Untargeted metabolomics investigating porcine reproductive and respiratory syndrome virus biomarkers of serum-derived exosomes in piglets infected with PRRSV NADC30-like CHsx1401

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most severe swine diseases in the pig industry. The identification of biomarkers for PRRSV infection is valuable for controlling, eliminating, and treating PRRSV. This study utilized the ultra-performance liquid chromatography–mass spectrometry metabolite profiling platform to identify differential metabolites in exosomes between the control and NADC30-like PRRSV strain infected pigs. Using multivariate analysis combined with univariate analysis, unsupervised principal component analysis and orthogonal partial least squares discriminant analysis models were constructed between the groups. A total of 41 differential metabolites were detected, with 14 upregulated and 27 downregulated metabolites with PRRSV infection. MetaboAnalyst and Kyoto Encyclopedia of Genes andGenomes were used to identify potentially relevant significant pathways, and a receiver operating characteristic curve was used to quantify the predictive performance of differential metabolites. The results indicated that tryptophan-related Lkynurenine, 5-hydroxytryptophan, and D (+)-tryptophan significantly increased among PRRSV infected groups, which may play an important role in the progression of PRRSV infection. Metabolites related to amino acid synthesis and metabolism, including 2-arachidonoylglycerol Lysopcs and phosphatidylcholines may also contribute to the lack of immune protection in piglets after PRRSV infection. Moreover, L-kynurenine and taurocholic acid may serve as potential biomarkers for early diagnosis or drug targeting of PRRSV. Overall, these findings provide an important reference to our understanding of PRRS pathogenesis and immune or protective responses during PRRSV acute infection in the host.

Keywords

biomarkers / exosomes / metabolomics / pig / PRRSV

References

[1] Zhou, L., Wang, Z., Ding, Y., Ge, X., Guo, X., & Yang, H. (2015). NADC30-like strain of porcine reproductive and respiratory syndrome virus, China. Emerging Infectious Diseases, 21(12), 2256-2257.

[2] Alenquer, M., & Amorim, M. J. (2015). Exosome biogenesis, regulation, and function in viral infection. Viruses, 7(9), 5066-5083.

[3] Wang, T., Fang, L., Zhao, F., Wang, D., & Xiao, S. (2018). Exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus. Journal of Virology, 92(4), e01734-17.

[4] Bryzgunova, O. E., Zaripov, M. M., Skvortsova, T. E., Lekchnov, E. A., Grigor’eva, A. E., Zaporozhchenko, I. A., Morozkin, E. S., Ryabchikova, E. I., Yurchenko, Y. B., Voitsitskiy, V. E., & Laktionov, P. P. (2016). Comparative study of extracellular vesicles from the urine of healthy individuals and prostate cancer patients. PLoS One, 11(6), e0157566.

[5] Tamkovich, S. N., Tutanov, O. S., & Laktionov, P. P. (2016). Exosomes: Generation, structure, transport, biological activity, and diagnostic application. Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 10(3), 163-173.

[6] Ibrahim, A., & Marbán, E. (2016). Exosomes: Fundamental biology and roles in cardiovascular physiology. Annual Review of Physiology, 78(1), 67-83.

[7] Whiteside, T. L. (2016). Exosomes and tumor-mediated immune suppression. Journal of Clinical Investigation, 126(4), 1216-1223.

[8] Huo, L., Du, X., Li, X., Liu, S., & Xu, Y. (2021). The emerging role of neural cell-derived exosomes in intercellular communication in health and neurodegenerative diseases. Frontiers in Neuroscience, 15, 738442.

[9] Chen, P., Wang, L., Fan, X., Ning, X., Yu, B., Ou, C., & Chen, M. (2021). Targeted delivery of extracellular vesicles in heart injury. Theranostics, 11(5), 2263-2277.

