Nuclear Hormone Receptor Regulation of MicroRNAs Controls Innate Immune Responses in C. elegans

Nuclear hormone receptors respond to small molecules such as retinoids or steroids and regulate development. Signaling in the conserved p38/PMK-1 MAP kinase pathway regulates innate immunity. In this study, we show that the Caenorhabditis elegans nuclear receptor DAF-12 negatively regulates the defense against pathogens via the downstream let-7 family of microRNAs, which directly target SKN-1, a gene downstream of PMK-1. These findings identify nuclear hormone receptors as components of innate immunity that crosstalk with the p38/PMK-1 MAP kinase pathway.


Introduction
Innate immunity is an evolutionarily conserved response to pathogens and forms the first line of defense for most organisms. When infected by pathogens, the nematode Caenorhabditis elegans mounts a rapid innate immune response and produces an array of anti-microbial genes, similar to other organisms throughout the animal kingdom [1], [2]. Several conserved signaling pathways that function in the perception of and defense against bacterial pathogens have been identified in C. elegans. These pathways include the NSY-1/PMK-1 MAP kinase signaling pathway, the DAF-2/DAF-16 insulin/insulin-like growth factor (IGF)-1 like signaling pathway, the DBL-1/transforming growth factor-b (TGF-b) signaling pathway and the BAR-1 b-catenin signaling pathway [1]- [4]. Although many conserved innate immune components have been identified in C. elegans using genetic and biochemical approaches, extensive characterization of the signaling networks that regulate the host response and outcome of infections is warranted.
Nuclear hormone receptors (NRs) are a class of transcription factors that are regulated by small lipophilic hormones. In all, 284 NRs have been identified in C. elegans, and approximately 20 of them have been genetically analyzed [5]. The dauer formation abnormal gene daf-12, a well-characterized nuclear hormone receptor, and the orphan receptors nhr-8 and nhr-48 are the conserved homologs of the mammalian vitamin D receptor and liver-X receptor [6], [7]. DAF-12 regulates developmental progression and arrest in response to environmental cues [6], [8]. In favorable conditions, the activation of TGF-b and insulin/ IGF-1 signaling cascades results in the production of the DAF-12 steroidal ligands, dafachronic acids (DAs). DAs are synthesized from cholesterol via a multi-step pathway involving the daf-36 Rieske-like oxygenase and the daf-9 cytochrome P450 enzyme, which promote a rapid progression through four larval stages (L1 to L4) to reproductive adults [9]- [14]. In unfavorable environments, DAs expression is suppressed, and DAF-12, without its ligand, binds to the co-repressor DIN-1, resulting in an arrest at a stress-resistant, long-lived alternative third larval stage, called the dauer diapauses (L3d) [15]. In addition, DAF-12 regulates the normal lifespan of worms and the longevity of germline-ablated animals [16]- [21]. However, the role of DAF-12 in the immune regulation of C. elegans remains unknown.
MicroRNAs (miRNAs) are small non-coding RNA molecules that repress target gene expression by base-pairing with partially complementary sequences in the 39-untranslated regions (39-UTR) of target mRNAs [22], [23]. MiRNAs influence molecular signaling pathways and regulate many biological processes, including immune function [24]. Originally discovered in C. elegans, lethal-7 (let-7) miRNA is conserved across species in both sequence and temporal expression [25], [26]. In C. elegans, the let-7 miRNA homologs mir-48, mir-84 and mir-241 (together referred to as let-7s) regulate developmental timing and promote cellular differentiation pathways [27], [28]. The human let-7-related miRNAs also have anti-proliferative functions, and the downregulation of let-7 levels is associated with a variety of cancers, such as lung, breast and colon cancer [27], [28]. DAF-12 and its steroidal ligands activate the expression of let-7s, which downregulate the heterochronic gene hbl-1, thus integrating environmental signals and developmental progression [29], [30]. However, the functional role of let-7 family of miRNAs in the innate immune responses to pathogens is largely unknown. Hence, we sought to investigate whether DAF-12 and the let-7 family of miRNAs play a role in the regulation of the innate immune responses to bacterial infection in C. elegans.

