Proline Catabolism Modulates Innate Immunity in Caenorhabditis elegans

Report

Proline Catabolism Modulates Innate Immunity in Caenorhabditis elegans Graphical Abstract

Authors Haiqing Tang, Shanshan Pang

Correspondence [email protected] (H.T.), [email protected] (S.P.)

In Brief Tang and Pang show that proline catabolism is a component of the innate immune system in C. elegans. In response to P. aeruginosa infection, proline catabolic enzymes modulate ROS homeostasis and subsequent SKN-1 activation, likely through metabolic intermediate P5C and dual-oxidase CeDuox1/BLI-3.

Highlights d

Mitochondrial proline catabolism is essential for pathogen resistance in C. elegans

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Proline catabolism regulates infection-induced SKN-1 activation through ROS

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Metabolic intermediate P5C may account for ROS production and SKN-1 activation

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Ce-Duox1/BLI-3 is required for proline catabolism-induced ROS and SKN-1 responses

Tang & Pang, 2016, Cell Reports 17, 2837–2844 December 13, 2016 ª 2016 The Author(s). http://dx.doi.org/10.1016/j.celrep.2016.11.038

Cell Reports

Report Proline Catabolism Modulates Innate Immunity in Caenorhabditis elegans Haiqing Tang1,* and Shanshan Pang1,2,* 1School

of Life Sciences, Chongqing University, Chongqing 401331, China Contact *Correspondence: [email protected] (H.T.), [email protected] (S.P.) http://dx.doi.org/10.1016/j.celrep.2016.11.038 2Lead

SUMMARY

Metabolic pathways are regulated to fuel or instruct the immune responses to pathogen threats. However, the regulatory roles for amino acid metabolism in innate immune responses remains poorly understood. Here, we report that mitochondrial proline catabolism modulates innate immunity in Caenorhabditis elegans. Modulation of proline catabolic enzymes affects host susceptibility to bacterial pathogen Pseudomonas aeruginosa. Mechanistically, proline catabolism governs reactive oxygen species (ROS) homeostasis and subsequent activation of SKN-1, a critical transcription factor regulating xenobiotic stress response and pathogen defense. Intriguingly, proline catabolism-mediated activation of SKN-1 requires cell-membrane dual-oxidase CeDuox1/BLI-3, highlighting the importance of interaction between mitochondrial and cell-membrane components in host defense. Our findings reveal how animals utilize metabolism of a single amino acid to defend against a pathogen and identify proline catabolism as a component of innate immune signaling. INTRODUCTION Cellular metabolism is regulated or reprogrammed to support immune functions (Ganeshan and Chawla, 2014; Pearce and Pearce, 2013). Metabolic reprogramming provides activated immune cells with sufficient ATP to match their considerable energetic demands. Metabolic pathways, such as glycolysis, glutaminolysis, and fatty acid oxidation, are preferentially utilized by various immune cells to generate energy (Ganeshan and Chawla, 2014). On the other hand, metabolic intermediates can act as signaling molecules in immune responses. For example, the rise of succinate, a metabolite from tricarboxylic acid (TCA) cycle, is identified as an endogenous danger signal to initiate proinflammatory responses in macrophages (Tannahill et al., 2013). The metabolism of amino acids, representing an essential pool of nutrients utilized for cellular energy production, also plays regulatory roles in immune functions. Catabolism of arginine and tryptophan is found to be critical for regulation of T cell functions

