Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells

Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial... Background: Drosophila is a powerful model for the study of factors modulating innate immunity. This study examines the effect of water-loss dehydration on innate immune responsiveness in the Drosophila renal system (Malpighian tubules; MTs), and how this leads to elevated host defense and contributes to immunosenescence. Results: A short period of desiccation-elevated peptidoglycan recognition protein-LC (PGRP-LC) expression in MTs, increased antimicrobial peptide (AMP) gene induction, and protected animals from bacterial infection. We show that desiccation increased ecdysone synthesis in MTs, while inhibition of ecdysone synthesis or ecdysone receptor expression, specifically within MTs, prevented induction of PGRP-LC and reduced protection from bacterial infection. Additionally, aged flies are constitutively water-stressed and have elevated levels of ecdysone and PGRP-LC. Conversely, adults aged at high relative humidity show less water loss and have reduced expression of PGRP-LC and AMPs. Conclusions: The Drosophila renal system is an important contributor to host defense and can modulate immune responses in an organ autonomous manner, responding to environmental changes such as desiccation. Desiccation primes immune responsiveness by elevating PGRP-LC expression specifically in MTs. In response to desiccation, ecdysone is produced in MTs and acts in a paracrine fashion to increase PGRP-LC expression, immune responsiveness, and improve host defense. This activity of the renal system may contribute to the immunosenescence observed in Drosophila. Keywords: Drosophila Malpighian tubules, desiccation, ecdysone, innate immune aging, immunosenescence Background kappa-light-chain-enhancer of activated B cells (NF-κB) sig- In Drosophila, the humoral immune response is character- naling pathways, the Toll and Imd pathways [1, 2]. ized by the rapid induction of a battery of antimicrobial The Imd pathway responds to diaminopimelic acid-type peptides (AMPs). These endogenous antimicrobials are peptidoglycan (DAP-type PGN) from the cell wall of expressed at nearly undetectable levels in the blood (or Gram-negative and certain types of Gram-positive bacteria. hemolymph) of healthy, young adult flies but are transcrip- DAP-type PGN is sensed by two receptors, both members tionally induced to massive levels following systemic micro- of the peptidoglycan recognition protein (PGRPs) family of bial infection. This response is triggered by microbial cell receptors. PGRP-LC is a transmembrane receptor found on walls, peptidoglycans from bacteria or beta-glucans from the cell surface, while PGRP-LE is a cytosolic receptor [3]. fungi, and is regulated by two nuclear factor DAP-type PGN ligation of either of these receptors is suffi- cient to activate Imd signaling and massive AMP gene in- * Correspondence: neal.silverman@umassmed.edu; Marc_Tatar@Brown.edu duction. In particular, these receptors trigger the formation Wenjing Zheng and Florentina Rus contributed equally to this work. of a functional amyloidal signaling platform, involving the Department of Medicine, Division of Infectious Diseases, University of factor Imd [4], which in turns leads to K63-ubiquitination, Massachusetts, Medical School, Worcester, MA, USA Division of Biology and Medicine, Brown University, Providence, RI, USA activation of the Drosophila kinases TGF-β activated kinase Full list of author information is available at the end of the article © Silverman et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. BMC Biology (2018) 16:60 Page 2 of 14 1 and I Kappa B Kinase, and ultimately the cleavage and and in dissected tubules [3, 17, 18]. In MTs and other nuclear translocation of the NF-κB precursor Relish [5–7]. immune responsive tissues, tracheal cytotoxin (TCT), a Relish directly induces AMP gene expression. monomeric fragment of DAP-type PGN, activates Imd Developmental and environmental signals modulate the signaling [3, 19]. Thus, to characterize the innate im- activity and sensitivity of the Imd immune response. In par- mune response in MTs, we challenged MTs excised from ticular, the steroid hormone 20-hydroxyecdysone (20E) wild-type (wDah) females with TCT and measured AMP controls the expression of the key PGN-sensing receptor gene expression using NanoString nCounter. As shown PGRP-LC [8]. In earlier work, we established that 20E, sig- here, TCT induced robust expression of multiple AMP naling through a canonical nuclear hormone receptor path- genes in MTs dissected from 7-day-old females (Fig. 1a, way, is required for PGRP-LC expression both in cultured Additional file 1: Figure S1). cells and in adult flies. Given the central role PGRP-LC In preliminary studies with this ex vivo MT assay, we plays in sensing systemic bacterial infections, steroid hor- noticed a high degree of variability in the amplitude of mone regulation provides a significant degree of modula- AMP induction. This led us to hypothesize that animals tion on the ability of cells or animals to respond to bacterial may be exposed to relatively brief periods of dehydration infection. It is clear from earlier literature that this regula- during handling on the CO anesthesia ‘fly pad’, which tory network provides developmental modulation to Imd could be affecting immune response amplitudes. An ex- responses. In particular, early third instar larvae are largely perimental protocol was designed to directly test this hy- unresponsive to challenge with DAP-type PGN-containing pothesis. Without anesthesia, 7-day-old females were microbes, while wandering larvae, which have received a transferred to vials and maintained in PBS (isotonic to large bolus of 20E as part of their developmental program, fly hemolymph) or in a dry vial (desiccation) for 2 h are highly responsive to these infections [9]. (both conditions without food). RNA was isolated from In adult flies, it has been suggested that 20E also serves as freshly dissected MTs (time 0), and from dissected MTs a stress-responsive steroid hormone, analogous to incubated for 4 h with or without TCT. Defensin and adrenal-produced, stress-responsive hormones (such as glu- Cecropin-A2 (Cec-A2) mRNAs were quantified. MTs cocorticoids) in mammals [10–13]. In this study, we have stimulated with TCT strongly induced Defensin and examined how 20E modulates the immune response follow- Cec-A2 compared to freshly dissected MTs or to ing the stress caused by a brief period of desiccation. Given mock-treated MTs (Fig. 1b). For Defensin, desiccation the physiological role for the Malpighian tubules (MTs, the triggered a modest increase in expression in the insect renal system) in solute homeostasis [14], this immune mock-treated samples and amplified the induction upon responsive organ was the focus of investigation. We found TCT treatment (two-way ANOVA, χ = (desiccation x TCT) that MT autonomously exhibited dehydration-induced 3.96, p < 0.05). For Cec-A2, the effect of desiccation steroid-mediated priming of the Imd pathway. This priming trended in the same direction, but did not reach statis- response required 20E-mediated upregulation of the innate tical significance (χ = 1.31, p = 0.31). In a (desiccation x TCT) immune recognition receptor PGRP-LC, thus termed second similarly designed experiment, RNA was ex- recognition-protein priming. Aged flies are known to natur- tracted from four biologically independent sets of MT, ally become dehydrated [15, 16], and experimental rehydra- isolated from control (PBS) or desiccated wDah animals, tion ameliorated the elevated Imd activity typically and analyzed by Nanostring nCounter for the expression observed in older animals, suggesting a link between of multiple AMPs and other immune related genes dehydration-induced recognition-protein priming and (Heat Map in Additional file 2: Figures S2). In this ana- immunosenescence. Moreover, dehydration stress-induced lysis, 13 AMP genes were significantly upregulated either steroid-mediated recognition-protein priming, specifically by desiccation alone or upon TCT-challenge following in the MTs, provided enhanced protection to systemic bac- desiccation. Many of these AMP genes showed signifi- terial infection in adult animals. These data demonstrate cant interactions between desiccation and TCT (side the important role of steroid-mediated recognition-protein table in Additional file 3: Figure S3, after Bonferroni cor- priming in modulating the immune response of adult Dros- rection for multiple comparisons). Together, these data ophila, shaping host defense based on life history and envir- demonstrate that desiccation robustly induces AMP gene onmental conditions. expression within the MT, and frequently increases the sensitivity to TCT. Results Desiccation increases immune sensitivity in the Desiccation triggers increased expression of the innate Drosophila renal system immune recognition protein via steroid hormone It was previously shown that Drosophila MTs are au- signaling tonomously immune responsive with robust AMP gene In MTs, TCT is a direct agonist of both the cell surface induction following immune challenge in intact animals receptor PGRP-LC and the cytosolic receptor PGRP-LE Zheng et al. BMC Biology (2018) 16:60 Page 3 of 14 Fig. 1 TCT and desiccation stress induce multiple AMP genes in Malpighian tubules (MTs). a Nanostring nCounter analysisofAMP transcriptsfrom isolated MTs of wDah females (7 days old) exposed to TCT for 4 h compared to unstimulated tubules. The mean of four independent biological replicates, harvested on separate days, is shown, and error bars represent standard error of the mean. Statistical significance was calculated by two-way ANOVA and Sidak’s multiple comparison test; **p < 0.01. b mRNA expression (means with SEM) for Defensin and Cecropin-A2 (Cec-A2) measured by qRT-PCR relative 2 2 to control (time 0, no TCT). TCT induces Defensin (Likelihood ratio test, DF = 1, χ = 9.32, p = 0.0023) and Cec-A2 (Likelihood ratio test, DF = 1, χ = 6.15, p = 0.013). Generalized Linear Model estimated by maximum likelihood with TCT and desiccation as main effects and TCT x desiccation as interaction effect, N = 12; desiccation and TCT significantly interact to amplify Defensin mRNA (TCT x desiccation χ =3.96 p = 0.046). The desiccation-induced increase for Cec-A2 did not reach significance (TCT x desiccation χ =1.03, p = 0.31). c qRT-PCR analysis of PGRP-LC and PGRP-LE expression from MTs, normalized to PBS-treated control. *p < 0.05; means with SEM, N =6; t test; PGRP-LC p = 0.038, PGRP-LE p =0.27 [19–23]. Desiccation caused a significant increase in expression [8]. Ecdysone also induces expression and syn- PGRP-LC mRNA expressed in MT, while PGRP-LE ex- thesis of its nuclear hormone receptor, ecdysone receptor pression was unchanged (Fig. 1c). Overall, MT from (EcR) [29]. Here, we found elevated EcR mRNA levels in adults held without water for a short duration increased MTs of desiccated flies, compared to PBS-treated or con- expression of PGRP-LC and their capacity to induce ventionally reared animals (Fig. 2a). In addition, ecdysone AMP gene expression. is known to activate an EcR-responsive reporter in adult Among its many functions [24], ecdysone modulates tissues including the MT [30]. Therefore, we tested if EcR AMP expression in larvae, cultured cells, pupae, and is required for desiccation to induce PGRP-LC in MTs. adults [8, 9, 25–28]. In particular, 20E regulates PGRP-LC EcR RNAi expressed in stellate cells (c724-Gal4) or Zheng et al. BMC Biology (2018) 16:60 Page 4 of 14 Fig. 2 Malpighian tubule (MT) EcR and PGRP-LC induced by desiccation and 20E feeding. Relative mRNA of (a) EcR and (b) PGRP-LC in control genotype (+/EcR(RNAi)) and with EcR(RNAi) expressed in stellate cells (c724>) or principal cells (c324>). c Relative mRNA of PGRP-LC in control genotype (+/eip75B(RNAi)) and when eip75B(RNAi) is expressed in stellate cells (c724>) or principal cells (c324>). a–c Mean with SEM of three independent biological samples for each genotype, normalized relative to food treatment, wild-type genotype. Within each genotype: one-way ANOVA with treatment as main effect (N = 9), *p < 0.05, **p < 0.01 and Tukey’s post hoc comparison. d Relative mRNA for PGRP-LC, EcR, Defensin, and Cec-A2 from MTs of adults fed 20E, each normalized to corresponding control. Mean of three independent biological samples (SEM); t test for each gene with Holm–Bonferroni Sequential correction *p < 0.05 principal cells (c324-Gal4) blocked the ability of desicca- within MTs, as well as the expression of Defensin and tion to induce EcR or PGRP-LC expression in MTs (Fig. 2a, Cec-A2 even without external immune challenge (Fig. 2d). b). In a complementary fashion, ecdysone induced protein Ecdysone signaling appears to drive the expression of 75 (Eip75), a negative regulator of ecdysone-mediated sig- PGRP-LC during desiccation stress, and thus prime tu- naling, was targeted via tissue-specific RNAi. Knockdown bules to express higher levels of AMPs (‘recognition-- in stellate cells (via c724-Gal4/UAS-eip75B(RNAi)) signifi- protein priming’). These results suggest that desiccation cantly amplified the ability of desiccation to increase stress should increase 20E levels. Measured from whole PGRP-LC mRNA relative to control genotype (β animals of two genetic backgrounds (yw and wDah), 20E genotype x = −0.018, χ = −3.50, p <0.01), while c324-Gal4/ was elevated after 2 h of desiccation (Fig. 3a). To deter- desiccation UAS-eip75B(RNAi) trended in the same direction, but did mine where this 20E is produced, the expression of ec- not reach statistical significance (Fig. 2c). dysone biosynthetic enzyme genes (Halloween Ecdysone has a myriad of activities beyond its classic role cytochrome P450s) was quantified from different tissues in controlling the insect life cycle. For example, 20E drives (Fig. 3b and Additional file 4: Figure S4, all expression PGRP-LC expression in S2* cells [8]. Additionally, 20E fed was