Yamaya Mutsuo, Shimotai Yoshitaka, Hatachi Yukimasa, Lusamba Kalonji Nadine, Tando Yukiko, Kitajima Yasuo, Matsuo Kaori, Kubo Hiroshi, Nagatomi Ryoichi, Hongo Seiji, Homma Morio, Nishimura Hidekazu
Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.
Department of Infectious Diseases, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan.
Pulm Pharmacol Ther. 2015 Aug;33:66-74. doi: 10.1016/j.pupt.2015.07.001. Epub 2015 Jul 10.
Serine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear.
Primary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2).
Camostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat.
These findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.
丝氨酸蛋白酶通过蛋白水解流感病毒的血凝素（HA）来促使流感病毒进入细胞，从而导致感染。然而，丝氨酸蛋白酶抑制剂对人气道上皮细胞感染流感病毒及其炎性细胞因子产生的抑制作用尚不清楚。
用人气管上皮细胞原代培养物分别用四种丝氨酸蛋白酶抑制剂（包括抑肽酶）处理，并用A/仙台-H/108/2009/（H1N1）甲型H1N1流感大流行毒株或A/纽约/55/2004（H3N2）感染。
抑肽酶减少了上清液中流感病毒的数量和细胞中病毒RNA的含量。它减少了流感病毒前体蛋白HA0裂解为亚基HA1。抑肽酶还降低了上清液中细胞因子白细胞介素（IL）-6和肿瘤坏死因子（TNF）-α的浓度。加贝酯和抑肽酶降低了细胞中的病毒滴度和RNA水平，抑肽酶降低了上清液中TNF-α的浓度。已知能裂解HA0并激活病毒的蛋白酶跨膜蛋白酶丝氨酸S1成员（TMPRSS）2和HAT（人胰蛋白酶样蛋白酶：TMPRSS11D）在细胞膜和细胞质中被检测到。在细胞中可检测到编码TMPRSS2、TMPRSS4和TMPRSS11D的mRNA，其表达水平不受抑肽酶影响。
这些发现表明人气道上皮细胞表达这些丝氨酸蛋白酶，丝氨酸蛋白酶抑制剂，尤其是抑肽酶，可能通过抑制这些蛋白酶的活性来减少流感病毒复制及由此产生的炎性细胞因子。
