key: cord-0303695-kf04tra6
authors: Sheffield, Lakbira; Sciambra, Noah; Evans, Alysa; Hagedorn, Eli; Delfeld, Megan; Goltz, Casey; Fierst, Janna L.; Chtarbanova, Stanislava
title: Age-dependent impairment of disease tolerance is associated with a robust transcriptional response following RNA virus infection in Drosophila
date: 2020-09-21
journal: bioRxiv
DOI: 10.1101/2020.09.21.307017
sha: e42985203f7dc733e23525ac2f833dfaf9dc6445
doc_id: 303695
cord_uid: kf04tra6

Advanced age in humans is associated with greater susceptibility to and higher mortality rates from infections, including infections with some RNA viruses. The underlying innate immune mechanisms, which represent the first line of defense against pathogens, remain incompletely understood. Drosophila melanogaster is able to mount potent and evolutionarily conserved innate immune defenses against a variety of microorganisms including viruses and serves as an excellent model organism for studying host-pathogen interactions. With its relatively short lifespan, Drosophila also is an organism of choice for aging studies. Despite numerous advantages that this model offers, Drosophila has not been used to its potential to investigate the response of the aged host to viral infection. Here we show that in comparison to younger flies, aged Drosophila succumb more rapidly to infection with the RNA-containing Flock House Virus (FHV) due to an age-dependent defect in disease tolerance. In comparison to younger individuals, we find that older Drosophila mount larger transcriptional responses characterized by differential regulation of more genes and genes regulated to a greater extent. Our results indicate that loss of disease tolerance to FHV with age possibly results from a stronger regulation of genes involved in apoptosis, activation of the Drosophila Immune deficiency (IMD) NF-kB pathway or from downregulation of genes whose products function in mitochondria and mitochondrial respiration. Our work shows that Drosophila can serve as a model to investigate host-virus interactions during aging and sets the stage for future analysis of the age-dependent mechanisms that govern survival and control of virus infections at older age.

Advanced age in humans is associated with greater susceptibility to and higher mortality rates 23 from infections, including infections with some RNA viruses. The underlying innate immune 24 mechanisms, which represent the first line of defense against pathogens, remain incompletely 25 understood. Drosophila melanogaster is able to mount potent and evolutionarily conserved 26 innate immune defenses against a variety of microorganisms including viruses and serves as 27 an excellent model organism for studying host-pathogen interactions. With its relatively short 28 lifespan, Drosophila also is an organism of choice for aging studies. Despite numerous 29 advantages that this model offers, Drosophila has not been used to its potential to investigate 30 the response of the aged host to viral infection. Here we show that in comparison to younger 31 flies, aged Drosophila succumb more rapidly to infection with the RNA-containing Flock House 32

Virus (FHV) due to an age-dependent defect in disease tolerance. In comparison to younger 33 individuals, we find that older Drosophila mount larger transcriptional responses characterized 34 by differential regulation of more genes and genes regulated to a greater extent. Our results 35

indicate that loss of disease tolerance to FHV with age possibly results from a stronger 36 regulation of genes involved in apoptosis, activation of the Drosophila Immune deficiency 37 (IMD) NF-kB pathway or from downregulation of genes whose products function in 38 mitochondria and mitochondrial respiration. Our work shows that Drosophila can serve as a 39 model to investigate host-virus interactions during aging and sets the stage for future analysis 40

Infectious diseases, including viral infections, represent an important burden among the 46 elderly. For instance, older age is a major risk factor for increased morbidity and mortality to 47 numerous viral pathogens including the Severe acute respiratory syndrome (SARS) associated 48 coronavirus-2 (SARS-CoV-2), the agent responsible for the current COVID-19 pandemic 49 (NIKOLICH-ZUGICH et al. 2020). Immunosenescence, a collective term used to describe the 50 progressive functional decline of the immune system over time, is associated with the 51 increased susceptibility to infections and lower responsiveness to vaccination observed in the 52 elderly (LENG AND GOLDSTEIN 2010). Considerable progress has been made in understanding 53 how aging affects both, the innate and adaptive immune systems, however, the causes 54 underlying immunosenescence remain incompletely elucidated. In particular, the age-55 dependent mechanisms leading to dysregulated innate immunity, which represents the first 56 In the present study, we conducted comparative analysis of survival, virus load and 106 gene expression between young and aged Drosophila following infection with the Flock House 107

