A Nutrition-Longevity Tradeoff Enforced by Innate Immunity.

Dietary restriction (DR) extends lifespan in multiple animal species, but the underlying molecular mechanisms remain poorly understood. A recent study published in Cell Metabolism by Wu et al. (2019) shows that DR represses an evolutionarily conserved p38 MAPK pathway involved in innate immunity, leading to diminished expression of p38 MAPK-regulated genes and extended lifespan.

Distinct members of the Caenorhabditis elegans CeMbio reference microbiota exert cryptic virulence that is masked by host defense.

Microbiotas are complex microbial communities that colonize specific niches in the host and provide essential organismal functions that are important in health and disease. Understanding the ability of each distinct community member to promote or impair host health, alone or in the context of the community, is imperative for understanding how differences in community structure affect host health and vice versa. Recently, a reference 12-member microbiota for the model organism Caenorhabditis elegans, known as CeMbio, was defined. Here, we show the differential ability of each CeMbio bacterial species to activate innate immunity through the conserved PMK-1/p38 MAPK, ACh-WNT, and HLH-30/TFEB pathways. Although distinct CeMbio members differed in their ability to activate the PMK-1/p38 pathway, the ability to do so did not correlate with bacterial-induced lifespan reduction in wild-type or immunodeficient animals. In contrast, most species activated HLH-30/TFEB and showed virulence toward hlh-30-deficient animals. These results suggest that the microbiota of C. elegans is rife with bacteria that can shorten the host's lifespan if host defense is compromised and that HLH-30/TFEB is a fundamental and key host protective factor.

Why worms watch their hemidesmosomes and why you should, too.

Hemidesmosomes are cellular attachment structures of great importance to the epidermis. In this issue of Immunity, Zhang et al. (2015) have discovered that in addition to having structural functions, invertebrate and human hemidesmosomes are actively monitored by the cell as a novel mechanism for detecting pathogenic infection.

Key Roles of MiT Transcription Factors in Innate Immunity and Inflammation.

Microphthalmia/TFE (MiT) transcription factors (TFs), such as transcription factor EB (TFEB) and transcription factor E3 (TFE3), are emerging as key regulators of innate immunity and inflammation. Rapid progress in the field requires a focused update on the latest advances. Recent studies show that TFEB and TFE3 function in innate immune cells to regulate antibacterial and antiviral responses downstream of phagocytosis, interferon (IFN)-γ, lipopolysaccharide (LPS), and adenosine receptors. Moreover, overexpression of TFEB or TFE3 can drive inflammation in vivo, such as in atherosclerosis, while in other scenarios they can perform anti-inflammatory functions. MiT factors may constitute potential therapeutic targets for a broad range of diseases; however, to harness their therapeutic potential, sophisticated ways to manipulate MiT factor activity safely and effectively must be developed.

Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes.

Animal host defense against infection requires the expression of defense genes at the right place and the right time. Understanding such tight control of host defense requires the elucidation of the transcription factors involved. By using an unbiased approach in the model Caenorhabditis elegans, we discovered that HLH-30 (known as TFEB in mammals) is a key transcription factor for host defense. HLH-30 was activated shortly after Staphylococcus aureus infection, and drove the expression of close to 80% of the host response, including antimicrobial and autophagy genes that were essential for host tolerance of infection. TFEB was also rapidly activated in murine macrophages upon S. aureus infection and was required for proper transcriptional induction of several proinflammatory cytokines and chemokines. Thus, our data suggest that TFEB is a previously unappreciated, evolutionarily ancient transcription factor in the host response to infection.

An image analysis toolbox for high-throughput C. elegans assays.

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.

Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids.

Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d-alanylation, α-O-GlcNAcylation, and ß-O-GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches ß-O-GlcNAc (ß-O-N-acetyl-D-glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to ß-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to ß-lactams, ΔtarS strains have no growth or cell division defects. Because neither α-O-GlcNAc nor ß-O-Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, ß-O-GlcNAc residues. These data suggest ß-O-GlcNAcylated WTAs scaffold factors required for MRSA resistance. The ß-O-GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to ß-lactams.

EGL-9 controls C. elegans host defense specificity through prolyl hydroxylation-dependent and -independent HIF-1 pathways.

