CpG ODN对肺纤维化形成的作用及其可能机制
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摘要
特发性肺纤维化(idiopathic lung fibrosis)是一种原因不明的慢性进行性肺成纤维细胞增生性疾病,新近研究发现其发生发展与Th2免疫反应增强导致Th1/Th2失衡有关,暗示调节免疫反应可能具有预防或延缓肺纤维化的作用。CpG ODN(CpG oligodeoxynucleotide)是一种人工合成的含有非甲基化胞嘧啶和鸟嘌呤二核苷酸(CpG)基序的单链脱氧寡核苷酸,通过识别Toll样受体9(TLR9)发挥天然免疫激活作用,而且大量研究显示其具有促进Thl免疫应答的作用,从而有望用于调节因Th2免疫反应过强引起的Th1/Th2免疫反应失衡。为了探讨CpG ODN是否能干预肺纤维化的形成和发展,根据A、B、C型三类CpG ODN的生物学特点,以本实验室相关研究为基础,选择一种具有本实验室自行设计的C型CpG ODN(CpG002)作为研究对象,观察其对小鼠肺纤维化的干预作用,结果发现：这种C型CpG ODN不但不能干预小鼠肺纤维化的形成,还对肺纤维化的发展有促进作用。为了解释这种作用的可能原因,考虑到腹腔应用CpG ODN可能导致其直接作用于受损部分的成纤维细胞,我们制造了小鼠皮肤受损模型,并局部应用CpG ODN,观察受损伤口的愈合情况,结果发现CpG ODN能促进小鼠皮肤伤口的愈合,愈合部位主要是成纤维细胞的增生。进一步以人胚肺成纤维细胞作为靶细胞,体外研究发现CpG ODN能促进肺成纤维细胞增生和Ⅰ型胶原分泌,而且多种CpG ODN具有这种活性。本研究结果提示：CpG ODN作为免疫调节剂虽然可能调节免疫失衡状态,但用药途径可能需要慎重选择,尤其直接用于受损组织可能造成局部纤维化,但CpG002促进伤口愈合的作用有望被开发成为一种促进皮肤伤口愈合的新型药物。
Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN), as an innate immune system agonist through Toll-like receptor 9 (TLR9), have been extensively studied in recent years. Based on functional characteristics, CpG ODNs are divided into A, B and C classes. A-class CpG ODN induces plasmacytoid dendritic cells (pDC) to secret large amount of interferon-a (IFNa) and indirectly activates NK cells and facilitates the induction of cytotoxic T lymphocytes (CTLs). B-class CpG ODN stimulates the proliferation of B cells and activates them. C-class CpG ODN shares both of the properties of A-class and B-class CpG ODN.
It is now clear that synthetic oligodeoxynucleotides containing CpG dinucleotide (CpG ODN) are potent immunostimulatory agents, and activate B cells, natural killer cells, and antigen-presenting cells including monocytes, macrophages, and dendritic cells. Over the past 10 years there has been a dramatic increase in our understanding of the molecular and cellular effects of CpG ODN, and its effects in vivo in animal models and in clinical trials. In phase III trials, CpG ODN was proved to enhance the efficacy of HBsAg in the immunocopprimised people. In aminal models, CpGODN was shown to stimulate the innate immunue responses against various virus infections. Used in combination with. CpGODN synergized the antitumor effect of monoclonal antibodies specific to CD20 by enhancing antibody-dependent cellular cytotoxicity in patients with B cell Iymphoma and leukemia. Due to its modulating effect on the immune response form Th2 bias reaction to a Thl biased one, CpG ODN was evaluated in the clinical trial for the treatment of allergy and asthma.
Wound healing, as a normal biological process in the human body, is achieved through five precisely and highly programmed phases:hemostasis. inflammation, proliferation, contraction and remodeling. For a wound to heal successfully, all five phases must occur in the proper sequence and time frame. Many factors such as inflammation of the wound, diabetes mellitus, malignant tumor et al can interfere with one or more phases of this process, thus causing improper or impaired wound healing.
Idiopathic pulmonary fibrosis (IPF) is a disease of unknown aetiology with a poor prognosis. Mean survival time after biopsy-confirmed diagnosis is 3 to 5 years. Traditional therapy involves the use of corticosteroids as nonspecific anti-inflammatory agents. This treatment produces an objective response in only 10 to 20% of patients and has a minimal effect on the fatal course of IPF. Thus, the need for new therapeutic modalities is evident.
