Simultaneous blockade of TLR4 and TNFR1 attenuates TLR2 sensitivity in LPS-stimulated macrophages through TNFR2-mediated pathway
Main Article Content
Abstract
Background: Recent studies have found TLR2 to be a significant player in initiating immune responses in the host during bacterial infection. Macrophage polarization is one of the vital factors in the amelioration of sepsis. It is well established that recognition and binding of LPS with cell surface TLR4 could induce the production of a wide array of pro-inflammatory cytokines that initiate an organism’s inflammatory responses. Recent studies claimed that augmented expression of TLR2 shows better responsiveness to LPS, thus increasing its affinity to the ligand. Objectives: Our study attempts to demonstrate the underlying mechanisms of how TLR2 sensitivity is altered during the simultaneous blocking of TLR4 and TNFR1 and how TLR2 contributes towards the phenotypic switching of macrophages. We were also interested to see whether blocking TLR4, in any way, affects the LPS/TLR2 interactions and influences some major cytokine receptors. Materials and Methods: Murine peritoneal macrophages (5×106 cells/ml) were pre-treated with TLR4 and TNFR1 antibody (alone or in combination) and then stimulated with LPS for 60 min. FACS analyses were performed to determine M1 and M2 polarized cell populations. Assays from the cell-free supernatant determined ROS generation, and the activities of antioxidant enzymes were determined from the cell-free lysate. Western blot analysis was used to determine receptor expressions. Results: Our results indicated that blocking both receptors markedly reduced ROS levels due to its scavenging by the elevated antioxidant enzymes. Western blot data confirmed that combinatorial blockade of TLR4 and TNFR1 augmented TLR2 and TNFR2 expression in contrast to the attenuation of IL-1R. Conclusion: Therefore, the regulation of TLR2 expression was found to be TLR4-dependent, and it can show reduced NF-κB activation in response to LPS in TLR4 and TNFR1 blocked macrophages. Moreover, dual blocking can promote M2 polarization by up-regulating TNFR2. This approach could be taken as an alternative therapeutic strategy to treat LPS-sepsis.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
References
REFERENCES
Love W, Dobbs N, Tabor L, Simecka JW. Toll-like receptor 2 (TLR2) plays a major role in innate resistance in the lung against murine mycoplasma. PloS one. 2010; 5(5): e10739. https://doi.org/10.1371/journal.pone.0010739
Hoppstädter J, Dembek A, Linnenberger R, et al. Toll-like receptor 2 release by macrophages: an anti-inflammatory program induced by glucocorticoids and lipopolysaccharide. Frontiers in immunology. 2019; 10: 1634. https://doi.org/10.3389/fimmu.2019.01634
Chang CP, Su YC, Hu CW, Lei HY. TLR2-dependent selective autophagy regulates NF-κB lysosomal degradation in hepatoma-derived M2 macrophage differentiation. Cell death and differentiation. (2013); 20(3): 515–523. https://doi.org/10.1038/cdd.2012.146
Zhang C, Yang M, Ericsson AC. Function of macrophages in disease: current understanding on molecular mechanisms. Frontiers in immunology. 2021; 12:620510. https://doi.org/10.3389/fimmu.2021.620510
Ramachandran G. Gram-positive and gram-negative bacterial toxins in sepsis: a brief review. Virulence. 2014; 5(1):213–218. https://doi.org/10.4161/viru.27024
Satoh T, Akira S. Toll-Like Receptor Signaling and Its Inducible Proteins. Microbiol Spectr. 2016; 4(6): 10.1128/microbiolspec.MCHD-0040-2016. doi:10.1128/microbiolspec.MCHD-0040-2016
Skinner NA, MacIsaac CM, Hamilton JA, Visvanathan K. Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens. Clin Exp Immunol. 2005; 141(2):270-278. doi: 10.1111/j.1365-2249.2005.02839.x.
Chen X, Andresen1 BT, Hill M, Zhang J, Booth F, Zhang C. Role of Reactive Oxygen Species in Tumor Necrosis Factor-alpha Induced Endothelial Dysfunction. Curr Hypertens Rev. 2008; 4(4): 245-255. doi:10.2174/157340208786241336
Herb M, Schramm M. Functions of ROS in macrophages and antimicrobial immunity. Antioxidants (Basel). 2021; 10(2): 313. doi: 10.3390/antiox10020313.
