對蝦素經由趨化顆粒球而調控發炎反應

Abstract

對蝦素(penaeidin)是蝦類的一種抗微生物肽，分子量介於5.5~6.6 kDa，等電點(pI)為9.1~9.8，由N端脯氨酸豐富區域(proline-rich domain, PRD)與C端半胱氨酸豐富區域(cysteine-rich domain, CRD)所構成，表現並儲存於顆粒型血球中。對蝦素可抑制格蘭氏陽性細菌及絲狀真菌生長，但抑制格蘭氏陰性細菌及酵母菌的效果較差甚或沒有抗菌性。草蝦胚胎發育至1-4個細胞期即表現對蝦素，成蝦在蛻殼前 (premoult)對蝦素表現量最低，蛻殼後對蝦素表現量逐漸上升，蛻殼間(intermoult)表現量最高；又對蝦素可與幾丁質結合，顯示對蝦素與蛻殼有關，是蝦子成長的指標之ㄧ。 本研究分兩部分：首先利用序列專一性小段干擾RNA (short-interfering RNA, siRNA)剔減(knockdown)初代培養血球細胞的對蝦素，証明對蝦素是一種細胞激素(cytokine)；進一步剔減活蝦體內對蝦素，証明對蝦素具促發炎細胞激素(pro-inflammatory cytokine)特性，經自分泌(autocrine)方式促進發炎反應。 離體研究中，以序列專一性、小段干擾RNA剔減初代培養血球的對蝦素後，發現血球吸附的相對數比對照組少20 %。在剔減對蝦素的血球培養液中補充重組對蝦素或化學合成PRD，都可恢復吸附血球的相對數目，但CRD則無影響。由Q-PCR檢測剔減對蝦素後10個與細胞吸附相關的基因表現，發現其中3個基因的表現顯著改變。整合素-β (integrin-β)與膠原蛋白(collagen)的表現分別下降91%與64%，而膠原蛋白酶(collagenase) 的表現則增加395%；添加化學合成對蝦素於被剔減血球中，上述3個基因表現轉趨常態。添加RGDS (同膠原蛋白分子內一小段結合區塊序列，可作為配體與整合素結合)於血球培養液，發現血球減少吸附的程度與對蝦素剔減後血球減少吸附的程度相若；但添加RGES (RGDS的無活性類似物)則無此效應，故推論對蝦素調控整合素的表現進而影響血球吸附。上述結果顯示對蝦素或對蝦素分子中的PRD具細胞激素性質，可促進血球吸附，CRD則否；對蝦素可調控減少膠原蛋白酶表現以保存膠原蛋白，再促進膠原蛋白和整合素表現、增加彼此結合，進而促進血球吸附。 由於整合素是細胞遷移的重要分子之一，故推測對蝦素可能參與發炎反應，本研究第二部分從活體切創引起的發炎反應中，探討對蝦素在其中的功能。 首先建立活體剔減對蝦素之技術：蝦子注射對蝦素序列專一性siRNA後再切創，Q-PCR和ELISA皆顯示剔減技術成功。即相較於未切創蝦(UT)，Q-PCR測量到切創後2小時傷口組織對蝦素表現量比UT顯著增加到1173%，剔減後則顯著減為446%；週邊循環血球的對蝦素表現量比UT顯著降為76%，剔減後再顯著降為32%；用ELISA測對蝦素濃度亦呈現相同變化。利用H&E染色並以影像分析計算剔減蝦與未剔減蝦的傷口組織單位面積內之總血球數目，結果顯示剔減蝦之傷口組織內總血球數顯著較少，添加重組對蝦素或化學合成PRD於傷口可恢復剔減所造成總血球數減少的現象，但CRD無此效果；顯示對蝦素或其PRD具趨化血球、集中傷口組織的活性。由於Q-PCR測得的整合素表現量隨對蝦素表現量而同步增減，因此推測血球可能藉由整合素遷移至傷口組織。利用兔抗PRD抗體可專一性的辨識對蝦素表現顆粒球；分別進行流式細胞測量(Flow Cytometry)以及免疫組織化學染色(immunohistochemistry, IHC)，發現雖然切創蝦和未切創蝦其單位體積血淋巴液中的總血球數目無顯著改變，每顆血球中平均對蝦素的表現量也未改變，但切創蝦較之未切創蝦血淋巴液中表現對蝦素的顆粒球佔全顆粒球的比例卻減少(86%減為62%)，呼應前述Q-PCR測量週邊循環血球中所顯示的切創蝦相較於未切創蝦對蝦素表現量顯著減少(76%)。上述兩項分析結果一致都指向因切創使血淋巴液中對蝦素表現顆粒球數目顯著減少；已知血淋巴液中顆粒球約佔全血球8~14%，但免疫組織化學染色卻顯示切創蝦傷口組織中表現對蝦素的顆粒球佔全血球的80%，因而推測血淋巴液中消失的對蝦素表現顆粒球出現在傷口組織。剔減後切創蝦傷口組織中對蝦素表現顆粒球顯著減少，添加化學合成PRD於傷口處可恢復剔減所造成對蝦素表現顆粒球減少的現象，並且具有劑量依存(dose dependent)的特性，故推論對蝦素或其PRD可作為促發炎反應細胞激素，並以自分泌的方式趨化對蝦素表現顆粒球至傷口組織；對蝦素與整合素的表現在活體研究中亦呈現一致性，故推測對蝦素表現顆粒球依靠整合素進行細胞遷移往創傷組織移動。 總結，對蝦素已知是一種抗微生物肽和細胞激素，對蝦素分子或其PRD尚具有促發炎反應細胞激素的特性，以自分泌作用趨化更多表現對蝦素的顆粒球，移往傷口部位，有利於創傷組織修補。

