Immunity & infection
Objective: Impact of Plasmodium bergheei ANKA infection on spleen and macrophages in mice model with special emphasis on the regulation of p62/sqstm1 molecule and it's impact on Nrf2-Keap1 pathway and subsequent effect on autophagy-proteasomal degradation pathways.
Methodology: Development of mice model of Plasmodium bergheei ANKA. Staining Spleen sections with Acridine Orange, MDC. Transmission electron Microscopy. Immunohistochemistry and immunofluorescence with LC3B, NRF2. Quantitative Real time PCR, RT PCR & Western Blot analysis of the marker genes. DCFDA method of ROS measurement & GSH-GST assay, FACS for detecting autophagosome positive cells, immunoprecipitation. Proteasomal degradation assay by Promega luciferase assay kit.
Results: The parasitemia load in spleen is increased. The MDC punct formation and the acridine orange staining is positive. The ubiquitin level increased. Data suggests upregulation of LC3B and Beclin. The FACS with autophagic dye indicates autophagic process. The Proteasomal degradation levels are markedly higher. Upregulation ROS and change in the anti-oxidant enzyme level noticed. Change in Nrf2 – keap1 pathway is observed. Autophagic Flux flow is studied in every step of it. The autophagosome-lysosome fusion occurs and GFP-RFP-LC3B transfection experiment suggests a block in autophagic pathway. Macrophage cells also got affected. The phosphorylation status of p62/sqstm1 changed with subsequent impact on nrf2-keap1 pathway by binding with keap1 molecule.
Conclusion: The initial studies depicts experimental cerebral malaria definitely brings about change in the autophagic & proteasomal degradation status in mice spleen and macrophage. P62/sqstm1 plays a critical role in balancing the above two aspects and regulates nrf2-keap1 pathway.