Fig 1.
An RNAi screen identifies a role for the nuclear hormone receptor nhr-86 in the induction of C. elegans immune effectors.
(A) C. elegans carrying either the Pirg-4(F08G5.6)::GFP or the Pirg-5(F35E12.5)::GFP immune reporter of the indicated genotypes were transferred at the L4 stage to media supplemented with either R24 or the solvent control (1% DMSO) for approximately 18 hours. Red pharyngeal expression is the Pmyo-2::mCherry co-injection marker, which confirms the presence of the Pirg-4::GFP transgene. Presence of the Pirg-5::GFP transgene was confirmed by assaying for the Rol phenotype. Photographs were acquired using the same imaging conditions for each immune reporter. (B) Model of the nhr-86 gene. Blue squares are exons. Black lines show the locations of the deletions in each of the nhr-86 mutants. (C) The expression of the C. elegans immune effector genes irg-4, irg-5 and irg-6 (C32H11.1) were analyzed by qRT-PCR in wild-type animals and in two different nhr-86 loss-of-function mutants (tm2590 and ums12), each exposed to either R24 or control for approximately 18 hours. Data for irg-6 is shown in S1B Fig. Data are the average of four independent replicates, each normalized to a control gene with error bars representing SEM. Data are presented as the value relative to the average expression from all replicates of the indicated gene in the baseline condition (wild-type animals exposed to control). The difference in induction of irg-4, irg-5 and irg-6 by R24 in wild-type animals compared to each of the two nhr-86 mutant strains is significant (**** p<0.0001 by 2-way ANOVA with Bonferroni multiple comparisons test).
Fig 2.
nhr-86 activates the transcription of innate immune response genes.
(A) All data from the mRNA-seq experiment are presented on scatter plots. Genes that were differentially regulated upon R24 treatment in wild-type animals are shown in black (Fold change> 2, PPEE<0.05). These same genes are also highlighted in black in the nhr-86(tm2590) and nhr-86(ums12) scatter plots. Genes involved in innate immunity by Gene Ontology (GO) term are highlighted in red. (B) Pearson correlation coefficients are presented for all samples in the mRNA-seq experiment. (C) Gene ontology enrichment of the nhr-86-dependent, R24-induced genes identified in the mRNA-seq experiment are shown. (D) Results of NanoString nCounter gene expression analysis for 118 C. elegans genes performed on wild-type and nhr-86(tm2590) animals exposed to either R24 or control. The 28 genes that were induced 5-fold or greater in wild-type animals by R24 are presented. Data are the average of three replicates, each of which was normalized to three control genes, with error bars representing standard deviation and are presented as the value relative to the average expression from the replicates of the indicated gene in the baseline condition (wild-type animals exposed to control). * p<0.05 by student’s t-test for the comparison of the R24-induced conditions.
Fig 3.
NHR-86 binds to the promoters of innate immune genes to drive their transcription.
(A) Venn diagram showing the number of nhr-86-dependent, R24-induced genes in the mRNA-seq experiment, the genes whose promoters were bound by NHR-86 following R24 treatment in the ChIP-seq experiment, and the overlap between these datasets. The overlap between these datasets is significantly more than is expected by chance alone (1.1 gene overlap expected by chance, hypergeometric p-value = 2x10-39). ChIP-seq profiles, mRNA-seq profiles and confirmatory ChIP-PCR are presented for the representative immune effectors irg-4 (B), irg-5 (C), mul-1 (F49F1.6) (D) and drd-50 (F49F1.1) (E) in animals of the indicated genotype exposed to R24 or the control. The y-axis is the number of reads (log2). A gene model shows the location of the exons (blue) of the indicated genes. ChIP was performed with an anti-GFP antibody in C. elegans wild-type and transgenic NHR-86::GFP animals. Final set of peaks were called if the difference in intensity values of samples had a significance level of p-value < 0.025 (see S3 Table) for the indicated comparison. In the ChIP-PCR data, the percent input for each condition was normalized to the abundance of a random intergenic region of chromosome four. ** p<0.01 and *** p<0.001 by 2-way ANOVA with Bonferroni multiple comparisons test for the indicated comparison. A region 200 bp upstream of irg-5 (C) and a random intergenic region on chromosome six (F) were not enriched by control or R24 treatment. Each data point in the ChIP-qPCR data is from an independent biological replicate.