normalized relative to the head sample in the food to adult Drosophila restores normal aging to otherwise control group). Desiccation upregulated Halloween long-lived EcR mutant females, restores courtship memory genes in several somatic tissues. The most striking ef- defects and promotes sleep [12, 31, 32]. Here, 20E fed to fe- fects (Fig. 3b) were observed for disembodied (dib), a males induced the expression of EcR and PGRP-LC mRNA cytochrome P450 with ecdysteroid 22-hydroxylase Zheng et al. BMC Biology (2018) 16:60 Page 5 of 14 Fig. 3 Water-loss dehydration increases ecdysone and Malpighian tubule (MT) expression of ecdysone biosynthetic genes in young females (7 days). a 20E titer (pg/fly) (mean and SEM among three biological replicates, each of 25–30 pooled females) from whole females of two genetic backgrounds (yw and wDah) with control (food or PBS) and desiccation treatments. Independent biological replicates were prepared on separate days. One-way ANOVA within each genotype, Tukey’s post hoc comparison: *p <0.05, **p <0.01. b Relative disembodied (dib) mRNA in female tissues from control (food or PBS) and desiccation treatments. All values normalized to mean value from food treatment, head sample; mean values of three biological replicates, each containing the specific tissue from 15 females. Statistical comparisons are made within each tissue by t test for difference between desiccation and PBS treatment: dib mRNA was significantly elevated in muscle and MT (*p <0.05 with Holm–Bonferroni Sequential correction). c Relative mRNA for EcR and (d) PGRP-LC in control genotype (+/dib(RNAi)) and when dib is depleted in stellate cells (c724>) or principal cells (c324>). Values are mean and SEM of three independent biological replicates, normalized relative to food treatment, wild-type for each mRNA type. For each genotype: one-way ANOVA with Tukey’s post hoc comparison. For the control genotype (+/dib RNAi) a significant difference was observed between the flies exposed to desiccation compared to those exposed to normal food or PBS (*p < 0.05, **p < 0.01). However, no significant differences were observed when EcR or PGRP-LC were knocked down (c324 >RNAi and c724 >RNAi); EcR-RNAi F = 0.52, p =0.62 and F = 4.43, p = 0.066; PGRP-LC-RNAi F = 0.39, p =0.70 and F = 0.54, p =0.61 activity; control-treated females expressed dib strongly significantly less dib mRNA in the fat body relative to food in ovaries (via follicle cells [33]) and modestly in fat controls (p = 0.019). This effect on fat body expression of body, while little dib mRNA expression was observed in dib appears to be due to a starvation response rather than a other tissues. However, upon desiccation, dib mRNA desiccation response. No significant differences in dib ex- was significantly increased in muscle and MTs. Accord- pression in ovaries were observed among treatments. ingly, when dib mRNA was reduced by RNAi in stellate or principal cells (Additional file 5: Figure S5), EcR and Aged flies exhibit desiccation-induced ecdysone-mediated PGRP-LC expression was not induced in MTs by desic- recognition-protein priming cation relative to food or PBS controls (Fig. 3c, d). Interestingly, water loss rates and AMP gene expression Together, these data show that desiccation controls ec- both increase in aged Drosophila [16, 34–36]. In addition dysone synthesis within MTs, which locally regulates to higher AMP expression at baseline, older flies also in- recognition-protein priming. On the other hand, females duce higher levels of AMP gene expression when chal- maintained for 2 h without food (PBS controls) produced lenged with live bacteria [28, 35], suggesting that Zheng et al. BMC Biology (2018) 16:60 Page 6 of 14 recognition receptor expression could be affected by age. expressed in ovaries of old, food-reared females, al- Indeed, expression of PGRP-LC as well as EcR was in- though in old females, fat body showed lower expression creased in MT from flies at age 40 days (Fig. 4a, b). Experi- under all conditions. Old females under control condi- mental dehydration did not further elevate PGRP-LC and tions (PBS) expressed somewhat more dib than young EcR mRNAs in aged animals, in contrast to their induction females in head (1.83 fold), muscle (1.71 fold), and gut in young flies. Ecdysone titers measured from aged flies (2.97 fold) (each tissue p < 0.03). Expression of dib was were also significantly elevated relative to young females in similarly low in the MT of young and old control fe- control (food) conditions, while desiccation increased 20E males, but significantly induced by desiccation in old in both young and old animals (Fig. 4c). These data suggest MTs (3.5 fold), as observed in young females, and con- that aging is associated with recognition-protein priming sistent with the ecdysone titers. mediated through elevated ecdysone. Aged females may produce ecdysone because they are in- To identify the source of ecdysone in aged flies, tissue trinsically water stressed. To explore this hypothesis, co- was dissected from 40 day females exposed to control horts of yw adults were maintained in normal food (food, PBS) and dehydration treatments. Dib mRNA was conditions in three levels of relative humidity (RH), namely quantified relative to young females. Relative to young 20% RH, 40% RH (incubator standard condition), and 80% females, old females expressed dib in similar but muted RH (Additional file 6: Figure S6). Water content was mea- patterns (Fig. 4d, compared to Fig. 3b). dib was still sured in flies sampled from each RH cohort across 7 weeks Fig. 4 Peptidoglycan recognition-protein priming increases with age. (a) EcR and (b) PGRP-LC mRNA from Malpighian tubules (MTs) of young (7 days) and old (40 days) yw females in control (food, PBS) and desiccation treatments. Expression was normalized relative to young flies in the food treatment control; mean of three independent biological samples with SEM; two-way ANOVA with age and treatment as main effects, Tukey’s post hoc comparison, *p < 0.05, **p < 0.01. c 20E extracted from 7- and 40-day-old wild-type females (yw and wDah) in control (food, PBS) and desiccation treatments; mean of three independent biological samples with SEM. Within each genotype, two-way ANOVA with age and treatment as main effects, Tukey’s post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001. d Disembodied (dib) mRNA from specific tissues of old females (40 days) in control (food, PBS) and desiccation treatments; normalized relative to food treatment, head tissue of young females; mean of three independent biological samples for each tissue type. Statistical comparisons within each tissue by t test for difference between desiccation and PBS treatment: dib mRNA was significantly elevated in MTs (*p<0.05 with Holm-Bonferroni Sequential correction) Zheng et al. BMC Biology (2018) 16:60 Page 7 of 14 of age (Fig. 5a). Consistent with reports where water loss Defensin in cohorts at 80% RH increased to an extent that rates weredirectlymeasured in aging Drosophila [16], was significantly less than observed at 20% RH (two-way 2 2 water content per fly declined with age in all cohorts (χ = ANOVA for age and RH, each mRNA: χ >4.3, (age x humidity) 102.9, p < 0.0001), and at a significantly greater rate at 20% p < 0.04). Thus, innate immune aging is blunted in flies RH compared to 80% RH (χ = 50.02, p < reared at RH that reduces intrinsic water stress, decreasing (age x humidity) 0.025). Lifespan was similar among the 40% and 80% RH recognition protein expression and AMP gene levels. This cohorts (median life span: 44 and 46 days respectively), but amelioration in water loss and immunosenescence also had significantly less at 20% RH (median life span: 40 days) a modest, but significant, effect on lifespan. (Fig. 5b). Remarkably, elevated RH sharply blunted the in- creased expression of PGRP-LC, EcR,and the AMP gene Desiccation-triggered ecdysone-mediated recognition- Defensin typically observed in the MT of aged adults protein priming leads to enhanced host defense (Fig. 5c–e). MTs from females at 20% RH strongly upregu- We were also interested to examine how recognition-protein lated PGRP-LC, EcR,and Defensin by age 6 weeks (age as priming might alter host defense. To test if main effect, each mRNA as independent variable: χ > 13.0, desiccation-induced recognition-protein priming affects sur- p < 0.001). In contrast, mRNA for PGRP-LC, EcR,and vival following bacterial infection, we exposed two wild-type Fig. 5 High relative humidity (RH) reduces water loss with age and slows innate immune aging. a Water content per adult female, cohorts sampled from age 1 to 7 weeks old; cohorts maintained at 20%, 40%, and 80% RH. Trajectory of water content among RH groups analyzed with Generalized Linear 2 2 Model Fit by maximum likelihood ratio test. For main effects: age affected water content (χ = 102.9, p < 0.0001) but humidity did not (χ = 1.23, p =0.54). For interaction effects: water content declined faster in 20% RH compared to 80% RH (χ = 50.0, p < 0.025); no significant difference in water loss rate between 40% and 80% RH (χ = 0.625, p = 0.43). All point estimates produced represent the mean from five independent biological samples comprised of 20 pooled females. b Survivorship of yw adult females aged under RH of 20%, 40%, and 80% (25 °C). Log-rank test of mortality: 20% versus 40%, p = 0.0003; 40% versus 80%, p = 0.616. Three cages of 125 flies used for each RH condition. c–e Relative PGRP-LC, EcR,and Defensin mRNA levels in MTs from yw females, mean and SEM, aged at 20%, 40%, and 80% RH (25 °C), sampled at 1, 2, 4, and 6 weeks; three samples of 15 females were censored from demography cages to produce three independent biological samples at each time point and condition. Analysis was performed with Generalized Linear 2 2 Model Fit by maximum likelihood ratio test for main(age, RH)and interaction(agex RH) effects. PGRP-LC:age, χ = 24.3, p < 0.001; RH, χ = 13.0, p = 0.0015; 2 2 2 2 2 2 age x RH, χ = 7.08, p = 0.029. EcR:age, χ = 13.3, p < 0.001; RH, χ = 11.3, p = 0.0035; age x RH, χ = 6.75, p = 0.034. Defensin:age, χ = 15.1, p < 0.001; RH, χ = 2.79, p = 0.095; age x RH, χ = 4.32, p = 0.038 Zheng et al. BMC Biology (2018) 16:60 Page 8 of 14 fly strains (wDah and yw) to 2 h desiccation and then immedi- improved their survival relative to all other infected cohorts, ately infected the adults with the Gram-negative pathogen including non-desiccated controls (Fig. 6a–c and Additional Erwinia carotovora carotovora 15 (Ecc15). Desiccated flies file 7: Figure S7). Importantly, this improved survival following were significantly more susceptible to Ecc15 infection com- septic infection required ecdysone signaling and PGRP-LC ex- pared to those without prior desiccation (Additional file 7:Fig- pression in the MT; knockdown of EcR or of ure S7). Given that PGRP-LC expression is elevated upon PGRP-LC in MT principal cells eliminated the sur- desiccation, these results were unexpected and led us to vival benefit of desiccation with recovery following hypothesize that some time, following desiccation, may be re- Ecc15 challenge (Fig. 6d, e). These results demon- quired for PGRP-LC mRNA to produce functional receptors. strate that MTs contribute to host defense, and that Accordingly, we allowed the desiccated flies to recover upon recognition-protein priming, specifically in this renal regularfoodfor 3or6hprior to Ecc15 challenge. Wild-type organ, improves outcomes following septic bacterial flies that were allowed to recover for 6 h significantly challenge. Fig. 6 Functional immunity enhanced by desiccation stress response requires EcR and PGRP-LC in Malpighian tubules (MTs). a–e Kaplan–Meier survival plots of adult females sterile pricked or infected with Ecc15, with or without prior desiccation (des) and either no recovery, 3 h recovery, or 6 h recovery. Genotypes: a yw; b +/EcR(RNAi); c +/PGRP-LC(RNAi); d c324-gal4/UAS-EcR(RNAi); e c324-gal4/UAS-PGRP-LC(RNAi). Each survival curve is from a combined lifetable of two cohorts tested at different times, each cohort initiated with 90 females. For survival analyses (right censored log-rank test) conducted with six pair-wise comparisons within each genotype; curves with different letters differ significantly by at least p < 0.02 (details in Additional file 9:Table S1). f Microbial load (colony-forming units) 48 h after Ecc15 infection of adult females across treatments by desiccation (0 or 2 h) and post-desiccation recovery (0, 3,6 h).Genotypes as in (a–e): wildtype controls (yw, +/EcR, +/PGRP-LC) and EcR or PGRP-LC knock-down in principal cells (c324 > RNAi). For each genotype, three independent samples were prepared from each treatment group, in both cohort trials (displaying mean and SD of all six observations). CFU compared by ANOVA with Sidak post-hoc comparison, bars indicate differences with p < 0.05, see details in Additional file 10:Table S2 Zheng et al. BMC Biology (2018) 16:60 Page 9 of 14 Improved survival upon septic bacterial challenge can dehydration induces MT to express ecdysone biosynthetic arise from tolerance to the infection as well as from immune genes and targeted reduction of dib within MT stellate or defenses that reduce microbial load, often referred to as re- principal cells prevented induction of PGRP-LC during sistance [37]. To determine whether recognition-protein water-loss dehydration. How MTs regulate dib and other priming affects tolerance or resistance, we measured Ecc15 Halloween gene expression in response to water loss is un- colony-forming units (CFUs) from flies treated as in the in- known. Desiccation can reduce Drosophila neuropeptide fection survival studies. Flies were initially inoculated with capa-1 (homolog of human Neuromedin U) and increase ~6.0 × 10 CFUs of Ecc15 (0 h, Additional file 8:FigureS8). short neuropeptide F and tachykinin in protocerebral neu- 2+ At 48 h post infection, wild-type flies harbored less Ecc15 if rosecretory cells [42–44]. Capa-1 controls intracellular Ca they had been desiccated and allowed to