Neuronal NPR-15 modulates molecular and behavioral immune responses via the amphid sensory neuron-intestinal axis in C. elegans

  1. Benson Otarigho
  2. Anna Frances Butts
  3. Alejandro Aballay

  • Curated by eLife

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    eLife assessment

    The important work by Aballay et al. significantly advances our understanding of how G protein-coupled receptors (GPCRs) regulate immunity and pathogen avoidance. The authors provide convincing evidence for the GPCR NPR-15 to mediate immunity by altering the activity of several key transcription factors. This work will be of broad interest to immunologists.

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Abstract

The survival of hosts during infections relies on their ability to mount effective molecular and behavioral immune responses. Despite extensive research on these defense strategies in various species, including the model organism Caenorhabditis elegans , the neural mechanisms underlying their interaction remain poorly understood. Previous studies have highlighted the role of neural G-protein-coupled receptors (GPCRs) in regulating both immunity and pathogen avoidance, which is particularly dependent on aerotaxis. To address this knowledge gap, we conducted a screen of mutants in neuropeptide receptor family genes. We found that loss-of-function mutations in npr-15 activated immunity while suppressing pathogen avoidance behavior. Through further analysis, NPR-15 was found to regulate immunity by modulating the activity of key transcription factors, namely GATA/ELT-2 and TFEB/HLH-30. Surprisingly, the lack of pathogen avoidance of npr-15 mutant animals was not influenced by oxygen levels. Moreover, our studies revealed that the amphid sensory neuron ASJ is involved in mediating the immune and behavioral responses orchestrated by NPR-15. Additionally, NPR-15 was found to regulate avoidance behavior via the TRPM (transient receptor potential melastatin) gene, GON-2, which may sense the intestinal distension caused by bacterial colonization to elicit pathogen avoidance. Our study contributes to a broader understanding of host defense strategies and mechanisms underlining the interaction between molecular and behavioral immune responses.

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  1. Version published to 10.7554/elife.90051.4 on eLife
  2. Version published to 10.7554/elife.90051 on eLife
  3. Version published to 10.7554/elife.90051.3 on eLife
  4. eLife

    Author Response

    eLife assessment

    The important work by Aballay et al. significantly advances our understanding of how G protein-coupled receptors (GPCRs) regulate immunity and pathogen avoidance. The authors provide convincing evidence for the GPCR NPR-15 to mediate immunity by altering the activity of several key transcription factors. This work will be of broad interest to immunologists.

    The authors express their sincere appreciation to Timothy Behrens (Senior Editor), the Reviewing Editor, and the original reviewers for their considerate and favorable assessment of our manuscript.

    Reviewer #1 (Public Review):

    Summary:

    Otarigho et al. presented a convincing 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.

    The authors are grateful for the thoughtful and insightful comments on our manuscript. Your feedback has been instrumental in refining our work, and we appreciate the time and expertise you have invested in evaluating our study.

    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.

    The authors express gratitude to the reviewer for recognizing that the present study revealed specific molecules and neuronal cells involved in regulating both molecular immune defense and behavioral defense against pathogen attacks. Additionally, the acknowledgment that the same neural circuit may oversee distinct defense mechanisms under different circumstances is appreciated.

    1. The 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.

    The authors thank the reviewer for appreciating that this study is supported by solid evidence.

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

    The authors thank the reviewer for appreciating the Gene differential expression, gene ontology, and pathway analyses performed in the study.

    1. 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).

    The author thanks the reviewer for appreciating the approaches used in this study.

    1. Statistical analyses were appropriate and adequate.

    The authors thank the reviewer for appreciating the Statistical analyses used in this study.

    Reviewer #2 (Public Review):

    Summary:

    The authors are studying the behavioral response to pathogen exposure. They and others have previously describe 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.

    The authors sincerely thank the reviewer for the thoughtful and positive review of our manuscript. We greatly appreciate the time and effort you dedicated to evaluating our work, and we are pleased that you find our study to be a rigorous and elegant contribution to the understanding of behavioral responses to pathogen exposure.

    Reviewer #1 (Recommendations For The Authors):

    The authors have adequately addressed my concerns and questions. I have no more comments or recommendations for the authors.