(Bronte and Zanovello, 2005; Bronte et al., 2003; Grohmann and Bronte, 2010). However, the regulatory roles for other amino acid metabolisms in immune responses, especially innate immunity, remain poorly understood. Proline is one of amino acids whose metabolism impacts multiple cellular functions. Mitochondrial proline dehydrogenase (PRODH), the first enzyme of proline catabolism, is a well-known tumor regulator involved in cancer cell autophagy, proliferation, and apoptosis (Liu et al., 2012a, 2012b, 2006, 2009). PRODH also promotes fat utilization in starved adipocytes to prevent cell death (Lettieri Barbato et al., 2014). Both we and others have shown that, under nutrient stress, C. elegans utilizes proline catabolism to promote stress responses, which impacts animal survival (Pang and Curran, 2014; Zarse et al., 2012). Despite the essential roles for proline catabolism in multiple fundamental cellular processes, its contribution to animal innate immunity is unclear. Innate immunity is an ancient and conserved system that serves as the first line of defense against invading pathogens. During infection, antimicrobial reactions are activated to directly eliminate invading pathogens. Meanwhile, host-resistant responses, such as detoxification, are exploited to limit the deleterious impact caused by pathogen infection (Schneider and Ayres, 2008). C. elegans provides a valuable genetic model for studying the conserved players of animal innate immune systems. One major factor responsible for host immunity in C. elegans is the Nrf protein ortholog SKN-1, a transcription factor that promotes host resistance by inducing genes involved in detoxification of reactive oxygen species (ROS) and harmful bacterial components (Hoeven et al., 2011; Papp et al., 2012). In response to bacterial pathogen, cell-membrane dual-oxidase Ce-Duox1/BLI3 generates ROS, which are important for pathogen resistance by acting as microbicidal molecules (Cha´vez et al., 2009). On the other hand, ROS could also function as signaling molecules to induce SKN-1 activation in a p38/PMK1-dependent manner (Hoeven et al., 2011); however, the signaling components of this key host immune pathway remain poorly defined. In this study, we investigated the role for mitochondrial proline catabolism in C. elegans host defense. We identified proline catabolism as part of innate immunity by governing ROS homeostasis and SKN-1 activation. Given that the function of amino acid metabolism is highly conserved, we propose that mechanisms identified here might be applied in other organisms, including mammals.

Cell Reports 17, 2837–2844, December 13, 2016 ª 2016 The Author(s). 2837 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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.

RESULTS AND DISCUSSION Proline Dehydrogenase Promotes Pathogen Resistance Mitochondrial proline catabolism comprises two consecutive enzymatic reactions. First, proline is catabolized to pyrroline-5carboxylate (P5C) through proline dehydrogenase (PRODH). P5C dehydrogenase (P5CDH) then further oxidizes P5C to glutamate, which is converted to a-ketoglutarate and fed into the TCA cycle (Figure 1A). Both prodh and p5cdh genes are encoded in the nuclear DNA. We studied the involvement of proline catabolism in pathogen resistance by using the C. elegans–P. aeruginosa host-pathogen system. First, the expression of proline catabolic enzymes was examined. qRT-PCR results revealed that the mRNA levels of prodh/B0513.5, the C. elegans PRODH-encoded gene, were significantly upregulated by P. aeruginosa infection, while the levels of alh-6/p5cdh, the C. elegans P5CDH-encoded gene, were unaffected (Figure 1B), implicating a possible role for PRODH in pathogen resistance. By using RNAi strategy to efficiently downregulate the expression of prodh during infection (Figure S1A), we found that knockdown of prodh increased the susceptibility of C. elegans to P. aeruginosa (Figure 1C; Table S1), indicating the requirement of this enzyme in host defense. Supporting this idea, proline supplementation promoted host survival during infection (Figure 1D; Table S1), and, more importantly, RNAi targeting host prodh largely

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suppressed the effects of proline (Figure 1E; Table S1), suggesting that exogenous proline acts, at least in part, through host PRODH to promote pathogen resistance. We also examined the involvement of PRODH in pathogen load. Congruent with pathogen resistance, prodh RNAi treatment increased P. aeruginosa load, while proline supplementation decreased pathogen load in the intestine (Figures S1B and S1C). PRODH Promotes ROS Production and SKN-1 Activation during Infection PRODH can promote pathogen resistance by either replenishing the TCA cycle intermediates to generate ATP (Figure 1A) or executing a regulatory function to certain immune pathways. Induction of PRODH is found to activate a mitochondrial ROS signal to promote lifespan in long-lived C. elegans (Zarse et al., 2012). ROS, generated from NADPH oxidase or mitochondria, are implicated in direct killing of pathogens as well as activation of essential immune pathways in innate immune cells (Bedard and Krause, 2007; Sena and Chandel, 2012). These findings inspired the hypothesis that infection-caused increases in ROS levels might be dependent on PRODH. By measuring the ratio of oxidized to reduced HyPer, an established indicator of hydrogen peroxide (Back et al., 2012; Knoefler et al., 2012; Pang and Curran, 2014), we found that P. aeruginosa PA14 infection indeed significantly increased host ROS levels (Figure 2A),

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 mm. 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.

