Mechanism Of Mast Cell-derived Exosomes In Cerebral Malaria

Cerebral malaria(CM)is the most severe neurological complication caused by Plasmodium falciparum infection.CM occurs mostly in children under 5 years old in Africa,accounting for 90% of total malaria deaths.In addition,~20% of CM survivors suffered a long-term neurological impairment,including seizures,convulsions,hemiplegia,delirium,epilepsy,and coma.Currently,restriction of routine malaria control in Africa caused by COVID-19 pandemic,emergence and spreading of anti-malaria drug resistance,as well as ineffective malaria vaccine raised a major challenge for malaria control and CM treatment,which makes it to be much more essential to elucidate the pathological mechanisms of CM.It was demonstrated that mast cells(MCs)and its mediators played a critical role in mediating malaria pathogenesis.Further studies showed that MCs could release exosomes,which were identified as a novel intercellular information mediator.However,the potential functions and pathological mechanisms of MCs-derived exosomes(MCs-Exo)impacting on CM pathogenesis remain largely unknown.Objective Herein,we utilized an experimental CM(ECM)model(C57BL/6 mice infected with P.berghei ANKA strain),and then intravenously(i.v.)injected MCsExo into P.berghei ANKA-infected mice to investigate the effect of MCs-Exo on ECM pathogenies.Methods 1.MCs-Exo isolation and characterization Exosomes were extracted from the supernatants of P815 cells by using commercial exosome extraction kit(from cell culture media).Negative-staining transmission electron microscopy(TEM)analysis of the size of MCs-Exo was performed.In addition,the exosomal markers(e.g.,CD9,CD63,and CD81)of MCs-Exo were detected by western blotting assay.2.Animal experiment The ECM model was established by infecting C57BL/6 mice with P.berghei ANKA,and experimental mice were randomly divided into 4 different groups,including Naive group(uninfected mice received daily i.v.injection of only PBS),MCs-Exo group(i.v.injection of MCs-Exo into uninfected mice),Pb infection group(mice with intraperitoneal injection of P.berghei ANKA)and Pb+MCsExo group(mice intraperitoneal injection of P.berghei ANKA and i.v.injection of MCs-Exo).The survival time,incidence of ECM,and parasitemia in each group of mice were analyzed.3.Toluidine blue staining for MCs Toluidine blue staining was performed to analyze the the MCs density and MCs degranulation levels in skin,CLN,and brain tissues of mice from different groups.4.Histopathological analysis H&E staining was used to analyze the pathological changes of liver and brain tissue of mice from different groups.Meanwhile,ELISA assay was performed to analyze the levels of AST/ALT in sera of mice from different groups.5.Th1 cytokines response in sera Blood samples were harvested from mice in different groups,the protein levels of Th1 cytokines(IFN-γ,TNF-α,IL-6,and IL-1α)in sera were measured by ELISA.6.Assessment of BBB permeability by Evans blue dye staining Animal received a tail vein injection of Evans blue dye,and Evans blue permeability assay was used to evaluate whether MCs-Exo altered BBB integrity in ECM mice.Meanwhile,Western blotting was performed to analyze the protein of ZO-1 and Claudin-5 of brain tissue of mice in different groups.7.Endothelial activation and dysfunction Immunohistochemistry assay was performed to analyze the the endothelial activation and dysfunction(e.g.,ICAM-1and VCAM-1)in brain tissues of mice in different groups.8.In vitro experiments Preparation of P.berghei ANKA infected-mice blood-stage soluble antigen(Pb Ag)was prepared.To investigate the effect of MCs-Exo on b End.3 cell viability,the cells were co-cultured with Pb Ag(20 μg/ m L),MCs-Exo(50 μg/m L),Pb Ag(20 μg/m L)plus MCs-Exo(50 μg/m L),or equal volume of complete medium,respectively.CCK-8 assay was performed.To assess the effect of MCsExo on the expression of ZO-1,Claudin-5,Ang-1,and Ang-2,as well as the release of chemokines(CCL2,CXCL1,and CXCL9),b End.3 cells were co-cultured with Pb Ag(20 μg/ m L),MCs-Exo(50 μg/m L),Pb Ag(20 μg/m L)plus MCs-Exo(50 μg/m L),or equal volume of complete medium and q PCR assay was performed.Results 1.Characteristics of MCs-Exo Negative-staining TEM analysis showed that P815 cells-derived exosomes displayed closed round like vesicles with a typical diameter of 30–150 nm.Western blotting analysis revealed that the exosomal markers(e.g.,CD9,CD63,and CD81)were highly expressed in exosomes,but rarely expressed in P815 cells lysates.Furthermore,nanoparticle tracking analysis indicated that the size of diameter in exosomes ranged from 30 to 150 nm,with a peak of 86 nm.2.MCs-Exo treatment could boost P.berghei ANKA-infected mice from ECM.I.v.injection of MCs-Exo shorten host survival time and elevated incidence of ECM.However,no significance of peripheral parasitemia in P.berghei ANKA-infected mice was observed from Pb group and Pb+MCs-Exo group at days 1–9 p.i..3.ECM mice had more MCs number and maintained higher level of MCs degranulation The results of toluidine blue staining for MCs showed that ECM mice had more MCs number and maintained higher level of MCs degranulation.However,there was no significant difference in MCs number or level of degranulated MCs in