石膏样小孢子菌感染小鼠的Dectin-1、TLR-2和TLR-4及细胞因子动态变化
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摘要
本试验旨在研究石膏样小孢子菌经皮肤感染小鼠后皮肤损伤组织中模式识别受体Dectin-1、TLR-2和TLR-4及细胞因子动态变化规律。通过对C57BL/6小鼠皮下接种大熊猫源石膏样小孢子菌,分别在第3、7和14天取病变皮肤组织,经PAS染色和HE染色观察孢子定植部位和病理损伤;分别在第3、6和12小时取病变皮肤组织,用荧光定量PCR检测受体mRNA的转录表达量;分别在第1、3、7和14天取样,用荧光定量PCR检测细胞因子mRNA的转录表达量。结果表明:石膏样小孢子菌感染小鼠后导致皮下脓肿,大量的炎性细胞浸润,并伴有角质层增厚。试验中模式识别受体Dectin-1、TLR-2和TLR-4的转录表达量增加(P<0.01),细胞因子IL-6、IL-1β、IL-23、 TGF-β、IL-17A、IL-17F和IL-22的转录表达量增加(P<0.01)。模式识别受体通过识别病原菌表面模式识别分子而引发免疫反应。本研究表明,机体感染石膏样小孢子菌后,模式识别受体表达显著增加,并通过识别菌体表面模式识别分子从而激活Th17途径,分化成熟的Th17细胞分泌细胞因子IL-22、IL-17A和IL-17F,从而发挥抗菌作用。
The aim of this study was to investigate the dynamic changes of Dectin-1, TLR-2 and TLR-4 pattern recognition receptors and cytokines in skin lesions of mice infected with Microsporum gypseum. M. gypseum from giant panda were subcutaneously inoculated into the C57 BL/6 mice. The skin tissues with pathological changes were collected on the 3 rd, 7 th and 14 th day, then the spore colonization sites and pathological damages were observed by PAS staining and HE staining. The transcription of pattern recognition receptors mRNA were detected by real-time PCR at the 3 rd, 6 th and 12 th hour. The transcription of cytokines mRNA were detected by real-time PCR at the 1 st, 3 rd, 7 th and 14 th day. The results showed that M. gypseum infected mice resulted in a small subcutaneous abscess, a large number of inflammatory cells infiltrated, and accompanied by thickening of the stratum corneum. The transcription of the pattern recognition receptors Dectin-1, TLR-2 and TLR-4 were increased(P<0.01), the transcription levels of cytokines IL-6, IL-1β, IL-23, TGF-β, IL-17 A, IL-17 F and IL-22 were increased(P<0.01). The pattern recognition receptor can recognize the surface pattern recognition molecules of M. gypseum and activate the Th17 pathway, and differentiate the mature Th17 cells into secret cell cytokines IL-22, IL-17 A and IL-17 F, thereby exerting resistance to M. gypseum.
The aim of this study was to investigate the dynamic changes of Dectin-1, TLR-2 and TLR-4 pattern recognition receptors and cytokines in skin lesions of mice infected with Microsporum gypseum. M. gypseum from giant panda were subcutaneously inoculated into the C57 BL/6 mice. The skin tissues with pathological changes were collected on the 3 rd, 7 th and 14 th day, then the spore colonization sites and pathological damages were observed by PAS staining and HE staining. The transcription of pattern recognition receptors mRNA were detected by real-time PCR at the 3 rd, 6 th and 12 th hour. The transcription of cytokines mRNA were detected by real-time PCR at the 1 st, 3 rd, 7 th and 14 th day. The results showed that M. gypseum infected mice resulted in a small subcutaneous abscess, a large number of inflammatory cells infiltrated, and accompanied by thickening of the stratum corneum. The transcription of the pattern recognition receptors Dectin-1, TLR-2 and TLR-4 were increased(P<0.01), the transcription levels of cytokines IL-6, IL-1β, IL-23, TGF-β, IL-17 A, IL-17 F and IL-22 were increased(P<0.01). The pattern recognition receptor can recognize the surface pattern recognition molecules of M. gypseum and activate the Th17 pathway, and differentiate the mature Th17 cells into secret cell cytokines IL-22, IL-17 A and IL-17 F, thereby exerting resistance to M. gypseum.