Virus (FHV). FHV is a small, non-enveloped virus, whose genome is composed of two positive, 108 single-stranded RNA molecules (VENTER AND SCHNEEMANN 2008). We report that older flies 109 succumb faster to FHV infection without accumulating higher virus loads, suggesting that a 110 tolerance mechanism becomes impaired with age. Additionally, we show that aged flies mount 111 a more robust transcriptional response to FHV than young flies, including the regulation of 112 innate immunity genes; response, which is different from the response of flies undergoing 113 aging. Genes encoding components of the apoptotic process are predominantly regulated in 114 aged, FHV-infected flies. Additionally, we show that several genes whose gene products 115 function in mitochondria and mitochondrial respiratory chain are specifically downregulated in 116 aged, FHV-infected flies. We also demonstrate that among genes that do not belong to specific 117 gene ontology categories, the expression of several encoding for non-coding RNAs (ncRNAs) 

To determine how age affects survival to infection with FHV, we injected 5-day old and 30-day Figure 1B) . Interestingly, although survival curves overlapped at 5 days of age between 138 both sexes ( Figure 1A and Figure S1A ), virus load was significantly lower in females in 139 comparison to males ( Figure 1B ). At 30 days of age, females showed significant, two-fold 140 decrease in virus load in comparison to males ( Figure 1B) , which was accompanied with 141 slightly better, although non-significantly different median survival to FHV (6.75±0.17 days for 142 males and 7.17±0.28 days for females, Figure S1A ). In support of the data obtained for 143

OregonR male flies, similar differences in survival between 5-and 30-day old flies, and 144

comparable FHV1 load at 72h p.i. between animals of the two age groups was observed for 145 males of another genotype, y1 w67c23 ( Figure S1B ). Additionally, we found non-significant 146 differences between FHV titers in circulating hemolymph (insect blood) of 5-and 30-day old 147 female w1118 flies 96h p.i. ( Figure S1C ). accounting for the observed increase in mortality. To test this, we performed transcriptomics 159 analysis using RNA sequencing (RNA-Seq) on 7-day old (young) and 25-day old (aged) male 160

OregonR Drosophila injected with either Tris or FHV at 24h and 48h following injection. This 161 sex was chosen because aged males showed more pronounced effect on survival than 162 females. The time points were chosen early in the infection process before differences in 163 survival between age groups were detected. As an additional control, we used non-infected 164 young and aged flies to control for the effects of aging alone in absence of infection. An 165 average of 95.4% of each RNA-Seq library (Table S1) aligned to the D. melanogaster genome 166 (Table S2) . We validated the RNA-Seq data for aging and the 48h post FHV infection time 167 point using specific primers and RT-qPCR analysis for four genes per experimental condition. 168

We confirmed that in aging flies Cpr67Fb and CG15199 were upregulated and Acp54A1 and 169

Lman III were downregulated. In young Drosophila, 48h after FHV infection, Upd2 and Ets21c 170 were upregulated and Rfabg and Diedel 3 were downregulated in comparison to Tris-injected 171 controls. In aged flies, FHV infection led to upregulation of Or85a and Upd3 and 172 downregulation of IM14 and GNBP-Like 3 ( Figure S2 ). 173

To evaluate the overall similarity and differences between treatments, we used principal Differential gene expression analysis following FHV infection revealed that more genes 186 were significantly regulated (p adj < 0.05) at least two-fold at 48h p.i. in comparison to 24h p.i. 187

in both age groups. More genes were differentially changed in aged FHV-infected flies in 188 comparison to young flies for both time points ( Figure 2B , Table S3 ). Overall, in young flies, 189 the expression of 505 genes was differentially regulated 24h p.i. vs 1,168 genes 48h p.i. In 190 aged flies, we observed differential regulation of 816 genes at 24h p.i. and 2,625 genes at 48h 191 p.i. The process of aging itself differentially regulated expression of 1,639 genes ( Figure 2B ). 192

We note that in aging flies, more genes are downregulated than upregulated, whereas in aged, 193 FHV-infected flies there are fewer downregulated than upregulated genes ( Figure 2B) . 194

Among the genes differentially regulated during aging, we observed a very small 195 Altogether, these results indicate that aged male flies mount a larger transcriptional 202 response following FHV infection than younger flies, a signature that is different from the 203 transcriptional changes taking place during the aging process itself. The fact that most of 204 commonly regulated genes between young and aged FHV-infected flies were found to overlap 205 as a function of time (86% of up-and 87% of down-regulated genes, Figure S3 ), is in support 206 of the hypothesis that the age-dependent defect in disease tolerance is unlikely to result from 207 the regulation of these genes. Rather, our data suggest that impaired tolerance in aged flies 208 could be due to differential regulation of the genes that are uniquely expressed in infected 209 young flies, uniquely expressed in infected aged flies or a combination of both. 210

