The Functional Role And Mechanism Of Cathepsin C Involved In LPS-induced Neuroinflammation

Objective:Neuroinflammation is a common pathological feature of central nervous system(CNS)injury and diseases.The main pathological changes of neuroinflammation include overactivation of glial cells,infiltration of inflammatory cells,overproduction of proinflammatory cytokines,increased blood-brain-barrier permeability and so on.Microglia(MG)which is the immune cell and serves as the first line of defense of innate immunity in the CNS is first activated and plays an important role in the initial stage and progression of neuroinflammation.Activated MG can be polarized into M1 and M2 phenotypes with very different morphology and function.M1 phenotype which is also named classical activation can eliminate pathogen while producing proinflammatory cytokines such as TNF-a(tumor necrosis factor-a,TNF-a,IL-lp(interleukin-1β,IL-1β)eta that can aggravate inflammation and tissue damage.M2 phenotype which is also named alternative activation,can clear cell debris and release a large number of protective factors,such as IL-10,IL-4 and IGF-I(insulin-like growth factor 1,IGF-I).These factors can promote the reconstruction and repairment of damaged tissue.Numerous studies have shown closely relationship between the polarization of MG and various CNS diseases.There are significant differences in the phenotypic characteristics of MG in different disease or distinct stages in the same disease.Finding the reason of polarization and controlling polarization state may become a new therapeutic strategy of CNS disease.The activation of MG in neuroinflammation generally accompanies expression changes of many genes.Among these genes cathepsin C(Cat C)is one of them which was found in our previous study.CatC belongs to cystein protease family and has a unique aminodipeptidyl peptidase activity which can progressively remove dipeptides from N-terminal of protein,therefore participates in variety of protein modification proceses.In the peripheral system,it is reported that Cat C is involved in rheumatoid arthritis,asthma,pulmonary fibrosis and many other inflammatory reactions by activating the serine proteases(SP)in cytotoxic T lymphocytes,mast cells and neutrophils.Especially in the relevant study for arteriosclerosis,it is found that Cat C is not only the marker of M1 but also affects macrophage polarization through macrophages autocrine,namely Cat C regulates macrophages polarization into M1 through macrophages autocrine positive feedback manner.The CNS expression of Cat C which has been considered as a very important molecule in the peripheral system inflammation was found in our preliminary work aiming to find out the differentially expressed genes in demyelinating diseases by gene chip.The further study showed that Cat C was highly expressed in activated MG in both demyelinating model and LPS(lipopolysaccharide,LPS)(5 mg/kg)induced neuroinflammation,indicating that Cat might participat in the process of neuroinflammation.However,the functional role and mechanism of Cat C involved in the neuroinflammation is unclear.In order to clarify the functional role of Cat C in neuroinflammation,we used conditional Cat C OE(Cat C over expression,Cat C OE)and Cat C KD(Cat C knock down,Cat C KD)transgenic mice and littermate wild type(WT)mice as the control.Intraperitoneal and intracerebroventricular injection of LPS were used to induce neuroinflammation in the brain,then the learning ability behavior and expression of proinflammatory cytokines were analyzed in different genotype mice and compared to each other.The mechanism of Cat C involved in neuroinflammation was further studied by in vitro experiments,and was confirmed in vivo by using neuroinflammation animal model.Materials and methods:1.Animal and processing methodsFor the in vivo experiments,C57BL/6J mice with genotypes of WT,conditional Cat C KD and Cat C OE weighing 20 to 25 g were used.In the experimental groups,mice with different genotype suffered LPS injection respectively(intraperitoneal injection 10Oug/kg or intracerebroventricular injection 1 mg/kg)to induce neuroinflammation of brain,and the WT mice were treated by equivolume 0.9%saline vehicle as the control group.For the in vitrol experiments,primary cultured MG were isolated from mice with different genotype then were incubated with LPS(50 ng/kg)or recombinant active Cat C(1,10,100 ng/ml)for 24 hours,and control MG isolated from WT mice were incubated with 2%FBS.Brain perfusion and frozen section were made as the common practice.The protein and RNA were extracted from tissue or culture cells by using reagent kit and trizol regent respectively.2.Navigation experiment of water mazeThe learning ability of mice was determined by Morris water maze test.After LPS intraperitoneal injection for 24 hours,the swimming speed and escape latency of different genotype mice were continuously recorded for 5 days.3.Immunohistochemical(IHC)and Immunocytochemical(ICC)stainingMG morphology was detected by Ibal IHC on the brain slides which come from the mice which are untreated or end of Morris water maze test or LPS intracerebroventricular injection for 24 hous.IL-Iβ and Cat C IHC were used to detect the expression of proinflammatory cytokines and ’Cat C respectively.After incubating with LPS for 24 hours,CD86 ICC which is Ml marker was performed in primary cultured MG from WT mice.4.In situ hybridization(ISH)The expression of proinflammatory cytokines mRNA were detected by TNF-a and IL-1(3 probes,the activation of MG and the expression of Cat C mRNA were detected by c-fins and Cat C probes by using the frozen brain sections mentioned before..5.Enzyme linked immunosorbent assay(ELISA)ELISA-sandwich technique was used to investigate TNF-a and IL-1β expression in brain and serum.For the expression in brain,the proteins were extracted from untreated,end of water maze test and LPS intracerebroventricular injection 24 hours mice respectively.For the expression of serum,the serum was collected at the end of water maze test.6.Realtime-PCRRealtime-PCR was used to detect the expression of TNF-aand IL-1β in above-mentioned mouse brain and the Ml polarization after primary cultured WT MG treated by recombinant active Cat C by using TNF-a,IL-lβ,CD86,CD 16,CD32 and IL-6 primers.7.Flow cytometryFlow cytometry was used to detect MG polarization after stimulating with