Cellular Physiology and Biochemistry

Original Paper

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Oxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy

Piippo N.a · Korhonen E.a · Hytti M.a · Kinnunen K.b,c · Kaarniranta K.b,c · Kauppinen A.a

Author affiliations

aSchool of Pharmacy, University of Eastern Finland, Kuopio, Finland
bDepartment of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland
cDepartment of Ophthalmology, Kuopio University Hospital, Kuopio, Finland

Corresponding Author

Anu Kauppinen

School of Pharmacy, Faculty of Health Sciences, University of Eastern

Finland P.O. Box 1627, 70211 Kuopio (Finland)

Tel. +358 40 3553216, Fax +358 17 162048, E-Mail anu.kauppinen@uef.f

Key Words: Nlrp3 inflammasomeActivation mechanismPotassium effluxCathepsin BROS

Related Articles for ""
Cell Physiol Biochem 2018;49:359–367
https://doi.org/10.1159/000492886

Abstract

Background/Aims: Previously, we demonstrated that blockade of the intracellular clearance systems in human retinal pigment epithelial (RPE) cells by MG-132 and bafilomycin A1 (BafA) induces NLRP3 inflammasome signaling. Here, we have explored the activation mechanisms behind this process. NLRP3 is an intracellular receptor detecting factors ranging from the endogenous alarmins and adenosine triphosphate (ATP) to ultraviolet radiation and solid particles. Due to the plethora of triggers, the activation of NLRP3 is often indirect and can be mediated through several alternative pathways. Potassium efflux, lysosomal rupture, and oxidative stress are currently the main mechanisms associated with many activators. Methods: NLRP3 inflammasomes were activated in human RPE cells by blocking proteasomes and autophagy using MG-132 and bafilomycin A1 (BafA), respectively. P2X7 inhibitor A740003, potassium chloride (KCl), and glyburide, or N-acetyl-L-cysteine (NAC), ammonium pyrrolidinedithiocarbamate (APDC), diphenyleneiodonium chloride (DPI), and mito-TEMPO were added to cell cultures in order to study the role of potassium efflux and oxidative stress, respectively. IL-1β was measured using the ELISA method. ATP levels and cathepsin B activity were examined using commercial kits, and ROS levels using the fluorescent dye 2´,7´-dichlorodihydrofluorescein diacetate (DCFDA). Results: Elevated extracellular potassium prevented the priming factor IL-1α from inducing the production of reactive oxygen species (ROS). It also prevented IL-1β release after exposure of primed cells to MG-132 and BafA. Inflammasome activation increased extracellular ATP levels, which did not appear to trigger significant potassium efflux. The activity of the lysosomal enzyme, cathepsin B, was reduced by MG-132 and BafA, suggesting that cathepsin B was not playing any role in this phenomenon. Instead, MG-132 triggered ROS production already 30 min after exposure, but treatment with antioxidants blocking NADPH oxidase and mitochondria-derived ROS significantly prevented IL-1β release after this activating signal. Conclusion: Our data suggest that oxidative stress strongly contributes to the NLRP3 inflammasome activation upon dysfunctional cellular clearance. Clarification of inflammasome activation mechanisms provides novel options for alleviating pathological inflammation present in aggregation diseases, such as age-related macular disease (AMD) and Alzheimer’s disease.

© 2018 The Author(s). Published by S. Karger AG, Basel



Related Articles:

