Dietary Supplementation of Aspirin Promotes Drosophila Defense against Viral Infection

Aspirin, also known as acetylsalicylic acid, is widely consumed as a pain reliever and an anti-inflammatory as well as anti-platelet agent. Recently, our studies using the animal model of Drosophila demonstrated that the dietary supplementation of aspirin renovates age-onset intestinal dysfunction and delays organismal aging. Nevertheless, it remains probable that aspirin plays functional roles in other biological activities, for instance antiviral defense reactions. Intriguingly, we observed that the replications of several types of viruses were drastically antagonized in Drosophila macrophage-like S2 cells with the addition of aspirin. Further in vivo experimental approaches illustrate that adult flies consuming aspirin harbor higher resistances to viral infections with respect to flies without aspirin treatment. Mechanistically, aspirin positively contributes to the Drosophila antiviral defense largely through mediating the STING (stimulator of interferon genes) but not the IMD (immune deficiency) signaling pathway. Collectively, our studies uncover a novel biological function of aspirin in modulating Drosophila antiviral immunity and provide theoretical bases for exploring new antiviral treatments in clinical trials.


Introduction
Viruses are a type of microorganism that may have a wide range of negative effects on the human body including damages to tissues/organs, immune system dysfunctions, respiratory illnesses, neurological symptoms, and increased risks of cancers. In order to prevent or treat viral infections, a large body of efforts have been dedicated to the field of "exploring antiviral drugs", which was obviously one of the hottest scientific topics during the global pandemic of COVID-19 (caused by the SARS-CoV-2 virus). In general, antiviral drugs function by targeting specific parts of viruses, thereby preventing them from reproducing and spreading throughout the body. When taken as directed, these drugs can help to reduce the severity and duration of viral illnesses and prevent complications (for details, please see reviews including [1][2][3][4][5][6][7]). To date, there has been a significant breakthrough in the development of antiviral drugs, as many of them, including nucleoside inhibitors, protease inhibitors, neuraminidase inhibitors, entry inhibitors, RNA polymerase inhibitors, and integrase inhibitors, have been successively adopted in clinical trials. Nevertheless, antiviral drugs are not always a promise due to the facts that (1) viruses are notoriously difficult to combat as they are able to mutate quickly and develop resistance to drugs; (2) novel viruses, for instance SARS-CoV-2, regularly emerge and cause outbreaks, but existing antiviral drugs are ineffective against them. Therefore, exploring new antiviral drugs/compounds is crucial for responding to the ongoing threats posed by viral diseases.
The fruit fly (Drosophila melanogaster) has long been used as an animal model for studying a wide range of biological processes and diseases, including viral infections. In recent decades, there have been growing interests in utilizing Drosophila for identifying and testing potential antiviral drugs [8,9]. One advantage of using Drosophila as a model organism is that Drosophila has a relatively simple and well-characterized immune system, which makes it quite easy to study the interactions between viruses and host cells. In addition, Drosophila is a type of cost-effective and easy-to-maintain insect that can be used in large-scale screening assays in order to identify potential antiviral compounds [10,11]. For instance, a study by Adamson and colleagues established a Drosophila model of influenza virus infection by stably expressing the M2 gene in specific tissues/organs [12]. They confirmed the antiviral efficiency of amantadine in restricting influenza titers, supporting the probable use of this system for high-throughput antiviral drug screening [12]. Another approach of infecting flies with HSV (herpes simplex virus) identified potential host factors that could be targeted by antiviral drugs [13]. Moreover, Lee and colleagues lately succeeded in expressing the Orf6 (open reading frame 6) gene of SARS-CoV-2 in fly tissues, which displayed corresponding disease phenotypes [14], making it even more promising to explore functional materials against COVID-19. Overall, the use of Drosophila as a model organism for exploring antiviral drugs is an active area of scientific research, and has the potential to identify new compounds and targets for treating viral infections.
Aspirin (acetylsalicylic acid), a type of well-known NSAID (nonsteroidal anti-inflammatory drug), is commonly consumed by patients in order to relieve pain, reduce inflammation, and lower fever (reviewed in [15,16]). Although it is not typically used as an antiviral drug, recent in vitro studies have shown that it may inhibit the replication of several types of viruses, including HCV (hepatitis C virus), influenza virus, HSV (herpes simplex virus), and HIV (human immunodeficiency virus) [17][18][19]. However, it remains largely unknown whether or not aspirin executes antiviral activity in vivo. We lately reported the beneficial effect of aspirin on preventing age-onset gut epithelial dysfunction and delaying aging in Drosophila [20]. Encouraged by this, our next objective is to explore the potential antiviral role of aspirin by utilizing a fruit fly animal model.
In the present study, we provide in vivo evidence displaying that the dietary consumption of aspirin positively contributes to the fly antiviral defense against various types of viruses. Of interest is that aspirin likely mediates the STING (stimulator of interferon genes) but not the IMD (immune deficiency) signaling pathway to exert its essential role in the host defense against viral infections. Collectively, our studies shed light on the physiological function of aspirin in antiviral immunity and provide bases for the clinical usage of aspirin as an antiviral drug/treatment.

