Infection By The Nematode Angiostrongylus Cantonensis Induces Differential Expression Of Mirnas In Mouse Brain

BackgroundsThe parasitic nematode Angiostrongylus cantonensis is an important causative agent of human eosinophilic meningitis.Found often in the pulmonary arteries of rodents,A.cantonensis is considered as an emerging zoonotic pathogen of human angiostrongyliasis worldwide.A.cantonensis is mainly endemic in the Pacific islands and South Asia,especially in Taiwan and mainland China;several southern provinces of China are natural foci of this nematode.In the past decade,several outbreaks of human angiostrongyliasis have been reported not only in these natural foci,but also in some northern cities,raising a significant public concern of this disease.In nature,snails are the primary intermediate hosts,where larvae develop until they are infective.Definitive hosts of A.cantonensis are wild rodents,especially the brown rat.Humans are incidental hosts and become infected through ingestion of parasitic larvae in raw or undercooked snails,or from contaminated water and vegetables.Within the hosts,larvae are transported via the blood to the brain,and commonly cause eosinophilic meningitis,a serious condition that can lead to death or permanent brain and nerve damage.Angiostrongyliasis is considered as a self-limiting disease in immunocompetent persons but could be a life-threatening disease in immunocompromised persons including the elderly,children and those with immunodeficiency diseases.Different from the definitive host rats,humans and mice are non-permissive hosts in which the larvae are naturally prevented from maturing and completing the life cycle and most of the worms terminate development at the young adult worm stage in the brain.It is reported that immune response against infection is important for eliminating the parasite.As resident immune cells of the central nervous system(CNS),microglia can be activated to react towards brain injury or induction of immune responses after A.cantonensis infection.Treated with A.cantonensis larvae antigen,mouse microglia can secrete a series of substances:protease such as iNOS,proinflammatory cytokines such as IL-6,IL-1?,TNF-?,IL-5,IL-13 and chemotactic factors such as eotaxin and Yml.The altered expression of these molecular mediators can induce the activation and infiltration of eosinophils.However,the molecular mechanism delineating the regulation of immune response remains unknown.MicroRNAs(miRNAs)are small non-coding RNAs with approximately 21-23 nucleotides(nt)in length.miRNAs direct the miRISC to its target mRNA and the consequence of miRNA:mRNA interaction results in the down regulation of protein expression by either translational repression,mRNA cleavage,or promotion of mRNA decay.It has been found that miRNAs are important not only for host physiology and but also for the development of parasites.There is emerging evidence indicating that parasite infection can alter host miRNAs and most of the differentially expressed miRNAs are relevant to the host defense against infection.To date,many miRNAs have been reported to undergo alterations between different hosts or in the same host following infection with parasites such as Schistosoma japonicum,Toxoplasma gondii and Cryptosporidim parvum.Several miRNAs including the miR-212/132 cluster were reported to be critical in the pathogenesis of inflammation-related disease,and they were confirmed to have a regulatory role in the inflammation of CNS in mice and rats induced by infection with A.cantonensis.However,the global changes in the miRNA expression profiles in the brains of non-permissive hosts during A.cantonensis infection have not been investigated.In this study,we performed a comprehensive analysis of miRNA expression in BALB/c mice brains following A.cantonensis infection using a miRNA array.This analysis revealed significant alterations in miRNAs expression following A.cantonensis infection.We further aimed to delineate the miRNA expression profile following infection with A.cantonensis and identify the possible role of these miRNAs in the immune response in non-permissive hosts.Among the miRNAs up-regulated after A.cantonensis infection,we identified that inhibiting the activation of miR-146a could increase the expression of TNF-a.ObjectivesThe present study is to investigate the expression profile of miRNAs in BALB/c mice brains following A.cantonensis infection,search for the possible functions of the differentially expressed miRNAs,provides a better understanding of the pathogen