    The authors thank the reviewer for the constructive comments on the manuscript

    Reviewer #2 (Recommendations For The Authors):

    The authors have adequately addressed my concerns.

    The authors express their appreciation to the reviewer for the valuable and constructive comments provided on the manuscript.

  5. eLife

    eLife assessment

    The important work by Aballay et al. significantly advances our understanding of how G protein-coupled receptors (GPCRs) regulate immunity and pathogen avoidance. The authors provide convincing evidence for the GPCR NPR-15 to mediate immunity by altering the activity of several key transcription factors. This work will be of broad interest to immunologists.

  6. eLife

    Reviewer #1 (Public Review):

    Summary:

    Otarigho et al. presented a convincing 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. The 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.

  7. eLife

    Reviewer #2 (Public Review):

    Summary:
    The authors are studying the behavioral response to pathogen exposure. They and others have previously describe 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.

  8. Version published to 10.1101/2023.07.27.550570v4 on bioRxiv
  9. Version published to 10.7554/elife.90051.2 on eLife
  10. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    We would like to thank the reviewers for their strong interest in our studies and their excellent suggestions for improvement.

    Reviewer #1:

    Weaknesses:

    Comment 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.

    Response to comment 1. We have removed the word “interplay.”

    Comment 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.

    Response to Comment 2. We’d like to clarify that it remains unclear whether OCTR-1 itself controls both immunity and behavior (PMID: 29117551). The reference study showed that OCTR-1-expressing neurons ASH and ASI control immunity and behavior, respectively. We modified the manuscript to make this point clearer: “While OCTR-1-expressing neurons ASI play a role in avoidance (34), the specific role of OCTR-1 in ASH and ASI neurons remains unclear. “

    Comment 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.

    Response to comment 3. We studied GON-2 and GTL-2 because a recent screen of intestinal TRPM genes showed that they are the only two involved in the control of pathogen avoidance. We modified the manuscript to make this rationale clearer: “Because transient receptor potential melastatin (TRPM) ion channels, GON-2 and GTL-2, are required for pathogen avoidance (32), we studied whether they may be part of the NPR-15 pathway that controls pathogen avoidance”

    Comment 3b. 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.

    Response to comment 3b. We agree that NPR-15 may broadly regulate multiple effectors in different tissues. Indeed, we have shown that the transcriptional activity of ELT-2, HLH-30, DAF-16, and PMK-1 is higher in npr-15 than in WT animals. We found that expression of NPR-15 only in ASJ cells rescues both the survival and behavioral phenotypes of npr-15 animals (Figs. 4F and 5C).

    Comment 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.

    Response to Comment 4. We agreed. We have removed the statements discussing conservation in that manner.

    Comment 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.

    Response to comment 5. We found that NPR-15 controls the expression of immune genes in the absence of an infection. Without further experiments, we think it would be too speculative to discuss the possibility of a temporal regulation. However, we modified the manuscript to address the control of both molecular and behavioral immunity by NPR-15. The revised discussion reads: “Our findings shed light on the role of NPR-15 in the control of the immune response. NPR-15 seems to suppress specific immune genes while activating pathogen avoidance behavior to minimize potential tissue damage and the metabolic energy cost associated with activating the molecular immune response against pathogen infections. Overall, the control of immune activation is essential for maintaining homeostasis and preventing excessive tissue damage caused by an overly aggressive and energy-costly response against pathogens (60-63).”

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

    Response to comment 6. We have removed repetitive concepts and modified the discussion as mentioned in the response to point 5.

    Comment 7. 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.

    Response to comment 7: We removed the statements that may have appeared to be overreaching and addressed the weakness raised by the reviewer. The revised discussion reads “Our findings shed light on the role of NPR-15 in the control of the immune response. NPR-15 seems to suppress specific immune genes while activating pathogen avoidance behavior to minimize potential tissue damage and the metabolic energy cost associated with activating the molecular immune response against pathogen infections. Overall, the control of immune activation is essential for maintaining homeostasis and preventing excessive tissue damage caused by an overly aggressive and energy-costly response against pathogens (60-63).”

