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EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY)

EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY). Andhika Yudistira, MD. INTRODUCTION. Tuberculosis as infection remains one of major health problems in Indonesia. Indonesia in 2009 : 100 / 100.000 populations in 2009 (5th Rank worldwide)

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EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY)

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  1. EFFECT OF MESENCHYMAL STEM CELLS ON MYCOBACTERIUM TUBERCULOSIS GROWTH (IN VITRO STUDY) Andhika Yudistira, MD

  2. INTRODUCTION • Tuberculosis as infection remains one of major health problems in Indonesia. • Indonesia in 2009 : 100 / 100.000 populations in 2009 (5th Rank worldwide) • Indonesia in 2011 : 0.4 – 0.5 millions cases (4th rank worldwide) *World Health Organization. Global Tuberculosis Report 2012. Diunduh dari: www.who.int/tb/publications/global_report/gtbr12_main.pdf *Perkumpulan Pemberantasan Tuberkulosis Indonesia. TBC di Indonesia Peringkat ke-4. 2012. www.ppti.info/2012/09/tbc-di-indonesia-peringkat-ke-5l

  3. Musculoskeletal tuberculosis accounts for 10% – 15% of all TB notifications in the non-industrialized world. • Spine is the most common site (50%), followed by pelvis (12%), hip and femur (10%), knee and tibia (10%), ribs (7%), and mulltiple sites (3%). 50% 10% Talbot J C, Bismil Q, Saralaya D, Newton DAG, Frizzel RM, Shaw DL. Musculoskeletal Tuberculosis in Bradford – A 6 Year Review. Ann R Coll Surg Engl. 2007. May; 89(4): 405-409. 3% 10%

  4. Pathophysiology Watts, H.G., Lifeso, R.M. (1996). Current Concepts Review-Tuberculosis of bones and Joints. JBJA Journal of Bone and Joint Surgery - American 1995 - 1998 February 1996, Volume 78-A: 2. Spiegel D A, Singh G K, Banskota, A K. Tuberculosis of the Musculoskeletal System. 2005. Techniques in Orthopaedics. Lippincott Williams & Wilkins,, Inc. Philadelphia. 20(2):167-178.

  5. Respond to Mycobacterium tuberculosis

  6. Mycobacterium tuberculosis has no exotoxins or destructing proteins • Macrophage dependent  Extended dormancy • MTB role in bone pathology : induce osteolytic activity through chaperonin 10 protein. • Chaperonin 10 as a bone resorption stimulator, and prevent osteoblast bone-forming cell pathway Meghji, S. White, P A. Nair, S P. Reddi, K. Heron, K. Henderson, B. Zalliani, A. Fossati, G. Mascagni, P. Hunt, J F. Roberts, M M, Coates, A. Mycobacterium tuberculosis Chaperonin 10 Stimulates Bone Resorption : A Potential Contributory Factor in Pott's Disease. 1997. J Exp Med, Rockefeller University Press. Volume 186 No. 8.

  7. Orthopaedic Management in Musculoskeletal TB • Establish Diagnosis • “Treating rather than Testing” • Chemotherapy – Anti Tuberculous Drugs • Surgical Debridemant • Joint Arthrodesis • Correction Deformity • Improve Function Spiegel D A, Singh G K, Banskota, A K. Tuberculosis of the Musculoskeletal System. 2005. Techniques in Orthopaedics. Lippincott Williams & Wilkins,, Inc. Philadelphia. 20(2):167-178.

  8. Mesenchymal Stem Cells • Discovered in 1968, by A.J. Friedenstein : “ MSCs are a subset of non-haematopoetic pluripotent cells found in adult bone marrow and are capable of differentiating into adipocytes, fibroblasts and even myoblasts” • Stem cell nature of these cells : ability to self renewal and differentiation into various mesenchymal elements. (bone, fat, cartilage, and muscle) • Over the years, MSCs are increasingly used in clinical practice for cell therapy. • There is growing understanding among scientific community that many of infectious diseases may be cured or controlled using stem cells. Augello A, Kurth TB, De Bari C. Mesenchymal Stem Cells : A Perspective from In Vitro Cultures to In Vivo Migration and Niches. 2010. European Cells and Materials Vol.20 : 121-133. Karl H. Kraus, Carl Kirker-Head, Vet MB. Mesenchymal Stem Cells And Bone Regeneration, Veterinary Surgery 35:232-242, 2006, The American College of Veterinary Surgeons.