To visualize biological processes regulated by aging and FHV infection in young and aged 214 flies, we performed gene ontology (GO) analysis. The number of genes with Flybase ID (FBgn 215 number) without a matching DAVID ID is listed in Table S4 . We note that most differentially 216 regulated genes with a DAVID ID were labeled as "Others" ( Figure S4 ). For instance, 76% of 217 differentially regulated genes for the Aging group did not match a specific biological process. 218

For Young FHV24h, Young FHV48h, Aged FHV24h and Aged FHV48h, these percentages are 219 53%, 59%, 60% and 59%, respectively ( Figure S4 ). 220

Our GO analysis revealed a complex signature. For instance, aging led to changes in 221 expression of genes belonging to 57 biological processes. Five of them ('defense response', 222

'response to bacterium', 'antibacterial humoral response', 'defense response to Gram-positive 223 bacterium' and 'oxidation-reduction') overlapped between all five experimental conditions. 224 'Mannose metabolic process' and 'protein refolding' were in common between Aging and Aged 225

FHV24h groups and 'sperm storage' between Aging and Aged FHV48h groups. Processes 226 identified in common between the Aging group and young and aged FHV-infected flies were 227 'circadian rhythm', 'multicellular organism reproduction' and 'proteolysis' (Figure 3 and Table  228 S5). In Drosophila, aging leads to both, deregulation of organismal reproduction (TATAR 2010) At 24h p.i., we identified more biological processes in young flies than in aged animals 240 (96 vs 80, respectively), among which five overlapped between the two age groups. 70 and 26 241 biological processes were specific to Young FHV24h and Aged FHV24h, respectively ( Figure 3  242 and Table S5 ). At 48h p.i., we found an opposite trend with 81 and 135 biological processes in 243 young and aged flies, respectively, among which 23 overlapped. We found 20 and 63 244 biological processes to be specific to the Young FHV48h and Aged FHV48h groups, 245 respectively ( Figure 3 and Table S5 ). 246

In both young and aged flies, FHV infection led to differential regulation of genes 247 involved in processes associated with the nervous system. Clustering analysis identified one 248 module of 'neurogenesis' genes that were strongly upregulated in the Aged FHV48h group and 249 regulated to a lesser extent in Young FHV48h and Aged FHV24h groups ( Figure S5A ). For 250 instance, among genes belonging to this GO category at 48h p.i., the gene midlife crisis 251 Table S3 ). Other biological processes linked to the nervous system 256 development and function for which genes were enriched in young and aged FHV-infected 257 groups were 'lateral inhibition', 'sleep' and 'ventral cord development' (Table S5 ). The 258 significance of this regulation is not known as FHV has not been previously demonstrated to 259 target the nervous system, but rather the Drosophila heart and fat body (ELEFTHERIANOS et al. several heat shock proteins belonging to the biological process 'response to heat' were 265 upregulated in both young and aged FHV-infected flies (Table S3 and Table S5 ). This 266 suggests that following FHV infection, this branch of antiviral immunity is functional in aged 267

Interestingly, genes belonging to additional categories associated with nervous system's 269 function such as 'neuromuscular synaptic transmission', 'transmembrane transport' and 270

'neurotransmitter secretion' were specifically found in the Young FHV24h group. On the other 271 hand, among processes specific to Aged FHV24h we found 'autophagic cell death', and 272 'regulation of autophagy' (Table S5) . Among processes specifically enriched 48h p.i., we found 273 'regulation of transcription, DNA-templated', 'transmembrane receptor protein tyrosine kinase 274 signaling pathway' and "protein ubiquitination" in young flies and 'phagocytosis', 'programmed 275 cell death' and 'peptidoglycan recognition protein signaling pathway' in aged flies. The latter 276 category contained multiple genes encoding for components of the Drosophila IMD pathway. 277

Finally, among the processes specifically regulated in aged flies at both 24h and 48h p.i., we 278 found 'apoptotic process', 'determination of adult lifespan' and 'chromatin remodeling' (Table  279 S5). 280

Overall, these results indicate that despite a large number of "other" genes, genes 281 belonging to identifiable common and distinct categories of biological processes are regulated 282 by aging and FHV infection of young and aged flies. Although our results identify specific 283 categories of biological processes for each experimental group (Figure 3) , at this stage we are 284 not able to determine whether the age-associated impairment of tolerance depends on the 285 regulation of genes that are specifically regulated in young or/and aged flies. Table S3 ). Interestingly, in both young and aged flies, FHV infection led to 298 strong downregulation of most AMP and IM genes, despite a robust upregulation of the mRNA 299 encoding the NF-κB factor Relish ( Figure 4A and Table S3 ). In aged FHV-infected flies, we 300 observed marked upregulation of IMD pathway components PGRP-LE, imd, key (IKKγ) and 301

AttD. This upregulation was to a greater extent in the Aged FHV48h group ( Figure 4A and 302 Figure S6 ). In comparison to aging and young FHV-infected Drosophila, we found dSTING, 303 whose product acts upstream of Relish to protect flies against infection with DCV and CrPV Figure 4A ). This suggests that older animals respond to injury by upregulating innate immunity 308 genes toa greater extent than younger flies. 

