Neuronal GPCR controls innate immunity by regulating noncanonical unfolded protein response genes

Abstract

The unfolded protein response (UPR), which is activated when unfolded or misfolded proteins accumulate in the endoplasmic reticulum, has been implicated in the normal physiology of immune defense and in several human diseases, including diabetes, cancer, neurodegenerative disease, and inflammatory disease. In this study, we found that the nervous system controlled the activity of a noncanonical UPR pathway required for innate immunity in Caenorhabditis elegans. OCTR-1, a putative octopamine G protein-coupled catecholamine receptor (GPCR, G protein-coupled receptor), functioned in sensory neurons designated ASH and ASI to actively suppress innate immune responses by down-regulating the expression of noncanonical UPR genes pqn/abu in nonneuronal tissues. Our findings suggest a molecular mechanism by which the nervous system may sense inflammatory responses and respond by controlling stress-response pathways at the organismal level.

Fig.1

C. elegans G-protein coupled receptor OCTR-1 is involved in immunity to P. aeruginosa. (A) Wild-type and octr-1(ok371) animals were exposed to P. aeruginosa. (B) Wild-type and octr-1(ok371) animals were exposed to heat-killed P. aeruginosa. (C) Wild-type and octr-1(ok371) animals were exposed to a full lawn of P. aeruginosa. The survival graphs represent combined results of two independent experiments, N=90 adult animals per strain. (D) Animals were placed on a small spot of P. aeruginosa in a 3.5cm plate and monitored over time for their presence or absence on the lawn. The graph represents combined results of three independent experiments, N=60 adult animals per strain. (E) Wild-type and octr-1(ok371) animals were exposed to P. aeruginosa expressing GFP for 48 hours and then visualized using a MZ FLIII Leica stereomicroscope. (F) Wild-type and octr-1(ok371) animals were exposed to P. aeruginosa expressing GFP for 48 hours and the colony forming units were quantified. Ten animals were used for each condition. The graph represents combined results of three independent experiments. Bars represent mean ± SEM.

Fig.2

OCTR-1 controls pathways required for C. elegans innate immunity. (A) Venn diagrams of genes that are up-regulated in octr-1(ok371) animals and positively regulated by daf-16, pmk-1, and ced-1. (B) Wild-type and octr-1(ok371) animals grown on double-stranded RNA (dsRNA) for vector control or dsRNA for pmk-1 were exposed to P. aeruginosa. (C) Wild-type and octr-1(ok371) animals grown on dsRNA for vector control or dsRNA for daf-16 were exposed to P. aeruginosa. The survival graphs represent combined results of two independent experiments, N=90 adult animals per strain. (D) Active PMK-1 was detected in wild-type and octr-1(ok371) animals. Animals were grown at 20°C until 1-day-old adult and whole-worm lysates were used to detect active PMK-1 with an antibody against human p38 from Promega (Madison, WI). Actin was used as loading control.

Fig. 3

pqn/abu genes are required for the enhanced immunity of octr-1(ok371) animals to P. aeruginosa. (A) Cluster of pqn/abu genes that are up-regulated in octr-1(ok371) mutants. (B) Quantitative reverse transcription-PCR analysis of abu-1, abu-7, abu-8, abu-12, abu-13, abu-14 and abu-15 expression in octr-1(ok371) relative to wild-type animals exposed to P. aeruginosa. N=3; bars represent mean ± SEM. (C) Wild-type animals grown on dsRNA for vector control and octr-1(ok371) animals grown on dsRNA for vector control or dsRNA for abu genes were exposed to P. aeruginosa. (D) Wild-type, octr-1(ok371), ced-1(e1735) and octr-1(ok371);ced-1(e1735) animals were exposed to P. aeruginosa. (E) Wild-type and octr-1(ok371) animals grown on dsRNA for vector control and octr-1(ok371);ced-1(e1735) animals grown on dsRNA for vector control or dsRNA for abu genes were exposed to P. aeruginosa. (F) Wild-type, octr-1(ok371), octr-1(ok371);ced-1(e1735), octr-1(ok371);abu-1::gfp(zcEx8), and octr-1(ok371);ced-1(e1735);abu-1::gfp(zcEx8) animals were exposed to P. aeruginosa. The survival graphs represent combined results of two independent experiments, N=90 adult animals per strain.

Fig.4

OCTR-1 expressing neurons ASH and ASI suppress innate immunity by controlling the expression of pqn/abu genes. (A) Wild-type, octr-1(ok371) and FY746 octr-1(ok371);grEx158[Psra-6::octr-1] animals were exposed to P. aeruginosa. (B) Wild-type, octr-1(ok371) and FY745 octr-1(ok371);grEx157 [Poctr-1::octr-1::gfp] animals were exposed to P. aeruginosa. The survival graphs represent combined results of two independent experiments, N=90 adult animals per strain. (C) Quantitative reverse transcription-PCR analysis of abu-1, abu-12, abu-13, abu-14, and abu-15 expression in octr-1(ok371) and FY746 octr-1(ok371);grEx158[Psra-6::octr-1] relative to wild-type animals exposed to P. aeruginosa. N=3; bars represent mean ± SEM.