  9. Mesenchymal Stem Cell has Immunomodulator Properties • MSCs are rare population of non-haematopoetic stromal cells, present in bone marrow and connective tissues. • MSCs as multipotent cells: bone, cartilage, adipose tissue, muscles. • MSC have potent immunosuppresive and immunomodulator capacity. • It was reported that MSCs posess antingen-presenting properties • Under inflammatory conditions, MSCs can act as proffesional antigen-presenting cells Chelluri, I. K. Prasad, C t. Vennila, A G. Gokhale, V. Adavi, R. Preliminary Report on Immunomodulation of Mesenchymal Stem Cells in M.tb Infection, 2010. The Internet Journal of Infectious Diseases. 2010 Vol 8 No.l. Shilpa R, Pawan Sharmaa, Sarman Singhb, Luc Van Kaerc, and Gobardhan Das. Mycobacterium tuberculosis evades host immunity by recruiting mesenchymcl stem cells, www.pnas.as.org/cgi/doi/l 0.1073/pnas. 1007967107. Hoogduijn M J, Popp F, Verbeek R, Masoodi M, Nicolaou A, Baan C, Dahlke M. The Immunomadulatory Properties of Mesenchymal Stem Cells and Their Use for Immunotherapy. 2010. International Immunopharmacology. Doi : 10.1016/j.intimp.2010.06.019.

  10. Effects of MSCs on Immunocytes MSC modulates immune response by interaction with : • T cells • B cells • Dendritic cells • Treg cells • NK cells • Γδ cells Shi M, Liu Z-W. Wang FS. Immunomodulatory Properties and Therapeutic Application of Mesenchymal Stem Cells. Clinical and Experimental Immunology. 2011. British Society for Immunology, Clinical and Experimental Immunology, 164: 1-8.

  11. MSCs possess immunosuppressive : inhibit T-cells, B-cells, NK cells proliferation and functions. • Modulate activities of DCs, induce Treg cells. • These properties is dependent on cell – cell contact or IDO, PGE2, NO, IFN-γ, TGF-β secretions • Inflammatory : MScs prevent naive CD4 cells into TH17

  12. Raghuvanshi : “MSCs are recruited to the site of MTB infection” • MSCs contains the pathogenic microorganisms but doesn’t eliminate them. • Inhibit T-cell atcivation by producing NO, TGF-β, or IDO • NO controls the growth of MTB organisms and confines them within granuloma-like structures • Persistent MTB infections !

  13. It is believed that MSCs can act as Antigen Presenting Cells under inflammatory conditions • MSCs secreted IL6, IL8, chmokines CCl2, TIMP2 • Pro inflammatory : attracts Monocytes, DCs, T cells and NK cells  antigen eradications actively or through T cells and NK cells, and NO. • Anti inflammatory : Chemotaxis targets neutrophils, monocytes. • MSCs have dual immunomodulatory capacity, depend on IFN-γ and TNF-α.

  14. MSCs role in TB Infection ????????? MSC

  15. MSCs role in immunomodulation is still questionable • Mensyuknil (2011) studied effect of MTB debris on MSCs growth (in vitro).  NO effect on MSCs growth by MTB debris • Interaction between living Mycobacterium tuberculosis and Mesenchymal Stem Cells in one single medium is required to evaluate. Mensyuknil, U. Rahyussalim Pengaruh Debris dan Supernataan Kuman Mycobacterium Tuberculosis yang Diambil Dari Lesi Spondylitis Terhadap Pertumbuhan Sel Punca (Studi in vitro). 2011. Program Studi Orthopaedi dan Traumatologi, Fakultas Kedokteran Ur.iversitas Indonesia, RS Cipto Mangunkusumo, Jakarta.

  16. Research Questions • Is there any effect on Mycobacterium tuberculosis growth by performing co-culture Mycobacterium tuberculosis and Mesenchymal Stem Cells ? • By performing co-culture  is there any effect on Mesenchymal Stem Cells growth ?

  17. Research Hypothesis • MSCs have immunoregulator properties and have direct eradication effect on Mycobacterium tuberculosis • Effect MSCs on eradication of Mycobacterium tuberculosis or MTB growth are seen in Acid fast staining, bacterial culture, and PCR.

