Insect immune resolution with EpOME/DiHOME and its dysregulation by their analogs leading to pathogen hypersensitivity

Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid: LA). They are metabolized into dihydroxyoctadecamonoenoic acids (DiHOMEs) in mammals. Unlike in mammals where they act as adipokines or lipokines, EpOMEs act as immunosuppressants in insects. However, the functional link between EpOMEs and pro-immune mediators such as PGE2 is not known. In addition, the physiological significance of DiHOMEs is not clear in insects. This study analyzed the physiological role of these C18 oxylipins using a lepidopteran insect pest, Spodoptera exigua. Immune challenge of S. exigua rapidly upregulated the expression of the phospholipase A2 gene to trigger C20 oxylipin biosynthesis, followed by the upregulation of genes encoding EpOME synthase (SE51385) and a soluble epoxide hydrolase (Se-sEH). The sequential gene expression resulted in the upregulations of the corresponding gene products such as PGE2, EpOMEs, and DiHOMEs. Interestingly, only PGE2 injection without the immune challenge significantly upregulated the gene expression of SE51825 and Se-sEH. The elevated levels of EpOMEs acted as immunosuppressants by inhibiting cellular and humoral immune responses induced by the bacterial challenge, in which 12,13-EpOME was more potent than 9,10-EpOME. However, DiHOMEs did not inhibit the cellular immune responses but upregulated the expression of antimicrobial peptides selectively suppressed by EpOMEs. The negative regulation of insect immunity by EpOMEs and their inactive DiHOMEs were further validated by synthetic analogs of the linoleate epoxide and corresponding diol. Furthermore, inhibitors specific to Se-sEH used to prevent EpOME degradation significantly suppressed the immune responses. The data suggest a physiological role of C18 oxylipins in resolving insect immune response. Any immune dysregulation induced by EpOME analogs or sEH inhibitors significantly enhanced insect susceptibility to the entomopathogen, Bacillus thuringiensis.

octadecadienoic acid: LA). They are metabolized into dihydroxyoctadecamonoenoic acids 23 (DiHOMEs) in mammals. Unlike in mammals where they act as adipokines or lipokines, EpOMEs 24 act as immunosuppressants in insects. However, the functional link between EpOMEs and pro-25 immune mediators such as PGE 2 is not known. In addition, the physiological significance of 26 DiHOMEs is not clear in insects. This study analyzed the physiological role of these C18 oxylipins 27 using a lepidopteran insect pest, Spodoptera exigua. Immune challenge of S. exigua rapidly 28 upregulated the expression of the phospholipase A 2 gene to trigger C20 oxylipin biosynthesis, 29 followed by the upregulation of genes encoding EpOME synthase (SE51385) and a soluble 30 epoxide hydrolase (Se-sEH). The sequential gene expression resulted in the upregulations of the 31 corresponding gene products such as PGE 2 , EpOMEs, and DiHOMEs. Interestingly, only PGE 2 32 injection without the immune challenge significantly upregulated the gene expression of SE51825 33 and Se-sEH. The elevated levels of EpOMEs acted as immunosuppressants by inhibiting cellular 34 and humoral immune responses induced by the bacterial challenge, in which 12,13-EpOME was 35 more potent than 9,10-EpOME. However, DiHOMEs did not inhibit the cellular immune responses 36 but upregulated the expression of antimicrobial peptides selectively suppressed by EpOMEs. The 37 negative regulation of insect immunity by EpOMEs and their inactive DiHOMEs were further 38 validated by synthetic analogs of the linoleate epoxide and corresponding diol. Furthermore, 39 inhibitors specific to Se-sEH used to prevent EpOME degradation significantly suppressed the 40 immune responses. The data suggest a physiological role of C18 oxylipins in resolving insect . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint

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In insects, these EpOMEs and DiHOMEs have been speculated to play a crucial role in immune 72 response [7]. Indeed, they were detected in all the developmental stages of the mosquito, Culex 73 quinquefasciatus [7]. Feeding sEH inhibitor increased EpOME levels in the mosquito midgut and 74 reduced the total number of bacteria in the lumen, suggesting that EpOMEs were associated with 75 insect immunity [8]. The role of EpOMEs in insect immune response was further analyzed in the 76 immune-challenged larvae of a lepidopteran insect, Spodoptera exigua, which exhibited relatively 77 high levels of 941.8 pg/g of 9,10-EpOME and 2,198.3 pg/g of 12,13-EpOME [9]. In the insect 78 species, a specific cytochrome P450 enzyme (CYP, SE51385) and a soluble epoxide hydrolase 79 (Se-sEH) mostly catalyzed the production and hydrolysis of EpOMEs, respectively. These two was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint 5 84 inflammatory response in the insect. However, little is known regarding the role of EpOMEs and 85 their association with pro-inflammatory agents. 86 DiHOMEs are also produced in neutrophils in humans following bacterial challenge [10][11].