We took a closer look at the differentially regulated genes, which were labeled as 'other' in our 359 GO analysis ( Figure S4 ). We observed that most of these genes are uncharacterized 360 (categorized as candidate genes, or CG); several are non-coding RNAs (ncRNA); and others 361 have previously described function but do not fit a specific DAVID GO category. Among We compared the number of ncRNAs differentially regulated at least two-fold in our 369

aged FHV-infected than in young FHV-infected flies (68 vs 42 genes 24h p.i. and 267 vs 111 371 genes 48h p.i.) ( Figure 6A , B and Figure S8A ). Aging itself regulated the expression of 202 372 ncRNA genes. As observed for the total number of transcripts, ncRNAs, which were regulated 373 by infection shared minimal overlap with aging ( Figure 6B and Figure S8A ). Among ncRNAs, 374 we identified the largest proportion to correspond to lncRNAs. For all experimental groups we 375 also found asRNAs and small nucleolar RNAs (snoRNAs). In young FHV-infected flies, a small 376 percentage of ncRNAs corresponded to stable intronic sequence RNAs (sisRNAs). 377

Specifically, in aged, FHV-infected flies we found differential regulation of ncRNAs that belong 378 to small nuclear (snRNAs) and small non-messenger RNAs (snmRNAs) ( Figure S8B) . 379

We compared the expression of CR45445 (an asRNA) and CR46083 (an lncRNA) 380 genes 48h p.i. by RT-qPCR. Consistent with the RNA-Seq data, we observed significant 381 increase in CR45445 and significant decrease in CR46038 expression in comparison to Tris-382 injected controls in aged, but not young flies ( Figure 6C ). Together, these results indicate that 383 both, aging and FHV infection affect the expression of genes encoding different categories of 384 ncRNAs, and that specific ncRNAs are regulated in the aged organism after FHV infection. 385

We used the highly tractable genetic model Drosophila melanogaster to investigate the 387 response of the aged organism following infection with the RNA(+) virus FHV. We found that 388 30-day old flies died faster than younger flies to FHV infection and that older, but not younger 389 males were more sensitive than females. Although for both sexes we did not observe a 390 difference in virus load as a function of age, our results indicate higher FHV titers in younger 391 males in comparison to younger females, for which survival curves overlap. Although we 392 cannot exclude genetic background-specific effects, our results raise the interesting question 393 of whether control of virus replication in the young organism represents a sexually dimorphic 394 trait. We observed that older males die faster than older females and contain twice the level of 395 FHV RNA1 transcript than females. This could potentially indicate that in comparison to 396 females, younger males are able to tolerate higher FHV loads, but that this ability becomes 397 impaired with age and results in more rapid death. Indeed, it is increasingly recognized that However, additional studies including small RNA sequencing during aging to compare the 420 abundance of siRNAs against the FHV genome, are needed to determine whether this is the 421

We cannot entirely rule out the possibility that aging impacts resistance mechanisms in 423 a tissue-specific way, differences which cannot necessarily be detected by measuring virus 424 load in whole flies. It therefore would be very informative to perform additional studies to 425 determine whether FHV differentially targets tissues at different ages and whether FHV load 426 differs among tissues as a function of age. For instance, it is appreciated that aging affects 427 gene expression differently in different tissues and in mammalian models differentially 428 expressed genes in a given tissue are often not genes specific to this tissue (RODWELL et al. One striking finding of this study is that aged flies infected with FHV mount a more 439 robust transcriptional response than younger flies. The fact that at 48h after FHV infection we 440

find an overlap between 93% of upregulated genes and 57% of downregulated genes in young 441 flies with genes regulated in aged flies, suggests that most of the transcriptional response to 442 FHV is maintained as a function of age. However, aged flies show extensive regulation of 443 additional genes. One possibility was that these additional genes are related to the process of 444 aging itself. We show, however, that the overlap between the transcriptional profiles of aging, Our transcriptomic analyses reveal that as FHV infection progresses in aged flies, 488 genes associated with mitochondrial respiratory chain become downregulated. Additionally, we 489 notice that several transcripts of genes encoded by the mitochondrial genome (Table S3) days-old (labeled as 7d-old), and aged flies were 22-29 days-old (labeled as 25d-old), 

