Ageing leads to nonspecific antimicrobial peptide responses in Drosophila melanogaster

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 immune-deregulation 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. In contrast, ageing led to a complete loss of such specificity, warranting the action of multiple AMPs across Imd- and Toll-pathways during infections. Moreover, use of diverse AMPs either had no survival benefits, or even accompanied survival costs post-infection. These features were also sexually dimorphic: females expressed a larger repertoire of AMPs than males, but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-pool was associated with downregulation of negative-regulators of the Imd-pathway and a potential damage to renal function, as features of poorly-regulated immunity, Overall, we could establish ageing as an important driver of nonspecific AMP responses, across sexes and bacterial infections.

renal function, as features of poorly-regulated immunity, Overall, we could establish ageing 48 as an important driver of nonspecific AMP responses, across sexes and bacterial infections. 2019). We also used '∆AMPs' flies where independent mutations were recombined into a 137 background lacking 10 inducible AMPs. However, we note that the impact of ∆AMPs could be 138 due to AMPs having specific effects or combinatorial action of multiple co-expressed AMPs. 139 To tease apart these effects, we also included various combined mutants where different 'young' and 'old' adults, respectively. We transferred the adults to fresh food vials every 3 157 days, during the entire experimental window. By screening the single mutants, along with 158 combined genotypes, we were able to compare the changes in specific immunity as a function into a bacterial suspension made from 5 mL overnight culture (optical density of 0.95, 168 measured at 600 nm) of either Providencia rettgeri or Pseudomonas entomophila adjusted to 169 9 OD of 0.1 and 0.05 respectively (See SI methods for details). In total, we infected 160-280 170 flies/sex/infection treatment/bacterial pathogen/age-group/fly genotypes and then held 171 them in food vials in a group 20 individuals (For each treatment, sex, age-group, pathogen 172 type, we thus had 8-14 replicate food vials). We carried out sham infection with a pin dipped 173 in sterile phosphate buffer solution (1X PBS). 174 We then recorded their survival every 4-hours (2) for 5 days. Due to logistical challenges of 175 handling a large number of flies, we infected each sex and age-groups with P. rettgeri (or P. 176 entomophila) separately in multiple batches, where they were handled as -(i) Groups AB, 177 BC, AC; (ii) Group-A, B & C; (iii) Imd-responsive and (iv) Toll-responsive single mutants for P. 178 rettgeri; or (i) Groups AB, BC, AC, A, B & C; (ii) Imd-responsive and (iii) Toll-responsive single 179 mutants for P. entomophila. Every time, we also assayed iso-w 1118 flies as a control to facilitate 180 a meaningful comparison across different batches. Therefore, although sexes and age-groups 181 for each mutant were not directly comparable, their relative effects with respect to control 182 iso-w 1118 were estimated across sexes, age-groups and pathogen types. Note that we 183 compared each mutant separately with iso-w 1118 flies, since we only wanted to capture their 184 changes in infection susceptibility relative to control flies. For each batch of flies, across 185 pathogen types, sexes and age-groups, we analysed the survival data with a mixed effects Cox 186 model, using the R package 'coxme' (Therneau, 2015). We specified the model as: survival ~ 187 fly lines (individual AMP mutant lines vs iso-w 1118 ) + (1|food vials), with fly lines as a fixed 188 effect and replicate food vials as a random effect. Since none of the fly lines had any mortality 189 after sham-infection, we were able to quantify the susceptibility of each infected mutant lines 190 (AMP knockouts) with respect to control flies (iso-w 1118 group) as the estimated hazard ratio 191 of infected AMP mutants versus control flies (hazard ratio = rate of deaths occurring in 192 infected AMP mutants /rate of deaths occurring in iso-w 1118 group). A hazard ratio 193 significantly greater than one indicated a higher risk of mortality in the AMP mutant 194

individuals. 195
Note that the above experimental design allowed us to repeat the assay for post-infection 196 survival of young and old iso-w 1118 flies infected with P. rettgeri (or P. entomophila) in 4 (or 3) 197 independently replicated experiments. We thus estimated the effects of ageing on their post-198 infection survival, using a mixed effects Cox model specified as: survival ~ age + (1|food vials), 199 with age as a fixed effect, and food vials as random effects. 200

