Neuronal GPCR NMUR-1 regulates energy homeostasis in response to pathogen infection.

A key question in current immunology is how the innate immune system generates high levels of specificity. Our previous study in Caenorhabditis elegans revealed that NMUR-1, a neuronal G protein-coupled receptor homologous to mammalian receptors for the neuropeptide neuromedin U (NMU), regulates distinct innate immune responses to different bacterial pathogens. Here, by using quantitative proteomics and functional assays, we discovered that NMUR-1 regulates F 1 F O ATP synthase and ATP production in response to pathogen infection, and that such regulation contributes to NMUR-1-mediated specificity of innate immunity. We further demonstrated that ATP biosynthesis and its contribution to defense is neurally controlled by the NMUR-1 ligand CAPA-1 and its expressing neurons ASG. These findings indicate that NMUR-1 neural signaling regulates the specificity of innate immunity by controlling energy homeostasis as part of defense against pathogens. Our study provides mechanistic insights into the emerging roles of NMU signaling in immunity across animal phyla.

Pathogen infection induces specific transgenerational modifications to gene expression and fitness in Caenorhabditis elegans.

How pathogen infection in a parental generation affects response in future generations to the same pathogen via epigenetic modifications has been the topic of recent studies. These studies focused on changes attributed to transgenerational epigenetic inheritance and how these changes cause an observable difference in behavior or immune response in a population. However, we questioned if pathogen infection causes hidden epigenetic changes to fitness that are not observable at the population level. Using the nematode Caenorhabditis elegans as a model organism, we examined the generation-to-generation differences in survival of both an unexposed and primed lineage of animals against a human opportunistic pathogen Salmonella enterica. We discovered that training a lineage of C. elegans against a specific pathogen does not cause a significant change to overall survival, but rather narrows survival variability between generations. Quantification of gene expression revealed reduced variation of a specific member of the TFEB lipophagic pathway. We also provided the first report of a repeating pattern of survival times over the course of 12 generations in the control lineage of C. elegans. This repeating pattern indicates that the variability in survival between generations of the control lineage is not random but may be regulated by unknown mechanisms. Overall, our study indicates that pathogen infection can cause specific phenotypic changes due to epigenetic modifications, and a possible system of epigenetic regulation between generations.

The longevity response to warm temperature is neurally controlled via the regulation of collagen genes.

Studies in diverse species have associated higher temperatures with shorter lifespan and lower temperatures with longer lifespan. These inverse effects of temperature on longevity are traditionally explained using the rate of living theory, which posits that higher temperatures increase chemical reaction rates, thus speeding up the aging process. Recent studies have identified specific molecules and cells that affect the longevity response to temperature, indicating that this response is regulated, not simply thermodynamic. Here, we demonstrate that in Caenorhabditis elegans, functional loss of NPR-8, a G protein-coupled receptor related to mammalian neuropeptide Y receptors, increases worm lifespan at 25°C but not at 20°C or 15°C, and that the lifespan extension at 25°C is regulated by the NPR-8-expressing AWB and AWC chemosensory neurons as well as AFD thermosensory neurons. Integrative transcriptomic analyses revealed that both warm temperature and old age profoundly alter gene expression and that genes involved in the metabolic and biosynthetic processes increase expression at 25°C relative to 20°C, indicating elevated metabolism at warm temperature. These data demonstrate that the temperature-induced longevity response is neurally regulated and also provide a partial molecular basis for the rate of living theory, suggesting that these two views are not mutually exclusive. Genetic manipulation and functional assays further uncovered that the NPR-8-dependent longevity response to warm temperature is achieved by regulating the expression of a subset of collagen genes. As increased collagen expression is a common feature of many lifespan-extending interventions and enhanced stress resistance, collagen expression could be critical for healthy aging.

Using single-worm RNA sequencing to study C. elegans responses to pathogen infection.

BACKGROUND: Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. RESULTS: In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. CONCLUSIONS: We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.

Protocol to measure bacterial intake and gut clearance of Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a common model organism for studies in immunity and host-pathogen interactions. In this protocol, we describe techniques for measuring pharyngeal pumping and defecation rates of adult C. elegans exposed to either pathogenic or non-pathogenic bacteria. Quantifying changes in pumping or defecation rates during pathogenic infection can account for behavioral changes that may affect survival. We also detail how to synchronize C. elegans and expose the synchronize animals to either pathogenic or non-pathogenic bacteria. For complete details on the use and execution of this protocol, please refer to Wibisono et al. (2022).

Neuronal GPCR NMUR-1 regulates distinct immune responses to different pathogens.

