This research is the first bidirectional comprehensive investigation of the interaction between activated microglia and MSCs through transcriptomic analysis.
Activated microglia increase migration of rBM-MSCs and that rBM-MSCs reduce inflammation of activated microglia at the cellular network level were shown. And bidirectional cellular interaction that occur between rBM-MSCs and LPS-stimulated microglia using transcriptomics (Figure 17). The results show that LPS-stimulated microglia help homing of rBM-MSCs via inducing transcriptomic changes and that rBM-MSCs reduce the inflammation of microglia by alternating the expression of the relevant genes.
Transplanted BM-MSCs migrate to the ischaemic border zone in animal models of stroke (34, 35), and to the substantia nigra (SN) induced by LPS and MPTP administration in animal models of Parkinson’s disease (23), in which inflammatory regions contain activated microglia. Additionally, BM-MSCs migrate to LPS-stimulated microglia in vitro in response to chemotactic factors (27). These reports indicate that activated microglia are a possible cause of movement of rBM-MSCs to the site of injury. However, detailed transcriptomic mechanisms of how activated microglia induce the movement of BM-MSCs are not well understood. In this study, the expression levels of 67 genes were significantly altered, and that network analysis predicted an increase in migration activity was shown. Meanwhile, CXCR4 receptor and its ligand stromal cell-derived factor-1 (SDF-1a) play an important role in homing of MSCs to brain lesions (36). However, there was no changes in microarray data and RT-PCR (data not shown). Further studies concerning the homing effects of rBM-MSC on microglia in coculture systems are needed.
The prediction was confirmed experimentally in rBM-MSCs cocultured with LPS-stimulated microglia compared with control and cells cocultured with microglia.
Especially, Mmp3 and 9 are essential enzymes for migratory activity and the corresponding genes are induced in inflammatory conditions via pro-inflammatory
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cytokines and oxidative substrates (37, 38). Thus, LPS-stimulated microglia might form a locally conditioned inflammatory region in the coculture system, and rBM-MSCs move toward this region.
Human BM-MSCs (hBM-MSCs) dramatically reduce neural damages in a LPS-stimulated mouse brain and reduce inflammation of microglia owing to the production of cytokines and trophic factorshave been reported (39). In addition, co-cultured BM-MSCs reduce the expression of TNF-α and iNOS and NO production in LPS-stimulated microglia compared with non-cocultured BM-MSCs (23, 27).
However, the detailed mechanism about how BM-MSCs down-regulate activated microglia has not been well studied. In this study, the expression levels of 64 genes were significantly altered, and gene network predicted the suppression of the inflammatory response were shown. This prediction was experimentally verified in LPS-stimulated microglia cocultured with rBM-MSCs. The results show that the levels of most pro-inflammatory genes were reduced when cocultured with rBM-MSCs. The expression levels of Ccl2, a key mediator of microglia activation (40, 41), were significantly decreased in the presence of rBM-MSCs, indicating anti-inflammatory effects by rBM-MSC. These phenomena may occur as a result of secreted molecules from both rBM-MSCs and LPS-stimulated microglia. Further studies are needed to identify the secreted molecules, i.e., the secretome, that increase invasive migratory activity of rBM-MSCs and decrease the inflammatory response of LPS-stimulated microglia. Therefore, detailed mechanism can be elucidated by connecting secretome of rBM-MSC with receptor of microglia and reverse condition.
There are several limitations in this study. First, ±1.2 fold-change in expression levels was used as a cut-off and identified low level of abundant genes due to the limitation in in vitro coculture system. In general, transwell coculture system has central limitations, such as cell density, fluid concentration, and cell-other cell direct interaction, compared to in vivo (42). However, the transcriptomic analysis for bidirectional effect of each cells and the above approach cannot be done in in vivo
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system, due to diversity of cells in organ. The implied biological effect in our results might be more strongly expressed in in vivo were expected. Second, fundamental limitations reside in microarray and bioinformatics analysis. Although microarray is performed with additional probe for compensation of inaccuracies and statistically analysed, the reduction of false positive or negative is a challenging task (43). In addition, bioinformatics tool is constructed with existing knowledge, hence novel finding is limited (44). As a result, unique pathway cannot be deduced in our analysis system and individual analysis for genes can be biased, which could bring about false positive or negative result. Thus, I focused more in cellular process than target molecule. Finally, analysing specific pathway/s or molecule/s are beyond our limitation in this study. To find novel pathway/s or molecule/s, responsible for attracting rBM-MSCs or inflammatory response of microglia identified, other methodological biomolecular approaches are required.
