Innate Sensors Trigger Regulated Cell Death to Combat Intracellular Infection.

Intracellular pathogens pose a significant threat to animals. In defense, innate immune sensors attempt to detect these pathogens using pattern recognition receptors that either directly detect microbial molecules or indirectly detect their pathogenic activity. These sensors trigger different forms of regulated cell death, including pyroptosis, apoptosis, and necroptosis, which eliminate the infected host cell niche while simultaneously promoting beneficial immune responses. These defenses force intracellular pathogens to evolve strategies to minimize or completely evade the sensors. In this review, we discuss recent advances in our understanding of the cytosolic pattern recognition receptors that drive cell death, including NLRP1, NLRP3, NLRP6, NLRP9, NLRC4, AIM2, IFI16, and ZBP1.

The Innate Immune Response to Mycobacterium tuberculosis Infection.

Infection with Mycobacterium tuberculosis causes >1.5 million deaths worldwide annually. Innate immune cells are the first to encounter M. tuberculosis, and their response dictates the course of infection. Dendritic cells (DCs) activate the adaptive response and determine its characteristics. Macrophages are responsible both for exerting cell-intrinsic antimicrobial control and for initiating and maintaining inflammation. The inflammatory response to M. tuberculosis infection is a double-edged sword. While cytokines such as TNF-α and IL-1 are important for protection, either excessive or insufficient cytokine production results in progressive disease. Furthermore, neutrophils-cells normally associated with control of bacterial infection-are emerging as key drivers of a hyperinflammatory response that results in host mortality. The roles of other innate cells, including natural killer cells and innate-like T cells, remain enigmatic. Understanding the nuances of both cell-intrinsic control of infection and regulation of inflammation will be crucial for the successful development of host-targeted therapeutics and vaccines.

cGLRs are a diverse family of pattern recognition receptors in innate immunity.

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

A phospho-switch constrains BTL2-mediated phytocytokine signaling in plant immunity.

Enabling and constraining immune activation is of fundamental importance in maintaining cellular homeostasis. Depleting BAK1 and SERK4, the co-receptors of multiple pattern recognition receptors (PRRs), abolishes pattern-triggered immunity but triggers intracellular NOD-like receptor (NLR)-mediated autoimmunity with an elusive mechanism. By deploying RNAi-based genetic screens in Arabidopsis, we identified BAK-TO-LIFE 2 (BTL2), an uncharacterized receptor kinase, sensing BAK1/SERK4 integrity. BTL2 induces autoimmunity through activating Ca2+ channel CNGC20 in a kinase-dependent manner when BAK1/SERK4 are perturbed. To compensate for BAK1 deficiency, BTL2 complexes with multiple phytocytokine receptors, leading to potent phytocytokine responses mediated by helper NLR ADR1 family immune receptors, suggesting phytocytokine signaling as a molecular link connecting PRR- and NLR-mediated immunity. Remarkably, BAK1 constrains BTL2 activation via specific phosphorylation to maintain cellular integrity. Thus, BTL2 serves as a surveillance rheostat sensing the perturbation of BAK1/SERK4 immune co-receptors in promoting NLR-mediated phytocytokine signaling to ensure plant immunity.

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 immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function.

Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens.

The interferon landscape along the respiratory tract impacts the severity of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites.

Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots.

Recognition of microbe-associated molecular patterns (MAMPs) is crucial for the plant's immune response. How this sophisticated perception system can be usefully deployed in roots, continuously exposed to microbes, remains a mystery. By analyzing MAMP receptor expression and response at cellular resolution in Arabidopsis, we observed that differentiated outer cell layers show low expression of pattern-recognition receptors (PRRs) and lack MAMP responsiveness. Yet, these cells can be gated to become responsive by neighbor cell damage. Laser ablation of small cell clusters strongly upregulates PRR expression in their vicinity, and elevated receptor expression is sufficient to induce responsiveness in non-responsive cells. Finally, localized damage also leads to immune responses to otherwise non-immunogenic, beneficial bacteria. Damage-gating is overridden by receptor overexpression, which antagonizes colonization. Our findings that cellular damage can "switch on" local immune responses helps to conceptualize how MAMP perception can be used despite the presence of microbial patterns in the soil.

TLR8 Is a Sensor of RNase T2 Degradation Products.

TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment, yet its mode of action is poorly understood. TLR8 engages two distinct ligand binding sites to sense RNA degradation products, although it remains unclear how these ligands are formed in cellulo in the context of complex RNA molecule sensing. Here, we identified the lysosomal endoribonuclease RNase T2 as a non-redundant upstream component of TLR8-dependent RNA recognition. RNase T2 activity is required for rendering complex single-stranded, exogenous RNA molecules detectable for TLR8. This is due to RNase T2's preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Thus-generated molecules constitute agonistic ligands for the first and second binding pocket of TLR8. Together, these results establish the identity and origin of the RNA-derived molecular pattern sensed by TLR8.

The conceptual foundations of innate immunity: Taking stock 30 years later.

While largely neglected over decades during which adaptive immunity captured most of the attention, innate immune mechanisms have now become central to our understanding of immunology. Innate immunity provides the first barrier to infection in vertebrates, and it is the sole mechanism of host defense in invertebrates and plants. Innate immunity also plays a critical role in maintaining homeostasis, shaping the microbiota, and in disease contexts such as cancer, neurodegeneration, metabolic syndromes, and aging. The emergence of the field of innate immunity has led to an expanded view of the immune system, which is no longer restricted to vertebrates and instead concerns all metazoans, plants, and even prokaryotes. The study of innate immunity has given rise to new concepts and language. Here, we review the history and definition of the core concepts of innate immunity, discussing their value and fruitfulness in the long run.

