NLRC5 senses NAD+ depletion, forming a PANoptosome and driving PANoptosis and inflammation.

NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5's role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.

NLRP12-PANoptosome activates PANoptosis and pathology in response to heme and PAMPs.

Cytosolic innate immune sensors are critical for host defense and form complexes, such as inflammasomes and PANoptosomes, that induce inflammatory cell death. The sensor NLRP12 is associated with infectious and inflammatory diseases, but its activating triggers and roles in cell death and inflammation remain unclear. Here, we discovered that NLRP12 drives inflammasome and PANoptosome activation, cell death, and inflammation in response to heme plus PAMPs or TNF. TLR2/4-mediated signaling through IRF1 induced Nlrp12 expression, which led to inflammasome formation to induce maturation of IL-1ß and IL-18. The inflammasome also served as an integral component of a larger NLRP12-PANoptosome that drove inflammatory cell death through caspase-8/RIPK3. Deletion of Nlrp12 protected mice from acute kidney injury and lethality in a hemolytic model. Overall, we identified NLRP12 as an essential cytosolic sensor for heme plus PAMPs-mediated PANoptosis, inflammation, and pathology, suggesting that NLRP12 and molecules in this pathway are potential drug targets for hemolytic and inflammatory diseases.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

The NLR family of innate immune and cell death sensors.

Nucleotide-binding oligomerization domain (NOD)-like receptors, also known as nucleotide-binding leucine-rich repeat receptors (NLRs), are a family of cytosolic pattern recognition receptors that detect a wide variety of pathogenic and sterile triggers. Activation of specific NLRs initiates pro- or anti-inflammatory signaling cascades and the formation of inflammasomes-multi-protein complexes that induce caspase-1 activation to drive inflammatory cytokine maturation and lytic cell death, pyroptosis. Certain NLRs and inflammasomes act as integral components of larger cell death complexes-PANoptosomes-driving another form of lytic cell death, PANoptosis. Here, we review the current understanding of the evolution, structure, and function of NLRs in health and disease. We discuss the concept of NLR networks and their roles in driving cell death and immunity. An improved mechanistic understanding of NLRs may provide therapeutic strategies applicable across infectious and inflammatory diseases and in cancer.

STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum.

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of the mechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms to promote immunity and survival after infection.

The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector.

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

Baker's yeast beta glucan supplementation was associated with an improved innate immune mRNA expression response after exercise.

Beta glucans are found in many natural sources, however, only Baker's Yeast Beta Glucan (BYBG) has been well documented to have structure-function effects that are associated with improved innate immune response to stressors (e.g., exercise, infection, etc.). The purpose was to identify a BYBG-associated mRNA expression pattern following exercise. Participants gave IRB-approved consent and were randomized to BYBG (Wellmune®; N=9) or Placebo (maltodextrin; N=10) for 6-weeks prior to performing 90 min of whole-body exercise. Paxgene blood samples were collected prior to exercise (PRE), after exercise (POST), two hours after exercise (2H), and four hours after exercise (4H). Total RNA was isolated and analyzed for the expression of 770 innate immune response mRNA (730 mRNA targets; 40 housekeepers/controls; Nanostring nCounter). The raw data were normalized against housekeeping controls and expressed as Log2 fold change from PRE for a given condition. Significance was set at p < 0.05 with adjustments for multiple comparisons and false discovery rate. We identified 47 mRNA whose expression was changed after exercise with BYBG and classified them to four functional pathways: 1) Immune Cell Maturation (8 mRNA), 2) Immune Response and Function (5 mRNA), 3) Pattern Recognition Receptors and DAMP or PAMP Detection (25 mRNA), and 4) Detection and Resolution of Tissue Damage (9 mRNA). The identified mRNA whose expression was altered after exercise with BYBG may represent an innate immune response pattern and supports previous conclusions that BYBG improves immune response to a future sterile inflammation or infection.

Pathological remodeling of reactive astrocytes: Involvement of DNA methylation and downregulation of homeostatic genes.

