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Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice

Nature Medicine volume 22, pages 1285–1293 (2016)Cite this article

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Abstract

Successful recovery from lung injury requires the repair and regeneration of alveolar epithelial cells to restore the integrity of gas-exchanging regions within the lung and preserve organ function. Improper regeneration of the alveolar epithelium is often associated with severe pulmonary fibrosis, the latter of which involves the recruitment and activation of fibroblasts, as well as matrix accumulation. Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to the lung repair process. The mechanisms that regulate AEC2 renewal are incompletely understood. We provide evidence that expression of the innate immune receptor Toll-like receptor 4 (TLR4) and the extracellular matrix glycosaminoglycan hyaluronan (HA) on AEC2s are important for AEC2 renewal, repair of lung injury and limiting the extent of fibrosis. Either deletion of TLR4 or HA synthase 2 in surfactant-protein-C-positive AEC2s leads to impaired renewal capacity, severe fibrosis and mortality. Furthermore, AEC2s from patients with severe pulmonary fibrosis have reduced cell surface HA and impaired renewal capacity, suggesting that HA and TLR4 are key contributors to lung stem cell renewal and that severe pulmonary fibrosis is the result of distal epithelial stem cell failure.

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Figure 1: Tlr4−/− mice demonstrate higher mortality and more severe fibrosis after bleomycin-induced lung injury.
Figure 2: Tlr4−/− deficiency leads to loss of AEC2s in mouse lungs after bleomycin-induced injury.
Figure 3: AEC2 differentiation and proliferation requires TLR4 signaling.
Figure 4: Has2-deficient AEC2s have lower colony-forming capacity.
Figure 5: IL-6 promotes AEC renewal and limits lung fibrosis.
Figure 6: Loss of cell surface HA and impaired renewal capacity of AEC2s from patients with IPF.

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References

  1. Rakoff-Nahoum, S., Hao, L. & Medzhitov, R. Role of toll-like receptors in spontaneous commensal-dependent colitis. Immunity 25, 319–329 (2006).

    CAS  PubMed  Google Scholar 

  2. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004).

    Article  CAS  Google Scholar 

  3. Fukata, M. et al. Cox-2 is regulated by Toll-like receptor 4 (TLR4) signaling: role in proliferation and apoptosis in the intestine. Gastroenterology 131, 862–877 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Hogan, B.L. et al. Repair and regeneration of the respiratory system: complexity, plasticity and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Kotton, D.N. & Morrisey, E.E. Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat. Med. 20, 822–832 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Desai, T.J., Brownfield, D.G. & Krasnow, M.A. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507, 190–194 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Barkauskas, C.E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Invest. 123, 3025–3036 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Hofer, C.C., Woods, P.S. & Davis, I.C. Infection of mice with influenza A/WSN/33 (H1N1) virus alters alveolar type II cell phenotype. Am. J. Physiol. Lung Cell. Mol. Physiol. 308, L628–L638 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, Y., Kumar, V.S., Zhang, W., Rehman, J. & Malik, A.B. Activation of type II cells into regenerative stem-cell-antigen-1+ cells during alveolar repair. Am. J. Respir. Cell Mol. Biol. 53, 113–124 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Rock, J.R. et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial-to-mesenchymal transition. Proc. Natl. Acad. Sci. USA 108, E1475–E1483 (2011).

    CAS  PubMed  Google Scholar 

  11. Jiang, D. et al. Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat. Med. 11, 1173–1179 (2005).

    CAS  PubMed  Google Scholar 

  12. Jiang, D., Liang, J., Li, Y. & Noble, P.W. The role of Toll-like receptors in non-infectious lung injury. Cell Res. 16, 693–701 (2006).

    CAS  PubMed  Google Scholar 

  13. Jiang, D., Liang, J. & Noble, P.W. Hyaluronan in tissue injury and repair. Annu. Rev. Cell Dev. Biol. 23, 435–461 (2007).

    CAS  PubMed  Google Scholar 

  14. Noble, P.W. & Jiang, D. Matrix regulation of lung injury, inflammation and repair: the role of innate immunity. Proc. Am. Thorac. Soc. 3, 401–404 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Jiang, D., Liang, J. & Noble, P.W. Hyaluronan as an immune regulator in human diseases. Physiol. Rev. 91, 221–264 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Camenisch, T.D. et al. Disruption of hyaluronan synthase 2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J. Clin. Invest. 106, 349–360 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. The joint statement of American Thoracic Society (ATS) and the European Respiratory Society (ERS). Am. J. Respir. Crit. Care Med. 161, 646–664 (2000).

