Anti-inflammatory mechanisms of human umbilical cord blood-derived mesenchymal stem cells on atopic dermatitis and rheumatoid arthritis = 아토피 피부염과 류마티스 관절염에 대한 인간 제대혈 유래 중간엽 줄기세포의 항염증 기전

다국어 초록 (Multilingual Abstract)

Atopic dermatitis (AD) and rheumatoid arthritis (RA) are representative intractable immune-related diseases characterized by complex allergic inflammatory responses and autoimmunity, respectively. Recently, extensive investigations of the pathogenic immune mechanisms in AD and RA have fueled the development of new biologic therapies that block specific cytokine networks or cellular pathways. Nonetheless, these agents have been reported to have limited efficacy as well as uncertainty in long term safety. Thus, there is a clear need to develop a novel treatment with efficacy and safety. Cell therapies utilizing mesenchymal stem cells (MSCs) have emerged as a promising alternative to replace current therapeutic approaches due to their unique immunomodulatory properties. However, the underlying mechanisms of MSCs on specific disease pathogenesis-related immune cells and the relevant efficacies are not fully elucidated. Therefore, the aims of this study are to investigate the therapeutic potency of hUCB-MSCs on AD and RA, and to provide the better understanding of anti-inflammatory mechanisms of MSCs on disease pathogenesis-related immune cells in allergic inflammatory responses and autoimmunity.
AD is a chronic and relapsing allergic skin disorder characterized by eczematous skin lesions and severe pruritus. Because AD is difficult to cure completely and its recurrence is frequent, no satisfactory therapeutics is currently available. Although few studies have shown the potential use of MSCs for the attenuation of AD, the precise mechanisms of interaction between MSCs and AD pathogenesis-related immune cells are barely verified. In the first study, I investigated the therapeutic efficacy of human umbilical cord blood-derived MSCs (hUCB-MSCs) against murine atopic dermatitis and explored distinct mechanisms that regulate their efficacy. AD was induced in mice by the topical application of Dermatophagoides farinae. Naïve or activated-hUCB-MSCs were administered to mice, and the severity of clinical symptoms was determined. The subcutaneous administration of nucleotide-binding oligomerization domain 2 (NOD2)-activated-hUCB-MSCs exhibited prominent protective effects against AD, and suppressed the infiltration and degranulation of mast cell (MC). A β-hexosaminidase assay was performed to evaluate the effect of hUCB-MSCs on MC degranulation. NOD2-activated MSCs reduced the MC degranulation via NOD2-cyclooxygenase 2 (COX2) signaling. In contrast to bone marrow-derived MSCs (BM-MSCs), hUCB-MSCs exerted a cell-to-cell contact-independent suppressive effect on MC degranulation through the higher production of prostaglandin E2 (PGE2). Additionally, transforming growth factor (TGF)-β1 production from hUCB-MSCs in response to IL-4 contributed to the attenuation of MC degranulation by down-regulating FcεRI expression in MCs. In conclusion, the subcutaneous application of NOD2-activated hUCB-MSCs can efficiently ameliorate AD, and MSC-derived PGE2 and TGF-β1 are required for the inhibition of MC degranulation.
RA is one of the most common chronic autoimmune diseases accompanied by progressive synovitis leading to joint impairment as well as systemic complications. Recent studies have reported that monocytic lineage cells and their derived cytokines play pivotal roles in the pathogenesis of RA. Although several studies have shown the therapeutic potency of MSCs for the attenuation of RA, the underlying mechanism of MSCs on monocyte/macrophage has not been clearly investigated. I thus examined the therapeutic efficacy of hUCB-MSCs against murine collagen-induced arthritis (CIA) and explored integrated mechanisms mainly focused on macrophage. The systemic administration of hUCB-MSCs significantly ameliorated the severity of CIA to a similar extent in anti-TNF-α-treated group. hUCB-MSCs exerted this therapeutic effect against CIA through the regulation of macrophage functions. To verify the regulatory effects, human and murine macrophages were co-cultured with hUCB-MSCs. Activation of COX-2 and tumor necrosis factor-stimulated gene (TSG)-6 in hUCB-MSCs by TNF-α stimuli polarized naïve macrophage toward M2 type. In addition, hUCB-MSCs down-regulated nucleotide binding domain and leucine-rich repeat pyrin 3 (NLRP3) inflammasome activation via paracrine loop of IL-1β signaling. These immune-balancing effects of hUCB-MSCs were reproducible in co-culture experiments using PBMCs isolated from patients with active RA. These data suggest that can exert simultaneous regulation of multiple cytokine pathways in response to elevated inflammatory cytokines in RA-related immune microenvironment, implying hUCB-MSCs can be an attractive candidate for refractory RA patient.
Taken together, these findings imply that (i) locally infused hUCB-MSCs exert therapeutic effect against AD by suppressing the MC degranulation and NOD2 activation in MSCs can enhance the immunosuppressive effect by increasing the release of PGE2, and that (ii) systemically delivered hUCB-MSCs can significantly ameliorate RA and they act as a cellular modulator of macrophages through the simultaneous regulation on the production of TNF-α and IL-1β by macrophages in response to disease-related inflammatory milieu. Consequently, this study suggests the possibility that hUCB-MSCs can be a promising strategy for the therapy of allergic disorders and autoimmune diseases.

