Immunoenhancing effects of Lactococcus lactis OTG1204 in RAW 264.7 macrophages and cyclophosphamide-induced immunosuppressed mice via activation of TLR2 pathways

다국어 초록 (Multilingual Abstract)

Immunoenhancing effects of Lactococcus lactis OTG1204 in RAW 264.7 macrophages and cyclophosphamide-induced immunosuppressed mice via activation of TLR2 pathways by Song HyeonA Master of Pharmacy in Fundamental Pharmaceutical Science Graduate School of Kyung Hee University Advised by Prof. Lee Kyung-Tae Probiotics can modulate the gut microbiota, conferring beneficial effects on immunity and chronic autoimmune diseases such as multiple sclerosis. In this study, we investigated the mechanisms of Lactococcus lactis OTG1204 (OTG1204) isolated from kimchi, a traditional Korean dish, on the activation of RAW 264.7 macrophages and assessed its immunomodulatory effects in cyclophosphamide (CTX)-induced immunosuppressed C57BL/6 mice model. In RAW 264.7 macrophages, OTG1204 significantly enhanced the production of NO, prostaglandin E2, and immunomodulatory cytokines [interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)]. OTG1204 treatment also activated NF-κB and MAPKs/AP-1 signaling. In addition, OTG1204 stimulated the expression of myeloid differentiation primary response 88, TNF receptor-associated factor 6, and transforming growth factor-β-activated kinase 1 via Toll-like receptor 2. Administration of OTG1204 (50, 100, 150, or 200 mg/kg/day, p.o.) restored the body weight as well as the spleen and mesenteric lymph node indices in CTX- induced mice. Additionally, OTG1204 restored CTX-induced reduction in natural killer cell activity, immune cell population, T helper 1 cell-related production of cytokines (TNF-α, interferon-γ, and IL-12), and T helper 2 cell-related cytokines (IL-4 and IL-6). OTG1204 treatment repaired the CTX-damaged histological structure of the colon tissue and the expression of mucin 2 and tight junction proteins. Furthermore, the OTG1204 treatment maintained gut homeostasis by regulating the microbiota composition, which was altered by CTX, similar to the control group’s composition. Our in vitro and in vivo data demonstrate the potential of OTG1204 as a supplement for enhancing immune health.

목차 (Table of Contents)

• Abstract Ⅴ
• 1. Introduction 1
• 2. Material and Methods 4
• 2.1. Materials 4
• 2.2. Preparation of OTG1204 4
• 2.3. In vitro OTG1204 treatment 4
• 2.4. Cell viability 5
• 2.5. Measurement of nitric oxide (NO) 5
• 2.6. Measurement of cytokine, prostaglandin E2 (PGE2) 5
• 2.7. Luciferase reporter assay using cellular transfection 6
• 2.8. Quantitative real-time PCR (qRT-PCR) 6
• 2.9. Western blot analysis 7
• 2.10. Animals 8
• 2.11. Cyclophosphamide-induced immunosuppression in mice 9
• 2.12. Hepatoxicity and nephrotoxicity analysis 10
• 2.13. Measurement of immunoglobulin G (IgG) production in serum 11
• 2.14. Spleen and MLN index 11
• 2.15. Splenocyte isolation 11
• 2.16. NK cell activity 12
• 2.17. Flow cytometry assay 13
• 2.18. Histological analysis 13
• 2.19. Gut microbiota analysis 13
• 2.20. Statistical analysis 14
• 3. Results 16
• 3.1 OTG1204 modulates the production and expression of immunostimulatory mediators in RAW 264.7 macrophages 16
• 3.2 OTG1204 activates NF-κB, AP-1, and MAPK signaling pathway in RAW 264.7 macrophages 18
• 3.3 OTG1204 upregulates TLR2 signaling pathway involving MyD88, TRAF6, and TAK1 in RAW 264.7 macrophages 20
• 3.4 OTG1204 does not induce hepatotoxicity or nephrotoxicity in CTX-induced mice 22
• 3.5 OTG1204 treatment recovers immune state in CTX-induced immunosuppression mice 24
• 3.6 OTG1204 treatment regulates the proportion of immune cells in CTX-induced immunosuppression mice 25
• 3.7 OTG1204 enhances cytokines secreted from T cells in splenocytes of CTX-induced immunosuppression mice 28
• 3.8 OTG1204 prevents gut barrier disruption in CTX-induced immunosuppression mice 30
• 3.9 OTG1204 regulates gut microbiota composition in CTX-induced immunosuppression mice 32
• 3.10 OTG1204 restores TLR2 mRNA and p-p65 protein expression in CTX-induced immunosuppression mice 34
• 4. Discussion 35
• 5. Conclusion 43
• 6. References 45