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CONCLUSION
In conclusion, bidirectional transcriptomic analysis of activated microglia and BM-MSCs were performed. The results indicate that activated microglia in a neuroinflammatory condition enhance the migration of MSCs, and that BM-MSCs reduce microglia mediated inflammation. This study improves understanding of the relationship between activated microglia and transplanted stem cells and may prime to a new therapeutic strategy using stem cell therapy for brain diseases.
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REFERENCES
1. Lee DY, Jin MS, Manavalan B, Kim HK, Song JH, Shin TH, et al. Bidirectional Transcriptome Analysis of Rat Bone Marrow-Derived Mesenchymal Stem Cells and Activated Microglia in an In Vitro Coculture System. Stem Cells Int. 2018;2018:6126413.
2. Jäkel S, Dimou L. Glial Cells and Their Function in the Adult Brain: A Journey through the History of Their Ablation. Frontiers in Cellular Neuroscience. 2017;11(24).
3. Benraiss A, Wang S, Herrlinger S, Li X, Chandler-Militello D, Mauceri J, et al.
Human glia can both induce and rescue aspects of disease phenotype in Huntington disease. Nature Communications. 2016;7(1):11758.
4. Masuda T, Sankowski R, Staszewski O, Prinz M. Microglia Heterogeneity in the Single-Cell Era. Cell Reports. 2020;30(5):1271-81.
5. Salter MW, Stevens B. Microglia emerge as central players in brain disease.
Nature medicine. 2017;23(9):1018.
6. Colonna M, Butovsky O. Microglia function in the central nervous system during health and neurodegeneration. Annual review of immunology. 2017;35:441-68.
7. Uccelli A, Benvenuto F, Laroni A, Giunti D. Neuroprotective features of mesenchymal stem cells. Best Practice & Research Clinical Haematology. 2011;24(1):59-64.
8. Beschorner R, Schluesener HJ, Gözalan F, Meyermann R, Schwab JM. Infiltrating CD14+ monocytes and expression of CD14 by activated parenchymal microglia/macrophages contribute to the pool of CD14+ cells in ischemic brain lesions.
Journal of neuroimmunology. 2002;126(1-2):107-15.
9. Inoue K, Tsuda M. Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential. Nature Reviews Neuroscience. 2018;19(3):138.
10. Xu L, He D, Bai Y. Microglia-mediated inflammation and neurodegenerative disease. Molecular neurobiology. 2016;53(10):6709-15.
11. McGeer PL, Itagaki S, Boyes BE, McGeer E. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology.
1988;38(8):1285-.
12. Thameem Dheen S, Kaur C, Ling E-A. Microglial activation and its implications in the brain diseases. Current medicinal chemistry. 2007;14(11):1189-97.
13. Ajmone-Cat MA, Bernardo A, Greco A, Minghetti L. Non-steroidal anti-inflammatory drugs and brain inflammation: effects on microglial functions.
- 46 - Pharmaceuticals. 2010;3(6):1949-65.
14. Jacobs BL, Van Praag H, Gage F. Adult brain neurogenesis and psychiatry: a novel theory of depression. Molecular psychiatry. 2000;5(3):262-9.
15. Feng Y-L, Tang X-L. Effect of glucocorticoid-induced oxidative stress on the expression of Cbfa1. Chemico-biological interactions. 2014;207:26-31.
16. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease.
Nature Reviews Immunology. 2008;8(9):726-36.
17. Oliveira JT, Mostacada K, de Lima S, Martinez AMB. Chapter Three - Bone Marrow Mesenchymal Stem Cell Transplantation for Improving Nerve Regeneration. In:
Geuna S, Perroteau I, Tos P, Battiston B, editors. International Review of Neurobiology.
108: Academic Press; 2013. p. 59-77.
18. Lee P, Kim J, Bang O, Ahn Y, Joo I, Huh K. Autologous mesenchymal stem cell therapy delays the progression of neurological deficits in patients with multiple system atrophy. Clinical Pharmacology & Therapeutics. 2008;83(5):723-30.
19. Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal stem cell transplantation in stroke patients. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society. 2005;57(6):874-82.
20. Venkataramana N, Pal R, Rao SA, Naik AL, Jan M, Nair R, et al. Bilateral transplantation of allogenic adult human bone marrow-derived mesenchymal stem cells into the subventricular zone of Parkinson’s disease: a pilot clinical study. Stem cells international. 2012;2012.
21. Shin TH, Phukan G, Shim JS, Nguyen D-T, Kim Y, Oh-Lee JD, et al. Restoration of polyamine metabolic patterns in in vivo and in vitro model of ischemic stroke following human mesenchymal stem cell treatment. Stem cells international. 2016;2016.
22. Matthay MA, Pati S, Lee JW. Concise review: mesenchymal stem (stromal) cells:
biology and preclinical evidence for therapeutic potential for organ dysfunction following trauma or sepsis. Stem Cells. 2017;35(2):316-24.
