Neuro-immune circuits in C. elegans

Abstract

The nervous and immune systems use bi-directional communication to control host responses against microbial pathogens. Recent studies at the interface of the two systems have highlighted important roles of the nervous system in the regulation of both microbicidal pathways and pathogen avoidance behaviors. Studies on the neural circuits in the simple model host Caenorhabditis elegans have significantly improved our understanding of the roles of conserved neural mechanisms in controlling innate immunity. Moreover, behavioral studies have advanced our understanding of how the nervous system may sense potential pathogens and consequently elicit pathogen avoidance, reducing the risk of infection. In this review, we discuss the neural circuits that regulate both behavioral immunity and molecular immunity in C. elegans.

Introduction

Metazoans have developed multiple strategies to deal with pathogenic microbes, including pathogen avoidance, resistance, and tolerance [1]. Different animals, ranging from simple nematodes to chimpanzees and humans, engage in many behaviors that reduce their exposure to pathogens [2–4]. These avoidance behaviors that protect against pathogen infections are referred to as the behavioral immune system [5]. In addition, microbial sensing mechanisms activate molecular immune pathways that provide resistance to pathogens by reducing pathogen burden and clearing the infection. Increasing evidence also indicates that those mechanisms involved in the initial activation of defense pathways also regulate the immune system. Thus, immune regulatory mechanisms play a critical role in maintaining immune homeostasis because disproportionate activation of immune pathways could be detrimental to the host.

The nervous system maintains control of homeostasis through bi-directional communication with peripheral physiological systems. Recent research at the interface of the nervous system and the immune system identified neural circuits that are triggered by and control immune pathways [6–10]. While the understanding of neuro-immune communications holds great therapeutic potential [11], the complex nervous and immune systems of higher organisms have limited our understanding of these connections. Studies in the model nematode Caenorhabditis elegans have advanced our understanding of neuro-immune connections. In this review, we describe these recent advances in C. elegans.

Neural circuits involved in behavioral immunity

Physical avoidance of pathogens is a critical defense strategy used by hosts to reduce pathogen infections [2,4]. In mammals, olfactory chemosensory neurons and nociceptor sensory neurons detect various bacterial products, such as toxins, quorum-sensing molecules, formyl peptides, and lipopolysaccharides, through distinct molecular mechanisms that lead to rapid avoidance behaviors [6,12–16]. Similarly, in the fruit fly Drosophila melanogaster, olfactory and gustatory neurons have been reported to detect geosmin (the smell of mold), phenol, and lipopolysaccharides via distinct molecular mechanisms, allowing the organism to avoid food contaminated with bacteria [17,18]. The simple and well-described nervous system of C. elegans has made it an important model for understanding how animals learn to avoid pathogens. Behavioral pathogen avoidance is a crucial defense response employed by C. elegans that significantly improves the survival of animals [19–22]. The animals are capable of sensing and avoiding pathogenic microbes by multiple mechanisms (Figure 1). C. elegans shows innate as well as learned avoidance of pathogenic microbes. In innate avoidance behavior, animals rapidly avoid a pathogen without any prior exposure. C. elegans shows innate aversion to several species of Streptomyces [23], which produce various nematicides. Streptomyces secreted dodecanoic acid is sensed by a C. elegans G-protein-coupled receptor (GPCR), SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons, leading to a rapid avoidance behavior that happens within seconds. This rapid detection of and escape from Streptomyces species, that secrete powerful toxins, is potentially important for the survival of nematodes [23]. In contrast to innate avoidance, in the learned avoidance behavior, animals are initially attracted towards a given pathogen and through a learning process eventually develop an aversive behavior [24].

Figure 1.

Different mechanisms of elicitation of microbial aversion behavior in C. elegans

Sensation of bacterial metabolites, intestinal infection leading to bloating, and disruption of core cellular components activate various neuroendocrine signals required for microbial aversion.

