Intestinal immunity in C. elegans is activated by pathogen effector-triggered aggregation of the guard protein TIR-1 on lysosome-related organelles.

Toll/interleukin-1/resistance (TIR)-domain proteins with enzymatic activity are essential for immunity in plants, animals, and bacteria. However, it is not known how these proteins function in pathogen sensing in animals. We discovered that the lone enzymatic TIR-domain protein in the nematode C. elegans (TIR-1, homolog of mammalian sterile alpha and TIR motif-containing 1 [SARM1]) was strategically expressed on the membranes of a specific intracellular compartment called lysosome-related organelles. The positioning of TIR-1 on lysosome-related organelles enables intestinal epithelial cells in the nematode C. elegans to survey for pathogen effector-triggered host damage. A virulence effector secreted by the bacterial pathogen Pseudomonas aeruginosa alkalinized and condensed lysosome-related organelles. This pathogen-induced morphological change in lysosome-related organelles triggered TIR-1 multimerization, which engaged its intrinsic NAD+ hydrolase (NADase) activity to activate the p38 innate immune pathway and protect the host against microbial intoxication. Thus, TIR-1 is a guard protein in an effector-triggered immune response, which enables intestinal epithelial cells to survey for pathogen-induced host damage.

Intestinal Epithelial Wnt Signaling Mediates Acetylcholine-Triggered Host Defense against Infection.

Regulated antimicrobial peptide expression in the intestinal epithelium is key to defense against infection and to microbiota homeostasis. Understanding the mechanisms that regulate such expression is necessary for understanding immune homeostasis and inflammatory disease and for developing safe and effective therapies. We used Caenorhabditis elegans in a preclinical approach to discover mechanisms of antimicrobial gene expression control in the intestinal epithelium. We found an unexpected role for the cholinergic nervous system. Infection-induced acetylcholine release from neurons stimulated muscarinic signaling in the epithelium, driving downstream induction of Wnt expression in the same tissue. Wnt induction activated the epithelial canonical Wnt pathway, resulting in the expression of C-type lectin and lysozyme genes that enhanced host defense. Furthermore, the muscarinic and Wnt pathways are linked by conserved transcription factors. These results reveal a tight connection between the nervous system and the intestinal epithelium, with important implications for host defense, immune homeostasis, and cancer.

Caenorhabditis elegans RIG-I-like receptor DRH-1 signals via CARDs to activate antiviral immunity in intestinal cells.

Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, the Caenorhabditis elegans RLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation in C. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling in C. elegans, highlighting unexpected parallels in RLR signaling between C. elegans and mammals.

Activation of a G protein-coupled receptor by its endogenous ligand triggers the innate immune response of Caenorhabditis elegans.

Immune defenses are triggered by microbe-associated molecular patterns or as a result of damage to host cells. The elicitors of immune responses in the nematode Caenorhabditis elegans are unclear. Using a genome-wide RNA-mediated interference (RNAi) screen, we identified the G protein-coupled receptor (GPCR) DCAR-1 as being required for the response to fungal infection and wounding. DCAR-1 acted in the epidermis to regulate the expression of antimicrobial peptides via a conserved p38 mitogen-activated protein kinase pathway. Through targeted metabolomics analysis we identified the tyrosine derivative 4-hydroxyphenyllactic acid (HPLA) as an endogenous ligand. Our findings reveal DCAR-1 and its cognate ligand HPLA to be triggers of the epidermal innate immune response in C. elegans and highlight the ancient role of GPCRs in host defense.

Structural damage in the C. elegans epidermis causes release of STA-2 and induction of an innate immune response.

The epidermis constantly encounters invasions that disrupt its architecture, yet whether the epidermal immune system utilizes damaged structures as danger signals to activate self-defense is unclear. Here, we used a C. elegans epidermis model in which skin-penetrating infection or injury activates immune defense and antimicrobial peptide (AMP) production. By systemically disrupting each architectural component, we found that only disturbance of the apical hemidesmosomes triggered an immune response and robust AMP expression. The epidermis recognized structural damage through hemidesmosomes associated with a STAT-like protein, whose disruption led to detachment of STA-2 molecules from hemidesmosomes and transcription of AMPs. This machinery enabled the epidermis to bypass certain signaling amplification and directly trigger AMP production when subjected to extensive architectural damage. Together, our findings uncover an evolutionarily conserved mechanism for the epithelial barriers to detect danger and activate immune defense.

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans.

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABAAR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse-muscular insulin-intestinal innate immunity in vivo.

