Sleep, aging, and lifespan in Drosophila

<p>Abstract</p> <p>Background</p> <p>Epidemiological studies in humans suggest that a decrease in daily sleep duration is associated with reduced lifespan, but this issue remains controversial. Other studies in humans also show that both sleep quantity and sleep quality decrease with age. <it>Drosophila melanogaster </it>is a useful model to study aging and sleep, and inheriting mutations affecting the potassium current Shaker results in flies that sleep less and have a shorter lifespan. However, whether the link between short sleep and reduced longevity exists also in wild-type flies is unknown. Similarly, it is unknown whether such a link depends on sleep amount per se, rather than on other factors such as waking activity. Also, sleep quality has been shown to decrease in old flies, but it remains unclear whether aging-related sleep fragmentation is a generalized phenomenon.</p> <p>Results</p> <p>We compared 3 short sleeping mutant lines (<it>Hk</it>¹, <it>Hk</it>^{<it>Y </it>}and <it>Hk</it>²) carrying a mutation in Hyperkinetic, which codes for the beta subunit of the Shaker channel, to wild-type siblings throughout their entire lifespan (all flies kept at 20°C). <it>Hk</it>¹and <it>Hk</it>^{<it>Y </it>}mutants were short sleeping relative to wild-type controls from day 3 after eclosure, and <it>Hk</it>²flies became short sleepers about two weeks later. All 3 <it>Hk </it>mutant lines had reduced lifespan relative to wild-type flies. Total sleep time showed a trend to increase in all lines with age, but the effect was most pronounced in <it>Hk</it>¹and <it>Hk</it>^{<it>Y </it>}flies. In both mutant and wild-type lines sleep quality did not decay with age, but the strong preference for sleep at night declined starting in "middle age". Using Cox regression analysis we found that in <it>Hk</it>¹and <it>Hk</it>^{<it>Y </it>}mutants and their control lines there was a negative relationship between total sleep amount during the first 2 and 4 weeks of age and hazard (individual risk of death), while no association was found in <it>Hk</it>²flies and their wild-type controls. <it>Hk</it>¹and <it>Hk</it>^{<it>Y </it>}mutants and their control lines also showed an association between total daily wake activity over the first 2 and 4 weeks of age and hazard. However, when both sleep duration and wake activity were used in the same regression, the effects of activity were much reduced, while most of the sleep effects remained significant. Finally, <it>Hk</it>¹flies and wild-type siblings were also tested at 25°C, and results were similar to those at 20°C. Namely, <it>Hk</it>¹mutants were short sleeping, hyperactive, and short lived relative to controls, and sleep quality in both groups did not decrease with age.</p> <p>Conclusions</p> <p>Different <it>Hk </it>mutations affect the sleep phenotype, and do so in an age-dependent manner. In 4 of the 6 lines tested sleep associates significantly with lifespan variation even after any effect of activity is removed, but activity does not associate significantly with lifespan after the effects of sleep are removed. Thus, in addition to environmental factors and genetic background, sleep may also affect longevity. Sleep quality does not necessarily decay as flies age, suggesting that aging-related sleep fragmentation may also depend on many factors, including genetic background and rearing conditions.</p