recover prior to in- and nitric oxide/guanosine 3′,5′-cyclic monophosphate sig- fection (Fig. 6f), consistent with their improved survival. In naling in MTs [42]. Further, tachykinin induces insulin pro- contrast, flies with EcR or PGRP-LC expression knock-down duction and insulin signaling within MTs [45]. Notably, specifically in their MT had a higher Ecc15 load at 48 h post insulin signaling has the capacity to stimulate ecdysone infection, and desiccation with recovery did not reduce infec- production in the prothoracic gland of developing larvae tion load. Thus, recognition-protein priming in adult MTs [46–48]. It remains to be determined whether similar neu- provides functional whole animal immune resistance to sep- roendocrine factors control Halloween gene expression and tic bacterial challenge, leading to improved survival. recognition-protein priming in MTs. Female Drosophila use cholesterol to produce ecdys- Discussion one in egg follicles. In the fat body, this ecdysone is con- Water-loss dehydration triggers recognition-protein verted by 20-hydroxylase CYP314A1 (shade) to 20E, priming within adult Drosophila MTs. This response is which activates nuclear hormone receptor EcR and stim- mediated by the local synthesis and signaling of ecdys- ulates yolk production [49, 50]. In the ovary, ecdysone one within MTs, which induces expression of the micro- also regulates germline stem cells, somatic cysts, and bial recognition protein PGRP-LC. Elevated PGRP-LC border cells [33]. Other adult tissues in female insects potentiates the expression of AMP mRNA in tubules. do not produce ecdysone de novo, although some may Increased AMP gene expression is expected to produce complete its final synthesis, including the step involving more mature AMPs, although the study of the peptides dib [51]. Ecdysone synthesis also occurs in adult males, is hampered by a lack of effective antibodies. Our ana- where its production may take place in accessory glands lysis of survival after bacterial challenge with desiccation [52–54]. We find that muscle, gut, and MT, express dib and recovery suggests that recognition-protein priming when adults are dehydrated; these tissues may thus pro- in the MT provides functional antimicrobial resistance duce ecdysone during physiological stress, or at least to the entire animal. Moreover, the load of infecting mi- complete its synthesis. Consistent with this idea, crobe is reduced in wild-type flies able to mount Rauschenbach et al. [10] and Terashima et al. [11] noted recognition-protein priming, but not in flies where that nutrient restriction and heat increased 20E levels in PGRP-LC or EcR expression is reduced specifically in adult Drosophila. MTs, highlighting a previously unknown role for this Ecdysone has many potential somatic targets within organ in whole animal antimicrobial defense. adult Drosophila [24, 30]. Notably, 20E can modulate fly Recognition-protein priming induced by water-loss de- aging. Males and females heterozygous for mutant EcR hydration may be an adaptive response if dehydration are long-lived, and resistant to the oxidizing agent para- coincides with infection. Observations on this topic are quat as well as to heat and starvation despite being sur- scarce. Virulence in the waxmoth Galleria mellonella prisingly fertile [31]. Tricoire et al. [55] subsequently was increased by infection with Beauveria bassiana that found that mild repression of EcR also extended male exogenously expressed diuretic hormone, which stimu- lifespan, while EcR inactivation decreased female lifespan lates host water loss [38]. In Drosophila, septic infection except when egg production was genetically suppressed. might increase if water-loss dehydration reduces barrier Besides control of egg production, ecdysone in adults protection of gut or cuticle [39, 40]. Alternatively, Imd has stress- and aging-related functions, although where signaling primed by water-loss dehydration could pro- and how these work remain poorly understood. vide novel immune protective or tolerance functions. Here, we studied Drosophila innate immune aging in For instance, Relish (NF-κB) regulates expression of the context of desiccation stress. Water loss may be a cathepsin-L required for remodeling fat body in the typical feature of fly aging. Aged Drosophila experience moth Helicoverpa armigera, and Har-Relish is modu- elevated water loss and are less resistant to desiccation lated by ecdysone signaling in this moth [41]. stress [56, 57], susceptibilities that may be caused by ac- In Drosophila, recognition-protein priming is mediated celerated cuticle water transpiration [16]. Cuticle lipids by ecdysone produced within the MT. Water-loss regulate barrier transpiration in adult insects and this Zheng et al. BMC Biology (2018) 16:60 Page 10 of 14 layer may deteriorate or change composition during caused a marked sensitivity to infection. How MTs con- aging [15, 58, 59]. We propose that recognition-protein tribute to immunity and host defense will be the focus priming and increased AMP response in the aged arise of future studies. in part because old flies experience high water loss. Con- In addition, aged flies also display symptoms consist- sistent with this idea, cohorts maintained at high RH re- ent with water-loss dehydration. Aged flies, reared at pressed the typical age-related increase in PGRP-LC, 20%, 40%, or 80% RH, were dehydrated, consistent with Defensin, and EcR expression. Conversely, flies at low earlier reports [58]. Aged flies also displayed increased RH were somewhat shorter lived, and previous work systemic 20E levels and increased expression of the in- suggests that chronic innate immune activation reduces nate immune recognition protein PGRP-LC and effec- life expectancy in an NF-κB-dependent manner [60]. tors. However, adults aged at high relative humidity The production of AMPs is costly, impacting physi- show less water loss, and have reduced expression of ology and fecundity [61], and is tightly regulated. AMP PGRP-LC, AMPs, and EcR in their tubules. Our study induction is robust but transient to minimize the dele- suggests that the steroid hormone ecdysone in old flies terious effects of immune activation. Likewise, the Imd is induced in response to age-related water imbalance, pathway, and AMP production, is regulated by ecdysone and this facilitates immunosenescence in Drosophila in the context of development and, as shown here, by renal MTs. stress. This steroid-mediated regulation likely optimizes the AMP response such that the benefits of this mech- Methods anism of host defense at least equal these costs. Steroid Drosophila strains modulation of AMP induction during development may Stocks were reared and maintained at 25 °C, 40% RH coordinate this feature of innate immunity with nutrient and 12-h light/dark cycles. Food media contained corn- demands and availability, which change during different meal (5.2%), sugar (11.0%), autolyzed yeast (2.5%; SAF growth phases. Alternatively, developmental steroid brand) and agar (0.79%) (w/v in 100 mL water) with regulation of AMP production may be an adaptation to 0.2% Tegosept (methyl 4-hydroxybenzoate, Sigma, St. the selective pressures associated with the different mi- Louis, MO, USA) as an antifungal agent. MT-specific crobial pathogens encountered with different develop- Gal4-driver stocks c724 (stellate cell) and c324 (principal mental stages. Furthermore, here we demonstrate that cell) were provided by Julian Dow (University of Glas- the adult flies use ecdysone to prime innate immune in- gow); second chromosomes containing these transgenes duction of AMPs in the renal organ in response to were crossed and balanced into a genetic background water-stress imbalance. Remarkably, the resulting ele- marked with yw, producing yw; c724-Gal4/Cyo and yw; vated expression of AMPs from these renal tissues ap- and c324-Gal4/Cyo. RNAi to reduce PGRP-LC, EcR, pears to protect adult animals from systemic, septic Eip75B, and dib was respectively provided from Vienna KK101636 infection. Stock Center as strains P{UAS-PRGP-LC RNAi}- GD1428 VIE-260B, P{UAS-EcR RNAi}v37058, P{UAS-Eip KK108982 KK106954 Conclusions 75B RNAi}VIE-260B, and P{UAS-dib R- In this study, we examined how water-loss dehydration NAi}VIE-260B. Genotypes (F1 offspring) were generated increases innate immune responsiveness in Drosophila from crosses of these UAS-RNAi stocks with MT MTs, and how this response improves outcomes follow- Gal4-drivers or by crosses with yw. wDah was provided ing infection and contributes to elevated innate immune by the laboratory of Linda Partridge (University College gene expression with age. In this model, the stress of London). water-loss dehydration increased systemic levels of 20E, which is responsible for the pattern recognition-protein 20E treatment priming observed in MTs. While it is not possible to 20E was fed to wDah females aged 7–10 days old by measure 20E levels in a tissue-specific manner, genetic transferring adults from standard laboratory diet to 1 g manipulations of both the 20E synthesis gene dib and fly instant food media with 2 mL of water (Genesee Sci- the EcR, demonstrate that the tubules are autonomously entific, San Diego, CA, USA) for 24 h containing vehicle responding to water-loss dehydration through the pro- control (20 μL ethanol) or vehicle with 20 μL 1 mM 20E duction of ecdysone and subsequent sensing of this hor- (Sigma cat H-5142). mone signal. Interestingly, the data presented here further argue that MTs serve a critical function in Desiccation protocol anti-bacterial immunity, wherein recognition-protein Prior to conducting a trial, adult flies were placed in priming in MTs is required for increased host defense vials with standard food at a density of approximately 15 observed after desiccation and recovery, and loss of per vial without anesthesia. Vials were maintained over- recognition-protein expression specifically in MTs night at 25 °C. To initiate each trial, flies from vials were Zheng et al. BMC Biology (2018) 16:60 Page 11 of 14 flipped without anesthesia into fresh vials that contained Quantitative RT–PCR standard food (food control), tissue soaked with 0.5 mL Total RNA was extracted from dissected tissue (MT, 0.5× PBS solution (PBS control), or nothing (desicca- head, muscle, gut, fat body, ovary) in Trizol reagent tion). MTs were dissected after 2 h of treatment from (Invitrogen, Carlsbad, CA, USA) and treated with DNase flies within 5 minutes, immediately transferred to Trizol, (Ambion). DNase-treated total RNA was quantified with and processed for RT-qPCR. Three replicate biological a NanoDrop ND-1000. cDNA was synthesized using samples of 15 MT (sampled from across five vials) were iScript cDNA Synthesis (Bio-Rad) and measured on an generated per treatment. ABI prism 7300 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). For tissue-specific ana- lysis of AMP, PGRP-LC, EcR, or Halloween genes, three MT stimulation with TCT independent biological replicates for each tissue and Females (wDah) aged 7 to 10 days post eclosion, were each treatment group were analyzed. Each replicate in- processed through the desiccation protocol (with PBS cluded RNA extracted from 10 flies for head, muscle, as the only control). Dissected MTs were transferred gut, fat body, and ovary, or 15–20 for MT. Three tech- for 4 h to S2 cell culture medium with TCT nical replicates were used for each biological replicate. (0.01 mM dissolved in water) or without TCT. Ex- mRNA abundance of each gene was normalized relative pression of defensin and Cec-A2 was measured by to ribosomal protein L32 (rp49) by comparative CT. qRT-PCR from RNA extracted from these tubules im- mediately upon dissection (0 h), and at 4 h with or Enzyme immunoassay (EIA) of 20E without TCT. Three replicate biological samples were Female flies (25–30 flies/sample) were homogenized in produced for each measure set. 250 μL of ice-cold 100% methanol and centrifuged for 15 min at 18,000 g at 4 °C. Supernatants were trans- NanoString nCounter expression analysis ferred to 6 × 50 mm borosilicate glass tubes; precipitates The mRNA content of dissected MTs was analyzed were suspended in 200 μL of aqueous 75% methanol and by NanoString methodology according to published kept on ice for 30 min (adapted from [63]). Samples procedures [62]. Total RNA (100 ng per sample) was were centrifuged and dried with a SpeedVac centrifuge. hybridized to the target-specific codeset overnight at The precipitates were dissolved in EIA buffer at 4 °C 65 °C. The codeset used in Additional file 1:Figure and each individual sample was analyzed in a technical S1 contained probes against a panel of 45 genes en- triplicate by competitive EIA using 20E EIA antiserum coding AMPs, Imd pathway components, and four (Cayman Chemical: 482202) and 20E AChE Tracer loading controls (nucleostemin, alpha tubulin, Rp49, (Cayman Chemical: 482200). Calibration curves were and Faf2), while that in Additional file 2: Figure S2 generated from commercial 20E (Sigma, H5142). utilized an expanded codeset of 113 immunity-related genes and three loading controls (nucleostemin, alpha RH cohorts tubulin,and Faf2). The hybridized reactions were Newly enclosed females (yw) were mated for 2 days and loaded onto the NanoString Prep station, which separated into replicate 1 L demography cages at 125 removes excess reporter, binds the reporter to the flies per cage in an environmentally controlled room set cartridge surface, and stretches the probes for scan- at 40% RH, 25 °C, and 12 h light: 12 h dark cycles. Three ning. Subsequently, the cartridges were loaded onto independent cages (plus supplemental cages for dissec- the NanoString Digital Analyzer and scanned. The tion samples) were placed in 60 L clear plastic, nCounter data were normalized in two steps. In the tight-lidded storage boxes that maintained RH at 20%, first, data was normalized to positive spiked-in con- 40%, and 80%. To produce 20% RH, boxes contained a trols, as per the manufacturer’sinstructions(Nano- solution