TLR9/NF-κB-mediated dendritic cell activation by neutrophil extracellular traps drives pathogenesis in experimental cerebral malaria

Cerebral malaria (CM) is the most severe complication of Plasmodium falciparum infection, and accounts for the majority of malaria-associated mortality. Reducing the overwhelming inflammatory responses in the early stage of infection is a key point to prevent death due to CM. In this study, we found that neutrophil mobilization occurred rapidly in response to Plasmodium berghei ANKA (PbA) infection in a murine CM model. Depletion of neutrophils protected the infected mice from neuropathology, with low infiltration and activation of CD8⁺ T cells in the brain, and attenuated activation of dendritic cell (DC) and parasite-specific T cell responses in the spleen. Flow cytometry analysis showed that following PbA infection the expression of TLR4, TLR7, and TLR9 were increased in splenic DC, while only TLR9 expression was reduced after the depletion of neutrophils. To validate the TLR9-dependent activation between neutrophils and DC, we used neutrophil extracellular traps (NETs) to stimulate bone marrow-derived DCs (BMDC) from WT and Tlr9^-/- mice. The results showed that the DNA component of NETs activates DCs through the TLR9/NF-κB signaling pathway, leading to upregulated expression of costimulatory molecules and the production of proinflammatory cytokines, which was abolished by DNase I. BMDC stimulated by NETs promoted CD8⁺ T cell activation with TLR9 dependence. Inhibiting NETs with Sivelestat effectively impeded the onset and progression of CM in the PbA infected mice. Collectively, our results indicated that neutrophil cell death (NETosis) induced TLR9-dependent DC activation and pathogenic CD8⁺ T cell responses, revealing that the NETs-TLR9/NF-κB-DC-CD8⁺ T cell axis may provide novel insights into the immunopathogenic mechanisms of CM.