Neutrophil extracellular traps aggravate intestinal epithelial necroptosis in ischaemia–reperfusion by regulating TLR4/RIPK3/FUNDC1-required mitophagy

Abstract

Neutrophil extracellular trap (NET) has been confirmed to be related to gut barrier injury during intestinal ischaemia–reperfusion (II/R). However, the specific molecular regulatory mechanism of NETs in II/R-induced intestinal barrier damage has yet to be fully elucidated. Here, we reported increased NETs infiltration accompanied by elevated inflammatory cytokines, cellular necroptosis and tight junction disruption in the intestine of human II/R patients. Meanwhile, NETs aggravated Caco-2 intestinal epithelial cell necroptosis, impairing the monolayer barrier in vitro. Moreover, Pad4-deficient mice were used further to validate the role of NETs in II/R-induced intestinal injury. In contrast, NET inhibition via Pad4 deficiency alleviated intestinal inflammation, attenuated cellular necroptosis, improved intestinal permeability, and enhanced tight junction protein expression. Notably, NETs prevented FUN14 domain-containing 1 (FUNDC1)-required mitophagy activation in intestinal epithelial cells, and stimulating mitophagy attenuated NET-associated mitochondrial dysfunction, cellular necroptosis, and intestinal damage. Mechanistically, silencing Toll-like receptor 4 (TLR4) or receptor-interacting protein kinase 3 (RIPK3) via shRNA relieved mitophagy limitation, restored mitochondrial function and reduced NET-induced necroptosis in Caco-2 cells, whereas this protective effect was reversed by TLR4 or RIPK3 overexpression. The regulation of TLR4/RIPK3/FUNDC1-required mitophagy by NETs can potentially induce intestinal epithelium necroptosis.

References

[1] Li G, Wang S, Fan Z. Oxidative stress in intestinal ischemia-reperfusion. Front Med (Lausanne). 2021;8:750731.

[2] Wang Z, Sun R, Wang G, et al. SIRT3-mediated deacetylation of PRDX3 alleviates mitochondrial oxidative damage and apoptosis induced by intestinal ischemia/reperfusion injury. Redox Biol. 2020;28:101343.

[3] Li Y, Feng D, Wang Z, et al. Ischemia-induced ACSL4 activation contributes to ferroptosis-mediated tissue injury in intestinal ischemia/reperfusion. Cell Death Differ. 2019;26(11):2284-2299.

[4] Jia Y, Cui R, Wang C, et al. Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway. Redox Biol. 2020;32:101534.

[5] Hayase N, Doi K, Hiruma T, et al. Recombinant thrombomodulin on neutrophil extracellular traps in murine intestinal ischemia-reperfusion. Anesthesiology. 2019;131(4):866-882.

[6] Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532-1535.

[7] Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol. 2018;18(2):134-147.

[8] Breda LCD, Breda CNS, de Almeida JRF, et al. Fonsecaea pedrosoi conidia and hyphae activate neutrophils distinctly: requirement of TLR-2 and TLR-4 in neutrophil effector functions. Front Immunol. 2020;11:540064.

[9] Yang S, Wang S, Chen L, et al. Neutrophil extracellular traps delay diabetic wound healing by inducing endothelial-to-mesenchymal transition via the hippo pathway. Int J Biol Sci. 2023;19(1):347-361.

[10] Honda M, Kubes P. Neutrophils and neutrophil extracellular traps in the liver and gastrointestinal system. Nat Rev Gastroenterol Hepatol. 2018;15(4):206-221.

[11] Zhan Y, Ling Y, Deng Q, et al. HMGB1-mediated neutrophil extracellular trap formation exacerbates intestinal ischemia/reperfusion-induced acute lung injury. J Immunol. 2022;208(4):968-978.

[12] Chen K, Shao LH, Wang F, et al. Netting gut disease: neutrophil extracellular trap in intestinal pathology. Oxid Med Cell Longev. 2021;2021:5541222.

[13] Wang S, Xie T, Sun S, et al. DNase-1 treatment exerts protective effects in a rat model of intestinal ischemia-reperfusion injury. Sci Rep. 2018;8(1):17788.

[14] Onishi M, Yamano K, Sato M, Matsuda N, Okamoto K. Molecular mechanisms and physiological functions of mitophagy. EMBO J. 2021;40(3):e104705.

[15] Chang X, Lochner A, Wang HH, et al. Coronary microvascular injury in myocardial infarction: perception and knowledge for mitochondrial quality control. Theranostics. 2021;11(14):6766-6785.

[16] Zhang Q, Liu XM, Hu Q, et al. Dexmedetomidine inhibits mitochondria damage and apoptosis of enteric glial cells in experimental intestinal ischemia/reperfusion injury via SIRT3-dependent PINK1/HDAC3/p53 pathway. J Transl Med. 2021;19(1):463.

[17] Kip AM, Soons Z, Mohren R, et al. Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death Dis. 2021;12(1):95.