Fig 4.
The immune response induced by nhr-86 protects C. elegans from P. aeruginosa infection.
(A) P. aeruginosa infection assays of C. elegans wild-type, nhr-86(tm2590) and nhr-86(ums12) treated with R24 or 1% DMSO (control) are shown. The difference in susceptibility to P. aeruginosa between R24-exposed wild-type and each of the mutant animals is significant (p<0.001 by the log-rank test). Data are representative of three trials. Sample sizes, mean lifespan, % lifespan extension conferred by R24 treatment in each background and p values for all trials are shown in S4A Table. Significance was determined using Kaplan-Meier survival curves and log-rank tests. (B) C. elegans carrying the Phsp-4::GFP reporter were exposed to the indicated RNAi bacteria and transferred at the L4 stage to media supplemented with either R24 or control for approximately 18 hours. Scale bar equals 100 μm. (C) qRT-PCR was used to measure the spliced (active) and total xbp-1 mRNA in animals of the indicated genotype exposed to R24 or control. Comparisons were calculated using 2-way ANOVA with Bonferroni multiple comparisons test and * p<0.05 and ** p<0.0001. Data are the average of four independent replicates, each normalized to a control gene with error bars representing SEM. Data are presented as the value relative to the average expression from all replicates of the indicated gene in the baseline condition (wild-type animals exposed to control).
Fig 5.
nhr-86 induces innate immune defenses independent of the p38 MAPK pmk-1.
(A) The expression of the C. elegans immune effector genes irg-4, irg-5, drd-50, mul-1 and irg-6 were analyzed by qRT-PCR in wild-type animals, pmk-1(km25), nhr-86(tm2590), and pmk-1(km25); nhr-86(tm2590) double mutants, each exposed to either R24 or the control for approximately 18 hours. Data for drd-50, mul-1 and irg-6 are shown in S5A Fig. Data are the average of six independent replicates, each normalized to a control gene with error bars representing SEM. Data are presented as the value relative to the average expression from all replicates of the indicated gene in the baseline condition (wild-type animals exposed to control). The difference in induction of irg-4, irg-5, drd-50, mul-1 and irg-6 by R24 in wild-type animals compared to each of the mutant strains is significant (p<0.0001 by 2-way ANOVA with Bonferroni multiple comparisons test). There is no significant difference between the expression of these genes in pmk-1(km25) animals exposed to control compared to either condition in the pmk-1(km25); nhr-86(tm2590). (B) P. aeruginosa infection assays of C. elegans wild-type, pmk-1(km25), nhr-86(tm2590), and pmk-1(km25); nhr-86(tm2590), each exposed to control or R24, are shown. The difference in susceptibility to P. aeruginosa between control and R24-exposed wild-type and pmk-1(km25) animals is significant (p<0.001). There is no significant difference between control and R24-exposed nhr-86(tm2590) and pmk-1(km25); nhr-86(tm2590) animals. Data are representative of three trials. Sample sizes, mean lifespan, % lifespan extension conferred by R24 treatment in each background and p values for all trials are shown in S4B Table. Significance was determined using Kaplan-Meier survival curves and log-rank tests. (C) Immunoblot analysis of lysates from L4 stage animals of the indicated genotype using antibodies that recognize the doubly-phosphorylated TGY motif of PMK-1 (⍺-Active PMK-1), the total PMK-1 protein (⍺-Total PMK-1) and tubulin (⍺-Tubulin). The total PMK-1 antibody detects total, but not active (phosphorylated) PMK-1. (D) The relative intensity of active PMK-1 and total PMK-1 was quantified from three biological replicates and is expressed as the average ratio of active to total PMK-1, relative to wild-type control. Error bars report SEM. There is no significant difference (n.s.) between these conditions (2-way ANOVA with Bonferroni multiple comparisons test).
Fig 6.
Model of NHR-86-mediated immune regulation in C. elegans.
The basal expression of immune effectors such as irg-4, irg-5, mul-1 and drd-50 are ensured by p38 MAPK PMK-1. Activated NHR-86 traffics to the promoters of these and other immune effectors to drive their induction and provide protection from bacterial infection.