Lentinan Has A Regulatory Effect On Immune Response Of The Early Stages For BALB/c Mice Infected With Plasmodium Yoelii

ObjectiveMalaria is one of the most prevalentinan infectious diseases worldwide,with over three billion people at risk of infection and 500 million clinical cases reported annually. Thus,the rational development of effective antimalarial vaccines and novel therapies to alleviate or prevent the symptoms of severe malaria infection requires a better understanding of the various mechanisms of immune responses to malaria parasite.Mice infected with rodent malaria parasites may provide a valuable animal model for dissecting protective immunity against human malaria.A series of studies have proved that immune effector mechanisms are required to eliminate malaria parasites, and Thl immune e.fectors are essential to control the early infection.Different genetic backgrounds of mice infection with the same parasite strain will show significant differences about their immune response.Acquired resistance in DBA/2 mice by P.y17XL involved induction of a effective Th1 response;The outbreak proliferation of blood-stage malaria parasites,which was caused by the inhibition of the establishment of effective Th1 responses,was the key points that cause the death of BALB/c mice infected by P.y17XL.Especially,CD4+Th1 immune effector is crucial for controlling the outbreak proliferation of malaria parasites during early infection by triggering cellular immune responses However,it is also apparent that quantitative and qualitative differences in the nature of the CD4+Th1 immune response have profound effects on disease progression and eventual outcome.Therefore,the regulation of Th1 response is very important to effectively establish protective immunity.At present the regulation of immune responses on anti-malaria have become one of hot topics in malaria research. These observations suggest that the development of immune escape or cerebral malaria is mediated by Tregs through inhibiting the establishment of Thl immunity and affecting the phase and intensity of immune responses to malaria infection.Potential regulatory mechanisms including regulatory T cells have been shown to significantly modify cellular immune responses to malaria.Recently,Tregs have also been reported to maintain the persistence of Plasmodium yoelii,Tregs depletion led to a modest delay in parasite growth in mice infected with P.berghei NK65.In vivo depletion of Tregs prevented the development of parasite-specific Th1 cells involved in the induction of cerebral malaria during P.berghei ANKA infection.And the number change of Tregs seemed significantly affect the final outcome of malaria infection.Moreover,in our previous study Tregs are central to regulation of Th1 responses during rodent malaria infections,and the potential regulatory mechanisms include IL-10-dependent manner and induction of CD4+T cells apoptosis.DCs are antigen-presenting cells that play a central role in both innate and adaptive immune responses Plasmodium blood-stage infection modulates the responses of DCs.Previous studies have shown that human malaria infections induce low parasite-specific T cell responses.A number of epidemiological studies suggest the existence of malaria-induced immune suppression.Studies using mouse malaria models have shown both Plasmodium-specific and unspecific T cell inhibition.Although different effects have been described depending on the parasite strain used or time after infection,the cytokine responses of DCs were markedly affected by Plasmodium either in vitro or in vivo.Recently,the parasite pigment hemozoin has been identified as a major cause of DCs modulation by Echabaudi In addition,IL-10 another well-characterized inhibitor of cellular immunity infection with P.yoelii and after incubation of human DCs with R falciparum-infected erythrocytes.Conversely,DCs secretion of IL-12,a Th1 cell activatory cytokine,is very low during late infection with P.yoelii after LPS stimulation or after incubation of human DCs with P. falciparum-infected erythrocytes.Lentinaninan,a(1-3)-beta-d-glucan extracted from the mushroom Lentinaninus edodes,is a