Proline Catabolism Modulates Innate Immunity in Caenorhabditis elegans Haiqing Tang, Shanshan Pang  Cell Reports  Volume 17, Issue 11, Pages 2837-2844 (December 2016) DOI: 10.1016/j.celrep.2016.11.038 Copyright © 2016 The Author(s) Terms and Conditions

Cell Reports 2016 17, 2837-2844DOI: (10.1016/j.celrep.2016.11.038) Copyright © 2016 The Author(s) Terms and Conditions

Figure 1 Proline Dehydrogenase Promotes Pathogen Resistance (A) Schematic of mitochondrial proline catabolism pathway. (B) The mRNA levels of prodh and p5cdh in worms exposed to E. coli OP50 and P. aeruginosa PA14. (C) Effects of prodh RNAi on host survival upon P. aeruginosa PA14 infection. (D) Proline supplementation promotes pathogen resistance. (E) Effects of prodh RNAi on pathogen resistance induced by exogenous proline. Data are presented as mean ± SEM. ∗p < 0.05 versus respective controls. See also Figure S1 and Table S1. Cell Reports 2016 17, 2837-2844DOI: (10.1016/j.celrep.2016.11.038) Copyright © 2016 The Author(s) Terms and Conditions

Figure 2 Proline Dehydrogenase Regulates ROS Generation and SKN-1 Activation during Infection (A) ROS levels in animals fed with E. coli OP50 and pathogen P. aeruginosa PA14. (B) prodh RNAi treatment reduces ROS levels in P. aeruginosa PA14-infected worms. (C) Effects of proline supplementation on ROS levels in worms fed with E. coli OP50 and P. aeruginosa PA14. (D) prodh RNAi reduces gst-4p::GFP expression in P. aeruginosa PA14-infected worms. Upper panel: representative images. Lower panel: quantification of GFP expression levels, scored as low (L), medium (M), and high (H). (E) Effect of prodh RNAi on proline-induced gst-4p::GFP expression upon P. aeruginosa infection. Left panel, representative images. Right panel, quantification of GFP expression levels. (F and G) Effects of prodh RNAi and proline supplementation on SKN-1::GFP nuclear localization induced by P. aeruginosa infection. ROS levels were measured by HyPer ratio. Scale bar, 100 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus respective controls unless specifically indicated. See also Figure S2. Cell Reports 2016 17, 2837-2844DOI: (10.1016/j.celrep.2016.11.038) Copyright © 2016 The Author(s) Terms and Conditions

Figure 3 P5CDH/ALH-6 Prevents Excessive ROS Production and SKN-1 Activation in Response to Infection (A) Survival of alh-6 mutants in response to P. aeruginosa PA14 infection. (B) ROS levels of WT and alh-6 mutants fed with E. coli OP50 and P. aeruginosa PA14. (C) The expression of gst-4p::GFP in WT and alh-6 mutants fed with E. coli OP50 and P. aeruginosa PA14. (D) Effects of NAC treatment on the survival of alh-6 mutants during infection. (E–G) Effects of RNAi targeting prodh and p5cr on ROS levels (E), gst-4p::GFP expression (F), and survival (G) of alh-6 mutants in response to P. aeruginosa PA14 infection. For gst-4p::GFP expression, left panel is representative images, and right panel is quantification of GFP expression levels. ROS levels were measured by HyPer ratio. Scale bar, 100 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as indicated. See also Figure S3 and Table S1. Cell Reports 2016 17, 2837-2844DOI: (10.1016/j.celrep.2016.11.038) Copyright © 2016 The Author(s) Terms and Conditions

Figure 4 Interaction between Proline Catabolism and Ce-Duox1/BLI-3 during Infection (A and B) Effects of bli-3 RNAi on ROS production (A) and gst-4p::GFP expression (B) induced by proline supplementation during infection. (C and D) Effects of bli-3 RNAi on ROS production (C) and gst-4p::GFP expression (D) in alh-6 mutants in response to P. aeruginosa PA14 infection. (E and F) Effects of bli-3 RNAi on the survival of wild-type worms treated with exogenous proline (E) and alh-6 mutants (F) upon infection. For gst-4p::GFP expression, left panel is representative images, and right panel is quantification of GFP expression levels. ROS levels were measured by HyPer ratio. Scale bar, 100 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as indicated. See also Figure S4 and Table S1. Cell Reports 2016 17, 2837-2844DOI: (10.1016/j.celrep.2016.11.038) Copyright © 2016 The Author(s) Terms and Conditions