The neurotransmitter serotonin is known to play an important role in the associative learning of pathogenic bacteria in C. elegans [24]. Exposure to pathogenic bacteria increases serotonin in ADF chemosensory neurons by transcriptional and post-transcriptional mechanisms. The tph-1 gene encodes the tryptophan hydroxylase that is required for a rate-limiting step in serotonin biosynthesis. C. elegans tph-1 mutants were found to be defective in avoidance of pathogenic bacteria [25]. In addition, tph-1 mutants were also found to be defective in avoidance of non-pathogenic bacteria under various forms of animal physiological perturbations [26,27]. However, some studies showed that TPH-1 might not be required for the avoidance of pathogens [28,29]. This suggests that the role of TPH-1 in eliciting avoidance behavior might be context-dependent. Some studies also suggest that the lack of pathogen avoidance in tph-1 mutant animals could be because of background mutations [28].

Transforming growth factor-β (TGF-β) superfamily ligands are secreted molecules that play critical roles in cell-to-cell communication, cell growth, differentiation, and death [30]. Two of the five TGF-β ligands in C. elegans, DBL-1 and DAF-7, signal through a canonical receptor-Smad signaling pathway [31]. Whereas DAF-7 regulates diverse functions such as the dauer developmental decision, foraging and aggregation behaviors, quiescence, metabolism, and longevity, DBL-1 controls body morphology, innate immunity, and reproductive aging [31]. In addition to the regulation of the aforementioned functions, both of these TGF-β ligands are also known to regulate pathogen avoidance behavior. Expression of DBL-1 and its receptor SMA-6 in the AVA command interneurons and hypodermis, respectively, is required for the avoidance behavior [32]. Similarly, expression of DAF-7 in the ASI and ASJ chemosensory neurons is important for the avoidance behavior [33]. Recently, it was shown that DAF-7/TGF-β is also required for transgenerational memory of pathogen avoidance [34]. Interestingly, expression of DAF-7 in the ASI neurons, but not in ASJ neurons, was found to be important for this transgenerational memory, suggesting that the circuits for pathogen avoidance might be different from those involved in transgenerational memory.

The GPCR NPR-1 is related to mammalian neuropeptide Y receptors. Along with its ligands, the FMRF-like peptides FLP-18 and FLP-21, NPR-1 controls several behavioral and physiological functions such as behavioral quiescence and social feeding [35,36]. NPR-1 is expressed in the body fluid neurons AQR, PQR, and URX and regulates innate immunity non-cell-autonomously [22,37]. In addition to the above functions, NPR-1 and its ligands, FLP-18 and FLP-21, also regulate the pathogen avoidance behavior [19,21,22,29]. The expression of NPR-1 in the neurons AQR, PQR, and URX is required for the avoidance behavior [38]. Various stimuli, such as mechanosensation via OLL neuron pair [39] or intestinal bloating caused by pathogen infection [29], modulate NPR-1 activity and thus, avoidance behavior.

The NPR-1 and DAF-7/TGF-β pathways are required for aerotaxis behavior and regulate hyperoxia avoidance in C. elegans. Inhibition of the NPR-1 and DAF-7/TGF-β pathways elicit avoidance of high oxygen (O₂), while higher activity of these pathways induces avoidance of low O₂ [33,36,40,41]. Recent studies have shown that C. elegans pathogen avoidance could also be mediated by avoidance of nitric oxide (NO) [42] and carbon dioxide (CO₂) [43]. The chemosensory neurons ASJ and BAG were found to be required for avoidance against NO and CO₂, respectively. Interestingly, both of these neurons are also important for sensing O₂ and, therefore, it is possible that animals integrate information about different gases to sense their environment. Because bacterial metabolism influences the local concentration of gases, these studies highlight that C. elegans microbial avoidance behavior is influenced by the animal’s ability to respond to different levels of gases. Consistent with this idea, animals lacking ASI neurons, that are involved in sensing O₂, were found to have dampened pathogen avoidance behavior [44].

It was recently shown that bloating of the intestine caused by pathogen infection induces microbial avoidance behavior [27,29,45]. Intestinal bloating also induced multiple neuroendocrine pathways that are known to regulate avoidance behavior, including the serotonin biosynthesis pathway, the DAF-7/TGF-β pathway, and the NPR-1/GPCR pathway [29]. These studies suggest that modulation of neuroendocrine pathways that lead to avoidance behavior might be driven by the sensation of physiological changes induced by pathogen infection, such as intestinal bloating, rather than direct chemosensation of pathogenic microbes. Therefore, physiological changes induced by pathogen infection might play an important role in the learning of pathogen avoidance. Indeed, intestinal expression of the neuropeptide INS-11, that is induced by Pseudomonas aeruginosa infection, modulates the pathogen avoidance behavior [28].

