause corneal NLRP3 activation.Part Ⅱ The expression and significance of NLRP3 inflammasome in a mouse model of FKObjective:To explore the expression of NLRP3 inflammasome in a mouse model of FK and the therapeutic effect of FK506 on FKMethods:C57BL/6 mice were used to establish FK model.Flow cytometry and ELISA were used to detect the expression of macrophage specific marker F4/80 and the secretion of inflammatory cytokines in the cornea.The expression of NLRP3 inflammasome and its downstream inflammatory cytokines in the mouse model of FK was measured by immunohistochemistry and western blot.The effects of FK506 on the activation of NLRP3 inflammasome and the expression of inflammatory factors were also determined by the above methods.Results:Compared with the corneal tissues of the normal control group,flow cytometry showed a high proportion of macrophages in the corneal region after 48h infection with aspergillus flavus and fusarium solanum in the cornea of C57BL/6 mice.The results of ELISA test showed that the expression of IFN-γ,TNF-α and IL-6 were markedly increased in the corneal tissues of FK mice.HE staining showed that the corneal tissue structure was obviously damaged,the cornea was significantly thickened,typical endothelial plaque was formed,and necrotic areas and microvessels were formed within the plaque.Immunohistochemical analysis showed stronger staining of NLRP3 after fungal infection.The staining intensity and range of NLRP3 inflammasomes in the corneas in F.solani group were higher than that in A.flavus group.Western blot analysis demonstrated that the protein expressions of NLRP3,ASC oligomerization(oligomer,trimer,dimer,and monomer forms),procaspase-1(p45),cleaved caspase-1(p10),pro-IL-1β,IL-1β,and IL-18 were markedly increased in corneas after A.flavus or F.solani infection compared to the control group.However,there was much less inflammation and edema in corneas of mice after FK506 or natamycin treatment or after FK506 combined with natamycin treatment.Similarly,protein expressions of ASC oligomerization(oligomer,trimer,dimer,and monomer forms),pro-caspase-1(p45),cleaved caspase-1(p10),pro-IL-1β,IL-1β,and IL-18 significantly decreased in corneas after treatment with FK506 alone or natamycin alone or combined treatment.Furthermore,the protein expression levels of these molecules were lower in combined treatment group than that in single-agent treatment group.Conclusion:After C57BL/6 mice were infected with corneal fungi,there were a large number of macrophages in the corneal tissue and a significant increase in the secretion of inflammatory cytokines and the protein levels of NLRP3,ASC,pro-caspase-1(p45),cleaved caspase-1(p10),pro-IL-1β,IL-1β,and IL-18.Combination treatment with FK506 and natamycin alleviates A.flavus and F.solani-induced activation of the NLRP3 inflammasome.Part III Mechanism of activation of NLRP3 by aspergillus flavus and fusarium solanumObjective:To explore the mechanism of activation of NLRP3 by aspergillus flavus and fusarium solanum on innate immunity of macrophages in vitro.Methods:We established an in vitro co-culture model of THP-1-derived macrophages with A.flavus or F.solani as well as phosphate buffer solution(PBS)and lipopolysaccharide(LPS)as negative and positive controls.After 6h fungal stimulation,cells and supernatant were collected.Western blot was used to detect the protein expression levels of NLRP3,ACS,caspases-1 p10,casases-1 p45,pro-IL-1β,IL-1β and IL-18.The secretion of IL-1β and IL-18 in supernatant was detected by ELISA.The phagocytic ability of macrophages was evaluated.PMA-induced differentiation of THP-1 macrophages were transfected with NC siRNA,NLRP3 siRNA,ACS siRNA and caspase-1 siRNA and then infected with A.flavus or F.solani for 6h.Cells and supernatant were collected for real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)analysis of transfection efficiency and ELISA assay for the secretion of IL-1β,IL-18,interferon(IFN)-γ,tumor necrosis factor(TNF)-α and IL-6.Results:Compared with the control group,LPS or fungi stimulus led to an increase in ASC,NLRP3,ASC oligomerization(oligomer,trimer,dimer,and monomer forms),procaspase-1(p45),cleaved caspase-1(p10),pro-IL-1β,IL-1β,and IL-18 as well as the secretion of IL-1β and IL-18 in THP-1 macrophages.Activated HTP-1 macrophages can effectively phagocytose more than 40%A.flavus and F.solani.Compared with the NC siRNA transfection group,the expression levels of IL-1β、IL-18,IFN-y,TNF-a and IL-6 in the cells of other interference group were significantly decreased.Besides,NLRP3 interference group showed the most significant decrease.Conclusion:LPS or fungal stimulation can activate NLRP3 inflammatory complex.Activation of NLRP3 inflammasome pathway induces macrophages to secrete downstream related cytokines IL-1β and IL-18.To control inflammation by inhibiting the activation of NLRP3 inflammasome. |
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