The Complex Roles Of Annexin A2 And TNFa In Regulating C. Neoformans-host Interactions

ObjectivesCryptococcus neoformans is an improtant opportnistic fungal pathogen which can breach host blood brain-barrier(BBB)and result in cryptococcal meningoencephalitis with high mortility.Initiation of the infection is trough inhalation followed by conlonization in lung niche.The immune respones during pulmonary infection is critrcal for prognosis of the disease as comprised immune systems often lead to replication and central nervous system dissemination of the pathogen.In this study,we mainly focused on the pathogenic mechanism of this fungal pathogen which can be concluded as two major questions: the mechanism of C.neoformans penetrate across the BBB and the regulation of host immune response towards pulmonary crytpococcosis.The objective of the first part is to dissect the multiple roles of Annexin A2,one member of Annexin family,in regualting the penetraion of C.neoformans cross host BBB.In the second and third part,we construted and tested the therapeitoc effects of a transgenic cryptococcal strain which can produce murine TNFa.MethodsPart I We dissected the roles of AnxA2 in regulating penetration and adhersion of C.neoformans to the BBB using an in vitro BBB model and C.neoformans adhersion model.Anx A2 phosphorylation site mutant cell lines and specilfic blockers were also used to illuminate the mechanism of dephosphorylation of AnxA2 during cryptococcal challenge.Part II The murine TNF? expression sequences were synthesised by PCR.The murine TNFa-producing strain were generated by transformation using a biolistic transformantion system.Part III We determined the therapeutic effects of the TNF? secreted by the transgenic strain T16 by looking into the survival time,CFU burden and pathology of T16-infected mice compared with wild type strain H99 infected mice using an intratracheal infection model.We also isolated total lung leukocytes from T16 or H99 infected mice which was followed by flow cytometry analysis to determine the frequecies and cell counts of each leukocyte subset.Cytokine productions and activation patterns of macrophages were also analyzed.ResultsPart I Up-regulation of AnxA2 expression was observed in endothelial cells upon C.neoformans challenge.AnxA2 depletion in endothelial cells via shRNA resulted in significant increase of fungal association activity but no effect on its transcytosis activity.Furthermore,AnxA2 dephosphorylation at Tyr23 and dephosphorylation of downstream cofilin were also required for cryptococcal transversal with endothelial cells,both of which might be primarily involved in the association of C.neoformans.Part II&III The murine TNF?-producing transgenic C.neoformans strain was successfully construcyed.Induction of TNFa by the transgenic strain T16 has protective effects in a mice model of pulmonary cryptococcosis,which is evidenced by elongated survival time and dramatically decreased fungal burden of infected mice compared with H99.The pathoglogy results showed that infection sites of T16 infected mice were surrounded by condensed inflammatory infiltraion.At innate phase,TNF? induced by T16 promoted classical activation thus enhanced fugicidal effects of macrophages.At adaptive phase,T16 infected mice showed a T cell-dominant inflammatory infitration patterm,increased Th1/Th2 ratio,improved CD103+ cDC and monocytes.ConclusionsPart I our findings demonstrate that host AnxA2 plays dual roles in C.neoformans traversal of the blood-brain-barrier via two distinct mechanisms.Dephosphorylation of AnxA2 leading to dephosphorylation of downstream cofilin in brain endthelial cells,which was essential for cryptococcal adhesion.On the other hand,AnxA2 might be dependent on binding with its ligand S100A10 to mediate fungal transcytosis across the endothelial cells.Part II&III The TNF? secreted by the transgenic strain T16 was proved to be able to induce protective immune responses in a pulmonary cryptococcosis mouse model.The underlying mechanisms are supposed to be improved fungicidal effects of macrophages at innate phase and Th1 biased immune responses at adaptive phase.