NLRP3 selectively drives IL-1ß secretion by Pseudomonas aeruginosa infected neutrophils and regulates corneal disease severity.

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-dependent, but GSDME independent IL-1ß secretion and pyroptosis. Here we examine inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1ß secretion by neutrophils requires the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT (ΔexoST) require NLRC4 for IL-1ß secretion. While IL-1ß release from ΔexoST infected neutrophils is also NLRC4-dependent, infection with PAO1 is instead NLRP3-dependent and driven by the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches using MCC950 reveal that NLRP3 is also essential for bacterial killing and disease severity in a murine model of P. aeruginosa corneal infection (keratitis). Overall, these findings reveal a function for ExoS ADPRT in regulating inflammasome subtype usage in neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

Beyond an inflammatory mediator: Interleukin-1 in neurophysiology.

NEW FINDINGS: What is the topic of this review? This review focuses on the physiological role of the cytokine interleukin-1ß in the CNS. What advances does it highlight? Traditionally, interleukin-1ß is known as a key mediator of inflammation and immunity. This review highlights the more recent findings describing how interleukin-1ß signalling is required to maintain homeostasis in the CNS. ABSTRACT: Since its discovery in the early 1940s, the interleukin-1 (IL-1) cytokine family has been associated primarily with acute and chronic inflammation. The family member IL-1ß is produced by different leucocytes, endothelial cells and epithelial cells. This cytokine has been characterized as a key modulator of inflammation and innate immunity because it induces the transcription of several downstream inflammatory genes. More recently, several groups have demonstrated that IL-1ß production is also required to maintain homeostasis in several organ systems. This review focuses on providing an overview of the more recently characterized role of IL-1ß in the physiology of the CNS. So far, IL-1ß signalling has been implicated in neuronal survival, neurite growth, synaptic pruning, synaptic transmission, neuroplasticity and neuroendocrine functions.

Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases.

Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1ß during infection of primary human monocytes.

IL-1ß is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1ß; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1ß via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1ß release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1ß transcripts and the production of pro-IL-1ß. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1ß production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1ß protein levels and IL-1ß release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1ß transcriptional activation. IL-1ß release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1ß to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1ß in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.

Mechanisms of Human Innate Immune Evasion by Toxoplasma gondii.

Toxoplasma gondii is an intracellular protozoan parasite of global importance that can remarkably infect, survive, and replicate in nearly all mammalian cells. Notably, 110 years after its discovery, Toxoplasmosis is still a neglected parasitic infection. Although most human infections with T. gondii are mild or asymptomatic, T. gondii infection can result in life-threatening disease in immunocompromised individuals and in the developing fetus due to congenital infection, underscoring the role of the host immune system in controlling the parasite. Recent evidence indicates that T. gondii elicits a robust innate immune response during infection. Interestingly, however, T. gondii has evolved strategies to successfully bypass or manipulate the immune system and establish a life-long infection in infected hosts. In particular, T. gondii manipulates host immunity through the control of host gene transcription and dysregulation of signaling pathways that result in modulation of cell adhesion and migration, secretion of immunoregulatory cytokines, production of microbicidal molecules, and apoptosis. Many of these host-pathogen interactions are governed by parasite effector proteins secreted from the apical secretory organelles, including the rhoptries and dense granules. Here, we review recent findings on mechanisms by which T. gondii evades host innate immunity, with a focus on parasite evasion of the human innate immune system.

Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1ß) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1ß synthesis in human peripheral blood neutrophils. IL-1ß suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1ß release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1ß and NLRP3 and reduced protein levels of pro-IL-1ß, mature IL-1ß, and the inflammasome sensor NLRP3. In T. gondii-infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKß, and phosphorylated IKKα/ß were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii-infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1ß involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1ß.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T. gondii infection, there is limited understanding of how human neutrophils respond to T. gondii Neutrophils control infectious pathogens by a variety of mechanisms, including the release of the cytokine IL-1ß, a major driver of inflammation during infection. This study reveals that T. gondii is able to inhibit IL-1ß production in human neutrophils by impairing the activation of the NF-κB signaling pathway and by inhibiting the inflammasome, the protein complex responsible for IL-1ß maturation. This two-pronged strategy of targeting the IL-1ß pathway may facilitate the survival and spread of T. gondii during acute infection.

NLRP3 and Potassium Efflux Drive Rapid IL-1ß Release from Primary Human Monocytes during Toxoplasma gondii Infection.

