The Possible Mechanism Of Anti-lung Fibrosis By Human Bone Marrow Mesenchymal Stem Cells And Synergistic Effects Of Flavonoids

Background and ObjectiveThe idiopathic interstitial pneumonias are a group of interstitial lung diseases characterized by varying patterns of inflammation and fibrosis. The two most common forms are idiopathic pulmonary fibrosis (IPF), characterized by a histologic pattern of usual interstitial pneumonia (UIP), and nonspecific interstitial pneumonia(NSIP). IPF is associated with an extremely poor prognosis for survival in most patients. Life expectancy after diagnosis varies, but is on average less than 5 years. It has been suggested that in some patients, NSIP may represent an early stage of UIP or, alternatively, that an NSIP pattern seen at biopsy may simply be indicative of relatively inactive UIP. Despite extensive research efforts over the past decades, no currently available therapy has been shown to either reverse or even halt the progression of this disorder.Recent evidence suggests that in more than 50% of patients diagnosed with iNSIP evidence of autoimmune diseases develop within 2 years, suggesting a likely link between the clinical entity of iNSIP and autoimmune disorders. As a bridge between innate and adaptive immunity, type ? NKT cells appears to play a pivotal role in the pathogenesis of iNSIP. NKT cells are a heterogeneous group of T lymphocytes that express NK and T cell markers, recognize the lipid antigens presented by the nonclassical MHC class I-like molecule CD1. NKT cells are known to functionally bridge innate and adaptive immune system in various immune diseases by cytotoxic effect and producing inflammatory factor L-4 and INF-γ.The immunomodulatory properties of bone marrow-derived mesenchymal stem cells(MSCs) have recently become exciting for investigators in terms of examining their potential implications in a variety of disease models. MSCs have been tested in rodent animal models to treat diseases where immunomodulation is thought to be the main operative mechanism. They can reverse autoimmune response disorder by multitarget modulating immunocyte subset. Their potentiality of multi-directional differentiation may benefit iNSIP patients with impaired alveolus and bring first light of morning for curing interstitial pulmonary fibrosis. However, it was uncertain whether MSCs could differentiate into myofibroblasts under inflammatory microenvironment in NSIP. Therefore, there is an urgent need to determine if MSCs take an active role in the management of NSIP but not initiate fibrotic process. Flavonoids are natural products of polyphenolic compounds, which display a variety of biological effects, such as antioxidation,anti-inflammation, anticancer. In addition, flavonoid compounds have been shown to regulate immune responses. Thus, flavonoids may have the potential to expand MSCs function for therapeutic applications in interstitial pulmonary fibrosis. In the present study, we aimed to investigate if MSCs can treat autoimmune response disorder by multitarget modulating immunocyte subset (e.g., NKT ), and to understand effects of MSCs functions in treatment of NSIP fibroblasts on pulmonary fibrosis and possible synergistic effect by the flavonoids from prescription of traditional Chinese medicine. Materrials and MethodsPARTⅠ. Preparation of human bone marrow mesenchymal stem cellsHuman bone marrow mesenchymal stem cells (MSCs) were isolated from 2-3 months aborted fetus and cultured using whole bone marrow culture method. Phenotypes of MSCs were identified by flow cytometry before the cells were transfected with green fluorescent protein(GFP) eukaryotic expression plasmid -PEGFPn2. MSCs expressing GFP (MSCs-GFP 1×106 cells/100 ul total volume) were slowly infused into SCID mouse via a tail venous canula. Mice were humanely killed within 24 hour after MSC infusion. Frozen sections of the mice lung were made rapidly, and then subjected to observation by confocal laser microscope.PARTⅡ. Effects of MSCs immunomodulation on PBMCs with active NKTPeripheral blood mononuclear cell (PBMC) were isolated from peripheral blood of health adult by lymphocyte isolation method, and cultured with addition of IFN-γ,CD3 antibody and IL-2 for 21 days. PBMCs containing high proportion of CD3~+CD56~+NKT cell were collected and cultured with P4-8 human bone marrow mesenchymal stem cells (ratio 20:1)for another 24 hour. The cultured supernatants were harvested and the cultured PBMCs were analyzed by flow cytometry. Samples were divided into 3 groups: NKT cell control, MSC-NKT cell co-culture and HFB-NKT cell co-culture group. ELISA or Luminex Assay was performed for measurement of levels in TGF-β1 ,IP-10 and other cytokines/chemokines released into the supernatants.PARTⅢ. Effects of MSCs immunomodulation on active