similar to E. faecalis infection (Cha´vez et al., 2009). The ROS increases during infection were also detected by 20 ,70 -dichlorofluorescein diacetate (DCF-DA) (Figure S2A), a well-known dye sensitive to ROS in C. elegans (Lee et al., 2010; Schulz et al., 2007; Yang and Hekimi, 2010). We next examined the involvement of PRODH in ROS production. Knockdown of prodh largely abrogated infection-induced ROS production (Figures 2B and S2B), implying that ROS reduction may, at least in part, be responsible for compromised survival in prodh RNAi-treated worms. Exogenous proline supplementation, on the other hand, further increased ROS levels, specifically during infection (Figures 2C and S2C). Prolineinduced ROS increases were important for pathogen resistance, as treatment with ROS inhibitor N-acetyl-L-cysteine (NAC) completely abolished the effects of proline on host survival upon infection (Figure S2D; Table S1). SKN-1, a central transcription factor involved in detoxification and innate immunity, is activated by ROS during infection (Hoeven et al., 2011). We therefore evaluated the possible function of PRODH on SKN-1 activation. By using SKN-1 transcriptional activity reporter gst-4p::GFP, we verified that P. aeruginosa

infection indeed activated the SKN-1 reporter gene (Figure S2E) (Hoeven et al., 2011; Papp et al., 2012). Congruent with a role for PRODH in ROS regulation, prodh RNAi significantly inhibited the expression of SKN-1 reporter gst-4p::GFP (Figure 2D), as well as the mRNA levels of other SKN-1 target genes (Figure S2F), suggesting that PRODH is a critical regulator of SKN-1 transcriptional activity during infection. On the other hand, exogenous proline supplementation further activated gst-4p::GFP (Figure 2E) as well as SKN-1 targets in infected worms (Figure S2G). More importantly, both host PRODH and ROS were required for proline-induced gst-4p::GFP expression (Figures 2E and S2H), indicating that the involvement of PRODH and ROS in prolineinduced SKN-1 targets activation. To further characterize SKN-1 activation, we examined SKN-1::GFP nuclear translocation (Hoeven et al., 2011; Papp et al., 2012). RNAi of prodh significantly inhibited SKN-1 nuclear accumulation upon infection (Figures 2F and S2I). However, we did not observe further increases in SKN-1 nuclear accumulation in proline-treated worms (Figure 2G), possibly due to saturated SKN-1 nuclear translocation, as previous reports showed that PA14 infection could induce SKN-1 nuclear accumulation as strong as the intense

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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 mm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as indicated. See also Figure S3 and Table S1.

SKN-1 activator paraquat (An and Blackwell, 2003; Hoeven et al., 2011). These data suggest that PRODH promotes ROS generation and SKN-1 activation in response to infection, which may confer host resistance to pathogen infection. P5CDH/ALH-6 Functions in an Opposite Way to PRODH in Regulating ROS Production and SKN-1 Activation To further explore the mechanism of proline catabolism-mediated immune responses, we evaluated the role for P5CDH (Figure 1A) in pathogen resistance. Our previous study has shown that mutation of alh-6, the worm ortholog of p5cdh, increases mitochondrial ROS levels in older gravid adults but not in larva or young adult worms (Pang and Curran, 2014). When we infected L4 stage worms with P. aeruginosa, alh-6 mutants showed reduced survival (Figure 3A; Table S1), indicating that alh-6 might have a similar function to that of prodh. However,

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alh-6 mutation increased ROS levels specifically in infected C. elegans (Figures 3B and S3A), which was opposite to prodh. Congruent with this finding, infected alh-6 mutants exhibited robust activation of SKN-1 target gst-4p::GFP in the intestine (Figure 3C), whereas uninfected mutants only showed moderate gst-4p::GFP activation specifically in the muscle (Figure 3C) as previously reported (Pang and Curran, 2014). RNAi targeting alh-6 phenocopied the activation of gst-4p::GFP upon infection (Figure S3B). skn-1 RNAi completely silenced the gst-4p::GFP activation in alh-6 mutants (Figure S3C). These data indicate that alh-6 mutation enhances infection-induced generation of ROS and transcriptional activation of SKN-1. We previously showed that gst-4p::GFP activation in muscle of alh-6 mutants was independent on ROS (Pang and Curran, 2014). Nevertheless, we found that treatment with ROS inhibitor NAC significantly silenced gst-4p::GFP hyper-activation in the