Astrocytes provide metabolic support to neurons, maintain ionic and water homeostasis, and uptake and recycle neurotransmitters. After exposure to the prototypical PAMP lipopolysaccharide (LPS), reactive astrocytes increase the expression of pro-inflammatory genes, facilitating neurodegeneration. In this study, we analyzed the expression of homeostatic genes in astrocytes exposed to LPS and identified the epigenetic factors contributing to the suppression of homeostatic genes in reactive astrocytes. Primary astrocytic cultures were acutely exposed to LPS and allowed to recover for 24, 72 h, and 7 days. As expected, LPS exposure induced reactive astrogliosis and increased the expression of pro-inflammatory IL-1B and IL-6. Interestingly, the acute exposure resulted in persistent hypermethylation of astroglial DNA. Similar hypermethylation was observed in highly reactive astrocytes from the traumatic brain injury (TBI) penumbra in vivo. Hypermethylation was accompanied by decreased expression of homeostatic genes including LDHA and Scl16a1 (MCT1) both involved in the lactate shuttle to neurons; glutamine synthase (GS) responsible for glutamate processing; Kcnj10 (Kir4.1) important for K+ homeostasis, and the water channel aquaporin-4 (Aqp4). Furthermore, the master regulator of DNA methylation, MAFG-1, as well as DNA methyl transferases DNMT1 and DNMT3a were overexpressed. The downregulation of homeostatic genes correlated with increased methylation of CpG islands in their promoters, as assessed by methylation-sensitive PCR and increased DNMT3a binding to the GS promoter. Treatment with decitabine, a DNMT inhibitor, prevented the LPS- and the HMGB-1-induced downregulation of homeostatic genes. Decitabine treatment also prevented the neurotoxic effects of these astrocytes in primary cortical cultures. In summary, our findings reveal that the pathological remodeling of reactive astrocytes encompasses not only the pro-inflammatory response but, significantly, also entails a long-term suppression of homeostatic gene expression with methylation of crucial CpG islands within their promoters.

Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses.

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity responses. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2, present in the apoplast, depends on ribonuclease catalytic residues. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

Vibrio cholerae Porin OmpU Activates Dendritic Cells via TLR2 and the NLRP3 Inflammasome.

OmpU is one of the major porins of Vibrio cholerae, a Gram-negative human pathogen. Previously, we showed that OmpU stimulates host monocytes and macrophages and induces the production of proinflammatory mediators via activation of the Toll-like receptor 1/2 (TLR1/2)-MyD88-dependent pathways. In the present study, we show that OmpU activates murine dendritic cells (DCs) via activation of the TLR2-mediated pathway and the NLRP3 inflammasome, leading to the production of proinflammatory cytokines and DC maturation. Our data reveal that although TLR2 plays an important role in providing both priming and the activation signal for the NLRP3 inflammasome in OmpU-activated DCs, OmpU is capable of activating the NLRP3 inflammasome, even in the absence of TLR2, if a priming signal is given. Furthermore, we show that the OmpU-mediated interleukin-1ß (IL-1ß) production in DCs depends on calcium flux and mitochondrial reactive oxygen species (mitoROS) generation. Interestingly, both OmpU translocation to the mitochondria of DCs as well as calcium signaling contribute to mitoROS production and prompt NLRP3 inflammasome activation. We also demonstrate that OmpU induces downstream signaling via activation of phosphoinositide-3-kinase (PI3K)-AKT, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and transcription factor NF-κB. Furthermore, our data reveal that OmpU-mediated activation of TLR2 induces signaling via PKC, MAPKs p38 and extracellular signal-regulated kinase (ERK), and transcription factor NF-κB; however, PI3K and MAPK Jun N-terminal protein kinase (JNK) are activated in TLR2 independent manner.

Exploring the heterogeneous transcriptional response of the CNS to systemic LPS and Poly(I:C).

Peripheral contact to pathogen-associated molecular patterns (PAMPs) evokes a systemic innate immune response which is rapidly relayed to the central nervous system (CNS). The remarkable cellular heterogeneity of the CNS poses a significant challenge to the study of cell type and stimulus dependent responses of neural cells during acute inflammation. Here we utilized single nuclei RNA sequencing (snRNAseq), serum proteome profiling and primary cell culture methods to systematically compare the acute response of the mammalian brain to the bacterial PAMP lipopolysaccharide (LPS) and the viral PAMP polyinosinic:polycytidylic acid (Poly(I:C)), at single cell resolution. Our study unveiled convergent transcriptional cytokine and cellular stress responses in brain vascular and ependymal cells and a downregulation of several key mediators of directed blood brain barrier (BBB) transport. In contrast the neuronal response to PAMPs was limited in acute neuroinflammation. Moreover, our study highlighted the dominant role of IFN signalling upon Poly(I:C) challenge, particularly in cells of the oligodendrocyte lineage. Collectively our study unveils heterogeneous, shared and distinct cell type and stimulus dependent acute responses of the CNS to bacterial and viral PAMP challenges. Our findings highlight inflammation induced dysregulations of BBB-transporter gene expression, suggesting potential translational implications on drug pharmacokinetics variability during acute neuroinflammation. The pronounced dependency of oligodendrocytes on IFN stimulation during viral PAMP challenges, emphasizes their limited molecular viral response repertoire.