Additional Aspirin Leads to Decreased Viral Titers in Drosophila S2 Cells
We first carried out experiments on Drosophila macrophage-like S2 cells to confirm the antiviral function of aspirin. Cultured S2 cells were pretreated with aspirin at various concentrations (0.1 and 0.5 mM, respectively), followed by infection from DCV (Drosophila C virus), which is one of the widely utilized viruses to infect S2 cells. As illustrated in Figure 1A, the viral titer of DCV was drastically decreased in S2 cells with additional aspirin (0.1 mM) compared to that in the control group. A more significant trend was observed when S2 cells were treated with a higher dose of aspirin (0.5 mM) ( Figure 1A). These results suggested that aspirin prevents viral replication in a concentration-dependent manner. We next examined the impact of aspirin on the loads of other types of viruses. A strong decrease in CrPV (cricket paralysis virus) RNA was noticed in S2 cells with the treatment of aspirin ( Figure 1B). A similar effect was also detected when S2 cells were infected with another RNA virus, VSV (vesicular stomatitis virus) ( Figure 1C). However, additional aspirin hardly affected the titers of either SINV (sindbis virus) ( Figure 1D) or Molecules 2023, 28, 5300 3 of 10 DNA virus IIV6 (invertebrate iridescent virus 6) ( Figure 1E). Taken together, our data support the notion that aspirin antagonizes viral replication in cultured Drosophila S2 cells largely depending on the specific type of virus. treatment of aspirin ( Figure 1B). A similar effect was also detected when S2 cells were infected with another RNA virus, VSV (vesicular stomatitis virus) ( Figure 1C). However additional aspirin hardly affected the titers of either SINV (sindbis virus) ( Figure 1D) o DNA virus IIV6 (invertebrate iridescent virus 6) ( Figure 1E). Taken together, our data sup port the notion that aspirin antagonizes viral replication in cultured Drosophila S2 cell largely depending on the specific type of virus.

Viral Proliferation Is Limited in Adult Flies with Dietary Supplementation of Aspirin
Encouraged by this, we then sought to illustrate whether or not aspirin executes an tiviral activity in vivo. To determine this, we first raised the wild-type (w 1118 or Canton-S flies with a medium supplemented with different concentrations of aspirin (0, 0.5, and 1 mg/L, respectively) and the dietary addition of aspirin hardly affected the food consump tion of tested flies [20]. We then infected these flies with DCV and detected the viral tite on the following days (day 0, 1, and 2). As demonstrated in Figure 2A and S1A, on day 1 after infection, the DCV RNA levels were markedly reduced in the flies consuming 0.5 mg/L of aspirin with respect to those in the control group. A greater reduction was de tected when flies were supplemented with a higher dose of aspirin (1 mg/L) (Figure 2A and S1A). Consistently, similar results were obtained when flies were collected for moni toring the viral titers on day 2 after infection ( Figure 2A and S1A), implying that aspirin harbors antiviral function in adult flies. To further confirm these observations, we infected w 1118 males with other types of viruses, including CrPV and VSV. Intriguingly, the repli cations of both viruses were limited in the host flies by aspirin in a dosage-dependen manner ( Figure 2B,C).

Viral Proliferation Is Limited in Adult Flies with Dietary Supplementation of Aspirin
Encouraged by this, we then sought to illustrate whether or not aspirin executes antiviral activity in vivo. To determine this, we first raised the wild-type (w 1118 or Canton-S) flies with a medium supplemented with different concentrations of aspirin (0, 0.5, and 1 mg/L, respectively) and the dietary addition of aspirin hardly affected the food consumption of tested flies [20]. We then infected these flies with DCV and detected the viral titer on the following days (day 0, 1, and 2). As demonstrated in Figure 2A and Figure S1A, on day 1 after infection, the DCV RNA levels were markedly reduced in the flies consuming 0.5 mg/L of aspirin with respect to those in the control group. A greater reduction was detected when flies were supplemented with a higher dose of aspirin (1 mg/L) ( Figure 2A and Figure  S1A). Consistently, similar results were obtained when flies were collected for monitoring the viral titers on day 2 after infection ( Figure 2A and Figure S1A), implying that aspirin harbors antiviral function in adult flies. To further confirm these observations, we infected w 1118 males with other types of viruses, including CrPV and VSV. Intriguingly, the replications of both viruses were limited in the host flies by aspirin in a dosage-dependent manner ( Figure 2B,C).

