Phenotypic and functional alterations in neutrophils during the preclinical stage of Plasmodium berghei ANKA infection

Malaria is a parasitic disease caused by Plasmodium parasites and remains a global public health challenge. While the innate immune response helps limit parasite growth, an unbalanced inflammatory response can contribute to severe manifestations, such as cerebral malaria (CM). Neutrophils are key players in host defense and produce inflammatory mediators that can amplify inflammation. Here, we investigated the phenotypic and functional profile of neutrophils during the preclinical stage of Plasmodium berghei ANKA (PbA) infection. C57BL/6 mice were infected with PbA-GFP-infected erythrocytes. The infection followed the expected course of experimental cerebral malaria (ECM), with infected animals dying at 6 days post-infection (dpi). At 5 dpi, prior to the onset of clinical signs, mice exhibited detectable parasitemia, mild weight loss, and splenomegaly. Neutrophils from the spleen and bone marrow were then analyzed by flow cytometry. Infection increased splenic neutrophil numbers and reduced bone marrow cellularity and neutrophil numbers. Neutrophils from both tissues showed increased expression of PD-L1, PD-1, and CD206, while splenic neutrophils exhibited reduced CD62L expression and reduced production of reactive oxygen species (ROS), nitric oxide (NO), IL-1β, IL-6, and IL-10, together with increased TNF production. Plasma levels of TNF, IL-6, IFN-γ, and IL-10 were increased in infected animals. These findings indicate marked phenotypic and functional changes in neutrophils during preclinical PbA infection, characterized by increased expression of regulatory markers and altered production of inflammatory mediators. Importantly, these alterations occurred in parallel with an enhanced systemic cytokine response. Together, these results highlight the complex involvement of neutrophils during ECM development. All experimental protocols were approved by the Animal Ethics Committee of IOC/Fiocruz (CEUA/IOC L-029/2020, L-029/2020-A3, L-022/2021, and L-022/2021-A3).