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Nebulization of RNS60, a Physically-Modified Saline, Attenuates the Adoptive Transfer of Experimental Allergic Encephalomyelitis in Mice: Implications for Multiple Sclerosis Therapy

An Author Correction to this article was published on 02 April 2018

This article has been updated

Abstract

Developing a new and effective therapeutic approach against multiple sclerosis (MS) is always an important area of research. RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor-Couette-Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60, administered through intraperitoneal injection, ameliorated clinical symptoms and disease progression of experimental allergic encephalomyelitis (EAE), an animal model of MS. Since the intravenous route is not preferred for treating a chronic condition, we tested if nebulization of RNS60 could attenuate the disease process of adoptively-transferred EAE in mice. Although we could not directly image RNS60 after nebulization, nebulized Alexa680 reached spleen, spinal cord and different parts of the brain. Nebulization of RNS60 starting from the acute phase attenuated clinical symptoms of relapsing-remitting EAE in female SJL/J mice. RNS60 nebulization also inhibited perivascular cuffing, maintained the integrity of blood–brain and blood–spinal cord barriers, suppressed inflammation, normalized the expression of myelin genes, and blocked demyelination in the CNS of EAE mice. On the immunomodulatory front, nebulization of RNS60 to EAE mice led to the enrichment of anti-autoimmune regulatory T cells (Tregs) and suppression of autoimmune Th17 cells. Together, these results suggest that nebulization of RNS60 may be used to control aberrant immune responses in MS and other autoimmune disorders.

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Change history

  • 02 April 2018

    The original version of this article unfortunately contained a mistake. The Figure 3, 4, 5 legends have been misplaced. The updated legends along with the figures are corrected with this erratum.

References

  1. 1.

    Absinta M, Sati P, Reich DS (2016) Advanced MRI and staging of multiple sclerosis lesions. Nat Rev Neurol 12:358–368

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Baxter AG (2007) The origin and application of experimental autoimmune encephalomyelitis. Nat Rev Immunol 7:904–912

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Mandolesi G, Gentile A, Musella A, Fresegna D, De Vito F, Bullitta S, Sepman H, Marfia GA, Centonze D (2015) Synaptopathy connects inflammation and neurodegeneration in multiple sclerosis. Nat Rev Neurol 11:711–724

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Paterson PY (1966) Experimental allergic encephalomyelitis and autoimmune disease. Adv Immunol 5:131–208

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Pahan K (2010) Neuroimmune pharmacological control of EAE. J Neuroimmune Pharmacol 5:165–167

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Mondal S, Martinson JA, Ghosh S, Watson R, Pahan K (2012) Protection of Tregs, suppression of Th1 and Th17 cells, and amelioration of experimental allergic encephalomyelitis by a physically-modified saline. PLoS ONE 7:e51869

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Khasnavis S, Jana A, Roy A, Mazumder M, Bhushan B, Wood T, Ghosh S, Watson R, Pahan K (2012) Suppression of nuclear factor-kappaB activation and inflammation in microglia by physically modified saline. J Biol Chem 287:29529–29542

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Choi S, Yu E, Kim DS, Sugimori M, Llinas RR (2015) RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production. Physiol Rep 3:e12261

    Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Mondal S, Pahan K (2015) Cinnamon ameliorates experimental allergic encephalomyelitis in mice via regulatory T cells: implications for multiple sclerosis therapy. PLoS ONE 10:e0116566

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Dasgupta S, Jana M, Zhou Y, Fung YK, Ghosh S, Pahan K (2004) Antineuroinflammatory effect of NF-kappaB essential modifier-binding domain peptides in the adoptive transfer model of experimental allergic encephalomyelitis. J Immunol 173:1344–1354

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Dasgupta S, Zhou Y, Jana M, Banik NL, Pahan K (2003) Sodium phenylacetate inhibits adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice at multiple steps. J Immunol 170:3874–3882

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Brahmachari S, Pahan K (2007) Sodium benzoate, a food additive and a metabolite of cinnamon, modifies T cells at multiple steps and inhibits adoptive transfer of experimental allergic encephalomyelitis. J Immunol 179:275–283

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Mondal S, Roy A, Pahan K (2009) Functional blocking monoclonal antibodies against IL-12p40 homodimer inhibit adoptive transfer of experimental allergic encephalomyelitis. J Immunol 182:5013–5023

