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

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.

Keywords: C. elegans; G-protein-coupled receptors; avoidance behavior; immunity; immunology; inflammation; neuropeptides; neuroscience.

Figure 1.. NPR-15 loss-of-function enhanced pathogen resistance and inhibited avoidance behavior.

(A) Wild-type (WT), npr-15(tm12539), and npr-15(tm12539);Pnp-15::npr-15 animals were exposed to P. aeruginosa partial lawn and scored for survival. (B) Colonization of WT, npr-15(tm12539), and npr-15(tm12539);Pnp-15::npr-15 animals by P. aeruginosa-GFP after 24 hr at 25°C. Scale bar, 200 μm. (C) Colony-forming units per animal (WT, npr-15(tm12539), npr-15(tm12539);Pnp-15::npr-15) grown on P. aeruginosa-GFP for 24 hr at 25°C. Bars represent means while error bars indicate the standard deviation (SD) of three independent experiments; ***p<0.001 and NS=not significant. (D) WT, npr-15(tm12539), and npr-15(tm12539);Pnp-15::npr-15 animals were exposed to S. enterica partial lawn and scored for survival. (E) WT, npr-15(tm12539), and npr-15(tm12539);Pnp-15::npr-15 animals were exposed to E. faecalis partial lawn and scored for survival. (F) WT, npr-15(tm12539), and npr-15(tm12539);Pnp-15::npr-15 animals were exposed to S. aureus partial lawn and scored for survival. (G) WT, npr-15(tm12539), and npr-15(tm12539);Pnp-15::npr-15 animals were exposed to live E. coli and scored for survival. (H) WT and npr-15(tm12539) animals were exposed to ultraviolet light (UV)-killed E. coli and scored for survival. (I) Schematics of avoidance behavior assay on S. aureus. (J) Lawn occupancy of WT C. elegans and npr-15(tm12539), and npr-15(tm1250);Pnpr-15::npr-15 animals on a partial lawn of S. aureus at 24 hr. Bars represent means while error bars indicate SD; **p<0.001 and NS=not significant.

Figure 1—figure supplement 1.. NPR-15 loss-of-function exhibited pathogen resistance independent of brood size and oxygen-independent avoidance behavior.

Related to Figure 1. (A) Wild-type (WT) and npr-15(ok1626) animals were exposed to P. aeruginosa partial lawn and scored for survival. (B) Lawn occupancy of WT and npr-15(tm12539) animals exposed to a partial lawn of S. aureus. ***p<0.001. The marked region shows re-occupancy. (C) Lawn occupancy of naïve and trained WT and npr-15(tm12539) animals exposed to a partial lawn of S. aureus. **p<0.001, ****p<0.0001. (D) Lawn occupancy of WT, npr-15(tm12539), npr-1(ad609), and npr-1(n1353) animals exposed to a partial lawn of S. aureus at 24 hr in 8% and atmospheric oxygen (~21%). Bars represent means while error bars indicate standard deviation (SD); ****p<0.0001, ***p<0.001, and NS=not significant. (E) WT, npr-15(tm12539), and npr-15(ok1626) animals were exposed to S. aureus full lawn and scored for survival. (F) WT, npr-15(tm12539), and npr-15(ok1626) animals were exposed to P. aeruginosa full lawn and scored for survival. (G) The pharyngeal pumping rate of WT and npr-15(tm12539) animals. Bars represent means while error bars indicate SD; p=NS. (H) Defecation cycle of WT and npr-15(tm12539) animals. Bars represent means while error bars indicate SD; p=NS.

Figure 2.. NPR-15 inhibits the expression of immune and aversion-related genes/pathways.

(A) Volcano plot of upregulated and downregulated genes in npr-15(tm12539) vs. wild-type (WT) animals. Red and blue dots represent significant upregulated and downregulated genes respectively, while the gray dots represent not significant genes. (B) Gene ontology analysis of upregulated genes in npr-15(tm12539) vs. WT animals. The result was filtered based on significantly enriched terms, with a q value <0.1. (C) Representation factors of immune pathways for the upregulated immune genes in npr-15(tm12539) vs. WT animals. (D) Venn diagram showing the upregulated immune genes in each pathway in npr-15(tm12539) vs. WT animals. (E) Quantitative reverse transcription-PCR (qRT-PCR) analysis of ELT-2-depenent immune gene expression in WT and npr-15(tm12539) animals. Bars represent means while error bars indicate standard deviation (SD) of three independent experiments; *p<0.05, **p<0.001, and ***p<0.0001. (F) qRT-PCR analysis of HLH-30-depenent immune gene expression in WT and npr-15(tm12539) animals. Bars represent means while error bars indicate SD of three independent experiments; *p<0.05, **p<0.001, and ***p<0.0001.