  18. Selman, M. et al. Idiopathic pulmonary fibrosis: pathogenesis and therapeutic approaches. Drugs 64, 405–430 (2004).

    CAS  PubMed  Google Scholar 

  19. Noble, P.W., Barkauskas, C.E. & Jiang, D. Pulmonary fibrosis: patterns and perpetrators. J. Clin. Invest. 122, 2756–2762 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Amin, R.S. et al. Surfactant protein deficiency in familial interstitial lung disease. J. Pediatr. 139, 85–92 (2001).

    CAS  PubMed  Google Scholar 

  21. Thomas, A.Q. et al. Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Am. J. Respir. Crit. Care Med. 165, 1322–1328 (2002).

    PubMed  Google Scholar 

  22. Garcia, C.K. Idiopathic pulmonary fibrosis: update on genetic discoveries. Proc. Am. Thorac. Soc. 8, 158–162 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Alder, J.K. et al. Telomere dysfunction causes alveolar stem cell failure. Proc. Natl. Acad. Sci. USA 112, 5099–5104 (2015).

    CAS  PubMed  Google Scholar 

  24. Chen, H. et al. Airway epithelial progenitors are region specific and show differential responses to bleomycin-induced lung injury. Stem Cells 30, 1948–1960 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Mummert, M.E., Mohamadzadeh, M., Mummert, D.I., Mizumoto, N. & Takashima, A. Development of a peptide inhibitor of hyaluronan-mediated leukocyte trafficking. J. Exp. Med. 192, 769–779 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, Y. et al. Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44. J. Exp. Med. 208, 1459–1471 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Matsumoto, K. et al. Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb. Development 136, 2825–2835 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Eblaghie, M.C., Reedy, M., Oliver, T., Mishina, Y. & Hogan, B.L. Evidence that autocrine signaling through Bmpr1a regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells. Dev. Biol. 291, 67–82 (2006).

    CAS  PubMed  Google Scholar 

  29. Gonzalez, R.F., Allen, L., Gonzales, L., Ballard, P.L. & Dobbs, L.G. HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells. J. Histochem. Cytochem. 58, 891–901 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Armstrong, L. et al. Expression of functional toll-like receptor–2 and -4 on alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 31, 241–245 (2004).

    CAS  PubMed  Google Scholar 

  31. Abreu, M.T. et al. Decreased expression of Toll-like receptor 4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J. Immunol. 167, 1609–1616 (2001).

    CAS  PubMed  Google Scholar 

  32. Neal, M.D. et al. Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53-upregulated modulator of apoptosis. J. Biol. Chem. 287, 37296–37308 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Pivarcsi, A. et al. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int. Immunol. 15, 721–730 (2003).

    CAS  PubMed  Google Scholar 

  34. Oh, K. et al. Epithelial transglutaminase 2 is needed for T cell interleukin-17 production and subsequent pulmonary inflammation and fibrosis in bleomycin-treated mice. J. Exp. Med. 208, 1707–1719 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Tadokoro, T. et al. IL-6–STAT3 promotes regeneration of airway ciliated cells from basal stem cells. Proc. Natl. Acad. Sci. USA 111, E3641–E3649 (2014).

    CAS  PubMed  Google Scholar 

  36. Tebbutt, N.C. et al. Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130-mutant mice. Nat. Med. 8, 1089–1097 (2002).

    CAS  PubMed  Google Scholar 

  37. Zhang, S. et al. Interleukin 6 mediates the therapeutic effects of adipose-derived stromal–stem cells in lipopolysaccharide-induced acute lung injury. Stem Cells 32, 1616–1628 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Le, T.T. et al. Blockade of IL-6 trans-signaling attenuates pulmonary fibrosis. J. Immunol. 193, 3755–3768 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. O'Donoghue, R.J. et al. Genetic partitioning of interleukin-6 signaling in mice dissociates Stat3 from Smad3-mediated lung fibrosis. EMBO Mol. Med. 4, 939–951 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Rafii, S. et al. Platelet-derived SDF-1 primes the pulmonary capillary vascular niche to drive lung alveolar regeneration. Nat. Cell Biol. 17, 123–136 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Cao, Z. et al. Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis. Nat. Med. 22, 154–162 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Guzy, R.D., Stoilov, I., Elton, T.J., Mecham, R.P. & Ornitz, D.M. Fibroblast growth factor 2 is required for epithelial recovery, but not for pulmonary fibrosis, in response to bleomycin. Am. J. Respir. Cell Mol. Biol. 52, 116–128 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. Chapman, H.A. et al. Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice. J. Clin. Invest. 121, 2855–2862 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Lee, J.H. et al. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4–NFATc1–thrombospondin-1 axis. Cell 156, 440–455 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Treutlein, B. et al. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature 509, 371–375 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Vaughan, A.E. et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 517, 621–625 (2015).