Understanding host defense against microbes is key to developing new and more effective therapies for infection and inflammatory disease. However, how animals integrate multiple environmental signals and discriminate between different pathogens to mount specific and tailored responses remains poorly understood. Using the genetically tractable model host Caenorhabditis elegans and pathogenic bacterium Staphylococcus aureus, we describe an important role for hypoxia-inducible factor (HIF) in defining the specificity of the host response in the intestine. We demonstrate that loss of egl-9, a negative regulator of HIF, confers HIF-dependent enhanced susceptibility to S. aureus while increasing resistance to Pseudomonas aeruginosa. In our attempt to understand how HIF could have these apparently dichotomous roles in host defense, we find that distinct pathways separately regulate two opposing functions of HIF: the canonical pathway is important for blocking expression of a set of HIF-induced defense genes, whereas a less well understood noncanonical pathway appears to be important for allowing the expression of another distinct set of HIF-repressed defense genes. Thus, HIF can function either as a gene-specific inducer or repressor of host defense, providing a molecular mechanism by which HIF can have apparently opposing roles in defense and inflammation. Together, our observations show that HIF can set the balance between alternative pathogen-specific host responses, potentially acting as an evolutionarily conserved specificity switch in the host innate immune response.

Distinct members of the C. elegans CeMbio reference microbiota exert cryptic virulence and infection protection.

Microbiotas are complex microbial communities that colonize specific niches in the host and provide essential organismal functions that are important in health and disease. A key aspect is the ability of each distinct community member to promote or impair host health, alone or in the context of the community, in hosts with varied levels of immune competence. Understanding such interactions is limited by the complexity and experimental accessibility of current systems and models. Recently, a reference twelve-member microbiota for the model organism C. elegans, known as CeMbio, was defined to aid the dissection of conserved host-microbiota interactions. Understanding the physiological impact of the CeMbio bacteria on C. elegans is in its infancy. Here, we show the differential ability of each CeMbio bacterial species to activate innate immunity through the conserved PMK-1/p38 MAPK, ACh/WNT, and HLH-30/TFEB pathways. Using immunodeficient animals, we uncovered several examples of bacterial 'cryptic' virulence, or virulence that was masked by the host defense response. The ability to activate the PMK-1/p38 pathway did not correlate with bacterial virulence in wild type or immunodeficient animals. In contrast, ten out of twelve species activated HLH-30/TFEB, and most showed virulence towards hlh-30-deficient animals. In addition, we identified Pseudomonas lurida as a pathogen in wild type animals, and Acinetobacter guillouiae as avirulent despite activating all three pathways. Moreover, short pre-exposure to A. guillouiae promoted host survival of infection with P. lurida, which was dependent on PMK-1/p38 MAPK and HLH-30/TFEB. These results suggest that the microbiota of C. elegans is rife with "opportunistic" pathogens, and that HLH-30/TFEB is a fundamental and key host protective factor. Furthermore, they support the idea that bacteria like A. guillouiae evolved the ability to induce host innate immunity to improve host fitness when confronted with pathogens, providing new insights into how colonization order impacts host health.

C. elegans orphan nuclear receptor NHR-42 represses innate immunity and promotes lipid loss downstream of HLH-30/TFEB.

In recent years, transcription factors of the Microphthalmia-TFE (MiT) family, including TFEB and TFE3 in mammals and HLH-30 in Caenorhabditis elegans, have emerged as important regulators of innate immunity and inflammation in invertebrates and vertebrates. Despite great strides in knowledge, the mechanisms that mediate downstream actions of MiT transcription factors in the context of innate host defense remain poorly understood. Here, we report that HLH-30, which promotes lipid droplet mobilization and host defense, induces the expression of orphan nuclear receptor NHR-42 during infection with Staphylococcus aureus. Remarkably, NHR-42 loss of function promoted host infection resistance, genetically defining NHR-42 as an HLH-30-controlled negative regulator of innate immunity. During infection, NHR-42 was required for lipid droplet loss, suggesting that it is an important effector of HLH-30 in lipid immunometabolism. Moreover, transcriptional profiling of nhr-42 mutants revealed wholesale activation of an antimicrobial signature, of which abf-2, cnc-2, and lec-11 were important for the enhanced survival of infection of nhr-42 mutants. These results advance our knowledge of the mechanisms by which MiT transcription factors promote host defense, and by analogy suggest that TFEB and TFE3 may similarly promote host defense via NHR-42-homologous nuclear receptors in mammals.

Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals.

A NOVEL NOX/PHOX-CD38-NAADP-TFEB AXIS IMPORTANT FOR MACROPHAGE ACTIVATION DURING BACTERIAL PHAGOCYTOSIS.

