Tiger shrimp (Penaeus monodon) penaeidin modulates the inflammatory response by chemotaxis of granulocytes

• Main Authors: Ching-Yu Li, 李靖宇
• Other Authors: Yen-Ling Song
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/84080639625186021484

博士 === 臺灣大學 === 動物學研究所 === 99 === 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.