Stress resistance and lifespan enhanced by downregulation of antimicrobial peptide genes in the Imd pathway

Imd downregulation in flies developed at 25°C enhances stress resistance and extends lifespan with reduced AMP expressions

To investigate how stress resistance in flies is influenced by the developmental temperatures, we compared the RNA expression levels between 2-day-old male flies developed at 18°C and 25°C by microarray analyses (Fig. 1). We first filtered out non-expressed genes that received both of Affymetrix Absent calls in the gene expression of 18°C and 25°C development and, next, removed highly variable genes for which the standard error was bigger than 1.32-fold from three independent experiments. The final 62% of the genes (11,768) among total gene probes (18,952) were considered to have statistically reliable expressions in the 2-day-old male flies developed at 18°C and 25°C (Fig. 1B). With a cut-off value of ± 1.5-fold changes [14-16], we found that 0.9% of the genes (176/18,952) are differentially expressed between the flies developed at 18°C and 25°C (Fig. 1B). The gene ontology analyses of these 176 genes revealed a possible link of those genes to the stress resistance and aging process, such as defense responses, extracellular proteins, and the polysaccharide metabolic process (Fig. 1C). Among the defense response genes, antimicrobial peptide (AMP) genes in the Imd pathway [9] were specifically downregulated with the development at a lower temperature 18°C (Fig. 1D). Imd AMP genes, DptB and AttA, were still downregulated even after 30 days of age at 25°C (Fig. 1E) when the higher stress resistance was sustained (Fig. 1A).

To test if the downregulation of imd (0.76-fold) and AMP genes contributed to higher stress resistance in the flies developed at 18°C (Fig. 1A), we reduced the expression of the imd gene with the hypomorphic imd mutants and compared the stress resistance between the wild-type and imd mutant flies, both developed at 25°C. The imdP mutant, in which a P-element is inserted into the imd promoter, reduced the imd expression (heterozygote imdP/+: 0.88-fold; homozygote imdP: 0.45-fold in Fig. 2A) and then decreased the expression of downstream AMP genes (Fig. 2A). Dependent on the reduced levels of imd and AMP expressions, the stress resistances were enhanced (Fig. 2B-C) with a higher SOD2 (MnSOD) activity (33.8% increase). Interestingly, lifespan of imdP male flies were extended up to 24.8% (Fig. 2D) and these longer lifespans were shown in both male and female flies (Fig. 2E). Previous studies have suggested that attaining higher resistance against stresses may extend the lifespan of fruit flies [17-19]. It was shown that several long-lived mutant flies are more resistant to stresses such as starvation, oxidation, and heat than wild-type flies [5], indicating that lifespan and stress resistance often correlate positively with each other [20]. When we measured the nutrients of fly bodies, only fat (triacylglycerol) content increased ~40% in both hetero and homozygous imdP flies (Fig. 2F), which is commonly observed in long-lived mutant animals including C. elegans, flies, and mice [7,8,21]. These nutrient storages are often interpreted as an adaptation mechanism for increased energy demands during a longer lifespan and against stress resistance [6,22].

Mild downregulation of the Imd pathway increases stress resistance and fat content in adult flies developed at 25°C

The imd1 mutant is known to be a stronger hypomorphic mutant than imdP in the immune response to E. coli infection [23]. Although the expression change of the imd gene was not shown due to the missense mutation of imd1 [24], the AMP genes in the Imd pathway were downregulated in the imd1 flies (Table 1). The heterozygous (imd1/+) flies exhibited enhanced stress resistances, higher MnSOD activity, longer lifespans, and increased fat content (Table 1), as shown in the imdP mutants (Fig. 2). However, in imd1 homozygous mutants, the stress resistance was not significantly changed under oxidation or was highly decreased when subjected to heat stress (Table 1). Another imdNP mutant, in which a P-element is inserted into the coding region of the imd gene, also showed the different phenotypes between the heterozygous and homozygous imdNP flies (Fig. S1). A possible explanation is that the JNK/basket pathway, which is activated by the Imd protein and then is subsequently responsible for upregulation of the stress response genes [10,11], is less active due to the severe loss of imd function. The JNK downstream genes, Thor and GstD1, are upregulated for stress tolerance [25]. As expected, their expressions were increased during oxidative stress (24 hours) of the wild-type flies (Fig. 3A). In contrast, AMP genes (DptB and AttC) were downregulated (Fig. 3A), which suggests that reducing Imd AMP expressions may be beneficial for stress tolerance. Compared to the wild-type flies, both imdP and imd1 mutant flies show more reduced DptB and AttC expressions after 24 hours of oxidative stress (Fig. 3B). However, the imd1 flies also showed significantly reduced expressions of Thor and GstD1 (Fig. 3B). Then, we found that the imdP flies were able to survive longer than the wild-type and imd1 flies under the oxidative stress (Fig. 3C), probably due to reduced AMP expressions, but still sustained expressions of Thor and GstD1.

AMP downregulations of the Imd pathway directly enhance stress resistance and extend lifespan

Next, to investigate if the expression changes of Imd-driven AMP genes are directly related to stress resistance and longevity, we tested AttC-P mutant flies in which AttC expression is completely removed without any change in the expression of another longevity-related gene, loco [26] (Fig. 4A). The AttC-P flies interestingly enhanced stress resistances against oxidation and heat, significantly (Fig. 4B-C), as did the imdP mutant flies (Fig. 2C). In addition, lifespan was 21% extended with the AttC downregulation (Fig. 4D). Using an UAS/Gal4 system [27], we examined the downregulation effect of another AMP gene (UAS-DptB-dsRNAi: DptBRi) with an arm-Gal4 driver that expresses a target UAS-gene in the whole body. The DptBRi/armGal4 flies also exhibited higher stress resistance (~21% increase) and longer lifespan (+13%) compared to the control DptBRi/+ flies (Fig. 4E-G), indicating that the expression levels of antimicrobial peptide genes in the Imd pathway are directly involved in the stress resistance and the longevity mechanism.

