Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis

Abstract

Antimicrobial peptides (AMPs) are innate immune effectors first studied for their role in host defence. Recent studies have implicated these peptides in the clearance of aberrant cells and in neurodegenerative syndromes. In Drosophila, many AMPs are produced downstream of Toll and Imd NF-κB pathways upon infection. Upon aging, AMPs are upregulated, drawing attention to these molecules as possible causes of age-associated inflammatory diseases. However, functional studies overexpressing or silencing these genes have been inconclusive. Using an isogenic set of AMP gene deletions, we investigated the net impact of AMPs on aging. Overall, we found no major effect of individual AMPs on lifespan, with the possible exception of Defensin. However, ΔAMP14 flies lacking seven AMP gene families displayed reduced lifespan. Increased bacterial load in the food of aged ΔAMP14 flies suggested that their lifespan reduction was due to microbiome dysbiosis, consistent with a previous study. Moreover, germ-free conditions extended the lifespan of ΔAMP14 flies. Overall, our results did not point to an overt role of individual AMPs in lifespan. Instead, we found that AMPs collectively impact lifespan by preventing dysbiosis during aging.

Keywords: Drosophila; Aging; Antimicrobial peptide; Host defence peptide; Inflammation; NF-κB.

Fig. 1.

Drosophila Nora virus significantly reduces lifespan of DrosDel iso w¹¹¹⁸ flies. Results for male flies are reported here and for female flies in Fig. S1. (A) Comparison of lifespan from early experiments using two wild-type strains (iso w¹¹¹⁸ and OR-R) alongside compound AMP mutants lacking Def, Dro, AttA, AttB, AttC, Mtk, DptA and DptB, which are deleted in ΔAMP Chr2 flies. (B) Effect of Nora clearance on lifespan of iso w¹¹¹⁸, OR-R, AttC, Bom and Group C (GrC) genotypes. Median lifespans are shown in the right panel (**P<0.01, ***P<0.001). Number of independent experiments (n^exp) is reported. (C) Nora titres measured by cycle threshold (CT) values in 18°C source stocks (Stocks) or Nora-positive flies aged 3+ weeks kept at densities of 20 flies per vial (Aged flies). CT values represent Nora titre from 5 ng total fly RNA per 10 μl qPCR reaction. Bars show the mean±s.d.

Fig. 2.

Individual AMP gene deletions do not drastically affect lifespan. Results for male flies are reported here and for female flies in Fig. S3. (A) Cumulative lifespans of flies with various genetic backgrounds. Of note, ATM⁸ data are based on fewer individuals per experiment (see Table S1). (B) Cumulative lifespans of single-gene/single-mutation AMP mutants. Most AMP mutant lifespans (black lines) cluster around the wild-type lifespan (blue line), except Def^SK3. Bom^Δ55C is also noted as an outlier, perhaps living slightly longer than iso w¹¹¹⁸, which was not seen in females (Fig. S3). (C) Median lifespans in which each data point represents one replicate experiment (cumulative of ∼20 males). Median lifespan analysis suggests that the only AMP mutation noticeably differing from iso w¹¹¹⁸ is Def^SK3. Of note, the impact of Def on lifespan was not corroborated using RNAi (Fig. S4). Bom^Δ55C median lifespans were not different from those of iso w¹¹¹⁸. Horizontal dotted lines indicate median lifespans of iso w¹¹¹⁸ (top), Def^SK3 (middle), and Rel^E20 (bottom). Statistical summaries (^xP<0.1; *P<0.05, **P<0.01, ***P<0.001) reflect comparisons to iso w¹¹¹⁸. (D) Climbing pass rates suggest most AMP mutants climb like wild-type flies, whereas methuselah mutants uniquely retain climbing competence into old age (also seen at 29°C, Fig. S2); Mtk and Drs males also show improved climbing with aging (but see text in File S1).

Fig. 3.

ΔAMP14 flies have significantly reduced lifespan. Results for male flies are reported here and for female flies in Fig. S6. (A) Survival curves of various compound AMP mutants. The lifespan of Rel^E20 from Fig. 2 is overlaid for direct comparison. ‘{’ annotations highlight major mortality events in ΔAMP14 flies. (B) Median lifespans of compound AMP mutants. Dotted lines indicate average median lifespans from Fig. 2 of iso w¹¹¹⁸ (top), Def^SK3 alone (middle), and Rel^E20 (bottom) for easier comparisons across figures. Statistical summaries (^xP<0.1; *P<0.05, **P<0.01, ***P<0.001) reflect comparisons to iso w¹¹¹⁸ data specific to Fig. 3. (C) Top: representative photonegative images of agar plates seeded by the microbiome found in the vial of 40-day-old flies. Thick bacterial films in ΔAMP14 vials are readily visualized by this method, which shows the significantly greater bacterial density (dark parts) compared to iso w¹¹¹⁸ vials. Bottom: representative photo of iso w¹¹¹⁸ and ΔAMP14 food vials revealing discoloured bacterial biofilm alongside a major mortality event (‘{’ in Fig. 3A). (D) Climbing pass rates of AMP group mutants, with climbing curves from genotypes in Fig. 2 overlaid for direction comparison. Group C is highlighted for having a slightly improved climbing over aging, although this improvement is still minor compared to the climbing competence of mth¹ flies (also see results for females in Fig. S6 and File S1).

Fig. 4.

Microbiome depletion rescues the ΔAMP14 fly lifespan. Results for male flies are reported here and for female flies in Fig. S7. (A) Survival curves, including lifespans for both antibiotic-reared (ABX) flies and also conventionally reared (CR) flies from previous figures as dotted lines for direct comparison. (B) Median lifespans, including both ABX and CR fly lifespans for direct comparison (conventionally reared iso w¹¹¹⁸ and Rel^E20 lifespans shown in Fig. 2C). *P<0.05. (C) Climbing pass rates of ABX (solid lines) and CR (dotted lines) flies at 5, 40, 50 and 60 days post eclosion.