Summary
Immunosenescence is considered an inevitable decline in immune function during aging. Here we show that genetic inhibition of the DAF-2/insulin/IGF-1 receptor drastically delays immunosenescence and rejuvenates immunity in C. elegans. We find that p38 mitogen-activated protein kinase 1 (PMK-1), a key determinant of immunosenescence, is dispensable for this rejuvenated immunity. Instead, we demonstrate that longevity-promoting DAF-16/FOXO and heat-shock transcription factor 1 (HSF-1) increase immunocompetence in old daf-2(-) animals. The upregulation of DAF-16/FOXO and HSF-1 decreases the expression of the zip-10/bZIP transcription factor, which in turn downregulates INS-7, an agonistic insulin-like peptide, resulting in further reduction of insulin/IGF-1 signaling (IIS). Thus, reduced IIS bypasses immunosenescence and rejuvenates immunity via the upregulation of anti-aging transcription factors that modulate an endocrine insulin-like peptide through a positive feedback mechanism. Because many functions of IIS are conserved across phyla, our study may lead to the development of strategies for human immune rejuvenation.
Introduction
An increase in mortality after infection is a key feature of aging. This age-dependent decline in immunity, i.e., immunosenescence, is nearly universal across phyla (Fulop et al., 2017; Goronzy and Weyand, 2013; Müller et al., 2013). Vertebrates display immunosenescence in adaptive immunity. In addition, old mice and humans have increased pro-inflammatory cytokine levels, termed “inflamm-aging”, because of the chronic activation of innate immune systems (Baylis et al., 2013; Franceschi and Campisi, 2014). However, the complexity of the immune systems and cell types in vertebrates impedes the dissection of the molecular mechanisms underlying immunosenescence.
Caenorhabditis elegans is an excellent model for research on aging and immunity, phenomena which are closely related with each other. Many long-lived C. elegans mutants display enhanced innate immunity at young age (Alper et al., 2010; Garsin et al., 2003; Yunger et al., 2017; Tiku et al., 2018). Aged C. elegans also display an increased susceptibility to pathogen infection, indicating that C. elegans can be used as a model of immunosenescence (Kurz and Tan, 2004; Laws et al., 2004). An age-dependent decline in PMK-1/p38 mitogen-activated protein kinase (MAPK) levels underlies C. elegans immunosenescence against the pathogenic bacteria Pseudomonas aeruginosa (PA14) (Youngman et al., 2011). Although these findings suggest the existence of a link between aging and immunity, the molecular mechanisms by which longevity affects immunosenescence remain largely unknown.
Insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) plays important roles in aging and immunity in various species, including C. elegans. In C. elegans, inhibition of IIS, for example, by mutations in daf-2/insulin/IGF-1 receptor or its agonist, ins-7, increases the lifespan and enhances resistance against pathogen-mediated death (Evans et al., 2008a; Evans et al., 2008b; Garsin et al., 2003; Kawli and Tan, 2008; Kenyon et al., 1993; Murphy et al., 2003). Reduced IIS activates several transcription factors, such as DAF-16/FOXO, heat-shock transcription factor 1 (HSF-1), and SKN-1/NRF, which upregulate the expression of various genes that contribute to longevity, stress resistance, and immunity (reviewed in Kenyon, 2010; Murphy and Hu, 2013; Riera et al., 2016). Despite extensive research on the roles of IIS in aging and immunity, its role in the regulation of immunosenescence remains unknown.
In this study, we investigated whether longevity interventions affected the rate of immunosenescence in C. elegans. Among the various longevity regimens tested here, genetic inhibition of daf-2/insulin/IGF-1 receptor substantially improved immunocompetence in old worms. We found that temporal inhibition of daf-2 starting in the middle age rejuvenated immunity in old adult animals. We then showed that delayed immunosenescence conferred by daf-2 mutations bypassed the functional requirement of PMK-1/p38 MAPK signaling, which is crucial for immunity. Instead, DAF-16/FOXO and HSF-1 increased immunocompetence in old daf-2 mutants by regulating the expression of various genes. In particular, DAF-16/FOXO and HSF-1 downregulated an agonistic insulin-like peptide, INS-7, via decreasing the expression of a bZIP transcription factor, ZIP-10, which in turn reduced IIS further and enhanced immunity at old age. Thus, daf-2 mutants appear to elude normal immunosenescence via a positive-feedback endocrine circuit that consists of the DAF-16/FOXO, HSF-1, and ZIP-10 transcription factors, together with INS-7, which is an agonist of the DAF-2/insulin/IGF-1 receptor.
Results
Genetic inhibition of the DAF-2/insulin/IGF-1 receptor improves immunity in aged worms
We wondered whether the rates of immunosenescence were proportionally affected by genetic mutations that promote longevity. Thus, we measured immunosenescence in various long-lived animals, including IIS-defective daf-2, sensory-defective osm-5, germline-deficient glp-1, dietary restriction mimetic eat-2, and mitochondrial respiration-impaired isp-1 mutants. Similar to the wild-type counterparts (Laws et al., 2004; Youngman et al., 2011), osm-5, glp-1, and eat-2 mutants exhibited an age-dependent increase in susceptibility to Pseudomonas aeruginosa (PA14) infection (Figures 1A-1E). Old isp-1 mutants displayed susceptibility to PA14 infection that was comparable to that of young animals; however, their overall survival rate was lower than that of wild-type worms (Figures 1A and 1F). Surprisingly, old (day 9) daf-2 mutants displayed substantially enhanced survival upon PA14 infection compared with young (day 1) daf-2 mutants and wild-type worms (Figures 1A and 1G). Consistent with daf-2 mutant data, old (day 9) daf-2(RNAi) worms also displayed enhanced PA14 resistance (Figure 1H), without defects in feeding rates (Figures 1I). Old (day 9) daf-2 mutant worms also survived longer than did young worms after infection with PAO1, another P. aeruginosa strain (Figures 1J and 1K). These data suggest that genetic inhibition of DAF-2 confers sustained immunocompetence against P. aeruginosa in old age, which was different from other longevity interventions.