Of worms and men: HLH-30 and TFEB regulate tolerance to infection.

In this issue of Immunity, Visvikis et al. (2014) use the model host Caenorhabditis elegans to discover a role in innate immunity for the basic helix-loop-helix transcription factor, HLH-30. The finding inspires study of the mammalian ortholog TFEB, in which a similar role in immune response is ascertained.

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.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans.

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen.

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.

Deficiency of Innate Immunity against Pseudomonas aeruginosa Enhances Behavioral Avoidance via the HECW-1/NPR-1 Module in Caenorhabditis elegans.

To antagonize infection of pathogenic bacteria in soil and confer increased survival, Caenorhabditis elegans employs innate immunity and behavioral avoidance synchronously as the two main defensive strategies. Although both biological processes and their individual signaling pathways have been partially elucidated, knowledge of their interrelationship remains limited. The current study reveals that deficiency of innate immunity triggered by mutation of the classic immune gene pmk-1 promotes avoidance behavior in C. elegans and vice versa. Restoration of pmk-1 expression using the tissue-specific promoters suggested that the functional loss of both intestinal and neuronal pmk-1 is necessary for the enhanced avoidance. Additionally, PMK-1 colocalized with the E3 ubiquitin ligase HECW-1 in OLL neurons and regulated the expressional level of the latter, which consequently affected the production of NPR-1, a G-protein-coupled receptor (GPCR) homologous to the mammalian neuropeptide Y receptor, in RMG neurons in a non-cell-autonomous manner. Collectively, our study illustrates that once the innate immunity is impaired when C. elegans antagonizes bacterial infection, the other defensive strategy of behavioral avoidance can be enhanced accordingly via the HECW-1/NPR-1 module, suggesting that GPCRs in neural circuits may receive the inputs from the immune system and integrate those two systems for better adapting to the real-time status.

Mitochondrial chaperone HSP-60 regulates anti-bacterial immunity via p38 MAP kinase signaling.

Mitochondria play key roles in cellular immunity. How mitochondria contribute to organismal immunity remains poorly understood. Here, we show that HSP-60/HSPD1, a major mitochondrial chaperone, boosts anti-bacterial immunity through the up-regulation of p38 MAP kinase signaling. We first identify 16 evolutionarily conserved mitochondrial components that affect the immunity of Caenorhabditis elegans against pathogenic Pseudomonas aeruginosa (PA14). Among them, the mitochondrial chaperone HSP-60 is necessary and sufficient to increase resistance to PA14. We show that HSP-60 in the intestine and neurons is crucial for the resistance to PA14. We then find that p38 MAP kinase signaling, an evolutionarily conserved anti-bacterial immune pathway, is down-regulated by genetic inhibition of hsp-60, and up-regulated by increased expression of hsp-60 Overexpression of HSPD1, the mammalian ortholog of hsp-60, increases p38 MAP kinase activity in human cells, suggesting an evolutionarily conserved mechanism. Further, cytosol-localized HSP-60 physically binds and stabilizes SEK-1/MAP kinase kinase 3, which in turn up-regulates p38 MAP kinase and increases immunity. Our study suggests that mitochondrial chaperones protect host eukaryotes from pathogenic bacteria by up-regulating cytosolic p38 MAPK signaling.

Neuroimmune regulation of antimicrobial peptide expression by a noncanonical TGF-beta signaling pathway in Caenorhabditis elegans epidermis.

After being infected by the fungus Drechmeria coniospora, Caenorhabditis elegans produces antimicrobial peptides in its epidermis, some regulated by a signaling cascade involving a p38 mitogen-activated protein kinase. Here we show that infection-induced expression of peptides of the Caenacin family occurred independently of the p38 pathway. The caenacin (cnc) genes enhanced survival after fungal infection, and neuronal expression of the transforming growth factor-beta homolog DBL-1 promoted cnc-2 expression in the epidermis in a dose-dependent paracrine way. Our results lead to a model in which antifungal defenses are coordinately regulated by a cell-autonomous p38 cascade and a distinct cytokine-like transforming growth factor-beta signal from the nervous system, each of which controls distinct sets of antimicrobial peptide-encoding genes in the epidermis.

Lactic acid bacteria that activate immune gene expression in Caenorhabditis elegans can antagonise Campylobacter jejuni infection in nematodes, chickens and mice.