intestine of alh-6 mutants (Figure S3D), suggesting an essential role for ROS in activating SKN-1 targets in P. aeruginosa-infected alh-6 mutants. Consistently, PMK-1/p38, a kinase mediating ROS-induced SKN-1 activation under oxidative and pathogenic stresses (Hoeven et al., 2011; Inoue et al., 2005), was also required for gst-4p::GFP activation caused by alh-6 mutation (Figure S3E). These results may reflect the mechanistic differences in SKN-1 activation under different circumstances. Together, these data indicate that PRODH and P5CDH/ALH-6, two consecutive enzymes in proline catabolism, act oppositely to govern ROS production and subsequent SKN-1 activation during infection. It is widely known that excessive ROS may cause cell damages and therefore be deleterious to the host. We previously showed that alh-6 mutation caused excessive accumulation of ROS and ultimately accelerated animal aging in C. elegans (Pang and Curran, 2014). We proposed that, during infection, mutation of alh-6 compromised host survival due to ROS accumulation. As expected, we found that treatment with ROS inhibitor NAC completely abolished the survival reduction in infected alh-6 mutants (Figure 3D; Table S1). Intriguingly, alh-6 mutation even increased host resistance to infection under NAC treatment (Figure 3D). During the aging process, low levels of ROS extend lifespan, whereas high levels of ROS shorten lifespan, which is defined as mitohormesis (Schulz et al., 2007). Our findings imply that it might also be the case during pathogen infection that PRODH acts to generate ROS to fight against pathogen invasion, while alh-6 mutation causes excessive accumulation of ROS, which are harmful to the host. Metabolic Intermediate P5C Likely Accounts for ROS Homeostasis and SKN-1 Activation How could two consecutive enzymes of proline catabolism function in opposite ways to modulate ROS levels and SKN-1 activation? Mutation of alh-6 causes accumulation of its substrate P5C during infection when prodh is induced (Figures 1A and 1B). Also, we previously found that accumulation of P5C induced mitochondrial stress and excessive ROS generation during aging (Pang and Curran, 2014). Given the differential effects of PRODH and P5CDH/ALH-6 on the levels of metabolic intermediate P5C, we proposed that accumulation of P5C might be the key event for initiating the downstream ROS and SKN-1 responses. If so, knockdown of prodh should alleviate accumulation of P5C and abolish the effects of alh-6 mutation. As expected, RNAi of prodh largely inhibited the ROS elevation (Figure 3E) as well as gst-4p::GFP activation (Figure 3F) in infected alh-6 mutants. Remarkably, although prodh RNAi shortened the survival of wild-type worms, it reversed the survival reduction of alh-6 mutants (Figure 3G; Table S1). These results suggest that PRODHgenerated P5C may account for observed phenotypes in alh-6 mutants. Excessive P5C due to alh-6 mutation may be converted back to proline through P5C reductase (P5CR), which forms intensive proline-P5C cycle (Figure 1A). This enhanced proline-P5C cycle concomitantly increases the transfer of electrons to mitochondrial electrons transfer chain (mETC), which supports ROS generation (Miller et al., 2009). However, we found that p5cr RNAi treatment, which disrupted intensive proline-P5C cycle but not P5C accumu-