To date, only one paper (Zeng M et al,2008) has been published to describe the effect of CpG ODN on idiopathic interstitial pneumonia (ⅡP), a human diseases of fibrosis. The data in the paper showed that the activation of TLR9 contributed to experimental chronic tissue remodeling and the TLR9 expression was increased in biopsies from the patients with IIP compared with normal lung biopsies. Confocal microscopy studies showed that TLR9 activation by CpG-ODN increased the expression of alpha smooth muscle actin. These data suggested that TLR9 expression might drive the abnormal tissue healing response in severe forms of IIP and promote the progression of disease during the terminal phase of IPF.
In this study, we tried to study the putative effect of CpG 002 on the activation of fibroblasts, wound healing and the lung fibrosis, with an aim to develop one of a biologics for improving the wound healing and treating lung fibrosis.
1. Establishment of mouse lung fibrosis model.
To establish a mouse model of pulmonary fibrosis, bleomycin was administrated in mice by intraperitoneal injection or intratracheal instillation. ICR mice were divided into three groups randomly, which were group P (bleomycin was injected intraperitoneally 5 times at a dose of 40mg/kg), group I (bleomycin was instilled intratracheally at a dose of 5 mg/kg) and group C (injected with saline). The mice were sacrificed on day 14.28 or 40. The pulmonary pathological changes and respiratory system symptoms were observed.
Data showed that the survival rate of mice in group? was 100%, whereas 50% mice in group I succumbed. From day 1 to 10, showing significant difference between the two groups (P< 0.05). After the BLM injection, body weigt of the mice aradually lost:5 g in P group mice,3 g in I group mice. After the 6th day post-the injectin of BLM, the body weight of the model mice became equability. The body weight of I group mice increased after 14th day. The body weight difference between the P group mice is smaller than the other two groups.
We counted the coughing and touching nose times per 5 minutes every morning to describe the respiratory system symptom. After analyzing the data, we found that the respiratory system symptom was more serious in group P than that of group I. When the mice were sacrificed on day 40, we calculated the pulmonary coefficient of the mice. The pulmonary coefficient showed a significant increase in group P, compared with group I.
In group I, on day 28 after BLM injection, fibrosis was widely and stably formed around the bronchia and bronchioles, especially near the pulmonary hilar area. However, the same changes were found under the pleura or perivascular pulmonary tissues in group P. The pathological score of pulmonary fibrosis was different between those two groups.
To explore the optimum times of BLM administration, ICR mice were randomly divided into four groups:bleomycin was injected intraperitoneal three, four or five times at a dose of 40mg/kg,and in the group control, liquor natrii chloridi isotonicus was injected intraperitoneal at a dose of 200μl for five times. The mice were sacrificed on day 28 and 40. The pulmonary pathological changes and respiratory system symptoms were observed.Different dose of bleomycine induced different change of the mouse's body weight. The results demonstrated that the.The body weight of BLM treated mice remarkably decreased in the five time BLM injection group compared with that in the other groups. Additionally, we found the more times bleomycin administrated, the higher pulmonary coefficient was induced. In conclusion, intraperitoneal injection with bleomycin for 5 times could establish mouse model of pulmonary fibrosis was more convenient. The sites of pulmonary fibrosis in the mice wen different between the mice induced with bleomycin.by intraperitoneal injection and intratracheal instillation. Intraperitoneal injection of BLM for five times was better for pulmonary fibrosis establishment.
2. CpG 002 interfere in pulmonary fibrosis
Upon the in-vitro analysis, to assess whether CpG 002 could induce the interference on pulmonary fibrosis in vivo, pulmonary fibrosis mouse model was established with a convenient method. After intraperitoneal injection of CpG002, the body weight of mice lost almost 9g and poor general status was fjound in them. The hypoxia such as cyanosis could be observed obviously. Pulmonary coefficient of the mice received CpG 002 was two times more than those in the other two groups. The lung fibrosis in the mice treated with CpG 002 showed more serious than the other groups.
3. Effects of CpG 002 on the wound healing in mice.
Since CpG 002 could promote the fibroblast proliferation and expression of typeⅠcollagen, CpG 002 was selected to test its activity on wound healing after CpG 002 or PBS was dropped onto the wound of mouse. We found that the wound length in CpG 002 treated mice was shorter than that in PBS treated mice., indicating CpG 002 could promote the wound healing in mice. Additionally, the wound of CpG 002 treated mice healed faster than that in the PBS treated mice. To further investigate the effect of CpG 002 on wound healing, we cut a piece of skin around the wound in mice, to observe the pathological changes by HE staining. Data showed that.there were more ECM in the CpG 002 group than control. These results demonstrated that CpG 002 could promote the wound healing in mice.
4. Screening of CpG ODNs which can promote the proliferation of HELF cell and the expression of typeⅠcollagen in HELF cell.
