臺灣鋏蠓(Forcipomyia taiwana)俗稱小黑蚊，是分布於福建、廣東、臺灣地區特有的鋏蠓科吸血鋏蠓，被叮咬後奇癢無比，異常嚴重的患部過敏及嚴重的發炎反應。儘管目前有藥物可以緩解搔癢但能沒有藥物可以抑制膿包或丘疹。而小黑蚊在叮咬的患部造成其嚴重發炎反應的相關分子及機制仍為未知。我們利用小黑蚊蛋白刺激角質細胞株、巨噬細胞以及人類周邊血液血球，並收取其細胞上清液檢測細胞發炎激素的表現量。結果顯示介白素-6、介白素-8、腫瘤壞死因子-α的表現量隨著時間而上升。接著為了進一步了解皮膚細胞和巨噬細胞在被小黑蚊蛋白刺激之後的交互作用，我們以小黑蚊蛋白刺激角質細胞將細胞培養液並轉移至巨噬細胞的細胞培養液中，並偵測細胞發炎激素的表現量。結果顯示在巨噬細胞的細胞培養液中，介白素-6及介白素-8因轉移了細胞培養液之後表現量都有顯著上升。先前有文獻提及，人類周邊血液在受到蚊子的唾液的刺激之後會改變細胞發炎激素的表現量，因此我們萃取出小黑蚊頭部和小黑蚊軀幹之蛋白質後，再分別去刺激角質細胞株、巨噬細胞株、人類周邊血液血球以及人類周邊血液單核球分化後之巨噬細胞。實驗結果表明角質細胞株在受到小黑蚊頭部或軀幹的蛋白刺激下會大量表現介白素-6和介白素-8，而由巨噬細胞株及人類周邊血液單核球分化後之巨噬細胞則僅會對由小黑蚊頭部蛋白刺激而表現大量的介白素-8和腫瘤壞死因子-α。先前的文獻也提及目前可能可以透過抑制類鐸受體4達到對於昆蟲叮咬的預防或是治療，因此我們也在利用小黑蚊頭部蛋白刺激角質細胞或是巨噬細胞株給予細胞類鐸受體4的抑制劑，再檢測細胞發炎激素的表現量。實驗結果顯示無論是在蛋白刺激前或後給予細胞抑制劑皆無法達到降低細胞發炎的現象。為了瞭解小黑蚊頭部蛋白引起的發炎機制是否可能透過其他路徑，我們以小黑蚊頭部蛋白刺激人類周邊血液單核球分化後之巨噬細胞後，抽取細胞之核醣核酸並分析其基因表現。基因分析的結果顯示巨噬細胞在受小黑蚊頭部蛋白刺激後可能透過核苷酸結合及寡聚化結構域樣受體引起細胞發炎機制。總結以上，人類在受到小黑蚊叮咬而引起相關症狀的致炎因子主要是來自小黑蚊頭部的蛋白質，並且此反應可能是透過寡聚化結構域樣受體所調控。

Forcipomyia taiwana (F. taiwana) is a native blood-sucking insect in Taiwan, which can induce allergic dermatitis in affected subjects, such as local itching, swelling, and blisters at the bitten site. Although there are drugs that can soothe the itching, they cannot alleviate the inflammatory response related to swelling and pruritus feelings. The protein factors that trigger the production of inflammatory cytokines on the skin during the bite of F. taiwana were unknown. Inflammatory cytokines, IL-6, IL-8, and TNF-α in the supernatants of human keratinocyte cells (HaCaT cells), peripheral blood mononuclear cells (PBMC), and macrophage-derived from THP-1 cells (pTHP-1) after stimulating with soluble proteins from the extract of F. taiwana (FT proteins) were increased as time-dependent. To investigate whether an enhancement effect may occur during the interactions between skin cells and immune cells, supernatants collected in HaCaT cells after stimulating with FT proteins were transferred to pTHP-1 cells, cells that have been induced to be macrophage-like. Results showed that the levels of IL-8 and IL-6 in pTHP-1 cells were increased after co-cultured with the supernatants of skin cells pre-treated with whole FT Proteins. Previous studies have shown that proteins in mosquito saliva had profound effects on the human immune system. HaCaT cells, pTHP-1 cells, PBMC, and monocyte-driven macrophages (MDM) were stimulated with FT proteins from the head and the body of F. taiwana, respectively. Both IL-8 and IL-6 were significantly increased in HaCaT cells after stimulating with both FT proteins from the head and the body. As for pTHP-1 cells and MDM, IL-8 and TNF-α increased after stimulating with FT proteins from the head only. In previous studies, TLR4 had been considering a therapeutic target against insect bites. Thus, we treated HaCaT cells and pTHP-1 cells with TLR4 inhibitor before or after the stimulation of FT head protein. Results showed that TLR4 inhibitor failed to inhibit the production of inflammatory cytokines induced by FT head protein. To get a further investigation of the inflammatory mechanism induced by FT head protein, we analyzed the gene expression of MDM stimulated by FT head protein through RNA Sequencing. Results indicated that the inflammatory responses induced by FT head protein may be regulated by NOD-like receptor signaling. In summary, FT proteins in the head of F. taiwana contain an inflammatory factor contributing to allergic dermatitis observed in affected individuals may be regulated by NOD-like receptor signaling pathway.

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