The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection.

Activation of endosomal Toll-like receptor (TLR) 7, TLR9, and TLR11/12 is a key event in the resistance against the parasite Toxoplasma gondii. Endosomal TLR engagement leads to expression of interleukin (IL)-12 via the myddosome, a protein complex containing MyD88 and IL-1 receptor-associated kinase (IRAK) 4 in addition to IRAK1 or IRAK2. In murine macrophages, IRAK2 is essential for IL-12 production via endosomal TLRs but, surprisingly, Irak2-/- mice are only slightly susceptible to T. gondii infection, similar to Irak1-/- mice. Here, we report that upon T. gondii infection IL-12 production by different cell populations requires either IRAK1 or IRAK2, with conventional dendritic cells (DCs) requiring IRAK1 and monocyte-derived DCs (MO-DCs) requiring IRAK2. In both populations, we identify interferon regulatory factor 5 as the main transcription factor driving the myddosome-dependent IL-12 production during T. gondii infection. Consistent with a redundant role of DCs and MO-DCs, mutations that affect IL-12 production in both cell populations show high susceptibility to infection in vivo.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells.

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ T cells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ T cells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

Alcohol-induced extracellular ASC specks perpetuate liver inflammation and damage in alcohol-associated hepatitis even after alcohol cessation.

BACKGROUND AIMS: Prolonged systemic inflammation contributes to poor clinical outcomes in severe alcohol-associated hepatitis (AH) even after the cessation of alcohol use. However, mechanisms leading to this persistent inflammation remain to be understood. APPROACH RESULTS: We show that while chronic alcohol induces nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in the liver, alcohol binge results not only in NLRP3 inflammasome activation but also in increased circulating extracellular apoptosis-associated speck-like protein containing a caspase recruitment domain (ex-ASC) specks and hepatic ASC aggregates both in patients with AH and in mouse models of AH. These ex-ASC specks persist in circulation even after the cessation of alcohol use. Administration of alcohol-induced-ex-ASC specks in vivo in alcohol-naive mice results in sustained inflammation in the liver and circulation and causes liver damage. Consistent with the key role of ex-ASC specks in mediating liver injury and inflammation, alcohol binge failed to induce liver damage or IL-1ß release in ASC-deficient mice. Our data show that alcohol induces ex-ASC specks in liver macrophages and hepatocytes, and these ex-ASC specks can trigger IL-1ß release in alcohol-naive monocytes, a process that can be prevented by the NLRP3 inhibitor, MCC950. In vivo administration of MCC950 reduced hepatic and ex-ASC specks, caspase-1 activation, IL-1ß production, and steatohepatitis in a murine model of AH. CONCLUSIONS: Our study demonstrates the central role of NLRP3 and ASC in alcohol-induced liver inflammation and unravels the critical role of ex-ASC specks in the propagation of systemic and liver inflammation in AH. Our data also identify NLRP3 as a potential therapeutic target in AH.

Malaria Resilience in South America: Epidemiology, Vector Biology, and Immunology Insights from the Amazonian International Center of Excellence in Malaria Research Network in Peru and Brazil.

The 1990s saw the rapid reemergence of malaria in Amazonia, where it remains an important public health priority in South America. The Amazonian International Center of Excellence in Malaria Research (ICEMR) was designed to take a multidisciplinary approach toward identifying novel malaria control and elimination strategies. Based on geographically and epidemiologically distinct sites in the Northeastern Peruvian and Western Brazilian Amazon regions, synergistic projects integrate malaria epidemiology, vector biology, and immunology. The Amazonian ICEMR's overarching goal is to understand how human behavior and other sociodemographic features of human reservoirs of transmission-predominantly asymptomatically parasitemic people-interact with the major Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, and with human immune responses to maintain malaria resilience and continued endemicity in a hypoendemic setting. Here, we will review Amazonian ICEMR's achievements on the synergies among malaria epidemiology, Plasmodium-vector interactions, and immune response, and how those provide a roadmap for further research, and, most importantly, point toward how to achieve malaria control and elimination in the Americas.

The IRAK4 scaffold integrates TLR4-driven TRIF and MYD88 signaling pathways.

Interleukin-1 receptor-associated kinases (IRAKs) -4, -2, and -1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-ß (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 signaling.

Lymphocyte crosstalk is required for monocyte-intrinsic trained immunity to Plasmodium falciparum.

Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum-infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell-depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum-trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum-trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.

Author Correction: Caspase-8 mediates inflammation and disease in rodent malaria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Caspase-8 mediates inflammation and disease in rodent malaria.

