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CFTR regulates phagosome acidification in macrophages and alters bactericidal activity

Abstract

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr−/− mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

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Figure 1: Whole-cell and single channel recordings of cAMP-evoked Cl current in murine and human alveolar macrophages.
Figure 2: CFTR expression in alveolar macrophages and related cells.
Figure 3: Capacity of macrophages to eliminate internalized bacteria is decreased in murine Cftr−/− alveolar macrophage cells.
Figure 4: Phagocytosis is independent of Cftr expression in macrophages.
Figure 5: Cftr expression regulates acidification of intracellular lysosome-like compartments and phagosomes.
Figure 6: Inhibition of cAMP-dependent kinase prevents lysosomal acidification in Cftr+/+ alveolar macrophages.
Figure 7: Contribution of lysosomal fusion to phagosomal acidification.
Figure 8: Schematic representation of a model summarizing the results of this study.

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References

  1. Vieira, O. V., Botelho, R. J. & Grinstein, S. Phagosome maturation: aging gracefully. Biochem. J. 366, 689–704 (2002).

    Article  CAS  Google Scholar 

  2. Lee, W. L., Harrison, R. E. & Grinstein, S. Phagocytosis by neutrophils. Microbes Infect. 5, 1299–1306 (2003).

    Article  CAS  Google Scholar 

  3. Aderem, A. & Underhill, D. M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17, 593–623 (1999).

    Article  CAS  Google Scholar 

  4. Scott, C. C., Botelho, R. J. & Grinstein, S. Phagosome maturation: a few bugs in the system. J. Membr. Biol. 193, 137–152 (2003).

    Article  CAS  Google Scholar 

  5. Russell, D. G. Mycobacterium tuberculosis: here today, and here tomorrow. Nature Rev. Mol. Cell Biol. 2, 569–577 (2001).

    Article  CAS  Google Scholar 

  6. Babior, B. M. NADPH oxidase. Curr. Opin. Immunol. 16, 42–47 (2004).

    Article  CAS  Google Scholar 

  7. Hara-Chikuma, M. et al. ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J. Biol. Chem. 280, 1241–1247 (2005).

    Article  CAS  Google Scholar 

  8. Gadsby, D. C., Nagel, G. & Hwang, T. C. The CFTR chloride channel of mammalian heart. Annu. Rev. Physiol. 57, 387–416 (1995).

    Article  CAS  Google Scholar 

  9. Bradbury, N. A. Intracellular CFTR: localization and function. Physiol. Rev. 79, S175–S191 (1999).

    Article  CAS  Google Scholar 

  10. Schwiebert, E. M., Benos, D. J., Egan, M. E., Stutts, M. J. & Guggino, W. B. CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79, S145–S166 (1999).

    Article  CAS  Google Scholar 

  11. McCarty, N. A. Permeation through the CFTR chloride channel. J. Exp. Biol. 203, 1947–1962 (2000).

    CAS  PubMed  Google Scholar 

  12. Ma, T. et al. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J. Clin. Invest. 110, 1651–1658 (2002).

    Article  CAS  Google Scholar 

  13. Jovov, B. et al. Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels. J. Biol. Chem. 270, 29194–29200 (1995).

    Article  CAS  Google Scholar 

  14. Roos, D. & Winterbourn, C. C. Immunology. Lethal weapons. Science 296, 669–671 (2002).

    Article  CAS  Google Scholar 

  15. Kobayashi, T. et al. A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils. Histochem. Cell Biol. 113, 251–257 (2000).

    Article  CAS  Google Scholar 

  16. Belhoussine, R., Morjani, H., Sharonov, S., Ploton, D. & Manfait, M. Characterization of intracellular pH gradients in human multidrug-resistant tumour cells by means of scanning microspectrofluorometry and dual-emission-ratio probes. Int. J. Cancer 81, 81–89 (1999).

    Article  CAS  Google Scholar 

  17. Bowman, E. J., Siebers, A. & Altendorf, K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl Acad. Sci. USA 85, 7972–7976 (1988).

    Article  CAS  Google Scholar 

  18. Lukacs, G. L., Rotstein, O. D. & Grinstein, S. Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)–ATPase activity, counterion conductance, and H+ “leak”. J. Biol. Chem. 266, 24540–24548 (1991).

    CAS  PubMed  Google Scholar 

  19. Makranz, C., Cohen, G., Reichert, F., Kodama, T. & Rotshenker, S. cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages. Glia 53, 441–448 (2006).

    Article  Google Scholar 

  20. Ydrenius, L., Majeed, M., Rasmusson, B. J., Stendahl, O. & Sarndahl, E. Activation of cAMP-dependent protein kinase is necessary for actin rearrangements in human neutrophils during phagocytosis. J. Leukoc. Biol. 67, 520–528 (2000).

    Article  CAS  Google Scholar 

  21. Oh, Y. K. & Straubinger, R. M. Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction. Infect. Immun. 64, 319–325 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Jensen, M. S. & Bainton, D. F. Temporal changes in pH within the phagocytic vacuole of the polymorphonuclear neutrophilic leukocyte. J. Cell. Biol. 56, 379–388 (1973).

    Article  CAS  Google Scholar 

  23. Styrt, B. & Klempner, M. S. Internal pH of human neutrophil lysosomes. FEBS Lett. 149, 113–116 (1982).

    Article  CAS  Google Scholar 

  24. Yates, R. M. & Russell, D. G. Phagosome maturation proceeds independently of stimulation of Toll-like receptors 2 and 4. Immunity 23, 409–417 (2005).