BACKGROUND: Campylobacter jejuni is the major micro-bacillary pathogen responsible for human coloenteritis. Lactic acid bacteria (LAB) have been shown to protect against Campylobacter infection. However, LAB with a good ability to inhibit the growth of C. jejuni in vitro are less effective in animals and animal models, and have the disadvantages of high cost, a long cycle, cumbersome operation and insignificant immune response indicators. Caenorhabditis elegans is increasingly used to screen probiotics for their anti-pathogenic properties. However, no research on the use of C. elegans to screen for probiotic candidates antagonistic to C. jejuni has been conducted to date. RESULTS: This study established a lifespan model of C. elegans, enabling the preselection of LAB to counter C. jejuni infection. A potential protective mechanism of LAB was identified. Some distinct LAB species offered a high level of protection to C. elegans against C. jejuni. The LAB strains with a high protection rate reduced the load of C. jejuni in C. elegans. The transcription of antibacterial peptide genes, MAPK and Daf-16 signalling pathway-related genes was elevated using the LAB isolates with a high protection rate. The reliability of the lifespan model of C. elegans was verified using mice and chickens infected with C. jejuni. CONCLUSIONS: The results showed that different LAB had different abilities to protect C. elegans against C. jejuni. C. elegans provides a reliable model for researchers to screen for LAB that are antagonistic to C. jejuni on a large scale.

Evolutionary plasticity in the innate immune function of Akirin.

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

NHR-49 Transcription Factor Regulates Immunometabolic Response and Survival of Caenorhabditis elegans during Enterococcus faecalis Infection.

Immune response to pathogens is energetically expensive to the host; however, the cellular source of energy to fuel immune response remains unknown. In this study, we show that Caenorhabditis elegans exposed to pathogenic Gram-positive and Gram-negative bacteria or yeast rapidly utilizes lipid droplets, the major energy reserve. The nematode's response to the pathogenic bacterium Enterococcus faecalis entails metabolic rewiring for the upregulation of several genes involved in lipid utilization and downregulation of lipid synthesis genes. Genes encoding acyl-CoA synthetase ACS-2, involved in lipid metabolism, and flavin monooxygenase FMO-2, involved in detoxification, are two highly upregulated genes during E. faecalis infection. We find that both ACS-2 and FMO-2 are necessary for survival and rely on NHR-49, a peroxisome proliferator-activated receptor alpha (PPARα) ortholog, for upregulation during E. faecalis infection. Thus, NHR-49 regulates an immunometabolic axis of survival in C. elegans by modulating breakdown of lipids as well as immune effector production upon E. faecalis exposure.

Neuroendocrine signals modulate the innate immunity of Caenorhabditis elegans through insulin signaling.

Communication between the immune and nervous systems, each of which is able to react rapidly to environmental stimuli, may confer a survival advantage. However, precisely how the nervous system influences the immune response and whether neural modulation of immune function is biologically important are not well understood. Here we report that neuronal exocytosis of neuropeptides from dense core vesicles suppressed the survival of Caenorhabditis elegans and their clearance of infection with the human bacterial pathogen Pseudomonas aeruginosa. This immunomodulatory function was mediated by INS-7, an insulin-like neuropeptide whose induction was associated with Pseudomonas virulence. INS-7 secreted from the nervous system functioned in a non-cell autonomous way to activate the insulin pathway and alter basal and inducible expression of immunity-related genes in intestinal cells.

Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection.

Metazoans identify and eliminate bacterial pathogens in microbe-rich environments such as the intestinal lumen; however, the mechanisms are unclear. Host cells could potentially use intracellular surveillance or stress response programs to detect pathogens that target monitored cellular activities and then initiate innate immune responses. Mitochondrial function is evaluated by monitoring mitochondrial protein import efficiency of the transcription factor ATFS-1, which mediates the mitochondrial unfolded protein response (UPR(mt)). During mitochondrial stress, mitochondrial import is impaired, allowing ATFS-1 to traffic to the nucleus where it mediates a transcriptional response to re-establish mitochondrial homeostasis. Here we examined the role of ATFS-1 in Caenorhabditis elegans during pathogen exposure, because during mitochondrial stress ATFS-1 induced not only mitochondrial protective genes but also innate immune genes that included a secreted lysozyme and anti-microbial peptides. Exposure to the pathogen Pseudomonas aeruginosa caused mitochondrial dysfunction and activation of the UPR(mt). C. elegans lacking atfs-1 were susceptible to P. aeruginosa, whereas hyper-activation of ATFS-1 and the UPR(mt) improved clearance of P. aeruginosa from the intestine and prolonged C. elegans survival in a manner mainly independent of known innate immune pathways. We propose that ATFS-1 import efficiency and the UPR(mt) is a means to detect pathogens that target mitochondria and initiate a protective innate immune response.