Osmoelectric siphon models for signal and water dispersal in wounded plants.

When attacked by herbivores, plants produce electrical signals which can activate the synthesis of the defense mediator jasmonate. These wound-induced membrane potential changes can occur in response to elicitors that are released from damaged plant cells. We list plant-derived elicitors of membrane depolarization. These compounds include the amino acid l-glutamate (Glu), a potential ligand for GLUTAMATE RECEPTOR-LIKE (GLR) proteins that play roles in herbivore-activated electrical signaling. How are membrane depolarization elicitors dispersed in wounded plants? In analogy with widespread turgor-driven cell and organ movements, we propose osmoelectric siphon mechanisms for elicitor transport. These mechanisms are based on membrane depolarization leading to cell water shedding into the apoplast followed by membrane repolarization and water uptake. We discuss two related mechanisms likely to occur in response to small wounds and large wounds that trigger leaf-to-leaf electrical signal propagation. To reduce jasmonate pathway activation, a feeding insect must cut through tissues cleanly. If their mandibles become worn, the herbivore is converted into a robust plant defense activator. Our models may therefore help to explain why numerous plants produce abrasives which can blunt herbivore mouthparts. Finally, if verified, the models we propose may be generalizable for cell to cell transport of water and pathogen-derived regulators.

Ubiquitination from the perspective of plant pathogens.

The constant battle of survival between pathogens and host plants has played a crucial role in shaping the course of their co-evolution. However, the major determinants of the outcome of this ongoing arms race are the effectors secreted by pathogens into host cells. These effectors perturb the defense responses of plants to promote successful infection. In recent years, extensive research in the area of effector biology has reported an increase in the repertoire of pathogenic effectors that mimic or target the conserved ubiquitin-proteasome pathway. The role of the ubiquitin-mediated degradation pathway is well known to be indispensable for various aspects of a plant's life, and thus targeting or mimicking it seems to be a smart strategy adopted by pathogens. Therefore, this review summarizes recent findings on how some pathogenic effectors mimic or act as one of the components of the ubiquitin-proteasome machinery while others directly target the plant's ubiquitin-proteasome system.

Suppression of NLR-mediated plant immune detection by bacterial pathogens.

The plant immune system is constituted of two functionally interdependent branches that provide the plant with an effective defense against microbial pathogens. They can be considered separate since one detects extracellular pathogen-associated molecular patterns by means of receptors on the plant surface, while the other detects pathogen-secreted virulence effectors via intracellular receptors. Plant defense depending on both branches can be effectively suppressed by host-adapted microbial pathogens. In this review we focus on bacterially driven suppression of the latter, known as effector-triggered immunity (ETI) and dependent on diverse NOD-like receptors (NLRs). We examine how some effectors secreted by pathogenic bacteria carrying type III secretion systems can be subject to specific NLR-mediated detection, which can be evaded by the action of additional co-secreted effectors (suppressors), implying that virulence depends on the coordinated action of the whole repertoire of effectors of any given bacterium and their complex epistatic interactions within the plant. We consider how ETI activation can be avoided by using suppressors to directly alter compromised co-secreted effectors, modify plant defense-associated proteins, or occasionally both. We also comment on the potential assembly within the plant cell of multi-protein complexes comprising both bacterial effectors and defense protein targets.

Exploring the immunogenic properties of SARS-CoV-2 structural proteins: PAMP:TLR signaling in the mediation of the neuroinflammatory and neurologic sequelae of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) produces an array of neurologic and neuropsychiatric symptoms in the acute and post-acute phase of infection (PASC; post-acute sequelae of SARS-CoV-2 infection). Neuroinflammatory processes are considered key factors in the etiology of these symptoms. Several mechanisms underpinning the development of inflammatory events in the brain have been proposed including SARS-CoV-2 neurotropism and peripheral inflammatory responses (i.e., cytokine storm) to infection, which might produce neuroinflammation via immune-to-brain signaling pathways. In this review, we explore evidence in support of an alternate mechanism whereby structural proteins (e.g., spike and spike S1 subunit) derived from SARS-CoV-2 virions function as pathogen-associated molecular patterns (PAMPs) to elicit proinflammatory immune responses in the periphery and/or brain via classical Toll-Like Receptor (TLR) inflammatory pathways. We propose that SARS-CoV-2 structural proteins might directly produce inflammatory processes in brain independent of and/or in addition to peripheral proinflammatory effects, which might converge to play a causal role in the development of neurologic/neuropsychiatric symptoms in COVID-19.

C-type lectin receptors in antifungal immunity: Current knowledge and future developments.