lation, did not affect ROS levels (Figure 3E), SKN-1 reporter expression (Figure 3F), or survival rate (Figure 3G; Table S1) of P. aeruginosa infected alh-6 mutants, ruling out a major role for enhanced proline-P5C cycle in these observed phenotypes. These results imply that, during infection, PRODH increases levels of P5C, which may act as an endogenous danger/toxic signal to induce ROS and SKN-1 activation. P5CDH/ALH-6, by catabolizing P5C to glutamate, inhibits excessive accumulation of P5C and therefore prevents ROS overproduction. Ce-Duox1/BLI-3 Is Required for ROS Production and SKN-1 Activation Induced by Modulation of Proline Catabolism Cell-membrane NADPH oxidase and mitochondria are two major sources of ROS production during infection. The crosstalk between NADPH oxidase and mitochondria in many pathophysiological processes has been reported, during which mitochondria may represent critical nodes in the activation of NADPH oxidase (Dikalov, 2011). Ce-Duox1/BLI-3 is a cell-membrane dual oxidase responsible for ROS production and SKN-1 activation in C. elegans. bli-3 RNAi-treated worms exhibit low levels of ROS and are hyper-sensitive to bacterial pathogen infection (Hoeven et al., 2011). Our finding that mitochondrial proline catabolism governs ROS homeostasis and SKN-1 activation during infection raises an interesting question: is there any crosstalk between mitochondrial proline catabolism and cell-membrane CeDuox1/BLI-3? Intriguingly, we found that bli-3 RNAi suppressed the ROS increases and SKN-1 reporter gst-4p::GFP expression in proline-treated animals (Figures 4A and 4B), suggesting an essential role for Ce-Duox1/BLI-3 in proline-induced ROS homeostasis. Supporting this idea, bli-3 RNAi also dramatically inhibited the ROS generation and gst-4p::GFP activation in alh-6 mutants (Figures 4C and 4D). Moreover, Ce-Duox1/BLI-3 was largely required for improved pathogen resistance promoted by proline supplementation (Figure 4E; Table S1). Mutation of alh-6 moderately reversed the hyper-susceptibility of bli-3 RNAi-treated animals to P. aeruginosa infection (Figure 4F; Table S1). These data implicate that mitochondrial proline catabolism may act through cell-membrane NAPDH oxidase in governing ROS homeostasis and SKN-1 activation (Figure S4). How does mitochondrial proline catabolism act upstream of NADPH oxidase to regulate ROS production? In mammalian endothelial cells, modulation of mitochondrial ROS levels could affect cell-membrane NADPH oxidase activity, as well as ROS production by NADPH oxidase, in response to angiotensin treatment (Dikalov, 2011; Dikalova et al., 2010). In addition, mitochondrial ROS signal was reported to stimulate NADPH oxidase activity and its production of ROS through a signaling cascade involving PI3K and Rac1 in human 293T cells (Lee et al., 2006). Given that studies have well established a role for proline catabolism in generation of mitochondrial ROS (Nishimura et al., 2012; Zarse et al., 2012), it is intriguing to postulate that, during infection, activation of PRODH promotes mitochondrial ROS production, which, in turn, acts as an initiating signal to activate NADPH oxidase (Ce-Duox1/BLI-3) to amplify the ROS pool (Figure S4). Further studies testing this model and identification of possible molecules linking mitochondria and NADPH oxidase would be of great interest.

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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 mm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as indicated. See also Figure S4 and Table S1.

Concluding Remarks In this study, we identify proline catabolism as a component of the C. elegans innate immune system. We reveal that, in response to infection, two proline catabolic enzymes, PRODH and P5CDH/ALH-6, act together to govern ROS homeostasis and SKN-1 activity. PRODH promotes ROS production and SKN-1 activation, likely through generation of metabolic interme-

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diate P5C. An interesting implication is that the rise of P5C may be recognized as an endogenous toxic/danger signal that initiates ROS and SKN-1-dependent detoxification response. P5CDH/ALH-6 catalyzes the conversion of P5C to glutamate, to prevent excessive elevation of P5C, and ultimately maintains ROS homeostasis. Disruption of the balance between PRODH and P5CDH/ALH-6, such as mutation of alh-6, leads to ROS

