PARP1 inhibition enhances reactive oxygen species on gut microbiota

Abstract Poly(ADP‐ribose) polymerase 1 (PARP1) plays a key role in genome stability by modulating DNA‐damage responses. Activated by DNA interruptions through ultraviolet (UV) exposure, PARylation is synthesized by PARP1 and serves as a survival mechanism for cancer and metabolic diseases. Several strategies including ROS and antimicrobial peptides (AMPs) function in host defenses, while the targeted tissue and mechanism under DNA damage are unknown. Here, we show that DNA damage induces responses specifically in the gut tissue. The knockdown of PARP1 reduces the activation of PARylation. Parp1 knockdown under DNA damage results in over‐accumulated ROS and secretion of AMPs through the regulation of Relish, a subunit of nuclear factor‐κB (NF‐κB). Double‐knockdown of Parp1 and Relish specifically in the gut inhibits AMP secretion. In conclusion, the host defense is achieved through ROS accumulation rather than the AMPs under DNA damage. In contrast, the knockdown of PARP1 exacerbates ROS accumulation to a harmful level. Under this circumstance, NF‐κb targeted AMP secretion is provoked for host defense. Microbiome and functional analysis provide evidence for the hazard of DNA damage and show variations in the metabolic pathways following Parp1 inhibition. Our findings suggest the notion that PARP1 inhibition contributes to ROS accumulation under DNA damage and its role in NF‐κb activation for host defense.


| INTRODUCTION
PARP1, an important member of the poly(ADP-ribose) polymerase (PARP) family (Jacobson et al., 2001), is activated by DNA interruptions as a DNA repair gene (Gibson & Kraus, 2012). Poly (ADP-ribosyl)ation (PARylation), synthesized by PARP1 (Jacobson et al., 2001), is a survival mechanism for ultraviolet (UV)-induced DNA damages (Lakatos et al., 2013). PARP1 inhibitors, such as Olaparib, Rucaparib, Niraparib, and Talazoparib, have been approved by the FDA to treat cancers (Faraoni & Graziani, 2018). However, the mechanism for PARP1 in the ROS and host defense under DNA damage is largely unknown. DNA damages, generated by environmental stress of UV (O'Donovan et al., 2005), activate the DNA repair genes and eventually result in deleterious effects on energy expenditure, with the generation of ROS which is a byproduct of mitochondrial energy metabolism (Finkel & Holbrook, 2000). Studies have focused on the whole body under DNA damage hazards while the reaction of gut has been masked by other tissues (Buchon et al., 2009).
Intestinal homeostasis is achieved through several strategies (Guo et al., 2014;Li et al., 2020): reactive oxygen species (ROS), the production of antimicrobial peptides (AMPs), melanization reaction, and phagocytosis. ROS, generated by NADPH enzymes Duox and Nox (Ha et al., 2009;, is a barrier of defense while the activation of AMPs, melanization reaction, and phagocytosis play complementary roles to microbicidal oxidants for defense (Ryu et al., 2006). However, the balance between ROS and other barriers of host defense under DNA damage is unclear.
Our study shows that the gut tissue responds to DNA damage.
Under DNA damage, the host defense is achieved through ROS accumulation rather than the AMPs. In contrast, the knockdown of PARP1 exacerbates ROS accumulation to a harmful level. Under this circumstance, nuclear factor-κB (NF-κb) targeted AMP secretion is provoked for host defense. Microbiome and functional analysis provide evidence for the hazard of DNA damage and show variations in the metabolic pathways following Parp1 inhibition.
For details, please see Supporting Information.

| Oxidative stress resistance assay
Flies were fed with 300 μl of 5% H 2 O 2 solution. The activity data were extracted at 1 h bin . Over 100 flies were used for each line (Belyi et al., 2020). For details, please see the Supporting Information.
2.3 | DNA damage by UV radiation DNA damage was generated by UV radiation and 20-25 flies were housed in quartz glass vail, through which UV irradiation was able to transmit (Y. L. Liu et al., 2022). Flies were exposed to 0.6 mW/cm 2 UV for 2 h. For details, please see Supporting Information.

| Quantitative real-time PCR
The quantitative real-time PCR was carried out as described in the previous study (Kong et al., 2018;Xiao et al., 2021). The standardized RP49 mRNA was used as the invariant control. Supporting Information: Table S1 lists the sequence of used primers in the study. For details, please see Supporting Information.

| ROS imaging
For dihydroethidium (DHE) staining, in situ ROS detection was performed using DHE (Beyotime, S0063). Images were captured and analyzed using the Olympus FV 1200 imaging system. For details, please see Supporting Information.