DAF-12 regulates pathogenic defense
We used an RNAi feeding method to search for the host components that influence the response of C. elegans to infection with Pseudomonas aeruginosa strain PA14, which is a human opportunistic pathogen that can also infect and kill C. elegans. Using 399 RNAi clones targeting transcription factors, we identified 17 transcriptional factors that affect the survival of worms on the P. aeruginosa lawns (Table S1). Among these candidates, treatment with daf-12 RNAi improved either the resistance of C. elegans to P. aeruginosa infection or its survival on an avirulent E. coli lawn (Fig. 1A, Fig. S1A). Transgenic daf-12(dhls26) worms containing daf-12::GFP were more susceptible to P. aeruginosa (Fig. S1B). DAF-12, along with NHR-8 and NHR-48, is a conserved homolog of the mammalian vitamin D/liver X receptor (LXR) in C. elegans [6], [7]. However, inhibition of nhr-8 and nhr-48 increased pathogenic susceptibility to P. aeruginosa infection (Fig. S2A), suggesting that nhr-8 and nhr-48 have roles opposite to that of daf-12 in innate immune regulation.
To further investigate the role of DAF-12 in the immune response to bacterial infection, we examined the survival rate and lifespan of daf-12 alleles that have been previously identified on the basis of development and aging [6], [8]. A daf-12 null mutant daf-12(rh61rh411) that contained two nonsense mutations affecting both DNA binding domain (DBD) and ligand binding domain (LBD) [6] and was more resistant to P. aeruginosa, had a shortened lifespan compared to wild-type N2 animals ( Fig. 1B and 1C). The daf-12(sa156) mutant containing a C121Y mutation in the zinc finger of DBD [6], which may interrupt the DNA binding activity of DAF-12, displayed a normal lifespan but increased resistance to P. aeruginosa infection ( Fig. 1B and 1C). In contrast, the two other two mutants, daf-12(m20), which has a nonsense mutation affecting DBD [6], and daf-12(m25), containing a M562I mutation in LBD [6], exhibited extended lifespans and normal pathogenic resistance to P. aeruginosa infection ( Fig. S3A and 3B). These results not only identify DAF-12 as a negative regulator of innate immune responses to the infection of P. aeruginosa but also suggest a cross-talk between developmental progression and host defense.
We have also examined whether DAF-12 is involved in C. elegans host defense to other different pathogens, and found that inhibition of daf-12 greatly increased the resistance of daf-12(sa156) mutants to Staphyloccocus aureus infection (Fig. S2B). We next performed transmission electron microscopy analysis to examine gut cells of wild-type worms or daf-12(sa156) worms fed P. aeruginosa or E. coli. When fed E. coli, both of the wild-type N2 and daf-12(sa156) worms display normal intestinal ultrastructure, whereas when infected by P. aeruginosa, the daf-12(sa156) worms exhibited less severely damaged gut cells and more intact microvilli than in the wild-type worms (Fig. 1D). To determine the cellular localization of DAF-12, we utilized previously generated transgenic daf-12(dhls26) worms containing daf-12::GFP [7] and showed a significant accumulation of DAF-12 in the nuclei of neurons and intestinal cells when worms were fed E. coli. However, when infected with P. aeruginosa, DAF-12 expression was not affected (Fig. S4C), but the associated GFP signal was diffusely distributed throughout both neuronal and intestinal cells ( Fig. S4A and S4B), suggesting that the P. aeruginosa infection suppresses nuclear localization of DAF-12 and promotes its translocation to the cytoplasm.