overproduction and therefore compromises host defense to infection (Figure S4). The regulation of ROS by proline catabolism is highly conserved throughout evolution (Lettieri Barbato et al., 2014; Liu et al., 2012b; Miller et al., 2009; Nomura and Takagi, 2004; Pang and Curran, 2014; Yoon et al., 2004; Zarse et al., 2012). More intriguingly, PRODH has been reported to contribute to pathogen defense in Arabidopsis thaliana (Cecchini et al., 2011; Qamar et al., 2015). Research in past years has revealed the mechanisms that animals and plants utilize to resist infection are surprisingly similar (Ausubel, 2005). Our finding that animals also utilize the proline catabolism pathway to defend against pathogen implicates an ancient and important role for this pathway in the host defense system. We propose that metazoan innate immunity could be governed by shared mechanisms. EXPERIMENTAL PROCEDURES C. elegans Growth Condition and Strains C. elegans were cultured using standard techniques at 20 C (Brenner, 1974). The following strains were used: wild-type N2 Bristol, SPC321[alh-6 (lax105)], CL2166[gst4-p::gfp], LD1[skn-1b/c::GFP], KU25[pmk-1 (km25)], and the HyPer expression strain jrIs1[Prpl-17::HyPer]. Double and triple mutants were generated by standard genetic techniques. Infection Assay Infection assay was performed as previously described (Tan et al., 1999). In brief, overnight-cultured P. aeruginosa strain PA14 bacteria were seeded on slow killing (SK) plates and incubated at 37 C for 24 hr followed by 25 C for 24 hr. For killing assay, L4 animals were added to those plates and incubated at 25 C for survival analysis. All survival data were repeated at least twice. Worms that died of vulva burst, bagging, or crawling off plates were censored. For proline and NAC treatment, proline and NAC were added to plates at a final concentration of 10 mM. Fluorescent Microscopy To analyze gst-4p::GFP expression, worms were infected by PA14 for 24 hr and paralyzed with 1 mM levamisole, and fluorescent microscopic images were taken after they were mounted on slides. The expression levels of GFP were scored as low, medium, and high. In brief, a dim green signal in the anterior or posterior of the intestine, a green signal throughout the intestine, and a bright signal throughout the intestine were categorized as low, medium, or high expression, respectively. To study SKN-1 nuclear localization, SKN-1B/ C::GFP worms were infected by PA14 for 6 hr and mounted on slides. The levels of GFP nuclear localization were scored as previously described (An and Blackwell, 2003). In brief, no nuclear GFP, GFP signal in the nucleus of anterior or posterior intestine cells, and nuclear GFP in all intestine cells were categorized as low, medium, or high expression, respectively. For P. aeruginosa load, worms were infected by PA14 GFP strain for 24 hr, mounted on slides and examined for GFP signal inside the intestine. Dim GFP signal throughout the intestine, bright GFP signal in the anterior or posterior of the intestine, and bright GFP throughout the intestine were scored as low, medium, or high expression, respectively. ROS Measurement ROS determined by HyPer ratio were measured as previously described (Back et al., 2012; Pang and Curran, 2014). In brief, worms of indicated genotypes and treatment were mounted on slides. The oxidized and reduced HyPer of individual worms were excited with GFP and CFP channels, respectively. Fluorescent density was calculated by using ImageJ software. The hydrogen peroxide levels were measured as the ratio of oxidized to reduced HyPer intensity. ROS determined by DCF-DA were measured as previously described (Lee et al., 2010), with modifications. In brief, about 600 worms of indicated genotypes and treatment were collected and washed twice in M9 to remove bacte-

ria. Worms were then homogenized in PBST (PBS with 0.1% Tween 20) and centrifuged at 15,000 rpm at 4 C. 50 mL supernatants were mixed with equal volume 100 mM DCF-DA prepared in PBS. Fluorescent intensity was measured at the excitation wavelength 488 nm and emission wavelength 535 nm after 1 hr incubation. The protein content in supernatant was determined by bicinchoninic acid (BCA) assay to normalize the fluorescent intensity. qRT-PCR qRT-PCR was performed as previously described (Pang et al., 2014). In brief, worms of indicated genotype and treatment were collected, washed in M9 buffer, and then homogenized in Trizol reagent (Life Technologies). RNA was extracted according to the manufacturer’s protocol. DNA contamination was digested with DNase I (Thermo Fisher Scientific), and subsequently RNA was reverse transcribed to cDNA by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qPCR was performed by using SYBR Green (Bio-Rad). The expression of ama-1 or pmp-3 was used to normalize samples. For measurement of prodh and alh-6, worms were cultured on PA14 plates for 24 hr. For measurement of SKN-1 targets, worms were collected 6 hr after treatment for RNA extraction. RNA Interference Treatment HT115 bacteria containing specific double-stranded RNA (dsRNA)-expression plasmids were seeded on nematode growth media (NGM) plates containing 5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG). RNAi was induced at room temperature for 24 hr. L1 worms were added to those plates to knock down indicated genes. For bli-3 RNAi, the bacterial strain was diluted at a ratio of 1:30 to the vector control, as previously described (Hoeven et al., 2011). Statistics Data were presented as mean ± SEM. Survival data were analyzed by using the log-rank (Mantel-Cox) test. The levels of fluorescent micrographs were analyzed by using chi-square and Fisher’s exact tests. p < 0.05 was considered significant. Other data were analyzed by using unpaired Student’s t test. SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.11.038. AUTHOR CONTRIBUTIONS H.T. and S.P. designed the study, performed the experiments, analyzed data, and wrote the manuscript. ACKNOWLEDGMENTS We thank Dr. Kun Zhu for providing the P. aeruginosa PA14 strain, Dr. Chenggang Zhou for providing the PA14 GFP strain, and Dr. B. Braeckman for providing the HyPer expression strain. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by the National Natural Science Foundation of China (grant no. 31571243 to S.P.) and the Fundamental Research Funds for the Central Universities (grant no. 106112015CDJRC291208 to H.T.). Received: March 1, 2016 Revised: September 28, 2016 Accepted: November 10, 2016 Published: December 13, 2016 REFERENCES An, J.H., and Blackwell, T.K. (2003). SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev. 17, 1882–1893. Ausubel, F.M. (2005). Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 6, 973–979.

