Innate immunity in Caenorhabditis elegans is regulated by neurons expressing NPR-1/GPCR

Abstract

A large body of evidence indicates that metazoan innate immunity is regulated by the nervous system, but the mechanisms involved in the process and the biological importance of such control remain unclear. We show that a neural circuit involving npr-1, which encodes a G protein-coupled receptor (GPCR) related to mammalian neuropeptide Y receptors, functions to suppress innate immune responses. The immune inhibitory function requires a guanosine 3',5'-monophosphate-gated ion channel encoded by tax-2 and tax-4 as well as the soluble guanylate cyclase GCY-35. Furthermore, we show that npr-1- and gcy-35-expressing sensory neurons actively suppress immune responses of nonneuronal tissues. A full-genome microarray analysis on animals with altered neural function due to mutation in npr-1 shows an enrichment in genes that are markers of innate immune responses, including those regulated by a conserved PMK-1/p38 mitogen-activated protein kinase signaling pathway. These results present evidence that neurons directly control innate immunity in C. elegans, suggesting that GPCRs may participate in neural circuits that receive inputs from either pathogens or infected sites and integrate them to coordinate appropriate immune responses.

Fig. 1

C. elegans G-protein coupled receptor NPR-1 is involved in immunity to P. aeruginosa. (A) C. elegans strains carrying mutations in GPCRs were screened for altered survival on P. aeruginosa. C56G3.1(ok1439) (P=0.0347), F57H12.4 (ok1504) (P=0.0071), and str-182(ok1419) (P=0.0342) had enhanced resistance to P. aeruginosa and npr-1(ad609) (P=0.0246) had enhanced susceptibility to P. aeruginosa. Shown is the time required for 50% of the nematodes to die (TD₅₀) as mean +/− SEM corresponding to at least three independent experiments, each of which used at least 40 adult nematodes per strain. (B) Wild-type N2 and npr-1(ad609) (P=0.0001) nematodes were exposed to P. aeruginosa and scored for survival over time. The graph represents combined results of four independent experiments, N≥40 adult nematodes per strain. (C) Wild-type N2 and npr-1(ad609) (P=0.1411) nematodes were exposed to heat-killed P. aeruginosa and scored for survival over time. The graph represents the combined results of two independent experiments, N=100 adult nematodes per strain. (D) Wild-type N2, npr-1(ad609) (P=0.0001), npr-1(ky13) (P=0.0001), npr-1(n1353) (P=0.0001), npr-1(ur89) (P=0.0001), npr-1(g320) (P=0.0001), and the wild isolate (WI) npr-1(g320) (P=0.0922) were exposed to P. aeruginosa and scored for survival over time. Shown is a representative assay of at least 3 independent experiments, N=48 adult nematodes per strain.

Fig. 2

Hyperoxia avoidance of NPR-1-deficient animals increases susceptibility to P. aeruginosa. (A) C. elegans wild-type N2 animals and (B) npr-1(ad609) mutants were propagated at 20°C as hermaphrodites on modified NG agar plates seeded with E. coli strain OP50 and then visualized using a Leica MZ FLIII stereomicroscope. The characteristic aggregate of npr-1(ad609) nematodes shown here is at the edge of the bacterial lawn. (C) Twelve wild-type N2 and (D) twelve npr-1(ad609) nematodes were exposed to P. aeruginosa for 24 hours under standard killing assay conditions and visualized using a Leica MZ FLIII stereomicroscope. Under these conditions, npr-1(ad609) nematodes do not form characteristic aggregates of the strain. (E) Wild-type N2 and npr-1(ad609) nematodes were exposed to either a full lawn or a center lawn of P. aeruginosa on a 3.5 cm in diameter plate and scored for survival over time. Under both conditions npr-1(ad609) animals were more susceptible to P. aeruginosa-mediated killing (P=0.0001). Wild-type animals on full lawns were more susceptible to P. aeruginosa-mediated killing than animals on center lawns (P=0.0001); npr-1(ad609) animals were equally susceptible (P=0.07). The graph represents combined results of three independent experiments, N≥40 adult nematodes per strain. (F) Wild-type N2 and npr-1(ad609) nematodes at exposed to P. aeruginosa at either 21% or 8% oxygen and scored for survival over time. Under both conditions npr-1(ad609) animals were more susceptible to P. aeruginosa-mediated killing (P=0.0001). npr-1(ad609) animals at 21% oxygen were more susceptible to P. aeruginosa-mediated killing than animals at 8% oxygen (P=0.0001); wild-type animals were equally susceptible (P=0.95). The graph represents combined results of two independent experiments, N=40 adult nematodes per strain.