DAF-12 regulates antimicrobial genes expression
We then examined the effect of P. aeruginosa infection on the expression of eight selected anti-microbial genes that are regulated by the NSY-1/PMK-1 pathway or the insulin/IGF-1-like pathway [31]. We found that in the daf-12(rh61rh411) and daf-12 RNAitreated worms infected with P. aeruginosa, expression levels of five of the eight anti-microbial genes were significantly higher compared to the wild-type control ( Fig. 2A). To further confirm the quantitative RT-PCR results, we treated the dod-22::gfp or F55G11.7::gfp transgenic worms with daf-12 RNAi, fed them E. coli or P. aeruginosa, and then subjected them to confocal image analysis. Treatment with daf-12 RNAi greatly increased the expression of dod-22::GFP and F55G11.7::GFP at both basal E. coli levels and in P. aeruginosa-induced levels (Fig. 2B, Fig. S5A and S5B).
Dafachronic acids (DAs) regulate pathogenic defense via DAF-12 DAF-12 is known to control C. elegans response to its environment. Under favorable conditions, the stimulation of the insulin/IGF-1 andTGF-b pathways leads to the production of sterol-derived dafachronic acids (DAs). D 4 -DA and D 7 -DA bind to DAF-12, leading to developmental progression [10], [11]. The substitution of dietary cholesterol with D 7 -DAs reduced the resistance of wild-type worms, but not daf-12(sa156) worms, to P. aeruginosa infection (Fig. 3A). An increased dose of D 7 -DA did not lead to further increases in pathogenic susceptibility of the wildtype N2 worm to P. aeruginosa infection (Fig. S6). DAs are derivatives of dietary cholesterols that are synthesized via several pathways involving the cytochrome P-450 DAF-9 and the SAMdependent methyltransferase STRM-1 [12], [13], [32]. Inhibition of DAF-9 expression by RNAi feeding increased the resistance of the worm to P. aeruginosa infection (Fig. 3B). In unfavorable environments, the downregulation of the insulin/IGF-1 and TGFb pathways suppresses DA production, and without its ligand, DAF-12 associates with the co-repressor DIN-1 to promote dauer