23. Kim YJ, Park HJ, Lee G, Bang OY, Ahn YH, Joe E, et al. Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti-inflammatory action. Glia. 2009;57(1):13-23.
24. Cheng Z, Peng H-l, Zhang R, Fu X-m, Zhang G-s. Bone marrow-derived innate macrophages attenuate oxazolone-induced colitis. Cellular immunology. 2017;311:46-53.
25. Yan K, Zhang R, Sun C, Chen L, Li P, Liu Y, et al. Bone marrow-derived
- 47 -
mesenchymal stem cells maintain the resting phenotype of microglia and inhibit microglial activation. PLoS One. 2013;8(12).
26. Jaimes Y, Naaldijk Y, Wenk K, Leovsky C, Emmrich F. Mesenchymal stem cell-derived microvesicles modulate lipopolysaccharides-induced inflammatory responses to microglia cells. Stem Cells. 2017;35(3):812-23.
27. Jose S, Ramasamy R, Vidyadaran S. Reciprocal interactions of mouse bone marrow-derived mesenchymal stem cells and BV2 microglia after lipopolysaccharide stimulation. Stem cell research & therapy. 2013;4(1):12.
28. Park HJ, Lee PH, Bang OY, Lee G, Ahn YH. Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson’s disease. Journal of neurochemistry. 2008;107(1):141-51.
29. Bachstetter AD, Van Eldik LJ, Schmitt FA, Neltner JH, Ighodaro ET, Webster SJ, et al. Disease-related microglia heterogeneity in the hippocampus of Alzheimer’s disease, dementia with Lewy bodies, and hippocampal sclerosis of aging. Acta neuropathologica communications. 2015;3(1):32.
30. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends in neurosciences. 1996;19(8):312-8.
31. Shin TH, Da Yeon Lee H-SL, Park HJ, Jin MS, Paik M-J, Manavalan B, et al.
Integration of metabolomics and transcriptomics in nanotoxicity studies. BMB reports.
2018;51(1):14.
32. Shin TH, Lee S, Choi KR, Kim Y, Paik MJ, Seo C, et al. Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buffered saline. Scientific reports. 2017;7(1):1-8.
33. de Lucas B, Pérez LM, Gálvez BG. Importance and regulation of adult stem cell migration. Journal of cellular and molecular medicine. 2018;22(2):746-54.
34. Kalladka D, Muir KW. Brain repair: cell therapy in stroke. Stem cells and cloning:
advances and applications. 2014;7:31.
35. Paik MJ, Li WY, Ahn YH, Lee PH, Choi S, Kim KR, et al. The free fatty acid metabolome in cerebral ischemia following human mesenchymal stem cell transplantation in rats. Clinica Chimica Acta. 2009;402(1-2):25-30.
36. Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem cells. 2007;25(11):2739-49.
- 48 -
37. Almalki SG, Agrawal DK. Effects of matrix metalloproteinases on the fate of mesenchymal stem cells. Stem cell research & therapy. 2016;7(1):129.
38. Warner RL, Bhagavathula N, Nerusu KC, Lateef H, Younkin E, Johnson KJ, et al.
Matrix metalloproteinases in acute inflammation: induction of MMP-3 and MMP-9 in fibroblasts and epithelial cells following exposure to pro-inflammatory mediators in vitro.
Experimental and molecular pathology. 2004;76(3):189-95.
39. Zhou C, Zhang C, Chi S, Xu Y, Teng J, Wang H, et al. Effects of human marrow stromal cells on activation of microglial cells and production of inflammatory factors induced by lipopolysaccharide. Brain research. 2009;1269:23-30.
40. Thacker MA, Clark AK, Bishop T, Grist J, Yip PK, Moon LD, et al. CCL2 is a key mediator of microglia activation in neuropathic pain states. European journal of pain.
2009;13(3):263-72.
41. Hinojosa AE, Garcia-Bueno B, Leza JC, Madrigal JL. CCL2/MCP-1 modulation of microglial activation and proliferation. Journal of neuroinflammation. 2011;8(1):77.
42. Carroll MJ, Stopfer LE, Kreeger PK. A simplified culture system to examine soluble factor interactions between mammalian cells. Chemical Communications.
2014;50(40):5279-81.
43. Tinker AV, Boussioutas A, Bowtell DD. The challenges of gene expression microarrays for the study of human cancer. Cancer cell. 2006;9(5):333-9.
44. Jin L, Zuo X-Y, Su W-Y, Zhao X-L, Yuan M-Q, Han L-Z, et al. Pathway-based analysis tools for complex diseases: a review. Genomics, proteomics & bioinformatics.
2014;12(5):210-20.