Ageing leads to reduced specificity of antimicrobial peptide responses in Drosophila melanogaster

Abstract

Evolutionary theory predicts a late-life decline in the force of natural selection, possibly leading to late-life deregulations of the immune system. A potential outcome of such deregulations is the inability to produce specific immunity against target pathogens. We tested this possibility by infecting multiple Drosophila melanogaster lines (with bacterial pathogens) across age groups, where either individual or different combinations of Imd- and Toll-inducible antimicrobial peptides (AMPs) were deleted using CRISPR gene editing. We show a high degree of non-redundancy and pathogen-specificity of AMPs in young flies: in some cases, even a single AMP could confer complete resistance. However, ageing led to drastic reductions in such specificity to target pathogens, warranting the action of multiple AMPs across Imd and Toll pathways. Moreover, use of diverse AMPs either lacked survival benefits or even accompanied survival costs post-infection. These features were also sexually dimorphic: females required a larger repertoire of AMPs than males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-repertoire was accompanied with ageing-induced downregulation of negative-regulators of the Imd pathway and damage to renal function post-infection, as features of poorly regulated immunity. Overall, we could highlight the potentially non-adaptive role of ageing in producing less-specific AMP responses, across sexes and pathogens.

Keywords: ageing; antimicrobial peptides; immune senescence; immune specificity; pathogen resistance; sexual dimorphism.

Figure 1.

Infection with Providencia rettgeri in multiple AMP-knockouts. The estimated hazard ratios, including their lower and upper limits of the 95% confidence interval, calculated from survival curves (160–180 flies/sex/infection treatment/age group/fly line; see electronic supplementary material, figure S2, S3), and bacterial load (n = 8–9 replicate groups/sex/treatment/age group/fly line) measured at 24 h after P. rettgeri infection across sexes and age groups. Hazard ratios for double combination of AMP-knockouts (i.e. group-AB, BC & AC; table 1 for details about the fly lines) in males (a) and females (e). Bacterial loads for double combination of AMP-knockouts in males (b) and females (f). Hazard ratios for single combination of AMP-knockouts (e.g. group-A, B & C) in males (c) and females (g). Bacterial load for single combination of compound of AMP-knockouts in males (d) and females (h). In panels (a,c,e,g), hazard ratios significantly greater than 1 (hazard ratio = 1; shown as horizontal dashed grey lines), indicated by asterisk (*), suggests higher infection susceptibility of mutant flies than the iso-w¹¹¹⁸ control flies. In panels (b,d,f,h), each data point represents the bacterial load of flies pooled in a group of 6. Mutant fly lines that had significantly different bacterial load from wild-type iso-w¹¹¹⁸ are indicated by asterisks. ns = not significant. Group A: flies lacking Defensin; Group B: flies lacking AMPs such as Drosocin, Diptericins and Attacins; Group C: flies lacking Metchnikowin and Drosomycin. Group A, B and C mutants were combined to generate flies lacking AMPs either from groups A and B (AB), or A and C (AC), or B and C (BC). (Online version in colour.)

Figure 2.

Infection with Providencia rettgeri in individual AMP-knockouts. The estimated hazard ratios, including their lower and upper limits of the 95% confidence interval, calculated from survival curves (160–180 flies/sex/infection treatment/ age group/fly line; see electronic supplementary material, figure S2, S3) and bacterial load (n = 8–9 replicate groups/sex/treatment/age group/fly line) measured at 24 h after P. rettgeri infection across sexes and age groups. Hazard ratios for Imd-responsive single AMP (e.g. Dpt, AttC, AttD, Dro; table 1 for details about the fly lines) and Dro-Att knockouts in males (a) and females (e). Bacterial load of Imd-responsive single AMP and Dro-Att knockouts in males (b) and females (f). Hazard ratios for Toll-responsive single AMP knockouts (e.g. Drs & Mtk) in males (c) and females (g). Bacterial loads of Toll-responsive single AMP knockouts in males (d) and females (h) respectively. In panels (a,c,e,g), hazard ratios significantly greater than 1 (hazard ratio = 1; shown as horizontal dashed grey lines), indicated by asterisk (*), suggests higher infection susceptibility of mutant flies than the iso-w¹¹¹⁸ control flies. In panels (b,d,f,h), each data point represents the bacterial load of flies pooled in a group of 6. Mutant fly lines that had significantly different bacterial load from wild-type iso-w¹¹¹⁸ are indicated by asterisks (*). ns = not significant. (Online version in colour.)