References

  1. Martinon F, Mayor A, Tschopp J: The inflammasomes: guardians of the body. Annu Rev Immunol 2009; 27: 229-265.
  2. Schroder K, Zhou R, Tschopp J: The NLRP3 inflammasome: a sensor for metabolic danger? Science 2010; 327: 5963: 296-300.
  3. Perregaux D, Gabel CA: Interleukin-1 beta maturation and release in response to ATP and nigericin: Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity. J Biol Chem 1994; 269: 15195-15203.
  4. Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA: Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 2001; 276: 125-132.
  5. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM: Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 2006; 440: 228-232.
  6. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J: Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ 2007; 14: 1583-1589.
  7. Munoz-Planillo R, Kuffa P, Martinez-Colon G, Smith BL, Rajendiran TM, Nunez G: K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 2013; 38: 1142-1153.
  8. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J: Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006; 440: 237-241.
  9. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J: Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 2008; 320: 674-677.
  10. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, Fitzgerald KA, Latz E: Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008; 9: 847-856.
  11. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT: The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol 2008; 9: 857-865.
  12. Cruz CM, Rinna A, Forman HJ, Ventura AL, Persechini PM, Ojcius DM: ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages. J Biol Chem 2007; 282: 2871-2879.
  13. Lopez-Castejon G, Theaker J, Pelegrin P, Clifton AD, Braddock M, Surprenant A: P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases. J Immunol 2010; 185: 2611-2619.
  14. Zhou R, Yazdi AS, Menu P, Tschopp J: A role for mitochondria in NLRP3 inflammasome activation. Nature 2011; 469: 221-225.
  15. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA, Ryter SW, Choi AM: Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 2011; 12: 222-230.
  16. Piippo N, Korkmaz A, Hytti M, Kinnunen K, Salminen A, Atalay M, Kaarniranta K, Kauppinen A: Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells. Biochim Biophys Acta 2014; 1843: 3038-3046.
  17. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.
  18. Lamkanfi M, Mueller JL, Vitari AC, Misaghi S, Fedorova A, Deshayes K, Lee WP, Hoffman HM, Dixit VM: Glyburide inhibits the Cryopyrin/Nalp3 inflammasome. J Cell Biol 2009; 187: 61-70.
  19. Kerur N, Hirano Y, Tarallo V, Fowler BJ, Bastos-Carvalho A, Yasuma T, Yasuma R, Kim Y, Hinton DR, Kirschning CJ, Gelfand BD, Ambati J: TLR-independent and P2X7-dependent signaling mediate Alu RNA-induced NLRP3 inflammasome activation in geographic atrophy. Invest Ophthalmol Vis Sci 2013; 54: 7395-7401.
  20. Sun SY: N-acetylcysteine, reactive oxygen species and beyond. Cancer Biol Ther 2010; 9: 109-110.
  21. Halasi M, Wang M, Chavan TS, Gaponenko V, Hay N, Gartel AL: ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors. Biochem J 2013; 454: 201-208.
  22. Hancock JT, Jones OT: The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages. Biochem J 1987; 242: 103-107.
  23. Bauernfeind F, Bartok E, Rieger A, Franchi L, Nunez G, Hornung V: Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J Immunol 2011; 187: 613-617.
  24. Guha S, Baltazar GC, Tu L, Lim JC, Eysteinsson T, Laties AM, Mitchell CH: P2X7 receptor stimulation raises lysosomal pH and triggers cytokine release from RPE cells. FASEB J 2011; 25: 1 Supplement: 906.2-906.2.
  25. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J: Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol 2010; 11: 136-140.
  26. Martinon F: Signaling by ROS drives inflammasome activation. Eur J Immunol 2010; 40: 616-619.
  27. Kauppinen A, Niskanen H, Suuronen T, Kinnunen K, Salminen A, Kaarniranta K: Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells-Implications for age-related macular degeneration (AMD). Immunol Lett 2012; 147: 29-33.
  28. Boya P, Kroemer G: Lysosomal membrane permeabilization in cell death. Oncogene 2008; 27: 6434-6451.
  29. Tseng WA, Thein T, Kinnunen K, Lashkari K, Gregory MS, D’Amore PA, Ksander BR: NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 2013; 54: 110-120.
  30. Brandstetter C, Mohr LK, Latz E, Holz FG, Krohne TU: Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage. J Mol Med (Berl) 2015; 93: 905-916.
  31. Longva KE, Blystad FD, Stang E, Larsen AM, Johannessen LE, Madshus IH: Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies. J Cell Biol 2002; 156: 843-854.
  32. Lee S, Sato Y, Nixon RA: Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy. J Neurosci 2011; 31: 7817-7830.
  33. Brojatsch J, Lima H, Kar AK, Jacobson LS, Muehlbauer SM, Chandran K, Diaz-Griffero F: A proteolytic cascade controls lysosome rupture and necrotic cell death mediated by lysosome-destabilizing adjuvants. PLoS One 2014; 9:e95032.
  34. Kaarniranta K, Sinha D, Blasiak J, Kauppinen A, Vereb Z, Salminen A, Boulton ME, Petrovski G: Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration. Autophagy 2013; 9: 973-984.
  35. Taylor DJ, Hobby AE, Binns AM, Crabb DP: How does age-related macular degeneration affect real-world visual ability and quality of life? A systematic review. BMJ Open 2016; 6:e011504-2016-011504.
  36. Tarallo V, Hirano Y, Gelfand BD, Dridi S, Kerur N, Kim Y, Cho WG, Kaneko H, Fowler BJ, Bogdanovich S, Albuquerque RJ, Hauswirth WW, Chiodo VA, Kugel JF, Goodrich JA, Ponicsan SL, Chaudhuri G, Murphy MP, Dunaief JL, Ambati BK, Ogura Y, Yoo JW, Lee DK, Provost P, Hinton DR, Nunez G, Baffi JZ, Kleinman ME, Ambati J: DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell 2012; 149: 847-859.
  37. Doyle SL, Campbell M, Ozaki E, Salomon RG, Mori A, Kenna PF, Farrar GJ, Kiang AS, Humphries MM, Lavelle EC, O’Neill LA, Hollyfield JG, Humphries P: NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components. Nat Med 2012; 18: 791-798.
  38. Kauppinen A, Paterno JJ, Blasiak J, Salminen A, Kaarniranta K: Inflammation and its role in age-related macular degeneration. Cell Mol Life Sci 2016; 73: 1765-1786.