Dietary Supplementation of Aspirin Enhances Fly Survival after Viral Infection
Since the viral load was decreased in flies consuming aspirin, one would expect th stronger resistance of these flies against viral infection. Indeed, when we conducted su vival rate assays after viral infections, we observed an increased viability of flies with th dietary addition of aspirin, compared to that of the controls ( Figure 2D,G).
In summary, our data indicate that aspirin behaves as an antiviral compound in th fly defense against viral infection.

Aspirin Modulates Fly Survival and Viral Titers after Infection in an IMD-Independent Manner
We then aimed to illustrate how aspirin benefits the antiviral defense of Drosophi Based upon our recent finding that aspirin delays fly aging via targeting Imd [20], whic is a key adaptor protein in the IMD (immune deficiency) innate immune pathway [21 and the fact that the IMD signal plays an essential role in Drosophila antiviral immuni

Dietary Supplementation of Aspirin Enhances Fly Survival after Viral Infection
Since the viral load was decreased in flies consuming aspirin, one would expect the stronger resistance of these flies against viral infection. Indeed, when we conducted survival rate assays after viral infections, we observed an increased viability of flies with the dietary addition of aspirin, compared to that of the controls ( Figure 2D,G).
In summary, our data indicate that aspirin behaves as an antiviral compound in the fly defense against viral infection.

Aspirin Modulates Fly Survival and Viral Titers after Infection in an IMD-Independent Manner
We then aimed to illustrate how aspirin benefits the antiviral defense of Drosophila. Based upon our recent finding that aspirin delays fly aging via targeting Imd [20], which is a key adaptor protein in the IMD (immune deficiency) innate immune pathway [21], and the fact that the IMD signal plays an essential role in Drosophila antiviral immunity [22], we would like to hypothesize that aspirin executes an antiviral function via mediating Imd. In this regard, we examined the antiviral role of aspirin in the imd LOF (loss of function) mutant flies. Unexpectedly, the dietary supplementation of aspirin still restricted the replication of injected DCV ( Figure 3A) and promoted the survival of the imd LOF mutants ( Figure 3B). These results implied that imd is largely not responsible for aspirin positively contributing to the fly antiviral defense.
Molecules 2023, 28, x FOR PEER REVIEW 5 of [22], we would like to hypothesize that aspirin executes an antiviral function via media ing Imd. In this regard, we examined the antiviral role of aspirin in the imd LOF (loss function) mutant flies. Unexpectedly, the dietary supplementation of aspirin still r stricted the replication of injected DCV ( Figure 3A) and promoted the survival of the im LOF mutants ( Figure 3B). These results implied that imd is largely not responsible for a pirin positively contributing to the fly antiviral defense.

Aspirin Relies on Sting and Relish to Regulate Fly Survival and Viral Replication after Infection
Pioneering studies have delineated that in addition to the IMD signaling cascade, the Toll [23], JAK-STAT (Janus kinase-signal transducer and activator of transcription) [24], RNAi (RNA interference) [25], autophagy [26], and STING (stimulator of interferon genes) [27] pathways are also involved in governing the antiviral immune defense system of Drosophila. We then sought to identify whether or not aspirin mediates one (or some) of them to unleash its antiviral activity. As shown in Figure 3C-J, the dif (dorsal-related immunity factor), hop (hopscotch), ago2 (argonaute 2), or atg7 (autophagy-related 7) LOF mutant flies supplemented with aspirin displayed decreased mortalities. The DCV loads in these flies were reduced after viral injection ( Figure 3C,J). However, when we dietarily supplemented the sting LOF mutants with aspirin, we observed that aspirin almost failed to affect fly survival and the viral titer upon DCV infection ( Figure 4A,B). Moreover, we obtained similar results when we performed these assays utilizing the rel (relish) LOF mutant flies ( Figure 4C,D). Collectively, our data indicated that aspirin benefits the fly antiviral defense largely in a STING-NF-kB (nuclear factor kappa B) signal-dependent manner.