potent immunostimulatory drug.The immunomodulatory effects of lentinaninan range from enhanced host resistance to bacterial,fungal,viral or parasitic infections to antitumor effects.Previous studies have shown that the protective effect of lentinaninan against infection is host mediated and due to potentiation of innate and adaptive immune responses.In general,the immunostimulatory effects of lentinaninan are attributed to macrophages and T cells.Several studies reported changes in cytokine production of macrophages or monocytes in mice and in humans,e.g.enhanced production of TNF-a or IL-1 after in vivo lentinaninan administration Lentinaninan treatment also enhances production of NO and the cytotoxic activity of macrophages Lentinaninan also influences the frequency of T-cell populations as well as the effector function of T cellsIn present study,we used rodent model of BALB/c infected by bloodstage Plasmodium yoelii 17XL to analyze the effect and potential regulatory mechanisms of Lentinaninan on immunity response.Assessing the experimental results,pretreatment with Lentinan at different time-points after normal infection can effectively control parasitaemia level,increase survival rate and promote the establishment of Th1 immunity.To pretreat with Lentinan,the proliferation and activation of CD4+T cells Played a key role in antimalarial immune responses of infection BALB/c with bloodstage Plasmodium yoelii 17XL by inhibiting Tregs and promoting the cell differentiation,maturity and function of DCs.So Tregs is very important to the induction of establishing effective immunity during early malaria infection.In summary, the protective effect of lentinaninan against infection is host mediated and due to potentiation of innate and adaptive immune responses.Materials1.Mouse,parasite,and experimental infection Female 6-8-weeks-old BABL/c mice were purchased from Beijing Animal Institute.P.y17XL were kindly provided by Dr.Motomi Torii(Department of Molecular Parasitology,Ehime University Graduate School of Medicine,Ehime,Japan).Infections were initiated by intraperitoneal (i.p.) injection of 1×10⁶ P.y17XL parasitized erythrocytes for BABL/c mice.Parasitemia was monitored by light microscope examination of Giemsa-stained,thin(tail) blood smears.Mortality was checked daily.All experiments were performed in compliance with local animal ethics committee requirements.2.Lentinan treatment.Lentinan used in this study was kindly provided by Ajinomoto(Japan).Lentinan was tested to be endotoxin free with the Limulus assay and did also not influence the parasite growth kinetics in the presence of up to 5μg/ml as measured by determination of the OD600(data not shown). Lentinan was dissolved in PBS before use and was added to spleen cell culture supematants at a concentration of 1 mg/ml or 5mg/ml for 24h or 48h..For animal experiments,lentinan was intraperitoneally injected three times at 48 h intervals with a dose of 1 mg/kg in 0.2 ml PBS starting on day 15 or 6 before infection.The control group received 0.2 ml PBS i.p.at the same time points. BALB/c mice were randomly divided into four groups(10 mice/group) that were intraperitoneally injected as follows:pre-15d PBS(0.2ml/mouse) in group 1(Control group),pre-15d Lent (0.1mg/ml) in group 2,pre-6d Lent(0.1mg/ml) in group 3 and Lent treated at the same time as infection(0.1 mg/ml) in group 4(Lent group).Administration,PBS or Lent(0.1 mg/ml) was injected i.p.three times at 48 h intervals with a dose of 1 mg/kg,continuous drug treatment until the cure or death of the mice3.The purify pRBCsheparinized blood was obtained via cardiac puncture from P.y17XL-infected B6 mice with 30-50%parasitemia and washed twice with PBS.Blood was diluted with PBS(1-2 ml),loaded onto a 74%Percoll(Sigma-Aldrich) density gradient,and centrifuged at 5000×g for 20 min at room temperature.The topband,containing＞96%pRBCs as determined by staining with Diff-Quik (American Scientific Products).Collected cells were washed twice with PBS and resuspended in cell culture medium. 