    Recommendations for the authors:

    Recommendations 1. The title, abstract and some statements in the main text need to be re-written to reflect the fact that regulation of the interplay between molecular and behavioral immune responses was not explored in this study.

    Response to recommendations 1. We modified the title and abstract accordingly.

    Recommendations 2. It should be mentioned in the manuscript that OCTR-1 is the first GPCR that was identified to regulate both immunity and avoidance behavior.

    Response to recommendation 2. We addressed this issue as discussed in the response to comment 2.

    Recommendations 3. Repetitive statements should be removed from Discussion.

    Response to recommendations 3. The statements were removed.

    Recommendations 4. It is surprising to see that pmk-1 RNAi did not affect the survival of npr-15(tm12539) animals against S. aureus because PMK-1 has a general role in defense against S. aureus infection.

    Response to recommendations 4. We agree. However, the RNAi studies were validated using mutants (Fig. S3B).

    Recommendations 4b. Also, the rationale for using skn-1 RNAi as a control was not given. These need to be explained adequately in the manuscript.

    Response to recommendations 4b. There’s no need to include skn-1 RNAi and we removed the data.

    Recommendations 5. The conclusion that the lack of avoidance behavior by NPR-15 loss-of-function is independent of immunity and neuropeptide genes was drawn entirely based on experiments with RNAi of individual genes. Functional redundancy among genes could render RNAi of individual genes ineffective, thus masking the dependence of avoidance behavior on these genes. More experiments are needed to support this conclusion, or the wording of the conclusion need to be changed.

    Response to recommendations 5. We modified the conclusion to address this issue: “Given the possibility of functional redundancy among these genes, we cannot rule out the possibility that different combinations may play a role in controlling avoidance behavior.”

    Recommendations 6. What is representation factor in Fig. 2B and 2C?

    Response to recommendations 5. Figure 2B shows significantly enriched terms with a Q value < 0.1, sorted by P values. Figure 2 C shows the representation factor that is calculated using a tool, http://nemates.org/MA/progs/overlap_stats.html. The calculation is based on the number of genes in set 1, the number of genes in set 2, and the Overlap between set 1 and set 2, as well as the number of genes in the genome.

    We corrected the Figure legends and included the corresponding information in Material and Methods.

    Recommendations 7. The legend of Fig. 6 was wrong and should be changed to 'GPCR/NPR-15 suppressed immune response and enhanced avoidance behavior via sensory neurons'.

    Response to recommendations 7. Thank you for pointing this out. We changed the legend.

    Reviewer #2:

    Comments 1. 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).

    Response to comment 1. We appreciate the observation. We did notice some variation in both the WT and npr-15(tm12539) animals during our study. Notably, the variation appeared to be more in the WT compared to the npr-15(tm12539) animals. However, it's important to note that these variations did not significantly affect the outcome of our findings. We calculated the means, standard deviation, and standard error across different experimental trials that are presented in the manuscript (Table S2) (new Table). It's worth noting that these variations did not significantly impact the observed differences in lawn occupancy between the wild-type (WT) and npr-15 mutant strains.

    We addressed this issue in the revised manuscript: “Interestingly, we noticed that the variation in lawn occupancy is greater in WT than in npr-15(tm12539) animals across experiments (Table S2), which suggests that the strong lack of avoidance of npr-15(tm12539) somehow counteracts the experimental variation”

    Comment 2. Does this reflect rates of migration or re-occupancy in WT?

    Response to comment 2. We did not observe any re-occupancy in either the WT or npr-15 animals at 24-hour time points (which we mostly use in this study) or beyond. To address the comment, we performed a new experiment and found that the re-occupancy of npr-15 mutants is comparable to that of WT animals at 4 hours post-exposure (Figure S1B).

    Comment 3. Does pathogen avoidance persist and/or the rate of avoidance differ in npr mutant worms?

    Response to comment 3. As illustrated in new Figure S1B, the avoidance behavior in response to pathogens remained consistent even when we extended our observations up to 48 hours (Figure S1B).

    Comment 4. 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?

