Abstract：
BACKGROUND: Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm.
OBJECTIVE: Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants.
METHODS: HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release.
RESULTS: We show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein.
CONCLUSIONS: These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs.
CONCLUSIONS: These findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.
OBJECTIVE: Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants.
METHODS: HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release.
RESULTS: We show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein.
CONCLUSIONS: These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs.
CONCLUSIONS: These findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.
摘要：
背景：内酶体区室是酸性的，含有低pH依赖性蛋白酶，这些条件被呼吸道病毒利用，如SARS-CoV-2和流感病毒，逃入胞质溶胶.此外，内溶酶体含有各种模式识别受体（PRR），通过产生促炎细胞因子/趋化因子来响应病毒来源的病原体相关分子模式（PAMPs）。然而,过度的促炎反应可导致潜在的致命性细胞因子风暴.
目的：在这里，我们研究了原发性人类小气道上皮细胞（HSAECs）中的内体PRR表达谱，以及内溶酶体酸化的阻断是否会在用病毒来源的兴奋剂攻击后影响其细胞因子/趋化因子的产生。
方法：将HSAECs暴露于模拟病毒来源的PAMPs的兴奋剂中，在不存在或存在导致内溶酶体酸化阻断的化合物的情况下，然后测量细胞因子的表达和释放。
结果：我们表明Toll样受体3（TLR3）是HSAECs表达的主要内体PRR，TLR3的表达是由TLR3激动剂强烈诱导的，但不是通过一系列其他PRR激动剂。我们还证明TLR3与其激动剂的结合引发了强烈的促炎细胞因子/趋化因子反应。通过内溶酶体酸化的阻断被深刻地抑制，巴弗洛霉素A1，莫能菌素，或者氯硝柳胺.使用TLR3报告细胞，证实了TLR3信号传导是由Poly(I:C)强烈诱导的,并且内溶酶体酸化的阻断有效地阻断了TLR3信号传导。最后,我们发现阻断内溶酶体酸化会导致TLR3mRNA和蛋白水平降低.
结论：这些研究结果表明，内溶酶体酸化的阻断抑制了HSAECs中TLR3依赖性细胞因子和趋化因子的产生。
结论：这些发现可用于治疗策略，旨在改善对呼吸道病毒感染的细胞因子风暴。
目的：在这里，我们研究了原发性人类小气道上皮细胞（HSAECs）中的内体PRR表达谱，以及内溶酶体酸化的阻断是否会在用病毒来源的兴奋剂攻击后影响其细胞因子/趋化因子的产生。
方法：将HSAECs暴露于模拟病毒来源的PAMPs的兴奋剂中，在不存在或存在导致内溶酶体酸化阻断的化合物的情况下，然后测量细胞因子的表达和释放。
结果：我们表明Toll样受体3（TLR3）是HSAECs表达的主要内体PRR，TLR3的表达是由TLR3激动剂强烈诱导的，但不是通过一系列其他PRR激动剂。我们还证明TLR3与其激动剂的结合引发了强烈的促炎细胞因子/趋化因子反应。通过内溶酶体酸化的阻断被深刻地抑制，巴弗洛霉素A1，莫能菌素，或者氯硝柳胺.使用TLR3报告细胞，证实了TLR3信号传导是由Poly(I:C)强烈诱导的,并且内溶酶体酸化的阻断有效地阻断了TLR3信号传导。最后,我们发现阻断内溶酶体酸化会导致TLR3mRNA和蛋白水平降低.
结论：这些研究结果表明，内溶酶体酸化的阻断抑制了HSAECs中TLR3依赖性细胞因子和趋化因子的产生。
结论：这些发现可用于治疗策略，旨在改善对呼吸道病毒感染的细胞因子风暴。