4,Spleen cell cultureSpleen cell culture was prepared as previously described[29].Briefly,spleen from each mice was removed aseptically and pressed through a sterile fine-wire mesh with 10 ml of RPMI 1640 (Life Technologies,Burlington,Ontario,Canada) supplemented with 5%heat-inactivated fetal calf serum(FCS)(Hyclone Laboratories,Inc.),25mM Hepes(Life Technologies),0.12%gentamicin (Schering,Montreal,Quebec,Canada) and 2mM glutamine(Life Technologies).Cell suspensions were centrifuged at 350g for 10 min at room temperature(rt).Erythrocytes were lysed with cold 0.17M NH₄Cl and the cells were washed twice with fresh medium.Viability of the spleen cells was determined by trypan blue exclusion and was always＞90%.Spleen cells were adjusted to a final concentration of 107cells/ml in RPMI1640 supplemented with 10%heat-inactivated FCS.Aliquots (500μl/well) of the cell suspension were incubated in 24-well plat-bottom tissue culture plates (FALCON) in triplicate for 48h at 37℃in a humidified 5%CO2 incubator.Then the 24-well plates were centrifuged at 350g for 10 min at rt,supernatants were collected and stored at -80℃until assayed for IL-12,IFN-γand IL-10 levels.5,Cytokines analysisLevels of Cytokines were measured by commercial enzymelinked immunosorbent assay (ELISA) kits according to the manufacturer's protocol(R&D Systems,Minneapolis,MN).The OD values were read in a microplate reader at 450 nm.The concentration of cytokines in samples was calculated against the standard curve generated using recombinant cytokine.6,CD4^CD25⁺Tcells by Flow cytometry analysisCells prepared as described above were incubated with anti-FoR antibody for 20 min at 4℃to block non-specific binding of fluorochrome-labelled antibodies and then stained with antibodies either produced in house or obtained from BD Biosciences as following:anti-CD4(mAb YTS 169.4) labelled with FITC,anti-CD25(HL3) labelled with PE.The antibodies were diluted in FACS buffer and incubation with the cells was done for 30 min on ice.FACS analyses were performed on a FACSCalibur instrument run with the CellQuest programme(both from Becton Dickinson) after gating out propidium-iodide-labelled dead cells. 7,CD4⁺ Tcells cell apoptosis in vivo by Flow cytometry analysisA flow cytometry apoptosis detection kit(Becton Dickinson Systems,McKinley,MN,USA) was used to identify programmed cell death.0,5-1×106 cells were stained in 100μl of PBS with anti-CD16/CD32 mAb to block unspecific binding of mAb to Fc receptors with 10μg/ml of a FITC-conjugated rnAb against the cell surface Ag of interest at 4℃for 30 min,followed by the PE-conjugated annexin V(R&D Systems,Minneapolis,MN,USA).7-Aminoactinomycin D(2.5μg/ml) were added to exclude nonviable cells.Cell suspensions were analyzed using a FACScan equipped with CellQuest software(BD Biosciences,Heidelberg,Germany).8,The coculture of Spleen cell with Lent in vitroSpleen cell suspensions were collected as described above and adjusted to a final concentration of 107cells/ml,then 500μl per well cell suspensions or 500μ1 per well pRBC(1×107/ml),10μl LPS (0.1mg/ml),10μl Lent(Img/ml) and 2μl Lent(5μg/ml) were seeded in 24-well culture plates respectively,incubated in complete medium only as nonpulsed controls.in hexaploid group,we separated them into two parts,one was incubated in 24-well plat-bottom tissue culture plates (FALCON) for 24h at 37℃in a humidified 5%CO2 incubator prior to flow cytometry..The other was incubated for 48h,then the 24-well plates were centrifuged at 350g for 10 min at rt, supernatants were collected and stored at -80℃until assayed for IL-12 level.9,Splenic DC maturation,cytokine production assayedFor studies to determine activation of DCs by expression of MHC class II,CD80,CD86 and TLRs,cells were labelled for flow cytometry Following overnight coculture of Spleen cell with pRBes or Lent,noningested red cells were removed by lysis with NH4CI lysing buffer,and Spleen cell were FcR blocked and then stained with FITC-labeled rnAb(BD Biosciences) to CD11c(clone HL3),stained with PE-labeled mAb(BD Biosciences) to MHC classâ…¡(M5/114.15.2),CD80 (16-10A1),CD86(GL1) and stained with PE-labeled mAb(eBioscience) to TLR2(6C2),TLR4 (MTS510) in sorting buffer.Cells were gated on the CFSE_population,and the percentages and mean fluorescence intensity(MFI) of