Antiviral Activity And Increased Host Defense Against Coxsackievirus B3 Elicited By The Mammalian Cathelicidins,CRAMP And Ll37

Antimicrobial peptides（AMPs）,also known as cationic host defense peptides（HDPs）,are crucial components of the innate immune system and possess broad-spectrum antibacterial,antiviral,and immune modulatory activities.They can contribute to the rapid clearance of biological agents through direct killing of the organisms,inhibition of pro-inflammatory mediators such as lipopolysaccharide,and by modulating the inflammatory response to infection.AMPs are evolutionarily conserved and categorized into two main families in humans;defensins and cathelicidins.Only one cathelicidin is found in human（LL-37）and mice（CRAMP）.AMP cathelicidins are mainly expressed in tissue cells（epithelial cells,keratinocytes,and pancreatic islet cells）and immune cells（neutrophils,macrophages,mast cells）.Cathelicidins arestored as inactive pro-forms,the active form is released comprising the cathelin domain and cathelicidins.Cathelicidins have broad spectrumantimicrobial activity against Gram-positive and Gram-negative bacteria,fungi and enveloped viruses The antimicrobial mechanism involvesdirect electrostatic interactions betweenthe cationic AMPs and anionic LPS（Gram-negative bacteria）or lipoteichoic acids（Gram-positive bacteria）of pathogens,leading to membrane disruption.Non-membrane disruptive AMPs can cross the membrane and block function of lipopolysaccharide（LPS）.AMPs can be new hope in developing novel,effective and safe therapeutics without antibiotic resistance including marketed AMPs,like Fuzeon?（Polymyxin）and Locilex?（pexiganan cream）,and clinical trialed ones like Iseganan and Omiganan.AMPs cathelicidins also possess immune modulatory function on neutrophils,monocytes,macrophages,eosinophils,mast cells,DCs and epithelial cells/keratinocytes,which allows them to function in different inflammatory immune disorders,including skin diseases,rheumatoid arthritis and diabetes mellitus.The cathelicidins have been well characterized to disrupt envelopes and particles of enveloped viruses（vaccinia virus,respiratory syncytial virus,influenza Virus,human immunodeficiency virus,and Hepatitis C virus）.However,the antiviral effects of AMPs on the non-enveloped viruses are rarely reported.Only one study indicated that LL-37inhibitedreplication of adenovirus and rhinovirus in vitro with no in vivo study and no mechanism.Whether AMPs cathelicidins play an antiviral effect on the infection of non-enveloped viruses?To answer this question,we focus on Coxsackievirus B3（CVB3）,a positive-stranded RNA virus,belonging to non-enveloped viruses.CVB3infects human and mice through fecal-oral way,and disseminates to infect cardiomyocytes and pancreatic cells,leading to acute myocarditis and pancreatitis associated with lethal dilated cardiomyopathy.Our preliminary data found that:1)CVB3 replication kinetics shows a peak on day3 post infection（p.i.）;2)CVB3 infection induced CRAMP expression in hearts of mice,the peaking time also on day3 p.i.;3)CVB3 infection induced a quick wave of neutrophil infiltration into the hearts of mice,which has been reported as an important producer for AMPs.Thus,we raise our hypothesis:as the natural antimicrobial molecules released by immune cells and tissue cells,AMPs cathelicidins might be up-regulated by CVB3 infection in the hearts of mice and play important roles in host innate defense against CVB3 infection and the pathogenesis of viral myocarditis.In this study,we detected the dynamic expression curve of murine cathelicidins（CRAMP）in susceptible organs of mice after CVB3 infection;and explored the role of CRAMP in CVB3 infection and CVB3-induced acute myocarditis by exogenous administration to mice with cathelicidins（CRAMP and LL-37）during CVB3infectionor CRAMP knockout mice.The antiviral molecular mechanism of cathelicidins against non-enveloped CVB3 was further explored.Part one:CVB3 infection induced up-regulated expression ofCRAMP in hearts of mice1.Establishment of murine model of CVB3-induced acute myocarditisC57 male mice were injected intraperitoneally with CVB3 at a dose of1.5x10³TCID50.The survival rate of mice by day7 was around 50%with weight loss as30%;Histopathology of the heart revealed myocardial cell necrosis and massive inflammatory cell infiltration.The viral replication increased to peak at 3 days p.i.in all tissues（heart,liver and pancreas）then declined to the bottom on day7.2.CVB3 significantly elevated CRAMP expression in hearts of miceDynamic expression of CRAMP in the heart,small intestine,liver and spleen was detected during acute CVB3 infection.It was found CRAMP expression in all tissues was significantly up-regulated after infection,most dominantly in the hearts.ELISA,Western blot and IHC assay all confirmed that CRAMP protein level was significantly elevated in the heart on day3,and then