We then determined the duration of the enhanced PA14 resistance conferred by daf-2 mutations, and found that daf-2 mutant or daf-2(RNAi) animals maintained the PA14 resistance up to day 15 of adulthood, at a level that was comparable to that of young (day 1) adult animals (Figures 1L and S1B). Wild-type animals display an age-dependent increase in the intestinal accumulation of PA14, which is a sign of immunosenescence (Youngman et al., 2011). Therefore, we measured the accumulation of PA14 in the intestinal lumens of daf-2 mutants and daf-2(RNAi) animals at different ages. We found that daf-2 mutants displayed substantially reduced PA14 levels in the intestine in both young (Evans et al., 2008a) and old animals (Figure 1M). In contrast, we observed an age-dependent increase in the intestinal accumulation of PA14 in daf-2 RNAi-treated animals (Figures 1N and S1C). As both old daf-2 mutants and old daf-2(RNAi) worms survived longer on PA14 than did young animals (Figures 1G and 1H), genetic inhibition of daf-2 appears to maintain immunocompetence in old age, at least in part, independently of PA14 intake and clearance.
Temporal inhibition of daf-2 specifically reverses immunosenescence
We next asked whether temporal genetic inhibition of daf-2 was sufficient to enhance immunocompetence against PA14 at old age. We treated wild-type animals with control RNAi or daf-2 RNAi starting at a middle age (day 4) and measured the survival at old age (day 8) (Figure 2A). Importantly, the temporal daf-2 RNAi-treated old (day 8) worms displayed enhanced PA14 resistance compared with middle-aged (day 4) worms (Figure 2B). In contrast, the same population of control RNAi-treated worms displayed a substantial decline in survival upon PA14 infection at day 8 adulthood (Figure 2B). These data suggest that temporal inhibition of daf-2 in wild-type worms during aging can rejuvenate immunity. In contrast to PA14 resistance, we found that the temporal daf-2 RNAi treatment from day 4 did not reverse the age-dependent decline in resistance against oxidative stress or heat stress (Figures 2C and 2D). Thus, temporal inhibition of daf-2 specifically enhances immunocompetence without increasing general stress resistance at old age.
Genetic inhibition of daf-2 in the intestine, hypodermis, muscle, and neurons decreases susceptibility to pathogenic bacteria in old worms
We then assessed in which tissues DAF-2 affected immunosenescence by performing tissue-specific RNAi experiments (Calixto et al., 2010; Qadota et al., 2007). First, we showed that intestine-, hypodermis-, muscle-, and neuron-specific daf-2 RNAi treatment significantly increased lifespan (Figures S2A-S2H), suggesting a role for reduced DAF-2 expression in life extension in these various tissues. We then found that intestine-, hypodermis-, muscle-, and neuron-specific daf-2 RNAi treatment significantly enhanced PA14 resistance at old age (day 9), whereas it did not increase survival upon PA14 infection at young age (day 1) (Figures 2E-2J, S3A-S3F). These data suggest that reduced DAF-2 expression in the intestine, muscle, hypodermis, and neurons collectively contributes to the enhancement of immunocompetence at old age.
daf-2 mutation bypasses the requirement of p38 MAPK signaling for enhanced immunity at old age
Next, we sought to identify downstream factors that mediate the delayed immunosenescence in daf-2 mutants. We first examined whether PMK-1/p38 MAPK, which is a major immune-regulatory factor and the critical determinant of immunosenescence in wild-type animals (Kim et al., 2002; Youngman et al., 2011), played a role in immunocompetence in old daf-2 mutants. Consistent with the results of a previous study (Troemel et al., 2006), the enhanced PA14 resistance observed in young daf-2 mutants was largely dependent on pmk-1 (Figure S4A). Unexpectedly, pmk-1 was only partially required for the enhanced immunity of old (day 9) daf-2(–) mutants (Figure S4B). Overall, old (day 9 or 15) daf-2(–); pmk-1(–) double mutants displayed a substantially increased survival upon PA14 infection compared with young (day 1) animals (Figure 3A). Similarly, old daf-2 mutants containing mutations in each of nsy-1/MAPKKK and sek-1/MAPKK, which act upstream of PMK-1 (reviewed in Irazoqui and Ausubel, 2010; Kim and Ewbank, 2018), survived longer on PA14 than young animals (Figures 3B, 3C, S4C-S4F). In addition, the levels of active phospho-PMK-1 proteins and the expression of pmk-1-regulated genes declined during aging in both wild-type and daf-2 mutant worms (Figures 3D and 3E), consistent with a previous report (Wu et al., 2019). These data suggest that daf-2 mutations bypass the functional requirement of MAPK-cascade components and act through other factors for maintaining immunity during aging.
DAF-16/FOXO and HSF-1 are required for delayed immunosenescence in daf-2 mutants
DAF-16/FOXO, HSF-1, and SKN-1/NRF are required for increased pathogen resistance in young daf-2 mutants (Garsin et al., 2003; Miyata et al., 2008; Papp et al., 2012; Singh and Aballay, 2006, 2009 and Figures S5A, S5C and S5E); however, it remains unknown whether these transcription factors played a role in daf-2(–)-mediated delay in immunosenescence. Importantly, we found that daf-16 mutations fully suppressed the PA14 resistance of daf-2 mutants at all ages tested and unveiled immunosenescence in daf-2 mutants (Figures 4A-4C, S5A and S5B). hsf-1 RNAi also largely suppressed the enhanced immunocompetence of old daf-2 mutants (Figures 4D-4F and S5D). In contrast, RNAi targeting skn-1 did not affect the enhanced immunocompetence of old daf-2 mutants (Figures 4G-4I and S5F). Together, these data suggest that DAF-16/FOXO and HSF-1 are responsible for drastically delayed immunosenescence in daf-2 mutants.