A key question in current immunology is how the innate immune system generates high levels of specificity. Using the Caenorhabditis elegans model system, we demonstrate that functional loss of NMUR-1, a neuronal G-protein-coupled receptor homologous to mammalian receptors for the neuropeptide neuromedin U, has diverse effects on C. elegans innate immunity against various bacterial pathogens. Transcriptomic analyses and functional assays reveal that NMUR-1 modulates C. elegans transcription activity by regulating the expression of transcription factors involved in binding to RNA polymerase II regulatory regions, which, in turn, controls the expression of distinct immune genes in response to different pathogens. These results uncover a molecular basis for the specificity of C. elegans innate immunity. Given the evolutionary conservation of NMUR-1 signaling in immune regulation across multicellular organisms, our study could provide mechanistic insights into understanding the specificity of innate immunity in other animals, including mammals.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode's thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Neuro-immune communication in C. elegans defense against pathogen infection.

The innate immune system is a complex collection of physical barriers and physiological defense responses to internal and external environmental assaults. Recent studies in the model organism Caenorhabditis elegans have highlighted how the nervous system interacts with the innate immune system to generate coordinated protective responses. Indeed, studies on neuro-immune interaction pathways have provided mechanistic insights into the roles of neuro-immune communication in modulating both immune and behavioral responses to pathogen attacks. The nervous system releases a variety of neurotransmitters, peptides, and hormones that regulate the innate immune response, while the innate immune system also relays information to the nervous system to affect learning and behavioral responses. Although these interactions still need further investigation, the knowledge that we have gained thus far has improved our understanding of how separate biological systems can act collectively for the survival and well-being of an organism. Here, we review recent studies on neuro-immune communication related to the survival and defense of C. elegans against pathogens.

A novel in vitro Caenorhabditis elegans transcription system.

BACKGROUND: Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system. RESULTS: By employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner. CONCLUSIONS: In this study, we developed an in vitro C. elegans transcription system that re-constitutes transcription reactions with nuclear extract of larval or adult worms and can both qualitatively and quantitatively detect transcription activity using non-radioactive approaches. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

Neuronal GPCR NPR-8 regulates C. elegans defense against pathogen infection.

Increasing evidence indicates that infection-triggered host defenses are regulated by the nervous system. However, the precise mechanisms of this regulation are not well understood. Here, we demonstrate that neuronal G protein-coupled receptor NPR-8 negatively regulates Caenorhabditis elegans defense against pathogen infection by suppressing cuticular collagen expression. NPR-8 controls the dynamics of cuticle structure in response to infection, likely through its regulation of cuticular collagen genes which, in turn, affects the nematode's defense. We further show that the defense activity of NPR-8 is confined to amphid sensory neurons AWB, ASJ, and AWC. It is generally believed that physical barrier defenses are not a response to infections but are part of the body's basic innate defense against pathogens. Our results challenge this view by showing not only that C. elegans cuticle structure dynamically changes in response to infection but also that the cuticle barrier defense is regulated by the nervous system.

Octopaminergic Signaling Mediates Neural Regulation of Innate Immunity in Caenorhabditis elegans.

Upon pathogen infection, the nervous system regulates innate immunity to confer coordinated protection to the host. However, the precise mechanisms of such regulation remain unclear. Previous studies have demonstrated that OCTR-1, a putative G protein-coupled receptor for catecholamine, functions in the sensory neurons designated "ASH" to suppress innate immune responses in Caenorhabditis elegans It is unknown what molecules act as OCTR-1 ligands in the neural immune regulatory circuit. Here we identify neurotransmitter octopamine (OA) as an endogenous ligand for OCTR-1 in immune regulation and show that the OA-producing RIC neurons function in the OCTR-1 neural circuit to suppress innate immunity. RIC neurons are deactivated in the presence of pathogens but transiently activated by nonpathogenic bacteria. Our data support a model whereby an octopaminergic immunoinhibitory pathway is tonically active under normal conditions to maintain immunological homeostasis or suppress unwanted innate immune responses but downregulated upon pathogen infection to allow enhanced innate immunity. As excessive innate immune responses have been linked to a myriad of human health concerns, our study could potentially benefit the development of more-effective treatments for innate immune disorders.IMPORTANCE Insufficient or excessive immune responses to pathogen infection are major causes of disease. Increasing evidence indicates that the nervous system regulates the immune system to help maintain immunological homeostasis. However, the precise mechanisms of this regulation are largely unknown. Here we show the existence of an octopaminergic immunoinhibitory pathway in Caenorhabditis elegans Our study results indicate that this pathway is tonically active under normal conditions to maintain immunological homeostasis or suppress unwanted innate immune responses but downregulated upon pathogen infection to allow enhanced innate immunity. As excessive innate immune responses have been linked to human health conditions such as Crohn's disease, rheumatoid arthritis, atherosclerosis, diabetes, and Alzheimer's disease, elucidating octopaminergic neural regulation of innate immunity could be helpful in the development of new treatments for innate immune diseases.