The Quick-RNA MiniPrep Kit (Zymo Research) was used to isolate total RNA following 563 manufacturer's instructions. RNA (1000ng) was converted to cDNA using the High Capacity 564 RNA-to-cDNA Kit (Applied Biosystems). RT-qPCR reaction was performed using 565

Power SYBR™ Green PCR Master Mix (Applied Biosystems) according to manufacturer's 566

instructions. Primer sequences are listed in Table S7 . For all assays, expression of RpL32 567 (Rp49) was used to normalize gene expression. Table S7 . 584

The authors affirm that all data necessary for confirming the conclusions of the article are 585 

Differential expression analysis for sequence count data

Sexual Dimorphisms in Innate 646 Immunity and Responses to Infection in Drosophila melanogaster

Decline in self-renewal factors contributes to 649 aging of the stem cell niche in the Drosophila testis

Mitochondria, Bioenergetics and Apoptosis in Cancer

Dnr1 mutations cause 652 neurodegeneration in Drosophila by activating the innate immune response in the brain

midlife crisis encodes a conserved zinc-finger protein 655 required to maintain neuronal differentiation in Drosophila

Drosophila C virus 657 systemic infection leads to intestinal obstruction

ATP-sensitive potassium 659 channel (K(ATP))-dependent regulation of cardiotropic viral infections

Evolution of 662 longevity improves immunity in Drosophila

Inflamm-aging. An 664 evolutionary perspective on immunosenescence

Essential 666 function in vivo for Dicer-2 in host defense against RNA viruses in drosophila

The interplay between immunity and aging in Drosophila

The Kinase IKKbeta Regulates a STING-671 and NF-kappaB-Dependent Antiviral Response Pathway in Drosophila

Phagocytic ability declines with age in adult Drosophila 676 hemocytes

Bioinformatics enrichment tools: paths 678 toward the comprehensive functional analysis of large gene lists

Systematic and integrative analysis of large 681 gene lists using DAVID bioinformatics resources

Broad RNA interference-mediated 683 antiviral immunity and virus-specific inducible responses in Drosophila

NF-kappaB Immunity in the

Brain Determines Fly Lifespan in Healthy Aging and Age-Related Neurodegeneration

Induced antiviral innate immunity in Drosophila

The host defense of Drosophila melanogaster

Impact of aging on viral infections

P53-mediated rapid induction 695 of apoptosis conveys resistance to viral infection in Drosophila melanogaster

Inflammation-698 Induced, STING-Dependent Autophagy Restricts Zika Virus Infection in the Drosophila 699 Brain

Senescence of the cellular immune response 701 in Drosophila melanogaster

Nucleic Acid Sensors and Programmed Cell Death

Disease Tolerance 705 as an Inherent Component of Immunity

The heat shock 708 response restricts virus infection in

Innate and intrinsic antiviral immunity in 710 Drosophila

The twilight of immunity: emerging concepts in aging of the immune 712 system

SARS-CoV-2 and 714 COVID-19 in older adults: what we may expect regarding pathogenesis, immune responses

Genome-wide 717 transcript profiles in aging and calorically restricted Drosophila melanogaster

Functional analysis of the Drosophila immune 720 response during aging

A transcriptional profile of aging 722 in the human kidney

Flock house virus induces apoptosis by depletion of 724 Drosophila inhibitor-of-apoptosis protein DIAP1

Comparative Flavivirus-Host Protein 726 Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis

From discovery to function: the expanding roles of long noncoding 729 RNAs in physiology and disease

Reproductive aging in invertebrate genetic models

Recent insights into the biology and biomedical 733 applications of Flock House virus

IIV-6 Inhibits NF-kappaB Responses in 735 Drosophila

Aging of the innate immune response in 737 Drosophila melanogaster

Temporal and spatial transcriptional 739 profiles of aging in Drosophila melanogaster

Mitochondrial respiratory chain complex IV

Mitochondrial respiratory chain complex I (ND15, ND-B12

We thank Dr. Annette Schneemann for FHV virus stock and the anti-FHV antibody used in this 592 study. We are grateful to Drs. David Wassarman and Grace Boekhoff-Falk for critical reading 593 of the manuscript. The Authors declare that they have no conflict of interest. C.

Up-regulated ncRNA genes Down-regulated ncRNA genes CR45445 CR46083