Preliminary Study On The Activation Mechanism Of NLRP3 Inflammasome In Jurkat T Cells By Titanium Ion

The innate immune system is the first barrier to protect the host from pathogens,andcanrecognizedangeroussignalsfrompathogenic microorganisms-pathogen-associated molecular patterns（PAMPs）and dangerous signals from the host’s own source-by means of germline-encoded pattern recognition receptors-damage-associated molecular patterns（DAMPs）.Titanium is a metal with excellent biocompatibility and is the most frequently used biomaterial.Its use in the construction of dental implants is mainly due to the formation of a layer of titanium dioxide（TiO₂）,which can produce high corrosion resistance.However,despite the high corrosion resistance and biocompatibility of titanium,dental implant corrosion can still occur in some cases in the oral environment.The corrosion process results in physicochemical changes in the surface of the implant,including destruction of the TiO₂ layer and promotion of titanium dissolution and release of ions.The inflammatory complex is a multiprotein signaling platform that controls the inflammatory response and coordinates the defense of the antimicrobial host.They are assembled by pattern recognition receptors to detect pathogenic microorganisms and dangerous signals in the cytosol of host cells,and they activate inflammatory caspase to produce the cytokine cytokines IL-1βand IL-18,promoting inflammation and inducting pyroptosis.To date,several relatively typical inflammasomes have been studied.These include NLRP1,AIM2,and NLRC4 inflammasomes that respond to specific activators,as well as NLRP3 inflammasome that sense diverse stimulus.The NLRP3 inflammasome is the most intensive inflammatory body currently studied.It consists of a protein scaffold formed by oligomerization of NLRP3 molecules,ASC（PYCARD）and procaspase-1.Upon detection of cellular stress,NLRP3 is oligomerized by homotypic interactions between the NACHT domains.The PYD of NLRP3 is then exposed to interact with the PYD of ASC.The ASC CARD in turn recruits pro-caspase 1 through the CARD-CARD interaction.Pro-caspase 1 aggregation on oligomerized NLRP3 results in caspase 1 autoactivation and caspase 1-dependent cytoplasmic target processing,including the pro-inflammatory cytokines IL-1βand IL-18,which mediate the repair response.NLRP3 inflammasome can be activated by intact pathogens and can also be activated by a variety of PAMPs and DAMPs as well as environmental stimuli,although they vary in structure.NLRP3 inflammasome can also detect metabolic stress signals such as elevated levels of extracellular sugars and autoimmune diseases.In addition,the NLRP3 inflammasome drives immune responses against many environmental stimuli,including silica,asbestos,ultraviolet radiation,and some skin irritants such as trinitrophenyl chloride,trinitrochlorobenzene,dinitrofluorobenzene,etc.Therefore,whether the titanium ions released around the implant may also be an environmental activator,thereby activating the NLRP3 inflammasome and promoting the secretion of IL-1β,remains unknown.Purpose1.To investigate whether titanium ions in Jurkat T cells cause activation of inflammasomes;2.To investigate whether the titanium ion acts as a cofactor in the activation of PHA in Jurkat T cells,and promotes the secretion of cytokines IL-1β;3.To investigate whether titanium ions pass through the changes of ROS levels in Jurkat T cells,thereby activating NLRP3 inflammasome;Method1.Real-time quantitative fluorescence PCR was used to detect the differential expression of NLRP1,AIM2,NLRP3 and NLRC4 in Jurkat T cells under different concentrations of titanium ions and PHA pre-activated;2.Real-time quantitative fluorescent PCR was used to detect the expression of NLRP3,Caspase-1 and ASC mRNA in NLRP3 inflammasome complex after Jurkat T cells were stimulated by titanium ion and PHA;3.Enzyme-linked immunosorbent assay（ELISA）detects the secretion of IL-1βby titanium ions and PHA activated Jurkat T cells;4.The changes of NLRP3 mRNA and IL-1βsecretion were detected by real-time quantitative fluorescent PCR and ELISA after adding ROS scavenger N-acetylcysteine（NAC）;5.Fluorescent microscopy and flow cytometry were used to detect the changes of ROS levels in Jurkat T cells under the action of titanium ions.Result1.Real-time PCR results showed that the expression of NLRP1,AIM2 and NLRC4 mRNA did not change and the expression of NLRP3 mRNA increased under different concentrations of titanium ions;2.Real-time PCR results showed that the expression of NLRP3,Caspase-1and ASC mRNA increased when titanium ion alone.Under the pre-activation of PHA,the expression of NLRP3 and Caspase-1 mRNA was higher after stimulation with titanium ions;3.Under the stimulation of titanium ion alone,the secretion of IL-1βdid not change,and the secretion of IL-1βincreased significantly after PHA activation;4.Under the action of ROS scavenger,the expression of NLRP3 mRNA and the secretion of IL-1βwere decreased;5.Confocal microscopy and flow cytometry showed that the level of intracellular ROS did not change significantly under the action of titanium ion alone.After PHA activation,the level of intracellular ROS increased.Conclusion1.Titanium ions activate NLRP3 inflammasome in the activated state of Jurkat T cells;2.Under the activation of Jurkat T cells,titanium ions promote the secretion of IL-1βthrough the NLRP3/Caspase-1/IL-1βinflammatory pathway;3.In the activation state of Jurkat T cells,ROS is involved in the activation mechanism of titanium ions on NLRP3 inflammasome;...