declined indicating that CRAMP may play roles against CVB3 infection.Part Two:Cathelicidins administration significantly restricted viral replication and viral myocarditis in mice1.Cathelicidins（CRAMP and LL-37）administration ameliorated disease severity of mice by lethal CVB3challengeTo clarify the effect of cathelicidins AMPs on CVB3 infection,mice were infected with CVB3 together with sCRAMP/sLL37 or CRAMP/LL37.Mice were infected with CVB3 on day 0,and injected intraperitoneally with 10 mg/kg of CRAMP or LL-37)on day 0,2 and 4..We found that treatment with scrambled AMPs or PBS had no effect on CVB3 pathogenesis,while CRAMP or LL37 treatment both significantly elevated the survival rate（50%-75%,p<0.05）of mice indicating a therapeutic role.2.Cathelicidins administration significantly reduced CVB3 replication in hearts of miceThen the viral replication was detected by RT-PCR and western blot at 3 and 7days p.i..Cathelicidins（CRAMP and LL-37）treatment significantly reduced viral RNA（+）and RNA（-）expression（p<0.05）in hearts of mice as compared to scrambled AMPs.In situ hybridization assay further confirmed a signifcant decrease of viral RNAin CRAMP-treated mice.The protein level of CVB3 capsid protein VP1 and the virus titer were significantly reduced（p<0.05）by CRAMP/LL37 treatment as demonstrated by WB and TCID₅₀ assay.The data suggest that cathelicidins AMPs have potent antiviral effect on the replication of the non-enveloped virus,CVB3.3.CRAMP treatment significantly ameliorated the severity of myocarditisTo investigate effect of CRAMP on CVB3-induced myocarditis,pathology of heart tissues of mice were analyzed on day 7.It revealed that as compared to control mice,CRAMP-treated mice showed significantly reduced immune infiltration and cardiac necrosis in the hearts（pathological score,3.2 to 2.0,P<0.05）.Levels of inflammatory cytokines（TNFαand IL-1β）in the hearts were significantly reduced while IL-10 level was significantly increased as compared to those in the control group（P<0.05）.It indicated that exogenous administration of CRAMP significantly reduced CVB3-induced cardiac inflammation and injury.4.CRAMP administration significantly ameliorated cardiac function of mice after CVB3 infectionUltrasonic diagnosis at day7 p.i..found that CVB3 infection significantly increased left ventricular systolic（LVIDs）and end diastolic diameter（LVIDd）but decreased ejection fraction（EF%）and short axis（FS%）of the hearts,indicating a cardiac systolic and diastolic dysfunction by CVB3 infection.Compared to that,CRAMP treatment significantly reduced LVIDs and LVIDd,elevated EF%and FS%of hearts,indicating an improvement of the cardiac function by AMPs cathelicidins.Part Three:CRAMP deficiency significantly enhanced susceptibility of mice to CVB3 infection1.CRAMP deficiency significantly worsened disease severityTo further confirm the protective role of cathelicidins against non-enveloped CVB3,CRAMP knockout mice were infected with CVB3.A significant reduction in7-days-survival rate and an increase in weight loss of mice were noticed（P<0.05）indicating a worsened disease progression.2.CRAMP deficiency enhanced in vivo CVB3 replicationCRAMP-deficient mice showed a significant increase in viral load（+RNA）and replication（-RNA）in hearts,livers and spleens of mice on day3 p.i.after CVB3infection compared to those of WT mice（P<0.05）.TCID50 assay also proved that the viral titer significantly increased(heart,from 10^4.5 to 10^5.5,p<0.05)in mice deficient for CRAMP.The data suggested that CRAMP deficiency exacerbated CVB3 replication in mice.3.CRAMP deficiency aggravated CVB3-myocarditisHE staining of the heart section of mice revealed that the magnitude of cardiac inflammatory infiltration and cardiomyocyte necrosis was significantly increased（pathological score 2.8 to 3.5,p<0.05）in CRAMP KO mice as compared to WT mice.Levels of cardiac IL-1βand TNFαwere significantly higher than those of WT mice indicating a protective role of CRAMP against CVB3-induced myocarditis.4.CRAMP deficiency decreased cardiac function of CVB3-infected miceUltrasound diagnosis of hearts of mice at 7 days p.i.revealed that compared to WT mice,CRAMP deficient mice had significantly reduced left ventricular EF%as well as elevated LVIDs and LVIDd（p<0.05）,suggesting that CRAMP deficiency significantlyincreased heart function damage by CVB3,again confirming the protective function of CRAMP in the pathogenesis of CVB3 infection.Part Four:Mechanism of CRAMP-mediatedCVB3 inhibition and myocarditis restrictionThe anti-viral mechanism of AMPs cathelicidins against enveloped viruses has been elucidated as envelop disruptive and non-envelop disruptive ones,including inference of viral protein functionand modulation of host immune response.However,the exact mechanism of cathelicidins against non-enveloped viruses remains unclear.1.CRAMP inhibited CVB3 replication in an indirect mannerTo see whether