of saturated potassium acetate (Fisher Science String Technologies). In the second, the housekeeping Cat # P171–500) and solid desiccant (Drierite, W.A. genes were used to normalize overall mRNA levels. Hammond Co.). To produce 40% RH, the box was left For Fig. 1a, AMP gene expression was further nor- ajar, permitting cages to equilibrate at 40% RH as main- malized amongst the four biological replicates by set- tained in the environmental room. To produce 80% RH, ting the highest value for each gene to 100% and boxes contained a solution of saturated ammonium sul- plotted with standard error of the mean. For heat fate (Sigma-Aldrich Cat # A4418-500G). Boxes were maps in Additional file 1:FigureS1and Additional managed every second day at room temperature and file 2: Figure S2, only genes with expression greater room RH, at which time dead flies were removed and than 50 normalized counts in one or more conditions counted. Saturated solutions (and Drierite) were replen- are shown, as lower counts are below the reliable de- ished weekly. HOBO data loggers (Onset Computer tection threshold for the nCounter. Corp) continuously monitored RH within boxes; after Zheng et al. BMC Biology (2018) 16:60 Page 12 of 14 maintenance, all boxes rapidly re-equilibrated to RH tar- analysis of AMP transcripts from isolated MTs of wDah females (7 days gets (Additional file 5: Figure S5). old) exposed to TCT for 4 h compared to unstimulated tubules. The mean of four independent biological replicates, harvested on separate Longevity analysis was conducted with JMP statistical days, is shown by heatmap. Scale as indicated in side bar. (PDF 797 kb) software (SAS Institute, Cary, NC, USA). Data from three Additional file 2: Figure S2. Heat map summarizing innate immune replicate cages were combined to produce genotype and gene expression profiles in desiccated Malpighian tubules (MTs). MTs of treatment cohort life tables. Mortality distributions were 7-day-old wDah flies exposed to 2 h desiccation or PBS control treatment were excised, and then either RNA was immediately isolated, or were compared by Log-Rank and proportional hazard analyses. treated for 4 h with TCT or mock treated, and then RNA was isolated. Flies to measure mRNA were maintained in parallel dem- These RNA samples were used to measure immune-related gene ography cages and sampled at 1, 2, 4, and 6 weeks. MTs expression by NanoString nCounter. Values shown by heat map represent the mean of four independent experiments. Scale as indicated in side were dissected directly from non-anesthetized adults and bar. (PDF 847 kb) subjected to RT-qPCR (15 females per biological replicate, Additional file 3: Figure S3. Desiccation stress upregulated and amplified three replicates per time point per humidity condition). TCT-induced expression of 13 AMP genes in Malpighian tubules. Analysis of in- Water content was determined from samples in a rep- dividual AMP gene expression (as indicated), data from nCounter experiments displayed by heat map in Additional file 2: Figure S2. Values represent the licate trial with cohorts at 20%, 40%, and 80% RH. Fe- mean of four biologically independent replicates, and error bars are standard males were sampled at 1, 2, 4, 6, and 7 weeks from each error of the mean. Statistical analysis was performed by two-way ANOVA and RH cohort. Point estimates for water content were each Sidak’s test for pair-wise comparison of desiccation to PBS treatment (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Side table displays statistical met- generated as the mean among five groups of 20 females; rics after Bonferroni correction for multiple comparisons for the interaction of these females were sampled from 8 demography cages, desiccation and TCT treatments from the same ANOVA analyses. (PDF 631 kb) each with 125 females for every RH condition. For each Additional file 4: Figure S4. mRNA expression of Halloween genes. group, females were weighed (Mettler Toledo Halloween genes (spook, phantom, shadow, and shade) were measured in adult tissues from young (7 days) and old (40 days) females. All values semi-micron balance), dried overnight at 65 °C, and normalized relative to head samples from young adults in food control reweighed. Age, RH treatment, and main-effect interac- group. The mean of three independent biological replicates is shown. tions were analyzed by generalized linear model likeli- (PDF 2424 kb) hood ratio test. Additional file 5: Figure S5. RNAi on disembodied (dib) in Malpighian tubules (MTs). Reduced dib mRNA in MTs when dib RNAi is driven in stellate (c724 > dib RNAi) or principal (c324 > dib RNAi) cells relative to Infection challenge survival control (ywT1/dib RNAi). Results shown represent the mean and SEM of three independent replicates. (PDF 897 kb) Adult survival to a controlled infection challenge after Additional file 6: Figure S6. Relative humidity (RH) and temperature desiccation treatment was determined using 100 to 120 recorded from demography chambers under different humidity flies per group (Additional file 5: Figure S5) or 180 flies conditions. Spikes indicate when chambers were opened to access cages per group (Fig. 6) (among two replicate vials). Females and show rapid homeostasis of the humidity control system. Blue tracings show realized RH, black tracing represents temperature. (PDF at 3 to 7 days old were exposed to food or PBS-only as 3450 kb) control, or to 2 h desiccation. Following these treat- Additional file 7: Figure S7. Impact of desiccation followed by ments, either immediately or after 3 and 6 h recovery on recovery on the survival of wild-type flies infected with Erwinia caroto- standard food, flies were pricked with a clean microsur- vora carotovora 15 (Ecc15). Kaplan–Meier survival of yw and wDah adult gery needle (control) or with a needle dipped into a con- females challenged with Ecc15. Uninfected flies were desiccated or not centrated pellet of (Ecc15)[64, 65]. Surviving flies were for 2 h and survival was monitored for 8 days. Other cohorts were chal- subsequently transferred daily to fresh vials and recorded lenged with Ecc15 infection with or without a prior 2 h desiccation treatment and recovery of 0, 3, or 6 h prior, as indicated. Plots represent as alive or dead for 8 days. Survival is represented as the survival kinetics of 100–120 files, combined from two separate trials, Kaplan–Meier plots with all pairwise differences evalu- for each genotype; cohorts with significantly different mortality are ated by log-rank test with significance adjusted for grouped with different letters (all cases, log-rank test p <0.05, see side multiple comparisons (p < 0.01). tables). (PDF 1741 kb) Additional file 8: Figure S8. Initial bacterial loads in Erwinia CFU counts carotovora carotovora 15 (Ecc15)-infected flies. Adult load (colony-forming units) of Ecc15 at 0 h post infection as a function of Bacterial load was determined in flies at time 0 and 48 h desiccation (0 or 2 h) and post-desiccation recovery (0, 3, 6 h); after infection with Ecc15. Individual flies were homoge- among three control genotypes (yw, +/EcR, +/PGRP-LC) and geno- nized in 200 μL of PBS. Homogenates were diluted in types where EcR or PGRP-LC were knocked down by RNAi in principal cells (c324 > RNAi), no significant differences were observed at 0 h. series and plated on LB-Ampicillin plates, incubated Results represent the mean of six assays and the error bars show overnight at 37 °C, and scored for CFU count. standard deviation. (PDF 1024 kb) Additional file 9: Table S1. Statistical analysis for the effect of desiccation on survival to Ecc15 infection with or without recovery treatment, supporting Additional files Fig. 6a-e.(DOCX 26 kb) Additional file 10: Table S2. Statistical analysis for the effect of desiccation Additional file 1: Figure S1. Heat map of differentially expressed innate on bacterial growth after Erwinia carotovora carotovora 15 infection with or immune transcripts in Malpighian tubules (MTs). Nanostring nCounter without recovery treatment, supporting Fig. 6f.(DOCX 15 kb) Zheng et al. BMC Biology (2018) 16:60 Page 13 of 14 Abbreviations 6. Chen L, Paquette N, Mamoor S, Rus F, Nandy A, Leszyk J, Shaffer SA, 20E: 20-hydroxyecdysone; AMP: antimicrobial peptide; Cec-A2: Cecropin-A2; Silverman N. Innate immune signaling in Drosophila is regulated by CFU: colony-forming unit; DAP-type PGN: diaminopimelic acid type- transforming growth factor beta (TGFbeta)-activated kinase (Tak1)-triggered peptidoglycan; dib: disembodied; Ecc15: Erwinia carotovora carotovora 15; ubiquitin editing. J Biol Chem. 2017;292(21):8738–49. EcR: Ecdysone receptor; Eip75: ecdysone induced protein 75; MTs: Malpighian 7. Stoven S, Silverman N, Junell A, Hedengren-Olcott M, Erturk D, Engstrom Y, tubules; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; Maniatis T, Hultmark D. Caspase-mediated processing of the Drosophila NF- PGRP: Peptidoglycan recognition protein; RH: relative humidity; TCT: tracheal kappaB factor Relish. Proc Natl Acad Sci U S A. 2003;100(10):5991–6. cytotoxin 8. Rus F, Flatt T, Tong M, Aggarwal K, Okuda K, Kleino A, Yates E, Tatar M, Silverman N. Ecdysone triggered PGRP-LC expression controls Drosophila Acknowledgements innate immunity. EMBO J. 2013;32(11):1626–38. We thank Julian Dow for sharing Malpighian tubule Drosophila strains as well 9. Meister M, Richards G. Ecdysone and insect immunity: the maturation of the as Rolf Bodmer and Paul Shaw for their contributions to the conceptual inducibility of the diptericine gene in Drosophila larvae. Insect Biochemistry work on functional aging. Mol Biol. 1996;26:155–60. 10. Rauschenbach IY, Sukhanova MZ, Hirashima A, Sutsugu E, Kuano E. Role of Funding the ecdysteroid system in the regulation of Drosophila reproduction under NS and MT were collaboratively supported by NIH grant PO1 AG033561. NS environmental stress. Dokl Biol Sci. 2000;375:641–3. was independently supported by RO1 AI099708. MT was independently 11. Terashima J, Takaki K, Sakurai S, Bownes M. Nutritional status affects 20- supported by R37 AG024360. The funders had no role in study design, data hydroxyecdysone concentration and progression of oogenesis in Drosophila collection and analysis, decision to publish, or preparation of the manuscript. melanogaster. J Endocrinol. 2005;187(1):69–79. 12. Ishimoto H, Kitamoto T. The steroid molting hormone Ecdysone regulates Availability of data and materials sleep in adult Drosophila melanogaster. Genetics. 2010;185(1):269–81. All data generated or analyzed during this study are included in this 13. Ishimoto H, Kitamoto T. Beyond molting–roles of the steroid molting published article and its supplementary information files. All material used, hormone ecdysone in regulation of memory and sleep in adult Drosophila. such as Drosophila strains, are available from public stock centers and other Fly. 2011;5(3):215–20. repositories. 14. Dow JA. Insights into the Malpighian tubule from functional genomics. J Exp Biol. 2009;212(Pt 3):435–45. Authors’ contributions 15. Nghiem D, Gibbs AG, Rose MR, Bradley TJ. Postponed aging and WZ designed, performed, and analyzed experiments. FR designed, desiccation resistance in Drosophila melanogaster. Exp Gerontol. 2000; performed, analyzed experiments, and contributed to the drafting and 35(8):957–69. revising of the manuscript. AH performed and analyzed some of the 16. Gibbs AG, Markow TA. Effects of age on water balance in Drosophila experiments presented in Fig. 1. PK designed and performed RH controlled species. Physiol Biochem Zool. 2001;74(4):520–30. experiments. WG provided critical reagents. NS and MT together conceived 17. Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM, Lemaitre B, of this study, contributed to experimental design and analysis and Hoffmann JA, Imler JL. Tissue-specific inducible expression of antimicrobial manuscript preparation, and supervised all aspects of this study. All authors peptide genes in Drosophila surface epithelia. Immunity. 2000;13(5):737–48. read and approved the final manuscript. 18. McGettigan J, McLennan RK, Broderick KE, Kean L, Allan AK, Cabrero P, Regulski MR, Pollock VP, Gould GW, Davies SA, et al. Insect renal tubules Competing interests constitute a cell-autonomous immune system that protects the organism The authors declare that they have no competing interests against bacterial infection. Insect Biochem Mol Biol. 2005;35(7):741–54. 19. Kaneko T, Goldman WE, Mellroth P, Steiner H, Fukase K, Kusumoto S, Harley W, Fox A, Golenbock D, Silverman N. Monomeric and polymeric gram- Publisher’sNote negative peptidoglycan but not purified LPS stimulate the Drosophila IMD Springer Nature remains neutral with regard to jurisdictional claims in pathway. Immunity. 2004;20(5):637–49. published maps and institutional affiliations. 20. Hoffmann JA. The immune response of Drosophila. Nature. 2003; 426(6962):33–8. Author details 21. Werner T, Liu G, Kang D, Ekengren S, Steiner H, Hultmark D. A family of Division of Biology and Medicine, Brown University, Providence, RI, USA. peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Department of Medicine, Division of Infectious Diseases, University of Proc Natl Acad Sci U S A. 2000;97(25):13772–7. Massachusetts, Medical School, Worcester, MA, USA. Department of 22. Chang CI, Ihara K, Chelliah Y, Mengin-Lecreulx D, Wakatsuki S, Deisenhofer J. Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, Structure of the ectodomain of Drosophila peptidoglycan-recognition USA. protein LCa suggests a molecular mechanism for pattern recognition. Proc Natl Acad Sci U S A. 2005;102(29):10279–84. Received: 21 July 2017 Accepted: 15 May 2018 23. Chang CI, Pili-Floury S, Herve M, Parquet C, Chelliah Y, Lemaitre B, Mengin- Lecreulx D, Deisenhofer J. A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D- References carboxypeptidase activity. 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Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells

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Copyright © 2018 by Silverman et al.