To study the effect of CpG ODNs on fibroblasts, human embryo pulmonary fibroblast (HELF) cells were cultured with CpG ODNs (including A class, B class and C class) for 24 or 48 hours. Then the proliferation of HELF cell was detected by crystal violet. Data showed that CpG 001 and CpG 002 could promote the proliferation of HELF cells.
CpG 001 and CpG 002 were chosen to investigate their effects on the expression of typeⅠcollagen by RT-PCR. The result showed that CpG 002 could increase the.expression of typeⅠcollagen in the earlier period.
Taken together, we analyzed the effects of CpG ODNs (different types) on the proliferation of HELF cells. C type CpG ODNs, designated as CpG 001 and CpG 002, which could activate human immune cells. Data showed that CpG 002 promoted the wound healing in mice. When CpG 002 was injected to mouse model of pulmonary fibrosis, the pulmonary fibrosis became more serious,indicating. CpG002 could speed up the process of pulmonary fibrosis and aggravate the fibrosis extent.
Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN), as an innate immune system agonist through Toll-like receptor 9 (TLR9), have been extensively studied in recent years. Based on functional characteristics, CpG ODNs are divided into A, B and C classes. A-class CpG ODN induces plasmacytoid dendritic cells (pDC) to secret large amount of interferon-a (IFNa) and indirectly activates NK cells and facilitates the induction of cytotoxic T lymphocytes (CTLs). B-class CpG ODN stimulates the proliferation of B cells and activates them. C-class CpG ODN shares both of the properties of A-class and B-class CpG ODN.
It is now clear that synthetic oligodeoxynucleotides containing CpG dinucleotide (CpG ODN) are potent immunostimulatory agents, and activate B cells, natural killer cells, and antigen-presenting cells including monocytes, macrophages, and dendritic cells. Over the past 10 years there has been a dramatic increase in our understanding of the molecular and cellular effects of CpG ODN, and its effects in vivo in animal models and in clinical trials. In phase III trials, CpG ODN was proved to enhance the efficacy of HBsAg in the immunocopprimised people. In aminal models, CpGODN was shown to stimulate the innate immunue responses against various virus infections. Used in combination with. CpGODN synergized the antitumor effect of monoclonal antibodies specific to CD20 by enhancing antibody-dependent cellular cytotoxicity in patients with B cell Iymphoma and leukemia. Due to its modulating effect on the immune response form Th2 bias reaction to a Thl biased one, CpG ODN was evaluated in the clinical trial for the treatment of allergy and asthma.
Wound healing, as a normal biological process in the human body, is achieved through five precisely and highly programmed phases:hemostasis. inflammation, proliferation, contraction and remodeling. For a wound to heal successfully, all five phases must occur in the proper sequence and time frame. Many factors such as inflammation of the wound, diabetes mellitus, malignant tumor et al can interfere with one or more phases of this process, thus causing improper or impaired wound healing.
Idiopathic pulmonary fibrosis (IPF) is a disease of unknown aetiology with a poor prognosis. Mean survival time after biopsy-confirmed diagnosis is 3 to 5 years. Traditional therapy involves the use of corticosteroids as nonspecific anti-inflammatory agents. This treatment produces an objective response in only 10 to 20% of patients and has a minimal effect on the fatal course of IPF. Thus, the need for new therapeutic modalities is evident.
To date, only one paper (Zeng M et al,2008) has been published to describe the effect of CpG ODN on idiopathic interstitial pneumonia (ⅡP), a human diseases of fibrosis. The data in the paper showed that the activation of TLR9 contributed to experimental chronic tissue remodeling and the TLR9 expression was increased in biopsies from the patients with IIP compared with normal lung biopsies. Confocal microscopy studies showed that TLR9 activation by CpG-ODN increased the expression of alpha smooth muscle actin. These data suggested that TLR9 expression might drive the abnormal tissue healing response in severe forms of IIP and promote the progression of disease during the terminal phase of IPF.
In this study, we tried to study the putative effect of CpG 002 on the activation of fibroblasts, wound healing and the lung fibrosis, with an aim to develop one of a biologics for improving the wound healing and treating lung fibrosis.
1. Establishment of mouse lung fibrosis model.
To establish a mouse model of pulmonary fibrosis, bleomycin was administrated in mice by intraperitoneal injection or intratracheal instillation. ICR mice were divided into three groups randomly, which were group P (bleomycin was injected intraperitoneally 5 times at a dose of 40mg/kg), group I (bleomycin was instilled intratracheally at a dose of 5 mg/kg) and group C (injected with saline). The mice were sacrificed on day 14.28 or 40. The pulmonary pathological changes and respiratory system symptoms were observed.