Article / Publication Details

First-Page Preview
Abstract of Original Paper

Received: January 02, 2018
Accepted: August 14, 2018
Published online: August 23, 2018
Issue release date: September 2018

Number of Print Pages: 9
Number of Figures: 6
Number of Tables: 0

ISSN: 1015-8987 (Print)
eISSN: 1421-9778 (Online)

For additional information: https://www.karger.com/CPB

References

  1. Martinon F, Mayor A, Tschopp J: The inflammasomes: guardians of the body. Annu Rev Immunol 2009; 27: 229-265.
  2. Schroder K, Zhou R, Tschopp J: The NLRP3 inflammasome: a sensor for metabolic danger? Science 2010; 327: 5963: 296-300.
  3. Perregaux D, Gabel CA: Interleukin-1 beta maturation and release in response to ATP and nigericin: Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity. J Biol Chem 1994; 269: 15195-15203.
  4. Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA: Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 2001; 276: 125-132.
  5. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM: Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 2006; 440: 228-232.
  6. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J: Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ 2007; 14: 1583-1589.
  7. Munoz-Planillo R, Kuffa P, Martinez-Colon G, Smith BL, Rajendiran TM, Nunez G: K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 2013; 38: 1142-1153.
  8. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J: Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006; 440: 237-241.
  9. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J: Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 2008; 320: 674-677.
  10. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, Fitzgerald KA, Latz E: Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008; 9: 847-856.
  11. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT: The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol 2008; 9: 857-865.
  12. Cruz CM, Rinna A, Forman HJ, Ventura AL, Persechini PM, Ojcius DM: ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages. J Biol Chem 2007; 282: 2871-2879.
  13. Lopez-Castejon G, Theaker J, Pelegrin P, Clifton AD, Braddock M, Surprenant A: P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases. J Immunol 2010; 185: 2611-2619.
  14. Zhou R, Yazdi AS, Menu P, Tschopp J: A role for mitochondria in NLRP3 inflammasome activation. Nature 2011; 469: 221-225.
  15. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA, Ryter SW, Choi AM: Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 2011; 12: 222-230.
  16. Piippo N, Korkmaz A, Hytti M, Kinnunen K, Salminen A, Atalay M, Kaarniranta K, Kauppinen A: Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells. Biochim Biophys Acta 2014; 1843: 3038-3046.
  17. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.
  18. Lamkanfi M, Mueller JL, Vitari AC, Misaghi S, Fedorova A, Deshayes K, Lee WP, Hoffman HM, Dixit VM: Glyburide inhibits the Cryopyrin/Nalp3 inflammasome. J Cell Biol 2009; 187: 61-70.
  19. Kerur N, Hirano Y, Tarallo V, Fowler BJ, Bastos-Carvalho A, Yasuma T, Yasuma R, Kim Y, Hinton DR, Kirschning CJ, Gelfand BD, Ambati J: TLR-independent and P2X7-dependent signaling mediate Alu RNA-induced NLRP3 inflammasome activation in geographic atrophy. Invest Ophthalmol Vis Sci 2013; 54: 7395-7401.