skin of ECM mice selected from Pb and Pb+MCs-Exo groups.4.MCs-Exo exacerbated liver and brain damage in ECM mice H&E staining showed that no or rare obvious inflammatory foci were detected in liver tissue of mice from Naive and MCs-Exo groups.However,severe damage with elevated number of inflammatory foci were observed in liver tissue of ECM mice selected from Pb group in comparison to those from Naive group.Remarkably,the administration of MCs-Exo led to more severe damage and higher number of inflammatory foci in liver tissue from ECM mice selected from Pb group.Similarly,serum concentrations of AST and ALT in mice were also determined by ELISA assay,and the data showed that a significant increase in sera AST and ALTfrom ECM mice selected from Pb+MCs-Exo group related to those from Pb group.H&E staining also showed no obvious signals of inflammation or i RBCs sequestration were observed in brain tissues of mice from Naive and MCs-Exo groups.As expected,ECM mice selected from Pb group obviously exhibited i RBCs sequestration,leukocyte infiltration in the parenchymal and pia vessels,and multifocal hemorrhages.The administration of MCs-Exo accumulated more mononuclear cells,higher i RBCs sequestration in cerebral blood vessels,and even resulted in more obvious hemorrhage in the brain tissues from ECM mice.The data showed that a significant increase in hemorrhage sites in brain tissue from ECM mice selected from Pb+MCs-Exo group related to those from Pb group.5.MCs-Exo promoted Th1 cytokine responses in ECM mice ELIAS assay was performed to analyze the protein levels of Th1 cytokines in sera among different groups.there was no significant change in protein levels of IFN-γ,TNF-α,IL-1α or IL-6 among uninfected mice.However,compared with uninfected control mice(Naive group),the protein levels of IFN-γ,TNF-α,IL-1α,and IL-6 were significantly increased in sera from ECM mice selected from Pb group,respectively.Notably,there was a significant increase in protein levels of IFN-γ,TNF-α,IL-1α and IL-6 of ECM mice selected from Pb+MCs-Exo group in comparison to those from Pb group.6.MCs-Exo promoted BBB leakage in ECM mice Evans blue dye cannot leave into the brain parenchyma in mice from Naive and MCs-Exo groups,whereas the breakdown of BBB was obviously observed in ECM mice selected from both Pb and Pb+MCs-Exo groups,as demonstrated by Evans blue dye crossed into the brain parenchyma.ECM mice selected from Pb+MCs-Exo group showed the raised level of Evans blue dye penetration related to those from Pb group.Similarly,the levels of BBB permeability markers(e.g.,ZO-1 and Claudin-5)were detected from different groups by western blotting assay.Our data showed that the expression of ZO-1 and Claudin-5 were significantly decreased in brain tissue of ECM mice selected from Pb+MCs-Exo group in comparison to those from Pb group,respectively.7.MCs-Exo could play a role in triggering brain vascular endothelial activation in ECM mice Immunohistochemistry for ICAM-1 and VCAM-1 were performed to assess whether MCs-Exo can trigger vascular endothelial activation in brain tissues of ECM mice.Bare ICAM-1-and VCAM-1-positively stained cells were observed in the brain tissue of mice from Naive and MCs-Exo groups,while there were obvious positively expression of ICAM-1 and VCAM-1 in brain tissues of ECM mice selected from both Pb and Pb+MCs-Exo groups.Further analysis showed that the IOD/area of ICAM-1 and VCAM-1 expression were dramatically increased in brain tissue of ECM mice selected from Pb+MCs-Exo group in comparison to those from Pb group.8.MCs-Exo could exacerbate the decline of b End.3 cells viability with Pb Ag treatment and promote endothelial activation in vitro.Under complete medium condition,MCs-Exo had no obvious effect on b End.3 cells viability and did not cause any cytotoxicity in b End.3 cells in vitro.Co-incubation of b End.3 cells with Pb Ag in vitro significantly decreased the OD value at 48 h or at 72 h in comparison to control group,respectively.After MCsExo treatment,the OD value of b End.3 cells was reduced by at 48 h or at 72 h in related to those with only Pb Ag stimulation,respectively.Compared with control group,co-incubation of b End.3 cells with Pb Ag in vitro cardinally upregulated the m RNA levels of Ang-2,CCL2,CXCL1,and CXCL9,whereas downregulated level of Ang-1,ZO-1,and Claudin-5.Moreover,the treatment of the cultures with MCs-Exo resulted in the increase of the mRNA levels of Ang-2,CCL2,CXCL1,and CXCL9,whereas led to the decline of the Ang-1,ZO-1,and Claudin-5 m RNA levels in b End.3 cells co-cultured with Pb Ag.Conclusion: The results of this study demonstrated that i.v.injection of MCs-Exo shortened host survival time,while exacerbated brain histopathological damage,and elevated incidence of ECM in Pb ANKA-infected C57BL/6 mice.Our data provided evidence that MCs-Exo may worsen pathogenesis of ECM,maybe due to promoting brain microvascular endothelial cells activation and BBB breakdown.However,the exclusive mechanism on how MCs-Exo trigger endothelial cells activation or BBB breakdown during ECM development still require further investigation.Our finding will provide new insights for the subsequent treatment of cerebral malaria with MCs-Exo as the therapeutic target.