Macrophage activation in the presence of bacterial cells and molecules entails complex programs of gene expression. How such triggers elicit specific gene expression programs is incompletely understood. We previously discovered that TFEB (transcription factor EB) is a key contributor to macrophage activation during bacterial phagocytosis. However, the mechanism linking phagocytosis of bacterial cells to TFEB activation and downstream pro-inflammatory cytokine induction remained unknown. We found that macrophages lacking both TFEB and TFE3 (transcription factor E3) were unable to mount a pro-inflammatory phenotype in response to bacterial infection. The NOX/PHOX (NADPH oxidase)-dependent oxidative burst was required for nuclear translocation of TFEB during phagocytosis of Gram-positive or -negative bacteria, and reactive oxygen species (ROS) were sufficient to trigger TFEB activation in a CD38- and NAADP (nicotinic acid adenine dinucleotide phosphate)-dependent manner. Consistent with the Ca2+-releasing activity of NAADP, intracellular Ca2+ chelation and PPP3/calcineurin inhibition prevented TFEB activation by phagocytosis and ROS (reactive oxygen species), impairing the induction of pro-inflammatory cytokines such as IL6 and TNF/TNFα. Therefore, here we describe a previously unknown pathway that links phagocytosis with macrophage pro-inflammatory polarization via TFEB and related transcription factor TFE3. These findings reveal that activation of TFEB and TFE3 is a key regulatory event for the activation of macrophages, and have important implications for infections, inflammation, cancer, obesity, and atherosclerosis.

Scaffold-mediated symmetry breaking by Cdc42p.

Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking. In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally. The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization. We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments. Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour. Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch.

Role for beta-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions.

We used the model nematode Caenorhabditis elegans infected with the human pathogen Staphylococcus aureus to identify components of epithelial immunity. Transcriptional profiling and reverse genetic analysis revealed that mutation of the C. elegans beta-catenin homolog bar-1 or the downstream homeobox gene egl-5 results in a defective response and hypersensitivity to S. aureus infection. Epistasis analysis showed that bar-1 and egl-5 function in parallel to previously described C. elegans immune-response pathways. Overexpression of human homologs of egl-5 modulated NF-kappaB-dependent TLR2 signaling in epithelial cells. These data suggest that beta-catenin and homeobox genes play an important and conserved role in innate immune defense.

A unifying hypothesis on the central role of reactive oxygen species in bacterial pathogenesis and host defense in C. elegans.

During intestinal infection, microbes induce ROS by various mechanisms in C. elegans. ROS can have beneficial roles, acting as antimicrobials and as signaling molecules that activate cytoprotective pathways. Failure to maintain appropriate levels of ROS causes oxidative stress and cellular damage. This review uses the Damage Response Framework to interpret several recent observations on the relationships between infection, host response, and host damage, with a focus on mechanisms mediated by ROS. We propose a unifying hypothesis that ROS drive a collapse in proteostasis in infected C. elegans, which results in death during unresolved infection. Because the signaling pathways highlighted here are conserved in mammals, the mentioned and future studies can provide new tools of hypothesis generation in human health and disease.

NHR-49/PPAR-α and HLH-30/TFEB cooperate for C. elegans host defense via a flavin-containing monooxygenase.

The model organism Caenorhabditis elegans mounts transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, the regulation of which is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature, which was almost completely abrogated by deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, fmo-2/FMO5 induction required the nuclear hormone receptor, NHR-49/PPAR-α, which controlled host defense cell non-autonomously. These findings reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal FMOs as important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α during S. aureus infection, with implications for host defense and inflammation in higher organisms.

Nervous system control of intestinal host defense in C. elegans.

Interplay between the nervous and immune systems is critical for homeostasis, and its dysfunction underlies pathologies such as multiple sclerosis, autism, leukemia, and inflammation. The nematode Caenorhabditis elegans provides an opportunity to define evolutionarily conserved mechanisms of regulation of host innate immunity and inflammation in a genetically tractable whole-animal system. In the past few years, the C. elegans nervous system has emerged as an integral part of host defense against pathogens, acting through diverse mechanisms to repress or induce protective transcriptional responses to infection in distal tissues. In this review, we discuss current knowledge of the mechanisms through which the C. elegans nervous system controls the expression of host defense genes in the intestinal epithelium. Although still incomplete, the insights derived from such work have broad implications for neural regulation of epithelial function at mucosal barriers in higher organisms in health and disease.

Opposing roles for actin in Cdc42p polarization.

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.

Transcription factor TFEB cell-autonomously modulates susceptibility to intestinal epithelial cell injury in vivo.

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn's disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1.