  20. Sun SY: N-acetylcysteine, reactive oxygen species and beyond. Cancer Biol Ther 2010; 9: 109-110.
  21. Halasi M, Wang M, Chavan TS, Gaponenko V, Hay N, Gartel AL: ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors. Biochem J 2013; 454: 201-208.
  22. Hancock JT, Jones OT: The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages. Biochem J 1987; 242: 103-107.
  23. Bauernfeind F, Bartok E, Rieger A, Franchi L, Nunez G, Hornung V: Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J Immunol 2011; 187: 613-617.
  24. Guha S, Baltazar GC, Tu L, Lim JC, Eysteinsson T, Laties AM, Mitchell CH: P2X7 receptor stimulation raises lysosomal pH and triggers cytokine release from RPE cells. FASEB J 2011; 25: 1 Supplement: 906.2-906.2.
  25. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J: Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol 2010; 11: 136-140.
  26. Martinon F: Signaling by ROS drives inflammasome activation. Eur J Immunol 2010; 40: 616-619.
  27. Kauppinen A, Niskanen H, Suuronen T, Kinnunen K, Salminen A, Kaarniranta K: Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells-Implications for age-related macular degeneration (AMD). Immunol Lett 2012; 147: 29-33.
  28. Boya P, Kroemer G: Lysosomal membrane permeabilization in cell death. Oncogene 2008; 27: 6434-6451.
  29. Tseng WA, Thein T, Kinnunen K, Lashkari K, Gregory MS, D’Amore PA, Ksander BR: NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci 2013; 54: 110-120.
  30. Brandstetter C, Mohr LK, Latz E, Holz FG, Krohne TU: Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage. J Mol Med (Berl) 2015; 93: 905-916.
  31. Longva KE, Blystad FD, Stang E, Larsen AM, Johannessen LE, Madshus IH: Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies. J Cell Biol 2002; 156: 843-854.
  32. Lee S, Sato Y, Nixon RA: Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy. J Neurosci 2011; 31: 7817-7830.
  33. Brojatsch J, Lima H, Kar AK, Jacobson LS, Muehlbauer SM, Chandran K, Diaz-Griffero F: A proteolytic cascade controls lysosome rupture and necrotic cell death mediated by lysosome-destabilizing adjuvants. PLoS One 2014; 9:e95032.
  34. Kaarniranta K, Sinha D, Blasiak J, Kauppinen A, Vereb Z, Salminen A, Boulton ME, Petrovski G: Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration. Autophagy 2013; 9: 973-984.
  35. Taylor DJ, Hobby AE, Binns AM, Crabb DP: How does age-related macular degeneration affect real-world visual ability and quality of life? A systematic review. BMJ Open 2016; 6:e011504-2016-011504.
  36. Tarallo V, Hirano Y, Gelfand BD, Dridi S, Kerur N, Kim Y, Cho WG, Kaneko H, Fowler BJ, Bogdanovich S, Albuquerque RJ, Hauswirth WW, Chiodo VA, Kugel JF, Goodrich JA, Ponicsan SL, Chaudhuri G, Murphy MP, Dunaief JL, Ambati BK, Ogura Y, Yoo JW, Lee DK, Provost P, Hinton DR, Nunez G, Baffi JZ, Kleinman ME, Ambati J: DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell 2012; 149: 847-859.
  37. Doyle SL, Campbell M, Ozaki E, Salomon RG, Mori A, Kenna PF, Farrar GJ, Kiang AS, Humphries MM, Lavelle EC, O’Neill LA, Hollyfield JG, Humphries P: NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components. Nat Med 2012; 18: 791-798.
  38. Kauppinen A, Paterno JJ, Blasiak J, Salminen A, Kaarniranta K: Inflammation and its role in age-related macular degeneration. Cell Mol Life Sci 2016; 73: 1765-1786.

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