Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma gondii

Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. IMPORTANCE Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.National Institutes of Health (U.S.) (Intramural Research Program of the NIH and NIAID)Crohn's and Colitis Foundation of America (Research Fellowship)Crohn's and Colitis Foundation of America (CCFA Helmsley Scholar)National Institutes of Health (U.S.) (NIH grant AI104170)National Institutes of Health (U.S.) (R01-AI080621)Pew Charitable Trusts (Pew Scholars Program in the Biomedical Sciences)Canadian Institute for Advanced Research (CIFAR Program for Integrated Microbial Biodiversity

Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii

Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages

Interaction of human blood platelets, lymphocytes and monocytes with vascular laminin isoforms

Inflammatory and immune responses play a fundamental role in both health and disease, and leukocytes are important actors in these processes. Migration of the leukocytes to sites of injury or inflammation is a crucial component of innate and adaptive immunity. It is currently accepted that leukocyte extravasation is a multistep process. However, a later step in this cascade, namely the interaction of leukocytes with components of extracellular matrices (ECM), such as the vascular endothelial basement membrane (BM) and the interstitium ECM, is poorly understood. There is also limited information concerning the role of vascular BM proteins in hemostasis and/or thrombosis. In this thesis, the interaction of blood platelets, lymphocytes and monocytes with vascular BM components, particularly the endothelial laminin isoforms, has been studied. Laminins (Lms), major components of all BMs, are a family of heterotrimeric molecules, each composed of á-, â-, and ã-chains. To date, five á-, three â-, and three ã-chains have been identified that associate to form at least 15 Lm isoforms. Lms regulate various cellular functions, such as adhesion, motility, differentiation and proliferation through various integrin and nonintegrin receptors. Lm-411 (á4â1ã1, laminin-8) and Lm-511 (á5â1ã1, laminin-10) are major Lm isoforms of vascular endothelial BMs. These BM components may participate in leukocyte extravasation and, following vascular injury, contribute to hemostasis and/or thrombosis when exposed to circulating platelets. First, commercially available placenta laminin preparations, often used in functional studies, were characterized. These preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. They also exhibited major functional differences between batches. In a following study, megakaryocytic cells were found to synthesize and platelets to secrete heterotrimeric á5-Lms. Lm-511 strongly promoted platelet adhesion, but not activation, via á6â1 integrin. Thereafter, the pivotal role of á5-Lm(s), expressed by high endothelial venules, in promoting adhesion and migration of blood lymphocytes via á6â1 integrin was demonstrated. Lm-511 was also able to co-stimulate T cell proliferation, and stimulated blood lymphocytes secreted both á4- and á5-laminins. The lymph node cell number in Lmá4-deficient mice compared to wild type did not differ significantly. Finally, Lm-411 and Lm-511 were found to mediate adhesion and chemokine-induced migration of monocytes via áMâ2 and áXâ2 integrins. Isolated Lmã1, but not Lmâ1, chain reproduced the effect of the Lm heterotrimers. Moreover, endogenous á4-Lm(s) mediated chemokine-induced, áMâ2- and áXâ2-integrin dependent monocyte migration on an albumin substrate. Altogether, the present studies illustrate the differential effects of laminin isoforms in the biology of platelets, lymphocytes and monocytes, and their potential contribution to hemostasis, and to the generation of immune and inflammatory responses