recombinant active Cat C 100ng/ml for 24 hours.CD86-FITC and CD206-APC were used for M1 and M2 detection,and CD16/32 was used for blocking Fc receptor to minimize nonspecific binding.8.Statistical analysisThe experimental results were expressed as mean ± standard error of the mean(SEM).Repeated measures multivariate ANOVA and multivariate analysis of variance were used for water maze test results analysis.Other data were evaluated for one-way ANOVA and linear regression.All statistical analyses were performed in the Statistical Package for Social Sciences 19.0.Differences with P<0.05 were considered as statistically significant difference.Results:1.The impact of Cat C differentiated expression on learning ability in LPS-induced neuroinflammation(1)The morphology of MG and the expression of proinflammatory cytokines had no significantly different in different genotype miceIn untreated position,the MG in Cat C KD and WT mice were in resting state,in Cat C OE mice were slightly activated.There were no significant differences between the three genotype mice in the expression of TNF-α and IL-1β in the brain.These results indicated that no spontaneous neuroinflammation and other inflammatory changes occurring in untreated conditional Cat C transgenic mice.(2)Cat C was involved in low dose of LPS intraperitoneal injection induced neuroinflammationAfter LPS injection,MG were activated in the whole brain,and TNF-α and IL-1βwere significantly upregulated in WT mice after LPS(100μg/kg)intraperitoneal injection.This result suggested that low dose of LPS intraperitoneal injection could induce neuroinflammation in mouse brains.Meanwhile,Cat C positive signals were found in the whole brain which were only found in the CAII region of hippocampus under physiological conditions,suggesting that Cat C is involved in the neuroinflammatory process.(3)Cat OE aggravated impairment of learning ability in neuroinflammatory miceMorris Water Maze test showed there was no difference between the three genotype mice on swimming speed in the navigation experiment,suggesting that the motor ability of the mice was not affected following LPS treatment.However,the escape latency of neuroinflammatory mice increased significantly from the second days,which indicated that the low dose of LPS injection could cause the decline of learning ability,and the scape latency was significantly longer in Cat C OE mice than that in Cat C KD mice on the fifth day,indicating that Cat C OE aggravated the impairment of learning ability following low dose LPS treatment.(4)Cat C OE aggravated inflammatory reactionAt the end of navigation experiment of water maze,the mice were sacrificed to detect the expression of TNF-a,IL-1(3 in brain and IL-1β in serum by ELISA.The results showed that the highest expression levels of these inflammatory factors were found in Cat C OE mice,then followed by WT mice,and the lowest were found in Cat CKD mice.These results indicate that Cat C OE aggravated the inflammatory response both in peripheral blood and brain,which might be the main causes of impaired learning ability.Cat C KD reversed this process.2.The impact of microglial Cat C differentiated expression on the activation of MG and expression of proinflammatory cytokines(1)LPS intracerebroventricular injection induced neuroinflammationIn order to eliminate the influence of systemic inflammation on the brain,we adopted LPS intracerebroventricular injection to establish neuroinflammation model.The activation of MG and the upregulated expression of TNF-a and IL-1β were found in the whole brain of mice,indicating the neuroinflammation model was successful established and can be used for further study.(2)Neuroinflammation induced the high expression of Cat CThe expression of microglial Cat C was significantly increased in neuroinflammation mouse brain,indicating that Cat C was involved in the induction of neuroinflammation following LPS intracerebroventricular injection.(3)Cat C OE aggravated neuroinflammation induced by LPS intracerebroventri-cular injection.After the mice were treated by LPS intracerebroventricular injection,severely activated MG pattern was found in Cat C OE mouse brain.Meanwhile,in Cat C OE mouse brain the expression of proinflammatory cytokines TNF-a and IL-1β increased significantly at the highest level compared with that in WT mice,and the lowest level was found in Cat C KD mice.These results indicated that Cat C OE aggravated LPS mediated neuroinflammatory reaction.In contrast,Cat C KD reduced this reaction.3.Cat C promoted MG polarization into M1 phenotype and aggravated neuroinflammation(1)Cat C affected on the activation status and the production of proinflammatory cytokines in LPS-treated primary cultured MG.The morphology of the three genotype primary cultured MG was similar.After LPS treatment,severely activated MG pattern was found in Cat C OE group,meanwhile the expression of proinflammatory cytokines TNF-a and IL-lβ also increased significantly at the highest level.In constrast,Cat C KD MG showed that at the lowest level.It suggest that Cat C OE enhanced the activation of primary cultured MG after LPS stimulation and production of proinflammatory cytokines,however,Cat C KD reduced them.(2)Cat C promoted primary cultured MG to polarize into Ml phenotypeIt was found the expression of M1 mRNA markers in primary cultured WT MG stimulated by recombinant active Cat C was elevated in a dose dependent manner,and M1 marker positive cells was also detected.The number of Ml phenotype MG was significantly increased following recombinant active Cat C stimulation by flow cytometry analysis.The results suggests that Cat C has the function that promoted the primary cultured MG to polarize into Ml phenotype.(3)Cat C promoted MG to polarize into Ml phenotype in vivoThe in vitro experiments were performed again in in vivo experiments.The results showed the specific markers of Ml and proinflammatory cytokines mRNA were significantly up-regulated in Cat C OE mice compared with that in WT mice treated by LPS,whereas down-regulated in Cat C KD,indicating that Cat C promoted MG to polarize into Ml phenotype in vivo.Conclusions:1.Cat C overexpression aggravates impairment of spatial learning ability in LPS-induced neuroinflammatory mice.2.Microglial Cat C OE/KD promotes/inhibits LPS-induced neuroinflammation.3.Cat C aggravates neuroinflammation by promoting microglial M1 polarization.