Neural regulation of molecular immunity

While the activities of immune pathways reduce pathogen burden and are important to resist infections, uncontrolled activation of immune pathways could be detrimental to the host. Therefore, activation of the immune system must be controlled to minimize the changes in organismal homeostasis that occur during the host’s response to pathogen attack. The nervous system of C. elegans seems to play an important role during the host response to infections as it can both activate and suppress the immune system through multiple circuits (Figure 2).

Figure 2.

Neural regulation of immunity in C. elegans

Different neural circuits either activate or inhibit immune responses in non-nervous tissues. Signals upstream of the neural circuits activating immune responses remain poorly defined.

Neural activation of immune pathways

Neural circuits that activate immune pathways in different tissues have been identified in C. elegans and are involved in defense responses against different pathogenic microbes. Infections of the fungus Drechmeria coniospora lead to increased production of antimicrobial peptides of the Caenacin family in the epidermis of C. elegans. The expression of caenacin genes is controlled by the neural TGF-β analog DBL-1 via its receptor SMA-6 and the cytosolic Smad SMA-3 in hypodermis [46]. In addition to its importance in defense against the fungus D. coniospora, DBL-1 signaling is also required for protection against bacterial pathogens that cause intestinal infections [47–49]. Because DBL-1 is expressed only in the nervous system [31], it is likely that additional circuits involving DBL-1 control the expression of antimicrobial peptides in the intestine. It will be interesting to study whether the DBL-1 receptors and downstream signaling components such as the receptor SMA-6 and the cytoplasmic Smad SMA-3 play any roles in intestinal immune regulation or whether other novel receptors are involved in this regulation.

In a recent study, it was shown that infection of the Gram-positive pathogen Staphylococcus aureus leads to the release of acetylcholine from neurons [50]. The infection-induced release of acetylcholine 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. Because infection with several pathogens as well as bloating of the intestine induce the same C-type lectin and lysozyme genes [29,51–53], it is important to delineate whether the release of acetylcholine is specific to S. aureus or is generally related to infection-induced damage in the host.

Neural suppression of immune pathways

Several neural circuits have been identified that suppress immune pathways in C. elegans. Animals defective in neural secretion, such as unc-13 and unc-31 mutants, were more resistant to P. aeruginosa infection [54]. This suggested that the nervous system secretes cues that are immunoinhibitory. Indeed, treatments that resulted in a sustained increase in neural secretion increased sensitivity to pathogens. One of the immunoinhibitory cues that is released by neurons is the insulin-like peptide INS-7. INS-7 activates insulin/IGF-1-receptor/DAF-2 signaling in the intestinal cells, resulting in cytoplasmic retention of the FOXO transcription factor DAF-16 and decreased expression of antimicrobial genes [54]. Interestingly, it appears that pathogens have evolved mechanisms to subvert this pathway to suppress host immunity [55]. Infection of C. elegans by P. aeruginosa induces ins-7 expression, which in turn suppresses immune gene expression by accelerating the exit of the FOXO transcription factor DAF-16 from nuclei in intestinal cells.

One of the first neural circuits identified that suppresses immune pathways involves NPR-1. C. elegans deficient in NPR-1 exhibit enhanced susceptibility to infections by P. aeruginosa, Salmonella enterica, and Enterococcus faecalis [22]. Expression of NPR-1 in the oxygen-sensing neurons AQR, PQR, and URX is sufficient to rescue the enhanced susceptibility to these pathogens. In addition, lack of AQR, PQR, and URX neurons partially rescued the enhanced susceptibility to pathogens of npr-1 loss of function animals, indicating that NPR-1 inhibits the activity of these neurons. Importantly, gene expression studies on NPR-1 lacking animals demonstrated that NPR-1 regulates expression of genes that are controlled by a conserved PMK-1/p38 mitogen-activated protein kinase signaling pathway in the intestine [22,37].