Fig. 3

The NPR-1 neural circuit regulates innate immunity. (A) Wild-type N2, npr-1(ad609) (P=0.0001), gcy-35(ok769) (P=0.0125), and gcy-35(ok769);npr-1(ad609) (P=0.0639) were exposed to P. aeruginosa. (B) Wild-type N2, npr-1(ad609) (P=0.0001), tax-4(p678) (P=0.1673), tax-4(p678);npr-1(ad609) (P=0.3611), were exposed to P. aeruginosa. (C) Wild-type N2, npr-1(ad609) (P=0.0001), tax-2(p691) (P=0.0930), tax-2(p691);npr-1(ad609) (P=0.0031) were exposed to P. aeruginosa. (D) Wild-type N2, npr-1(ad609) (P=0.0001), qaIS2241 (P=0.0042), a strain which lacks AQR, PQR, and URX neurons, and npr-1(ad609); qaIS2241 (P=0.0001) were exposed to P. aeruginosa. The graphs represent combined results of at least three independent experiments, N≥40 adult nematodes per strain. (E) Wild-type N2, npr-1(ad609) (P=0.0001), pgcy-32::npr-1; npr-1(ad609) (P=0.0001), and pnpr-1::npr-1; npr-1(ad609) (P=0.1939) were exposed to P. aeruginosa. The graphs represent combined results of at least two independent experiments, N≥100 adult nematodes per strain. Killing assays were performed at 17°C, as low temperatures are known to increase the resolution of killing assays involving P. aeruginosa. (F) Model of the neural control of innate immunity in C. elegans: NPR-1 inhibits the activity of AQR, PQR, URX and additional neuron(s) designated YYY that suppress innate immunity, while GCY-35, TAX-2, and TAX-4 are required for the activation of AQR, PQR and URX neurons.

Fig. 4

The NPR-1 neural circuit regulates expression of innate immune genes. (A–J) Quantitative reverse transcription–PCR analysis of C01B4.6/Y19D10A.16, F56A4.9/Y19D10A.7, C01B4.7/Y19D10A.4, F56A4.12/Y19D10A.11, abf-1, dod-24, F36F12.8, F46F2.3, gst-24, and T28F2.2 expression in npr-1(ad609) and gcy-35(ok769);npr-1(ad609) nematodes relative to wild-type nematodes exposed to P. aeruginosa. Data were analyzed by normalization to pan-actin (act-1,-3,-4) and relative quantification using the comparative cycle threshold method. Student’s exact t-test indicates differences among the groups are significantly different; bar graphs correspond to mean ± SEM. Point graphs correspond to gene quantification in independent isolations of npr-1(ad609)(N=6) and gcy-35(ok769);npr-1(ad609)(N=3). (K) The Venn diagram lists the genes identified by microarray analysis to be regulated by both NPR-1 and one or more known innate immune pathways in C. elegans. Genes that lie within two or three circles are regulated by multiple innate immune pathways in addition to NPR-1. Twenty-six genes have not been previously connected to any of the innate immune pathways and are depicted in the solitary circle. (L) Immunological detection of active PMK-1. Active PMK-1 was detected in wild-type N2, npr-1(ad609) and gcy-35(ok769);npr-1(ad609). Animals were grown at 20°C until 1 day old adult and whole worm lysates were used to detect active PMK-1 by Western blotting using an anti-human p38 antibody from Promega, Inc. Actin was detected using a polyclonal antibody from SIGMA. BioRad Quantity One Analysis Software was used to scan and analyze the Western blot.