p38 MAPK regulates expression of immune response genes and contributes to longevity in C. elegans

Abstract

The PMK-1 p38 mitogen-activated protein kinase pathway and the DAF-2-DAF-16 insulin signaling pathway control Caenorhabditis elegans intestinal innate immunity. pmk-1 loss-of-function mutants have enhanced sensitivity to pathogens, while daf-2 loss-of-function mutants have enhanced resistance to pathogens that requires upregulation of the DAF-16 transcription factor. We used genetic analysis to show that the pathogen resistance of daf-2 mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there was essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they form parallel pathways to promote immunity. We found that PMK-1 controls expression of candidate secreted antimicrobials, including C-type lectins, ShK toxins, and CUB-like genes. Microarray analysis demonstrated that 25% of PMK-1 positively regulated genes are induced by Pseudomonas aeruginosa infection. Using quantitative PCR, we showed that PMK-1 regulates both basal and infection-induced expression of pathogen response genes, while DAF-16 does not. Finally, we used genetic analysis to show that PMK-1 contributes to the enhanced longevity of daf-2 mutants. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2-DAF-16 pathway appears to regulate immunity as part of a more general stress response. The contribution of the PMK-1 pathway to the enhanced lifespan of daf-2 mutants suggests that innate immunity is an important determinant of longevity.

Figure 1. The PMK-1 p38 MAPK Pathway Is Required for daf-2 Enhanced Pathogen Resistance

(A) pmk-1(km25) suppresses daf-2(e1368) enhanced pathogen resistance. (B) sek-1(km4) suppresses daf-2(e1370) enhanced pathogen resistance. Slow killing assays were performed with P. aeruginosa strain PA14 under standard conditions, and each graph shows the average of three plates for each strain, with each plate containing 20–30 worms. Results are representative of 3 independent assays. See Table S1 for more assays and statistical analysis.

Figure 2. PMK-1 and DAF-16 Upregulate Distinct Genes

(A) Intensity plot of genes upregulated by PMK-1 in daf-2(e1368) mutant. Plot represents signal intensity of all 22,500 sequences on Affymetrix C. elegans GeneChip. The x-axis shows expression level in daf-2(e1368);pmk-1(km25) animals and the y-axis shows expression level in daf-2(e1368) animals. Sequences colored red are considered upregulated by wild-type PMK-1 and sequences colored green are considered downregulated by wild-type PMK-1 (using Resolver; p <0.01). Diagonal lines delineate a 2-fold change. See Table S2 for a complete list of genes differentially expressed between daf-2 and daf-2;pmk-1. (B) Pie chart of gene classes upregulated by PMK-1. The top 36 genes induced are represented. (C and D) qRT-PCR comparing expression in daf-2(e1368) versus daf-2(e1368);pmk-1(km25), and daf-2(e1368) versus daf-2(e1368);daf-16(mgDf47). Results are the average of two biological replicates, each replicate measured in duplicate and normalized to a control gene. Error bars are SEM. (C) shows genes upregulated by PMK-1 and (D) shows genes upregulated by DAF-16.

Figure 3. P. aeruginosa Induces Expression of Genes in Similar Gene Classes as PMK-1–Induced Genes

(A) PA14 slow killing assay comparing survival of fer-15;fem-1 young adult animals on gacA and wild-type PA14. Animals were raised in the same way as for harvest for microarrays. (B) Intensity plot of genes regulated by PA14 versus OP50 at 4 h. Sequences colored red are considered upregulated by PA14 and sequences colored green are considered downregulated by wild-type PA14 (using Resolver; p <0.01). Diagonal lines delineate a 2-fold change. See Table S4 for a complete list of genes differentially expressed between PA14 and OP50. (C) Venn diagram of overlap between genes induced in PA14 versus OP50 at 4 h comparison and PA14 versus gacA at 4 h comparison. (D) Pie chart of gene classes induced by PA14 versus OP50. The genes (59) that are upregulated greater than 5-fold are represented.

Figure 4. PMK-1 Regulates Basal and Inducible Expression of P. aeruginosa–Induced Genes

(A) Venn diagram of overlap between genes regulated by PMK-1 and P. aeruginosa. (B) (C) qRT-PCR analysis of PA14-induced gene expression in wild-type animals and in pmk-1 mutants. Results are the average of two biological replicates, each replicate measured in duplicate and normalized to a control gene. Error bars are SEM. (D) Diagram of different gene classes regulated by PMK-1 and/or P. aeruginosa. PMK-1 is required for basal and inducible regulations of class A genes. PMK-1 is required for basal, but not inducible expression of class B genes. PMK-1 is required for inducible but not basal expression of class C genes. PMK-1 is required for neither basal nor inducible expression of class D genes. PMK-1 regulates basal expression of class E genes, but these genes are not induced by P. aeruginosa.

Figure 5. The PMK-1 p38 MAPK Pathway Is Partially Required for Increased Longevity of daf-2

(A) pmk-1(km25) partially suppresses enhanced longevity of daf-2(e1368). (B) sek-1(km4) partially suppresses enhanced longevity of daf-2(e1370). See Table S7 for quantitative data.

Figure 6. A Model for Regulation of Immunity/Longevity by the PMK-1 p38 MAPK Pathway and the DAF-2–DAF-16 Insulin-Signaling Pathway in C. elegans

We propose that the PMK-1 p38 MAPK pathway plays an active role in response to bacterial pathogens. PMK-1 controls basal levels of pathogen response genes on E. coli and also mediates induction of these genes upon infection. In contrast, DAF-16 controls a separate group of immune genes and does not mediate the infection response, but does turn down expression of some pathogen-induced genes. Certain pathogen response genes are induced independently of PMK-1 or DAF-16, indicating the existence of a third pathway. PMK-1 is required for the enhanced pathogen resistance of daf-2 mutants because it is the predominant pathway contributing to immunity. Finally, we suggest that PMK-1 contributes to enhanced longevity of daf-2 mutants because intact immune function promotes longevity.