[18] Huang Z, Wang S, Yang Y, et al. Mitochondrial dysfunction promotes the necroptosis of Purkinje cells in the cerebellum of acrylamide-exposed rats. Food Chem Toxicol. 2023;171:113522.

[19] Zhao M, Wang Y, Li L, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021;11(4):1845-1863.

[20] Li X, Wang C, Zhu J, et al. Sodium butyrate ameliorates oxidative stress-induced intestinal epithelium barrier injury and mitochondrial damage through AMPK-mitophagy pathway. Oxid Med Cell Longev. 2022;2022:3745135.

[21] Wang J, Toan S, Zhou H. Mitochondrial quality control in cardiac microvascular ischemia-reperfusion injury: new insights into the mechanisms and therapeutic potentials. Pharmacol Res. 2020;156:104771.

[22] Samson AL, Fitzgibbon C, Patel KM, et al. A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells. Cell Death Differ. 2021;28(7):2126-2144.

[23] Chenxu G, Minxuan X, Yuting Q, et al. Loss of RIP3 initiates annihilation of high-fat diet initialized nonalcoholic hepatosteatosis: a mechanism involving toll-like receptor 4 and oxidative stress. Free Radic Biol Med. 2019;134:23-41.

[24] Cao M, Chen F, Xie N, et al. c-Jun N-terminal kinases differentially regulate TNF- and TLRs-mediated necroptosis through their kinase-dependent and -independent activities. Cell Death Dis. 2018;9(12):1140.

[25] Liu H, Zang C, Yuan F, et al. The role of FUNDC1 in mitophagy, mitochondrial dynamics and human diseases. Biochem Pharmacol. 2022;197:114891.

[26] Zhou H, Zhu P, Wang J, Toan S, Ren J. DNA-PKcs promotes alcohol-related liver disease by activating Drp1-related mitochondrial fission and repressing FUNDC1-required mitophagy. Signal Transduct Target Ther. 2019;4:56.

[27] Zhou H, Zhu P, Guo J, et al. Ripk3 induces mitochondrial apoptosis via inhibition of FUNDC1 mitophagy in cardiac IR injury. Redox Biol. 2017;13:498-507.

[28] Ding W, Wang K, Liu B, et al. Open abdomen improves survival in patients with peritonitis secondary to acute superior mesenteric artery occlusion. J Clin Gastroenterol. 2017;51(9):e77-e82.

[29] Gubernatorova EO, Perez-Chanona E, Koroleva EP, Jobin C, Tumanov AV. Murine model of intestinal ischemia–reperfusion injury. J Vis Exp. 2016;111:53881.

[30] Li Y, Cao Y, Xiao J, et al. Inhibitor of apoptosis-stimulating protein of p53 inhibits ferroptosis and alleviates intestinal ischemia/reperfusion-induced acute lung injury. Cell Death Differ. 2020;27(9):2635-2650.

[31] Caudrillier A, Kessenbrock K, Gilliss BM, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. J Clin Invest. 2012;122(7):2661-2671.

[32] Chu C, Yang C, Wang X, et al. Early intravenous administration of tranexamic acid ameliorates intestinal barrier injury induced by neutrophil extracellular traps in a rat model of trauma/hemorrhagic shock. Surgery. 2020;167(2):340-351.

[33] Sun S, Duan Z, Wang X, et al. Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway. Cell Death Dis. 2021;12(6):606.

[34] Najmeh S, Cools-Lartigue J, Giannias B, Spicer J, Ferri LE. Simplified human neutrophil extracellular traps (NETs) isolation and handling. J Vis Exp. 2015;98:52687.

[35] Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg. 1970;101(4):478-483.

[36] Tan Y, Zhang Y, He J, et al. Dual specificity phosphatase 1 attenuates inflammation-induced cardiomyopathy by improving mitophagy and mitochondrial metabolism. Mol Metab. 2022;64:101567.

[37] Wang Y, Jasper H, Toan S, Muid D, Chang X, Zhou H. Mitophagy coordinates the mitochondrial unfolded protein response to attenuate inflammation-mediated myocardial injury. Redox Biol. 2021;45:102049.

[38] Li S, Zhou Y, Gu X, Zhang X, Jia Z. NLRX1/FUNDC1/NIPSNAP1-2 axis regulates mitophagy and alleviates intestinal ischaemia/reperfusion injury. Cell Prolif. 2021;54(3):e12986.

[39] Tsourouktsoglou TD, Warnatsch A, Ioannou M, Hoving D, Wang Q, Papayannopoulos V. Histones, DNA, and citrullination promote neutrophil extracellular trap inflammation by regulating the localization and activation of TLR4. Cell Rep. 2020;31(5):107602.

[40] Clark SR, Ma AC, Tavener SA, et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med. 2007;13(4):463-469.

[41] Boufenzer A, Carrasco K, Jolly L, et al. Potentiation of NETs release is novel characteristic of TREM-1 activation and the pharmacological inhibition of TREM-1 could prevent from the deleterious consequences of NETs release in sepsis. Cell Mol Immunol. 2021;18(2):452-460.