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

Rodent inflammasome activation by Toxoplasma gondii

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.Toxoplasma gondii is an obligate intracellular pathogen capable of chronically infecting nearly all warm-blooded animals, including humans. The chronic stage is characterized by the presence of semi-dormant cysts in brain and muscle tissues. These cysts are crucial in the success of Toxoplasma as they are orally infectious and allow for the transmission of the parasite between hosts. As the host immune response drives cyst formation, the establishment of this chronic infection relies on the parasite's ability to find a balance between activation of a host immune response and evasion of parasiticidal mechanisms. This balance is achieved through the modulation of host cell processes by parasite proteins secreted from specialized secretory organelles known as rhoptries and dense granules. Here, we report that Toxoplasma activates the inflammasomes in mice and rats. The inflammasomes are a set of cytoplasmic pattern recognition receptors (PRRs). Activation of the inflammasomes results in caspase-1 activation and the cleavage and release of the pro-inflammatory cytokines, Interleukin (IL)-1[beta] and IL-18. IL-1p is an important mediator of local inflammation and neutrophil recruitment. IL- 18 induces Interferon (IFN)-[gamma], which is a critical cytokine in the control of Toxoplasma. A form of cell death, termed pyroptosis, can accompany inflammasome activation. The NLRP3 inflammasome is activated in mouse macrophages, leading to the secretion of IL-1[beta] in vitro. The NLRP1 and NLRP3 inflammasomes play a major role in mouse survival and control of parasite replication in vivo. The NLRPI inflammasome is activated in infected macrophages from rats that are able to completely clear infection. Toxoplasma infection leads to the secretion of active IL-I[beta] and IL-18. Activation of the NLRP1 inflammasome leads to pyroptosis, a programmed form of cell death. Pyroptosis prevents parasite replication within the host cell and likely promotes clearance by nearby immune cells. Using a chemical mutagenesis screen, we identified three Toxoplasma dense granule proteins (GRAs), GRA18, GRA27 and GRA28, essential for NLRP1 inflammasome activation and pyroptosis in rat macrophages. Our work has identified Toxoplasma gondii as a novel activator of the rodent inflammasomes and demonstrated host cell death as a mechanism to control parasite replication. We have also identified three novel parasite proteins required for this activation, providing insight into interactions between parasite and host, which may aid in the treatment of human infection.by Kimberly M. Cirelli.Ph. D

Three Toxoplasma gondii Dense Granule Proteins Are Required for Induction of Lewis Rat Macrophage Pyroptosis.

Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis rat to Toxoplasma Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro, Δgra35, Δgra42, and Δgra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane.IMPORTANCE Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite's replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite

Summary flow diagram for mapping of rat macrophage sensitivity to four candidate genes.

<p>Methods for reducing the number of candidates at each stage are listed to the right and explained in detail in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003927#s3" target="_blank">Results</a> section. Detailed SNPs and gene lists for each stage can be found in Supporting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003927#ppat.1003927.s003" target="_blank">Figures S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003927#ppat.1003927.s004" target="_blank">S4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003927#ppat.1003927.s009" target="_blank">Dataset S1</a>.</p

NLRP1-variant dependent macrophage death depends on parasite invasion and controls parasite proliferation.

<p>(A) Viability of LEW BMDMs infected with Mycalolide-treated (3 µM, 15 min) RH tachyzoites (MOI 1∶1) after 24 h as measured by MTS assay (P-value comparing Mycalolide group to untreated = 0.0002). (B, C) Radiance emission analyses of metabolically active, viable Type II <i>Toxoplasma</i> 76K parasites (B, graph MOI 3∶1, 6 h; inset shows representative plate from one experiment) or Type I RH parasites (C, MOI 1∶1 over 48 h) in BMDMs from different rat strains. P-value comparing NLRP1^{variant 1,2} expressing strains to NLRP1^{variant 5} expressing strains are <0.01 in I by t-test and <0.0001 in J by two-way ANOVA. (D) Number of parasites/vacuole in infected BMDMS (24 h, 3∶1 MOI) as assessed by microscopy is shown. CDF, BN infections were with 76K, and SD, LEW infections were with RH. Between 50–100 vacuoles counted per experiment. Average values from 3 experiments are shown for all strains, except SD (n = 2). P-values are <0.01 (two-way ANOVA) when comparing NLRP1^{variant 1, 2} expressing strains to NLRP1^{variant 5} expressing strains. (E) Left panels show light microscopy images of CDF and LEW monolayers infected with 76K (MOI 6∶1, 6 h). Right panels show fluorescence microscopy image of single SD and LEW BMDMs infected with RH (MOI 1∶1, 2 h). Blue is Hoechst stained nucleus, green are GFP-expressing parasites. Dividing parasites in SD cells (upper right) or a single parasite in LEW cells (lower right) are shown. (F) LEW BMDMs were infected with PRU (MOI 3;1) and at 5 h post infection culture supernatants from dying cells was spun, filtered and transferred to similarly infected (PRU, MOI 3∶1) CDF BMDMs. Viability of CDF BMDMs was assessed at 10 h post-infection by MTT staining. All values were calculated relative to uninfected control BMDMs (G) SD BMDMs were infected with RH parasites (2 h, MOI 1∶1), washed with PBS and medium replaced with fresh media, media from RH-infected (24 h, MOI 1∶1) or uninfected LEW BMDMs. Parasites/vacuole counted at 24 h. P-values >0.1 (ns) for comparison of any of three groups for 1, 2, 4 and 8 parasites/vacuole counts (by two-way ANOVA).</p