cells expressing MHC classâ…¡and costimulatory molecules were determined by flow cytometry.DCs were analysed for MHC classâ…¡,CD80,CD86 expression using reagents purchased from Pharmingen/Becton Dickinson and or TLR2,TLR4 expression using reagents purchased from eB.Samples were analysed on FACS Calibur(BD Biosciences).Supernatants of Spleen cell incubated with pRBCs or Lent for 48 h at 37℃were analyzed for IL-12 production by ELISA as described previously.10,Statistical analysisStatistical significance of the differences was analyzed by One-way ANOVA(SPSS 12.0).A value of P＜0.05 was considered significant.Results1.Immunoloregulation effect of lentinan on Th1-type immune responses of BALB/c mice during P.y17XL infectionTo explore the influence of lentinan on process infection and infection outcome of BALB/c mice byP.y17XL,we first investigated the effect of lentinan on parasitaemia level and survival rate.In experiment we observed that erythrocytes with parasitaemia were found in the peripheral blood for different treatment groups at day 2.All infected mice died at day 6 p.i.or the next day because parasitemia in control group(p.i.PBS) increased rapidly.However,in pre-15d Lent group parasitemia increased gently and parasitemia only reached 20%at day 8 p.i.,followed by a gradual decrease.At day 15, all treated mice cured.And in pre-6d Lent group,parasitemia reached a peak 28%at day 5 p.i.,then declined and 66.7%of treated mice survived.Compared with control group,Z-Lent group parasitemia increased slowly and reached a peak by day 7(40%) then to 30%at day 9 and increased abruptly to 56%at day 10,all infected mice died, but the life span was significantly prolonged(Fig.1A,B).To further investigate the influence of lentinan on Thl-type immune responses of infection BALB/c mice byP.y17XL in blood stage,the effect of lentinan on the production of IL-12,which is an important stimulator of the T-cell response,the level of IFN-γand NO were tested by us.All the experiment results were measured by class-specific ELISA. IL-12 is an important stimulator of the T-cell response and links innate and adaptive immunity.The increases of pre-Lent groups were more rapid and pronounced. All experimental groups gradually decreased IL-12 production during P.y17XL infection.Compared with control group,pre-15d Lent and pre-6d Lent groups exhibited meaningful increases at day 1,3 and 5 p.i.(P＜0.01).In the Lent group,IL-12 secretion levels exhibited a significant increase at day 1 and 3 p.i.respectively(P＜0.05).But at day 5 p.i.,there were not any meaningful changes(Fig.2.A)As shown in Fig.2.B,the level of IFN-γincreased slowly and reached a peak on day 3 pi and then declined.Compared with untreated group,the increase of pre-15d Lent group was pronounced with IFN-γat day 1 p.i.(P＜0.05),day3 and 5 p.i. (P＜0.01).The pre-6 Lent group showed meaningful increases at day 3 and 5 p.i. (P＜0.01).And the Z-Lent group only up-regulated the IFN-γlevel at day 3 p.i. (P＜0.05) IFN-γ(Fig.2.B).We measured the concentration of nitrite(NO²) of BALB/c mice spleen cell supematants after infection,as a marker of NO synthesis.After lentinan treatment,NO synthesis increased significantly in all groups(P＜0.05) and reached a peak by day 5. The pre-15d Lent and pre-6d Lent groups kept a meaningful increase of NO synthesis at day 3 and 5 p.i.(P＜0.01) compared to untreated mice,but Z-Lent group did not exhibit any meaningful change(Fig.2.C).Taken together,these results indicate that lentinan improves antimalarial protection by stimulating the CD4⁺Th1 specific immune response in infected mice2,Effect of lentinan on the Tregs response of BALB/c mice during P.y 17XL infectionPrevious studies have shown that Tregs proliferation was causally associated with the suppression of Thl responses during early malaria infection,leading to increase parasitemia and mortality in BALB/c mice,so we next investigated the regulation mechanism of Lent on Th1-type immune responses,including of the effect on the quantitative change of IL-10-secreting Tregs,the level of IL-10 and the apoptosis population of CD4+T cells,and we measured the results by FACS and ELISA.The percentage of Treg in CD4+T cells At day 5 p.i.,the level of Tregs was significantly increased and showed a peak in CD4+T cells of all experimental groups. The pre-15d Lent and pre-6d Lent groups' percentage of Tregs in CD4+T cells kept a meaningful decrease at day 3 and 5 p.i.