以小鼠模式探討年齡和腸道菌因子以及肝臟巨噬細胞對B型肝炎病毒容忍性之影響

The role of liver macrophages in age and gut microbiota dependent HBV-persistent C3H/HeN mice

鄭吉宏(Chi-Hung Cheng)

指導教授 ： 陳培哲

國立臺灣大學/醫學院/微生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02932

摘要

慢性B型肝炎在全球約有3億的慢性帶原者，患者發生感染時的年齡與慢性B肝型肝炎的發展有很大的關聯性，其中有高於90%的新生兒會發展成慢性帶原者，而5歲以後成為帶原者的機率只有5~7%。在過去的研究中，我們發現利用高壓尾靜脈注射使C3H/HeN小鼠模擬B型肝炎病毒感染，可以依據小鼠年齡產生兩種不同的免疫反應。6周歲的小鼠對B型肝炎病毒產生免疫容忍，無法產生有效的免疫反應清除病毒，12周歲小鼠則可在6周內清除病毒，然而12周歲小鼠清除B型肝炎病毒的能力會因為使用抗生素去除腸道菌而降低。觀察小鼠的腸道菌發現，腸道菌的菌相會隨著年齡變化，到12周歲時會達到穩定。許多研究顯示，肝臟的免疫環境會根據外來抗原產生變化，特別是從腸道來的細菌代謝產物會使肝臟成為免疫容忍狀態，降低外來抗原引起的免疫反應，因此我們推論在C3H/HeN小鼠模型中，6周歲小鼠因為當時的腸道菌抗原造成較強的免疫容忍使其無法有效清除B型肝炎病毒，而12周歲小鼠的腸道菌因為組成和6周歲小鼠不同，其產生的抗原組成也有差異，進而造成肝臟的免疫環境足以產生有效的免疫反應來清除病毒。庫佛氏細胞在許多的研究中被認為是造成肝臟免疫容忍的重要免疫細胞，它會過濾外來抗原並產生免疫調控因子來調節免疫反應。我們將6周歲C3H/HeN小鼠的庫佛氏細胞剃除後，發現小鼠有效地清除病毒。接著我們觀察6周歲和12周歲小鼠在接觸B型肝炎後肝臟巨噬細胞族群的變化情形，發現在12周歲小鼠以高壓尾靜脈注射打入B型肝炎病毒質體後第二天，庫佛氏細胞會大量消失，而6周歲小鼠仍保有許多庫佛氏細胞。6周歲C3H/HeJ小鼠為4號類鐸受體突變並且失去功能的品系，我們推測其可能具有較差的肝臟免疫容忍，並證實其的有快速清除HBV的能力。同時我們也觀察到6周歲C3H/HeJ小鼠在高壓尾靜脈注射B型肝炎病毒質體後的18小時以及第2天，庫佛氏細胞也大量消失。由此結果推論，小鼠會藉由剔除庫佛氏細胞來破壞肝臟免疫容忍，進而使肝臟的環境易於產生較強的免疫反應來對抗病毒。最後我們發現腸道菌剃除的12周歲C3H/HeN小鼠，其庫佛氏細胞在高壓尾靜脈注射B型肝炎病毒質體後的第三天大量消失，此時間點在6周和12周歲C3H/HeN小鼠以及6周歲C3H/HeJ小鼠，庫佛氏細胞族群已經回復大多數。因此我們推論高壓尾靜脈注射B型肝炎病毒質體後的第三天，新增的庫佛氏細胞可能和原庫佛氏細胞具有不同的功能，進而幫助清除B型肝炎病毒。
綜合以上結果，我們發現庫佛氏細胞的消失對於小鼠清除B型肝炎病毒可能為必要的過程。B型肝炎病毒透過何種抗原引起庫佛氏細胞的消失，以及庫佛氏細胞是如何接收到B型肝炎病毒的訊息是未來重要的研究方向。

關鍵字

B型肝炎 ； 巨噬細胞 ； 庫佛氏細胞

並列摘要

Approximately 300 million people are HBV carrier. Mechanisms leading to hepatitis B virus (HBV) persistence is still unclear. One of important factor is the age of HBV infection. More than 90% of newborns or infants who acquire HBV infection become HBV carriers. The risk is reduced to 25~30% in 0~5 years old child and 5~7% after 5 years old. In our previous studies, we established age-related HBV clearance mice model on C3H/HeN mice and indicated that gut microbiota might be an important factor in HBV clearance. It is known that the composition of gut microbiota can change with age and become stable in adult C3H/HeN mice. We suppose that foreign antigens especially derived from gut microbiota can shape liver immune environment into immune tolerance to HBV in young C3H/HeN mice. Whereas, Gut microbiota in adult C3H/HeN provides difference signals from young C3H/HeN mice to influence liver environment and contribute to interrupt liver tolerance at the exposure to HBV. Kupffer cell (KC) is known for its immune regulatory effect in liver tolerance. Therefore we depleted KCs of young and adult C3H/HeN mice before transfection of HBV and found that the depletion of KCs can promote HBV clearance in young C3H/HeN mice. It indicates that the interrupt of liver tolerance may be an important pathway to clear HBV. Next, we investigated the population of liver macrophages in C3H/HeN mice. The population of liver macrophages can be divided into two subset, infiltrating macrophages and KCs, by CD11b and F4/80. We found that KCs dramatically decreased and massive macrophage infiltration after hydrodynamic injection (HDI) of pAAV/HBV1.2 in adult C3H/HeN mice. The change of liver macrophage population was completely restored at day 9 after HDI. However, the reduction of KC in young C3H/HeN mice was not notable than adult C3H/HeN mice and mainly caused by the effect of HDI. These results support our hypothesis that adult C3H/HeN mice have the ability to interrupt liver tolerance by reduction of KC at the exposure to HBV. We have the similar result on C3H/HeJ mice which are TLR4-deficient strand. Young C3H/HeJ mice have the ability to clear HBV that may be due to the weak liver tolerance causing by the loss of TLR4 signaling. As our previous study, the ability of HBV clearance can be partially disrupted by antibiotic treatment in adult C3H/HeN mice. We further investigated the population of liver macrophages in antibiotic-treated adult C3H/HeN mice and found that the population of KCs was significantly reduced compared with young and adult C3H/HeN mice which didn’t received antibiotic treatment. It indicates that the function of KCs at day 3 after the exposure of HBV are opposite to the original KCs before HDI and may promote HBV clearance.
In conclusion, our results suggest that the reduction of KCs is important to promote HBV clearance. It may be due to the interruption of liver tolerance that make liver environment trend to induce stronger innate immune response to against HBV. In the future, to find out what antigens of HBV promote the reduction of KCs is a top priority. That will provide a clue to further investigate the mechanism of age-related immune response to HBV.

並列關鍵字

HBV ； macrophage ； kupffer cell

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