Neuronal NPR-15 controls the interplay between molecular and behavioral immune responses through the amphid sensory neuron-intestinal axis in C. elegans

Reviewer #1 (Public Review):

Summary:

Otarigho et al. presented a solid study revealing that in C. elegans, the neuropeptide Y receptor GPCR/NPR-15 mediates both molecular and behavioral immune responses to pathogen attack. Previously, three npr genes were found to be involved in worm defense. In this study, the authors screened mutants in the remaining npr genes against P. aeruginosa-mediated killing and found that npr-15 loss-of-function improved worm survival. npr-15 mutants also exhibited enhanced resistance to other pathogenic bacteria but displayed significantly reduced avoidance to S. aureus, independent of aerotaxis, pathogen intake and defecation. The enhanced resistance in npr-15 mutant worms was attributed to upregulation of immune and neuropeptide genes, many of which were controlled by the transcription factors ELT-2 and HLH-30. The authors found that NPR-15 regulates avoidance behavior via the TRPM gene, GON-2, which has a known role in modulating avoidance behavior through the intestine. The authors further showed that both NPR-15-dependent immune and behavioral responses to pathogen attack were mediated by the NPR-15-expressing neurons ASJ. Overall, the authors discovered that the NPR-15/ASJ neural circuit may regulate distinct defense mechanisms against pathogens under different circumstances. This study provides novel and useful information to researchers in the fields of neuroimmunology and C. elegans research.

Strengths:

1. This study uncovered specific molecules and neuronal cells that regulate both molecular immune defense and behavior defense against pathogen attack and indicate that the same neural circuit may regulate distinct defense mechanisms under different circumstances. This discovery is significant because it not only reveals regulatory mechanisms of different defense strategies but also suggests how C. elegans utilize its limited neural resources to accomplish complex regulatory tasks.

2. Most conclusions in this study are supported by solid evidence, which are often derived from multiple approaches and/or experiments. Multiple pathogenic bacteria were tested to examine the effect of NPR-15 loss-of-function on immunity; the impacts of pharyngeal pumping and defecation on bacterial accumulation were ruled out when evaluating defense; RNA-seq and qPCR were used to measure gene expression; gene inactivation was done in multiple strains to assess gene function.

3. Gene differential expression, gene ontology and pathway analyses were performed to demonstrate that NPR-15 controls immunity through regulating immune pathways.

4. Elegant approaches were employed to examine avoidance behavior (partial lawn, full lawn, and lawn occupancy) and the involvement of neurons in regulating immunity and avoidance (the use of a diverse array of mutant strains).

5. Statistical analyses were appropriate and adequate.

Weaknesses:

1. The authors identified NPR-15 and ASJ neurons that are involved in both molecular and behavioral responses to pathogen attack. This finding, by itself, is significant. However, how the NPR-15/ASJ circuit regulates the interplay between the two defense strategies was not explored. Therefore, emphasizing the interplay in the title and the abstract is misleading.

2. Although the discovery of a single GPCR regulating both immunity and avoidance behavior is significant and novel, NPR-15 is not the first GPCR identified with these functions. Previously, the same lab reported that the GPCR OCTR-1 also regulates immunity and avoidance behavior through ASH and ASI neurons respectively (PMID: 29117551). This point was not mentioned in the current manuscript.

3. The authors discovered that NPR-15 regulates avoidance behavior via the TRPM gene, GON-2. Only two factors (GON-2 and GTL-2) were examined in this study, and GON-2 happens to function through the intestine. It is possible that NPR-15 may broadly regulate multiple effectors in multiple tissues. Confining the regulation to the amphid sensory neuron-intestinal axis, as stated in the title and elsewhere in the manuscript, is not accurate.

4. The C. elegans nervous system is simple, and hermaphrodites only have 302 neurons. Individual neurons possessing multiple regulatory functions is expected. Whether this is conserved in mammals and other vertebrates is unknown, because in higher animals, neurons and neuronal circuits could be more specialized.

5. A key question, that is, why would NPR-15 suppress immunity (which is bad for defense) but enhance avoidance behavior (which is good for defense), is not addressed or explained. This could be due to temporal regulation, for example, upon pathogen exposure, NPR-15 could regulate behavior to avoid the pathogen, but after infection, NPR-15 could suppress excessive immune responses or quench the responses for the resolution of infection.

6. The discussion appears timid in scope and contains some repetitive statements. Point 5 can be addressed in the Discussion.

Overall, the authors presented an impactful study that identified specific molecules and neuronal cells that regulate both molecular and behavioral immune responses to pathogen attack. Most conclusions are supported by solid evidence. However, some statements are overreaching, for example, regulation of the interplay between molecular and behavioral immune responses was emphasized but not explored. Nonetheless, this study reported a significant and novel discovery and has laid a foundation for investigating such an interplay in the future.