III.
Assay for bacterial clearance 201 Mortality of control flies (iso-w 1118 ) injected with the experimental infection dose began 202 around 24-hours and 20-hours after infection with P. rettgeri and P. entomophila respectively 203 ( Fig. S2A). We therefore used these time-points to estimate the bacterial load across the age-204 groups as a measure of the pathogen clearance ability across AMPs (see SI methods for 205 detailed protocol). We homogenized flies in a group of 6 in the sterile PBS (n= 8-15 replicate 206 groups/sex/treatment/age-group/fly lines), followed by plating them on Luria agar. Due to 207 logistical challenges with large number of experimental flies, we handled each sex, age-group 208 and pathogen type separately and in multiple batches as described above. 209 Also, similar to post-infection survival data, we were only interested in comparing the changes 210 in bacterial load for each mutant line relative to control iso-w 1118 flies across experimental 211 groups. We thus analysed the bacterial load data of each mutant genotype with iso-w 1118 flies 212 separately across age-groups, sexes and pathogen types. Since residuals of bacterial load data 213 were non-normally distributed (confirmed using Shapiro-Wilks's test), we log-transformed 214 the data, but residuals were still non-normally distributed. Subsequently, we analysed the 215 log-transformed data, using a generalised linear model best fitted to gamma distribution, with 216 fly lines (i.e., control iso-w 1118 line vs individual AMP knockout line) as a fixed effect. 217

IV.
Assay for the Malpighian tubule activity, as a proxy for immunopathological 218 damage 219 Malpighian tubules (MTs), the fluid-transporting excretory epithelium in all insects, are prone 220 to increased immunopathology following an immune activation due to their position in the 221 body and the fact that they cannot be protected with an impermeable membrane due to their 222 functional requirement (Dow et al., 1994;Khan et al., 2017). Previous experiments have 223 shown that risk of such immunopathological damage can increase further with ageing in 224 mealworm beetle Tenebrio molitor (Khan et al., 2017). It is possible that nonspecific AMP 225 responses with ageing in Drosophila was also associated with increased immunopathological 226 damage to MTs. We thus estimated the fluid transporting capacity of functional MTs 227 dissected from experimental females at 4-hours after immune challenge with 0.1 OD P. 228 rettgeri (n=12-20 females/ infection treatment/age-group), using a modified 'oil drop' 229 technique as outlined in previous studies (Dow et al. 1994;Li et al., 2020) (also see SI 230

methods). 231
This method provides a functional estimate of their physiological capacity by assaying the 232 ability to transport saline across the active cell wall into the tubule lumen. The volume of the 233 secreted saline droplet is negatively correlated with the level of immunopathological damage 234 to MTs. Since we collected the flies across the age-groups on different days, we analysed the 235 MT activity data as a function of infection status for each age-group separately, using a 236 generalized linear mixed model best fitted to a quasibinomial distribution. 237

V.
Gene expression assay 238 Finally, we note that transcription of negative regulators of Imd-pathway such as pirk and 239 caudal are important to ensure an appropriate level of immune response following infection 240 with gram-negative bacterial pathogens, thereby avoiding the immunopathological effects 241 al., 2018) (also see SI methods). We analysed the gene expression data using ANOVA (see SI 254 methods section-iv for details). 255

I.
Ageing leads to an expansion of the required AMP repertoire against P. rettgeri 271 infection 272 To gain a broad understanding of how AMP specificity changes with age, we first tested 273 mutants lacking different groups of AMPs either from Imd-(e.g., group B) or Toll-pathways 274 (e.g., group C) (pathway-specific), or combined mutants lacking pathway-specific mutants in 275 different combinations (e.g., group AB, BC or AC) (See Fig. S1 for description of mutants). As lacking group-AB and -BC AMPs were highly susceptible to P. rettgeri infection ( Fig. 1A; Table  278 S2), and this was generally associated with 10-100-fold increased bacterial loads in these 279 mutants relative to the iso-w 1118 control ( Fig. 1B; Table S3). Subsequent assays with pathway-280 specific (i.e., Imd-or Toll-pathway) AMP combinations (group A, B or C) confirmed that such 281 effects were primarily driven by Imd-regulated group-B AMPs that were shared between both 282 AB and BC combinations ( Fig. 1C; Fig. S3; Table S2), and equally driven by increased bacterial 283 load ( Fig. 1D; Table S3). We found a comparable pattern in young females as well, except that 284 flies lacking group BC combinations of AMPs were not negatively affected by infection (Fig.  285 1E, 1F, 1G, 1H; Fig. S4; Table S2, S3). 286 In contrast to young flies, most of the pathway-specific or combined mutants became highly 287 susceptible to P. rettgeri infection with age, except females of group-A mutants flies lacking 288 Def. This would suggest a possible sexually dimorphic effect of Defensin in P. rettgeri infection, 289 which appear to be important for males, but not females (Fig. 1C, 1G; Fig. S3, S4; Table S2).  Table S2) and increased bacterial growth (Fig. 1D, 1H; Table S3) 296 therein clearly indicated that other AMPs responsive to Gram-positive bacteria (e.g., Def) or 297 fungal pathogens (e.g., Mtk, Drs) might be needed as well. 298