Author Summary
When infected by the Pseudomonas aeruginosa, the nematode Caenorhabditis elegans invokes an innate immune response that protects the worm from pathogenic attack. The appropriate level of immune response in C. elegans requires the accurate regulation of multiple signal pathways, especially signals of repression, which attenuate the expression of pathogen-responsive genes. In the current study, we identified the nuclear hormone receptor DAF-12 and its downstream let-7 family of microRNAs, mir-84 and mir-241, are required for the regulation of C. elegans innate immunity against P. aeruginosa infection. Dafachronic acids, as DAF-12 ligands, can dramatically suppress the resistance of C. elegans to P. aeruginosa infection. Inhibition of the conserved PMK-1/p38 MAP kinase pathway can markedly attenuate the promoted resistance of daf-12 and let-7 family of microRNAs mutants to P. aureginosa infection. However, neither daf-12 nor let-7 family of microRNAs affect the activation of PMK-1/p38. Moreover, our data also reveals the role of SKN-1 in integrating the signals from the PMK-1/p38 MAPK and DAF-12-let-7s pathways to mediate the C. elegans innate immune response. RNAi-treated worms exposed to P. aeruginosa (PA) relative to control RNAi-treated worms (P,0.0001). (B) P. aeruginosa killing assay of wild-type N2, daf-12(sa156) (P,0.001) and daf-12 (rh61rh411) (P,0.001) worms. (C) Lifespan curve of N2, daf-12(sa156) (P = 0.4118) and daf-12(rh61rh411) (P = 0.0019) worms. (D) Transmission electron micrographs (TEM) of a gut section from wild-type N2 and daf-12(sa156) worms fed E. coli or P. aeruginosa for 48 hours. Red arrows indicate microvillis. (Scale bars 1000 nm) All data shown above are representative of at least three independent experiments (n$50 adult nematodes per strain). doi:10.1371/journal.ppat.1003545.g001  programs [15]. Thus, we next examined the role of DIN-1, a corepressor of DAF-12, in the immune response of C. elegans. The inhibition of din-1 did not affect the survival of wild-type worms on a P. aeruginosa lawn, but moderately attenuated the extended pathogenic resistance of daf-12 RNAi worms (Fig. S7A), suggesting that DAF-12 regulating the immune response of C. elegans may be partially dependent on din-1.
Inhibition of DAF-12-mediated immunity by NSY-1/PMK-1 Several conserved signaling pathways, including the NSY-1/ PMK-1 pathway and the insulin/IGF-1-like pathway, are involved in the pathogenic defense of C. elegans [2]. The loss of function of the insulin receptor DAF-2 activates the downstream target DAF-16, which triggers the expression of anti-microbial genes in response to pathogenic infection [3], [33]. However, daf-16 RNAi had no effect on the prolonged survival of daf-12(sa156) worms infected with P. aeruginosa (Fig. S7B). We then tested whether the NSY-1/PMK-1 pathway is involved in the enhanced resistance of daf-12 mutants to P. aeruginosa. Either inhibition of nsy-1 by RNAi or mutation of pmk-1 attenuated the enhanced pathogenic resistance of daf-12(sa156) worms or daf-12 RNAi-treated worms, respectively ( Fig. 4A and 4B), suggesting that DAF-12 may target the PMK-1 pathway to regulate the C. elegans immune response against P. aeruginosa infection. However, daf-12 RNAi did not markedly change the P. aeruginosa-stimulated phosphorylation of PMK-1 (Fig. 4C), suggesting that DAF-12 might act upstream or parallel to PMK-1 to suppress the PMK-1/p38 MAPK pathway.
MicroRNAs let-7s regulate both DAF-12 and NSY-1/PMK-1-mediated pathogenic defense MicroRNAs are approximately 20-to 22-nucleotide-long RNA molecules that bind to the 39 untranslated region (39UTR) of target messenger RNAs (mRNAs) and that decrease their expression [34], [35]. DAF-12 activates the expression of the let-7 miRNA homologs mir-84 and mir-241 (referred to as let-7s) to control developmental progression [29], [30]. To test whether mir-84 or mir-241 play a role in pathogenic defense, we infected the strains mir-84(n4037) and mir-241(n4316) with P. aeruginosa. Both mir-84(n4037) and mir-241(n4316) worms were more resistant to P. aeruginosa infection than the wild type (Fig. 5A). Likewise, both mir-84(n4037) and mir-241(n4316) worms had slightly longer lifespans than wild-type animals (Fig. 5B). We then employed the quantitative real-time PCR method to detect miRNA expression and found that P. aeruginosa infection of wild-type worms induced higher levels of mir-84 and mir-241 compared to E. coli. However, the daf-12 mutation markedly reduced the expression of both mir-84 and mir-241 (Fig. 5C). Confocal microscopic imaging of mir-84p::gfp also indicated that the mir-84 expression was highly upregulated in P. aeruginosa-infected wild-type worms, but not in the daf-12(rh61rh411) worms (Fig. 5D, Fig. S5C). To further determine the role of let-7s miRNAs in C. elegans innate immunity, we tested the function of mir-48, another let-7 relative, in P. aeruginosa infection and found that the mir-48(n4097) mutant exhibited decreased resistance to P. aeruginosa (Fig. S8A), suggesting that the let-7s miRNAs may target different regulators of C. elegans innate immunity. Quantitative real-time PCR results showed that the expression of daf-12-tergeted antimicrobial genes was also upregulated in let-7s miRNAs mutants (Fig. S9A), suggesting that these genes are also targeted by let-7s miRNAs. We then fed the daf-12 and let-7s mutants a GFP-tagged P. aeruginosa PA-14 strain and examined the bacterial burdens in worm intestines by confocal microscopy. We found there were significantly fewer accumulated bacteria in daf-12(sa156) and mir-241(n4316) worm intestines (Fig. S10A and S10B), suggesting that the inhibition of daf-12 and mir-241 may suppress bacterial accumulation through antimicrobial gene expression. The nsy-1 RNAi also counteracted the pathogenic defense of mir-84 mutant worms, and mutations of mir-84 or mir-241 did not affect the phosphorylation of PMK-1, suggesting that the let-7s miRNAs function downstream of DAF-12 to suppress the PMK-1/p38 MAPK signaling pathway (Fig. 5E, Fig. S8B).

SKN-1 is a direct functional target of let-7s
The finger protein hbl-1 is one target of miRNAs let-7s, and the expression of hbl-1 is regulated by daf-12 and let-7s [29]. The inhibition of hbl-1 by RNAi reduced the pathogenic resistance, but not the lifespan of C. elegans (Fig. S11A and S11B). To identify other target genes of let-7 family of miRNAs, we performed a bioinformatics analysis, determining that skn-1 is a potential target (Fig. 6A). To determine whether let-7s miRNAs could bind to the 39-UTR of the skn-1 mRNA and suppress it, we fused the 39-UTR region of the C. elegans skn-1 mRNA to the 39-end of a luciferase reporter gene and co-transfected it with synthesized dsRNAs mimicking let-7s miRNAs (let-7s mimics) into HEK293T cells. In contrast to the luciferase activity in the 39-UTR seed region mutants (skn-1 39-UTR (mut)), which could not bind with and respond to let-7s miRNAs, the luciferase activity of the skn-1 39-UTR decreased by approximately 30% in response to mir-48 mimics or mir-84 mimics and by approximately 10% in response to mir-241 mimics (Fig. 6B). Western blot results also showed that the SKN-1 protein expression could be upregulated by inhibition of daf-12, mir-84 and mir-241 (Fig. 6C, Fig. S11C). These results suggested that skn-1 is a target of mir-84 and mir-241.