IL-1ß is produced by myeloid cells and acts as a critical mediator of host defense during infection and injury. We found that the intracellular protozoan parasite Toxoplasma gondii induced an early IL-1ß response (within 4 h) in primary human peripheral blood monocytes isolated from healthy donors. This process involved upregulation of IL-1ß, IL-1RN (IL-1R antagonist), and NLRP3 transcripts, de novo protein synthesis, and the release of pro- and mature IL-1ß from infected primary monocytes. The released pro-IL-1ß was cleavable to mature bioactive IL-1ß in the extracellular space by the protease caspase-1. Treatment of primary monocytes with the NLRP3 inhibitor MCC950 or with extracellular potassium significantly reduced IL-1ß cleavage and release in response to T. gondii infection, without affecting the release of TNF-α, and indicated a role for the inflammasome sensor NLRP3 and for potassium efflux in T. gondii-induced IL-1ß production. Interestingly, T. gondii infection did not induce an IL-1ß response in primary human macrophages derived from the same blood donors as the monocytes. Consistent with this finding, NLRP3 was downregulated during the differentiation of monocytes to macrophages and was not induced in macrophages during T. gondii infection. To our knowledge, these findings are the first to identify NLRP3 as an inflammasome sensor for T. gondii in primary human peripheral blood cells and to define an upstream regulator of its activation through the release of intracellular potassium.

Crotoxin, a rattlesnake toxin, down-modulates functions of bone marrow neutrophils and impairs the Syk-GTPase pathway.

Neutrophils have a critical role in the innate immune response; these cells represent the primary line of defense against invading pathogens or tissue injury. Crotoxin (CTX), the major toxin of the South American rattlesnake (Crotalus durissus terrificus) venom, presents longstanding anti-inflammatory properties, inhibiting neutrophil migration and phagocytosis by peritoneal neutrophils for 14 days. Herein, to elucidate these sustained inhibitory effects induced by CTX, we performed in vitro and in vivo studies evaluating the functionality of bone marrow neutrophils and possible molecular mechanisms associated with these effects. CTX inhibited the processes of chemotaxis, adhesion to fibronectin, and phagocytosis of opsonized particles; however, it did not affect ROS production or degranulation in bone marrow neutrophils. To understand the molecular mechanisms that orchestrate this effect, we investigated the expression of CR3 on the neutrophil surface and the total expression and activity of signaling proteins from the Syk-GTPase pathway, which is involved in actin polymerization. CTX down-regulated both subunits of CR3, as well as, the activity of Syk, Vav1, Cdc42, Rac1 and RhoA, and the expression of the subunit 1B from Arp2/3. Together, our findings demonstrated that CTX inhibits the functionally of bone marrow neutrophils and that this effect may be associated with an impairment of the Syk-GTPase pathway. This study demonstrates, for the first time, that the sustained down-modulatory effect of CTX on circulating and peritoneal neutrophils is associated with functional modifications of neutrophils still in the bone marrow, and it also contributes to a better understanding of the anti-inflammatory effect of CTX.

Crotoxin, a rattlesnake toxin, induces a long-lasting inhibitory effect on phagocytosis by neutrophils.

Crotalus durissus terrificus snake venom (CdtV) has long-lasting anti-inflammatory properties and inhibits the spreading and phagocytic activity of macrophages. Crotoxin (CTX), the main component of CdtV, is responsible for these effects. Considering the role of neutrophils in the inflammatory response and the lack of information about the effect of CdtV on neutrophils, the aim of this study was to investigate the effect of CdtV and CTX on two functions of neutrophils, namely phagocytosis and production of reactive oxygen species, and on the intracellular signaling involved in phagocytosis, particularly on tyrosine phosphorylation and rearrangements of the actin cytoskeleton. Our results showed that the incubation of neutrophils with CdtV or CTX, at different concentrations, or the subcutaneous injection of CdtV or CTX in rats two hours or one, four or 14 days before or one hour after the induction of inflammation inhibited the phagocytic activity of neutrophils. Furthermore, these in vitro and in vivo effects were associated with CdtV and CTX inhibition of tyrosine phosphorylation and consequently actin polymerization. Despite the inhibitory effect on phagocytosis, this study demonstrated that CdtV and CTX did not alter the production of the main reactive oxygen species. Therefore, this study characterized, for the first time, the actions of CdtV on neutrophils and demonstrated that CTX induces a long-lasting inhibition of tyrosine phosphorylation and consequently phagocytosis. We suggest that CTX represents a potential natural product in controlling inflammatory diseases, since a single dose exerts a long-lasting effect on intracellular signaling involved in phagocytosis by neutrophils.