Organismal regulation of XBP-1-mediated unfolded protein response during development and immune activation

Abstract

The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1--a G protein-coupled catecholamine receptor expressed in neurons--suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.

Figure 1

XBP-1-mediated UPR pathway is regulated by OCTR-1 at the adult stage. The expression of XBP-1-dependent and XBP-1-independent genes was studied in L4 (A,B,C) and 1-day-old animals (D,E,F). (A,D) Quantitative reverse transcription polymerase chain reaction analysis of abu-1, abu-12, hsp-4, Y41C4A.11, F45E4.1, K01D12.11, and F20D6.4 expression in octr-1(ok371) relative to wild-type (WT) animals exposed to Pseudomonas aeruginosa PA14. N=3 independent experiments; bar graphs correspond to mean±s.e.m. (B,E) Image of xbp-1 splicing in WT and octr-1(ok371) animals exposed to Escherichia coli OP50 or P. aeruginosa PA14 and/or treated with tunicamycin (Tm). Actin was used as a loading control. (C,F) Bar graph represents the mean ratio of spliced xbp-1/total xbp-1 transcript, multiplied by a factor of 100, and in three independent experiments. Error bars represent s.e.m. UPR, unfolded protein response; XBP-1, X-box binding protein 1.

Figure 2

Loss of OCTR-1 signalling enhances the activation of XBP-1-mediated UPR pathway. (A) Images of Phsp-4::GFP(zcIs4) and octr-1(ok371);Phsp-4::GFP(zcIs4) 1-day-old animals exposed to Pseudomonas aeruginosa PA14. Animals that best represent the fluorescence level of the population were shown. (B). GFP quantification from Phsp-4::GFP(zcIs4) and octr-1(ok371);Phsp-4::GFP(zcIs4) 1-day-old animals exposed to P. aeruginosa PA14. Binary mean intensity of the region of interest (ROI) that corresponds to an entire animal was measured by NIS-Elements AR 3.2 software. N=10–20, error bars represent s.e.m. octr-1(ok371);Phsp-4::GFP(zcIs4) versus Phsp-4::GFP(zcIs4) on PA14: P<0.05. (C) GFP fluorescence intensity (FLU-2) of Phsp-4::GFP(zcIs4) and octr-1;Phsp-4::GFP(zcIs4) 1-day-old animals exposed to P. aeruginosa was plotted against animal size, measured as time of flight (TOF). Each dot represents an individual animal. GFP, green fluorescent protein; UPR, unfolded protein response; XBP-1, X-box binding protein-1.

Figure 3

OCTR-1 controls XBP-1-mediated UPR in infected young adult animals. (A) Images of Phsp-4::GFP(zcIs4) and octr-1(ok371);Phsp-4::GFP(zcIs4) egg, L1, L2, L3, L4, and young adult (YA) animals grown on Pseudomonas aeruginosa PA14. Animals that best represent the fluorescence level of the population were shown. (B) GFP quantification from Phsp-4::GFP(zcIs4) and octr-1(ok371);Phsp-4::GFP(zcIs4) egg, L1, L2, L3, L4, and YA animals exposed to P. aeruginosa PA14. Binary mean intensity of the region of interest that corresponds to an entire animal was measured by NIS-Elements AR 3.2 software. N=10–20, error bars represent s.e.m. Asterisk indicates significant difference. octr-1(ok371);Phsp-4::GFP(zcIs4) YA versus Phsp-4::GFP(zcIs4) YA on PA14: P<0.05. GFP, green fluorescent protein; UPR, unfolded protein response; XBP-1, X-box binding protein-1.

Figure 4

Inhibition of XBP-1-mediated UPR suppresses the enhanced resistance to Pseudomonas aeruginosa infection of octr-1(ok371) animals. (A) WT, octr-1(ok371), xbp-1(zc12), and xbp-1(zc12);octr-1(ok371) animals were exposed to P. aeruginosa PA14 and scored for survival over time. P-values are relative to octr-1(ok371) animals: WT (P<0.0001), xbp-1(zc12) (P<0.0001), xbp-1(zc12);octr-1(ok371) (P<0.0001). Shown is a representative assay of four independent experiments. (B) WT and octr-1(ok371) animals grown on double-stranded RNA (dsRNA) for vector control or dsRNA for xbp-1 were exposed to P. aeruginosa PA14 and scored for survival over time. P-values are relative to octr-1(ok371)+vector: WT+vector (P<0.0001), WT+xbp-1 RNAi (P<0.0001), octr-1(ok371)+xbp-1 RNAi (P<0.0001). Shown is a representative assay of three independent experiments. (C) WT and octr-1(ok371) animals grown on double-stranded RNA (dsRNA) for vector control or dsRNA for Y41C4A.11 were exposed to P. aeruginosa PA14 and scored for survival over time. P-values are relative to octr-1(ok371)+vector: WT+vector (P<0.0001), WT+Y41C4A.11 RNAi (P<0.0001), octr-1(ok371)+Y41C4A.11 RNAi (P<0.01). Shown is a representative assay of two independent experiments. RNAi, RNA-mediated interference; UPR, unfolded protein response; WT, wild-type; XBP-1, X-box binding protein-1.