[10] Channon, L. M., Tyma, V. M., Xu, Z., Greening, D. W., Wilson, J. S., Perera, C. J., & Apte, M. V. (2022). Small extracellular vesicles (exosomes) and their cargo in pancreatic cancer: Key roles in the hallmarks of cancer. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1877(3), 188728.

[11] Tao, L., Zhou, J., Yuan, C., Zhang, L., Li, D., Si, D., Xiu, D., & Zhong, L. (2019). Metabolomics identifies serum and exosomes metabolite markers of pancreatic cancer. Metabolomics, 15(6), 86.

[12] Marzan, A. L., Nedeva, C., & Mathivanan, S. (2021). Extracellular vesicles in metabolism and metabolic diseases. Subcellular Biochemistry, 97, 393-410.

[13] Williams, C., Palviainen, M., Reichardt, N.-C., Siljander, P. R.-M., & Falcón-Pérez, J. M. (2019). Metabolomics applied to the study of extracellular vesicles. Metabolites, 9(11), 276.

[14] Cheng, F., Wang, H., Zhou, L., Lan, G., Yang, H., Wang, L., Wang, L., & Liang, J. (2023). Systematic identification and comparison of the expressed profiles of exosomal MiRNAs in pigs infected with NADC30-like PRRSV strain. Animals, 13(5), 876.

[15] Ibsen, S. D., Wright, J., Lewis, J. M., Kim, S., Ko, S.-Y., Ong, J., Manouchehri, S., Vyas, A., Akers, J., Chen, C. C., Carter, B. S., Esener, S. C., & Heller, M. J. (2017). Rapid isolation and detection of exosomes and associated biomarkers from plasma. ACS Nano, 11(7), 6641-6651.

[16] Pulliam, L., Sun, B., Mustapic, M., Chawla, S., & Kapogiannis, D. (2019). Plasma neuronal exosomes serve as biomarkers of cognitive impairment in HIV infection and Alzheimer’s disease. Journal of NeuroVirology, 25(5), 702-709.

[17] Lin, J., Li, J., Huang, B., Liu, J., Chen, X., Chen, X.-M., Xu, Y.-M., Huang, L.-F., & Wang, X.-Z. (2015). Exosomes: Novel biomarkers for clinical diagnosis. The Scientific World Journal, 2015, 657086-657088.

[18] Wu, P., Tang, Y., Jin, C., Wang, M., Li, L., Liu, Z., Shi, H., Sun, Z., Hou, X., Chen, W., Xu, W., & Qian, H. (2022). Neutrophil membrane engineered HucMSC sEVs alleviate cisplatin-induced AKI by enhancing cellular uptake and targeting. Journal of Nanobiotechnology, 20(1), 353.

[19] Chu, X., Yang, Y., & Tian, X. (2022). Crosstalk between pancreatic cancer cells and cancer-associated fibroblasts in the tumor microenvironment mediated by exosomal microRNAs. International Journal of Molecular Sciences, 23(17), 9512.

[20] Aoi, W., & Tanimura, Y. (2021). Roles of skeletal muscle-derived exosomes in organ metabolic and immunological communication. Frontiers in Endocrinology, 12, 697204.

[21] Malgarin, C., MacPhee, D., & Harding, J. (2020). Fetal metabolomic alterations following porcine reproductive and respiratory syndrome virus infection. Frontiers in Molecular Biosciences, 11(7), 559688.

[22] Wu, Q., Han, Y., Wu, X., Wang, Y., Su, Q., Shen, Y., Guan, K., Michal, J., Jiang, Z., Liu, B., & Zhou, X. (2022). Integrated time-series transcriptomic and metabolomic analyses reveal different inflammatory and adaptive immune responses contributing to host resistance to PRRSV. Frontiers in Immunology, 20(13), 960709.

[23] Ring, E. K., Markert, J. M., Gillespie, G. Y., & Friedman, G. K. (2017). Checkpoint proteins in pediatric brain and extracranial solid tumors: Opportunities for immunotherapy. Clinical Cancer Research, 23(2), 342-350.

