Mechanisms underlying malaria-associated immune hyporesponsiveness and pathogenesis remain unclear . This investigation relates leukocyte differential counts, circulating NETs, cytokine profiles, and ANA levels to help elucidate these mechanisms in falciparum malaria pathogenesis in young children residing in sub-Saharan African pockets of endemicity. For this investigation, World Health Organization-trained drug sensitivity testing team members  prepared fingerprick peripheral blood smears and obtained blood samples before and seven days after treatment with sulfadoxine-pyrimethamine (SP) from 21 Nigerian northern plateau children five years old and younger diagnosed with clinically-uncomplicated, slide-positive falciparum malaria.
The peripheral slide analysis revealed that all 21 children exhibited some degree of leukocyte pathology including abnormal neutrophils. In the innate immune response, neutrophils can use phagocytosis to kill microbes intracellularly or NET formation to kill microbes extracellularly. To form a NET, the nucleus of an activated neutrophil loses its classic lobular appearance as the nuclear membrane dissolves mixing chromatin with cytoplasmic granules. The neutrophil plasma membrane then ruptures, releasing the chromatin and adherent granule proteins as a coherent NET or in some cases as less organized extracellular fibers . NETs composed of a chromatin scaffold decorated with granule proteins have been shown to kill gram positive and gram negative bacteria as well as some fungi [37, 38, 40, 42, 53, 54]. In this investigation, various stages of NET formation with bound falciparum parasites were found on the peripheral blood smear slides from the falciparum-infected children. Evidence for NET circulation and involvement in the immune response to falciparum infection has not been previously reported. It remains to be determined whether NET association leads to falciparum parasite destruction.
In addition to possibly killing the parasites, release of neutrophil self-DNA in the NETs found in this investigation could induce autoimmunity, which in turn may contribute to acute severe pathogenesis in young children. In systemic lupus erythematosus, for example, the adjuvant activity of released self-DNA contributes to production of ANA either through an acute response involving complement-fixing antibodies against DNA or nucleoprotein-DNA complexes [19, 23, 55] or through a Th2-mediated IgG-induced humoral response [55–57]. General ANA levels, as well as IgG anti-dsDNA ANA diagnostic of an autoimmune response, were found in samples collected during the season of high malaria transmission from all the children in this study (86% pre-treatment and 100% post-treatment, 81% pre-treatment and 81% post-treatment; general ANA versus specific IgG dsDNA ANA, respectively). Autoimmune reactions to self-DNA from neutrophil shadow cells of Gumprecht and lymphocyte smudge cells contribute to certain noncancerous leukemias [50, 51]. The elevated number of metamyelocytes found in some of the falciparum-infected children in this study may reflect autoimmune activity.
Once produced, ANA may contribute to malarial pathogenesis through formation of immune complexes that induce renal anomalies and hyperuraemia [58–60] or impair leukocyte function . Moreover, NET adherence to parasitized and nonparasitized erythrocytes, as found in this investigation, could generate a break in tolerance and induce a carrier effect anti-erythrocyte Th1 cell-mediated activity leading to severe anaemia [31, 61]. Although all the children in this study had a packed red cell volume greater than 25% and, therefore, were not classified as anaemic , the presence of burr cells, acanthocytes, helmet cells, and schistocytes on the peripheral blood slides depicted a predisposition to anaemia. As the disease progresses, anti-erythrocyte activity could cause erythrocyte sequestration contributing to development of the microvascular occlusion associated with fatal cerebral malaria .
The elevated TNF levels found in this study were coincident with NETs. It is possible that the distinct cytokine profile (the high TNF/Il-6/Il-10 triad relationship with high levels of persistent TGF-β and CRP in the absence of Il-2) and the consistent presence of ANAs found here may represent the NET immunologic response to malaria in this sub-Saharan pocket of endemicity .
A moderately strong inverse correlation between TGF-β and CRP was found before and after treatment in this investigation. TGF-β-treated neutrophils produce reactive oxygen species (ROS), which are necessary for NET formation , while CRP inhibits neutrophil chemotaxis and production of ROS . CRP participates in opsonization and increased clearance of damaged cells, including opsonized erythrocytes and/or damaged leukocytes, by the classical pathway [20, 22, 64]. In children with low CRP levels, lack of such protection may permit a lack of clearance of debris and a progression from uncomplicated to severe malaria. Further studies are needed to determine the extent of the inverse relationship between TGF-β and CRP in other age groups within this population to determine whether there is a critical ratio threshold where the balance between the normal activities of TGF-β  and CRP [63, 66] is lost. In the future, TGF-β activity will be confirmed by measuring levels of IgA resulting from immunoglobulin class-switch.
NET formation and ANA production in older individuals with more adapted immune systems in a population chronically exposed through high seasonal malaria transmission could produce a very different outcome. In the adult population over time, the novel presentation of the parasites captured by NETs could induce a carrier-hapten type of humoral and cellular immune induction similar to that produced by the adjuvant effect of certain bacterial and parasite DNAs . Self-DNA interaction with Toll-like receptor 9 (TLR9), which stimulates IL-12-IFN-γ-dependent activation of Th1 cells, could stimulate immune response not only to the DNA complexes but also to the captured falciparum parasites. Such an adaptive mechanism of protection is consistent with the observation that elevated IL-12 levels are associated with less severe malaria in adults . Cross reacting activation of Th1 and Th2 mechanisms through toll-like receptors has been associated with CpG adjuvant activity .
Transition of infected children from autoimmune pathogenesis to autoimmune protection may depend on developing the ability to mount a T cell independent pathogen associated molecular pattern (PAMP) response. The lack of detectable IL-2 in the children with uncomplicated falciparum malaria in this study could be because it is bound to a soluble IL-2 receptor and missed by the assay . Alternatively, it may represent a lack of Th1 and Th2 clonal expansion, with a shift to NET-induced activation  through Toll-like receptors. If NET DNA binds to TLR9 and the ANA that is present in the children binds to IgG receptors, dendritic cells may cross present both major histocompatibility complexes I and II, thereby enhancing both humoral- and cell-mediated immune responses. As the ability of the child to mount a PAMP immune response develops, this autoimmune activity may become protective against the parasite by activating T cytotoxic cell activity . Development of NET-induced PAMP activity therefore could explain the paradox that high IFN-γ levels are associated with severe malaria in young children who have low IL-10 levels  but also associated with decreased malarial morbidity in adults who have elevated IL-12 levels associated with an adaptive Th1  and Th2 response .
Ironically, development of ANA, as found in the children assessed in this investigation, may bind well enough to CpG to neutralize its adjuvant activity. This could contribute to immune hyporesponsiveness  and affect the outcome in CpG adjuvant-based vaccines .