목차 (Table of Contents)

• CHAPTER I. Concerted Action of PGE2 and TGF-β1 from Human Umbilical Cord Blood-derived Mesenchymal Stem Cells Alleviates Atopic Dermatitis by Reducing Mast Cell Degranulation 1
• 1.1 INTRODUCTION 2
• 1.2 MATERIALS AND METHODS 3
• 1.2.1 Isolation and culture of MSCs 3
• 1.2.2 Atopic dermatitis induction and treatment 4
• 1.2.3 Assessment of clinical severity 5
• 1.2.4 Histologic evaluation 5
• 1.2.5 Cell tracking 6
• 1.2.6 Mast cell culture 6
• 1.2.7 Mast cell degranulation 6
• 1.2.8 Western blotting 7
• 1.2.9 Cytokine production 8
• 1.2.10 Flow cytometric analysis 8
• 1.2.11 RNA interference 9
• 1.2.12 Statistical analysis 9
• 1.3 RESULTS 10
• 1.3.1 Protective effect of NOD2-activated hUCB-MSCs 10
• 1.3.2 Inhibitory effect of NOD2-activated MSCs on MCs 14
• 1.3.3 Therapeutic effect of NOD2-activated hUCB-MSCs 17
• 1.3.4 NOD2 signaling for the inhibition of MC degranulation 19
• 1.3.5 PGE2 as a key regulatory factor from hUCB-MSCs 23
• 1.3.6 TGF-β1-mediated inhibitory effect of MSCs 26
• 1.3.7 IL-4-STAT6 pathway for the regulation of FcεRI 29
• 1.4 DISCUSSION 33
• CHAPTER II. Systemic Delivery of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells Ameliorates Rheumatoid Arthritis via Regulation of NLRP3 inflammasome in Macrophages 38
• 2.1 INTRODUCTION 39
• 2.2 MATERIALS AND METHODS 42
• 2.2.1 Isolation and culture of hUCB-MSCs 42
• 2.2.2 CIA induction and treatment 42
• 2.2.3 Histological evaluation 43
• 2.2.4 Generation and stimulation of macrophages 44
• 2.2.5 Flow cytometric analysis 47
• 2.2.6 Cytokine profile 47
• 2.2.7 Patients and ex vivo experiments 48
• 2.2.8 Reagents 48
• 2.2.9 Cell tracking 49
• 2.2.10 RNA interference 49
• 2.2.11 Western blot 50
• 2.2.12 Statistical analysis 50
• 2.3 RESULTS 52
• 2.3.1 Efficacy and safety of systemically injected MSCs 52
• 2.3.2 Migration and distribution of injected MSCs in mice 58
• 2.3.3 Anti-inflammatory effect of MSCs on macrophages 61
• 2.3.4 Suppressive effect of MSCs on NLRP3 inflammasome activation in macrophages 67
• 2.3.5 Immunologic profiling of serum and PBMCs 71
• 2.3.6 Regulatory effect of MSCs on ex vivo immune cells 73
• 2.4 DISCUSSION 75
• GENERAL CONCLUSION 79
• REFERENCES 83
• ABSTRACT IN KOREAN 112