Aspirin Relies on Sting and Relish to Regulate Fly Survival and Viral Replication after Infection
Pioneering studies have delineated that in addition to the IMD signaling cascade, the Toll [23], JAK-STAT (Janus kinase-signal transducer and activator of transcription) [24], RNAi (RNA interference) [25], autophagy [26], and STING (stimulator of interferon genes) [27] pathways are also involved in governing the antiviral immune defense system of Drosophila. We then sought to identify whether or not aspirin mediates one (or some) of them to unleash its antiviral activity. As shown in Figure 3C-J, the dif (dorsal-related immunity factor), hop (hopscotch), ago2 (argonaute 2), or atg7 (autophagy-related 7) LOF mutant flies supplemented with aspirin displayed decreased mortalities. The DCV loads in these flies were reduced after viral injection ( Figure 3C,J). However, when we dietarily supplemented the sting LOF mutants with aspirin, we observed that aspirin almost failed to affect fly survival and the viral titer upon DCV infection ( Figure 4A,B). Moreover, we obtained similar results when we performed these assays utilizing the rel (relish) LOF mutant flies ( Figure  4C,D). Collectively, our data indicated that aspirin benefits the fly antiviral defense largely in a STING-NF-kB (nuclear factor kappa B) signal-dependent manner. Dietary supplementation of aspirin limits DCV load in Canton S flies (Supplementary Materials).

Additional Aspirin Increases the Expressions of STING-Induced Genes
To obtain more evidence, we examined whether or not the expressions of STING downstream antiviral effectors are modulated by aspirin. When we infected the wild-type flies with DCV under normal rearing conditions, we detected enormous inductions of nazo and srg1 (STING-regulated gene 1) ( Figure 4E,F), which were consistent with previous findings [27,28]. Of interest is that the dietary supplementation of aspirin drastically increased

Additional Aspirin Increases the Expressions of STING-Induced Genes
To obtain more evidence, we examined whether or not the expressions of STING downstream antiviral effectors are modulated by aspirin. When we infected the wild-type flies with DCV under normal rearing conditions, we detected enormous inductions of nazo and srg1 (STING-regulated gene 1) ( Figure 4E,F), which were consistent with previous findings [27,28]. Of interest is that the dietary supplementation of aspirin drastically increased the transcript levels of both nazo and srg1, but this was not the case in the sting LOF mutants ( Figure 4E,F). These results indicate that aspirin positively participates in Drosophila antiviral defense reactions through mediating the STING-NF-kB signaling cascade.

Discussion
In the present study, we conducted both in vitro and in vivo experimental approaches to identify the antiviral role of aspirin. We provided compelling evidence showing that the dietary supplementation of aspirin not only lowers the mortality rate of Drosophila upon the infection of specific viruses, but also prevents the viral loads in the host flies. Of note is that the loss of sting or rel reverses the advantageous assessment of aspirin in the fly antiviral defense, implying that aspirin benefits Drosophila antiviral immunity in a STING-NF-kB-dependent manner. Thus, our studies demonstrate the functional role of aspirin against viral infection in vivo.
Aspirin has long been well-known for its anti-inflammation and anti-platelet function, and widely utilized in medical treatments [15,16]. It was lately shown to harbor an antiviral function [17][18][19]29], but these studies were mostly conducted in vitro systems. As a result, the prior objective of this research was to explore the potential involvement of aspirin in modulating the host antiviral defense reaction in vivo. To address this issue, we paid attention to the insect Drosophila, for which we have already succeeded in establishing the model of dietary consumption of aspirin and illustrating the anti-aging role of aspirin [20]. As expected, the dietary supplementation of aspirin significantly enhanced the expressions of antiviral effectors and the survival of flies upon viral infections. In this regard, our studies not only confirm the antiviral role of aspirin that was suggested in the previous literature [17][18][19]29], but also provide for the first-time in vivo evidence to support this notion. Of interest is that aspirin enables the antagonization of several types of viruses, making it possible to become a broad antiviral drug in clinical medication.
How does aspirin benefit the Drosophila antiviral defense? Our recent findings illustrated that aspirin down-regulates the K63 (63rd lysine)-linked ubiquitination of the Imd protein, thereby contributing to gut immune homeostasis and epithelial function [20]. Therefore, we first explored whether or not aspirin is involved in the fly antiviral reaction via modulating Imd. Unexpectedly, dietary aspirin supplementation still enhanced the antiviral immunity in the imd LOF mutates. These observations encouraged us to further examine the antiviral role of aspirin in flies including dif, hop, ago2, atg7, or sting LOF mutants, where the known antiviral pathways (Toll, Jak-STAT, RNAi, autophagy, and STING-NF-κB, respectively) were blocked separately. Intriguingly, the STING-NF-κB signal turned out to be responsible for the antiviral activity of aspirin in adult flies. Nevertheless, knowledge regarding how aspirin modulates the STING-NF-κB signaling pathway is still lacking. Based upon our experience and knowledge with regard to the molecular mechanism by which aspirin can mediate protein ubiquitination, one mechanism we would like to propose is that the ubiquitination modification of a (some) key factor(s) in the STING-NF-κB signaling cascades is (are) regulated by aspirin.
Another question that needs to be addressed is that on which tissue/organ aspirin exerts its antiviral effect. Since we raised experimental flies with the dietary supplementation of aspirin, it is probable that aspirin functions in the digestive system to enhance fly metabolism, thereby contributing to the host antiviral response. In agreement with this, a previous transcriptomic analysis showed that aspirin can alter the expression of a series of genes involved in the metabolic pathway [30]. On the other hand, aspirin may circulate into fat body (the main immune tissue/organ during systemic infection), where it upregulates STING-NF-κB activity. It would thus be worthwhile in the future to explore (1) the potential regulatory relationship between aspirin and the Sting protein; (2) the antiviral function of aspirin in a tissue/organ-specific manner, for instance with the use of flies with the gut or fat body-specific RNAi of sting.