    CAS  PubMed  Google Scholar 

  47. Takeuchi, O. et al. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11, 443–451 (1999).

    CAS  PubMed  Google Scholar 

  48. Kawai, T., Adachi, O., Ogawa, T., Takeda, K. & Akira, S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11, 115–122 (1999).

    CAS  PubMed  Google Scholar 

  49. Jeannotte, L. et al. Unsuspected effects of a lung-specific Cre deleter mouse line. Genesis 49, 152–159 (2011).

    PubMed  Google Scholar 

  50. Morales-Nebreda, L.I. et al. Lung-specific loss of α3-laminin worsens bleomycin-induced pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 52, 503–512 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Dong, Y. et al. Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice. J. Exp. Med. 212, 235–252 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Lovgren, A.K. et al. β-arrestin deficiency protects against pulmonary fibrosis in mice and prevents fibroblast invasion of extracellular matrix. Sci. Transl. Med. 3, 74ra23 (2011).

    PubMed  PubMed Central  Google Scholar 

  53. Jiang, D. et al. Regulation of pulmonary fibrosis by chemokine receptor CXCR3. J. Clin. Invest. 114, 291–299 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Liang, J. et al. A macrophage subpopulation recruited by CC chemokine ligand 2 clears apoptotic cells in non-infectious lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 302, L933–L940 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Liang, J. et al. Role of hyaluronan and hyaluronan-binding proteins in human asthma. J. Allergy Clin. Immunol. 128, 403–411.e3 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Jiang, D. et al. Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan 4. J. Clin. Invest. 120, 2049–2057 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Leonado, Y. Jung and V. Hu for technical support. This work was supported by US National Institute of Health (NIH) grants P01 HL108793 (P.W.N.), R01 HL060539 (P.W.N.), AI052201 (P.W.N.) and R01 HL122068 (D.J.), and California Institute for Regenerative Medicine grant RB5-07302 (P.W.N.). We thank B.L.M. Hogan for critical reading of the manuscript and for providing invaluable comments. We are grateful to S. Akira (University of Osaka, Japan), Y. Yamaguchi (Sanford–Burnham Medical Research Institute) and L. Dobbs (University of California, San Francisco) for providing critical reagents for the study.

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  1. Yanli Zhang & Huaiyong Chen

    Present address: Present addresses: Department of Biochemistry and Molecular Biology, Peking Union Medical College, Tsinghua University, Beijing, China (Y.Z.); Tianjin Haihe Hospital, Tianjin, China and Tianjin Institute of Respiratory Diseases, Tianjin, China (H.C.).,

Authors and Affiliations

  1. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA

    Jiurong Liang, Yanli Zhang, Ting Xie, Ningshan Liu, Yan Geng, Adrianne Kurkciyan, Jessica Monterrosa Mena, Barry R Stripp, Dianhua Jiang & Paul W Noble

  2. Women's Guild Lung Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA

    Jiurong Liang, Yanli Zhang, Ting Xie, Ningshan Liu, Yan Geng, Adrianne Kurkciyan, Jessica Monterrosa Mena, Barry R Stripp, Dianhua Jiang & Paul W Noble

  3. Department of Medicine, Duke University School of Medicine, Durham, North Carolina, USA

    Huaiyong Chen

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Contributions

All authors participated in the design, execution and interpretation of the study. P.W.N., D.J. and J.L. conceived and designed the study; J.L. performed the majority of experiments and analyzed data; Y.Z., T.X., N.L., H.C., Y.G., A.K., J.M.M. and D.J. performed experiments and analyzed data; B.R.S. contributed to the design and interpretation of the study, and provided critical reagents; J.L., D.J. and P.W.N. wrote the manuscript; and P.W.N. and D.J. approved the submission.

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Correspondence to Dianhua Jiang or Paul W Noble.

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Liang, J., Zhang, Y., Xie, T. et al. Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice. Nat Med 22, 1285–1293 (2016). https://doi.org/10.1038/nm.4192

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