Age-associated gene expression changes are similar between wild-type and daf-2 mutant worms
Next, we investigated which transcriptional changes were associated with and responsible for the enhanced immunocompetence observed in aged daf-2 mutants. We conducted an mRNA sequencing (RNA seq) analysis using young (day 1) and old (day 9) daf-2(–) and wild-type animals. Hierarchical clustering indicated a clear separation of transcriptomes in accordance with ages and genotypes (Figure 5A), and a principal component analysis (PCA) revealed that the effect of age was larger than that of genotype (Figure S6A). Subsequently, we found that the age-dependent gene expression changes detected in wild-type worms displayed a positive correlation with those observed in daf-2(–) mutants (Figure 5B). We also showed that the mRNA levels of PMK-1 targets were decreased during aging in wild-type and daf-2(–) animals (Figure S6B), consistent with our Western blotting and qRT–PCR analysis (Figures 3D and 3E). In contrast, the mRNA levels of three selected DAF-16 targets, mtl-1, hsp-16.2, and sod-3, displayed age-dependent increases in wild-type worms, which tended to increase further in aged daf-2(–) mutants (Figures S6C-S6E). These data are consistent with our results showing the requirement of DAF-16/FOXO for the enhanced immunity observed in old daf-2 mutants.
Genetic inhibition of several genes whose expression is age-dependently altered enhances immunity in old age
We then asked which specific genes, whose expression was changed in an age-dependent manner, were responsible for the delayed immunosenescence of daf-2(–) mutants. Aged daf-2(–) mutants displayed increased immunity, which is the opposite of the impaired immunity observed in aged wild-type worms. Therefore, we focused our RNA seq analysis on genes that exhibited expression levels regulated in an opposite manner between wild-type and daf-2(–) worms during aging (Table S1). We found that the mRNA levels of 72 genes were increased with age in wild-type worms but were decreased with age in daf-2(–) animals (Figure 5B, red dots; fold change > 1.5; Benjamini and Hochberg (BH)-adjusted p value < 0.1 with 19,327 genes). Conversely, 14 genes exhibited expression levels that were decreased with age in wild-type worms and increased in daf-2(–) animals (Figure 5B, blue dots; fold change > 1.5; BH-adjusted p value < 0.1). A gene set enrichment analysis (GSEA) revealed that “immune/defense response” genes were significantly downregulated during aging in daf-2(–) worms but were marginally increased in wild-type animals (Figure 5C-5D, q value (BH-adjusted p value) < 0.1); thus, these genes may underlie the distinct immunity phenotypes observed between aged wild-type and aged daf-2(–) animals. We therefore performed immunosenescence assays using mutants that carried mutations in each of the 13 genes that were selected among the 72 candidate genes (Figure 5E), whose expression was specifically increased during wild-type aging and decreased during daf-2(–) aging. We found that the pmp-1/peroxisomal ABC transporter ABCD3, ins-7/an insulin-like peptide, zip-10/bZIP transcription factor, valv-1/Cysteine-Rich Intestinal Protein (CRIP), and lips-10/lipase loss-of-function mutants displayed enhanced immunocompetence at day 6 of adulthood compared with their wild-type counterparts (Figure 5E).
An age-dependent decrease in ins-7 expression contributes to enhanced immunocompetence in old daf-2 mutants
Interestingly, among the genes that displayed an opposite age-dependent change in expression between daf-2 mutants and wild-type animals, ins-7 was negatively regulated by both DAF-16/FOXO (Figure S6F) and HSF-1 (Figure S6G). The ins-7 gene encodes an agonist of DAF-2 that acts as a positive feedback regulator of IIS (Murphy et al., 2007; Murphy et al., 2003). Using qRT–PCR assays, we confirmed our RNA seq results by showing that the ins-7 mRNA level was increased with age in wild-type worms, but not in daf-2 mutants (Figures 6A and 6B). Consistent with previous reports (Lee et al., 2009; Murphy et al., 2007; Murphy et al., 2003), ins-7 expression was increased by daf-16(–) (Figure 6C). In addition, hsf-1 RNAi robustly elevated the level of the ins-7 mRNA (Figure 6D). These data suggest that ins-7 is downregulated by DAF-16/FOXO and HSF-1 in daf-2 mutants but is upregulated during aging in wild-type worms. We then asked whether ins-7 contributed to immunocompetence in old animals. Consistent with previous reports (Evans et al., 2008b; Kawli and Tan, 2008), ins-7 mutation enhanced pathogen resistance at young age (day 1 adulthood) (Figures 6E and S7A). Notably, mutations in ins-7 enhanced immunocompetence in adults at days 6 and 9 (Figures 6F-6G, S7B and S7C), indicating the immune-suppressive roles of INS-7 during aging. Taken together, these data suggest that the upregulation of DAF-16/FOXO and HSF-1 decreases ins-7 expression and enhances immunocompetence further in old daf-2 mutants.