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Back, P., De Vos, W.H., Depuydt, G.G., Matthijssens, F., Vanfleteren, J.R., and Braeckman, B.P. (2012). Exploring real-time in vivo redox biology of developing and aging Caenorhabditis elegans. Free Radic. Biol. Med. 52, 850–859. Bedard, K., and Krause, K.-H. (2007). The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology. Physiol. Rev. 87, 245–313. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Bronte, V., and Zanovello, P. (2005). Regulation of immune responses by L-arginine metabolism. Nat. Rev. Immunol. 5, 641–654. Bronte, V., Serafini, P., Mazzoni, A., Segal, D.M., and Zanovello, P. (2003). L-arginine metabolism in myeloid cells controls T-lymphocyte functions. Trends Immunol. 24, 302–306. Cecchini, N.M., Monteoliva, M.I., and Alvarez, M.E. (2011). Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiol. 155, 1947–1959.

Liu, W., Le, A., Hancock, C., Lane, A.N., Dang, C.V., Fan, T.W.-M., and Phang, J.M. (2012a). Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC. Proc. Natl. Acad. Sci. USA 109, 8983–8988. Liu, W., Glunde, K., Bhujwalla, Z.M., Raman, V., Sharma, A., and Phang, J.M. (2012b). Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments. Cancer Res. 72, 3677–3686. Miller, G., Honig, A., Stein, H., Suzuki, N., Mittler, R., and Zilberstein, A. (2009). Unraveling delta1-pyrroline-5-carboxylate-proline cycle in plants by uncoupled expression of proline oxidation enzymes. J. Biol. Chem. 284, 26482–26492. Nishimura, A., Nasuno, R., and Takagi, H. (2012). The proline metabolism intermediate D1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast. FEBS Lett. 586, 2411–2416. Nomura, M., and Takagi, H. (2004). Role of the yeast acetyltransferase Mpr1 in oxidative stress: Regulation of oxygen reactive species caused by a toxic proline catabolism intermediate. Proc. Natl. Acad. Sci. USA 101, 12616–12621.

Cha´vez, V., Mohri-Shiomi, A., and Garsin, D.A. (2009). Ce-Duox1/BLI-3 generates reactive oxygen species as a protective innate immune mechanism in Caenorhabditis elegans. Infect. Immun. 77, 4983–4989.

Pang, S., and Curran, S.P. (2014). Adaptive capacity to bacterial diet modulates aging in C. elegans. Cell Metab. 19, 221–231.

Dikalov, S. (2011). Cross talk between mitochondria and NADPH oxidases. Free Radic. Biol. Med. 51, 1289–1301.

Pang, S., Lynn, D.A., Lo, J.Y., Paek, J., and Curran, S.P. (2014). SKN-1 and Nrf2 couples proline catabolism with lipid metabolism during nutrient deprivation. Nat. Commun. 5, 5048.

Dikalova, A.E., Bikineyeva, A.T., Budzyn, K., Nazarewicz, R.R., McCann, L., Lewis, W., Harrison, D.G., and Dikalov, S.I. (2010). Therapeutic targeting of mitochondrial superoxide in hypertension. Circ. Res. 107, 106–116.

} ti, C. (2012). A role for SKN-1/Nrf in pathogen Papp, D., Csermely, P., and So resistance and immunosenescence in Caenorhabditis elegans. PLoS Pathog. 8, e1002673.

