Age is an intrinsic driver of inflammatory responses to malaria

Age is a critical factor influencing the host immune response and disease pathogenesis. In malaria, the risk of severe disease increases with age in non-immune individuals. Malaria disease is in part driven by inflammation, but the specific cells and mechanisms contributing to age-dependent disease risk are incompletely understood. Here, we assessed inflammatory cytokines in malaria in non-immune children and adults, and the phenotypic, functional and transcriptional differences of innate immune cell responders to malaria parasites in malaria-naïve children and adults. During naturally acquired malaria, age was associated with increased plasma levels of inflammatory chemokines CCL2, CCL3, CXCL8, CXLC9, along with CRP, and IDO, which were associated with clinical symptoms. In malaria naïve individuals, classical monocyte and Vd2+ ?d T cell responses from adults were characterized by increased inflammatory cytokine production, and higher transcriptional activation following stimulation with malaria parasites. Classical monocyte responses in adults were dominated by CCL2 production, while in children the response had increased IL-10 production and enrichment in IL-10 signaling pathways upon parasite stimulation. This heightened inflammatory response in adults was not mitigated by regulatory T cells (Tregs). Taken together, these findings identify cellular mechanisms of age-dependent host responses that play crucial roles in driving inflammatory responses in malaria. Overall design: PBMCs were co-cultured at 37 °C, 5% CO2 in a 96-well U-bottom plate for 4 hours at a 1:1 cell ratio with 1x106 mature trophozoite stage pRBCs. After incubation classical monocytes and Vd2 T cells subsets were FACS sorted using the BD FACSAria™ III Cell Sorter. RNA was extracted from isolated cell population lysates using the QIAGEN PicoPureTM RNA isolation kit (Applied Biosystems™, KIT0204), and RNA quality confirmed with the 2200 TapeStation system (G2964AA) by High Sensitivity RNA ScreenTape (5067- 5579). RNA sequencing libraries were constructed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (E6420S) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (E6440S). The libraries were sequenced using a NextSeq 550 system