The ZIP-10/bZIP transcription factor acts as a positive regulator of ins-7 for inducing immunosenescence
Next, we focused our functional characterization on the role of the ZIP-10/bZIP transcription factor in the regulation of immunosenescence with respect to INS-7 for the following reasons. First, a previous report indicated that ZIP-10 is a pathogen-responsive gene (Shapira et al., 2006). Second, the changes in the mRNA expression of zip-10 were similar to those of ins-7 in aged wild-type versus daf-2 mutants (Figure 5B). Third, both ins-7 and zip-10 expression was increased by daf-16 mutations and hsf-1 RNAi in daf-2 mutants (Figures 6C, 6D, 7A and 7B). Therefore, we wondered whether ZIP-10 acted together with INS-7 to regulate immunosenescence. We first showed that zip-10 mutations suppressed the age-dependent increase in ins-7p::GFP expression (Figures 7C and 7D), suggesting that ZIP-10 induces ins-7. We then conducted a converse analysis using isy-1(–) mutants, which exhibit zip-10 upregulation (Jiang et al., 2018). We found that ins-7 was among the nine genes upregulated by both isy-1(–) and aging specifically in wild-type worms (Figure 7E). Therefore, ZIP-10 appears to upregulate ins-7 during aging in wild-type worms, but not in daf-2 mutants. We also showed that zip-10 mutation increased the PA14 resistance at old age, but not at young age (Figures 7F and 7G). Conversely, isy-1 mutations significantly reduced immune competence in old worms, but not in young worms (Figures 7H and 7I). Altogether these data suggest that daf-2 mutations enhance immunocompetence in old age by downregulating ZIP-10; this decreases INS-7 expression, which normally elicits immunosenescence in wild-type worms.
Discussion
Reduced IIS appears to reverse immunosenescence via a positive endocrine feedback loop
Despite the close association between aging and immunity, whether and how longevity interventions affect immunosenescence remains largely unexplored. Here, we determined a relationship between longevity and immunosenescence using C. elegans. We found that reduction of IIS greatly delayed and even rejuvenated immunity at some point during aging. This retarded immunosenescence conferred by inhibition of the DAF-2/insulin/IGF-1 receptor was dependent on DAF-16/FOXO and HSF-1, whereas PMK-1/p38 MAPK signaling and SKN-1/NRF were dispensable. These data suggest that the activation of DAF-16/FOXO and/or HSF-1 is sufficient to reverse immunosenescence, whereas the activation of p38 MAPK is not a limiting factor for delaying immunosenescence. We also showed that daf-2 mutations suppressed the age-dependent upregulation of the ZIP-10/bZIP transcription factor, which led to the downregulation of INS-7 and boosted immunity. Because INS-7 is an agonist of DAF-2 (Murphy et al., 2003), this in turn appears to act as a positive feedback circuit to enhance immunity further in old daf-2 mutants. We propose that daf-2 mutations can uncouple aging and the age-dependent declines in immune function by increasing the activities of DAF-16/FOXO and HSF-1, but not those of p38 MAPK signaling, via a positive-feedback mechanism during aging.
Pharyngeal pumping does not appear to be a major cause of delayed immunosenescence in daf-2(–) animals
PA14 colonizes the C. elegans intestinal lumen, and exposure to PA14 ceases pharyngeal pumping in C. elegans (Tan et al., 1999). We wondered whether the reduction of pharyngeal pumping with age caused increased pathogen resistance in daf-2(–) mutants. Although animals with reduced daf-2 functions displayed age-dependent decreases in pharyngeal pumping after infection with the pathogen (Figure 1I and S1A), several lines of evidence indicated that pharyngeal pumping is not the main cause of the increased immunocompetence observed in old daf-2(–) mutants. First, aged wild-type worms displayed a reduced pumping rate but showed increased susceptibility to PA14. Second, eat-2(–) mutants with defective pharyngeal pumping displayed an age-dependent reduction in pathogen resistance. Third, daf-2 RNAi treatment significantly improved pharyngeal pumping and survival upon PA14 infection compared with wild-type worms. Fourth, daf-2 RNAi treatment in post-reproductive, middle-aged, wild-type animals was sufficient to enhance immunocompetence in old age. Overall, these data suggest that delayed immunosenescence can be achieved without defects in feeding rates.
The intestine and neurons are important tissues for daf-2(–)-mediated delayed immunosenescence, enhanced immunity, and longevity
Previous reports have shown that the expression of DAF-16/FOXO and HSF-1 in various tissues contributes to the longevity of daf-2 mutants (Douglas et al., 2015; Libina et al., 2003; Morley and Morimoto, 2004; Son et al., 2018; Zhang et al., 2013). In addition, tissue-specific transgenic rescue analyses indicate that the loss of daf-2 function in neurons is required for longevity (Wolkow et al., 2000). However, the specific tissues in which reduced DAF-2 function is sufficient for extending lifespan remain unknown. Here, we demonstrated that reducing daf-2 in all tissues tested (intestine, hypodermis, muscle, and neurons) contributed to longevity (Figure S2). Importantly, we also showed that intestine-, hypodermis-, muscle-, and neuron-specific daf-2 RNAi enhanced immunocompetence at old age (day 9 adulthood). Our results are consistent with those of previous reports, which showed that the intestine, hypodermis, and neurons are tissues important for the regulation of immunity (Irazoqui and Ausubel, 2010; Kim and Ewbank, 2018). The intestine and neurons were shown to be the main tissues that express ins-7 (Chen et al., 2013; Matsunaga et al., 2016; Murphy et al., 2007; Murphy et al., 2003). Thus, our data raise the possibility that the expression and subsequent secretion of INS-7 in the intestine and neurons act in a cell-non-autonomous manner on DAF-2 in multiple tissues, thus modulating immunosenescence and longevity.
Downregulation of the INS-7/insulin-like peptide may delay immunosenescence
The secretion of INS-7 by the nervous system negatively regulates immunity-related factors in intestinal cells via the DAF-16/FOXO transcription factor (Evans et al., 2008b; Kawli and Tan, 2008; Murphy et al., 2003). Our data suggest that the age-dependent increase in ins-7 expression is associated with immunosenescence in C. elegans. Consistent with previous reports (Murphy et al., 2007; Murphy et al., 2003), we showed that ins-7 expression increased with age in wild-type worms. Notably, we found that ins-7 expression decreased slightly with age in daf-2(–) mutants. Moreover, we demonstrated that genetic depletion of ins-7 enhanced pathogen resistance in old worms. Overall, these data suggest that inhibition of INS-7 during aging is an effective way of reversing immunity in old age.
