Skip to main content

Advertisement

SpringerLink
Log in

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis.

Methods

We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs).

Results

Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1 was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2.

Conclusions

Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein–protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

The data generated and included in this study can be made available from the corresponding author on reasonable request.

References

  1. Ahamad N, Rath PC (2019) Bone marrow stem cells, aging, and age-related diseases. In: Models, molecules and mechanisms in biogerontology: physiological abnormalities, diseases and interventions. Springer, Singapore, pp 321–352

    Google Scholar 

  2. Goudarzi N, Shabani R, Ebrahimi M et al (2020) Comparative phenotypic characterization of human colostrum and breast milk-derived stem cells. Hum Cell 33:308–317

    Article  CAS  PubMed  Google Scholar 

  3. Bacakova L, Zarubova J, Travnickova M et al (2018) Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells: a review. Biotechnol Adv 36:1111–1126

    Article  PubMed  Google Scholar 

  4. Clevers H (2015) Stem cells: what is an adult stem cell? Science 350:1319–1320

    Article  CAS  PubMed  Google Scholar 

  5. Crisan M, Yap S, Casteilla L et al (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3:301–313

    Article  CAS  PubMed  Google Scholar 

  6. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74

    Article  CAS  PubMed  Google Scholar 

  7. Conget PA, Minguell JJ (1999) Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181:67–73

    Article  CAS  PubMed  Google Scholar 

  8. Trivanović D, Jauković A, Popović B et al (2015) Mesenchymal stem cells of different origin: comparative evaluation of proliferative capacity, telomere length and pluripotency marker expression. Life Sci 141:61–73

    Article  PubMed  Google Scholar 

  9. Sacchetti B, Funari A, Remoli C et al (2016) No identical “mesenchymal stem cells” at different times and sites: human committed progenitors of distinct origin and differentiation potential are incorporated as adventitial cells in microvessels. Stem Cell Rep 6:897–913

    Article  CAS  Google Scholar 

  10. Wang Z, Chai C, Wang R et al (2021) Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity. Clin Transl Med 11:e650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147

    Article  CAS  PubMed  Google Scholar 

  12. Friedenstein A, Chailakhjan R, Lalykina K (1970) The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Prolif 3:393–403

    Article  CAS  Google Scholar 

  13. Colter DC, Class R, DiGirolamo CM, Prockop DJ (2000) Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci 97:3213–3218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Colter DC, Sekiya I, Prockop DJ (2001) Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci 98:7841–7845

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cai H, Kondo M, Sandhow L et al (2022) Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression. Blood 139:3040–3057

    Article  CAS  PubMed  Google Scholar 

  16. Xin T, Greco V, Myung P (2016) Hardwiring stem cell communication through tissue structure. Cell 164:1212–1225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Comazzetto S, Shen B, Morrison SJ (2021) Niches that regulate stem cells and hematopoiesis in adult bone marrow. Dev Cell 56:1848–1860

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Anjos-Afonso F, Bonnet D (2008) Isolation, culture, and differentiation potential of mouse marrow stromal cells. Curr Protoc Stem Cell Biol 7:31–311

    Article  Google Scholar 

  19. Zhu H, Guo Z-K, Jiang X-X et al (2010) A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc 5:550–560

    Article  CAS  PubMed  Google Scholar 

  20. Russell KC, Phinney DG, Lacey MR, Barrilleaux BL, Meyertholen KE, O’Connor KC (2010) In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem cells 28:788–798

    Article  CAS  PubMed  Google Scholar 

  21. Park JW, Fu S, Huang B, Xu RH (2020) Alternative splicing in mesenchymal stem cell differentiation. Stem Cells 38:1229–1240

    Article  PubMed  Google Scholar 

  22. Chaudhary JK, Rath PC (2017) A simple method for isolation, propagation, characterization, and differentiation of adult mouse bone marrow-derived multipotent mesenchymal stem cells. J Cell Sci Therapy 8:261. https://doi.org/10.4172/2157-7013.1000261

    Article  CAS  Google Scholar 

  23. Chaudhary JK, Rath PC (2017) Microgrooved-surface topography enhances cellular division and proliferation of mouse bone marrow-derived mesenchymal stem cells. PLoS ONE 12:e0182128

    Article  PubMed  PubMed Central  Google Scholar 

  24. Dominici M, Le Blanc K, Mueller I et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement. Cytotherapy 8:315–317

    Article  CAS  PubMed  Google Scholar 

  25. Chaudhary JK, Saini D, Chaudhary PK et al (2022) Exploring the immunomodulatory aspect of mesenchymal stem cells for treatment of severe coronavirus disease. Cells 19:11

