- 學位論文
以活體外模式探討可能協同因子於第二型豬環狀病毒致病機 制扮演之角色
The pathogenesis of PMWS by evaluating the effect and interaction of PCV2 and/or other possible co-factors, PRRSV or immune stimulator, on porcine immune cells in vitro
摘要
豬離乳後多系統消耗性症候群（postweaning multisystemic wasting syndrome﹔PMWS）為一遍及全球的新興豬病，已成為臺灣及許多養豬國家經濟上關切之疾病。迄今，由於此豬病之致病機轉仍未被完全了解，因此並無針對本病的有效控制方法。豬第二型環狀病毒（porcine circovirus type 2; PCV2）一般被認為為本病的主要致病原，然而PCV2存在於豬隻並不全然導致豬隻發生PMWS，因而PMWS之致病機制被認為可能需多因子之參與。PMWS豬隻常伴隨有細菌或豬生殖與呼吸道症候群病毒（porcine reproductive and respiratory syndrome virus; PRRSV）之共同感染，而這些共同感染的豬隻在臨床上呈現有加重病情及豬隻消耗性死亡之情形。本論文的主要目的為以活體外模式研究PCV2單獨或與其它可能的協同因子，如：PRRSV，以及含細菌內毒素酯多醣（lipopolysaccharide; LPS）及phorbol-12-myristate-13-acetate （PMA）的免疫活化因子，對豬肺臟巨噬細胞（alveolar macrophages; AMs）、脾臟巨噬細胞（splenic macrophages; SMs）、與共同培養的淋巴細胞之影響及交互作用。試驗發現PCV2雖易於在AMs及SMs細胞質內被偵測到，但並不會造成這些細胞之死亡（Chapters II, III, IV, V），因此推論其角色如同特洛伊木馬（Trojan horse），單核巨噬細胞可能為PCV2的重要保毒細胞，除可協助PCV2規避宿主的免疫監控外並協助PCV2在宿主體內之散播。單獨PCV2接種可減弱AMs的殺菌力並影響AMs及SMs多種細胞激素的表現（Chapters II, III, IV, V），此結果可能導致豬肺臟局部防禦力的受損，進而引發二次性及伺機性病原的感染。單獨PRRSV接種，雖僅造成AMs持續且低的感染率，但顯著造成細胞存活率降低及增加細胞凋亡率（Chapters III, IV），此結果顯示除了PRRSV的直接影響外，造成細胞死亡的現象可能與其他尚未闡明之非病毒性間接因素有關。另外，PRRSV亦可干擾AMs的吞噬及殺菌能力並誘導AMs產生IL-8及TNF-a（Chapters III, IV）﹔在SMs則造成IL-1b、IL-8及TNF-a表現量的顯著增加，及促進因Fas/FasL作用所導致之淋巴細胞死亡（Chapter V），因此推測PRRSV可能藉由促進淋巴細胞活化而導致活化誘導之淋巴細胞凋亡，這些現象可能與PRRS患豬常伴隨有免疫抑制、間質性肺炎及淋巴細胞流失現象之致病機制有關。在PCV2與PRRSV共同接種的AMs（Chapters III, IV），當先有或同時有PCV2的存在，PRRSV的感染率及其所造成的細胞死亡及凋亡率有減少的情形，此應與PCV2可誘導AMs產生大量IFN-a進而抑制PRRSV在AMs的感染及其所導致之細胞病變有關（Chapter III）。當先有PRRSV感染，雖然在接種PCV2後仍可誘導大量IFN-a的產生，然而一旦PRRSV已先行建立感染，後續PCV2所誘導之大量IFN-a並無法抑制PRRSV在AMs的感染及其所導致之細胞病變（Chapter VI），此PCV2及PRRSV因不同的感染順序所導致的不同交互現象，或許可解釋為何臨床上在不同豬場之間，甚至在同一豬場內，不同豬隻常發現不同的PMWS病徵程度及發生情形。相較於未接種或單獨接種病毒之AMs或SMs，同時有PCV2及PRRSV存在，不論接種的順序，在AMs皆可促進IL-8、TNF-a、IFN-a及FasL的大量表現（Chapter IV）﹔在SMs則可顯著增加IL-1b、IL-8、IL-10及TNF-a表現及促進Fas/FasL作用所導致之淋巴細胞死亡情形（Chapter V），這些結果顯示，PCV2及PRRSV共同感染單核巨噬細胞時，可協同誘導其產生大量炎症中介物，並促進Fas/FasL作用及加重活化誘導之淋巴細胞凋亡（activation-induced cell death; AICD），此等結果可能與在PCV2及PRRSV共同感染之PMWS豬隻所呈現嚴重且典型之間質性肺炎與淋巴細胞流失之致病機制相關。此外，以活體外方式證實細菌內毒素LPS，但非PMA，可誘導PCV2由AMs的細胞質內進入細胞核並進行複製（Chapter V），這結果顯示格蘭氏陰性菌可促進PCV2複製及增加患豬的PCV2病毒負載，因此推論格蘭氏陰性菌二次性感染，可能為造成豬場典型PMWS之一重要共同致病因子。 綜合以上結果，本論文證實單獨PCV2或單獨PRRSV的感染皆能削減豬肺臟防禦機制，並誘導產生炎症中介物，進而導致輕度肺炎的發生。雖然PCV2在PRRSV尚未建立感染前可藉由誘導AMs產生大量IFN-a而干擾PRRSV的複製，然而當PCV2與PRRSV同時感染時，不論先後順序，更能有效的促進AMs產生大量炎症中介物並導致細胞死亡及抗菌能力減弱，這結果可能與PCV2與PRRSV同時感染的PMWS豬隻在臨床上所呈現較嚴重並顯著的肺炎及易有二次性或伺機性病原感染之致病機制有關。另外，由於PMWS豬隻常見有淋巴細胞流失之病理變化，在本論文，我們也證實單獨PRRSV感染單核巨噬細胞，可促進Fas/FasL交互作用及細胞激素產生，並導致淋巴細胞發生AICD﹔雖然單獨PCV2感染單核巨噬細胞並無法導致淋巴細胞發生ACID，但同時有PCV2與PRRSV共同感染時，則PCV2能顯著的加重因PRRSV感染單核巨噬細胞所導致之淋巴細胞發生AICD的現象，此結果可用以解釋在PCV2與PRRSV同時感染的PMWS豬隻常呈現較嚴重並顯著的淋巴流失現象。由以上結果推測單獨PCV2或單獨PRRSV在肺臟所造成的影響為輕微且侷限的，但皆能削減豬肺臟防禦機制，進而導致易有二次性病原感染﹔若有二次性的細菌感染，尤其是格蘭氏陰性菌的感染，可釋放LPS以活化負載有PCV2之巨噬細胞進而促進PCV2的複製。另外，當同時有PCV2與PRRSV共同感染，PCV2可協同PRRSV在肺臟及淋巴組織誘導大量炎症中介物，並促進Fas/FasL作用及加重活化誘導之淋巴細胞發生AICD，由此推測雖然PCV2一般被認為為PMWS必要病原，然而其主要角色並非在於直接導致PMWS嚴重疾病之發生，而是作用在抑制豬隻免疫能力，進而造成二次性病原感染，及促進因PRRSV感染或免疫活化所導致之AICD，進而造成嚴重之淋巴流失現象，這些結果不但可用以解釋PMWS之致病機制及免疫抑制之現象，也支持了一般所認同之PMWS致病機制除了PCV2外需多因子參與之說法。
並列摘要
Postweaning multisystemic wasting syndrome (PMWS) has been recognized globally and become a major economic concern in many pig-producing countries, including Taiwan. To date, since the pathogenesis of the emerging syndrome is not yet completely understood, there is no effective method to control this disease. It is believed that porcine circovirus type 2 (PCV2) is the primary aetiological agent of PMWS, however, the existence of PCV2 does not definitively lead to PMWS in pigs. The pathogenesis of PMWS is considered multi-factorial. Bacteria or porcine reproductive