IIS may play roles in mammalian immunosenescence
Previous studies have shown that IIS regulates innate immunity in various other species. FOXO, which is a conserved transcription factor that acts downstream of IIS, promotes innate immunity directly in Drosophila and mammalian cells (Becker et al., 2010). Reduced IGF-1 signaling promotes the renewal of hematopoietic stem cells (HSCs), which are crucial for the generation of cells important for proper immune functions (Cheng et al., 2014). In addition, inhibition of target of rapamycin, which intersects IIS, increases vaccination response in elderly people and enhances the self-renewal of HSCs in mice (Chen et al., 2009; Mannick et al., 2014). In the current study, we identified systemic positive feedback mechanisms by which IIS rejuvenates immunity in C. elegans. Because the functions of IIS in various physiological processes, including aging and immunity, are conserved across phyla, the findings reported here for C. elegans may lead to the development of therapeutic strategies against immunosenescence in humans.
Materials and Methods
C. elegans strains and maintenance
All strains were maintained as previously described (Stiernagle, 2006). Some strains were obtained from Caenorhabditis Genetics Center, which is funded by the NIH National Center for Resources (p40 OD010440), or National Bio-Resource Project (NBRP). Strains used in this study are as follows: N2 wild-type, CF1041 daf-2(e1370) III, CF2553 osm-5(p813) X, CF1903 glp-1(e2141) III, CF2172 isp-1(qm150) IV, IJ173 eat-2(ad1116) II obtained by outcrossing DA1116 four times to Lee-laboratory N2, WM27 rde-1(ne219) V, NR222 rde-1(ne219) V; kzls9[lin-26p::nls::gfp, lin-26p::rde-1; rol-6D], NR350 rde-1(ne219) V; kzls20[hlh-1p::rde-1; sur-5p::nls::gfp], VP303 rde-1(ne219) V; kbls7[nhx-2p::rde-1; rol-6D], NL3321 sid-1(pk3321) V, TU3401 sid-1(pk3321) V; uIs69[myo-2p::mCherry; unc119p::sid-1], IJ130 pmk-1(km25) IV obtained by outcrossing KU25 four times to Lee-laboratory N2, IJ1147 daf-2(e1370) III; pmk-1(km25) IV, IJ128 sek-1(km4) X obtained by outcrossing KU4 four times to Lee-laboratory N2, IJ1299 daf-2(e1370) III; sek-1(km4) X, IJ131 nsy-1(ok593) II obtained by outcrossing VC390 four times to Lee-laboratory N2, IJ1298 nsy-1(ok593) II; daf-2(e1370) III, CF1042 daf-16(mu86) I, CF1085 daf-16(mu86) I; daf-2(e1370) III, IJ1733 ins-7(tm1907) obtained by outcrossing FX01907 four times to Lee-laboratory N2, IJ1010 ins-7(tm2001) obtained by outcrossing QL126 four times to Lee-laboratory N2, ZC1436 yxIs13[ins-7p::gfp(70ng/ul); unc-122p::dsred] a gift from Yun Zhang laboratory, IJ1873 daf-2(e1370) III; yxIs13[ins-7p::gfp(70ng/ul); unc-122p::dsred]. IJ1871 zip-10(ok3462) obtained by outcrossing RB2499 five times to Lee-laboratory N2, IJ1919 isy-1(dma50) obtained by outcrossing DMS175, a gift from Dengke Ma laboratory. RB908 pmp-1(ok773), RB1262 cpr-1(ok1344), RB1415 capt-3(ok1612), RB2452 acs-14(ok3391), RB2473 cpr-4(ok3413), RB2499 zip-10(ok3462), RB686 fmo-3(ok354), FX06726 valv-1(tm6726), FX07601 lips-10(tm7601), FX01907 ins-7(tm1907), CF2078 sgk-1(ok538), VC2249 dod-19(ok2679), and VC2200 grl-22(ok2704). All strains except daf-2(e1370); sek-1(km4) were maintained on NGM plates seeded with Escherichia coli OP50 strain as a food source at 20°C. daf-2(e1370); sek-1(km4) was maintained and synchronized at 15°C because of a semi-sterility phenotype at 20°C.
Immunosenescence assays
Immunosenescence assays were performed as described previously (Youngman et al., 2011) with minor modifications. Gravid adult worms were allowed to lay eggs overnight to synchronize progeny on nematode growth media (NGM) plates seeded with E. coli OP50 or RNAi bacteria. When the progeny reached L4 or young adult stage (day 0), the worms were transferred onto NGM plates treated with 50 μM of 5-fluoro-2’-deoxyuridine (FUdR, Sigma, St. Louis, MO, USA). Worms were maintained on these plates at 20°C until they were used for experiments at day 1, 3, 6, 9, 12, or 15 of adulthood. For the immunosenescence assays using temperature-sensitive glp-1(e2141) mutants, N2 and glp-1(e2141) worms were synchronized by allowing gravid adults to lay eggs on E. coli OP50-seeded plates for 12 hrs at 20°C and the progeny developed at 25°C until the worms reached L4 stage. For the immunosenescence assays using daf-2(e1370); sek-1(km4) mutants, which were semi-sterile at 20°C, worms were first cultured at 15°C for obtaining a sufficient number of animals and transferred to 20°C for allowing to age, when they reached adulthood. Worms were transferred every other day until N2 worms stopped producing progeny. Contaminated or E. coli-depleted plates over time were discarded. PA14 standard slow killing assays were performed as previously described (Jeong et al., 2017). Briefly, 5 μl of overnight P. aeruginosa liquid culture was seeded onto the center of 0.35% peptone NGM solid media. Plates were incubated at 37°C for 24 hrs and subsequently at room temperature for over 8 hrs before use. Different ages of worms were simultaneously infected with PA14 on plates containing 50 μM of FUdR at 25°C. After infection (time = 0 on survival curves), worms were scored more than once a day by gently prodding with a platinum wire to determine life or death. Statistical analysis of survival data was performed by using OASIS (http://sbi.postech.ac.kr/oasis), which calculates p values using log-rank (Mantel-Cox method) test (Yang et al., 2011). For Figure 5E, worms were synchronized on OP50-seeded NGM plates and transferred to 50 μM FUdR-treated NGM plates when they became young (day 0) adults. After 6 days, worms were infected with PA14. After 48 hrs of infection, live and dead worms were counted.
