NALP1 is a member of the NOD-like receptor (NLR) family of proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of proinflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor.Toxoplasma gondiiis an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines that are important in innate immunity. In this study, susceptibility alleles for human congenital toxoplasmosis were identified in the NALP1 gene. To investigate the role of the NALP1 inflammasome during infection withT. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression ofT. gondiiinfection, with accelerated host cell death and eventual cell disintegration. In line with this observation, upregulation of the proinflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-12 uponT. gondiiinfection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses toT. gondiiinfection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, this is the first report, to our knowledge, on the crucial role of the NALP1 inflammasome in the pathogenesis ofT. gondiiinfections in humans.

Toxoplasmosis is a healthcare problem in pregnant women and immunocompromised patients. Like humans, rats usually develop a subclinical chronic infection. LEW rats exhibit total resistance toToxoplasma gondiiinfection, which is expressed in a dominant mode. A genome-wide search carried out in a cohort of F₂progeny of susceptible BN and resistant LEW rats led to identify on chromosome 10 a major locus of control, which we calledToxo1. Using reciprocal BN and LEW lines congenic for chromosome 10 genomic regions from the other strain,Toxo1was found to govern the issue ofT. gondiiinfection whatever the remaining genome. Analyzes of rats characterized by genomic recombination withinToxo1, reduced the interval down to a 1.7-cM region syntenic to human 17p13.In vitrostudies showed that theToxo1-mediated refractoriness toT. gondiiinfection is associated with the ability of the macrophage to impede the proliferation of the parasite within the parasitophorous vacuole. In contrast, proliferation was observed in fibroblasts whatever the genomic origin ofToxo1. Furthermore,ex vivostudies indicate that macrophage controls parasitic infection spreading by aToxo1-mediated mechanism. This forward genetics approach should ultimately unravel a major pathway of innate resistance to toxoplasmosis and possibly to other apicomplexan parasitic diseases.

Natural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1β. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-β secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance. Toxoplasmosis is a ubiquitous parasitic infection causing a wide spectrum of diseases. It is usually asymptomatic but can lead to severe ocular and neurological disorders. The host factors that determine natural resistance to toxoplasmosis are yet poorly characterized. Among the animal models to study susceptibility to toxoplasmosis, rats develop like humans a subclinical chronic infection. The finding of a total resistance in the LEW rat strain has allowed genetic studies leading to the identification of Toxo1, a unique locus that controls the outcome of toxoplasmosis. In this report, a panel of recombinant inbred rat strains was used to genetically reduce the Toxo1 locus, on chromosome 10, to a limited region containing 29 genes. This locus is highly conserved among five resistant, by comparison to four susceptible, rat strains, indicating that refractoriness to toxoplasmosis could be predicted. The Toxo1-controlled refractoriness depends on the ability of macrophages to restrict parasite proliferation and the rapid death of both T. gondii and host macrophages in vitro. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. Our data represent new insights towards the identification of a major pathway of innate immunity that protects from toxoplasmosis.

Fas (CD95)-induced hepatocyte apoptosis and cytotoxic activity of neutrophils infiltrating the injured liver are two major events leading to hepatitis. Because it has been reported that opioids, via a direct interaction, sensitize splenocytes to Fas-mediated apoptosis by upregulating Fas messenger RNA (mRNA) and modulated neutrophil activity, we assumed that opioids may participate in the pathophysiology of hepatitis. Using the hepatitis model induced by agonistic anti-Fas antibody in mice, we showed that opioid receptor blockade reduced liver damage and consequently increased the survival rate of animals when the antagonist naltrexone was injected simultaneously or prior to antibody administration. Treatment of mice with morphine enhanced mortality. Naloxone methiodide—a selective peripheral opioid antagonist—had a protective effect, but the absence of opioid receptors in the liver, together with lack of morphine effect in Fas-induced apoptosis of primary cultured hepatocytes, ruled out a direct effect of opioids on hepatocytes. In addition, the neutralization of opioid activity by naltrexone did not modify Fas mRNA expression in the liver as assessed with real-time quantitative polymerase chain reaction. Injured livers were infiltrated by neutrophils, but granulocyte-depleted mice were not protected against the enhancing apoptotic effect of morphine. In conclusion, opioid receptor blockade improves the resistance of mice to Fas-induced hepatitis via a peripheral mechanism that does not involve a down-modulation of Fas mRNA in hepatocytes nor a decrease in proinflammatory activity of neutrophils. (Hepatology 2004.)

Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RC^high and CD45RC^low, that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RC^high CD8 T cells produce IL-2 and IFN-γ while CD45RC^low CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RC^high/CD45RC^low CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RC^high/CD45RC^low CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F₂(LEW × BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RC^high/CD45RC^low CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RC^high/CD45RC^low CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [ P , <0.000309], rs312462 [ P , <0.028499], rs6502998 [ P , <0.029794], and rs434473 [ P , <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12 . In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.