Discussion
We have identified DAF-12 as a novel negative regulator of innate immune signaling pathways in C. elegans. DAF-12, along with NHR-8 and NHR-48, is a conserved homolog of the mammalian vitamin D/liver X receptor (LXR) in C. elegans [6], [7]. The functions of vitamin D and LXR in mammalian innate immunity have been extensively investigated [40], [41]. In a variety of human innate immune cell types (i.e., macrophages and monocytes), vitamin D stimulates antibacterial activity by increasing the expression of antimicrobial genes and by promoting autophagic mechanisms. Furthermore, vitamin D insufficiency, which is a global health issue, may increase the risk of many infectious diseases [40]. Whereas daf-12 negatively regulates the innate immunity of C. elegans, a mutation of either of nhr-8 or nhr-48 impairs the C. elegans host defense (Fig. S2A), suggesting that the regulatory role of nuclear hormone receptors may depend on their ligands and their target genes.
Hormone binding to nuclear hormone receptors regulates the C. elegans reproductive life cycle and entry into dauer diapauses [42]. We have shown that the sterol-derived dafachronic acids (DAs), the ligand of DAF-12, negatively regulate the pathogenic resistance of C. elegans in a DAF-12-dependent manner. In response to pathogenic infection, DAF-12 translocated from the nucleus into the cytoplasm. Although the regulation of DAF-12 translocation is not fully understood, the binding of the DAs and their co-factors is hypothesized to lead to the translocation of DAF-12. DAF-9, which synthesizes DAs, has also been shown to regulate C. elegans antibacterial activity, suggesting that the sterol hormones might be the key sensors of pathogenic infection and important regulators of pathogenic defense.
DAF-12 is known to activate let-7s miRNAs and thus regulate the developmental progression through downstream target hlb-1 [29], [30]. In this study, we showed that a P. aeruginosa infection induces the intestinal expression of mir-84 and mir-241, which is consistent with Kudlow et al.'s earlier findings that multiple miRNAs accumulate in the intestinal miRISCs upon infection [43] and that DAF-12-mediated immunity is dependent on the activation of its downstream miRNA let-7s. Although our understanding of the role of miRNAs in the molecular signaling pathways of the immune response is rapidly expanding [24], to the best of our knowledge, this is the first evidence of the involvement of miRNAs in the innate immune regulation in C. elegans. We have also observed that more DAF-12 accumulate in the nuclei of neurons or intestinal cells when worms were fed E. coli but in a diffuse distribution in P. aeruginosa-infected worms. However, there may be still enough DAF-12 present in the nuclei of the intestine cell, which would be responsible for the induction of let-7s miRNAs.
Furthermore, we have demonstrated that SKN-1 is a direct target of let-7s miRNAs. SKN-1 accumulate in the nuclei of intestinal cells of worms infected with P. aeruginosa, but not E. coli, which is consistent with Papp et al. and Haeven et al.'s reports that exposure to P. aeruginosa leads to SKN-1 accumulation in intestinal nuclei [38], [39]. Their data have also shown that PA14 infection triggers the transcriptional activation of gcs-1 and gst-4, two downstream target gene of SKN-1. However, data in our experiments suggested that infection of P. aeruginosa may induce more let-7s miRNAs and thus downregulate the production of SKN-1. One possible explanation is that even if the SKN-1 production is downregulated during the infection, the activity of SKN-1 is more dependent on its protein modification rather than its quantity. Regulation of SKN-1 at both the level of its activity and quantity precisely modulate the innate immune response to microbial infection.. Furthermore, we found that the inhibition of skn-1 by RNAi markedly reduced DAF-12/let-7s-mediated pathogenic defense. These findings provide evidence that nuclear hormone receptors control let-7s miRNAs regulation of the C. elegans innate immunity, suggesting that DAF-12 may couple developmental progression and the response to pathogenic infection in order to coordinate appropriate immune responses.
The oxidative stress response is an evolutionally conserved response to reactive oxygen species (ROS), which are produced by mitochondrial respiration, toxins and pathogen virulence factors [44]. SKN-1 is required for the proper response of C. elegans to oxidative stress, which is mediated by the NSY-1/PMK-1 and DAF-2 insulin-like signaling pathways [36], [37]. Our present findings demonstrate an essential role of SKN-1 in the pathogenic resistance of C. elegans, an observation that is consistent with two other independent studies [38], [39]. Thus, SKN-1 appears to integrate longevity, stress resistance and pathogenic resistance. Although the molecular pathway by which SKN-1 regulates the innate response to pathogens remains unclear, an SKN-1-mediated oxidative stress response could potentially protect the worms from the peroxidation damage caused by ROS during pathogenic infection. Further investigation of the common downstream target of various SKN-1 actions is required to elucidate the role of SKN-1 in the pathogenic resistance of C. elegans.