  18. Aim • To evaluate interaction between living Mycobacterium tuberculosis and Mesenchymal Stem Cells in co-culture growth

  19. Theoretical Framework MTB MSCs MSCs recruited by MTB (APC) Macrophage coated MTB MHC I _CD8+ T eff MHC II – CD4+ Th Adaptive immune MHC I  Teff MHC II  B cells Activate NK - DCs IFN-γ & TNF-α Pro infllamatory mediators Th1  Makrofag Th2  B cells NK cells NO Immune Exhaustion Granuloma IDO, NO, PGE2 HGF, TGF-β IFNγ Granuloma degradation Replications & Activations Immunosuppresive Effects Inhibit MTB replications MTB eradications (-) Tissue Necrosis

  20. Conceptual Framework MSC MTB Co-Culture MTB & MSC • AFB • Culture • PCR • AFB • Culture • PCR • AFB • Culture • PCR

  21. Research Method • Exclusion • Contamination (+) • RPMI (-)

  22. Operational Definition

  23. Study Protocol MTB culture in LJ MSC culture in RPMI 5000 cells/cm2 1 McF = 108 cfu/ml 104 cfu/ml 5000 cells/cm2 MTB in RPMI 2,5cc MTB + 2,5 cc RPMI Co-Culture MSC+MTB in RPMI 2,5cc MTB + 2,5 cc MSC MSC in RPMI 2,5cc MSC + 2,5 cc RPMI Incubation in 5% CO2, Temp 37°C • AFB • Culture • PCR • MSC counting • AFB • Culture • PCR • MSC counting • AFB • Culture • PCR • MSC counting Day 3 Day 7 Day 9 Day 3 Day 7 Day 9 Day 3 Day 7 Day 9

  24. Co-Culture Transferring MTB from 3 days optimization RPMI (108 cfu/ml)  centrifuge  (104cfu/ml) • 2.5 cc of RPMI • 2.5cc of MTB in RPMI • 2.5cc of MSC in RPMI

  25. 25cc Tc-Flask • 3 flasks of culture of MSC • 2 flasks of culture of MTB • 3 flasks of co culture Incubation in CO2 incubator 5%, Temp 37°C

  26. Evaluation Each Group Each flask is being observed under inverted microscope Flasks from 3 groups are taken out from the incubator

  27. MTB Evaluation • MTB preparation • MTB samples for PCR • MTB Culture in LJ • MTB AFB • 1 cc for PCR exam • 2 cc for AFB and Bacterial culture in LJ medium

  28. Real Time PCR assay

  29. MSC counting • Add trypsin 2cc  incubate 37°C for 7 min • Add inactivating medium 4cc • Collect to 15cc tube  centrifuge 1200rpm for 4 min • Debris are taken  haematocytometer • Microscope counting

  30. ResultsDay 3 Flasks under Microscope MSC culture MTB culture MSC-MTB co culture AFB Ziehl Neelsen MSC-MTB co culture MTB culture

  31. LJ medium for culture MTB culture MSC-MTB co culture PCR Realtime Cyclus Threshold

  32. ResultsDay 7 Flasks under Microscope MSC culture MTB culture MSC-MTB co culture AFB Ziehl Neelsen MSC-MTB co culture MTB culture

  33. LJ medium for culture PCR Realtime Cyclus Threshold MSC-MTB co culture MTB culture

  34. Results Day 9 Flasks under Microscope MSC culture MTB culture MSC-MTB co culture AFB Ziehl Neelsen MSC-MTB co culture MTB culture