    Google Scholar 

  26. Song N, Scholtemeijer M, Shah K (2020) Mesenchymal stem cell immunomodulation: mechanisms and therapeutic potential. Trends Pharmacol Sci 41:653–664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Soliman H, Theret M, Scott W et al (2021) Multipotent stromal cells: one name, multiple identities. Cell Stem Cell 28:1690–1707

    Article  CAS  PubMed  Google Scholar 

  28. Mullen AC, Wrana JL (2017) TGF-beta Family Signaling in Embryonic and Somatic Stem-Cell Renewal and Differentiation. Cold Spring Harb Perspect Biol 9:89

    Article  Google Scholar 

  29. Chen Q, Shou P, Zheng C, Jiang M, Cao G, Yang Q, Cao J, Xie N, Velletri T, Zhang X, Xu C, Zhang L, Yang H, Hou J, Wang Y, Shi Y (2016) Fate decision of mesenchymal stem cells: adipocytes or osteoblasts? Cell Death Differ 7:1128–1139

    Article  Google Scholar 

  30. Jang JH, Jung JS, Im YB, Kang KS, Choi JI, Kang SK (2012) Crucial role of nuclear Ago2 for hUCB-MSCs differentiation and self-renewal via stemness control. Antioxid Redox Signal 16:95–111

    Article  CAS  PubMed  Google Scholar 

  31. Ong S-G, Lee WH, Kodo K, Wu JC (2015) MicroRNA-mediated regulation of differentiation and trans-differentiation in stem cells. Adv Drug Deliv Rev 88:3–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Orkin SH, Hochedlinger K (2011) Chromatin connections to pluripotency and cellular reprogramming. Cell 145:835–850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Blank U, Karlsson G, Karlsson S (2008) Signaling pathways governing stem-cell fate. Blood J Am Soc Hematol 111:492–503

    CAS  Google Scholar 

  34. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676

    Article  CAS  PubMed  Google Scholar 

  35. Dalskov L, Gad HH, Hartmann R (2023) Viral recognition and the antiviral interferon response. EMBO J 42:e112907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Negishi H, Taniguchi T, Yanai H (2018) The interferon (IFN) class of cytokines and the IFN regulatory factor (IRF) transcription factor family. Cold Spring Harb Perspect Biol 10:8

    Article  Google Scholar 

  37. Ekmekcioglu S, Mumm JB, Udtha M, Chada S, Grimm EA (2008) Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24. Cytokine 43:34–44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ahamad N, Rath PC (2019) Expression of interferon regulatory factors (IRF-1 and IRF-2) during radiation-induced damage and regeneration of bone marrow by transplantation in mouse. Mol Biol Rep 46:551–567

    Article  CAS  PubMed  Google Scholar 

  39. Battistini A (2009) Interferon regulatory factors in hematopoietic cell differentiation and immune regulation. J Interferon Cytokine Res 29:765–780

    Article  CAS  PubMed  Google Scholar 

  40. Zhu K-C, Guo H-Y, Zhang N et al (2019) Functional characterization of interferon regulatory factor 2 and its role in the transcription of interferon a3 in golden pompano Trachinotus ovatus (Linnaeus 1758). Fish Shellfish Immunol 93:90–98

    Article  CAS  PubMed  Google Scholar 

  41. Kraus TA, Lau JF, Parisien J-P, Horvath CM (2003) A hybrid IRF9-STAT2 protein recapitulates interferon-stimulated gene expression and antiviral response. J Biol Chem 278:13033–13038

    Article  CAS  PubMed  Google Scholar 

  42. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N (2001) IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 19:623–655

    Article  CAS  PubMed  Google Scholar 

  43. Yanai H, Negishi H, Taniguchi T (2012) The IRF family of transcription factors: Inception, impact and implications in oncogenesis. Oncoimmunology 1:1376–1386

    Article  PubMed  PubMed Central  Google Scholar 

  44. Lohoff M, Duncan GS, Ferrick D et al (2000) Deficiency in the transcription factor interferon regulatory factor (IRF)-2 leads to severely compromised development of natural killer and T helper type 1 cells. J Exp Med 192:325–336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sato T, Onai N, Yoshihara H, Arai F, Suda T, Ohteki T (2009) Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon–dependent exhaustion. Nat Med 15:696–700

    Article  CAS  PubMed  Google Scholar 

  46. Masumi A, Hamaguchi I, Kuramitsu M et al (2009) Interferon regulatory factor-2 induces megakaryopoiesis in mouse bone marrow hematopoietic cells. FEBS Lett 583:3493–3500