Xu Q, Choksi S, Qu J, et al. NADPH oxidases are essential for macrophage differentiation. J Biol Chem. 2016; 291(38): 20030-20041. doi: 10.1074/jbc.M116.731216.
Oeckinghaus A, Hayden MS, Ghosh S. Crosstalk in NF-Κbsignaling pathways. Nat Immunol. 2011; 12(8):695-708. doi: 10.1038/ni.2065
Ciesielska A, Matyjek M, Kwiatkowska K. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci. 2020; 78(4): 1233-1261. doi: 10.1007/s00018-020-03656-y.
Hwanga EH, Kim TH, Park JY, et al. TLR2 contributes to trigger immune response of pleural mesothelial cells against Mycobacterium bovis BCG and M. tuberculosis infection. Cytokine. 2017; 95: 80-87.doi: 10.1016/j.cyto.2017.02.021
Mukherjee S, Karmakar S, Babu SP. TLR2 and TLR4 mediated host immune responses in major infectious diseases: a review. Braz J Infect Dis. 2016; 20(2):193-204. doi: 10.1016/j.bjid.2015.10.011.
Liu S, Gallo DJ, Green AM, et al. Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide. Infect Immun. 2002; 70(7): 3433-3442. doi: 10.1128/IAI.70.7.3433-3442.2002.
Opal SM, Huber CE. Bench-to-bedside review: Toll-like receptors and their role in septic shock. Crit Care. 2002; 6(2): 125-136. doi: 10.1186/cc1471
Savva A, Roger T. Targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases. Front Immunol. 2013; 4: 387. doi: 10.3389/fimmu.2013.00387
Fitzgerald KA, Kagan JC. Toll-like receptors and the control of immunity. Cell. 2020; 180(6): 1044-1066. doi: 10.1016/j.cell.2020.02.041.
Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev. 2009; 22(2): 240-273. doi: 10.1128/CMR.00046-08.
McGettrick AF, O'Neill LA. The expanding family of MyD88-like adaptors in Toll-like receptor signal transduction. Mol Immunol. 2004; 41(6-7): 577-582. doi: 10.1016/j.molimm.2004.04.006.
Ozato K, Tsujimura H, Tamura T. Toll-like receptor signaling and regulation of cytokine gene expression in the immune system. Biotechniques. 2002; 70: 66-68.
Prauchner CA. Oxidative stress in sepsis: pathophysiological implications justifying antioxidant co-therapy. Burns: journal of the International Society for Burn Injuries. 2017; 43(3): 471–485. https://doi.org/10.1016/j.burns.2016.09.023
Younus H. Therapeutic potentials of superoxide dismutase. International journal of health sciences. 2018; 12(3): 88–93.
Li F, Yan R, Wu J, et al. An Antioxidant Enzyme Therapeutic for Sepsis. Front Bioeng Biotechnol. 2021; 9: 800684. doi:10.3389/fbioe.2021.800684.
Kalliolias GD, Ivashkiv LB. TNF biology, pathogenic mechanisms and emerging therapeutic strategies. Nat Rev Rheumatol. 2015; 12(1): 49-62. doi: 10.1038/nrrheum.2015.169.
Menegatti S, Bianchi E, Rogge L. Anti-TNF therapy in spondyloarthritis and related diseases, impact on the immune system and prediction of treatment responses. Front Immunol. 2019; 10: 382. doi: 10.3389/fimmu.2019.00382.
Yang S, Wang J, Brand DD, Zheng SG. Role of TNF-TNF receptor 2 signal in regulatory T cells and its therapeutic implications. Front Immunol. 2018; 9: 784. doi: 10.3389/fimmu.2018.00784.
Fischer R, Kontermann RE, Pfizenmaier K. Selective targeting of TNF receptors as a novel therapeutic approach. Front Cell Dev Biol. 2020; 8: 401. doi: 10.3389/fcell.2020.00401.
Uysal H, Chavez-Galan L, Vesin D, et al. Transmembrane TNF and partially TNFR1 regulate TNFR2 expression and control inflammation in mycobacterial-induced pleurisy. Int J Mol Sci. 2018; 19(7):1959. doi: 10.3390/ijms19071959.
