CpG-ODN可增強經培養之鼻腔表皮細胞的細胞激素分泌 -模擬慢性鼻竇炎急性發作的模式

Abstract

研究背景： 慢性鼻竇炎定義為鼻腔及鄰近鼻竇黏膜的感染或發炎。臨床上，慢性鼻竇炎患者可能會出現化膿性鼻涕、鼻塞、面部腫脹和嗅覺喪失等症狀。抗生素、以生理食鹽水沖洗、類固醇和抗白三烯素(anti-leukotrienes)等藥物，可以減輕症狀。不過，頻繁地急性發作及症狀復發常發生在這些病人身上，並造成結構和功能上不可逆的一些改變。目前慢性鼻竇炎急性發作的病理生理學尚不清楚，使得處理上尤具挑戰性。CpG-ODN，是一串連續序列，包含數個未甲基化的胞嘧啶(cytosine)和鳥嘌呤核苷(guanosine)，在大部分細菌和病毒的基因體較常見。CpG-ODN被認為是細菌生物膜(biofilm)結構的一個主要元件，會造成慢性、持久性的感染。臨床上，不易治療的、常復發的慢性鼻竇炎與生物膜的形成有關；生物膜的持續存在，亦可能導致慢性鼻竇炎手術的效果不佳。CpG-ODN在慢性鼻竇炎病人體內的分子生物學意義仍不清楚，我們希望研究CpG-ODN的免疫調節作用，並釐清慢性鼻竇炎病人體內生物膜所含有的CpG-ODN之分子生物學意義。 研究假說： 1.CpG-ODN的存在可能使鼻腔及鼻竇黏膜保持低量細胞激素的產生，並在慢性鼻竇炎的持久性發炎中扮演重要角色。 2.在慢性鼻竇炎的急性發作時期，CpG–ODN會對鼻腔表皮細胞的免疫發炎反應，有加乘的作用，因而增強鼻內細胞激素的釋放，對慢性鼻竇炎急性發作的嚴重程度及後續的併發症，具有關鍵的影響。 研究材料與方法：共二十名診斷為慢性鼻竇炎經手術的病人納入研究。手術前至少一個月，他們都未接受任何治療。本研究排除具過敏體質、支氣管哮喘、過敏性鼻炎的病人。病人經手術後，收集其鼻息肉的上皮細胞，並以氣液介面(air-liquid interface)進行培養，以模擬實際鼻腔黏膜表皮的生理狀況。在培養二十一天以後，分別以CpG-ODN, interleukin-1β(IL-1β) 和tumor necrosis factor-α(TNF-α)刺激鼻上皮細胞，或是同時加入這些製劑。經24 小時刺激後，收集培養基的細胞液，並立即儲存在-80℃。其後，以酵素免疫分析法(ELISA)進行IL- 6、IL- 8 及Monocyte chemoattractant protein-1 ( MCP-1)的測量，並以RT-PCR的方法驗證。 結果： 1.在此培養系統中，CpG-ODN只造成MCP-1的增加；而在文獻上，MCP-1被認為和許多慢性細菌性感染相關。所以CpG-ODN的存在可能使鼻腔及鼻竇粘膜保持某些低量細胞激素的產生，並在慢性鼻竇炎的持久性發炎中扮演重要角色。 2.以外加的IL-1β和TNF-α去刺激鼻上皮細胞，會造成IL-6、IL-8 及 MCP-1等細胞激素的大量釋放；模擬鼻腔黏膜在一個急性發作的狀況。其中，尤其以外加的IL-1β，相對於TNF-α，更能造成IL-6、IL-8 及 MCP-1等細胞激素的大量釋放。 3.在IL-1β和TNF-α的存在下，CpG-ODN能更加乘IL-6及 MCP-1等細胞激素釋放的反應，而這樣的一個作用，可以模擬慢性鼻竇炎病人在急性發作時期，CpG-ODN，會加乘這些細胞激素大量釋放的效果，而對疾病的嚴重程度及後續的併發症，具有關鍵的影響。 結論： CpG-ODN和鼻竇炎的慢性化相關。清除具有CpG-ODN的生物膜，對於慢性鼻竇炎的治療是絕對有益處的。在模擬慢性鼻竇炎急性發作時期，CpG-ODN會增加鼻內細胞激素的釋放，而使症狀加劇或造成併發症；因此治療時，針對CpG-ODN須盡可能移除這些有害的發炎物質；而在已發炎的狀況下，可以考慮使用短效的免疫抑制劑，以停止這細胞激素大量釋放的惡性循環。