The catecholamine GPCR OCTR-1 suppresses PMK-1/p38 mitogen-activated protein kinase signaling as well as canonical and non-canonical unfolded protein responses (UPR) in the ER, which are important for innate immunity [56,57]. OCTR-1 activity in the ASH neuron suppresses the AIA interneuron, which is required for release of NLP-20, a neuropeptide that regulates the induction of UPR and innate immune genes controlled by PMK-1 signaling [44]. The neurotransmitter octopamine, that is produced by RIC neurons, is the endogenous ligand for OCTR-1 and is involved in the suppression of innate immunity [58]. Interestingly, the RIC neurons are deactivated in the presence of pathogens but transiently activated by nonpathogenic bacteria. Thus, the activation of the octopaminergic pathway on nonpathogenic bacteria potentially suppresses unwanted innate immune responses. On the other hand, the inactivation of octopaminergic pathway on pathogenic bacteria likely helps in the activation of appropriate immune responses.

Pharmacological inhibition of dopamine signaling activates the PMK-1/p38 mitogen-activated protein kinase signaling pathway in the intestine [59,60]. The immunoinhibitory effects of the dopamine signaling are mediated via the D1-like dopamine receptor, DOP-4, in C. elegans. This function of dopamine originates in CEP neurons and requires active DOP-4 in downstream ASG neurons [60]. It will be interesting to study whether the dopaminergic signaling is differentially active on pathogenic vs. nonpathogenic bacteria.

The gastrin-releasing peptide receptor homolog, NPR-9, inhibits innate immune response [61]. Animals lacking NPR-9 show reduced colonization, enhanced survival on pathogen, and enhanced immune gene expression. NPR-9 is expressed in the interneuron AIB and antagonizes the activity of this neuron. Channelrhodopsin-2(ChR2)-mediated activation of the AIB interneuron enhances immune response that is suppressed by overexpression of NPR-9. However, it remains to be studied how pathogens modulate the NPR-9 pathway and the activity of the AIB interneurons.

Intersection of behavioral and molecular immune regulation

Several neural regulators that control molecular immunity also modulate avoidance behavior. For example, NPR-1 in the AQR, PQR, and URX neurons not only regulates innate immunity but also controls the avoidance behavior [22,38]. Similarly, DBL-1 controls both of these defense responses via a single circuit involving the TGF-β receptor SMA-6 and the cytoplasmic Smad SMA-3 in the hypodermis [32,46]. The modulation of both molecular immunity and avoidance behavior by the same neural circuits could be because of the common role of these mechanisms in improving the defense and survival of animals against pathogens. It is also likely that animals use common upstream signals to activate both avoidance behavior and molecular immunity. Indeed, bloating of the intestine caused by pathogen infection or microbial colonization enhances expression of immune genes and elicits microbial avoidance behavior [27,29]. Moreover, disruption of core cellular activities induces both avoidance behavior and immune gene expression [26]. These studies suggest that common upstream signals can modulate both behavioral and molecular immunity via either the same or different neural circuits.

Conclusion and future perspectives

Studies on the neural control of immunity in C. elegans have greatly improved our understanding of conserved neuro-immune connections. The roles of several conserved neurotransmitters such as serotonin, dopamine, octopamine, and acetylcholine in regulating immunity have been described. In addition, the roles of different TGF-β pathways as well as conserved neuropeptides in controlling immunity have been delineated. Despite these advances, it is still not understood if and how the neural circuits are activated by bacterial components and/or signals from the non-nervous tissues. Moreover, the signals from the nervous system that modulate immunity in non-nervous tissues remain to be understood. Developing an understanding of different signals involved in the communication across tissues, as well as signals from pathogens, would improve our knowledge of the mechanisms involved in the maintenance of organismal homeostasis. Because C. elegans neural circuits have both stimulatory and inhibitory actions on immunity, it is important to determine how the different neural circuits are fine-tuned to obtain an optimal immune response upon pathogen infection.

Highlights.

• Neural circuits regulate immune pathways in non-nervous tissues.

• Neural circuits control behavioral and molecular immunity in C. elegans.

• Conserved neuromodulators inhibit or activate immune responses in C. elegans.