C-type lectin receptors (CLRs) constitute a category of innate immune receptors that play an essential role in the antifungal immune response. For over two decades, scientists have uncovered what are the fungal ligands recognized by CLRs and how these receptors initiate the immune response. Such studies have allowed the identification of genetic polymorphisms in genes encoding for CLRs or for proteins involved in the signalisation cascade they trigger. Nevertheless, our understanding of how these receptors functions and the full extent of their function during the antifungal immune response is still at its infancy. In this review, we summarize some of the main findings about CLRs in antifungal immunity and discuss what the future might hold for the field.

BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer.

OBJECTIVE AND DESIGN: BacSp222 bacteriocin is a bactericidal and proinflammatory peptide stimulating immune cells to produce selected cytokines and NO in NF-ĸB dependent manner. This study aims to identify the receptor which mediates this activity. METHODS: We applied fluorescently labeled BacSp222 and a confocal microscopy imaging to analyze the direct interaction of the bacteriocin with the cells. Reporter HEK-Blue cells overexpressing human toll-like receptors (TLR2, TLR4, TLR5 or TLR2/TLR1 and TLR2/TLR6 heterodimers) were stimulated with BacSp222, and then the activity of NF-ĸB-dependent secreted embryonic alkaline phosphatase (SEAP) was measured. In turn, formylated peptide receptor (FPR) or TLR2 antagonists were used to verify bacteriocin-stimulated TNF production by murine monocyte-macrophage cell lines. RESULTS: BacSp222 undergoes internalization into cells without disturbing the cell membrane. FPR antagonists do not affect TNF produced by BacSp222-stimulated murine macrophage-like cells. In contrast, BacSp222 stimulates NF-ĸB activation in HEK-Blue overexpressing TLR2 or TLR2/TLR6 heterodimer, but not TLR2/TLR1, TLR4 or TLR5 receptors. Moreover, TLR2-specific antagonists inhibit NF-ĸB signaling in BacSp222-stimulated HEK-Blue TLR2/TLR6 cells and reduce TNF release by BacSp222-treated RAW 264.7 and P388.D1. CONCLUSIONS: BacSp222 is a novel ligand for TLR2/TLR6 heterodimer. By binding TLR complex the bacteriocin undergoes internalization, inducing proinflammatory signaling that employs MyD88 and NF-ĸB pathways.

Diverse pathogen-associated molecular patterns affect transcription of genes in the toll-like receptor signaling pathway in goat blood.

Pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), Polyinosinic-polycytidylic acid (poly I:C), and CpG Oligodeoxynucleotides (ODN) are recognized by Toll-like receptors (TLR). This study aimed to investigate the effect of diverse PAMPs on the transcription of TLR signaling pathway genes in goat blood. Whole blood was collected from 3 female BoerXSpanish goats and treated with the following PAMPs: 10 µg/ml LPS, PGN, CpG ODN (2216), CpG ODN (2006), and 12.5 µg/ml Poly I:C. Blood-treated PBS served as a control. The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array (Qiagen) was evaluated using real-time PCR. Treatment with PBS affected the expression of 74 genes, Poly I:C affected the expression of 40 genes, t ODN 2006 affected the expression of 50 genes, ODN 2216 affected the expression of 52 genes, LPS affected the expression of 49 genes, while PGN affected the expression of 49 genes. Our results show that PAMPs modulated and increased the expression of genes in the TLR signaling pathway. These results highlight important insights into how the host responds to different pathogens and may help design adjuvants for therapeutics and vaccines that target different.

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a ß-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

Flagellin-Induced Immune Response in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Pathogen-associated molecular patterns (PAMPs) are involved in the pathogenesis of septic cardiomyopathy through a toll-like receptor (TLR)-mediated immune response. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can reflect the innate immune abilities of cardiomyocytes. Therefore, hiPSC-CMs may provide an attractive tool with which to study PAMP-induced alterations in cardiomyocytes. HiPSC-CMs from two different healthy donors were exposed to the PAMP flagellin (FLA) at different doses and exposure times. Alterations in the expression levels of distinct inflammation-associated cytokines, intracellular inflammation pathways including TLR5 downstream signaling, reactive oxygen species levels and surface antigen composition were assessed using PCR, ELISA and FACS techniques. Higher doses of flagellin increased the expression levels of inflammation-associated cytokines like TNFα (p < 0.01) and downstream signaling molecules like caspase-8 (p < 0.05). TLR5 expression (p < 0.01) and TLR5 fluorescence proportion (p < 0.05) increased in hiPSC-CMs after prolonged FLA exposure. FLA-induced innate immune response processes in cardiomyocytes might be detectable with an hiPSC-CMs-based in vitro model.