Papazian I, Tsoukala E, Boutou A, et al. Fundamentally different roles of neuronal TNF receptors in CNS pathology: TNFR1 and IKKβ promote microglial responses and tissue injury in demyelination while TNFR2 protects against excitotoxicity in mice. J Neuroinflammation. 2021; 18(1): 222. doi: 10.1186/s12974-021-02200-4.
Ruiz A, Palacios Y, Garcia I, Chavez-Galan L. Transmembrane TNF and its receptors TNFR1 and TNFR2 in mycobacterial infections. Int J Mol Sci. 2021; 22(11): 5461. doi: 10.3390/ijms22115461.
Dinarello CA. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev. 2018; 81(1): 8-27. doi: 10.1111/imr.12621
Guven-Maiorov E, Keskin O, Gursoy A, et al. The architecture of the TIR domain signalosome in the Toll-like receptor-4 signaling pathway. Sci Rep. 2015; 5: 13128. doi: 10.1038/srep13128.
Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008; 14:14.1.1-14.1.14. doi: 10.1002/0471142735.im1401s83
Meltzer MS. Peritoneal mononuclear phagocytes from small animals. In: Adams D, Edelson PJ, Koren HS, editors. Methods for studying mononuclear phagocytes. New York: Academic Press; 1981. pp- 63–68.
Leon CG, Tory R, Jia J, Sivak O, Wasan KM. Discovery and development of toll-like receptor 4 (TLR4) antagonists: a new paradigm for treating sepsis and other diseases. Pharm Res. 2008; 25(8):1751-1761. doi: 10.1007/s11095-008-9571-x.
Dutta P, Sultana S, Dey R, Bishayi B. Regulation of Staphylococcus aureus-induced CXCR1 expression via inhibition of receptor mobilization and receptor shedding during dual receptor (TNFR1 and IL-1R) neutralization. Immunol Res. 2019; 67(2-3): 241-260 doi: 10.1007/s12026-019-09083-x.
Watanabe K, Jose PJ, Rankin SM. Eotaxin-2 generation is differentially regulated by lipopolysaccharide and IL-4 in monocytes and macrophages. J Immunol. 2002; 168(4):1911-1918. doi: 10.4049/jimmunol.168.4.1911
Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva A. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochem. 2012; 114(8): 785-796. doi: 10.1016/j.acthis.2012.01.006.
Jablonski KA, Amici SA, Webb LM, et al. Novel markers to delineate murine M1 and M2 macrophages. PLoS One. 2015; 10(12): e0145342. doi: 10.1371/journal.pone.0145342.
Leijh PC, van Zwet TL, van den Barselaar MT, van Furth R. The extracellular stimulation of intracellular killing by phagocytes. Adv Exp Med Biol. 1982; 141:139-149. doi:10.1007/978-1-4684-8088-7_14.
Watanabe I, Ichiki M, Shiratsuchi A, Nakanishi Y. TLR2-mediated survival of Staphylococcus aureus in macrophages: a novel bacterial strategy against host innate immunity. J Immunol. 2007; 178 (8): 4917-4925. doi: 10.4049/jimmunol.178.8.4917.
Buege JA, Steven DA. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302-310. doi:10.1016/s0076-6879(78)52032-6.
Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem. 1968; 25(1): 192-205. doi: 10.1016/0003-2697(68)90092-4.
Paoletti F, Aldinucci D, Mocali A, Caparrini A. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal Biochem. 1986; 154: 536-541. doi: 10.1016/0003-2697(86)90026-6.
Aebi H, Wyss SR, Scherz B, Skvaril F. Heterogeneity of erythrocyte catalase II. Isolation and characterization of normal and variant erythrocyte catalase and their subunits. Eur J Biochem. 1974; 48(1): 137-145. doi: 10.1111/j.1432-1033.1974.tb03751.x.
Goldberg DM, Spooner RJ. Assay of Glutathione Reductase. In: Bergmeyen, HV, editor. Methods of Enzymatic Analysis. Deerfiled Beach, Verlog Chemie. 1983; pp. 258-265.
Weisser SB, McLarren KW, Kuroda E, Sly LM. Generation and characterization of murine alternatively activated macrophages. Methods Mol Biol. 2013; 946: 225-239. doi:10.1007/978-1-62703-128-8_14.