(P＜0.05) compared to untreated mice,but Z-Lent group did not exhibit any meaningful changes(Fig.3.A)To investigate immunoregulatory roles of Tregs in the development of Th1 response during early P.y17XL infection,the population change of CD4+T cell apoptosis was studied.The apoptosis population of CD4+T cells appeared significant increase in all experimental groups,and peaked at day 5 p.i..Remarkably,pre-15d Lent and pre-6d Lent groups kept a meaningful decrease of the apoptosis population of CD4+T cells by day 3 and 5 p.i.compared to untreated mice(P＜0.05).There was no significant difference between the Z-Lent group and Co group(Fig.3.13).To investigate the source of IL-10 and the possible modulating mechanism of Tregs,we analyzed the population change of IL-10-secreting Tregs by intracytoplasmic staining.As shown in Fig.2,IL-10 secretion levels of spleen cell supernatants firstly increased and then fell after infection,the peak of secretion level was shown at day 3 p.i except the pre-15 Lent group..Compared with Co group,the IL-10 level of pre-6d Lent group went down by 25%approximately(P＜0.01) at day 1 and 3 p.i., corresponding the pre-15d Lent group exhibited a significant decrease(P＜0.05), especially at day 3 after infection a remarkable decline was shown(P＜0.01).The Z-Lent group showed no statistical significance(Fig.3.C).In summary these results show that Lent inhibited the Th-1 response,by a mechanism of decreasing the percentage of Tregs in CD4⁺T cells,the level of IL-10 and the apoptosis population of CD4⁺T cells.3,The regulatory mechanisms of Lent to DCs of BALB/c mice during P.y17XL infectionThe CD4⁺T cells play an irreplaceable role in antimalarial immune and need to contact with antigen which need the antigen-presenting cells MHC.Therefore,we further wanted to know whether Lent affects the cell differentiation,maturity and antigen presentation of DCs.In this study,contents change of IL-12 and expression of MHC-â…¡,CD80,CD86,TLR2 and TLR4 on DCs surface of infected BABL/c mice were observed by means of FACS and ELISA.The splenic DCs cocultured with Lent in vitro for 24h,and the expression of MHC-â…¡,CD80 and CD86 on surface of DCs was confirmed by flow cytometry,LPS was used as positive control..As shown in4 Fig A-C,expressions of MHC-â…¡,CD80 and CD86 were significantly increased in spleens of high-concentration lentinan(5mg/ml) pre-treated and LPS groups(P＜0.05).Pretreatment of mice with different concentrations of lentinan resulted in a concentration-dependence,namely proper concentration of Lent can accelerate the maturation of DCs.The splenic DCs cocultured with Lent in vitro for 24h,and determination of TLR2 and TLR4 on DCs surface was performed by flow cytometry,LPS was used as positive control.In Fig.D-E,interestingly,high-concentration lentinan(5mg/ml) treatment group significantly up-regulated TLR2 of DCs(P＜0.05),but the expression of TLR2 had little meaningful changes for the other two groups In Fig.E LPS and high-concentration lentinan(5mg/ml) treatment resulted in a pronounced increase of TLR4 compared to untreated mice(P＜0.05).Therefore,proper concentration of Lent treatment can promote the expression of TLRs on the surface of DCs.Since IL-12 is a specific cytokine from the stimulation of the DCs,we next investigated the contents changes of IL-12 after pre-treatment with lentinan.Pre-Lent treatment can increase the level of IL-12 in DCs culture supernatant,LPS was used as positive control.ELISA detection showed that untreated DCs(namely Co group) can only excrete a limited amount of IL-12,however large amount of IL-12 can be secreted by high-concentration Lent(5mg/ml) and LPS treated DCs(P＜0.05)(Fig.4.F)..The basal cytokine levels before infection were not different between the two groups. Furthermore,the expression of co-stimulatory molecules and MHC-â…¡on DCs showed increases after Lent treatment.Taken together,these results