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Although DiHOMEs are the metabolites of EpOMEs, they act as mediators of neutrophil 88 chemotaxis [12]. High concentrations of DiHOMEs under physiological stress such as following 89 severe skin burn or COVID infection can lead to toxicity by acting as isoleukotoxins, resulting in 90 severe dysfunction of the innate immune responses [6]. DiHOMEs also suppress excessive 91 production of reactive oxygen species in neutrophils [4]. However, studies investigating the 92 physiological role of DiHOMEs in insects have yet to be reported.

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This study investigated the physiological role of C18 oxylipins in attenuating the immune 94 response in insects by analyzing the functional relationship between EpOMEs and pro-immune 95 mediators as well as EpOME metabolism. The physiological roles of the resulting DiHOMEs in 96 insect immunity were also investigated. The physiological role of C18 oxylipins in immune 97 resolution was determined by analyzing the effects of DiHOME analogs or sEH inhibitors on 98 dysregulation of the immune responses and susceptibility to an insect pathogen. Bacterial challenge induced the expressions of PLA 2 , EpOME synthase (SE51385), and Se-sEH 104 genes in S. exigua larvae ( Fig 1A). However, their expression profiles differed (F = 3.96; df = 2, 105 9; P = 0.0009), and the maximum levels were detected during peak times. The maximal expression 106 peak of PLA 2 was observed at 1 h after the bacterial challenge while the peaks of SE51385 and . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. Se-sEH were detected 4 h post-challenge. In addition, SE51385 was expressed earlier (F = 5.70; 108 df = 1, 9; P < 0.0001) than Se-sEH. Interestingly, only PGE 2 injection induced the gene expression 109 of SE51385 and Se-sEH ( Fig 1B). 110 LC-MS/MS was used to analyze the concentrations of PGE 2 , EpOMEs, and DiHOMEs in 111 immune-challenged larvae at different time points (Fig 1C). To avoid exogenous dietary 112 contamination, the entire gut was removed from the whole body preparation before extracting the 113 oxylipins. The immune challenge significantly increased the levels of PGE 2 and 114 EpOMEs/DiHOMEs. The induced levels of PGE 2 were detected earlier (0.5  2 h) than those (> 115 12 h) of EpOMEs/DiHOMEs. The induced levels of the two EpOMEs did not differ significantly 116 (F = 0.26; df = 1, 9; P = 0.6138). However, the concentrations of the two DiHOMEs showed 117 statistically significant differences (F = 17.46; df = 1, 9; P < 0.0001) following induction, and 118 12,13-DiHOME was detected at higher levels than 9,10-DiHOME. 119 120 2.2 EpOMEs, but not DiHOMEs, suppress immune response 121 To investigate the role of EpOMEs/DiHOMEs in insect immunity, hemocyte-spreading behavior 122 was assessed by adding them to the hemocyte suspension (left panel in Fig 2A). Control hemocytes 123 spread via cytoplasmic extensions along with increased levels of F-actin. However, the addition 124 of 12,13-EpOME inhibited hemocyte spread in a dose-dependent manner (Fig 2B). Treatment with 125 LA, its biosynthetic precursor, also slightly suppressed the hemocyte spread. However, 12,13-126 DiHOME did not suppress even at the highest test concentration. Compared with 9,10-EpOME, 127 12,13-EpOME exhibited higher suppression of hemocyte-spreading behavior. However, none of 128 the regioisomeric DiHOMEs inhibited (F = 2.92; df = 1, 4; P = 0.1625) the spread of hemocytes.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. Nodule formation (right panel in Fig 2A) was assessed to test the effect of oxylipin on the cellular 130 immune response of S. exigua. Bacterial challenge resulted in the formation of about 55 nodules 131 per larva in the hemocoel (Fig 2C). Both regioisomeric EpOMEs significantly suppressed the 132 nodule formation, although 12,13-EpOME was more potent than 9,10-EpOME and exhibited dose- ). However, 12,13-EpOME significantly suppressed the PO activity, whereas 12,13-DiHOME 139 did not alter PO activity (Fig 2E) 140 To test the influence of DiHOMEs on the humoral immune responses, 10 AMP genes induced 141 by immune challenge were assessed ( Fig 2F). Both EpOME regioisomers suppressed the 142 expression of a few AMPs following immune challenge. The regioisomer 9,10-EpOME 143 suppressed attacin 1 and cecropin genes while 12,13-EpOME suppressed attacin 1, attacin 2, and 144 gloverin. Interestingly, both DiHOMEs upregulated the expression of the four AMPs suppressed 145 by the EpOMEs. In addition, 9,10-DiHOMEs upregulated the expression of defensin and 146 gallerimycin genes while 12,13-DiHOME upregulated gloverin and lysozyme genes. The analysis of hemocyte behavior in response to 12,13-EpOME treatment revealed cellular 150 blebbing. (Fig 3A). However, 12,13-DiHOME treatment had limited effect on cell blebbing in 151 hemocytes. The cell blebbing was further analyzed under different concentrations of . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. EpOMEs/DiHOMEs (Fig 3B). Both EpOMEs significantly induced cell blebbing: 12,13-EpOME 153 was more potent than 9,10-EpOME and exhibited hemolytic activity in a dose-dependent manner.