mechanisms of A.cantonensis infection in molecular level,and would contribute to the prevention and control of A.cantonensis.MethodsAnimal challenge and tissue preparationSix weeks old BALB/c mice were orally infected with 40 infective-stage larvae per mice and sacrificed for the brains 21 days following infection(dpi).Intracranial worms were harvested and processed in order to release the soluble antigen.MicroRNA Array analysisExtracted total RNA of infected brain tissues and reversed transcribed in a small RT reaction.MicroRNA Array was performed using Taqman(?)low density arrays.Microglia transient transfection of miR-146a inhibitor and control miRNAinhibitorTransfection with miR-146a inhibitor or the control miRNA inhibitor were performed using reverse-transfection way.The culture medium was changed 36h after transfection,and LSA was added at a concentration of 50?g/ml.Microglia stimulation assayMicroglia cell line N9 was cultured.Stimulation was performed in culture medium containing LSA(50?g/ml),culture medium with LPS(1?g/ml)was used for positive control.Cells were collected at Oh,4h,8h,12h and 24h after stimulation and total RNAs were isolated.Real-time PCR analysis for mature miRNAsComparative real-time PCR was performed in an Applied Biosystem 7500 Sequence Detection System using miScript SYBR Green PCR.Expression of genes was normalized to that of U6 small nuclear RNA.Real-time PCR for cytokine expressionThe total RNA samples of microglia were reverse transcribed with RevertAidTM First Strand cDNA Synthesis kit.Real-time PCR analyses for mRNA of TNF-a and P-actin were performed by using SYBR-TM Select Master Mix(Ambion,USA).Expression of genes was normalized to that of ?-actin.Gene ontology and KEGG pathway analysisThe target genes were predicted using three online software packages:Mireap,Targetscan and MiRanda.The predicted genes were subsequently classified in terms of their GO categories and KEGG pathway.ResultDifferentially expressed miRNAs in BALB/c mice brains following infection30 miRNAs were differentially expressed among three groups.Although a great majority of the host miRNAs is expressed in both uninfected and infected BALB/c mice,21 miRNAs had shown significantly difference.Of the 21 differentially expressed miRNAs,a total of 19 miRNAs were significantly up-regulated and 2 showed decrease.Verification of miRNA Array data with Real-time PCR analysisIncreased expression of miR-19,miR-21,miR-142,miR-146 and miR-155 was detected in the mice brain tissues following A.cantonensis infection,while miR-181a shown no significantly difference.In the in vitro model,the six selected miRNAs displayed up-regulated at 12h to 24h,but not in the early time points.The results further confirm the accuracy of the array data.Target genes prediction,GO enrichment analysis and KEGG pathway analysisAll of the 21 differentially expressed miRNAs following A.cantonensis infection were used for target gene analysis and were all successfully predicted to 905 target genes.The GO analyses of these predicted target genes revealed that some of the target genes potentially had important biological functions during the host-parasite interaction under A.cantonensis infection.The targets for differentially expressed miRNAs have important roles in axon guidance,signal pathway induction,immune regulation and apoptosis.Analysis of the biological function of the differentially expressed miRNAs in BALB/c mice brain under infection statusAmong the 21 miRNAs up-regulated following infection,five were modulated in immune response,among the miRNAs shown up-regulated,the functions also involved in central nervous system development.MiR-146a negatively regulates expression of TNF-? following LSA stimulationTNF-a rapidly up-regulated at 4h,then decreased at 8-24h following stimulation.The alteration of TNF-a following stimulation was exactly opposite to the expression of selected miRNAs.MiR-146a continued to be downregulate at 4h and 24h after LSA stimulation,the expression of TNF-a and Traf6 displayed upregulation from 4h to 24h in microglia transient transfected with miR-146a inhibitor.ConclusionsThe differentially expressed miRNAs are mainly involved in the immune response of infected mice.Our study further confirmed that MiR-146a may modulate the expression of TNF-a at least via decreasing the expression of Traf6.These data demonstrated a key role for miRNAs modulation against A.cantonensis infection and may provide new insights into general mechanisms of the regulation of anti-A.cantonensis immunity in mice.