Malaria infection alters the expression of B-cell activating factor resulting in diminished memory antibody responses and survival

Malaria is a major cause of morbidity worldwide with reports of over 200-500 million infected individuals and nearly 1 million deaths each year. Antibodies have been shown to play a critical role in controlling the blood stage of this disease; however, in malaria-endemic areas antibody immunity is slow to develop despite years of exposure to Plasmodium spp. the causative parasite. Using rodent Plasmodium yoelii YM, we provide evidence that malarial infections result in a decrease in the proportion of DCs that express the B-cell survival factor, BAFF, resulting in a decreased ability of these DCs to support memory B-cell differentiation into antibody secreting cells (ASCs) and/or the survival of ASCs. Further, compared with infected WT mice, ASC numbers were significantly increased in malaria-infected transgenic mice that either overexpressed BAFF or mice with BAFF-independent B-cell survival (B-cell-restricted TRAF3 deletion). Remarkably, BAFF-overexpressing mice were protected from lethal malaria infections, indicating the significance of the role BAFF plays in determining the outcome of malaria infections. These findings describe a previously unappreciated mechanism by which Plasmodium spp. can depress the generation of protective antibody responses

The mammalian PYHIN gene family: Phylogeny, evolution and expression

Background: Proteins of the mammalian PYHIN (IFI200/HIN-200) family are involved in defence against infection through recognition of foreign DNA. The family member absent in melanoma 2 (AIM2) binds cytosolic DNA via its HIN domain and initiates inflammasome formation via its pyrin domain. AIM2 lies within a cluster of related genes, many of which are uncharacterised in mouse. To better understand the evolution, orthology and function of these genes, we have documented the range of PYHIN genes present in representative mammalian species, and undertaken phylogenetic and expression analyses

A novel flow cytometric method to assess inflammasome formation

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments

AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and-1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2-and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome 'specks' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1