and respiratory syndrome virus (PRRSV) is a common complicating factor of PMWS and is clinically important based on the observation of increases in postweaning mortality when co-infection is present. The objectives of the thesis were to evaluate the effect and interaction of PCV2 and/or other possible co-factors such as PRRSV and immune stimulator, including bacterial lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA), on porcine alveolar macrophages (AMs) and splenic macrophages (SMs) and the subsequent effect on co-cultured lymphocytes by in vitro inoculation and tried to correlate the findings to the pathogenesis of PMWS. It was demonstrated that PCV2 could be easily detected in the cytoplasm of AMs and SMs but no cell death occurred in these cells in vitro following the inoculation of PCV2 (Chapters II, III, IV, V). Thus, somewhat similar to the “Trojan horse” the monocyte/macrophage lineage cells (MLCs) may function as an important reservoir not only to help PCV2 to evade the immunosurveillance but also to assist PCV2 to spread. Moreover, PCV2 alone may impair AMs’ microbicidal capability and disturb various cytokine expressions in AMs and SMs (Chapters II, III, IV, V). The alterations may be potentially harmful to the local pulmonary defense system leading to secondary or opportunistic pathogen infection with subsequent development of mild to moderate pneumonia. When PRRSV was inoculated alone, a low but constant infectious rate accompanied with markedly reduced cell viability and significantly increased apoptosis was revealed in AMs (Chapters III, IV). The result suggests the presence of a remained clarified non-viral indirect mechanism in PRRSV-induced cytopathic effect on AMs. It was revealed that PRRSV also might impair phagocytosis and microbial killing ability of AMs and induce IL-8 and TNF-a production in AMs (Chapters III, IV). Significant up-regulation of Fas/FasL-mediated cell death and apoptosis of co-cultured lymphocytes and increase in IL-1b, IL-8, and TNF-a production were also demonstrated in PRRSV alone-inoculated SMs (Chapter V). It is suggested that PRRSV might trigger lymphocyte activation and subsequent apoptosis by the enhancement of Fas/FasL interaction. The finding may explain the pathogenesis of immunosuppression, interstitial pneumonia, and lymphoid depletion in PRRS-affected pigs. In PCV2 and PRRSV co-inoculated AMs (Chapters III, IV), it was observed that PCV2 inoculation occurring before or simultaneously with PRRSV inoculation could cause variable reductions in the PRRSV-associated infectious rate, cell death, and apoptosis when compared with PRRSV inoculation alone. The reduction in PRRSV infection in AMs was further proven being mediated by IFN-a generated by PCV2 inoculation (Chapter III). However, in AMs inoculated with PRRSV prior to the addition of PCV2, despite being inoculated with both PRRSV and PCV2 and also releasing a significant amount of IFN-a once PCV2 was added, PCV2 couldn’t reduce PRRSV infection in AMs once PRRSV has established its infection (Chapter IV). The dissimilar outcomes of the interaction of PCV2 and PRRSV in different inoculation orders in swine AMs may explain in part the variable morbidity of PMWS among pigs in the field, or even in the same pig herd. When PCV2 and PRRSV were co-inoculated, up-regulation of IL-8, TNF-a, and IFN-a, and significant increase in the level of FasL expression were seen in AMs as compared with mock- or those single virus-inoculated AMs, regardless of their inoculation orders (Chapter IV). There was a significant increase in Fas/FasL-mediated