[24] Song, H., Park, H., Kim, Y. S., Kim, K. D., Lee, H. K., Cho, D. H., Yang, J. W., & Hur, D. Y. (2011). L-kynurenine-induced apoptosis in human NK cells is mediated by reactive oxygen species. International Immunopharmacology, 11(8), 932-938.

[25] Campesato, L. F., Budhu, S., Tchaicha, J., Weng, C.-H., Gigoux, M., Cohen, I. J., Redmond, D., Mangarin, L., Pourpe, S., Liu, C., Zappasodi, R., Zamarin, D., Cavanaugh, J., Castro, A. C., Manfredi, M. G., McGovern, K., Merghoub, T., & Wolchok, J. D. (2020). Blockade of the AHR restricts a Treg-macrophage suppressive axis induced by L-Kynurenine. Nature Communications, 11(1), 4011.

[26] Feng, Y., Guo, X., Tian, H., He, Y., Li, Y., Jiang, X., Zheng, H., & Xiao, S. (2022). Induction of HOXA3 by porcine reproductive and respiratory syndrome virus inhibits type I interferon response through negative regulation of HO-1 transcription. Journal of Virology, 96(3), e0186321.

[27] Takeuchi, O., & Akira, S. (2010). Pattern recognition receptors and inflammation. Cell, 140(6), 805-820.

[28] Crowl, J. T., Gray, E. E., Pestal, K., Volkman, H. E., & Stetson, D. B. (2017). Intracellular nucleic acid detection in autoimmunity. Annual Review of Immunology, 35(1), 313-336.

[29] Hu, M.-M., & Shu, H.-B. (2018). Cytoplasmic mechanisms of recognition and defense of microbial nucleic acids. Annual Review of Cell and Developmental Biology, 34(1), 357-379.

[30] Seth, R. B., Sun, L., Ea, C.-K., & Chen, Z. J. (2005). Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF3. Cell, 122(5), 669-682.

[31] Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K. J., Takeuchi, O., & Akira, S. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nature Immunology, 6(10), 981-988.

[32] Hu, M.-M., He, W.-R., Gao, P., Yang, Q., He, K., Cao, L.-B., Li, S., Feng, Y.-Q., & Shu, H.-B. (2019). Virus-induced accumulation of intracellular bile acids activates the TGR5-β-arrestin-SRC axis to enable innate antiviral immunity. Cell Research, 29(3), 193-205.

[33] Turcotte, C., Chouinard, F., Lefebvre, J. S., & Flamand, N. (2015). Regulation of inflammation by cannabinoids, the endocannabinoids 2-arachidonoyl-glycerol and arachidonoyl-ethanolamide, and their metabolites. Journal of Leukocyte Biology, 97(6), 1049-1070.

[34] Stefano, G. B., Bilfinger, T. V., Rialas, C. M., & Deutsch, D. G. (2000). 2-arachidonyl-glycerol stimulates nitric oxide release from human immune and vascular tissues and invertebrate immunocytes by cannabinoid receptor 1. Pharmacological Research, 42(4), 317-322.

[35] Rockwell, C. E., Snider, N. T., Thompson, J. T., Vanden Heuvel, J. P., & Kaminski, N. E. (2006). Interleukin-2 suppression by 2-arachidonyl glycerol is mediated through peroxisome proliferator-activated receptor gamma independently of cannabinoid receptors 1 and 2. Molecular Pharmacology, 70, 101-111.

[36] Signorello, M. G., & Leoncini, G. (2014). Effect of 2-arachidonoylglycerol on myosin light chain phosphorylation and platelet activation: The role of phosphatidylinositol 3 kinase/AKT pathway. Biochimie, 105, 182-191.

[37] Holmsen, H., Hindenes, J. O., & Fukami, M. (1992). Glycerophospholipid metabolism: Back to the future. Thrombosis Research, 67(3), 313-323.