Corraliza IM, Campo ML, Soler G, Modolell M. Determination of arginase activity in macrophages: a micromethod. J Immunol Methods. 1994; 174(1-2): 231-235. doi: 10.1016/0022-1759(94)90027-2.
Font MD, Thyagarajan B, Khanna AK. Sepsis and septic shock - basics of diagnosis, pathophysiology and clinical decision making. Med Clin North Am. 2020; 104(4): 573-585. doi: 10.1016/j.mcna.2020.02.011.
Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol. 2017; 39(5): 517-528. doi: 10.1007/s00281-017-0639-8.
Gotts JE, Matthay MA. Sepsis: pathophysiology and clinical management. BMJ. 2016; 353: i1585. doi: 10.1136/bmj.i1585.
Lima CX, Souza DG, Amaral FA, et al. Therapeutic effects of treatment with anti-TLR2 and anti-TLR4 monoclonal antibodies in polymicrobial sepsis. PLoS One. 2015; 10(7): e0132336. doi: 10.1371/journal.pone.0132336.
Zhang X, Kimura Y, Fang C, et al. Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood. 2007; 110(1): 228–236. https://doi.org/10.1182/blood-2006-12-063636.
Matsuguchi T, Musikacharoen T, Ogawa T, Yoshikai Y. Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages. J Immunol. 2000; doi: 10.4049/jimmunol.165.10.5767.
Good DW, George T, Watts BA. Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb. J Biol Chem. 2012; 287(24): 20208-202020. doi: 10.1074/jbc.M111.336255.
Oliveira-Nascimento L, Massari P, Wetzler LM. The role of TLR2 in infection and immunity. Front Immunol. 2012; 3: 79. doi: 10.3389/fimmu.2012.00079.
Carl VS, Brown-Steinke K, Nicklin MJ, Smith MF Jr. Toll-like receptor 2 and 4 (TLR2 and TLR4) agonists differentially regulate secretory interleukin-1 receptor antagonist gene expression in macrophages. J Biol Chem. 2002; 277(20): 17448-17456. doi: 10.1074/jbc.M111847200.
van Bergenhenegouwen J, Kraneveld AD, Rutten L, Garssen J, Vos AP, Hartog A. Lipoproteins attenuate TLR2 and TLR4 activation by bacteria and bacterial ligands with differences in affinity and kinetics. BMC Immunol. 2016; 17(1): 42. doi: 10.1186/s12865-016-0180-x.
Sato S, Nomura F, Kawai T, et al. Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways. J Immunol. 2000; 165(12): 7096-7101. doi: 10.4049/jimmunol.165.12.7096.
Zhou S, Wang G, Zhang W. Effect of TLR4/MyD88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response. Exp Ther Med. 2018; 15(4): 3376-3384. doi: 10.3892/etm.2018.5815.
Chang C, Hu L, Sun S, et al. Regulatory role of the TLR4/JNK signaling pathway in sepsis-induced myocardial dysfunction. Mol Med Rep. 2021; 23(5): 334. doi: 10.3892/mmr.2021.11973.
Mussbacher M, Salzmann M, Brostjan C, et al. Cell type-specific roles of NF-κB linking inflammation and thrombosis. Front Immunol. 2019; 10: 85. doi: 10.3389/fimmu.2019.00085.
Sawoo R, Dey R, Ghosh R, Bishayi B. TLR4 and TNFR1 blockade dampen M1 macrophage activation and shifts them towards an M2 phenotype. Immunol Res. 2021; 69(4): 334-351. doi: 10.1007/s12026-021-09209-0.
Kuzmich NN, Sivak KV, Chubarev VN, Porozov YB, Savateeva-Lyubimova TN, Peri F. TLR4 signaling pathway modulators as potential therapeutics in inflammation and sepsis. Vaccines (Basel). 2017; 5(4): 34. doi: 10.3390/vaccines5040034.
Wajant H, Siegmund D. TNFR1 and TNFR2 in the control of the life and death balance of macrophages. Front Cell Dev Biol. 2019; 7: 91. doi: 10.3389/fcell.2019.00091.
Legarda D, Justus SJ, Ang RL, et al. CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN. Cell Rep. 2016; 15(11): 2449-2461. doi: 10.1016/j.celrep.2016.05.032.