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Jana A, Modi KK, Roy A, Anderson JA, van Breemen RB, Pahan K (2013) Up-regulation of neurotrophic factors by cinnamon and its metabolite sodium benzoate: therapeutic implications for neurodegenerative disorders. J Neuroimmune Pharmacol 8:739–755

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Kundu M, Mondal S, Roy A, Martinson JL, Pahan K (2016) Sodium benzoate, a food additive and a metabolite of cinnamon, enriches regulatory t cells via stat6-mediated upregulation of TGF-beta. J Immunol 197:3099–3110

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Brahmachari S, Pahan K (2009) Suppression of regulatory T cells by IL-12p40 homodimer via nitric oxide. J Immunol 183:2045–2058

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Brahmachari S, Pahan K (2010) Myelin basic protein priming reduces the expression of Foxp3 in T cells via nitric oxide. J Immunol 184:1799–1809

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Hafler DA, Weiner HL (1989) MS: a CNS and systemic autoimmune disease. Immunol Today 10:104–107

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Steinman L (1996) Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell 85:299–302

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Traugott U, Reinherz EL, Raine CS (1983) Multiple sclerosis: distribution of T cell subsets within active chronic lesions. Science 219:308–310

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Sakaguchi S (2005) Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6:345–352

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Sakaguchi S, Powrie F (2007) Emerging challenges in regulatory T cell function and biology. Science 317:627–629

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    El-behi M, Rostami A, Ciric B (2010) Current views on the roles of Th1 and Th17 cells in experimental autoimmune encephalomyelitis. J Neuroimmune Pharmacol 5:189–197

    Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, Rudensky AY (2009) CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 326:986–991

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Shi G, Cox CA, Vistica BP, Tan C, Wawrousek EF, Gery I (2008) Phenotype switching by inflammation-inducing polarized Th17 cells, but not by Th1 cells. J Immunol 181:7205–7213

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Cohen JA, Reingold SC, Polman CH, Wolinsky JS (2012) Disability outcome measures in multiple sclerosis clinical trials: current status and future prospects. Lancet Neurol 11:467–476

    Article  PubMed  Google Scholar 

  27. 27.

    Miller A (1997) Current and investigational therapies used to alter the course of disease in multiple sclerosis. South Med J 90:367–375

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Werner MH, Huang D (2016) Natalizumab-treated patients at high risk for PML persistently excrete JC polyomavirus. J Neurovirol 22:871–875

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Gyang TV, Hamel J, Goodman AD, Gross RA, Samkoff L (2016) Fingolimod-associated PML in a patient with prior immunosuppression. Neurology 86:1843–1845

    Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Venken K, Hellings N, Thewissen M, Somers V, Hensen K, Rummens JL, Medaer R, Hupperts R, Stinissen P (2008) Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level. Immunology 123:79–89

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Paust S, Cantor H (2005) Regulatory T cells and autoimmune disease. Immunol Rev 204:195–207

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Ziegler SF (2006) FOXP3: of mice and men. Annu Rev Immunol 24:209–226

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Wang D, Ghosh D, Islam SM, Moorman CD, Thomason AE, Wilkinson DS, Mannie MD (2015) IFN-beta facilitates neuroantigen-dependent induction of CD25+ FOXP3+ regulatory T cells that suppress experimental autoimmune encephalomyelitis. J Immunol 197(8):2992–3007

    Google Scholar 

  34. 34.

    Niedbala W, Wei XQ, Cai B, Hueber AJ, Leung BP, McInnes IB, Liew FY (2007) IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur J Immunol 37:3021–3029

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Coffer PJ, Burgering BM (2004) Forkhead-box transcription factors and their role in the immune system. Nat Rev Immunol 4:889–899

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Xu D, Liu H, Komai-Koma M, Campbell C, McSharry C, Alexander J, Liew FY (2003) CD4+ CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection, and colitis in mice. J Immunol 170:394–399

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

This study was supported by Revalesio Corporation (Tacoma, WA).

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Correspondence to Kalipada Pahan.

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Mondal, S., Rangasamy, S.B., Ghosh, S. et al. Nebulization of RNS60, a Physically-Modified Saline, Attenuates the Adoptive Transfer of Experimental Allergic Encephalomyelitis in Mice: Implications for Multiple Sclerosis Therapy. Neurochem Res 42, 1555–1570 (2017). https://doi.org/10.1007/s11064-017-2214-z

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Keywords

  • Physically-modified saline
  • Nebulization
  • EAE
  • Immunomodulation
  • Regulatory T cells