The Preliminary Study Of Dectin-1 Mediated Innate Immune Response Against Sporothrix Schenckii Yeast In THP-1 Macrophages

Chapter I Study on the Sporothrix schenckii yeasts-induced phagocytosis and reactive oxygen species production of THP-1 macrophagesObjective To investigate the phagocytosis and reactive oxygen species(ROS)production of THP-1 macrophages induced by Sporothrix schenckii yeasts.Methods THP-1 monocytes were induced differentiation into macrophages by incubation in the medium with 100 ?g/L of phorbol 12-myristate-13-acetate(PMA)for 48 h.THP-1 macrophages were incubated with S.schenckii yeasts and then,the levels of phagocytosis and ROS production of THP-1 macrophages were examined.Results The morphology of THP-1 monocytes becomes adherent and polymorphic,undergoing physiological changes by incubating with PMA.Firstly,the phagocytosis of S.schenckii yeasts was observed by inverted fluorescence microscopy.The CFW-stained S.schenckii yeasts internalized in THP-1 macrophages shown slightly weake fluorescent blue under ultraviolet radiation.For further evaluation,THP-1 macrophages challenged with DHR-labeled live S.schenckii yeasts at indicated time points were detected by flow cytometer,showing that the phagocytic rate increased from 9%at 0.5-hour coculture time to 30%at 3-hours but increased slightly with further prolonged incubation.Lastly,we found that the level of ROS production of THP-1 macrophages induced by S.schenckii yeasts also increased with the extended incubation time.Conclusions The THP-1 macrophages could swallow S.schenckii yeasts and produce ROS to fight against S.schenckii infection.Chapter II Study on the effect of Sporothrix schenckii yeasts on the generation of proinflammatory cytokines in THP-1 macrophages.Objective To investigate the generation and secretion of proinflammatory cytokines(TNF-a and IL-6)in THP-1 macrophages after incubating with S.schenckii yeasts.Methods THP-1 macrophages were challenged with heat-killed S.schenckii yeasts(107 CFU/ml)and positive stimulator curdlan(100 ?g/mL)respectively in vitro.After incubation for different durations,the mRNA expression of TNF-a and IL-6 was detected by quantitative real-time reverse-transcription polymerase chain reaction(QPCR),and the secretions of TNF-a and IL-6 in the culture supernatant of THP-1 macrophages were measured by enzyme-linked immunosorbent assay(ELISA).Results The levels of TNF-? mRNA and IL-6 mRNA of THP-1 macrophages induced by different stimulators make differences(both P<0.001)and S.schenckii yeasts up-regulated the levels of TNF-? mRNA and IL-6 mRNA in a time dependent manner(both P<0.001).After exposure to heat-killed S.schenckii yeasts for 24 hours,THP-1 macrophages increased secretion of TNF-a(4610.419 ± 121.501 pg/mL,P<0.001)and IL-6(59.96± 18.16 pg/L,P=0.004).Conclusion THP-1 macrophages could release TNF-a and IL-6 in a time dependent manner after exposure to S.schenckii yeasts infection.Chapter III Study on the effect of Sporothrix schenckii yeasts on the expression of Dectin-1 and the activation of its signaling pathways downstream in THP-1 macrophagesObjective To explore the expression of Dectin-1 and the activation of spleen tyrosine kinase(Syk),p38 MAPK and NF-?B signaling pathways in THP-1 macrophages after exposure to S.schenckii yeasts.Methods Flowcytometry analysis was used to assess the protein expression of Dectin-1 on the surface of THP-1 macrophages after exposure to S.schenckii yeasts for different indicated duration.The phosphorylation profiles of Syk,I?B? and p38 MAPK in THP-1 macrophages challenged with S.schenckii yeasts for different indicated duration was analyzed by western blotting.NF-?B nuclear translocation was observed using confocal microscopy after THP-1 macrophages stimulated with S.schenckii yeasts for 16 hours.Results S.schenckii yeasts highly up-regulated the expression of Dectin-1 which reached a peak at 3 hours and still maintained the elevated level at 24 hours post stimulation.Besides,S.schenckii yeasts elicited Syk,I?B? and p38 MAPK phosphorylation in a time-dependent mannar but without increased Syk and p3 8 MAPK expression.In addition,an increase in NF-?B subunit p65 was observed in THP-1 nuclei stimulated with S.schenckii yeasts for 16 hours using confocal microscopy.Conclusions S.schenckii yeasts could induce THP-1 macrophages to up-regulate the protein expression of Dectin-1 and enhance the activation of Syk,p38 MAPK and NF-?B signaling pathways.Chapter IV Study on the mechanism of Dectin-l-mediated innate immune responses in THP-1 macrophages against Sporothrix schenckii yeasts infectionObjective Our previous studies have shown that S.schenckii yeasts could induce THP-1 macrophages to up-regulate Dectin-1 expression,enhance the activation of Syk,p38MAPK and NF-?B signaling pathways,phagocytose fungi,produce ROS and secrete proinflammatory cytokines(TNF-a and IL-6).To further investigate the mechanism of Dectin-l-mediated innate immune responses against S.schenckii yeasts infection.Methods Specific small interfering RNA(siRNA)transfection method was used to gain the THP-1 macrophages of low Dectin-1 expression.Then,Flow cytometry and quantitative real-time reverse-transcription polymerase chain reaction were used to assay the implication of Dectin-1 on S.schenckii yeasts induced phagocytosis,ROS production and the secretion of TNF-a and IL-6.Before stimulated with S.schenckii yeasts,we pretreated THP-1 macrophages with Piceatannol or Dexamethasone respectively.Thereafter,western blot was used to analyze the phosphorylation of Syk,I?B? and p38 MAPK in those THP-1 macrophages stimulated with S.schenckii yeasts.Phagocytosis,ROS production and the secretion of TNF-a and IL-6 were also detected.Results Knockdown of Dectin-1 in THP-1 macrophages notably decreased the internalization of S.schenckii yeast,the formation of ROS and the mRNA expression of TNF-a and IL-6 induced by S.schenckii yeasts.Inhibition of Syk using piceatannol blocked the phosphorylation of I?B-?,attenuated ROS production and the expression of TNF-a and IL-6,but did not affect phagocytosis of S.schenckii yeasts.Dexamethasone of the NF-?B and p38MAPK inhibitor blocked the phosphorylation of I?B? and p38 MAPK and attenuated the mRNA expression of TNF-? and IL-6 induced by S.schenckii yeasts,but did not affect phagocytosis and ROS production.Conclusions Dectin-1 activation is required for phagocytosis,ROS production and pro-inflammatory cytokines(TNF-a and IL-6)release in THP-1 macrophages challenged with S.schenckii yeasts.Syk activation is tiggered with Dectin-1-mediated ROS production and proinflammatory cytokines release but not phagocytosis in S.schenckii yeast-infected THP-1 macrophages.NF-?B and p38 MAPK signaling pathways downstream of Dectin-1/Syk triggered the release of proinflammatory cytokines in S.schenckii yeast-infected THP-1 macrophages.