[42] Kim SJ, Li J. Caspase blockade induces RIP3-mediated programmed necrosis in toll-like receptor-activated microglia. Cell Death Dis. 2013;4:e716.

[43] Yao RQ, Ren C, Xia ZF, Yao YM. Organelle-specific autophagy in inflammatory diseases: a potential therapeutic target underlying the quality control of multiple organelles. Autophagy. 2021;17(2):385-401.

[44] Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev. 2014;94(2):329-354.

[45] Tahir M, Arshid S, Fontes B, et al. Phosphoproteomic analysis of rat neutrophils shows the effect of intestinal ischemia/reperfusion and preconditioning on kinases and phosphatases. Int J Mol Sci. 2020;21(16):5799.

[46] Wu MCL, Lee JD, Ruitenberg MJ, Woodruff TM. Absence of the C5a receptor C5aR2 worsens ischemic tissue injury by increasing C5aR1-mediated neutrophil infiltration. J Immunol. 2020;205(10):2834-2839.

[47] Mutua V, Gershwin LJ. A review of neutrophil extracellular traps (NETs) in disease: potential anti-NETs therapeutics. Clin Rev Allergy Immunol. 2021;61(2):194-211.

[48] Liu L, Mao Y, Xu B, et al. Induction of neutrophil extracellular traps during tissue injury: involvement of STING and toll-like receptor 9 pathways. Cell Prolif. 2019;52(3):e12579.

[49] Dinallo V, Marafini I, Di Fusco D, et al. Neutrophil extracellular traps sustain inflammatory signals in ulcerative colitis. J Crohns Colitis. 2019;13(6):772-784.

[50] Fu ZJ, Wang ZY, Xu L, et al. HIF-1alpha-BNIP3-mediated mitophagy in tubular cells protects against renal ischemia/reperfusion injury. Redox Biol. 2020;36:101671.

[51] Zhou H, Ren J, Toan S, Mui D. Role of mitochondrial quality surveillance in myocardial infarction: from bench to bedside. Ageing Res Rev. 2021;66:101250.

[52] Zhang H, Yan Q, Wang X, et al. The role of mitochondria in liver ischemia-reperfusion injury: from aspects of mitochondrial oxidative stress, mitochondrial fission, mitochondrial membrane permeable transport pore formation, mitophagy, and mitochondria-related protective measures. Oxid Med Cell Longev. 2021;2021:6670579.

[53] Tong M, Zablocki D, Sadoshima J. The role of Drp1 in mitophagy and cell death in the heart. J Mol Cell Cardiol. 2020;142:138-145.

[54] Zhu P, Wan K, Yin M, et al. RIPK3 induces cardiomyocyte necroptosis via inhibition of AMPK-parkin-mitophagy in cardiac remodelling after myocardial infarction. Oxid Med Cell Longev. 2021;2021:6635955.

[55] Zhang X, Wu J, Liu Q, et al. mtDNA-STING pathway promotes necroptosis-dependent enterocyte injury in intestinal ischemia reperfusion. Cell Death Dis. 2020;11(12):1050.

[56] Wen S, Li X, Ling Y, et al. HMGB1-associated necroptosis and Kupffer cells M1 polarization underlies remote liver injury induced by intestinal ischemia/reperfusion in rats. FASEB J. 2020;34(3):4384-4402.

[57] Zhu P, Hu S, Jin Q, et al. Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: a mechanism involving calcium overload/XO/ROS/mPTP pathway. Redox Biol. 2018;16:157-168.

[58] Zhou H, Li D, Zhu P, et al. Inhibitory effect of melatonin on necroptosis via repressing the Ripk3-PGAM5-CypD-mPTP pathway attenuates cardiac microvascular ischemia-reperfusion injury. J Pineal Res. 2018;65(3):e12503.

[59] Xian H, Liou YC. Functions of outer mitochondrial membrane proteins: mediating the crosstalk between mitochondrial dynamics and mitophagy. Cell Death Differ. 2021;28(3):827-842.

[60] Wu X, Zheng Y, Liu M, et al. BNIP3L/NIX degradation leads to mitophagy deficiency in ischemic brains. Autophagy. 2021;17(8):1934-1946.

[61] Wu W, Tian W, Hu Z, et al. ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy. EMBO Rep. 2014;15(5):566-575.

[62] Edwards NJ, Hwang C, Marini S, et al. The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury. FASEB J. 2020;34(12):15753-15770.

[63] Shao S, Fang H, Dang E, et al. Neutrophil extracellular traps promote inflammatory responses in psoriasis via activating epidermal TLR4/IL-36R crosstalk. Front Immunol. 2019;10:746.

[64] Yazdani HO, Roy E, Comerci AJ, et al. Neutrophil extracellular traps drive mitochondrial homeostasis in tumors to augment growth. Cancer Res. 2019;79(21):5626-5639.

[65] Speir M, Lawlor KE. RIP-roaring inflammation: RIPK1 and RIPK3 driven NLRP3 inflammasome activation and autoinflammatory disease. Semin Cell Dev Biol. 2021;109:114-124.

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2023 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.