    Article  CAS  PubMed  Google Scholar 

  47. Pleyer L, Valent P, Greil R (2016) Mesenchymal stem and progenitor cells in normal and dysplastic hematopoiesis—masters of survival and clonality? Int J Mol Sci 17:1009

    Article  PubMed  PubMed Central  Google Scholar 

  48. Chen JM, Huang QY, Zhao YX, Chen WH, Lin S, Shi QY (2021) The latest developments in immunomodulation of mesenchymal stem cells in the treatment of intrauterine adhesions. Both Allogeneic Autologous Front Immunol 12:785717

    Article  CAS  PubMed  Google Scholar 

  49. Zappia E, Casazza S, Pedemonte E et al (2005) Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 106:1755–1761

    Article  CAS  PubMed  Google Scholar 

  50. Waterman RS, Tomchuck SL, Henkle SL, Betancourt AM (2010) A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype. PLoS ONE 5:e10088

    Article  PubMed  PubMed Central  Google Scholar 

  51. Shirjang S, Mansoori B, Solali S, Hagh MF, Shamsasenjan K (2017) Toll-like receptors as a key regulator of mesenchymal stem cell function: an up-to-date review. Cell Immunol 315:1–10

    Article  CAS  PubMed  Google Scholar 

  52. Najar M, Krayem M, Meuleman N, Bron D, Lagneaux L (2017) Mesenchymal stromal cells and toll-like receptor priming: a critical review. Immune Netw 17:89–102

    Article  PubMed  PubMed Central  Google Scholar 

  53. Fitzgerald KA, Kagan JC (2020) Toll-like receptors and the control of immunity. Cell 180:1044–1066

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Fathi E, Mesbah-Namin SA, Vietor I, Farahzadi R (2022) Mesenchymal stem cells cause induction of granulocyte differentiation of rat bone marrow C-kit+ hematopoietic stem cells through JAK3/STAT3, ERK, and PI3K signaling pathways. Iran J Basic Med Sci 25:1222–1227

    PubMed  PubMed Central  Google Scholar 

  55. Sen B, Guilluy C, Xie Z et al (2011) Mechanically induced focal adhesion assembly amplifies anti-adipogenic pathways in mesenchymal stem cells. Stem cells 29:1829–1836

    Article  CAS  PubMed  Google Scholar 

  56. Kammerer W, Osmond D (1978) Surface morphology of bone marrow lymphocytes I: scanning electron microscopy of small lymphocytes bone marrow and spleen. Anat Rec 192:423–433

    Article  CAS  PubMed  Google Scholar 

  57. Pevsner-Fischer M, Morad V, Cohen-Sfady M et al (2007) Toll-like receptors and their ligands control mesenchymal stem cell functions. Blood 109:1422–1432

    Article  CAS  PubMed  Google Scholar 

  58. Krampera M, Galipeau J, Shi Y, Tarte K, Sensebe L (2013) Therapy MSCCotISfC: immunological characterization of multipotent mesenchymal stromal cells: the International Society for Cellular Therapy (ISCT) working proposal. Cytotherapy 15:1054–1061

    Article  PubMed  Google Scholar 

  59. Wei X, Shen CY (2011) Transcriptional regulation of oct4 in human bone marrow mesenchymal stem cells. Stem Cells Dev 20:441–449

    Article  CAS  PubMed  Google Scholar 

  60. Guillot PV, Gotherstrom C, Chan J, Kurata H, Fisk NM (2007) Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells 25:646–654

    Article  CAS  PubMed  Google Scholar 

  61. Batsali AK, Georgopoulou A, Mavroudi I, Matheakakis A, Pontikoglou CG, Papadaki HA (2020) The role of bone marrow mesenchymal stem cell derived extracellular vesicles (MSC-EVs) in normal and abnormal hematopoiesis and their therapeutic potential. J Clin Med 9:856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Cai H, Kondo M, Sandhow L, Xiao P, Johansson AS, Sasaki T, Zawacka-Pankau J, Tryggvason K, Ungerstedt J, Walfridsson J, Ekblom M, Qian H (2022) Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression. Blood 139:3040–3057

    Article  CAS  PubMed  Google Scholar 

  63. Chaudhary JK, Rath PC (2020) Stem cells and aging. In: Models, molecules and mechanisms in biogerontology: cellular processes, metabolism and diseases. Springer, Singapore, pp 213–234

  64. Tamura T, Tailor P, Yamaoka K et al (2005) IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J Immunol 174:2573–2581