cell death of the co-cultured lymphocytes with the increase in IL-1b, IL-8, IL-10, and/or TNF-a expressions in PCV2 and PRRSV co-inoculated groups (Chapter V) when compared with mock- or those single virus-inoculated groups. The results imply that PCV2 and PRRSV may work synergistically on MLCs in the induction of overwhelming production of inflammatory mediators and intensification of Fas/FasL-mediated activation-induced cell death (AICD) in pigs with PCV2 and PRRSV dual infection. The observations may contribute, at least partially, to the more apparent interstitial pneumonia and lymphoid depletion seen in PMWS-affected pigs with PCV2 and PRRSV dual infection. Furthermore, it was found that the bacterial endotoxin, LPS, but not PMA, can induce translocation and transcription of PCV2 in AMs in vitro (Chapter VI). The result implies that co-infection with Gram negative bacteria as well as stimulation of macrophages may have the potential to promote PCV2 replication and increase PCV2 load in pigs. Thus, it is speculated that secondary Gram negative bacterial infection may induce PCV2 replication in PCV2-infected pigs and also be one of the important co-factors contributing to the full development of PMWS in the field. In conclusion, we have demonstrated that PCV2 alone and PRRSV alone have the ability to impair pulmonary defense mechanism and induce inflammatory mediators in the lungs resulting in the development of pneumonia. Although PCV2 can interfere with PRRSV infection by PCV2-induced IFN-a before PRRSV has established its infection, dual infection with PCV2 and PRRSV, regardless of their inoculation orders, will drastically enhance the production of inflammatory mediators and cause damage to AMs, including cell viability and anti-microbial activity. The alterations certainly will result in more apparent pneumonic lesion and more frequent secondary or opportunistic pathogen infection in PMWS-affected pigs with PCV2 and PRRSV dual-infection. Lymphoid depletion is a common pathological change seen in PMWS-affected pigs. Our results indicate that PRRSV alone may promote Fas/FasL and/or cytokine-mediated AICD in lymphocytes via infected MLCs. Although PCV2 itself was unable to induce Fas/FasL and/or cytokine-mediated AICD in lymphocytes, it could intensify the PRRSV-induced AICD in lymphocytes. This may explain, at least partially, why lymphoid depletion is more apparent in PMWS-affected pigs with dual infection of PCV2 and PRRSV. Based on the findings, it is assumed that the effect of PCV2 alone or PRRSV alone seems to be mild and restricted; however, both viruses can impair the pulmonary defense mechanism leading to higher susceptible to secondary infection. The secondary bacterial infection, especially Gram negative bacteria, may subsequently induce the replication of PCV2 via stimulation and activation of macrophages by the release of LPS. When PCV2 co-infected with PRRSV, PCV2 can work synergistically to intensify PRRSV-induced AICD in lymphoid organs and to significantly enhance cytokine production in lungs and lymphoid organs. It is suggested that although PCV2 is essential for the development of PMWS, it mainly acts as an immunosuppressor resulting in secondary infection and an enhancer in intensifying PRRSV- or immune activation-induced AICD rather than directly leads to the disease in pigs. The result may explain, at least partially, the pathogenesis and immunosuppression noted in PMWS-affected pigs. It also supports the general point of view that the mechanism for the development of full-spectrum PMWS is multi-factorial.