Background : Chronic rhinosinusitis (CRS) is defined as inflammation and infection involving the nasal mucosa and the adjacent sinus cavities. Clinically, the patients with CRS may suffer from symptoms of mucopurulent discharge, nasal obstruction, facial pain, and hyposmia. Antibiotics, saline irrigations, steroids, and anti-leukotrienes may be given for symptoms relief. However, episodes of recurrence or acute exacerbation occurred to these patients, and caused some irreversible changes in airway structure and function. The pathophysiology of frequent acute exacerbation in these patients is still unclear, making treatments particularly challenging. CpG-ODN refers to sequences that include an unmethylated cytosine and guanosine ,which are relatively common in the genomes of most bacteria and DNA viruses. CpG-ODN had been implicated as a major structural component of bacterial biofilm, and contributed to the persistence of chronic infection. Clinically, recalcitrant CRS is associated with biofilm formation, and biofilm may cause the unfavorable outcomes of surgery for CRS. However, the molecular significance of CpG-ODN in CRS is still unclear. We aim to investigate the immune-modulation effects of CpG-ODN ,and elucidate the role of biofilm in CRS. Hypothesis : 1.The existence of CpG-ODN may maintain a low profile of cytokines production in the sinus mucosa, and play a role in the persistence of inflammation in chronic rhinosinusitis. 2.During CRS with acute exacerbation, CpG–ODN synergistically up-regulate the immuno-reactions of pro-inflammatory mediators, augments intranasal cytokines release, and contribute to the complications and severity of CRS with acute exacerbation. Materials and Methods: Twenty patients who are diagnosed as CRS and underwent surgery were included prospectively. None of them receives any treatment at least one month before operation. The one who has atopy, asthma, or allergic rhinitis was previously excluded. The nasal epithelial cells harvested from nasal polyps were collected for ALI (air-liquid interface) culture. At the end of culture (Day 20 after confluence), CpG-ODN , IL-1β , and TNF-α were added in single regimen or in combination to stimulate the nasal epithelial cells. After 24 hours-treatment, the culture medium of each well was collected and immediately stored at -80℃for later IL-6,IL-8 and MCP-1 measurement using ELISA. Statistical analysis : The paired samples test was used for evaluation of differences between experiments and controls. ANOVA was used when there is a multiple sample comparison. P values less than 0.05 was considered significant. Results : 1.A model mimicking the in vivo microenvironment of stable CRS was created. 2.Intrinsic IL-1β and TNF-α levels were undetectable in this model, and IL-6 , IL-8 ,MCP-1 levels were detectable during 21-days culture period, with a trend toward homogeneity . 3.Extrinsic IL-1β and TNF-α were used to stimulate the cultured nasal epithelial cells, to initiate a condition resembles CRS with acute exacerbation. IL-1βinduced IL-6,IL-8 and MCP-1 expressions in a dose-dependent fashion, while TNF-α only induced these cytokines release insignificantly. 4. CpG–ODN induced MCP-1 expression with a dose-dependent fashion, but not in IL-6 and IL-8. 5. CpG-ODN synergistically potentiate IL-6,IL-8, and MCP-1 release in the presence of IL-1 β and TNF-α. Conclusion : CpG-ODN is associated with the chronicity of CRS. Elimination of such substances in the management of CRS is mandatory. CpG-ODN augments intranasal cytokines release in patients with acute exacerbated sinusitis. Strategies such as local debridement or short-term immuno-suppressants are needed to stop the vicious circle.