Malaria continues to be a profound medical problem world wide, despite new and effective public health strategies that have clearly diminished morbidity and mortality. A sterilizing vaccine for the disease is still not within site, perhaps because there is still much that we do not know concerning plasmodial immunity. This application is to renew a highly productive RO1 on innate immunity in malaria. Our overall hypothesis is that the innate immune response to malaria is driven by two highly synergistic parasitic products: the malarial crystal hemozoin (Hz) and plasmodial DNA. Our evidence suggests that DNA gets into innate immune cells via three major routes: within the intact parasite (which may or may not be alive at the time of entry), on the surface of Hz, or as part of an immune complex. Together, these Pathogen-associated molecular patterns (PAMPS) engage a variety of endolysosomal and cytosolic nucleotide receptors, including (but not limited to) TLR9. In addition, plasmodial derived PAMPs activate multiple inflammasome complexes, including NLRP3, NLRP12 and AIM2. In order to better characterize the responses to plasmodial infections, we propose three specific aims.
The first aim i s based on our hypothesis that cytosolic DNA receptors are activated during infection. These receptors appear, in part, to recognize parasite DNA via a unique AT-rich motif that we have recently characterized. We propose to explore this hypothesis using a combination of approaches, focusing first on four knockout mice that we have recently generated [CNBP, Mtr4/Skiv2L2, IFI16 and the enzyme cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)]. We have also worked out LSMS/MS methodologies for examining the production of cGAMP from monocytes purified from the blood of malaria patients. The identification of cGAMP in febrile patients would be strong evidence that there is a DNA driven innate immune response in human disease.
Aim 2 is based on the hypothesis that inflammasomes drive the pathogenesis of malaria. Most of these studies have been performed in mice, or mouse-derived cells, although we have good preliminary data in cells from febrile patients as well. We first propose to extend these studies using loss of function approaches in human cells and cell lines. More importantly, we will purify inflammasomes from the phagocytic cells of patients and subject these complexes to proteomic analysis, in an unbiased effort to find identify inflammasomes not yet realized to be in involved in disease. Finally, we propose to quantify the number of cells expressing assembled inflammasomes and relate these findings to pyroptotic cell death, as our preliminary data suggest that pyroptosis may be extensive (up to 25% of circulating monocytes). In our final Aim, we will test the hypothesis that inflammation in malaria is regulated to a degree not previously recognized by long non-coding RNAS (lincRNAs), similar to the response to LPS and other TLR ligands. We will begin by focusing on lncRNA-Cox2, which globally controls responses to TLR ligands in mice. We will perform RNA-sequencing analysis to identify novel lincRNAs using cells stimulated in vitro with plasmodial PAMPs and cells harvested from patients, to identify lincRNAs actually involved in malaria. Finally, we will identify protein bindng partners and genomic targets of identified lncRNAs using established techniques. We believe that this ambitious research plan will add significantly to our understanding of innate immunity in malaria and in hopefully give rise to novel approaches and vaccine strategies that can be used to reduce the global burden of disease.
Malaria is a major cause of sickness and death in the world today; even American citizens contract malaria at a rate of 1500-2000 individuals per year. Here, we propose to study the basic immune mechanisms that patients with malaria to get sick and die, both by studying mice with known defects in immune receptors and testing white blood cells taken from infected patients who come to our clinics for therapy. The knowledge we gain from this work has the potential to improve therapies for the disease as well as to help design a better vaccine for individuals who live in a malarial region.
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