Temporal daf-2 RNAi experiments
Wild-type worms were synchronized on control RNAi-treated plates and transferred onto new control RNAi plates when they became day 0 adults. Day 4 adult worms were transferred to control or daf-2 RNAi bacteria-containing plates, respectively, and maintained at 20°C for 4 days. Day 8 adult worms were moved to plates with PA14 (Figure 2A), or OP50 plates with 7.5 mM t-BOOH (tert-Butyl hydroperoxide solution, Sigma, St Louis, MO, USA) (Figure 2B), or placed in a 35°C incubator (Figure 2C) and live worms were scored over time. OP50-seeded NGM plates were used for the heat and oxidative stress resistance assays with temporal daf-2 RNAi-treated animals to avoid continuous RNAi induction and to match the RNAi conditions used for PA14 survival assays. Worms that reached young (day 0) adulthood were treated with 50 μM FUdR until finishing the survival assays. Statistical analysis of survival data was performed by using OASIS (http://sbi.postech.ac.kr/oasis), which calculates p values using log-rank (Mantel-Cox method) test (Yang et al., 2011).
RNAi induction
Each RNAi clone-expressing HT115 bacteria were cultured in Luria broth (LB) containing 50 μg/ml ampicillin (USB, Santa Clara, CA, USA) overnight at 37°C. One hundred microliter of RNAi culture was seeded onto NGM containing 50 μg/ml ampicillin, and incubated overnight at 37°C. One mM isopropylthiogalactoside (IPTG, Gold biotechnology, St Louis, MO, USA) was added and incubated at room temperature for over 24 hrs before use.
Lifespan assays
Lifespan assays were conducted as previously described (Son et al., 2017). Young (day 0) adult worms that were grown on control RNAi and daf-2 RNAi plates were transferred onto 5 μM FUdR-containing control and daf-2 RNAi plates, respectively. The animals that did not respond to gentle touch were considered as dead. The animals that crawled off the plates, missing, burrowed, or displayed vulval protrusion or internal hatching were censored but included in the subsequent statistical analysis. Statistical analysis of lifespan data was performed by using OASIS (http://sbi.postech.ac.kr/oasis), which calculates p values using log-rank (Mantel-Cox method) test (Yang et al., 2011).
Intestinal PA14-GFP accumulation assays
Intestinal PA14-GFP accumlation assays were performed as previously described (Jeong et al., 2017; Kim et al., 2002), with minor modifications. Day 1 and day 9 adult N2 and daf-2(e1370) worms were infected with PA14 that expresses GFP (PA14-GFP) for 24 hrs and subsequently imaged using a Zeiss Axio Scope A1 compound microscope (Zeiss Corporation, Jena, Germany). For control and daf-2 RNAi treatments, worms were infected with PA14-GFP for 100 hrs.
Measurements of pharyngeal pumping
Pharyngeal pumping rates were determined as previously described (Cao and Aballay, 2016). PA14 bacterial lawns were prepared as described above. Aged animals were transferred onto PA14 lawns, and incubated at 25°C. The number of contractions of the terminal bulb was counted for 15 sec. The pumping rates for ten adults were measured for each condition.
Microscopy
Worms were prepared at indicated ages and transferred on a 2% agarose pad with 100 mM sodium azide (DAEJUNG, Siheung, South Korea) or 2 mM levamisole (tetramisole; Sigma). The images of worms were captured by using an AxioCam HRc CCD digital camera (Zeiss Corporation, Jena, Germany) with a Zeiss Axio Scope A1 compound microscope (Zeiss Corporation, Jena, Germany). ImageJ (https://imagej.nih.gov/ij/) was used for the quantification of fluorescent images.
RNA extraction and quantitative RT-PCR
RNA extraction and qRT-PCR were performed as previously described with minor modifications (Son et al., 2017). When worms that were fed with OP50 or RNAi bacteria reached L4 or young adult stage (day 0), 50 μM FUdR was supplemented. After 1 or 9 days, worms were harvested by washing with M9 buffer 2-3 times to remove residual bacteria. Plates with bactrial or fungal contamination, or with starved worms because of depletion of foods, were discarded. If OP50 was almost depleted, soaked freeze-dried OP50 (LabTIE, Rosmalen, Netherlands) was added onto the plates to prevent starvation. Total RNA in the animals was extracted using RNAiso plus (Takara, Seta, Kyoto, Japan) and cDNA was synthesized by ImProm-II™ Reverse Transcriptase kit (Promega, Madison, WI, USA). qRT-PCR was performed by using StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR green dye (Applied Biosystems, Foster City, CA, USA). Comparative CT method was used for quantitative PCR analysis. Two technical repeats were averaged per biological sample and analyzed for all data sets. For all biological data sets, ama-1 (an RNA polymerase II large subunit), and/or pmp-3 (a peroximal membrane protein) were used as reference genes for normalization (Hoogewijs et al., 2008; Taki and Zhang, 2013)). Sequences of primers used for qRT-PCR are as follows.