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Life Sciences; Life Sciences, general
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Abstract

Background: Drosophila is a powerful model for the study of factors modulating innate immunity. This study examines the effect of water-loss dehydration on innate immune responsiveness in the Drosophila renal system (Malpighian tubules; MTs), and how this leads to elevated host defense and contributes to immunosenescence. Results: A short period of desiccation-elevated peptidoglycan recognition protein-LC (PGRP-LC) expression in MTs, increased antimicrobial peptide (AMP) gene induction, and protected animals from bacterial infection. We show that desiccation increased ecdysone synthesis in MTs, while inhibition of ecdysone synthesis or ecdysone receptor expression, specifically within MTs, prevented induction of PGRP-LC and reduced protection from bacterial infection. Additionally, aged flies are constitutively water-stressed and have elevated levels of ecdysone and PGRP-LC. Conversely, adults aged at high relative humidity show less water loss and have reduced expression of PGRP-LC and AMPs. Conclusions: The Drosophila renal system is an important contributor to host defense and can modulate immune responses in an organ autonomous manner, responding to environmental changes such as desiccation. Desiccation primes immune responsiveness by elevating PGRP-LC expression specifically in MTs. In response to desiccation, ecdysone is produced in MTs and acts in a paracrine fashion to increase PGRP-LC expression, immune responsiveness, and improve host defense. This activity of the renal system may contribute to the immunosenescence observed in Drosophila. Keywords: Drosophila Malpighian tubules, desiccation, ecdysone, innate immune aging, immunosenescence Background kappa-light-chain-enhancer of activated B cells (NF-κB) sig- In Drosophila, the humoral immune response is character- naling pathways, the Toll and Imd pathways [1, 2]. ized by the rapid induction of a battery of antimicrobial The Imd pathway responds to diaminopimelic acid-type peptides (AMPs). These endogenous antimicrobials are peptidoglycan (DAP-type PGN) from the cell wall of expressed at nearly undetectable levels in the blood (or Gram-negative and certain types of Gram-positive bacteria. hemolymph) of healthy, young adult flies but are transcrip- DAP-type PGN is sensed by two receptors, both members tionally induced to massive levels following systemic micro- of the peptidoglycan recognition protein (PGRPs) family of bial infection. This response is triggered by microbial cell receptors. PGRP-LC is a transmembrane receptor found on walls, peptidoglycans from bacteria or beta-glucans from the cell surface, while PGRP-LE is a cytosolic receptor [3]. fungi, and is regulated by two nuclear factor DAP-type PGN ligation of either of these receptors is suffi- cient to activate Imd signaling and massive AMP gene in- * Correspondence: neal.silverman@umassmed.edu; Marc_Tatar@Brown.edu duction. In particular, these receptors trigger the formation Wenjing Zheng and Florentina Rus contributed equally to this work. of a functional amyloidal signaling platform, involving the Department of Medicine, Division of Infectious Diseases, University of factor Imd [4], which in turns leads to K63-ubiquitination, Massachusetts, Medical School, Worcester, MA, USA Division of Biology and Medicine, Brown University, Providence, RI, USA activation of the Drosophila kinases TGF-β activated kinase Full list of author information is available at the end of the article © Silverman et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. BMC Biology (2018) 16:60 Page 2 of 14 1 and I Kappa B Kinase, and ultimately the cleavage and and in dissected tubules [3, 17, 18]. In MTs and other nuclear translocation of the NF-κB precursor Relish [5–7]. immune responsive tissues, tracheal cytotoxin (TCT), a Relish directly induces AMP gene expression. monomeric fragment of DAP-type PGN, activates Imd Developmental and environmental signals modulate the signaling [3, 19]. Thus, to characterize the innate im- activity and sensitivity of the Imd immune response. In par- mune response in MTs, we challenged MTs excised from ticular, the steroid hormone 20-hydroxyecdysone (20E) wild-type (wDah) females with TCT and measured AMP controls the expression of the key PGN-sensing receptor gene expression using NanoString nCounter. As shown PGRP-LC [8]. In earlier work, we established that 20E, sig- here, TCT induced robust expression of multiple AMP naling through a canonical nuclear hormone receptor path- genes in MTs dissected from 7-day-old females (Fig. 1a, way, is required for PGRP-LC expression both in cultured Additional file 1: Figure S1). cells and in adult flies. Given the central role PGRP-LC In preliminary studies with this ex vivo MT assay, we plays in sensing systemic bacterial infections, steroid hor- noticed a high degree of variability in the amplitude of mone regulation provides a significant degree of modula- AMP induction. This led us to hypothesize that animals tion on the ability of cells or animals to respond to bacterial may be exposed to relatively brief periods of dehydration infection. It is clear from earlier literature that this regula- during handling on the CO anesthesia ‘fly pad’, which tory network provides developmental modulation to Imd could be affecting immune response amplitudes. An ex- responses. In particular, early third instar larvae are largely perimental protocol was designed to directly test this hy- unresponsive to challenge with DAP-type PGN-containing pothesis. Without anesthesia, 7-day-old females were microbes, while wandering larvae, which have received a transferred to vials and maintained in PBS (isotonic to large bolus of 20E as part of their developmental program, fly hemolymph) or in a dry vial (desiccation) for 2 h are highly responsive to these infections [9]. (both conditions without food). RNA was isolated from In adult flies, it has been suggested that 20E also serves as freshly dissected MTs (time 0), and from dissected MTs a stress-responsive steroid hormone, analogous to incubated for 4 h with or without TCT. Defensin and adrenal-produced, stress-responsive hormones (such as glu- Cecropin-A2 (Cec-A2) mRNAs were quantified. MTs cocorticoids) in mammals [10–13]. In this study, we have stimulated with TCT strongly induced Defensin and examined how 20E modulates the immune response follow- Cec-A2 compared to freshly dissected MTs or to ing the stress caused by a brief period of desiccation. Given mock-treated MTs (Fig. 1b). For Defensin, desiccation the physiological role for the Malpighian tubules (MTs, the triggered a modest increase in expression in the insect renal system) in solute homeostasis [14], this immune mock-treated samples and amplified the induction upon responsive organ was the focus of investigation. We found TCT treatment (two-way ANOVA, χ = (desiccation x TCT) that MT autonomously exhibited dehydration-induced 3.96, p < 0.05). For Cec-A2, the effect of desiccation steroid-mediated priming of the Imd pathway. This priming trended in the same direction, but did not reach statis- response required 20E-mediated upregulation of the innate tical significance (χ = 1.31, p = 0.31). In a (desiccation x TCT) immune recognition receptor PGRP-LC, thus termed second similarly designed experiment, RNA was ex- recognition-protein priming. Aged flies are known to natur- tracted from four biologically independent sets of MT, ally become dehydrated [15, 16], and experimental rehydra- isolated from control (PBS) or desiccated wDah animals, tion ameliorated the elevated Imd activity typically and analyzed by Nanostring nCounter for the expression observed in older animals, suggesting a link between of multiple AMPs and other immune related genes dehydration-induced recognition-protein priming and (Heat Map in Additional file 2: Figures S2). In this ana- immunosenescence. Moreover, dehydration stress-induced lysis, 13 AMP genes were significantly upregulated either steroid-mediated recognition-protein priming, specifically by desiccation alone or upon TCT-challenge following in the MTs, provided enhanced protection to systemic bac- desiccation. Many of these AMP genes showed signifi- terial infection in adult animals. These data demonstrate cant interactions between desiccation and TCT (side the important role of steroid-mediated recognition-protein table in Additional file 3: Figure S3, after Bonferroni cor- priming in modulating the immune response of adult Dros- rection for multiple comparisons). Together, these data ophila, shaping host defense based on life history and envir- demonstrate that desiccation robustly induces AMP gene onmental conditions. expression within the MT, and frequently increases the sensitivity to TCT. Results Desiccation increases immune sensitivity in the Desiccation triggers increased expression of the innate Drosophila renal system immune recognition protein via steroid hormone It was previously shown that Drosophila MTs are au- signaling tonomously immune responsive with robust AMP gene In MTs, TCT is a direct agonist of both the cell surface induction following immune challenge in intact animals receptor PGRP-LC and the cytosolic receptor PGRP-LE Zheng et al. BMC Biology (2018) 16:60 Page 3 of 14 Fig. 1 TCT and desiccation stress induce multiple AMP genes in Malpighian tubules (MTs). a Nanostring nCounter analysisofAMP transcriptsfrom isolated MTs of wDah females (7 days old) exposed to TCT for 4 h compared to unstimulated tubules. The mean of four independent biological replicates, harvested on separate days, is shown, and error bars represent standard error of the mean. Statistical significance was calculated by two-way ANOVA and Sidak’s multiple comparison test; **p < 0.01. b mRNA expression (means with SEM) for Defensin and Cecropin-A2 (Cec-A2) measured by qRT-PCR relative 2 2 to control (time 0, no TCT). TCT induces Defensin (Likelihood ratio test, DF = 1, χ = 9.32, p = 0.0023) and Cec-A2 (Likelihood ratio test, DF = 1, χ = 6.15, p = 0.013). Generalized Linear Model estimated by maximum likelihood with TCT and desiccation as main effects and TCT x desiccation as interaction effect, N = 12; desiccation and TCT significantly interact to amplify Defensin mRNA (TCT x desiccation χ =3.96 p = 0.046). The desiccation-induced increase for Cec-A2 did not reach significance (TCT x desiccation χ =1.03, p = 0.31). c qRT-PCR analysis of PGRP-LC and PGRP-LE expression from MTs, normalized to PBS-treated control. *p < 0.05; means with SEM, N =6; t test; PGRP-LC p = 0.038, PGRP-LE p =0.27 [19–23]. Desiccation caused a significant increase in expression [8]. Ecdysone also induces expression and syn- PGRP-LC mRNA expressed in MT, while PGRP-LE ex- thesis of its nuclear hormone receptor, ecdysone receptor pression was unchanged (Fig. 1c). Overall, MT from (EcR) [29]. Here, we found elevated EcR mRNA levels in adults held without water for a short duration increased MTs of desiccated flies, compared to PBS-treated or con- expression of PGRP-LC and their capacity to induce ventionally reared animals (Fig. 2a). In addition, ecdysone AMP gene expression. is known to activate an EcR-responsive reporter in adult Among its many functions [24], ecdysone modulates tissues including the MT [30]. Therefore, we tested if EcR AMP expression in larvae, cultured cells, pupae, and is required for desiccation to induce PGRP-LC in MTs. adults [8, 9, 25–28]. In particular, 20E regulates PGRP-LC EcR RNAi expressed in stellate cells (c724-Gal4) or Zheng et al. BMC Biology (2018) 16:60 Page 4 of 14 Fig. 2 Malpighian tubule (MT) EcR and PGRP-LC induced by desiccation and 20E feeding. Relative mRNA of (a) EcR and (b) PGRP-LC in control genotype (+/EcR(RNAi)) and with EcR(RNAi) expressed in stellate cells (c724>) or principal cells (c324>). c Relative mRNA of PGRP-LC in control genotype (+/eip75B(RNAi)) and when eip75B(RNAi) is expressed in stellate cells (c724>) or principal cells (c324>). a–c Mean with SEM of three independent biological samples for each genotype, normalized relative to food treatment, wild-type genotype. Within each genotype: one-way ANOVA with treatment as main effect (N = 9), *p < 0.05, **p < 0.01 and Tukey’s post hoc comparison. d Relative mRNA for PGRP-LC, EcR, Defensin, and Cec-A2 from MTs of adults fed 20E, each normalized to corresponding control. Mean of three independent biological samples (SEM); t test for each gene with Holm–Bonferroni Sequential correction *p < 0.05 principal cells (c324-Gal4) blocked the ability of desicca- within MTs, as well as the expression of Defensin and tion to induce EcR or PGRP-LC expression in MTs (Fig. 2a, Cec-A2 even without external immune challenge (Fig. 2d). b). In a complementary fashion, ecdysone induced protein Ecdysone signaling appears to drive the expression of 75 (Eip75), a negative regulator of ecdysone-mediated sig- PGRP-LC during desiccation stress, and thus prime tu- naling, was targeted via tissue-specific RNAi. Knockdown bules to express higher levels of AMPs (‘recognition-- in stellate cells (via c724-Gal4/UAS-eip75B(RNAi)) signifi- protein priming’). These results suggest that desiccation cantly amplified the ability of desiccation to increase stress should increase 20E levels. Measured from whole PGRP-LC mRNA relative to control genotype (β animals of two genetic backgrounds (yw and wDah), 20E genotype x = −0.018, χ = −3.50, p <0.01), while c324-Gal4/ was elevated after 2 h of desiccation (Fig. 3a). To deter- desiccation UAS-eip75B(RNAi) trended in the same direction, but did mine where this 20E is produced, the expression of ec- not reach statistical significance (Fig. 2c). dysone biosynthetic enzyme genes (Halloween Ecdysone has a myriad of activities beyond its classic role cytochrome P450s) was quantified from different tissues in controlling the insect life cycle. For example, 20E drives (Fig. 3b and Additional file 4: Figure S4, all expression PGRP-LC expression in S2* cells [8]. Additionally, 20E fed was normalized relative to the head sample in the food to adult Drosophila restores normal aging to otherwise control group). Desiccation upregulated Halloween long-lived EcR mutant females, restores courtship memory genes in several somatic tissues. The most striking ef- defects and promotes sleep [12, 31, 32]. Here, 20E fed to fe- fects (Fig. 3b) were observed for disembodied (dib), a males induced the expression of EcR and PGRP-LC mRNA cytochrome P450 with ecdysteroid 22-hydroxylase Zheng et al. BMC Biology (2018) 16:60 Page 5 of 14 Fig. 3 Water-loss dehydration increases ecdysone and Malpighian tubule (MT) expression of ecdysone biosynthetic genes in young females (7 days). a 20E titer (pg/fly) (mean and SEM among three biological replicates, each of 25–30 pooled females) from whole females of two genetic backgrounds (yw and wDah) with control (food or PBS) and desiccation treatments. Independent biological replicates were prepared on separate days. One-way ANOVA within each genotype, Tukey’s post hoc comparison: *p <0.05, **p <0.01. b Relative disembodied (dib) mRNA in female tissues from control (food or PBS) and desiccation treatments. All values normalized to mean value from food treatment, head sample; mean values of three biological replicates, each containing the specific tissue from 15 females. Statistical comparisons are made within each tissue by t test for difference between desiccation and PBS treatment: dib mRNA was significantly elevated in muscle and MT (*p <0.05 with Holm–Bonferroni Sequential correction). c Relative mRNA for EcR and (d) PGRP-LC in control genotype (+/dib(RNAi)) and when dib is depleted in stellate cells (c724>) or principal cells (c324>). Values are mean and SEM of three independent biological replicates, normalized relative to food treatment, wild-type for each mRNA type. For each genotype: one-way ANOVA with Tukey’s post hoc comparison. For the control genotype (+/dib RNAi) a significant difference was observed between the flies exposed to desiccation compared to those exposed to normal food or PBS (*p < 0.05, **p < 0.01). However, no significant differences were observed when EcR or PGRP-LC were knocked down (c324 >RNAi and c724 >RNAi); EcR-RNAi F = 0.52, p =0.62 and F = 4.43, p = 0.066; PGRP-LC-RNAi F = 0.39, p =0.70 and F = 0.54, p =0.61 activity; control-treated females expressed dib strongly significantly less dib mRNA in the fat body relative to food in ovaries (via follicle cells [33]) and modestly in fat controls (p = 0.019). This effect on fat body expression of body, while little dib mRNA expression was observed in dib appears to be due to a starvation response rather than a other tissues. However, upon desiccation, dib mRNA desiccation response. No significant differences in dib ex- was significantly increased in muscle and MTs. Accord- pression in ovaries were observed among treatments. ingly, when dib mRNA was reduced by RNAi in stellate or principal cells (Additional file 5: Figure S5), EcR and Aged flies exhibit desiccation-induced ecdysone-mediated PGRP-LC expression was not induced in MTs by desic- recognition-protein priming cation relative to food or PBS controls (Fig. 3c, d). Interestingly, water loss rates and AMP gene expression Together, these data show that desiccation controls ec- both increase in aged Drosophila [16, 34–36]. In addition dysone synthesis within MTs, which locally regulates to higher AMP expression at baseline, older flies also in- recognition-protein priming. On the other hand, females duce higher levels of AMP gene expression when chal- maintained for 2 h without food (PBS controls) produced lenged with live bacteria [28, 35], suggesting that Zheng et al. BMC Biology (2018) 16:60 Page 6 of 14 recognition receptor expression could be affected by age. expressed in ovaries of old, food-reared females, al- Indeed, expression of PGRP-LC as well as EcR was in- though in old females, fat body showed lower expression creased in MT from flies at age 40 days (Fig. 4a, b). Experi- under all conditions. Old females under control condi- mental dehydration did not further elevate PGRP-LC and tions (PBS) expressed somewhat more dib than young EcR mRNAs in aged animals, in contrast to their induction females in head (1.83 fold), muscle (1.71 fold), and gut in young flies. Ecdysone titers measured from aged flies (2.97 fold) (each tissue p < 0.03). Expression of dib was were also significantly elevated relative to young females in similarly low in the MT of young and old control fe- control (food) conditions, while desiccation increased 20E males, but significantly induced by desiccation in old in both young and old animals (Fig. 4c). These data suggest MTs (3.5 fold), as observed in young females, and con- that aging is associated with recognition-protein priming sistent with the ecdysone titers. mediated through elevated ecdysone. Aged females may produce ecdysone because they are in- To identify the source of ecdysone in aged flies, tissue trinsically water stressed. To explore this hypothesis, co- was dissected from 40 day females exposed to control horts of yw adults were maintained in normal food (food, PBS) and dehydration treatments. Dib mRNA was conditions in three levels of relative humidity (RH), namely quantified relative to young females. Relative to young 20% RH, 40% RH (incubator standard condition), and 80% females, old females expressed dib in similar but muted RH (Additional file 6: Figure S6). Water content was mea- patterns (Fig. 4d, compared to Fig. 3b). dib was still sured in flies sampled from each RH cohort across 7 weeks Fig. 4 Peptidoglycan recognition-protein priming increases with age. (a) EcR and (b) PGRP-LC mRNA from Malpighian tubules (MTs) of young (7 days) and old (40 days) yw females in control (food, PBS) and desiccation treatments. Expression was normalized relative to young flies in the food treatment control; mean of three independent biological samples with SEM; two-way ANOVA with age and treatment as main effects, Tukey’s post hoc comparison, *p < 0.05, **p < 0.01. c 20E extracted from 7- and 40-day-old wild-type females (yw and wDah) in control (food, PBS) and desiccation treatments; mean of three independent biological samples with SEM. Within each genotype, two-way ANOVA with age and treatment as main effects, Tukey’s post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001. d Disembodied (dib) mRNA from specific tissues of old females (40 days) in control (food, PBS) and desiccation treatments; normalized relative to food treatment, head tissue of young females; mean of three independent biological samples for each tissue type. Statistical comparisons within each tissue by t test for difference between desiccation and PBS treatment: dib mRNA was significantly elevated in MTs (*p<0.05 with Holm-Bonferroni Sequential correction) Zheng et al. BMC Biology (2018) 16:60 Page 7 of 14 of age (Fig. 5a). Consistent with reports where water loss Defensin in cohorts at 80% RH increased to an extent that rates weredirectlymeasured in aging Drosophila [16], was significantly less than observed at 20% RH (two-way 2 2 water content per fly declined with age in all cohorts (χ = ANOVA for age and RH, each mRNA: χ >4.3, (age x humidity) 102.9, p < 0.0001), and at a significantly greater rate at 20% p < 0.04). Thus, innate immune aging is blunted in flies RH compared to 80% RH (χ = 50.02, p < reared at RH that reduces intrinsic water stress, decreasing (age x humidity) 0.025). Lifespan was similar among the 40% and 80% RH recognition protein expression and AMP gene levels. This cohorts (median life span: 44 and 46 days respectively), but amelioration in water loss and immunosenescence also had significantly less at 20% RH (median life span: 40 days) a modest, but significant, effect on lifespan. (Fig. 5b). Remarkably, elevated RH sharply blunted the in- creased expression of PGRP-LC, EcR,and the AMP gene Desiccation-triggered ecdysone-mediated recognition- Defensin typically observed in the MT of aged adults protein priming leads to enhanced host defense (Fig. 5c–e). MTs from females at 20% RH strongly upregu- We were also interested to examine how recognition-protein lated PGRP-LC, EcR,and Defensin by age 6 weeks (age as priming might alter host defense. To test if main effect, each mRNA as independent variable: χ > 13.0, desiccation-induced recognition-protein priming affects sur- p < 0.001). In contrast, mRNA for PGRP-LC, EcR,and vival following bacterial infection, we exposed two wild-type Fig. 5 High relative humidity (RH) reduces water loss with age and slows innate immune aging. a Water content per adult female, cohorts sampled from age 1 to 7 weeks old; cohorts maintained at 20%, 40%, and 80% RH. Trajectory of water content among RH groups analyzed with Generalized Linear 2 2 Model Fit by maximum likelihood ratio test. For main effects: age affected water content (χ = 102.9, p < 0.0001) but humidity did not (χ = 1.23, p =0.54). For interaction effects: water content declined faster in 20% RH compared to 80% RH (χ = 50.0, p < 0.025); no significant difference in water loss rate between 40% and 80% RH (χ = 0.625, p = 0.43). All point estimates produced represent the mean from five independent biological samples comprised of 20 pooled females. b Survivorship of yw adult females aged under RH of 20%, 40%, and 80% (25 °C). Log-rank test of mortality: 20% versus 40%, p = 0.0003; 40% versus 80%, p = 0.616. Three cages of 125 flies used for each RH condition. c–e Relative PGRP-LC, EcR,and Defensin mRNA levels in MTs from yw females, mean and SEM, aged at 20%, 40%, and 80% RH (25 °C), sampled at 1, 2, 4, and 6 weeks; three samples of 15 females were censored from demography cages to produce three independent biological samples at each time point and condition. Analysis was performed with Generalized Linear 2 2 Model Fit by maximum likelihood ratio test for main(age, RH)and interaction(agex RH) effects. PGRP-LC:age, χ = 24.3, p < 0.001; RH, χ = 13.0, p = 0.0015; 2 2 2 2 2 2 age x RH, χ = 7.08, p = 0.029. EcR:age, χ = 13.3, p < 0.001; RH, χ = 11.3, p = 0.0035; age x RH, χ = 6.75, p = 0.034. Defensin:age, χ = 15.1, p < 0.001; RH, χ = 2.79, p = 0.095; age x RH, χ = 4.32, p = 0.038 Zheng et al. BMC Biology (2018) 16:60 Page 8 of 14 fly strains (wDah and yw) to 2 h desiccation and then immedi- improved their survival relative to all other infected cohorts, ately infected the adults with the Gram-negative pathogen including non-desiccated controls (Fig. 6a–c and Additional Erwinia carotovora carotovora 15 (Ecc15). Desiccated flies file 7: Figure S7). Importantly, this improved survival following were significantly more susceptible to Ecc15 infection com- septic infection required ecdysone signaling and PGRP-LC ex- pared to those without prior desiccation (Additional file 7:Fig- pression in the MT; knockdown of EcR or of ure S7). Given that PGRP-LC expression is elevated upon PGRP-LC in MT principal cells eliminated the sur- desiccation, these results were unexpected and led us to vival benefit of desiccation with recovery following hypothesize that some time, following desiccation, may be re- Ecc15 challenge (Fig. 6d, e). These results demon- quired for PGRP-LC mRNA to produce functional receptors. strate that MTs contribute to host defense, and that Accordingly, we allowed the desiccated flies to recover upon recognition-protein priming, specifically in this renal regularfoodfor 3or6hprior to Ecc15 challenge. Wild-type organ, improves outcomes following septic bacterial flies that were allowed to recover for 6 h significantly challenge. Fig. 6 Functional immunity enhanced by desiccation stress response requires EcR and PGRP-LC in Malpighian tubules (MTs). a–e Kaplan–Meier survival plots of adult females sterile pricked or infected with Ecc15, with or without prior desiccation (des) and either no recovery, 3 h recovery, or 6 h recovery. Genotypes: a yw; b +/EcR(RNAi); c +/PGRP-LC(RNAi); d c324-gal4/UAS-EcR(RNAi); e c324-gal4/UAS-PGRP-LC(RNAi). Each survival curve is from a combined lifetable of two cohorts tested at different times, each cohort initiated with 90 females. For survival analyses (right censored log-rank test) conducted with six pair-wise comparisons within each genotype; curves with different letters differ significantly by at least p < 0.02 (details in Additional file 9:Table S1). f Microbial load (colony-forming units) 48 h after Ecc15 infection of adult females across treatments by desiccation (0 or 2 h) and post-desiccation recovery (0, 3,6 h).Genotypes as in (a–e): wildtype controls (yw, +/EcR, +/PGRP-LC) and EcR or PGRP-LC knock-down in principal cells (c324 > RNAi). For each genotype, three independent samples were prepared from each treatment group, in both cohort trials (displaying mean and SD of all six observations). CFU compared by ANOVA with Sidak post-hoc comparison, bars indicate differences with p < 0.05, see details in Additional file 10:Table S2 Zheng et al. BMC Biology (2018) 16:60 Page 9 of 14 Improved survival upon septic bacterial challenge