The fat body, the fruit fly analogue of mammalian liver and adipose tissues, is known to function in metabolic homeostasis, stress tolerance, growth, and longevity in Drosophila [28,29]. For the antimicrobial response to bacterial infection, the antimicrobial peptide genes are mainly synthesized from fat body and secreted into the hemolymph [13]. We tested how downregulation of AMP genes in the fat body affects stress resistance and lifespan. We found that fat-body-specific (r4-Gal4 [30],) downregulations of imd, DptB, and AttC genes enhanced heat resistances significantly, between 20% and 32% (Fig. 5A-B), compared to the downregulation of another longevity gene, rpd3 [31,32], in the fat body (1.5% in Fig. 5B). Surprisingly, the AttCRi/r4Gal4 flies could exhibit up to 25% extended lifespan compared to the control AttCRi/+ flies (Fig. 5C), indicating that a manipulation of AMP expression in fat body tissues affects longevity of the whole body. Our data propose that the Imd pathway regulates the longevity mechanism through two downstream branches (Fig. 5D): 1) positively by stress tolerance genes of p-JNK [25,29,33,34] and 2) negatively by antimicrobial peptide genes of NF-kB that function positively in the immune response to bacterial infection [9,35].

Fly genotypes and aging assay

The imdP (P{EPgy2}imd^EY08573), AttC-P (Mi{ET1}AttC^MB05438), UAS-DptBRi (P{TRiP.HM05186}attP2), arm-Gal4, and r4-Gal4 flies were obtained from the Bloomington Drosophila stock center. The UAS-dsRNAi stocks (AttCRi: V47041; imdRi: V9253) and imdNP (P{GawB}imd^NP1182) flies were obtained from Vienna and Kyoto stock centers, respectively. The imd1 (imd¹) was kindly provided by D. Kimbrell [23]. The y¹ w¹ (Bloomington) flies were used as wild-type control and the flies obtained outside were six times isogenized with y¹ w¹ before the stress resistance and aging tests. Virgin flies were collected from a bottle in which larval density was controlled in a standard cornmeal medium, and were used for all fly experiments including stress response, aging, and nutritional content studies [26]. For the aging test, 200 virgin flies (20 flies per vial) were counted and transferred to fresh standard cornmeal vials every 3-4 days [5]. Mean lifespan was calculated from the lifespan curves and averaged with standard error of mean (SEM) from 2 ~ 4 independent experiments.

Stress response assays

To measure stress responses, 100 newly eclosed flies (20 flies per vial) were kept on a standard cornmeal medium at 25°C for 2 days [3,5,26]. For the starvation test, these 2-day-old adult flies were transferred to new vials (2.5 × 9.3 cm) containing two filter circles (2.4-cm diameter, Fisher Scientific) soaked in 300 μl of distilled water, and were maintained at 25°C under moist conditions with 100 μl of water added every 12 hrs. For the oxidative stress test, the 2-day-old adult flies were starved for 6 hrs at 25°C as described above. The flies were then transferred to new vials containing two filter circles wetted with 300 μl of 20 mM methyl viologen hydrate (Paraquat, Fisher Scientific) in a 5% sucrose solution and maintained at 25°C. For the heat test, the 2-day-old adult flies were transferred to new vials containing standard cornmeal medium and maintained at 37°C with 30% humidity. The median survival times of flies under each stress (starvation, oxidation, or heat) were calculated from the survival curves of 4 ~ 8 independent experiments.

Nutrients and MnSOD assays

All 20 flies were weighed and homogenized with specific buffers for nutrients (fat, protein, and glycogen: 0.01 M KH₂PO₄, 1 mM EDTA pH 7.4) [37] and MnSOD assays (20 mM HEPES, pH 7.2, 1 mM EGTA, 210 mM mannitol, 70 mM sucrose). The supernatant was recovered by centrifugation at 4,000g for 10 min at 4°C and used for nutrient measurement (triacylglycerol: Cayman Chemical; protein: Pierce; glycogen: BioVision). For measuring MnSOD activity, the recovered supernatant above was centrifuged again at 10,000g for 15 min at 4°C to pellet down the mitochondrial fraction containing MnSOD. This pellet was homogenized with the same buffer and used for the MnSOD assay (Cayman Chemical) in the presence of 2 mM KCN (Cu/ZnSOD inhibitor). Each assay was repeated and averaged (± SEM) from the 5 ~ 20 independent samples.

Microarray and RT-PCR analyses

Total RNA was extracted with the TRIzol Reagent (Invitrogen) from a whole body of male adult flies and used as probes for the hybridization reaction on Affymetrix Drosophila 2.0 microarray chips in the EOHSI facility (http://eohsi.rutgers.edu/). The expression levels of genes were calculated by the RMA (robust multichip analysis) normalization method. For RT-PCR, oligo dT-primed cDNAs (Superscript II RT, Invitrogen) were made from 5 μg total RNA of adult flies and used as templates for the quantitative real-time PCR, which was performed with power SYBR green PCR mix (Applied Biosystems). The rp49 gene was used as an internal reference for normalizing the quality of total RNA purified from each flies. The fold changes in gene expression were determined by comparative C_T method (ABI Prism 7700 Sequence Detection System User Bulletin #2, Applied Biosystems) and then, were averaged with SEM from 4 ~ 8 independent experiments using at least two different RNA batches.