CRAMP could inhibit CVB3 replication directly,we did in vitro infection experiment on cardiomyocytes.First,we cultured primary cardiomyocytes from neonatal rat which yielded about 85-90%purity.Then,CVB3 virus（MOI=10）was added onto the surface of primary cardiomyocytes for 1hr infection.During this process,CRAMP,sCRAMP,LL-37 and sLL-37 peptides（20μg/ml）were added to cells either before CVB3 infection（CRAMP-cell-pre）or 1hr after CVB3 infection（CRAMP maint）,or mixed with CVB3 for 1hr and altogether added to cells.It was found that neither CRAMP-CVB3 mixture nor pretreatment with CRAMP influenced viral replication in primary cardiomyocytes;only treatment with CRAMP or LL-37 after CVB3 infection generated viral inhibitory effect in vitro.The RT-PCR,WB and TCID50assay all gave out the similar result indicating CRAMP displayed its anti-CVB3activity in an indirect manner.2.CRAMP down-regulated CAR expression after CVB3 infectionCVB3 infection of the polarized intestinal epithelial cells depends on virus attachment to decay-accelerating factor（DAF）and then interaction with Coxsackie-adenovirus receptor（CAR）.However,CVB3 infection of the heart,pancreas and lymphocytes is mediated by murine CAR.Therefore,since CRAMP administration did not inhibit viral replication directly,we next examine if CRAMP could change the expression profile of CAR on cardiomyocytes during CVB3 infection.When cardiomyocytes were infected by CVB3 alone,it was found that CVB3 infection could down-regulated the CAR protein expression by 50-60%at 50 min after infection as compared to non-infected cardiomyocytes;if CRAMP or LL37 were added to the system,the CAR protein level were significantly reduced at 20 min after infection and could be hardly detected at 50 min after infection indicating that CRAMP and LL37could significantly down-regulated the expression of CAR,the viral receptor,on cardiomyocytes which partially explained why AMPscathelicidins treatment could inhibit CVB3 replication in vitro and in vivo.3.CRAMP altered peripheral innate immune cell profile following CVB3infectionWe next determine whether CRAMP treatment could affect the innate immune cell profile during CVB3 infection.The numbers of macrophages,Dendritic cells（DCs）,monocytes and neutrophils in the blood and hearts of virus-infected mice were investigated by flow cytometry at day 3 and 7 p.i..We found thatCVB3 infection significantly reduced the CD45+CD11b-lymphoid cells but increased CD45+CD11b+myeloid cells at day3 p.i.indicating a robust lysis of peripheral lymphocytes and proliferation of myeloid cells.Compared to that,CRAMP administration significantly reduced CD45+CD11b+myeloid cell population（81%to 18%on day3;31%to 16%on day7,p<0.05）in the peripheral blood of mice including neutrophils（76%to 17.6%on day3,16.8%to 8.9%on day7,p<0.05）and inflammatory monocytes（3.5%to 2.7%on day3,5.1%to 2.1%,p<0.05）.Taken together,the numbers of blood CD11b+myeloid cells,neutrophils and Ly6C^high inflammatory monocytes were significantly reduced after CRAMP treatment which comprise the main early cardiac infiltrated immune cells.We also found that CRAMP treatment significantly increased total numbers of DCs in blood and mesenteric lymphoid node（MLNs）at day 3 p.i.which might enhance viral antigen presentation and T response activation.4.CRAMP treatment reduced cardiac myeloid and T infiltrationMyeloid and T cells are main infiltrated immune cells during acute CVB3 infection.We then examined the influence of CRAMP on the percentage and numbers of CD11b+and T cells in the hearts at day 3 and day7 p.i..CRAMP treatment did not change the percentage or numbers of CD3⁺T and subsets in the blood;however,it significantly reduced cardiac infiltrated CD45+lymphocytes（20%to 12%,p<0.05）and CD11b+myeloid cells（85%to 65%,p<0.05,day 3）especially neutrophils（19%to 7%）and Ly6C^high pro-inflammatory monocytes（14%to 4%,p<0.05）as compared to non-treated mice while CD3+T percentage was somehowup-regulated although total T cell numbers reduced in the hearts.It indicated that CRAMP treatment reduce myocarditis through blocking viral replication and reducing intracardiac enrichment of pro-inflammatory myeloid cells and T cells.Taken together,in this study,we tried to elucidate the possible role of AMPs cathelicidins in host defense against non-enveloped RNA virus,CVB3.Cathelicidins are rapidly up-regulated in hearts of mice upon CVB3 infection which significantly inhibit the viral replication in multi-organs via a CAR-down-regulating mechanism.Cathelicidins also alter the innate immune profile wherein mice treated with CRAMP have increased DCs,and reduced CD11b+neutrophils/monocytes.The innate defense by AMPs cathelicidins may control the initial viral burden and trigger subsequent anti-viral immune responses.Our data first suggest an important role for antimicrobial peptidescathelicidinsin host defense against non-enveloped Coxsackievirus.