fibroblasts of NSIP patientHuman bone MSCs were subjected to co-culture with the cultured pulmonary fibroblasts isolated from NSIP patient for 24 hour using Transwell. The cytokines/chemokines patterns in the supernatants were tested by using the same methods as above. Cell proteins were extracted from NSIP-fibroblasts after co-culture and then subjected to western blotting for detection ofα-SMA (Smooth Muscle Actin ). PARTⅣ. Synergistic effect of flavonoids extract from Chinese herbs (Tian-Long) with MSCs immunomodulation on protection of pulmonary cells from NKT-mediated damage and fibrosisHuman bronchial epithelial cell line 16HBE was seeded in 96 well cell culture plate overnight, and then flavonoids extract from Chinese herbs(Tian-Long) were added into the cell culture with different concentration(sfrom 12.5ug/ml to 200ug/ml), for another 24 hour culture. Cell proliferation analysis was performed for the 16HBE cultured with the flavonoids extract by Cell Counting Kit-8.CD3~+CD56~+NKT cell were pretreated by culture with human bone marrow mesenchymal stem cell or human fibroblasts for 24 hour (ratio 20:1)before being co-cultured with 16HBE cells in presence or absence of flavonoids extract(25ug/ml). At 4 hr after the pretreatment, 16HBE cells were subjected to culture with or without flavonoids extract(25ug/ml) for another 8hr, followed by cell survival counting using the same CCK8 assay. ResultsWe confirmed that Human bone marrow mesenchymal stem cells(BMMSC) were cultured successfully by flow cytometry analysis. After GFP- MSC infusion ,we observed human BMMSC engrafted in the lung tissue of mice by confocal laser microscope.It was shown that human bone marrow mesenchymal stem cells significantly decreased radio of NKT cell in the cultured PBMCs(.20.33±1.05%in NKT cell control or 19.93±1.64 %in HFB-NKT- group v.s 15.17±1.75% in MSC-NKT-group, P<0.05)., while decreased ratio of CD3~~+CD8~~+T lymphocyte cell in the PBMC culture, (70.37±1.44% in control group , 69.97±2.23% in HFB group v.s 66.93±1.69%in MSC group P<0.05). In contrast, Human bone marrow mesenchymal stem cell increased ratio of CD3~~+CD4~~+T and CD4~~+CD25~~+CD127(Low/-)Treg lymphocytes in the cultured PBMCs cell (24.66±1.40 % CD3~~+CD4~~+T lymphocyte/19.63±1.38 %CD4~~+CD25~~+CD127(Low/-)Treg lymphocyte in control group, 25.32±1.06 %CD3~~+CD4~~+T lymphocyte/19.17±1.06%CD4~~+CD25~~+CD127(Low/-)Treg lymphocyte in HFB group v.s 29.75±1.80 % CD3~~+CD4~~+T lymphocyte/24.50±1.98 %CD4~~+CD25~~+CD127(Low/-)Treg lymphocyte in MSC group cell, P<0.05). Protection effect of MSCs was related with their ability to secrete TGF-β1and IP-10, (22.45±3.37pg/ml and 21.38±3.97pg/ml v.s 238.36±19.24pg/ml P<0.01)and(51.23±2.45pg/ml and 49.78±6.55pg/ml v.s 1512.92±149.72pg/ml,P<0.01).MSC also decreased levels of IFN-γ,TNF-αin the culture.The levels of TGF-β1 and IP-10 were detected in the culture of Human bone marrow mesenchymal stem cell with human fibroblasts of NSIP patient as bellow: TGF-β1 39.44±3.67 pg/ml in NSIP group ,41.23±7.27 pg/ml in HFB-NSIP group and 154.13±11.72 pg/ml in MSC-NSIP group; IP-10 32.78±4.51 pg/ml in NSIP group, 34.63±6.87 pg/ml in HFB-NSIP group and 67.43±8.91 pg/ml in MSC-NSIP group (P<0.01). Wherein MSC significantly decreased levels of IL-6,IL-8,MCP-1 in MSC-NSIP co-culture. Total protein of NSIP-fibroblast extracted 48 hr after the co-culture with MSC was detected with decreasedα-SMA. Human BMMSC increased 16HBE cell survival following challenge by the NKT cell prior to HFB or MSC(16HBE cell death percentage is 36.05±5.63%by NKT cell, 34.64±5.81% by HFB-NKT, and 25.87±4.12%by MSC-NKT(P<0.05). The flavonoids( 25ug/ml) addition can increase 16HBE cell survival further; cell death rate of NKT-16HBE group, NKT-flavonoid-16HBE group, MSC-NKT-16HBE group and MSC–NKT-flavonoid-16HBE group was 30.57±3.63%,25.84±3.01%,22.63±2.12%,15.21±1.39% respectively. TGF-β1 level was 45.72±5.18 pg/ml,33.21±2.94 pg/ml, 201.34±18.11 pg/ml and 157.73±14.61 pg/ml respectively and IP-10 level was 34.83±2.96pg/m , 35.14±4.19 pg/ml,568.30±47.64pg/ml and 571.43±48.66pg/ml respectively.Conclusion1,Human bone marrow mesenchymal stem cells can engraft in mice lung .2,MSCs have immunomodulatory ability for T lymphocyte subset to down-regulate high proportional NKT cell in PBMC, and to increase Treg cells with the decreased levels of IFN-γand TNF-α. Effects of MSC immunomodulation on T lymphocyte subset are contributing to induction of TGF-β1 and IP-10.3,Human BMMSC can restrainα-SMA high level expression in the pulmonary fibroblasts of NSIP patient and inhibit the increased levels in cytokines/chemokines of NSIP fibroblasts.4,Flavonoids extract from the Chinese herbs(Tian long ) has the synergistic effects with MSCs immunomodulation on protection of pulmonary cells from NKT-mediated damage and fibrosis.