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1ß and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling.

BACKGROUND & AIMS: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. METHODS: We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. RESULTS: The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. CONCLUSIONS: TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

The immunology of Plasmodium vivax malaria.

Plasmodium vivax infection, the predominant cause of malaria in Asia and Latin America, affects ~14 million individuals annually, with considerable adverse effects on wellbeing and socioeconomic development. A clinical hallmark of Plasmodium infection, the paroxysm, is driven by pyrogenic cytokines produced during the immune response. Here, we review studies on the role of specific immune cell types, cognate innate immune receptors, and inflammatory cytokines on parasite control and disease symptoms. This review also summarizes studies on recurrent infections in individuals living in endemic regions as well as asymptomatic infections, a serious barrier to eliminating this disease. We propose potential mechanisms behind these repeated and subclinical infections, such as poor induction of immunological memory cells and inefficient T effector cells. We address the role of antibody-mediated resistance to P. vivax infection and discuss current progress in vaccine development. Finally, we review immunoregulatory mechanisms, such as inhibitory receptors, T regulatory cells, and the anti-inflammatory cytokine, IL-10, that antagonizes both innate and acquired immune responses, interfering with the development of protective immunity and parasite clearance. These studies provide new insights for the clinical management of symptomatic as well as asymptomatic individuals and the development of an efficacious vaccine for vivax malaria.

NLRP3 inflammasome activation drives tau pathology.

Alzheimer's disease is characterized by the accumulation of amyloid-beta in plaques, aggregation of hyperphosphorylated tau in neurofibrillary tangles and neuroinflammation, together resulting in neurodegeneration and cognitive decline1. The NLRP3 inflammasome assembles inside of microglia on activation, leading to increased cleavage and activity of caspase-1 and downstream interleukin-1ß release2. Although the NLRP3 inflammasome has been shown to be essential for the development and progression of amyloid-beta pathology in mice3, the precise effect on tau pathology remains unknown. Here we show that loss of NLRP3 inflammasome function reduced tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. Tau activated the NLRP3 inflammasome and intracerebral injection of fibrillar amyloid-beta-containing brain homogenates induced tau pathology in an NLRP3-dependent manner. These data identify an important role of microglia and NLRP3 inflammasome activation in the pathogenesis of tauopathies and support the amyloid-cascade hypothesis in Alzheimer's disease, demonstrating that neurofibrillary tangles develop downstream of amyloid-beta-induced microglial activation.

Platelet-activating factor (PAF) mediates NLRP3-NEK7 inflammasome induction independently of PAFR.

The role of lipids in inflammasome activation remains underappreciated. The phospholipid, platelet-activating factor (PAF), exerts multiple physiological functions by binding to a G protein-coupled seven-transmembrane receptor (PAFR). PAF is associated with a number of inflammatory disorders, yet the molecular mechanism underlying its proinflammatory function remains to be fully elucidated. We show that multiple PAF isoforms and PAF-like lipids can activate the inflammasome, resulting in IL-1ß and IL-18 maturation. This is dependent on NLRP3, ASC, caspase-1, and NEK7, but not on NLRC4, NLRP1, NLRP6, AIM2, caspase-11, or GSDMD. Inflammasome activation by PAF also requires potassium efflux and calcium influx but not lysosomal cathepsin or mitochondrial reactive oxygen species. PAF exacerbates peritonitis partly through inflammasome activation, but PAFR is dispensable for PAF-induced inflammasome activation in vivo or in vitro. These findings reveal that PAF represents a damage-associated signal that activates the canonical inflammasome independently of PAFR and provides an explanation for the ineffectiveness of PAFR antagonist in blocking PAF-mediated inflammation in the clinic.

The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.

Inflammasome-derived cytokine IL18 suppresses amyloid-induced seizures in Alzheimer-prone mice.

Alzheimer's disease (AD) is characterized by the progressive destruction and dysfunction of central neurons. AD patients commonly have unprovoked seizures compared with age-matched controls. Amyloid peptide-related inflammation is thought to be an important aspect of AD pathogenesis. We previously reported that NLRP3 inflammasome KO mice, when bred into APPswe/PS1ΔE9 (APP/PS1) mice, are completely protected from amyloid-induced AD-like disease, presumably because they cannot produce mature IL1ß or IL18. To test the role of IL18, we bred IL18KO mice with APP/PS1 mice. Surprisingly, IL18KO/APP/PS1 mice developed a lethal seizure disorder that was completely reversed by the anticonvulsant levetiracetam. IL18-deficient AD mice showed a lower threshold in chemically induced seizures and a selective increase in gene expression related to increased neuronal activity. IL18-deficient AD mice exhibited increased excitatory synaptic proteins, spine density, and basal excitatory synaptic transmission that contributed to seizure activity. This study identifies a role for IL18 in suppressing aberrant neuronal transmission in AD.