Figure 2—figure supplement 1.. Downregulated biological process and upregulated immune pathways/gene number in npr-15(tm12539) animals.

Related to Figure 2. Gene ontology analysis of downregulated genes in npr-15(tm12539) vs. wild-type (WT) animals. The result was filtered based on significantly enriched terms, with a q value <0.1.

Figure 3.. NPR-15 loss-of-function enhances immunity via ELT-2 and HLH-30 when exposed to S. aureus.

(A) Wild-type (WT) and npr-15(tm12539) animals fed with elt-2 RNAi were exposed to S. aureus full lawn and scored for survival. EV, empty vector RNAi control. (B) WT and npr-15(tm12539) animals fed with hlh-30 RNAi were exposed to S. aureus full lawn and scored for survival. EV, empty vector RNAi control. (C) WT and npr-15(tm12539) animals fed with hlh-30 and elt-2 RNAi were exposed to S. aureus full lawn and scored for survival. EV, empty vector RNAi control. (D) WT and npr-15(tm12539) animals fed with pmk-1 RNAi and animals were exposed to S. aureus full lawn and scored for survival. EV, empty vector RNAi control. (E) WT and npr-15(tm12539) animals fed with daf-16 RNAi were exposed to S. aureus full lawn and scored for survival. EV, empty vector RNAi control.

Figure 3—figure supplement 1.. NPR-15 loss-of-function enhances immunity via ELT-2 and HLH-30 when exposed to S. aureus.

Related to Figure 3. (A) Wild-type (WT), hlh-30(tm1978), npr-15(tm12539), and hlh-30(tm1978);npr-15(tm12539) animals were exposed to S. aureus full lawn and scored for survival. (B) WT, pmk-1(km25), npr-15(tm12539), npr-15(tm12539);pmk-1(km25) animals were exposed to S. aureus full lawn and scored for survival. (C) WT, daf-16(mu86), npr-15(tm12539), and daf-16(mu86);npr-15(tm12539) animals were exposed to S. aureus full lawn and scored for survival. (D) WT, hlh-30(tm1978), npr-15(tm12539), and hlh-30(tm1978);npr-15(tm12539) animals were exposed to P. aeruginosa full lawn and scored for survival. (E) WT, pmk-1(km25), npr-15(tm12539), npr-15(tm12539);pmk-1(km25) animals were exposed to P. aeruginosa full lawn and scored for survival. (F) WT, daf-16(mu86), npr-15(tm12539), and daf-16(mu86);npr-15(tm12539) animals were exposed to P. aeruginosa full lawn and scored for survival.

Figure 4.. NPR-15 suppresses molecular immune response via sensory neurons, ASJ.

(A) Neuronal connectome of NPR-15-expressing neurons. (B) Wild-type (WT), npr-15(tm12539), and npr-15(tm12539);Prab-3::npr-15 animals were exposed to S. aureus full lawn and scored for survival. (C) WT, npr-15(tm12539), ASJ(-), and ASJ(-);npr-15(tm12539) animals were exposed to S. aureus full lawn and scored for survival. (D) WT, npr-15(tm12539), ASG(-), and ASG(-);npr-15(tm12539) animals were exposed to S. aureus full lawn and scored for survival. (E) WT, npr-15(tm12539), ASE(-), and ASE(-);npr-15(tm12539) animals were exposed to S. aureus full lawn and scored for survival. (F) WT, npr-15(tm12539), npr-15(tm12539);Ptrx-1::npr-15 animals were exposed to S. aureus full lawn and scored for survival. (G) Quantitative reverse transcription-PCR (qRT-PCR) analysis of ELT-2- and HLH-30-depenent immune gene expression in WT and ASJ(-) animals. Bars represent means while error bars indicate standard deviation (SD) of three independent experiments; *p<0.05, **p<0.001, and ***p<0.0001.

Figure 4—figure supplement 1.. Survival of other NPR-15-expressing neuron-ablated strains in S. aureus infection.

Related to Figure 4. (A) Wild-type (WT) and MGH171 animals treated with npr-15 RNAi, exposed to S. aureus full lawn alongside npr-15(tm12539) animals, and scored for survival. EV, empty vector RNAi control. (B) WT and TU3401 animals treated with npr-15 RNAi, exposed to S. aureus full lawn alongside npr-15(tm12539) animals, and scored for survival. EV, empty vector RNAi control. (C) WT, npr-15(tm12539), AFD(-), and AFD(-);npr-15(tm12539) animals were exposed to S. aureus and scored for survival. (D) WT, npr-15(tm12539), ASI(-), and ASI(-);npr-15(tm12539) animals were exposed to S. aureus and scored for survival. (E) WT, npr-15(tm12539), AWC(-), and AWC(-);npr-15(tm12539) animals were exposed to S. aureus and scored for survival.