ama-1: 5’-TGGAACTCTGGAGTCACACC and 5’-CATCCTCCTTCATTGAACGG;
pmp-3: 5’-GTTCCCGTGTTC ATCACTCAT and 5’-ACACCGTCGAGA AGCTGTAGA;
T24B8.5: 5’-TGTTAGACAATGCCATGATGAA and 5’-ATTGGCTGTGCAGTTGTACC;
C17H12.8: 5’-GAACAATAGTGTCAAGCCGATCTGC and 5’-TTCTGAATGATGAATGCATGTTTAC;
mtl-1: 5’-GACTGCTGAAATTAAGAAATCATG and 5’-GTCTCCACTGCATTCACATTTGTC;
hsp-16.1/11: 5’-CTCATGAGAGATATGGCTCAG and 5’-CATTGTTAACAATCTCAGAAG;
ins-7: 5’-GTGGAAGAAGAATACATTCG and 5’-CTTCAGTATTCGATTCGCATG.
zip-10: 5’-TCGAGATGCTCTTCAACTG and 5’-CTAACTGCTTGCCGGAG
mRNA library preparation for RNA seq analysis and data acquisition
Synchronized N2 and daf-2(e1370) animals were treated with 50 μM FUdR when worms reached L4 or young adult stage on OP50. At indicated ages (day 1 and day 9 adulthoods), worms were harvested with M9 buffer, and washed 2-3 times, and frozen in liquid nitrogen. Three biological repeats of the samples were used for all conditions. For RNA seq analysis using hsf-1 RNAi, daf-2(e1370) mutants were synchronized on control or hsf-1 RNAi bacterial plates. Day 1 adult daf-2(e1370) animals on control or hsf-1 RNAi plates were harvested with M9 buffer. Two biological repeats of samples were used. RNA extraction for RNA seq samples was carried out as described above (see RNA extraction section). TruSeq (unstranded) mRNA libraries (Illumina, CA, USA) were constructed and paired-end sequencing of Illumina HiSeq2500 was performed by Macrogen Inc. (Seoul, South Korea).
RNA seq analysis
Sequencing pairs were aligned to the C. elegans genome WBcel235 (ce11) and Ensembl transcriptome (release 97) by using STAR (v.2.7.0e). Aligned pairs on genes or transcripts were quantified by using RSEM (v.1.3.1). Detailed parameters of alignment and quantification were calculated by following guidelines of ENCODE long RNA seq processing pipeline. Differentially expressed genes (fold change > 1.5 and adjusted p value < 0.1) were identified by using DESeq2 (v.1.22.2). Wald test p values are adjusted for multiple testing using the procedure of BH. Gene ontology (GO) terms generally changed in a certain comparison were identified by GSEA (v.4.0.1) using read counts of all expressed genes with a parameter ‘Permutation type: gene_set’. Terms with q value (BH-adjusted p value) less than 0.1 were considered as significantly changed. R (v.3.6.1, http://www.r-project.org) was used for plotting results. Raw data and processed data are available in Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo,GSE137861).
Western blot assays
Synchronized N2, daf-2(e1370) and pmk-1(km25) animals were treated with 50 μM FUdR when worms reached L4 or young adult stage (day 0) on OP50. At indicated ages (day 1, 9, 12 and 15), worms were harvested with M9 buffer, and washed 2-3 times, and frozen in liquid nitrogen with 2X SDS sample buffer. Western blot assays were conducted as described previously (Jeong et al., 2017).
Supplemental Figure Legends
Figure S1. Pharyngeal pumping rates, age-dependent survival on PA14 and the accumulation of PA14 of animals with genetic inhibition of daf-2.(A) Pharyngeal pumping rates of day 1, 6 and 9 adult wild-type (WT) and daf-2(e1370) [daf-2(-)] animals on PA14. (B) Normalized mean survival of day 1, 9, 15, and 30 adult daf-2 RNAi-treated worms on PA14. Error bars represent the SEM from five independent survival assay data for day 1 and day 9 old worms, and two independent survival assay data for day 15 and 30 old worms. (C) daf-2 RNAi-treated worms displayed an age-dependent increase in PA14-GFP accumulation. Shown are representative images of worms that were pre-treated with daf-2 RNAi, after PA14-GFP exposure for 100 hrs. * indicates PA14-GFP (scale bar: 100 μm). See Figure 1N for Semi-quantification of PA14-GFP levels shown in panel C.
Figure S2. Genetic inhibition of daf-2 in the intestine, hypodermis and neurons increases lifespan. (A-C) daf-2 RNAi increased the lifespan of wild-type (WT, N2) (A), but did not influence the lifespan of RNAi-defective rde-1(ne219) [rde-1(-)] (B) animals. (C-E) Intestine-(C), hypodermis-(D), and muscle-(E) specific daf-2 RNAi significantly increased lifespan. (F-G) daf-2 RNAi did not increase the lifespan of systemic RNAi-defective sid-1(pk3321) [sid-1(-)] mutants (F), while increasing the lifespan of neuron-specific RNAi strain (G). rde-1(-) mutant animals that expressed rde-1 under the control of an intestine-specific nhx-2 promoter, a hypodermis-specific lin-26 promoter, or a muscle-specific hlh-1 promoter, and sid-1(-) mutant animals that expressed sid-1 driven by a neuron-specific unc-119 promoter were used for tissue-specific RNAi experiments. (H) Normalized mean lifespan. Error bars represent the standard error of mean (SEM) (two-tailed Student’s t-test., *p<0.05, **p<0.01). Two independent lifespan assays were performed. See Table S4 for additional repeats and statistics.