    Article  CAS  PubMed  Google Scholar 

  65. Stellacci E, Testa U, Petrucci E et al (2004) Interferon regulatory factor-2 drives megakaryocytic differentiation. Biochem J 377:367–378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Gupta M, Rath PC (2014) Interferon regulatory factor-1 (IRF-1) interacts with regulated in development and DNA damage response 2 (REDD2) in the cytoplasm of mouse bone marrow cells. Int J Biol Macromol 65:41–50

    Article  CAS  PubMed  Google Scholar 

  67. Lu XB, Wang ZX, Liu SB, Zhang XY, Lu LF, Li S, Chen DD, Nie P, Zhang YA (2019) Interferon regulatory factors 1 and 2 play different roles in MHC II expression mediated by CIITA in grass carp. Ctenopharyngodonidella Front Immunol 10:1106

    Article  CAS  Google Scholar 

  68. Chae M, Kim K, Park SM, Jang IS, Seo T, Kim DM, Kim IC, Lee JH, Park J (2008) IRF-2 regulates NF-kappaB activity by modulating the subcellular localization of NF-kappaB. Biochem Biophys Res Commun 370:519–524

    Article  CAS  PubMed  Google Scholar 

  69. Zhang F, Wang C, Wang H, Lu M, Li Y, Feng H, Lin J, Yuan Z, Wang X (2013) Ox-LDL promotes migration and adhesion of bone marrow-derived mesenchymal stem cells via regulation of MCP-1 expression. Mediators Inflamm 2013:691023

    Article  PubMed  PubMed Central  Google Scholar 

  70. Wei L, Xu Y, Zhang L, Yang L, Zhao RC, Zhao D (2023) Mesenchymal stem cells promote wound healing and effects on expression of matrix metalloproteinases-8 and 9 in the wound tissue of diabetic rats. Stem Cells Dev 32:25–31

    Article  CAS  PubMed  Google Scholar 

  71. Wei Y, Zhang L, Chi Y, Ren X, Gao Y, Song B, Li C, Han Z, Zhang L, Han Z (2020) High-efficient generation of VCAM-1+ mesenchymal stem cells with multidimensional superiorities in signatures and efficacy on aplastic anaemia mice. Cell Prolif 53:e12862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Wang B, Lin Y, Hu Y, Shan W, Liu S, Xu Y, Zhang H, Cai S, Yu X, Cai Z, Huang H (2017) mTOR inhibition improves the immunomodulatory properties of human bone marrow mesenchymal stem cells by inducing COX-2 and PGE2. Stem Cell Res Ther 8:292

    Article  PubMed  PubMed Central  Google Scholar 

  73. Kurte M, Vega-Letter AM, Luz-Crawford P et al (2020) Time-dependent LPS exposure commands MSC immunoplasticity through TLR4 activation leading to opposite therapeutic outcome in EAE. Stem Cell Res Ther 11:416

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge the N JRF/SRF fellowships from the Govt. of India- Indian Council of Medical Research (ICMR) to JKC and the University Grants Commission (UGC) to NA. The Advanced Instrumentation Research Facility (AIRF) of JNU is acknowledged for scanning electron microscopy.

Funding

This study was supported by the Government of India: the Department of Biotechnology (DBT)-Builder (Grant No. BT/INF/22/SP45382/2022) and the Department of Science and Technology (DST)-FIST-II [Grant No. SR/FST/LSII-046/2016(C)] to the School of Life Sciences, J.N.U. and the Junior/Senior Research Fellowships (JRF/SRF) to JKC from the Indian Council of Medical Research (ICMR) and to NA from the University Grants Commission (UGC), respectively.

Author information

Authors and Affiliations

  1. Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

    Jitendra Kumar Chaudhary, Naseem Ahamad & Pramod C. Rath

Authors
  1. Jitendra Kumar Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Naseem Ahamad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pramod C. Rath
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The conception of the idea, design of the experiments, data-analysis, its interpretation and writing of the manuscript were carried out by JKC, NA and PCR. JKC and NA performed the experiments and compiled the data. PCR played the role of the P.I. of the laboratory.

Corresponding author

Correspondence to Pramod C. Rath.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest regarding this publication.

Ethical approval

All experiments conducted in this study were approved by the Institutional Animal Ethics Committee (Ref: 26/2018) of the Jawaharlal Nehru University, New Delhi.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 14 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chaudhary, J.K., Ahamad, N. & Rath, P.C. Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2. Mol Biol Rep 51, 97 (2024). https://doi.org/10.1007/s11033-023-09025-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11033-023-09025-9

Keywords

Search

Navigation