| Genomics DNA extraction and library construction
For each group, 8 samples of female flies were examined and each sample with 25 guts was respectively collected and sent to the BGI.
The microbial community DNA was extracted using MagPure Stool DNA KF kit B. DNA was quantified and the quality was checked.
Variable regions V3-V4 of bacterial 16S rRNA gene were amplified with degenerate PCR primers. For details, please see Supporting Information.

| DNA damage alters the intestine microbiome under Parp1 knockdown
Intestinal homeostasis is represented by the direct variations in the host commensal community. DNA damaged flies were examined by 16S rDNA sequencing. One domain, 1 kingdom, 9 phyla, 14 classes, 28 orders, 51 families, 103 geneses, and 163 species were detected.
The tub-gal4 ts >Luciferase RNAi flies (Group C) served as the control.
The results of α diversity analysis showed that DNA damage affected negatively the diversity of the microbiome by comparing the Group C with exposed tub-gal4 ts > Luciferase RNAi flies (Group CUV; Supporting Information: Figure S3A). The tub-gal4 ts > Parp1 RNAi flies (Group P) and exposed flies (Group PUV) showed an increase in the α

| DNA damage alters the microbial function under Parp1 knockdown
The functional prediction results showed that the COG functional composition of the four groups was different (Supporting Information: Figure S3B). Group PUV flies revealed the least functional abundance in amino acid transport and metabolism, inorganic ion transport, and metabolism but the highest abundance in replication, recombination, and repair together with translation, ribosomal structure, and biogenesis.

| Parp1-specific knockdown exacerbates ROS
To refine the tissue-specific physiology, we used intestinal stem cells and enteroblasts (esg-gal4 ts ) driver to knock down Parp1 expression specifically in the gut (gPARPKD) and the mRNA levels of Parp1  However, the expression of Nox showed no significant difference following the Parp1 knockdown (Supporting Information: Figure S4A). Antioxidant genes, including SOD1, SOD2, CAT, and GS, significantly decreased in gPARPKD flies with DNA damage (Supporting Information: Figure S4B). These data suggested that Parp1 inhibition and DNA damage additively contribute to ROS accumulation in the gut tissue and eventually become harmful to the individual vitality.

| DNA damage induces suppression in AMPs
Intestinal homeostasis is achieved through ROS and other several strategies (Guo et al., 2014;Vesala et al., 2020): the production of AMPs, the melanization reaction, and phagocytosis. DNA damage did not exert enhancement on the melanization reaction and phagocytosis. The expression of phagocytic receptors and opsonin in the gut of exposed w1118 flies, including the scavenger receptor Croquemort Collectively, these data suggested that under DNA damage, the host defense is achieved neither by the inactivated melanization reaction, phagocytosis or by the inhibited AMPs. Accumulation of ROS played a dominant role.

| Parp1-specific knockdown contributes to AMP-secretion through IMD/Rel
The production of AMPs after DNA damage in gPARPKD flies was examined. Similar to w1118 flies, there was a reduction in the Relish

| DISCUSSION
Besides its well-known application in the genetic region, Drosophila has emerged as a tool in studies for ROS, given that flies share the majority of mitochondrial and energy metabolic pathways with humans for the source of ROS generation. The simplicity in anatomy enables Drosophila to be employed in ROS studies and provides an immediate strategy for ROS quantification through unfixed staining (Vaccaro et al., 2020). By counting the death events, Drosophila offers an advantage over mammals in quantifying endogenous oxidative stress and vitality.
Biologically relevant doses of UV generate ROS in vitro (O'Donovan et al., 2005). Under DNA damage, we observed ROS accumulation in the gut tissue rather than other organs, which leads us to focus on the gut tissue in this study. Individual homeostasis is achieved through several strategies (Guo et al., 2014;Li et al., 2020): ROS, the production of AMPs, the melanization reaction, and phagocytosis. Usually, ROS serves as a first line of defense while AMPs and other strategies of defense act as eliminating antioxidant pathogens.
Studies have focused on the whole body while the reaction of the gut has been masked (Buchon et al., 2009). ROS is a byproduct of mitochondrial energy metabolism and is generated under DNA damage (Finkel & Holbrook, 2000). A significant fraction of ROS is made through the action of two conserved enzymes, Nox and Duox (Ha et al., 2009).  | 4177 2022). Also, the activity of another antioxidant enzyme, catalase, was reduced to UV light in the model organism . The imbalance between the generation and endogenous antioxidant systems in the gut causes ROS accumulation and could eventually be fatal (Ha, Oh, Ryu, et al., 2005).
The intestinal commensal community of Drosophila, which shares a large part of overlap with humans, represents intestinal homeostasis and has been employed to better define the relationship of microbiota with host defense and the molecular basis of pathophysiological traits. We observed independent and synergistic effects of