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LA also induced cell blebbing but its activity was substantially lower than that of EpOMEs. 155 However, 9,10-DiHOME did not exhibit hemolytic activity. In contrast, 12,13-DiHOME was 156 slightly hemolytic but significantly weaker than LA.

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The cytotoxicity manifested by cellular blebbing was further analyzed by TUNEL assays to 158 determine whether the hemolytic activity was caused by apoptosis ( Fig 3C). Incubation of 159 hemocytes with 12,13-EpOME led to DNA fragmentation, which was observed by DNA end 160 labeling with BrdU. The DNA fragmentation increased in a dose-dependent manner following 161 incubation with 12,13-EpOME. Treatment with 9,10-EpOME also induced DNA fragmentation 162 but less than in 12,13-EpOME treatment ( Fig 3D). Treatment with Ac-DEVD-AMC and Caspase-163 3 inhibitor (Ac-DEVD-CHO) significantly rescued the DNA fragmentation induced by 12,13-164 EpOME. LA also induced the DNA fragmentation but its activity was substantially lower than that 165 of EpOMEs. Both DiHOMEs also exhibited apoptotic activity at high concentrations but their 166 activities were significantly lower than those of EpOMEs. spreading behavior ( Fig 4A) and nodule formation ( Fig 4B). They also inhibited PO activation 174 ( Fig 4C). The diEpOME significantly suppressed the induction of some AMP genes ( Fig 4D). In . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint 9 175 addition, diEpOME showed high levels of cytotoxicity against hemocytes by inducing apoptosis 176 ( Fig. 4E and 4F). In contrast, THOME, similar to DiHOMEs, did not inhibit the cellular immune 177 response upon immune challenge (Fig 4A and 4D). Similar to DiHOMEs, it induced humoral 178 immune response by further upregulating the gene expression of some AMPs induced by the 179 bacterial infection (Fig 4D). Interestingly, THF-diols slightly retained the inhibitory activities 180 against cellular and humoral immune responses but were substantially weaker than those of the 181 epoxide metabolites. Both THOME and the THF-diol were less cytotoxic to hemocytes compared 182 with diEpOME ( Fig 4E and 4F).

Effect of sEH inhibitors on immune responses
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint 10 197 The immunosuppressive activity of EpOMEs suggested that sEH inhibitors suppress the immune 198 responses of S. exigua by elevating the endogenous EpOME levels following the immune 199 challenge. Six urea-based sEH inhibitors were assessed to test the hypothesis. All sEH inhibitors 200 significantly inhibited the cellular (Fig 6A-6C) or humoral responses ( Fig 6D). The sEH inhibitors 201 AUDA and CUDA were most potent inhibitors. These two sEH inhibitors were also highly 202 cytotoxic to hemocytes and induced cellular blebbing ( Fig 6E) and apoptosis ( Fig 6F). The immunosuppressive and hemolytic activities of EpOME derivatives ( Fig 7A) and sEH 207 inhibitors suggested their potential role in enhancing the virulence of entomopathogenic microbes.