Reviewer #2 (Public Review):

Summary:

The authors are studying the behavioral response to pathogen exposure. They and others have previously described the role that the G-protein coupled receptors in the nervous system plays in detecting pathogens, and initiating behavioral patterns (e.g. avoidance/learned avoidance) that minimize contact. The authors study this problem in C. elegans, which is amenable to genetic and cellular manipulations and allow the authors to define cellular and signaling mechanisms. This paper extends the original idea to now implicate signaling and transcriptional pathways within a particular neuron (ASJ) and the gut in mediating avoidance behaviour.

Strengths:

The work is rigorous and elegant and the data are convincing. The authors make superb use of mutant strains in C. elegans, as well tissue specific gene inactivation and expression and genetic methods of cell ablation. to demonstrate how a gene, NPR15 controls behavioral changes in pathogen infection. The results suggest that ASJ neurons and the gut mediate such effects. I expect the paper will constitute an important contribution to our understanding of how the nervous system coordinates immune and behavioral responses to infection.

Fig. 1/S1. Authors selected a mutant for further study, npr-15, which showed resistance to various pathogens, and less colonization. Data are convincing. Data also suggest that in response to S. aureus, where wt animals exhibit avoidance behavior measured as numbers of animals that move off a focal spot of bugs, the npr-15 mutants do not. The effect was abrogated when a full lawn was used, at least for S. aureus, where there was no place to run. The conclusion is that the NPR-15 mediates behavioral changes resulting in pathogen avoidance.

Comments: There is some variance in lawn occupancy of wt strains between the different trials in WT animals (e.g. in Fig. 1: 25 for wt vs 60% for npr mutant; S1c 5% for wt and 60% for npr mutant). Does this reflect rates of migration or re-occupancy in WT? Does pathogen avoidance persist and/or the rate of avoidance differ in npr mutant worms, and if animals were exposed then re-exposed, could the authors to determine whether a learned avoidance was similarly affected by this mutation by assessing rate changes?

Fig. 2/S2. NPR inhibits expression of immune and aversion pathway genes (ELT-2, HLH-30, PMK-1, and DAF-2/DAF-16). No concerns.

Comment: Is there any difference in gene expression of animals that have migrated off the lawn to those remaining on the lawn (e.g. in partial lawn expts?)

Fig. 3/S3. Let-2RNAi or hlh-30 RNAi abrogates immunity in both WT and npr mutants. Similar effects with mutants. pmk and daf-16 inactivation were without effect.

Comment. No concerns but the P values in the legends are a pain to read. Why not put them in figures as in the above figures.

Fig. 4. Using neuronal and gut specific RNAi, the authors implicate the ASJ neurons in NPR-15 effects (ie in WT animals npr15 RNAi resulted in a pathogen resistance phenotype similar to that of the mutant animals. Specific expression of NPR-15 in the enhanced survival of the npr-15 mutants, an effect rescued by neuronal expression of NPR-15. Using strains lacking particular neurons, they found that strains lacking ASJ- strains phenocopies the npr mutant. Finally, sealing things nicely, they rescued NPR-15 in the mutant on an ASJ-specific Ptrx promoter.

Fig. 5. explores the dependence of pathogen avoidance on ASJ neurons and gut effects. Fig 5 shows that mutation of NPR in ASJ neuron alone phenocopies pathogen avoidance of the global npr mutant, indicating NPR expression in this and only this neurons is required. Fig. 5 also demonstrates that the loss of the ion channel GON-2 phenocopies the npr-15 mutant.

Comments: The authors suggest that the ASJ/NPR15 effect to limit avoidance acts via inhibition of GON-2 in the intestine. The observation that GON-2 inhibition effects on pathogen avoidance occur independently of neurons could suggest that it is a redundant way of accomplishing the same thing, which then makes one ask what the connection exists between the neuron and the gut. The effect of ASJ via NPR on pathogen avoidance is not neuropeptide dependent, which they show. So how does the neuronal-gut communication works. Specific Transmitters... perhaps. Since ASJ neurons control entry into dauer, perhaps isn't surprising that DAF-16 showed up as an NPR-15. induced factor (and dauer worms are resistant to a lot of stressors); that said dauer hormones might be involved as well. Is there any evidence that DAF-16 down-regulates GON-2 expression (see Murphy, Kenyon et al. 2005), and along these lines would GON-2 RNAi work in a DAF-16 mutant? I think addressing these issues are in my view the subject of future studies.

Weaknesses: The paper is solid and elegantly defines the genetic basis of behavioral avoidance via neurons and gut. The neuronal gut connection is shown, but how they are connected remains unsolved. I wouldn't suggest this is a weakness as much as an invitation for future work.