Figure S3. Inhibition of daf-2 in single tissues did not increase the PA14 resistance of young (day 1) adult worms. (A-B) daf-2 RNAi significantly increased the survival of young (day 1) wild-type (WT) animals on PA14 (A), but not that of RNAi-defective rde-1(-) mutants (B). (C-F) Intestine- (C), hypodermis- (D), muscle- (E) or neuron- (F) specific daf-2 RNAi did not increase the survival of young (day 1) adult worms. RNAi-defective rde-1(ne219) [rde-1(-)] mutant animals that expressed rde-1 under the control of an intestine-specific nhx-2 promoter, a hypodermis-specific lin-26 promoter or a muscle-specific hlh-1 promoter were used for tissue-specific RNAi experiments. Systemic RNAi-defective sid-1(pk3321) [sid-1(-)] mutants were used as a control for neuron-specific RNAi experiments. Two independent experiments were performed and pooled for analysis. See Table S4 for additional repeats and statistics.
Figure S4. PMK-1 cascade signaling is required for enhanced pathogen resistance of daf-2 mutants at young age. (A-B) pmk-1(km25) [pmk-1(-)] mutations fully suppressed the enhanced PA14 resistance of young (day 1) adult daf-2(e1370) [daf-2(-)] animals (A), but partly suppressed the PA14 resistance of old (day 9) worms (B). (C-D) nsy-1(ok593) [nsy-1(-)] mutations fully suppressed the enhanced PA14 resistance of daf-2(e1370) [daf-2(-)] animals at day 1 adulthood (C), but partially suppressed that at day 9 adulthood (D). (E-F) sek-1(km4) [sek-1(-)] mutations decreased the PA14 resistance of wild-type (WT) and daf-2(-) worms at day 1 (E) and day 9 (F) adulthoods. See Table S5 for additional repeats and statistics.
Figure S5. DAF-16/FOXO and HSF-1 are required for the enhanced pathogen resistance of daf-2 mutants at old age. (A-B) daf-16(mu86) [daf-16(-)] mutation fully suppressed the enhanced PA14 resistance of daf-2(e1370) [daf-2(-)] animals at day 1 (A) and day 9 (B) adulthoods. (C-D) hsf-1(RNAi) partly reduced the pathogen resistance of day 1 adult daf-2(-) animals (C), but fully suppressed that of day 9 adult daf-2(-) worms (D). (E-F) skn-1 RNAi slightly decreased the PA14 resistance of wild-type (WT) and daf-2(-) worms at day 1 adulthood (E), but did not affect that at day 9 adulthood (F). See Table S5 for additional repeats and statistics.
Figure S6. Several DAF-16/FOXO targets are further upregulated in aged daf-2 mutants. (A) Principal component analysis (PCA) of RNA seq data sets for wild-type (WT) and daf-2(e1370) [daf-2(-)] worms at day 1 and day 9 adulthoods (n = 3). (B) Representative PMK-1-regulated genes (T24B8.5, C17H12.5 and K08D8.5) were downregulated with age in WT and daf-2(-) worms. Error bars represent SEM (two-tailed Student’s t-test, n = 3) from RNA seq data. (C) mRNA levels of three selected DAF-16 targets, mtl-1, hsp-16.2 and sod-3, in WT and daf-2(-) worms at day 1 and day 9 adulthoods in our RNA seq data (n = 3). (D-E) qRT–PCR analysis data confirmed that the mRNA levels of mtl-1 (D) and hsp-16.1/11 (E) were further upregulated with age in daf-2(-) compared to that in WT animals (n= 4). ama-1 was used as a normalization control. (F-G) Venn diagram represents a significant overlap between genes whose expression levels were oppositely regulated during aging between WT and daf-2(-) animals (labeled as “DEGs with age”) and genes whose expression was negatively regulated by DAF-16/FOXO (Riedel et al., 2013) (F) and by HSF-1 (this study) (G) in daf-2(-) mutant backgrounds. RF: representation factor. p values were calculated with hypergeometric probability test.
Figure S7. ins-7 mutations enhanced immunocompetence at young and old ages. ins-7(tm2001) [ins-7(-)2] mutation increased the survival of day 1 (A), day 6 (B), and day 9 (C) adult worms on PA14. See Table S5 for additional repeats and statistical analysis.
Supplemental Table Legends
Table S1. Genes whose expression was oppositely regulated with age between wild-type and daf-2(-) animals. (A) Genes whose expression was age-dependently upregulated in wild-type but downregulated in daf-2(-) animals. (B) Genes whose expression was age-dependently downregulated in wild-type but upregulated in daf-2(-) animals.
Table S2. Statistical analysis and additional repeats of immunosenescence assays with longevity mutants, or RNAi (related to Figure 1).
Table S3. Statistical analysis and additional repeats of stress resistance assays with and without temporaldaf-2 RNAi treatment (related to Figures 2).
Table S4. Statistical analysis and additional repeats of immunosenescence or lifespan assays using tissue-specific RNAi strains (related to Figure 2 and Supplemental Figures S2 and S3).
Table S5. Statistical analysis and additional repeats of immunosenescence assays (related to Figures 3 through 7 and Supplemental Figures S4 through S7).
Acknowledgments
We thank Drs. Dennis H. Kim, You-Hee Cho, Emily Troemel, Yun Zhang, Dengke Ma and the Caenorhabditis Genetics Center, for providing some bacteria and C. elegans strains. We also thank all Lee laboratory members and Dr. Kyuhyung Kim for help and discussion. This study is supported by the Korean Government (MSIT) through the National Research Foundation of Korea (NRF) (NRF-2016R1E1A1A01941152 and NRF-2019R1A3B2067745) to S-J.V.L. C.T.M. is the Director of the Glenn Center for Aging Research at Princeton University.