Significance and Limitations of this Study
Our studies uncover the functional involvement of aspirin in the fly defense against several types of viruses. Even though the genetic evidence implies an intersection between aspirin and the STING-NF-κB signaling pathway, the underlying molecular mechanism is still not fully addressed. Further experimental approaches at the levels of molecular biology and biochemistry are required to identify the substrate of aspirin and to illustrate how aspirin modulates the Drosophila STING-NF-κB signaling cascades.
To prepare the fly medium supplemented with aspirin, we first dissolved aspirin (#A2093, Sigma-Aldrich, St. Louis, USA) in sterile H 2 O (37 • C) at final concentrations of 0.5 and 1 g/L. Then, 1 mL of each aspirin solution was added into 1 L of the fly medium to prepare food with 0.5 or 1 mg/L of aspirin.

S2 Cell Manipulation
S2 cells were cultured in an insect medium (Hyclone, Amersham, UK) supplemented with 10% FBS (fetal bovine serum, Hyclone) and Penicillin/Streptomycin (Invitrogen, Carlsbad, USA). The MycoAlert TM kit (Lonza, Basel, Switzerland) was used for the mycoplasma detection and validation of the authenticity of S2 cells. For aspirin treatment in S2 cells, we first prepared a 5 and 10 mM aspirin solution (dissolved in sterile H 2 O). Then, 0.1 mL of each solution was added into S2 cells (0.9 mL) to reach the final concentration of aspirin, this being 0.5 or 1 mM, respectively. An equal volume of sterile H 2 O was added in the control group. Six hours after aspirin treatment, S2 cells were infected with DCV (MOI 1), CrPV (MOI 0.1), FHV (MOI 1), VSV (MOI 1), or IIV6 (MOI 1).

Viral Infection in Flies and Survival Rate Assays
Male flies (3 to 5 days old) were injected at the thorax with 4.6 nL of the viral particle solution (500 pfu per fly for DCV and VSV; 50 pfu per fly for CrPV) using a nanoliter injector (Nanoject III, Drummond, Broomall, USA). An equal volume of Tris-HCl solution (10 mM, pH = 7.5) was injected in the control group. Flies were then counted daily for survival. Flies that (<10%) died within 2 h were not considered in this assay.

Statistical Analyses
All statistical analyses were performed using GraphPad Prism 8. The statistical significance in Figure 1A,E, Figure 2A,C, Figure 3A,C,E,G,I, Figure 4A,C,E and Figure S1A was determined using the ANOVA or Mann-Whitney tests. In Figure 2D,F, Figure 3B,D,F,H,J and Figure 4B,D,F, the log-rank test (Kaplan-Meier method) was used for statistical analysis. A p value of less than 0.05 was considered statistically significant. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; ns indicates non-significance.
Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules28145300/s1. Figure S1: Dietary supplementation of aspirin limits DCV load in Canton S flies.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data supporting the findings of this study are available from the corresponding author upon reasonable request.