  35. LJ medium for culture PCR Realtime Cyclus Threshold MSC-MTB co culture MTB culture

  36. PCR - Cyclus Threshold P 0.04 P 0.07 P 0.07 *Kruskal Wallis test p 0.08 p 0.77 p 0.56 *Mann-Whitney test

  37. MSC : Cell Counting Cells : viable cells x 2 x 104 x volume (cc)

  38. Cell Counting MSC P 0.05 P 0.05 P 0.04 *Kruskal Wallis test P 0.037 P 0.046 P 0.046 *Mann-Whitney test

  39. DISCUSSION Why RPMI ? • DMEM (for MSCs) and MGIT/LJ (for MTB) are best for each MSC and MTB. But MSCs can’t be cultured in MGIT/LJ as well as MTB in DMEM • Roswell Park Memorial Institute Medium is a kind of medium in culturing cells and tissues. • RPMI-1640 as a medium to culture leucocytes, bone marrow, and hybridoma. • RPMI can be used in culturing Mycobacterium tuberculosis* • RPMI can be used in culturing MSCs, though DMEM is still superior compared to RPMI** *Zhang M, Gong J, Lin Y, Barnes P F. Growth of Virulent and Avirulent Mycobacterium tuberculosis Strains in Human Macrophages. 1998. Journal of Infection and Immunity. V.66(2) : 794-799. **Pittenger MF. Mesenchymal Stem Cells from Adult Bone Marrow. 2008. Methods in Molecular Biology, vol.449. Humana Press, Totowa, New Jersey.

  40. Discussion Mesenchymal Stem Cells in Tc-Flask • Using capped Tc-Flask  Biosafety • MSCs seeding invitro  Various amount • Eyckman : compared seeding 5000/cm2 – 25000 /cm2 – 85000/cm2 • 5000/cm2 results in more efficient in cell attachments compared to the others. * Eyckmans J, Lin GL, Chen CS. Adhesive and Mechanical Regulation of Mesenchymal Stem Cell Differentiation in Human Bone Marrow and Periosteum-Derived Progenitor Cells. 2012. Biology Open 000, 1-11.

  41. Discussion Effect MSCs on Mycobacterium tuberculosis growth • All evaluations for MTB existence/growth in co culture (AFB – Bacterial cuture – PCR) confirmed that MSCs has no eradication effect on MTB. • On AFB staining : coculture group showed more densed MTB compared to MTB group especially in day 7 and day 9. • On bacterial culture : Both MTB group and coculture group defines the same positive (+) result • On PCR assay : p 0.04 / 0.07 / 0.07 (3 groups) and p 0.08 / 0.56 / 0.77 (MTB : Coculture)  showed no difference in MTB existence.

  42. Discussion MSCs Immunomodulator Properties ? (Previous Studies) : • MSCs might support eradication of MTB (microorganism) infection through its immunomodulator properties* • MSCs inhibit T cells and provide immune tolerance for Mycobacterium tuberculosis (evade host immunity) ** • MSCs immunomodulation effect depends on surrounding inflammatory environment.***  In Vitro (-) *Rahyussalim. Transplantasi Sel Punca Mesenkimal Pada Defek Spondilitis Tuberkulosis : Pengaruh Terhadap Perbaikan Pembentukan Tulang Baru Dan Eradikasi Infeksi Pada Model Kelinci. Fakultas Kedokteran Universitas Indonesia. 2013. ** Shilpa R, Pawan Sharmaa, Sarman Singhb, Luc Van Kaerc, and Gobardhan Das. Mycobacterium tuberculosis evades host immunity by recruiting mesenchymcl stem cells, www.pnas.as.org/cgi/doi/l 0.1073/pnas. 1007967107. *** Hoogduijn M J, Popp F, Verbeek R, Masoodi M, Nicolaou A, Baan C, Dahlke M. The Immunomadulatory Properties of Mesenchymal Stem Cells and Their Use for Immunotherapy. 2010. International Immunopharmacology. Doi : 10.1016/j.intimp.2010.06.019.

  43. Discussion Coculture Study Mycobacterium tuberculosis – Mesenchymal Stem Cells • MSCs viabe cells are hardly seen in the coculture group • MSCs are believed to have Antigen Presenting Cellrole in its immunomodulator properties • Absence of “real” inflammatory mediators in invitro study. • However, MTB are known not to possess/produce exotoxins • Is it exotoxins ? / or “Medium competition” ? • Novak : “MTB demonstrated polyketide synthesis gense, indicating that MTB produces a related toxin”. • Finding an MTB homolog of this toxin will be a focus of future research Novak K. Tuberculosis Toxic Avengers. 1999. Science; 283:854. http://www.nature.com/nm/focus/tb/research_news.html

  44. Conclusions • Mesenchymal Stem Cells have no effect in Mycobacterium tuberculosis eradication (In Vitro) • Mycobacterium tuberculosis somehow suppress Mesenchymal Stem Cells growth ( In Vitro) However, effect of Mycobacterium tuberculosis on MSCs growth (In Vitro) creates another question ? Is it just “Medium Competition” or “MTB prouce toxins or virulence factors” ?

  45. THANK YOU

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