Ganeshan, K., and Chawla, A. (2014). Metabolic regulation of immune responses. Annu. Rev. Immunol. 32, 609–634. Grohmann, U., and Bronte, V. (2010). Control of immune response by amino acid metabolism. Immunol. Rev. 236, 243–264. Hoeven, Rv., McCallum, K.C., Cruz, M.R., and Garsin, D.A. (2011). Ce-Duox1/ BLI-3 generated reactive oxygen species trigger protective SKN-1 activity via p38 MAPK signaling during infection in C. elegans. PLoS Pathog. 7, e1002453. Inoue, H., Hisamoto, N., An, J.H., Oliveira, R.P., Nishida, E., Blackwell, T.K., and Matsumoto, K. (2005). The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Genes Dev. 19, 2278–2283. Knoefler, D., Thamsen, M., Koniczek, M., Niemuth, N.J., Diederich, A.-K., and Jakob, U. (2012). Quantitative in vivo redox sensors uncover oxidative stress as an early event in life. Mol. Cell 47, 767–776. Lee, S.B., Bae, I.H., Bae, Y.S., and Um, H.D. (2006). Link between mitochondria and NADPH oxidase 1 isozyme for the sustained production of reactive oxygen species and cell death. J. Biol. Chem. 281, 36228–36235. Lee, S.-J., Hwang, A.B., and Kenyon, C. (2010). Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity. Curr. Biol. 20, 2131–2136. Lettieri Barbato, D., Aquilano, K., Baldelli, S., Cannata, S.M., Bernardini, S., Rotilio, G., and Ciriolo, M.R. (2014). Proline oxidase-adipose triglyceride lipase pathway restrains adipose cell death and tissue inflammation. Cell Death Differ. 21, 113–123. Liu, Y., Borchert, G.L., Surazynski, A., Hu, C.-A., and Phang, J.M. (2006). Proline oxidase activates both intrinsic and extrinsic pathways for apoptosis: The role of ROS/superoxides, NFAT and MEK/ERK signaling. Oncogene 25, 5640– 5647. Liu, Y., Borchert, G.L., Donald, S.P., Diwan, B.A., Anver, M., and Phang, J.M. (2009). Proline oxidase functions as a mitochondrial tumor suppressor in human cancers. Cancer Res. 69, 6414–6422.

2844 Cell Reports 17, 2837–2844, December 13, 2016

Pearce, E.L., and Pearce, E.J. (2013). Metabolic pathways in immune cell activation and quiescence. Immunity 38, 633–643. Qamar, A., Mysore, K.S., and Senthil-Kumar, M. (2015). Role of proline and pyrroline-5-carboxylate metabolism in plant defense against invading pathogens. Front. Plant Sci. 6, 503. Schneider, D.S., and Ayres, J.S. (2008). Two ways to survive infection: What resistance and tolerance can teach us about treating infectious diseases. Nat. Rev. Immunol. 8, 889–895. Schulz, T.J., Zarse, K., Voigt, A., Urban, N., Birringer, M., and Ristow, M. (2007). Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab. 6, 280–293. Sena, L.A., and Chandel, N.S. (2012). Physiological roles of mitochondrial reactive oxygen species. Mol. Cell 48, 158–167. Tan, M.W., Rahme, L.G., Sternberg, J.A., Tompkins, R.G., and Ausubel, F.M. (1999). Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA 96, 2408–2413. Tannahill, G.M., Curtis, A.M., Adamik, J., Palsson-McDermott, E.M., McGettrick, A.F., Goel, G., Frezza, C., Bernard, N.J., Kelly, B., Foley, N.H., et al. (2013). Succinate is an inflammatory signal that induces IL-1b through HIF1a. Nature 496, 238–242. Yang, W., and Hekimi, S. (2010). A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol. 8, e1000556. Yoon, K.-A., Nakamura, Y., and Arakawa, H. (2004). Identification of ALDH4 as a p53-inducible gene and its protective role in cellular stresses. J. Hum. Genet. 49, 134–140. Zarse, K., Schmeisser, S., Groth, M., Priebe, S., Beuster, G., Kuhlow, D., Guthke, R., Platzer, M., Kahn, C.R., and Ristow, M. (2012). Impaired insulin/ IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal. Cell Metab. 15, 451–465.