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.

The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila.

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.

Targeting innate immune pathways for cancer immunotherapy.

The innate immune system is critical for inducing durable and protective T cell responses to infection and has been increasingly recognized as a target for cancer immunotherapy. In this review, we present a framework wherein distinct innate immune signaling pathways activate five key dendritic cell activities that are important for T cell-mediated immunity. We discuss molecular pathways that can agonize these activities and highlight that no single pathway can agonize all activities needed for durable immunity. The immunological distinctions between innate immunotherapy administration to the tumor microenvironment versus administration via vaccination are examined, with particular focus on the strategies that enhance dendritic cell migration, interferon expression, and interleukin-1 family cytokine production. In this context, we argue for the importance of appreciating necessity vs. sufficiency when considering the impact of innate immune signaling in inflammation and protective immunity and offer a conceptual guideline for the development of efficacious cancer immunotherapies.

Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans.

Distinguishing infectious pathogens from harmless microorganisms is essential for animal health. The mechanisms used to identify infectious microbes are not fully understood, particularly in metazoan hosts that eat bacteria as their food source. Here, we characterized a non-canonical pattern-recognition system in Caenorhabditis elegans (C. elegans) that assesses the relative threat of virulent Pseudomonas aeruginosa (P. aeruginosa) to activate innate immunity. We discovered that the innate immune response in C. elegans was triggered by phenazine-1-carboxamide (PCN), a toxic metabolite produced by pathogenic strains of P. aeruginosa. We identified the nuclear hormone receptor NHR-86/HNF4 as the PCN sensor in C. elegans and validated that PCN bound to the ligand-binding domain of NHR-86/HNF4. Activation of NHR-86/HNF4 by PCN directly engaged a transcriptional program in intestinal epithelial cells that protected against P. aeruginosa. Thus, a bacterial metabolite is a pattern of pathogenesis surveilled by nematodes to identify a pathogen in its bacterial diet.

Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer.

Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.

Cytoplasmic Mechanisms of Recognition and Defense of Microbial Nucleic Acids.

Microbial nucleic acids are major signatures of invading pathogens, and their recognition by various host pattern recognition receptors (PRRs) represents the first step toward an efficient innate immune response to clear the pathogens. The nucleic acid-sensing PRRs are localized at the plasma membrane, the cytosol, and/or various cellular organelles. Sensing of nucleic acids and signaling by PRRs involve recruitment of distinct signaling components, and PRRs are intensively regulated by cellular organelle trafficking. PRR-mediated innate immune responses are also heavily regulated by posttranslational modifications, including phosphorylation, polyubiquitination, sumoylation, and glutamylation. In this review, we focus on our current understanding of recognition of microbial nucleic acid by PRRs, particularly on their regulation by organelle trafficking and posttranslational modifications. We also discuss how sensing of self nucleic acids and dysregulation of PRR-mediated signaling lead to serious human diseases.

IKKß primes inflammasome formation by recruiting NLRP3 to the trans-Golgi network.

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKß, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKß recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKß-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKß activity.

The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis.

The microbiota are vital for immune homeostasis and provide a competitive barrier to bacterial and fungal pathogens. Here, we investigated how gut commensals modulate systemic immunity and response to viral infection. Antibiotic suppression of the gut microbiota reduced systemic tonic type I interferon (IFN-I) and antiviral priming. The microbiota-driven tonic IFN-I-response was dependent on cGAS-STING but not on TLR signaling or direct host-bacteria interactions. Instead, membrane vesicles (MVs) from extracellular bacteria activated the cGAS-STING-IFN-I axis by delivering bacterial DNA into distal host cells. DNA-containing MVs from the gut microbiota were found in circulation and promoted the clearance of both DNA (herpes simplex virus type 1) and RNA (vesicular stomatitis virus) viruses in a cGAS-dependent manner. In summary, this study establishes an important role for the microbiota in peripheral cGAS-STING activation, which promotes host resistance to systemic viral infections. Moreover, it uncovers an underappreciated risk of antibiotic use during viral infections.

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection.

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.

The RIG-I receptor adopts two different conformations for distinguishing host from viral RNA ligands.

RIG-I is an essential innate immune receptor for detecting and responding to infection by RNA viruses. RIG-I specifically recognizes the unique molecular features of viral RNA molecules and selectively distinguishes them from closely related RNAs abundant in host cells. The physical basis for this exquisite selectivity is revealed through a series of high-resolution cryo-EM structures of RIG-I in complex with host and viral RNA ligands. These studies demonstrate that RIG-I actively samples double-stranded RNAs in the cytoplasm and distinguishes them by adopting two different types of protein folds. Upon binding viral RNA, RIG-I adopts a high-affinity conformation that is conducive to signaling, while host RNA induces an autoinhibited conformation that stimulates RNA release. By coupling protein folding with RNA binding selectivity, RIG-I distinguishes RNA molecules that differ by as little as one phosphate group, thereby explaining the molecular basis for selective antiviral sensing and the induction of autoimmunity upon RIG-I dysregulation.