Penaeidins are shrimp-specific anti-microbial peptides (AMPs) with molecular weight of 5.6~6.6 and pI of 9.1~9.8. Two domains could be distinguished from the amino acids composition of penaeidins and these are the N-terminal proline-rich domain (PRD) and the C-terminal cysteine-rich domain (CRD). Granulocytes are the major cell type that express and store penaeidins. The major targets of penaeidins are gram-positive bacteria and filamentous fungi; gram-negative bacteria and yeast are less targeted. Expression of tiger shrimp (Penaeus monodon) penaeidin fluctuates during developmental and moulting stages. Penaeidin could be detected as early as the 1~4 cells embryo. In adult, penaeidin reaches the highest level during the inter-molting stage, drops to the lowest level in the premoult stage and then gradually increases until the next moulting cycle. Moreover, direct binding of penaeidin and chitin indicates the correlation between penaeidin and shrimp growth. There are two parts in this research. First, applying siRNA (short-interfering RNA) to knockdown of penaeidin in the hemocytic primary culture of tiger shrimp and to prove that penaeidin could also function as a cytokine. Second, to knockdown penaeidin in vivo to prove its pro-inflammation cytokine function and autocrine feature. Shrimp hemocytes in primary culture were transfected with penaeidin-specific siRNA and a concomitant 20% reduction in adhesive hemocytes compared with mock-transfected cells was observed. Addition of biosynthesized or chemically synthesized penaeidin or PRD to the culture medium of penaeidin knockdown hemocytes led to a full recovery in the number of adhesive hemocytes. The effect of penaeidin knockdown on the expression of 10 tiger shrimp cell adhesion-associated molecules were examined using real-time Q-PCR. Results demonstrated 91% and 64% decreases in the expression of integrin-β and collagen, respectively, and 396% increase in the expression of collagenase. The addition of chemically synthesized penaeidin after penaeidin knockddown hemocytes normalized the expression of these three genes. The addition of the integrin-β ligand competitor RGDS to mock-transfected hemocytes decreased the number of adhesive hemocytes similar to penaeidin knockdown. In conclusion, PRD of penaeidin possesses an integrin-β-mediated cytokine feature that promotes shrimp granulocyte and semi-granulocyte adhesion but not CRD. Penaeidin regulates hemocyte adhesion through the regulation of CAM (integrin-β) and ECMs (collagen and collagenase) expression. Not only integrin is a key molecule in cell migration, we propose that penaeidin functions in wound-induced inflammatory responses of shrimps in the second part of this research. We established the in vivo knockdown technique of penaeidin first. Penaeidin was successfully knockdown after injection of penaeidin-specific siRNA by Q-PCR and ELISA examination. Comparing with the untreated shrimp (UT), penaeidin in the wound tissue increased to 1173% at 2 h post-wound but only 446% after penaeidin knockdown and wound. In the peripheral hemolymph penaeidin decreased to 76% at 2 h post-wound and to 32% after penaeidin knockdown and wound. ELISA presented parallel results with Q-PCR in penaeidin expression level after treatments. The wound tissue sections in H&E stain were compared in penaeidin-normal and penaeidin-knockdown shrimps. Only the shrimps at normal penaeidin expression level presented the concentration of hemocytes phenomenon in the wound tissue at 2 h post-wound. This phenomenon was recovered in the penaeidin knockdown shrimps by adding recombinant penaeidin or PRD to the wound tissue, but not with CRD. Penaeidin-positive granulocytes could be specifically identified through the rabbit anti-PRD antibody and the composition of hemocytes was tested in peripheral hemolymph by flow cytometry and wound tissue by immunohistochemistry. Total hemocytes count did not present significant variation before or after wounding, but the number of penaeidin-positive granulocytes decreased in the peripheral hemolymph post-wound (from 86% of total granulocytes decreasing to 62%). Furthermore, the average expression of penaeidin in each granulocyte did not change. In the previous study, 8~14% of total hemocytes belongs to granulocyte and semi-granulocytes. The hemocytes that concentrated in the wound tissue were 80% penaeidin-positive granulocytes. We suggest that the decrease in penaeidin-positive granulocytes in the peripheral hemolymph was the reason for cell migration toward the wound tissue. Penaeidin-positive granulocytes in the wound tissue decreased after knockdown of penaeidin, but could be recovered by adding PRD. Penaeidin was also found to be simultaneously expressed with integrin in vivo. We suggest that penaeidin-positive granulocytes migrated to the wound tissue through integrin-dependent cell migration. This indicates that penaeidin possesses autocrine activity through integrin-dependent cell migration. In conclusion, penaeidin is an AMP with cytokine function. PRD is the cytokine functional domain of penaeidin, which acts as a pro-inflammatory cytokine to attract penaeidin-positive granulocytes to the wound tissue thus functioning as autocrine to repair the damaged tissue.