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Each of these compounds showed significant insecticidal activities against S. exigua larvae, and 209 diEpOME was more potent than the sEH inhibitor ( Fig 7B). To test the enhanced virulence 210 hypothesis, B. thuringiensis (= an entomopathogenic bacterium) was treated with each of the 211 potent immunosuppressants ( Fig 7C). All three treatments significantly enhanced the bacterial 212 virulence, and AUDA and the EpOME-mimic A841 were the most potent compounds. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint 11 220 also rich (7-25%) in aquatic insects, but are rare in terrestrial insects due to evolutionary adaptation to generate EpOME and DiHOME isomers presumably to shut down the induced immune response.

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The relative fatty acid abundance triggers inflammation via prostaglandin synthesis. The was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint 12 243 EpOME isomers against cellular and humoral immune responses of S. exigua. Among the two 244 regioisomers, 12,13-EpOME was more potent than 9,10-EpOME. This supports the previous study 245 [9] demonstrating the role of EpOMEs as immune suppressors. In addition, the current study 246 showed that DiHOMEs had limited inhibitory activity against cellular immune responses. These  Compared with DiHOMEs, EpOMEs were highly cytotoxic against hemocytes of S. exigua. 259 The cytotoxic activity of EpOMEs was attributed to apoptosis based on cellular blebbing and DNA 260 fragmentation in hemocytes exposed to EpOMEs [18]. Treatment with a caspase inhibitor 261 significantly rescued the hemocytes from the cytotoxic activity of 12,13-EpOME. Apoptosis is a . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. The immunosuppressive activity of EpOMEs was further supported by their alkoxide 281 derivatives or agonists such as sEH inhibitors, which prevented their degradation into DiHOMEs.

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The alkoxides are synthetic derivatives obtained by replacing the epoxide in EpOME with a 283 methoxy, ethoxy, propoxy or (iso)propoxy group. The alkoxy ethers cannot be hydrolyzed by she; 284 however, as shown by the insect growth regulator methoprene, alkoxides can mimic epoxides in 285 several biological systems. This significantly modifies the biological activities to suppress 286 different immune responses, suggesting the critical importance of the epoxide structure in the 287 immunosuppressive activity of 12,13-EpOME. Interestingly, A841 (= the methyl ester propoxy 288 EpOME mimic) was the most potent among the eight alkoxides tested and even more potent than . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023.   was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint 16 335 4.2 Chemicals 336 9,10-EpOME, 12,13-EpOME, 9,10-DiHOME, 12,13-DiHOME, LA, and prostaglandin E 2 (PGE 2 ) 337 were purchased from Cayman (Ann Arbor, MI, USA). A caspase inhibitor Z-DEVD-FMK (DEVD) 338 was purchased from Sigma-Aldrich Korea (Seoul, Korea). Other EpOME derivatives (diEpOME, 339 THOME, and THF-diol) were prepared as described in previous studies involving the synthesis of

Hemocyte-spreading behavior assay 354
Two-day-old L4 larvae of S. exigua were used to analyze the hemocyte-spreading behavior.

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Approximately 250 µL of hemolymph was collected from five larvae and mixed with 750 µL of 356 cold ACB, and incubated on ice for 20 min. The diluted hemolymph was then centrifuged at 800×g 357 for 5 min at 4°C to obtain the pellet, which was re-suspended in 300 μL of filter-sterilized TC-. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. template for quantitative PCR (qPCR) using gene-specific primers (Table S1). The qPCR was 380 performed using SYBR Green real-time PCR master mixture (Toyobo, Osaka, Japan) as described . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023.  insects were also injected with test compound(s) along with E. coli to assess any effect on AMP 394 induction. Total RNA was collected as described above 8 h after treatment. After cDNA synthesis, 395 qPCR was performed as described above using gene-specific primers (Table S1). The ribosomal 396 protein gene, RL32, was amplified as the housekeeping gene serving as the internal standard in the 397 qPCR assay. All samples were analyzed in triplicate. with PBS, and treated with 0.3% Triton-X in PBS, followed by incubation for 2 min at RT. After 426 blocking with 5% bovine serum albumin in PBS for 10 min, cells were incubated with mouse anti-. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023.   was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023.        . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023.           was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 7, 2023. ; https://doi.org/10.1101/2023.07.07.548078 doi: bioRxiv preprint