    Article  CAS  Google Scholar 

  25. Kasper, D. et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J. 24, 1079–1091 (2005).

    Article  CAS  Google Scholar 

  26. Barasch, J. et al. Defective acidification of intracellular organelles in cystic fibrosis. Nature 352, 70–73 (1991).

    Article  CAS  Google Scholar 

  27. Sonawane, N. D., Thiagarajah, J. R. & Verkman, A. S. Chloride concentration in endosomes measured using a ratioable fluorescent Cl- indicator: evidence for chloride accumulation during acidification. J. Biol. Chem. 277, 5506–5513 (2002).

    Article  CAS  Google Scholar 

  28. Sonawane, N. D. & Verkman, A. S. Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands. J. Cell Biol. 160, 1129–1138 (2003).

    Article  CAS  Google Scholar 

  29. Barg, S. et al. Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. J. Cell Sci. 114, 2145–2154 (2001).

    CAS  PubMed  Google Scholar 

  30. DeCoursey, T. E. During the respiratory burst, do phagocytes need proton channels or potassium channels, or both? Sci STKE pe21 (2004).

  31. Harrison, R. E., Touret, N. & Grinstein, S. Microbial killing: oxidants, proteases and ions. Curr. Biol. 12, R357–R359 (2002).

    Article  CAS  Google Scholar 

  32. Reeves, E. P. et al. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416, 291–297 (2002).

    Article  CAS  Google Scholar 

  33. Chandy, G., Grabe, M., Moore, H. P. & Machen, T. E. Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells. Am. J. Physiol. Cell Physiol. 281, C908–C921 (2001).

    Article  CAS  Google Scholar 

  34. al-Awqati, Q., Barasch, J. & Landry, D. Chloride channels of intracellular organelles and their potential role in cystic fibrosis. J. Exp. Biol. 172, 245–266 (1992).

    CAS  PubMed  Google Scholar 

  35. Barasch, J. & al-Awqati, Q. Defective acidification of the biosynthetic pathway in cystic fibrosis. J. Cell Sci. 17, 229–233 (1993).

    Article  CAS  Google Scholar 

  36. Poschet, J., Perkett, E. & Deretic, V. Hyperacidification in cystic fibrosis: links with lung disease and new prospects for treatment. Trends Mol. Med. 8, 512–519 (2002).

    Article  CAS  Google Scholar 

  37. Gibson, G. A., Hill, W. G. & Weisz, O. A. Evidence against the acidification hypothesis in cystic fibrosis. Am. J. Physiol. Cell Physiol. 279, C1088–C1099 (2000).

    Article  CAS  Google Scholar 

  38. Luckie, D. B., Singh, C. N., Wine, J. J. & Wilterding, J. H. CFTR activation raises extracellular pH of NIH3T3 mouse fibroblasts and C127 epithelial cells. J. Membr. Biol. 179, 275–284 (2001).

    Article  CAS  Google Scholar 

  39. Morris, M. R., Doull, I. J., Dewitt, S. & Hallett, M. B. Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis. Clin. Exp. Immunol. 142, 68–75 (2005).

    Article  CAS  Google Scholar 

  40. Moraes, T. J. et al. Abnormalities in the pulmonary innate immune system in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 34, 364–374 (2006).

    Article  CAS  Google Scholar 

  41. Di, A., Krupa, B. & Nelson, D. J. Calcium-G protein interactions in the regulation of macrophage secretion. J. Biol. Chem. 276, 37124–37132 (2001).

    Article  CAS  Google Scholar 

  42. Newman, J. Novel buffer systems for macromolecular crystallization. Acta. Crystallogr. D Biol. Crystallogr. 60, 610–612 (2004).

    Article  Google Scholar 

  43. Hamill, O. P., Marty, A., Neher, E. & Sakmann, B. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391, 85–100 (1981).

    Article  CAS  Google Scholar 

  44. Naren, A. P. et al. Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms. Nature 390, 302–305 (1997).

    Article  CAS  Google Scholar 

  45. Hamrick, T. S., Havell, E. A., Horton, J. R. & Orndorff, P. E. Host and bacterial factors involved in the innate ability of mouse macrophages to eliminate internalized unopsonized Escherichia coli. Infect. Immun. 68, 125–132 (2000).

    Article  CAS  Google Scholar 

  46. Tesciuba, A. G. et al. Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease. J. Immunol. 167, 1996–2003 (2001).

    Article  CAS  Google Scholar 

  47. Grisham, M. B., Engerson, T. D., McCord, J. M. & Jones, H. P. A comparative study of neutrophil purification and function. J. Immunol. Methods 82, 315–320 (1985).

    Article  CAS  Google Scholar 

  48. Fu, J., Ji, H. L., Naren, A. P. & Kirk, K. L. A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating. J. Physiol. 536, 459–470 (2001).

    Article  CAS  Google Scholar 

  49. Li, C., Roy, K., Dandridge, K. & Naren, A. P. Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane. J. Biol. Chem. 279, 24673–24684 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by National Institutes of Health (NIH) and the National Institute of General Medical Sciences (NIGMS) (R01 GM36823), the Cystic Fibrosis Foundation (Nelson03G0) and the University of Chicago DDRCC (DK42086). The authors wish to thank L. Lester, Director of the Cystic Fibrosis Center at the University of Chicago for many helpful discussions.

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A.D., M.E.B., L.V.D., C.L., F.L.S., Y.C., P.H. and J.K. performed experiments, A.P.N. and V.B. designed and performed immunochemical and fluorescence labelling experiments, H.C.P. and D.J. designed the study and wrote the paper with input from the other authors.

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Correspondence to Deborah J. Nelson.

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Di, A., Brown, M., Deriy, L. et al. CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8, 933–944 (2006). https://doi.org/10.1038/ncb1456

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