Deng Y, Ren X, Yang L, Lin Y, Wu X. A JNK-dependent pathway is required for TNFalpha-induced apoptosis. Cell. 2003; 115(1): 61-70. doi: 10.1016/s0092-8674(03)00757-8.
Tan HY, Wang N, Li S, Hong M, Wang X, Feng Y. The reactive oxygen species in macrophage polarization: reflecting its dual role in progression and treatment of human diseases. Oxid Med Cell Longev. 2016; 2016: 2795090. doi: 10.1155/2016/2795090.
Zhang L, Pavicic PG Jr, Datta S, et al. Unfolded protein response differentially regulates TLR4-induced cytokine expression in distinct macrophage populations. Front Immunol. 2019; 10: 1390. doi: 10.3389/fimmu.2019.01390.
Pushpakumar S, Ren L, Kundu S, Gamon A, Tyagi SC, Sen U. Toll-like Receptor 4 deficiency reduces oxidative stress and macrophage mediated inflammation in hypertensive kidney. Sci Rep. 2017; 7(1): 6349. doi: 10.1038/s41598-017-06484-6.
Fischer R, Maier O. Interrelation of oxidative stress and inflammation in neurodegenerative disease: role of TNF. Oxid Med Cell Longev. 2015; 2015: 610813. doi: 10.1155/2015/610813.
Li Y, Deng SL, Lian ZX, Yu K. Roles of Toll-like receptors in nitroxidative stress in mammals. Cells. 2019; 8(6): 576. doi: 10.3390/cells8060576.
Rath M, Müller I, Kropf P, Closs EI, Munder M. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front Immunol. 2014; 5: 532. doi: 10.3389/fimmu.2014.00532.
Caldwell RW, Rodriguez PC, Toque HA, Narayanan SP, Caldwell RB. Arginase: a multifaceted enzyme important in health and disease. Physiol Rev. 2018; 98(2): 641-665. doi: 10.1152/physrev.00037.2016.
Francisco S, Billod JM, Merino J, et al. Induction of TLR4/TLR2 interaction and heterodimer formation by low endotoxic atypical LPS. Front Immunol. 2022; 12: 748303. doi: 10.3389/fimmu.2021.748303.
Fu W, Hu W, Yi YS, et al. TNFR2/14-3-3ε signaling complex instructs macrophage plasticity in inflammation and autoimmunity. J Clin Invest. 2021; 131(16): e144016. doi: 10.1172/JCI144016.
Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem. 2009; 390(3): 191-214. doi: 10.1515/BC.2009.033.
Zhao H, Wu L, Yan G, et al. Inflammation and tumor progression: signaling pathways and targeted intervention. Signal transduction and targeted therapy. 2021; 6(1):263. https://doi.org/10.1038/s41392-021-00658-5.
Ighodaro OM, Akinloye OA. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alexandria J Med. 2018; 54(4): 287-293.
Glushkova OV, Parfenyuk SB, Khrenov MO, et al. Inhibitors of TLR-4, NF-κB, and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells. J Immunotoxicol. 2013; 10(2): 133-140. doi: 10.3109/1547691X.2012.700652.
Aquilano K, Baldelli S, Ciriolo MR. Glutathione: new roles in redox signaling for an old antioxidant. Front Pharmacol. 2014; 5: 196. doi: 10.3389/fphar.2014.00196.
Espinosa-Diez C, Miguel V, Mennerich D, et al. Antioxidant responses and cellular adjustments to oxidative stress. Redox Biol. 2015; 6: 183-197. doi: 10.1016/j.redox.2015.07.008.
Gabay C, Lamacchia C, Palmer G. IL-1 pathways in inflammation and human diseases. Nat Rev Rheumatol. 2010; 6(4): 232-241. doi: 10.1038/nrrheum.2010.4.
Yang Y, Wang Y, Guo L, Gao W, Tang TL, Yan M. Interaction between macrophages and ferroptosis. Cell Death Dis. 2022; 13(4): 355. doi: 10.1038/s41419-022-04775-z.
Matsumura T, Ito A, Takii T, Hayashi H, Onozaki K. Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J Interferon Cytokine Res. 2000; 20(10): 915-921. doi: 10.1089/10799900050163299.