indicate that Lent can promote maturation of DCs Function.In conclusion,there is growing evidence that DCs play an important role in the host defense against blood-stage malaria infection by increasing the expression of co-stimulatory molecules,MHC-â…¡on DCs and the secretion level of IL-12.ConclusionsThe production of IL-12 was induced by stimulating of parasites to DC and macrophage,and can augment the cell differentiation of CD4⁺T cells.Then the parasite-specific IFN-γresponses by CD4⁺T cells increased significantly,as an important immunological regulation factor IFN-γ,can enhance the production of NO largely by activated macrophage.,and NO decrease or deactivate significantly infectivity of schizozoite to erythrocyte.In the establishment of protective immunity responses against infection,the secretion of Th1 immune effectors such as IL-12,IFN-γand NO play key roles[2].Activation of Th1 responses can effectively control parasitaemia level,increase survival rate and essentially clear parasitemia.Our data clearly show that pre-15d and pre-6d Lentinan treatments can suppress the peak parasitemia,improve clinical symptom and prolong the live time of infection mice,the suppression of parasitemia was more effective with pre-15d Lentinan group compared to the other one,and parasite-infected BALB/c mice developed a moderate parasitemia which maintained approximately 20%and all of the mice were completely cured by day 15 p.i..The secretion level of IL-12,IFN-γand NO showed increases in pre-15d Lentinan and pre-6d Lentinan groups(P＜0.05),these results indicated that the cumulative effect of lentinan played the decisive role in induction of type 1 adaptive immune responses in blood stage of infection.In previous study,we have demonstrated that the resolution of a malaria infection depends on the effective establishment of Th1 immune response and a successful switch to Th2.And the number change of Tregs seemed significantly affect the final outcome of malaria infection.Moreover,our previous results showed that Tregs are central to regulation of Thl responses during rodent malaria infections,and the potential regulatory mechanisms include IL-10-dependent manner and induction of CD4⁺T cells apoptosis.After lentinan treatment the percentage of Tregs in CD4⁺T cells, the IL-10 level and specific CD4⁺T cells apoptosis showed significantly decrease at different time points(P＜0.05),our present study show that lentinan reestablishes protective immune responses of host by inhibiting the Tregs in early infection.In the untreated group,the protective immunity to P.y17XL infection was not observed,outcome of malaria infection correlated well with inhibition of the IL-2 production by activated DC and up-regulation of Th1 immune response.The data are highly consistent with the result that Lentinan can promote DC maturation in vitro. After pre-treatment with Lentinan(5mg/ml),We found significant up-regulation of the co-stimulation molecules(CD40,CD54,and CD86) on the surface of CD11c⁺cells. In addition,it secreted IL-12 in a high-level,resulted in DC activation and induction of protective Th1 cell-mediated immune responses compared to LPS group.These results confirm the important role of DC in initiating protective immunity to blood-stage malaria and also provide novel evidence for early interaction between mouse DC and pRBCs.Here,our above results further confirm Lentinan is in a prime position to enhance Thl response.In addition,we find pre-Lentinan via various administrations seem significantly affect the final outcome of malaria infection,all effects may be explained by the extent of medicine accumulation in vivo.In early stage of blood, immunomodulator assist the establishment of Thl immune response.Proper accumulation of Lentinan has important significance to anti-malaria.In summary,our data clearly demonstrate that Lentinan can induce protective immune responses and control the proliferation of malaria parasites during blood-stage of P.y17XL infection.The immune stimulating effects of lentinan play an important role in promoting DC maturation,inhibiting Tregs response,stimulating Th1 response and cleating parasitemia finally.