引文
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[15] WU Q,MARTIN R J,RINO J G,et al.IL-23-dependent IL-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory Mycoplasma pneumoniae infection[J].Microbes Infect,2007,9(1):78-86.
[16] ZHU J F,WANG Y Y,YANG F L,et al.IL-33 alleviates DSS-induced chronic colitis in C57BL/6 mice colon lamina propria by suppressing Th17 cell response as well as Th1 cell response[J].Int Immunopharmacol,2015,29(2):846-853.
[17] CHERMETTE R,FERREIRO L,GUILLOT J.Dermatophytoses in animals[J].Mycopathologia,2008,166(5-6):385-405.
[18] GUARNER J,BRANDT M E.Histopathologic diagnosis of fungal infections in the 21st century[J].Clin Microbiol Rev,2011,24(2):247-280.
[19] DE MATOS BALTAZAR L,SANTOS P C,DE PAULA T P,et al.IFN-γ impairs Trichophyton rubrum proliferation in a murine model of dermatophytosis through the production of IL-1β and reactive oxygen species[J].Med Mycol,2014,52(3):293-302.
[20] BALDO A,MONOD M,MATHY A,et al.Mechanisms of skin adherence and invasion by dermatophytes[J].Mycoses,2012,55(3):218-223.
[21] SMIJS T G M,BOUWSTRA J A,SCHUITMAKER H J,et al.A novel ex vivo skin model to study the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment in different growth phases[J].J Antimicrob Chemother,2007,59(3):433-440.
[22] VAN DE VEERDONK F L,KULLBERG B J,VAN DER MEER J W,et al.Host-microbe interactions:innate pattern recognition of fungal pathogens[J].Curr Opin Microbiol,2008,11(4):305-312.
[23] CAMBIER L C,HEINEN M P A L,BAGUT E T,et al.Overexpression of TLR-2 and TLR-4 mRNA in feline polymorphonuclear neutrophils exposed to Microsporum canis[J].Vet Dermatol,2016,27(2):78-81e22.
[24] VAUTIER S,DA GLóRIA SOUSA M,BROWN G D.C-type lectins,fungi and Th17 responses[J].Cytokine Growth Factor Rev,2010,21(6):405-412.
[25] SABAT R,GRüTZ G,WARSZAWSKA K,et al.Biology of interleukin-10[J].Cytokine Growth Factor Rev,2010,21(5):331-344.
[26] WOLK K,KUNZ S,WITTE E,et al.IL-22 increases the innate immunity of tissues[J].Immunity,2004,21(2):241-254.
[27] CONTI H R,GAFFEN S L.IL-17-mediated immunity to the opportunistic fungal pathogen candida albicans[J].J Immunol,2015,195(3):780-788.
[28] GAFFEN S L.An overview of IL-17 function and signaling[J].Cytokine,2008,43(3):402-407.
[2] DEGREEF H.Clinical forms of dermatophytosis (ringworm infection)[J].Mycopathologia,2008,166(5-6):257-265.
[3] NARDONI S,MUGNAINI L,PAPINI R,et al.Canine and feline dermatophytosis due to Microsporum gypseum:a retrospective study of clinical data and therapy outcome with griseofulvin[J].J Mycol Med,2013,23(3):164-167.
[4] BONFIM C V,MAMONI R L,BLOTTA M H S L.TLR-2,TLR-4 and dectin-1 expression in human monocytes and neutrophils stimulated by Paracoccidioides brasiliensis[J].Med Mycol,2009,47(7):722-733.
[5] SAIJO S,IKEDA S,YAMABE K,et al.Dectin-2 recognition of α-mannans and induction of Th17 cell differentiation is essential for host defense against Candida albicans[J].Immunity,2010,32(5):681-691.
[6] PATIN E C,THOMPSON A,ORR S J.Pattern recognition receptors in fungal immunity[J].Semin Cell Dev Biol,2019,89:24-33.
[7] HEINEN M P,CAMBIER L,FIEVEZ L,et al.Are Th17 cells playing a role in immunity to dermatophytosis?[J].Mycopathologia,2017,182(1-2):251-261.
[8] YOSHIKAWA F S,YABE R,IWAKURA Y,et al.Dectin-1 and Dectin-2 promote control of the fungal pathogen Trichophyton rubrum independently of IL-17 and adaptive immunity in experimental deep dermatophytosis[J].Innate Immun,2016,22(5):316-324.