can dehydration induces MT to express ecdysone biosynthetic arise from tolerance to the infection as well as from immune genes and targeted reduction of dib within MT stellate or defenses that reduce microbial load, often referred to as re- principal cells prevented induction of PGRP-LC during sistance [37]. To determine whether recognition-protein water-loss dehydration. How MTs regulate dib and other priming affects tolerance or resistance, we measured Ecc15 Halloween gene expression in response to water loss is un- colony-forming units (CFUs) from flies treated as in the in- known. Desiccation can reduce Drosophila neuropeptide fection survival studies. Flies were initially inoculated with capa-1 (homolog of human Neuromedin U) and increase ~6.0 × 10 CFUs of Ecc15 (0 h, Additional file 8:FigureS8). short neuropeptide F and tachykinin in protocerebral neu- 2+ At 48 h post infection, wild-type flies harbored less Ecc15 if rosecretory cells [42–44]. Capa-1 controls intracellular Ca they had been desiccated and allowed to recover prior to in- and nitric oxide/guanosine 3′,5′-cyclic monophosphate sig- fection (Fig. 6f), consistent with their improved survival. In naling in MTs [42]. Further, tachykinin induces insulin pro- contrast, flies with EcR or PGRP-LC expression knock-down duction and insulin signaling within MTs [45]. Notably, specifically in their MT had a higher Ecc15 load at 48 h post insulin signaling has the capacity to stimulate ecdysone infection, and desiccation with recovery did not reduce infec- production in the prothoracic gland of developing larvae tion load. Thus, recognition-protein priming in adult MTs [46–48]. It remains to be determined whether similar neu- provides functional whole animal immune resistance to sep- roendocrine factors control Halloween gene expression and tic bacterial challenge, leading to improved survival. recognition-protein priming in MTs. Female Drosophila use cholesterol to produce ecdys- Discussion one in egg follicles. In the fat body, this ecdysone is con- Water-loss dehydration triggers recognition-protein verted by 20-hydroxylase CYP314A1 (shade) to 20E, priming within adult Drosophila MTs. This response is which activates nuclear hormone receptor EcR and stim- mediated by the local synthesis and signaling of ecdys- ulates yolk production [49, 50]. In the ovary, ecdysone one within MTs, which induces expression of the micro- also regulates germline stem cells, somatic cysts, and bial recognition protein PGRP-LC. Elevated PGRP-LC border cells [33]. Other adult tissues in female insects potentiates the expression of AMP mRNA in tubules. do not produce ecdysone de novo, although some may Increased AMP gene expression is expected to produce complete its final synthesis, including the step involving more mature AMPs, although the study of the peptides dib [51]. Ecdysone synthesis also occurs in adult males, is hampered by a lack of effective antibodies. Our ana- where its production may take place in accessory glands lysis of survival after bacterial challenge with desiccation [52–54]. We find that muscle, gut, and MT, express dib and recovery suggests that recognition-protein priming when adults are dehydrated; these tissues may thus pro- in the MT provides functional antimicrobial resistance duce ecdysone during physiological stress, or at least to the entire animal. Moreover, the load of infecting mi- complete its synthesis. Consistent with this idea, crobe is reduced in wild-type flies able to mount Rauschenbach et al. [10] and Terashima et al. [11] noted recognition-protein priming, but not in flies where that nutrient restriction and heat increased 20E levels in PGRP-LC or EcR expression is reduced specifically in adult Drosophila. MTs, highlighting a previously unknown role for this Ecdysone has many potential somatic targets within organ in whole animal antimicrobial defense. adult Drosophila [24, 30]. Notably, 20E can modulate fly Recognition-protein priming induced by water-loss de- aging. Males and females heterozygous for mutant EcR hydration may be an adaptive response if dehydration are long-lived, and resistant to the oxidizing agent para- coincides with infection. Observations on this topic are quat as well as to heat and starvation despite being sur- scarce. Virulence in the waxmoth Galleria mellonella prisingly fertile [31]. Tricoire et al. [55] subsequently was increased by infection with Beauveria bassiana that found that mild repression of EcR also extended male exogenously expressed diuretic hormone, which stimu- lifespan, while EcR inactivation decreased female lifespan lates host water loss [38]. In Drosophila, septic infection except when egg production was genetically suppressed. might increase if water-loss dehydration reduces barrier Besides control of egg production, ecdysone in adults protection of gut or cuticle [39, 40]. Alternatively, Imd has stress- and aging-related functions, although where signaling primed by water-loss dehydration could pro- and how these work remain poorly understood. vide novel immune protective or tolerance functions. Here, we studied Drosophila innate immune aging in For instance, Relish (NF-κB) regulates expression of the context of desiccation stress. Water loss may be a cathepsin-L required for remodeling fat body in the typical feature of fly aging. Aged Drosophila experience moth Helicoverpa armigera, and Har-Relish is modu- elevated water loss and are less resistant to desiccation lated by ecdysone signaling in this moth [41]. stress [56, 57], susceptibilities that may be caused by ac- In Drosophila, recognition-protein priming is mediated celerated cuticle water transpiration [16]. Cuticle lipids by ecdysone produced within the MT. Water-loss regulate barrier transpiration in adult insects and this Zheng et al. BMC Biology (2018) 16:60 Page 10 of 14 layer may deteriorate or change composition during caused a marked sensitivity to infection. How MTs con- aging [15, 58, 59]. We propose that recognition-protein tribute to immunity and host defense will be the focus priming and increased AMP response in the aged arise of future studies. in part because old flies experience high water loss. Con- In addition, aged flies also display symptoms consist- sistent with this idea, cohorts maintained at high RH re- ent with water-loss dehydration. Aged flies, reared at pressed the typical age-related increase in PGRP-LC, 20%, 40%, or 80% RH, were dehydrated, consistent with Defensin, and EcR expression. Conversely, flies at low earlier reports [58]. Aged flies also displayed increased RH were somewhat shorter lived, and previous work systemic 20E levels and increased expression of the in- suggests that chronic innate immune activation reduces nate immune recognition protein PGRP-LC and effec- life expectancy in an NF-κB-dependent manner [60]. tors. However, adults aged at high relative humidity The production of AMPs is costly, impacting physi- show less water loss, and have reduced expression of ology and fecundity [61], and is tightly regulated. AMP PGRP-LC, AMPs, and EcR in their tubules. Our study induction is robust but transient to minimize the dele- suggests that the steroid hormone ecdysone in old flies terious effects of immune activation. Likewise, the Imd is induced in response to age-related water imbalance, pathway, and AMP production, is regulated by ecdysone and this facilitates immunosenescence in Drosophila in the context of development and, as shown here, by renal MTs. stress. This steroid-mediated regulation likely optimizes the AMP response such that the benefits of this mech- Methods anism of host defense at least equal these costs. Steroid Drosophila strains modulation of AMP induction during development may Stocks were reared and maintained at 25 °C, 40% RH coordinate this feature of innate immunity with nutrient and 12-h light/dark cycles. Food media contained corn- demands and availability, which change during different meal (5.2%), sugar (11.0%), autolyzed yeast (2.5%; SAF growth phases. Alternatively, developmental steroid brand) and agar (0.79%) (w/v in 100 mL water) with regulation of AMP production may be an adaptation to 0.2% Tegosept (methyl 4-hydroxybenzoate, Sigma, St. the selective pressures associated with the different mi- Louis, MO, USA) as an antifungal agent. MT-specific crobial pathogens encountered with different develop- Gal4-driver stocks c724 (stellate cell) and c324 (principal mental stages. Furthermore, here we demonstrate that cell) were provided by Julian Dow (University of Glas- the adult flies use ecdysone to prime innate immune in- gow); second chromosomes containing these transgenes duction of AMPs in the renal organ in response to were crossed and balanced into a genetic background water-stress imbalance. Remarkably, the resulting ele- marked with yw, producing yw; c724-Gal4/Cyo and yw; vated expression of AMPs from these renal tissues ap- and c324-Gal4/Cyo. RNAi to reduce PGRP-LC, EcR, pears to protect adult animals from systemic, septic Eip75B, and dib was respectively provided from Vienna KK101636 infection. Stock Center as strains P{UAS-PRGP-LC RNAi}- GD1428 VIE-260B, P{UAS-EcR RNAi}v37058, P{UAS-Eip KK108982 KK106954 Conclusions 75B RNAi}VIE-260B, and P{UAS-dib R- In this study, we examined how water-loss dehydration NAi}VIE-260B. Genotypes (F1 offspring) were generated increases innate immune responsiveness in Drosophila from crosses of these UAS-RNAi stocks with MT MTs, and how this response improves outcomes follow- Gal4-drivers or by crosses with yw. wDah was provided ing infection and contributes to elevated innate immune by the laboratory of Linda Partridge (University College gene expression with age. In this model, the stress of London). water-loss dehydration increased systemic levels of 20E, which is responsible for the pattern recognition-protein 20E treatment priming observed in MTs. While it is not possible to 20E was fed to wDah females aged 7–10 days old by measure 20E levels in a tissue-specific manner, genetic transferring adults from standard laboratory diet to 1 g manipulations of both the 20E synthesis gene dib and fly instant food media with 2 mL of water (Genesee Sci- the EcR, demonstrate that the tubules are autonomously entific, San Diego, CA, USA) for 24 h containing vehicle responding to water-loss dehydration through the pro- control (20 μL ethanol) or vehicle with 20 μL 1 mM 20E duction of ecdysone and subsequent sensing of this hor- (Sigma cat H-5142). mone signal. Interestingly, the data presented here further argue that MTs serve a critical function in Desiccation protocol anti-bacterial immunity, wherein recognition-protein Prior to conducting a trial, adult flies were placed in priming in MTs is required for increased host defense vials with standard food at a density of approximately 15 observed after desiccation and recovery, and loss of per vial without anesthesia. Vials were maintained over- recognition-protein expression specifically in MTs night at 25 °C. To initiate each trial, flies from vials were Zheng et al. BMC Biology (2018) 16:60 Page 11 of 14 flipped without anesthesia into fresh vials that contained Quantitative RT–PCR standard food (food control), tissue soaked with 0.5 mL Total RNA was extracted from dissected tissue (MT, 0.5× PBS solution (PBS control), or nothing (desicca- head, muscle, gut, fat body, ovary) in Trizol reagent tion). MTs were dissected after 2 h of treatment from (Invitrogen, Carlsbad, CA, USA) and treated with DNase flies within 5 minutes, immediately transferred to Trizol, (Ambion). DNase-treated total RNA was quantified with and processed for RT-qPCR. Three replicate biological a NanoDrop ND-1000. cDNA was synthesized using samples of 15 MT (sampled from across five vials) were iScript cDNA Synthesis (Bio-Rad) and measured on an generated per treatment. ABI prism 7300 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). For tissue-specific ana- lysis of AMP, PGRP-LC, EcR, or Halloween genes, three MT stimulation with TCT independent biological replicates for each tissue and Females (wDah) aged 7 to 10 days post eclosion, were each treatment group were analyzed. Each replicate in- processed through the desiccation protocol (with PBS cluded RNA extracted from 10 flies for head, muscle, as the only control). Dissected MTs were transferred gut, fat body, and ovary, or 15–20 for MT. Three tech- for 4 h to S2 cell culture medium with TCT nical replicates were used for each biological replicate. (0.01 mM dissolved in water) or without TCT. Ex- mRNA abundance of each gene was normalized relative pression of defensin and Cec-A2 was measured by to ribosomal protein L32 (rp49) by comparative CT. qRT-PCR from RNA extracted from these tubules im- mediately upon dissection (0 h), and at 4 h with or Enzyme immunoassay (EIA) of 20E without TCT. Three replicate biological samples were Female flies (25–30 flies/sample) were homogenized in produced for each measure set. 250 μL of ice-cold 100% methanol and centrifuged for 15 min at 18,000 g at 4 °C. Supernatants were trans- NanoString nCounter expression analysis ferred to 6 × 50 mm borosilicate glass tubes; precipitates The mRNA content of dissected MTs was analyzed were suspended in 200 μL of aqueous 75% methanol and by NanoString methodology according to published kept on ice for 30 min (adapted from [63]). Samples procedures [62]. Total RNA (100 ng per sample) was were centrifuged and dried with a SpeedVac centrifuge. hybridized to the target-specific codeset overnight at The precipitates were dissolved in EIA