    Response to comment 4. We conducted the proposed experiment and observed that the WT animals learned to avoid the pathogen but not npr-15(tm12539) mutants (Figure S1C). The revised manuscript reads: “We also found that npr-15(tm12539) exhibited reduced learned avoidance compared to WT animals (Figure S1C).”

    Comment 5: 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 experiments?).

    Response to comment 5. This is an interesting question that has not been addressed in the field yet. While we think the study is exciting, we believe that it is outside the scope of our work. All the gene expression studies performed here are in non-avoiding conditions.

    Comment 6. No concerns but the P values in the legends are a pain to read. Why not put them in figures as in above figures.

    Response to comment 6. We included the P values as suggested.

    Recommendations for the authors:

    Recommendation 1. Fig. 1/S1. 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).

    Response to recommendation 1. We addressed this issue as discussed in the response to comment 1.

    Recommendation 2. Fig. 1/S1. Comments. Does this reflect rates of migration or re-occupancy in WT?

    Response to recommendation 2. We have responded to this issue in comment 2.

    Recommendations 3. Fig. 1/S1. Comments. Does pathogen avoidance persist and/or the rate of avoidance differ in npr mutant worms.

    Response to recommendation 3. We have responded to this issue in comment 3.

    Recommendation 4. Fig. 1/S1. Comments B. 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?

    Response to recommendation 4: We have responded to this issue in comment 4 above.

    Recommendation 5. Fig. 2/S2. 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?).

    Response to recommendation 5. We have responded to this issue in comment 5 above.

    Recommendation 6. Fig. 3/S3. Comment. No concerns but the P values in the legends are a pain to read. Why not put them in figures as in above figures.

    Response to recommendation 6. We included the P values.

    Recommendation 7. Fig. 5. 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 wonder if or what the connection is 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 the neuronal-gut communication works. Specific Transmitters... perhaps.

    Response to Recommendation 7 Fig. 5. Thanks for this observation. To address the recommendation, we modified the discussion: “Our research additionally indicates that the regulation of NPR-15-mediated avoidance is not influenced by intestinal immune and neuropeptide genes. Given the potential for functional redundancy and our focus on genes upregulated in the absence of NPR-15, we cannot entirely rule out the possibility that unexamined immune effectors or neuropeptides, not transcriptionally controlled by NPR-15, might be involved. Different intestinal signals may also participate in the NPR-15 pathway that controls pathogen avoidance.”

    Recommendation 8. Comment. 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 the subject of future studies.

    Response to recommendation 8. We checked the data in the study by Murphy, Kenyon et al., and found that the gon-2 gene was not downregulated.

    Recommendation 9. Minor: Regarding the description to Fig. 5. "Consistently with our previous findings, we found that only " The adverb form of consistent should not be used here.

    Response to recommendation 9. Thank you for pointing this out. The description of Figure 5 was corrected.

  11. eLife

    eLife assessment

    The important work by Aballay et al. significantly advances our understanding of how G protein-coupled receptors (GPCRs) regulate immunity and pathogen avoidance. The authors provide convincing evidence for the GPCR NPR-15 to mediate immunity by altering the activity of several key transcription factors. This work will be of broad interest to immunologists.

  12. eLife

    Reviewer #1 (Public Review):

    Summary:

    Otarigho et al. presented a convincing 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. The 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.

  13. eLife

    Reviewer #2 (Public Review):

    Summary:
    The authors are studying the behavioral response to pathogen exposure. They and others have previously describe 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.

  14. Version published to 10.1101/2023.07.27.550570v3 on bioRxiv
  15. Version published to 10.1101/2023.07.27.550570v2 on bioRxiv
  16. Version published to 10.7554/elife.90051.1 on eLife
  17. eLife

    eLife assessment

    This important work by Aballay et al. advances our understanding of how G protein-coupled receptors (GPCRs) regulate immunity and pathogen avoidance. The authors provide convincing evidence for the GPCR NPR-15 to mediate immunity by altering the activity of several key transcription factors. Nevertheless, both reviewers have raised questions to be addressed, at which time this work will be of broad interest to immunologists specifically, and biologists in general.

  18. eLife

    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.

  19. eLife

    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.

  20. Version published to 10.1101/2023.07.27.550570v1 on bioRxiv