Abstract：
BACKGROUND: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo.
METHODS: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR.
RESULTS: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05).
CONCLUSIONS: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling.
METHODS: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR.
RESULTS: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05).
CONCLUSIONS: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling.
摘要：
背景：糖尿病患者特别容易感染嗜肺军团菌（LP），但糖尿病患者LP感染的确切发病机制尚不完全清楚。在这里，我们在体外和体内研究了糖尿病对LP感染期间免疫功能的影响。
方法：在体外检查了正常和高糖（HG）条件下巨噬细胞中LP感染的时程。蛋白质印迹用于确定核苷酸结合寡聚化结构域1（NOD1），激酶1/2（ERK1/2），丝裂原活化蛋白激酶p38(MAPKp38),和c-JunN末端激酶(JNK)。酶联免疫吸附试验（ELISA）用于评估肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌。细胞计数试剂盒-8(CCK8)测定评估了用不同浓度的高糖培养基和ML130(NOD1抑制剂)处理细胞后的U937细胞活力。对于体内研究，正常和链脲佐菌素诱导的糖尿病豚鼠感染LP6、24和72小时，之后NOD1，MAPK相关信号，TNF-α,和IL-6在肺组织中的表达使用免疫组织化学，westernblot,和RT-PCR。
结果：与暴露于正常葡萄糖水平的LP感染细胞相比，HG减弱了由LP引起的NOD1表达的上调，并减少了TNF-α和IL-6的分泌（所有p<0.05）。在糖尿病豚鼠中，与对照猪相比，HG抑制了由LP感染引起的肺组织中NOD1表达的上调以及p38，ERK1/2和cJNK的激活（均p<0.05）。
结论：HG通过抑制NOD1的上调和MAPK信号的激活来减弱巨噬细胞对LP感染的反应。
方法：在体外检查了正常和高糖（HG）条件下巨噬细胞中LP感染的时程。蛋白质印迹用于确定核苷酸结合寡聚化结构域1（NOD1），激酶1/2（ERK1/2），丝裂原活化蛋白激酶p38(MAPKp38),和c-JunN末端激酶(JNK)。酶联免疫吸附试验（ELISA）用于评估肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌。细胞计数试剂盒-8(CCK8)测定评估了用不同浓度的高糖培养基和ML130(NOD1抑制剂)处理细胞后的U937细胞活力。对于体内研究，正常和链脲佐菌素诱导的糖尿病豚鼠感染LP6、24和72小时，之后NOD1，MAPK相关信号，TNF-α,和IL-6在肺组织中的表达使用免疫组织化学，westernblot,和RT-PCR。
结果：与暴露于正常葡萄糖水平的LP感染细胞相比，HG减弱了由LP引起的NOD1表达的上调，并减少了TNF-α和IL-6的分泌（所有p<0.05）。在糖尿病豚鼠中，与对照猪相比，HG抑制了由LP感染引起的肺组织中NOD1表达的上调以及p38，ERK1/2和cJNK的激活（均p<0.05）。
结论：HG通过抑制NOD1的上调和MAPK信号的激活来减弱巨噬细胞对LP感染的反应。