[9] CAMBIER L,WEATHERSPOON A,DEFAWEUX V,et al.Assessment of the cutaneous immune response during Arthroderma benhamiae and A.vanbreuseghemii infection using an experimental mouse model[J].Br J Dermatol,2014,170(3):625-633.
[10] CONTI H R,SHEN F,NAYYAR N,et al.Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis[J].J Exp Med,2009,206(2):299-311.
[11] ZELANTE T,DE LUCA A,BONIFAZI P,et al.IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance[J].Eur J Immunol,2007,37(10):2695-2706.
[12] LEE S J,HAN J I,LEE G S,et al.Antifungal effect of eugenol and nerolidol against Microsporum gypseum in a guinea pig model[J].Biol Pharm Bull,2007,30(1):184-188.
[13] 张悦天,马晓平,古玉,等.大熊猫源石膏样小孢子菌的分离鉴定与致病性研究[J].中国兽医科学,2015,45(6):551-559.ZHANG Y T,MA X P,GU Y,et al.Identification and pathogenicity of Microsporum gypseum isolated from Ailuropoda melanoleuca[J].Chinese Veterinary Science,2015,45(6):551-559.(in Chinese)
[14] KNIGHT V,LOURENSZ D,TCHONGUE J,et al.Cytoplasmic domain of tissue factor promotes liver fibrosis in mice[J].World J Gastroenterol,2017,23(31):5692-5699.
[15] WU Q,MARTIN R J,RINO J G,et al.IL-23-dependent IL-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory Mycoplasma pneumoniae infection[J].Microbes Infect,2007,9(1):78-86.
[16] ZHU J F,WANG Y Y,YANG F L,et al.IL-33 alleviates DSS-induced chronic colitis in C57BL/6 mice colon lamina propria by suppressing Th17 cell response as well as Th1 cell response[J].Int Immunopharmacol,2015,29(2):846-853.
[17] CHERMETTE R,FERREIRO L,GUILLOT J.Dermatophytoses in animals[J].Mycopathologia,2008,166(5-6):385-405.
[18] GUARNER J,BRANDT M E.Histopathologic diagnosis of fungal infections in the 21st century[J].Clin Microbiol Rev,2011,24(2):247-280.
[19] DE MATOS BALTAZAR L,SANTOS P C,DE PAULA T P,et al.IFN-γ impairs Trichophyton rubrum proliferation in a murine model of dermatophytosis through the production of IL-1β and reactive oxygen species[J].Med Mycol,2014,52(3):293-302.
[20] BALDO A,MONOD M,MATHY A,et al.Mechanisms of skin adherence and invasion by dermatophytes[J].Mycoses,2012,55(3):218-223.
[21] SMIJS T G M,BOUWSTRA J A,SCHUITMAKER H J,et al.A novel ex vivo skin model to study the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment in different growth phases[J].J Antimicrob Chemother,2007,59(3):433-440.
[22] VAN DE VEERDONK F L,KULLBERG B J,VAN DER MEER J W,et al.Host-microbe interactions:innate pattern recognition of fungal pathogens[J].Curr Opin Microbiol,2008,11(4):305-312.
[23] CAMBIER L C,HEINEN M P A L,BAGUT E T,et al.Overexpression of TLR-2 and TLR-4 mRNA in feline polymorphonuclear neutrophils exposed to Microsporum canis[J].Vet Dermatol,2016,27(2):78-81e22.
[24] VAUTIER S,DA GLóRIA SOUSA M,BROWN G D.C-type lectins,fungi and Th17 responses[J].Cytokine Growth Factor Rev,2010,21(6):405-412.
[25] SABAT R,GRüTZ G,WARSZAWSKA K,et al.Biology of interleukin-10[J].Cytokine Growth Factor Rev,2010,21(5):331-344.
[26] WOLK K,KUNZ S,WITTE E,et al.IL-22 increases the innate immunity of tissues[J].Immunity,2004,21(2):241-254.
[27] CONTI H R,GAFFEN S L.IL-17-mediated immunity to the opportunistic fungal pathogen candida albicans[J].J Immunol,2015,195(3):780-788.
[28] GAFFEN S L.An overview of IL-17 function and signaling[J].Cytokine,2008,43(3):402-407.