Nematode methods
The C. elegans strains used in this study are listed in Table S2. All of the strains were maintained at 20uC using standard methods unless otherwise noted.
Lifespan and P. aeruginosa killing assay Lifespan and P. aeruginosa killing assays were conducted at least three times, as previously described [46]. A P value less than or equal to 0.05 was considered statistically significant. Statistical analysis of lifespan and P. aeruginosa killing assay is shown in Table  S3, S4, S6 and S7.

Screening of a transcription factor RNAi library
RNAi of candidate transcription factors in N2 worms was carried out using standard bacterial feeding methods. For all feeding assays, worms were exposed to RNAi bacteria from the time of hatching. Synchronized young adult animals were transferred to P. aeruginosa lawns supplemented with 50 mg/ml 5fluorodeoxyuridine (FUDR, Sigma). P. aeruginosa killing assays were performed as described above.

Confocal microscopy assay
Worms were washed from their plates with M9, anaesthetized with M9 containing 0.1% NaN 3 , fixed in the 2% soft agar and subjected to confocal imaging assay. Images were captured using Leica TCS SP5.

Transmission electron microscopy assay
Wild-type N2 and daf-12(sa156) young adults were fed P. aeruginosa or E. coli for 48 hours. Worms were rinsed from plates with M9 buffer, and anaesthetized in 8% alcohol in M9. Fixation and Sectioning was performed with a conventional two-steps method as described in Worm Method. Photographs were captured using HITACHI H-7650.

Dafachronic acids assay
Wild-type N2 and daf-12(sa156) young adults were removed from plates and bathed with D4-DAs, D7-DAs or cholesterol (400 nM) in M9 8 hours before killing assay. The killing assay was performed on P. aeruginosa plates supplemented with D 4 -DAs, D 7 -DAs or cholesterol (400 nM).

Luciferase assay
A 0.5-kb region of the skn-1 39 UTR containing the predicted miRNA let-7s binding sites was cloned into the psi-CHECK2 to obtain the skn-1 39 UTR-luc construct. The skn-1 39 UTR (mut)-luc construct was obtained from skn-1 39UTR construct by mutating the complementary sequence of let-7s' seed region(TACCTCA to TAGGTGA). Constructs were co-transfected with synthesized dsRNAs mimicking the let-7s miRNAs to HEK293T cells and the luciferase assay was performed using the dual-luciferase reporter assay system (Promega).

Quantitative real-time PCR of antimicrobial peptide expression
Synchronized C. elegans animals were treated essentially as described above for the killing assays except for the omission of FUDR. Infected samples were compared to control samples fed on the same medium with E. coli OP50-1. Total RNA was extracted as described [31] and reverse transcribed using the ReverTra Ace Q-PCR RT kit (Toyobo). cDNA was subjected to qRT-PCR analysis as described [31]. The primer sequences are listed in Table S5. All values were normalized to act-1. One-tailed t-tests were performed with GraphPad Prism4. A P value less than or equal to 0.05 was considered significant.

Quantitative real-time PCR for microRNA let-7s
Synchronized worms were collected in TRIzol (Invitrogen) and treated as described [47]. The miRNeasy Mini kit (QIAGEN) and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) were used for total RNA and cDNA preparation, respectively. qRT-PCR was performed with Power SYBR Green master mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Sno-RNA U18 was used as an internal control. The primer sequences were gifted from Prof. Adam Antebi from the Max Planck Institute for Biology of Ageing. One-tailed t-tests were performed with GraphPad Prism4. A P value less than or equal to 0.05 was considered significant.