[38] Wang, C., Li, L., Guan, H., Tong, S., Liu, M., Liu, C., Zhang, Z., Du, C., & Li, P. (2013). Effects of taurocholic acid on immunoregulation in mice. International Immunopharmacology, 15(2), 217-222.

[39] Ueno, Y., Francis, H., Glaser, S., Demorrow, S., Venter, J., Benedetti, A., Fava, G., Marzioni, M., & Alpini, G. (2007). Taurocholic acid feeding prevents tumor necrosis factor-alpha-induced damage of cholangiocytes by a PI3K-mediated pathway. Experimental Biology and Medicine (Maywood, NJ, United States), 232, 942-949.

[40] Yang, Q., Zhang, Q., Tang, J., & Feng, W.-H. (2015). Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection. Virology, 484, 170-180.

[41] Buck, J., Grün, F., Derguini, F., Chen, Y., Kimura, S., Noy, N., & Hämmerling, U. (1993). Anhydroretinol: A naturally occurring inhibitor of lymphocyte physiology. Journal of Experimental Medicine, 178(2), 675-680.

[42] Blomhoff, H. K., Smeland, E. B., Erikstein, B., Rasmussen, A. M., Skrede, B., Skjønsberg, C., & Blomhoff, R. (1992). Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells. Journal of Biological Chemistry, 267(33), 23988-23992.

[43] van Bennekum, A. M., Wong Yen Kong, L. R., Gijbels, M. J., Tielen, F. J., Roholl, P. J., Brouwer, A., & Hendriks, H. F. (1991). Mitogen response of B cells, but not T cells, is impaired in adult vitamin A-deficient rats. Journal of Nutrition, 121(12), 1960-1968.

[44] Tokuyama, H., & Tokuyama, Y. (1999). The regulatory effects of all-trans-retinoic acid on isotype switching: Retinoic acid induces IgA switch rearrangement in cooperation with IL-5 and inhibits IgG1 switching. Cellular Immunology, 192(1), 41-47.

[45] Hess, D., Chisholm, J. W., & Igal, R. A. (2010). Inhibition of stearoylCoA desaturase activity blocks cell cycle progression and induces programmed cell death in lung cancer cells. PLoS One, 5(6), e11394.

[46] Ishida, T., Nishiumi, S., Tanahashi, T., Yamasaki, A., Yamazaki, A., Akashi, T., Miki, I., Kondo, Y., Inoue, J., Kawauchi, S., Azuma, T., Yoshida, M., & Mizuno, S. (2013). Linoleoyl ethanolamide reduces lipopolysaccharide-induced inflammation in macrophages and ameliorates 2,4-dinitrofluorobenzene-induced contact dermatitis in mice. European Journal of Pharmacology, 699(1-3), 6-13.

[47] Jirak, P., Mirna, M., Wernly, B., Paar, V., Hoppe, U. C., & Lichtenauer, M. (2020). Assessment of cardiac remodeling - A chance for novel cardiac biomarkers? Journal of Clinical Medicine, 9(7), 2087.

[48] Davey, M. G., Hynes, S. O., Kerin, M. J., Miller, N., & Lowery, A. J. (2021). Ki-67 as a prognostic biomarker in invasive breast cancer. Cancers, 13(17), 4455.

[49] Lima, M. F., Amaral, A. G., Moretto, I. A., Paiva-Silva, F. J. T. N., Pereira, F. O. B., Barbas, C., Dos Santos, A. M., Simionato, A. V. C., & Rupérez, F. J. (2022). Untargeted metabolomics studies of H9c2 cardiac cells submitted to oxidative stress, β-adrenergic stimulation and doxorubicin treatment: Investigation of cardiac biomarkers. Frontiers in Molecular Biosciences, 9, 898742.

RIGHTS & PERMISSIONS

2023 The Authors. Animal Research and One Health published by John Wiley & Sons Australia, Ltd on behalf of Institute of Animal Science, Chinese Academy of Agricultural Sciences.