Data showed that the survival rate of mice in group? was 100%, whereas 50% mice in group I succumbed. From day 1 to 10, showing significant difference between the two groups (P< 0.05). After the BLM injection, body weigt of the mice aradually lost:5 g in P group mice,3 g in I group mice. After the 6th day post-the injectin of BLM, the body weight of the model mice became equability. The body weight of I group mice increased after 14th day. The body weight difference between the P group mice is smaller than the other two groups.
We counted the coughing and touching nose times per 5 minutes every morning to describe the respiratory system symptom. After analyzing the data, we found that the respiratory system symptom was more serious in group P than that of group I. When the mice were sacrificed on day 40, we calculated the pulmonary coefficient of the mice. The pulmonary coefficient showed a significant increase in group P, compared with group I.
In group I, on day 28 after BLM injection, fibrosis was widely and stably formed around the bronchia and bronchioles, especially near the pulmonary hilar area. However, the same changes were found under the pleura or perivascular pulmonary tissues in group P. The pathological score of pulmonary fibrosis was different between those two groups.
To explore the optimum times of BLM administration, ICR mice were randomly divided into four groups:bleomycin was injected intraperitoneal three, four or five times at a dose of 40mg/kg,and in the group control, liquor natrii chloridi isotonicus was injected intraperitoneal at a dose of 200μl for five times. The mice were sacrificed on day 28 and 40. The pulmonary pathological changes and respiratory system symptoms were observed.Different dose of bleomycine induced different change of the mouse's body weight. The results demonstrated that the.The body weight of BLM treated mice remarkably decreased in the five time BLM injection group compared with that in the other groups. Additionally, we found the more times bleomycin administrated, the higher pulmonary coefficient was induced. In conclusion, intraperitoneal injection with bleomycin for 5 times could establish mouse model of pulmonary fibrosis was more convenient. The sites of pulmonary fibrosis in the mice wen different between the mice induced with bleomycin.by intraperitoneal injection and intratracheal instillation. Intraperitoneal injection of BLM for five times was better for pulmonary fibrosis establishment.
2. CpG 002 interfere in pulmonary fibrosis
Upon the in-vitro analysis, to assess whether CpG 002 could induce the interference on pulmonary fibrosis in vivo, pulmonary fibrosis mouse model was established with a convenient method. After intraperitoneal injection of CpG002, the body weight of mice lost almost 9g and poor general status was fjound in them. The hypoxia such as cyanosis could be observed obviously. Pulmonary coefficient of the mice received CpG 002 was two times more than those in the other two groups. The lung fibrosis in the mice treated with CpG 002 showed more serious than the other groups.
3. Effects of CpG 002 on the wound healing in mice.
Since CpG 002 could promote the fibroblast proliferation and expression of typeⅠcollagen, CpG 002 was selected to test its activity on wound healing after CpG 002 or PBS was dropped onto the wound of mouse. We found that the wound length in CpG 002 treated mice was shorter than that in PBS treated mice., indicating CpG 002 could promote the wound healing in mice. Additionally, the wound of CpG 002 treated mice healed faster than that in the PBS treated mice. To further investigate the effect of CpG 002 on wound healing, we cut a piece of skin around the wound in mice, to observe the pathological changes by HE staining. Data showed that.there were more ECM in the CpG 002 group than control. These results demonstrated that CpG 002 could promote the wound healing in mice.
4. Screening of CpG ODNs which can promote the proliferation of HELF cell and the expression of typeⅠcollagen in HELF cell.
To study the effect of CpG ODNs on fibroblasts, human embryo pulmonary fibroblast (HELF) cells were cultured with CpG ODNs (including A class, B class and C class) for 24 or 48 hours. Then the proliferation of HELF cell was detected by crystal violet. Data showed that CpG 001 and CpG 002 could promote the proliferation of HELF cells.
CpG 001 and CpG 002 were chosen to investigate their effects on the expression of typeⅠcollagen by RT-PCR. The result showed that CpG 002 could increase the.expression of typeⅠcollagen in the earlier period.
Taken together, we analyzed the effects of CpG ODNs (different types) on the proliferation of HELF cells. C type CpG ODNs, designated as CpG 001 and CpG 002, which could activate human immune cells. Data showed that CpG 002 promoted the wound healing in mice. When CpG 002 was injected to mouse model of pulmonary fibrosis, the pulmonary fibrosis became more serious,indicating. CpG002 could speed up the process of pulmonary fibrosis and aggravate the fibrosis extent.
引文
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