II.
Dpt-specificity against P. rettgeri infection is sex-specific and disappears with age 299 Next, we decided to test the role of individual AMPs deleted in the pathway-specific or 300 compound mutants across age-groups and sexes. Interestingly, Dpt provided complete 301 protection against P. rettgeri only in young males, but not in females or older males ( Fig. 2A, 302 2E; Table S5). This was verified by using fly lines where DptA and DptB are introduced on an 303 AMP-deficient background (∆AMPs +Dpt ). Dpt reintroduction could fully restore survival as that 304 of wild-type flies only in young males, and this was associated with a decrease in CFUs 305 compared to the Dpt deletion mutant (Fig. 2B, Table S6). However, reintroduction of 306 functional DptA and DptB (∆AMPs +Dpt ) in young or old females flies did not result in lower 307 CFUs ( Fig. 2F; Table S6) and these flies remained highly susceptible to P. rettgeri ( Fig. 2E; Table  308 S2). Young (or old) females also showed increased bacterial loads and associated higher 309  Table S5, S6). Older ∆AMPs +Dpt males, on the other hand, could limit the bacterial 313 burden as low as that of the control iso-w 1118 flies ( Fig. 2B; Table S6), but still showed very 314 high post-infection mortality ( Fig. 2A; Table S5). These results from older males thus 315 suggested that the ability to clear pathogens might not always translate into an improved 316 ability to survive after infection ( Fig. 2A, 2B; Table S5, S6). 317 Why did females always require AMPs other than Dpt after P. rettgeri infection? Although the 318 mechanisms behind sex-specific expansion of AMP repertoire are unknown, a possible 319 explanation is that females show inherently lower expression level of Dpt relative to males . is not yet experimentally validated. 326 Also, both males and females showed further extension to a Toll-responsive AMP repertoire 327 with ageing. In addition to the role of Def (included in group-A) as described above in older  Taken together, these results describe ageing as a major driver behind the loss of specificity 333 of AMP responses. 334 Additionally, we also note that a few other mutations such as deletion of Dro and Dro-Att, 335 which otherwise had no effects on the survival of P. rettgeri-infected young males, caused 336 significant increase in the bacterial load ( Fig. 2A, 2B; Table S5, S6). Together, these results not 337 only underscored the multifaceted role of AMPs, but also provided functional resolution at 338 the level of single AMPs such as Dpt which in addition to playing the canonical role in resisting 339 the infection, also aided in withstanding the effects of increased pathogen growth, caused by 340 the dysfunction of other AMPs ( Fig. 2A, 2B; Table S5, S6). 341

III.
Expansion of the required AMP repertoire does not improve, and even reduces, 342 survival in both older males and females infected with P. entomophila 343 To test if age-related loss of AMP specificity was specific to P. rettgeri, or also occurred with  Table S7) and yet, died faster than young flies (old vs young: 4-fold vs 2-347 fold; Fig. 3A, 3C). In contrast to younger flies, where only group-B, -AB and -BC mutants were 348 susceptible to P. entomophila infection, all the other pathway-specific or combined mutants 349 of older males and females were also highly sensitive to infection (Fig. 3A, 3C; Table S7).  Table  355 S9), though it is striking that that increased mortality was not associated with increased 356 microbe loads relative to iso-w 1118 in this case (Fig. 4D, 4H; Table S10). Overall, this is 357 comparable to P. rettgeri infection where potential crosstalk between Toll & Imd immune-358 signalling pathways has already been implicated with ageing ( Fig. 2; Table S5, S6). Also, the 359 broad similarity between age-specific expansion and cross-reactivity of AMP repertoire 360 against two different pathogens indicated the possibility where non-specificity can indeed be 361 a generalised feature of an ageing immunity. Moreover, the increased mortality in older flies 362 infected with P. entomophila, despite involving a higher number of AMPs, was perhaps an 363 indication of their exacerbated cytotoxic effects with age (Badinloo et al., 2018). 364

IV.
Ageing-induced expansion of the required AMP repertoire was associated with 365 However, regardless of sex and pathogen, ageing led to a more drastic expansion of AMP 408 repertoire-instead of deploying only canonical expression of Imd-responsive AMPs to 409 counter Gram-negative bacterial infections, older males and females also used AMPs from 410 Toll pathways. 411 Surprisingly, despite using more diverse AMPs, late-life expansion either did not confer any 412 survival benefits (during P. rettgeri infection in older males) or was associated with survival 413 costs (after P. entomophila infection). We thus speculate that the nonspecific use of AMPs 414 with ageing was unnecessary, perhaps indicating an immune system failing to control over- We thank the grant supplement from SERB-DST India (No. ECR/2017/003370) to I. Khan Table S1 for qPCR primers used 738 in this study). For each cDNA sample across gene of interests, we had two technical replicates. We