Targeting the IL33-NLRP3 axis improves therapy for experimental cerebral malaria.

Cerebral malaria (CM) is a serious neurological complication caused by Plasmodium falciparum infection. Currently, the only treatment for CM is the provision of antimalarial drugs; however, such treatment by itself often fails to prevent death or development of neurological sequelae. To identify potential improved treatments for CM, we performed a nonbiased whole-brain transcriptomic time-course analysis of antimalarial drug chemotherapy of murine experimental CM (ECM). Bioinformatics analyses revealed IL33 as a critical regulator of neuroinflammation and cerebral pathology that is down-regulated in the brain during fatal ECM and in the acute period following treatment of ECM. Consistent with this, administration of IL33 alongside antimalarial drugs significantly improved the treatment success of established ECM. Mechanistically, IL33 treatment reduced inflammasome activation and IL1ß production in microglia and intracerebral monocytes in the acute recovery period following treatment of ECM. Moreover, treatment with the NLRP3-inflammasome inhibitor MCC950 alongside antimalarial drugs phenocopied the protective effect of IL33 therapy in improving the recovery from established ECM. We further showed that IL1ß release from macrophages was stimulated by hemozoin and antimalarial drugs and that this was inhibited by MCC950. Our results therefore demonstrate that manipulation of the IL33-NLRP3 axis may be an effective therapy to suppress neuroinflammation and improve the efficacy of antimalarial drug treatment of CM.

Cutting Edge: Plasmodium falciparum Induces Trained Innate Immunity.

Malarial infection in naive individuals induces a robust innate immune response. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed training) or hyporespond (tolerance) to subsequent immune challenge. Previous work in both mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with Plasmodium falciparum-infected RBCs or the malaria crystal hemozoin induced human adherent PBMCs to hyperrespond to subsequent ligation of TLR2. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that the induction of trained immunity by malaria and its ligands may occur via a previously unrecognized mechanism(s).

Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-ß induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites.

Activation of the NLRP3 inflammasome in microglia: the role of ceramide.

Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia-derived factors that play an essential role in the immune response. IL-1ß is a pro-inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase-1, pro-IL-1ß is cleaved to the mature cytokine following NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL-1ß using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild-type and apoptosis-associated speck-like protein containing a CARD knockout (ASC-/- ) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL-1ß secretion in microglia. Exposure of microglia to the serine palmitoyl transferase-inhibitor l-cycloserine significantly prevented PA-induced IL-1ß secretion. Application of the ceramide analogue C2 and the sphingosine-1-phosphate-receptor agonist Fingolimod (FTY720) up-regulated levels of IL-1ß and cleaved caspase-1 in wild-type microglia, whereas ASC-/- microglia were unaffected. HPA-12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL-1ß.

miR-718 represses proinflammatory cytokine production through targeting phosphatase and tensin homolog (PTEN).

Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5' UTR of the interleukin 1 receptor-associated kinase (IRAK1) gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during Neisseria gonorrhoeae infection and alters the infection dynamics of N. gonorrhoeae in vitro Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-κB.

Microglia-derived ASC specks cross-seed amyloid-ß in Alzheimer's disease.

The spreading of pathology within and between brain areas is a hallmark of neurodegenerative disorders. In patients with Alzheimer's disease, deposition of amyloid-ß is accompanied by activation of the innate immune system and involves inflammasome-dependent formation of ASC specks in microglia. ASC specks released by microglia bind rapidly to amyloid-ß and increase the formation of amyloid-ß oligomers and aggregates, acting as an inflammation-driven cross-seed for amyloid-ß pathology. Here we show that intrahippocampal injection of ASC specks resulted in spreading of amyloid-ß pathology in transgenic double-mutant APPSwePSEN1dE9 mice. By contrast, homogenates from brains of APPSwePSEN1dE9 mice failed to induce seeding and spreading of amyloid-ß pathology in ASC-deficient APPSwePSEN1dE9 mice. Moreover, co-application of an anti-ASC antibody blocked the increase in amyloid-ß pathology in APPSwePSEN1dE9 mice. These findings support the concept that inflammasome activation is connected to seeding and spreading of amyloid-ß pathology in patients with Alzheimer's disease.