Figure 3.

Infection with Pseudomonas entomophila in multiple AMP-knockouts. The estimated hazard ratios, including their lower and upper limits of the 95% confidence interval, calculated from survival curves (180–280 flies/treatment/age groups/sex/fly line; see electronic supplementary material figure S4, S5) and bacterial load (n = 9–15 replicate groups/sex/treatment/age group/fly line) measured at 20 h after P. entomophila infection across sexes and age groups. Hazard ratios for double (i.e. group-AB, BC & AC) and single combination (i.e. group-A, B, C) of AMP-knockouts in males (a) and females (c). Bacterial loads for double and single combination of AMP-knockouts in males (b) and females (d). In panels (a,c) hazard ratios significantly greater than 1 (hazard ratio = 1; shown as horizontal dashed grey lines), indicated by asterisk (*), suggests higher infection susceptibility of mutant flies than the iso-w¹¹¹⁸ control flies. In panels (b,d) each data point represents the bacterial load of flies pooled in a group of 6. Mutant fly lines that had significantly different bacterial load from wild-type iso-w¹¹¹⁸ are indicated by asterisks. ns = not significant. table 1 or the main text for the description of different fly groups. (Online version in colour.)

Figure 4.

Infection with Pseudomonas entomophila in individual AMP-knockouts. The estimated hazard ratios, including their lower and upper limits of the 95% confidence interval, calculated from survival curves (180–280 flies/sex/infection treatment/ age group/fly line; see electronic supplementary material, figures S4 and S5) and bacterial load (n = 9–15 replicate groups/sex/treatment/age group/fly line) measured at 20 h after P. entomophila infection across sexes and age groups. Hazard ratios for Imd-responsive single AMP (e.g. AttC, AttD, Dro; table 1 for details about the fly lines) and Dro-Att knockouts in males (a) and females (e). Bacterial load of Imd-responsive single AMPs and Dro-Att knockouts in males (b) and females (f). Hazard ratios for Toll-responsive single AMP knockouts (e.g. Drs & Mtk) in males (c) and females (g). Bacterial loads of Toll-responsive single AMP knockouts in males (d) and females (h) respectively. In panels (a,c,e,g), hazard ratios significantly greater than 1 (hazard ratio = 1; shown as horizontal dashed grey lines), indicated by asterisk (*), suggests higher infection susceptibility of mutant flies than the iso-w¹¹¹⁸ control flies. In panels (b,d,f,h), each data point represents the bacterial load of flies pooled in a group of 6. Mutant fly lines that had significantly different bacterial load from wild-type iso-w¹¹¹⁸ are indicated by asterisks (*). n.s. = not significant. (Online version in colour.)

Figure 5.

Ageing-associated immune dysregulation and immunopathology. (a) Malpighian tubule (MT) activity (n = 12–20 females/infection treatment/age group), as a proxy for immunopathological damage, measured at 4 h after infection with 0.1 OD of P. rettgeri. Statistically significant difference between groups are indicated by asterisk (*). (b) Expression of positive (Relish, PGRP-LC) and negative (Caudal, Pirk) regulators of Imd pathway across sexes and age groups after P. rettgeri infection, relative to an internal control rp49 (n = total 15–21 flies homogenized in Trizol in a group of 3/Infection treatment/age group/sex-combination). n.s. = not significant. (Online version in colour.)