Figure 5.. NPR-15 loss-of-function inhibits pathogen avoidance behavior in an ASJ-dependent manner.

(A) Wild-type (WT), npr-15(tm12539), and npr-15(tm12539);Prab-3::npr-15 animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the standard deviation (SD) of three independent experiments; ***p<0.0001 and NS=not significant. (B) WT, npr-15(tm12539), ASJ(-), and ASJ(-);npr-15(tm12539) animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001 and NS=not significant. (C) WT, npr-15(tm12539), and npr-15(tm12539);Ptrx-1::npr-15 animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001 and NS=not significant. (D) WT, npr-15(tm12539), ASE(-), and ASE(-);npr-15(tm12539) animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001 and NS=not significant. (E) WT, npr-15(tm12539), ASI(-), and ASI(-);npr-15(tm12539) animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the SD of three independent experiments; **p<0.001, ***p<0.0001, and NS=not significant. (F) WT, npr-15(tm12539), and AFD(-), and AFD(-);npr-15(tm12539) animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001 and NS=not significant. (G) WT, npr-15(tm12539), and AWC(-), and AWC(-);npr-14(tm12539) animals were exposed to partial lawn of S. aureus and scored for lawn occupancy. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001 and NS=not significant. (H) WT and npr-15(tm12539) fed with gon-2 RNAi and were exposed to the partial lawn of S. aureus and scored for lawn occupancy. EV, empty vector RNAi control. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001. (I) WT and npr-15(tm12539) fed with gtl-2 RNAi and were exposed to the partial lawn of S. aureus and scored for lawn occupancy. EV, empty vector RNAi control. Bars represent means while error bars indicate the SD of three independent experiments; **p<0.001, ***p<0.0001, and NS=not significant. (J) WT, npr-15(tm12539), RNAi intestine-specific strain MGH171, MGH171; npr-15(tm12539) fed with gon-2 RNAi and were exposed to the partial lawn of S. aureus and scored for lawn occupancy. EV, empty vector RNAi control. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001 and NS=not significant. (K) WT, npr-15(tm12539), ASJ(-), ASJ(-);npr-15(tm12539) fed with gon-2 RNAi and were exposed to the partial lawn of S. aureus and scored for lawn occupancy. EV, empty vector RNAi control. Bars represent means while error bars indicate the SD of three independent experiments; ***p<0.0001.

Figure 5—figure supplement 1.. NPR-15 controls avoidance behavior independent of immunity, neuropeptide pathways, and oxygen.

Related to Figure 5. (A) Lawn occupancy of wild-type (WT) and npr-15(tm12539) animals treated with elt-2 RNAi before exposure to a partial lawn of S. aureus at 24 hr. Bars represent means while error bars indicate standard deviation (SD); ***p<0.0001, and NS=not significant. (B) Lawn occupancy of WT and npr-15(tm12539) animals treated with pmk-1 RNAi before exposure to a partial lawn of S. aureus at 24 hr. Bars represent means while error bars indicate SD; ***p<0.0001, and NS=not significant. (C) Lawn occupancy of WT and npr-15(tm12539) animals treated with daf-16 RNAi before exposure to a partial lawn of S. aureus at 24 hr. Bars represent means while error bars indicate SD; ***p<0.001, and NS=not significant. (D) Lawn occupancy of WT and npr-15(tm12539) animals treated with hlh-30 RNAi before exposure to a partial lawn of S. aureus at 24 hr. Bars represent means while error bars indicate SD; ***p<0.001, and NS=not significant. (E) Lawn occupancy of WT and npr-15(tm12539) animals treated with different neuropeptide gene RNAi before exposure to a partial lawn of S. aureus at 24 hr. Bars represent means while error bars indicate SD; ****p<0.0001 and NS=not significant.

Figure 5—figure supplement 2.. The transient receptor potential melastatin (TRPM) channel GON-2 control avoidance is independent of the nervous system.

Related to Figure 5. Wild-type (WT), TU3401, and npr-15(tm12539) fed with gon-2 RNAi and were exposed to the partial lawn of S. aureus and scored for lawn occupancy. EV, empty vector RNAi control. Bars represent means while error bars indicate standard deviation (SD); ****p<0.0001 and NS=not significant.

Figure 6.. G-protein-coupled receptor (GPCR)/NPR-15 suppressed immune response and enhanced avoidance behavior via sensory neurons, ASJ.

The immune response control is dependent on ELT-2 and HLH-30 transcription factors, while NPR-15 controls avoidance behavior to S. aureus via intestinal-expressed transient receptor potential melastatin (TRPM) channel, GON-2.