buffer at 4 °C 65 °C. The codeset used in Additional file 1:Figure and each individual sample was analyzed in a technical S1 contained probes against a panel of 45 genes en- triplicate by competitive EIA using 20E EIA antiserum coding AMPs, Imd pathway components, and four (Cayman Chemical: 482202) and 20E AChE Tracer loading controls (nucleostemin, alpha tubulin, Rp49, (Cayman Chemical: 482200). Calibration curves were and Faf2), while that in Additional file 2: Figure S2 generated from commercial 20E (Sigma, H5142). utilized an expanded codeset of 113 immunity-related genes and three loading controls (nucleostemin, alpha RH cohorts tubulin,and Faf2). The hybridized reactions were Newly enclosed females (yw) were mated for 2 days and loaded onto the NanoString Prep station, which separated into replicate 1 L demography cages at 125 removes excess reporter, binds the reporter to the flies per cage in an environmentally controlled room set cartridge surface, and stretches the probes for scan- at 40% RH, 25 °C, and 12 h light: 12 h dark cycles. Three ning. Subsequently, the cartridges were loaded onto independent cages (plus supplemental cages for dissec- the NanoString Digital Analyzer and scanned. The tion samples) were placed in 60 L clear plastic, nCounter data were normalized in two steps. In the tight-lidded storage boxes that maintained RH at 20%, first, data was normalized to positive spiked-in con- 40%, and 80%. To produce 20% RH, boxes contained a trols, as per the manufacturer’sinstructions(Nano- solution of saturated potassium acetate (Fisher Science String Technologies). In the second, the housekeeping Cat # P171–500) and solid desiccant (Drierite, W.A. genes were used to normalize overall mRNA levels. Hammond Co.). To produce 40% RH, the box was left For Fig. 1a, AMP gene expression was further nor- ajar, permitting cages to equilibrate at 40% RH as main- malized amongst the four biological replicates by set- tained in the environmental room. To produce 80% RH, ting the highest value for each gene to 100% and boxes contained a solution of saturated ammonium sul- plotted with standard error of the mean. For heat fate (Sigma-Aldrich Cat # A4418-500G). Boxes were maps in Additional file 1:FigureS1and Additional managed every second day at room temperature and file 2: Figure S2, only genes with expression greater room RH, at which time dead flies were removed and than 50 normalized counts in one or more conditions counted. Saturated solutions (and Drierite) were replen- are shown, as lower counts are below the reliable de- ished weekly. HOBO data loggers (Onset Computer tection threshold for the nCounter. Corp) continuously monitored RH within boxes; after Zheng et al. BMC Biology (2018) 16:60 Page 12 of 14 maintenance, all boxes rapidly re-equilibrated to RH tar- analysis of AMP transcripts from isolated MTs of wDah females (7 days gets (Additional file 5: Figure S5). old) exposed to TCT for 4 h compared to unstimulated tubules. The mean of four independent biological replicates, harvested on separate Longevity analysis was conducted with JMP statistical days, is shown by heatmap. Scale as indicated in side bar. (PDF 797 kb) software (SAS Institute, Cary, NC, USA). Data from three Additional file 2: Figure S2. Heat map summarizing innate immune replicate cages were combined to produce genotype and gene expression profiles in desiccated Malpighian tubules (MTs). MTs of treatment cohort life tables. Mortality distributions were 7-day-old wDah flies exposed to 2 h desiccation or PBS control treatment were excised, and then either RNA was immediately isolated, or were compared by Log-Rank and proportional hazard analyses. treated for 4 h with TCT or mock treated, and then RNA was isolated. Flies to measure mRNA were maintained in parallel dem- These RNA samples were used to measure immune-related gene ography cages and sampled at 1, 2, 4, and 6 weeks. MTs expression by NanoString nCounter. Values shown by heat map represent the mean of four independent experiments. Scale as indicated in side were dissected directly from non-anesthetized adults and bar. (PDF 847 kb) subjected to RT-qPCR (15 females per biological replicate, Additional file 3: Figure S3. Desiccation stress upregulated and amplified three replicates per time point per humidity condition). TCT-induced expression of 13 AMP genes in Malpighian tubules. Analysis of in- Water content was determined from samples in a rep- dividual AMP gene expression (as indicated), data from nCounter experiments displayed by heat map in Additional file 2: Figure S2. Values represent the licate trial with cohorts at 20%, 40%, and 80% RH. Fe- mean of four biologically independent replicates, and error bars are standard males were sampled at 1, 2, 4, 6, and 7 weeks from each error of the mean. Statistical analysis was performed by two-way ANOVA and RH cohort. Point estimates for water content were each Sidak’s test for pair-wise comparison of desiccation to PBS treatment (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Side table displays statistical met- generated as the mean among five groups of 20 females; rics after Bonferroni correction for multiple comparisons for the interaction of these females were sampled from 8 demography cages, desiccation and TCT treatments from the same ANOVA analyses. (PDF 631 kb) each with 125 females for every RH condition. For each Additional file 4: Figure S4. mRNA expression of Halloween genes. group, females were weighed (Mettler Toledo Halloween genes (spook, phantom, shadow, and shade) were measured in adult tissues from young (7 days) and old (40 days) females. All values semi-micron balance), dried overnight at 65 °C, and normalized relative to head samples from young adults in food control reweighed. Age, RH treatment, and main-effect interac- group. The mean of three independent biological replicates is shown. tions were analyzed by generalized linear model likeli- (PDF 2424 kb) hood ratio test. Additional file 5: Figure S5. RNAi on disembodied (dib) in Malpighian tubules (MTs). Reduced dib mRNA in MTs when dib RNAi is driven in stellate (c724 > dib RNAi) or principal (c324 > dib RNAi) cells relative to Infection challenge survival control (ywT1/dib RNAi). Results shown represent the mean and SEM of three independent replicates. (PDF 897 kb) Adult survival to a controlled infection challenge after Additional file 6: Figure S6. Relative humidity (RH) and temperature desiccation treatment was determined using 100 to 120 recorded from demography chambers under different humidity flies per group (Additional file 5: Figure S5) or 180 flies conditions. Spikes indicate when chambers were opened to access cages per group (Fig. 6) (among two replicate vials). Females and show rapid homeostasis of the humidity control system. Blue tracings show realized RH, black tracing represents temperature. (PDF at 3 to 7 days old were exposed to food or PBS-only as 3450 kb) control, or to 2 h desiccation. Following these treat- Additional file 7: Figure S7. Impact of desiccation followed by ments, either immediately or after 3 and 6 h recovery on recovery on the survival of wild-type flies infected with Erwinia caroto- standard food, flies were pricked with a clean microsur- vora carotovora 15 (Ecc15). Kaplan–Meier survival of yw and wDah adult gery needle (control) or with a needle dipped into a con- females challenged with Ecc15. Uninfected flies were desiccated or not centrated pellet of (Ecc15)[64, 65]. Surviving flies were for 2 h and survival was monitored for 8 days. Other cohorts were chal- subsequently transferred daily to fresh vials and recorded lenged with Ecc15 infection with or without a prior 2 h desiccation treatment and recovery of 0, 3, or 6 h prior, as indicated. Plots represent as alive or dead for 8 days. Survival is represented as the survival kinetics of 100–120 files, combined from two separate trials, Kaplan–Meier plots with all pairwise differences evalu- for each genotype; cohorts with significantly different mortality are ated by log-rank test with significance adjusted for grouped with different letters (all cases, log-rank test p <0.05, see side multiple comparisons (p < 0.01). tables). (PDF 1741 kb) Additional file 8: Figure S8. Initial bacterial loads in Erwinia CFU counts carotovora carotovora 15 (Ecc15)-infected flies. Adult load (colony-forming units) of Ecc15 at 0 h post infection as a function of Bacterial load was determined in flies at time 0 and 48 h desiccation (0 or 2 h) and post-desiccation recovery (0, 3, 6 h); after infection with Ecc15. Individual flies were homoge- among three control genotypes (yw, +/EcR, +/PGRP-LC) and geno- nized in 200 μL of PBS. Homogenates were diluted in types where EcR or PGRP-LC were knocked down by RNAi in principal cells (c324 > RNAi), no significant differences were observed at 0 h. series and plated on LB-Ampicillin plates, incubated Results represent the mean of six assays and the error bars show overnight at 37 °C, and scored for CFU count. standard deviation. (PDF 1024 kb) Additional file 9: Table S1. Statistical analysis for the effect of desiccation on survival to Ecc15 infection with or without recovery treatment, supporting Additional files Fig. 6a-e.(DOCX 26 kb) Additional file 10: Table S2. Statistical analysis for the effect of desiccation Additional file 1: Figure S1. Heat map of differentially expressed innate on bacterial growth after Erwinia carotovora carotovora 15 infection with or immune transcripts in Malpighian tubules (MTs). Nanostring nCounter without recovery treatment, supporting Fig. 6f.(DOCX 15 kb) Zheng et al. BMC Biology (2018) 16:60 Page 13 of 14 Abbreviations 6. Chen L, Paquette N, Mamoor S, Rus F, Nandy A, Leszyk J, Shaffer SA, 20E: 20-hydroxyecdysone; AMP: antimicrobial peptide; Cec-A2: Cecropin-A2; Silverman N. Innate immune signaling in Drosophila is regulated by CFU: colony-forming unit; DAP-type PGN: diaminopimelic acid type- transforming growth factor beta (TGFbeta)-activated kinase (Tak1)-triggered peptidoglycan; dib: disembodied; Ecc15: Erwinia carotovora carotovora 15; ubiquitin editing. J Biol Chem. 2017;292(21):8738–49. EcR: Ecdysone receptor; Eip75: ecdysone induced protein 75; MTs: Malpighian 7. Stoven S, Silverman N, Junell A, Hedengren-Olcott M, Erturk D, Engstrom Y, tubules; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; Maniatis T, Hultmark D. Caspase-mediated processing of the Drosophila NF- PGRP: Peptidoglycan recognition protein; RH: relative humidity; TCT: tracheal kappaB factor Relish. Proc Natl Acad Sci U S A. 2003;100(10):5991–6. cytotoxin 8. Rus F, Flatt T, Tong M, Aggarwal K, Okuda K, Kleino A, Yates E, Tatar M, Silverman N. Ecdysone triggered PGRP-LC expression controls Drosophila Acknowledgements innate immunity. EMBO J. 2013;32(11):1626–38. We thank Julian Dow for sharing Malpighian tubule Drosophila strains as well 9. Meister M, Richards G. Ecdysone and insect immunity: the maturation of the as Rolf Bodmer and Paul Shaw for their contributions to the conceptual inducibility of the diptericine gene in Drosophila larvae. Insect Biochemistry work on functional aging. Mol Biol. 1996;26:155–60. 10. Rauschenbach IY, Sukhanova MZ, Hirashima A, Sutsugu E, Kuano E. Role of Funding the ecdysteroid system in the regulation of Drosophila reproduction under NS and MT were collaboratively supported by NIH grant PO1 AG033561. NS environmental stress. Dokl Biol Sci. 2000;375:641–3. was independently supported by RO1 AI099708. MT was independently 11. Terashima J, Takaki K, Sakurai S, Bownes M. Nutritional status affects 20- supported by R37 AG024360. The funders had no role in study design, data hydroxyecdysone concentration and progression of oogenesis in Drosophila collection and analysis, decision to publish, or preparation of the manuscript. melanogaster. J Endocrinol. 2005;187(1):69–79. 12. Ishimoto H, Kitamoto T. The steroid molting hormone Ecdysone regulates Availability of data and materials sleep in adult Drosophila melanogaster. Genetics. 2010;185(1):269–81. All data generated or analyzed during this study are included in this 13. Ishimoto H, Kitamoto T. Beyond molting–roles of the steroid molting published article and its supplementary information files. All material used, hormone ecdysone in regulation of memory and sleep in adult Drosophila. such as Drosophila strains, are available from public stock centers and other Fly. 2011;5(3):215–20. repositories. 14. Dow JA. Insights into the Malpighian tubule from functional genomics. J Exp Biol. 2009;212(Pt 3):435–45. Authors’ contributions 15. Nghiem D, Gibbs AG, Rose MR, Bradley TJ. Postponed aging and WZ designed, performed, and analyzed experiments. FR designed, desiccation resistance in Drosophila melanogaster. Exp Gerontol. 2000; performed, analyzed experiments, and contributed to the drafting and 35(8):957–69. revising of the manuscript. AH performed and analyzed some of the 16. Gibbs AG, Markow TA. Effects of age on water balance in Drosophila experiments presented in Fig. 1. PK designed and performed RH controlled species. Physiol Biochem Zool. 2001;74(4):520–30. experiments. WG provided critical reagents. NS and MT together conceived 17. Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM, Lemaitre B, of this study, contributed to experimental design and analysis and Hoffmann JA, Imler JL. Tissue-specific inducible expression of antimicrobial manuscript preparation, and supervised all aspects of this study. All authors peptide genes in Drosophila surface epithelia. 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