ROLE OF CXCL5 IN BACTERIAL PNEUMONIA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的目标是确定趋化因子CXCL5在嗜肺乳杆菌感染期间中性粒细胞重新聚集到肺中的相对重要性。我们将通过以下具体目标实现目标： 瞄准。评估CXCL5在嗜肺乳杆菌肺炎中性粒细胞募集中的作用。假设：CXCL5对嗜肺乳杆菌感染后中性粒细胞在肺内聚集起关键作用。 次目标1.利用特异性中和抗体确定CXCL5/lix在嗜中性粒细胞募集和细菌清除中的作用 实验方案：为评价CXCL5/lix对嗜肺乳杆菌(10^7只/只)感染的C57BL/6动物的免疫应答的贡献，将对三组动物进行如下研究： 1)气管内注射大鼠抗小鼠CXCL5/lix抗体(75ug/75ul/只小鼠)紧随其后的是IT。嗜肺性乳杆菌 2)同型相合对照抗体(75ug/75ul/只)I.T.紧随其后的是IT。嗜肺性乳杆菌 3)生理盐水(75ul/只)it.紧随其后的是IT。嗜肺性乳杆菌 将使用上述组确定以下参数： (1)肺白细胞和中性粒细胞内流：在这组实验中，我们将通过BAL细胞计数、分化、MPO活性和组织学来确定嗜中性粒细胞在感染后早期(6h)和晚期(24-72h)的募集情况。 (Ii)细菌清除：我们会在感染后6-72小时内检查肺部、血液和脾中的嗜肺乳杆菌菌落。 我们将使用这一时间框架进行白细胞流入和细菌清除，因为1)嗜肺乳杆菌诱导的中性粒细胞聚集在这一时间框架内非常显著[图6]；以及2)我们[图9和11]和其他人(21)观察到I.T.嗜肺杆菌感染后肺部大量细菌CFU以及细菌在血液和脾中的扩散。 进展/未来实验：我们的初步结果表明，CXCL5在宿主对嗜肺乳杆菌的防御中起重要作用。然而，KC、MIP-2和CXCL5在宿主对嗜肺乳杆菌的防御中起着重要作用。我们正在重复这些实验。 次目标2.利用CXCL5/lix基因缺陷小鼠，确定CXCL5/lix在嗜肺乳杆菌和大肠杆菌肺炎中性粒细胞聚集和细菌清除中的作用。 实验方案：CXCL5/lix，而不是KC或MIP-2，对嗜肺性乳杆菌的宿主防御是必需的，而CXCL5/lix，KC和MIP-2是宿主对大肠杆菌的防御[图12]。为了更确切地检验CXCL5/lix对革兰氏阴性细菌病原体的先天反应的贡献，我们将使用嗜肺乳杆菌(每只小鼠存活10^8只，其他参数10^7只)与以下组进行比较： 1.CXCL5/lix-/-小鼠接种I.T.嗜肺性乳杆菌 2.给CXCL5/lix+/+小鼠接种I.T.嗜肺性乳杆菌 3.CXCL5/lix-/-小鼠接种I.T.大肠杆菌 4.接种I.T.的CXCL5/lix+/+小鼠(窝鼠对照)。大肠杆菌 5.免疫接种CXCL5/lix-/-小鼠。生理盐水 6.免疫CXCL5/lix+/+小鼠。生理盐水 我们将评估以下参数： (I)存活：动物将被注射108个细菌，存活将被监测到15天。 (Ii)肺白细胞和中性粒细胞内流：在这组实验中，我们将通过细菌接种后的BAL细胞计数、分化、MPO活性和组织学来确定中性粒细胞在早期(6h)和晚期(24-72h)的聚集。 (3)细胞因子和趋化因子反应：我们将在细菌攻击(10^7只/只小鼠)后6-72小时内测定肺组织和肺组织中细胞因子(TNF-α和IL-6)和趋化因子(KC、MIP-2和CXCL5/lix)的水平和蛋白水平。 (4)细菌清除：在感染后6-72小时内(每只小鼠10^7)检测肺、血和脾中嗜肺杆菌和大肠埃希菌的菌落数。 进展/未来实验：我们成功地产生了CXCL5/lix基因破坏的小鼠。这些小鼠处于随机杂交/嵌合背景(C57BL/6 X129 SV)。我们的ICAUC方案最近获得批准。我们将研究CXCL5/lix在中性粒细胞最初进入时(6h)和随后的时间点(24-72h)的作用。我们还将评估CXCL5/lix在6-72小时内对肺、血和脾的细菌清除的影响，因为我们观察到I.T.后细菌大量扩散到脾。嗜肺乳杆菌感染(10^7只/只)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to determine the relative importance of a chemokine, CXCL5 in neutrophil recruitment to the lung during L. pneumophila infection. We will accomplish our goals by the following Specific Aim: Aim. Assess the contribution of CXCL5 to neutrophil recruitment in L. pneumophila pneumonia. Hypothesis: CXCL5 is critical for neutrophil accumulation in the lung after L. pneumophila infection. Sub-aim 1. To determine the contribution of CXCL5/LIX to neutrophil recruitment and bacterial clearance in L. pneumophila pneumonia using specific neutralizing antibodies Experimental protocol: To assess the contribution of CXCL5/LIX to immune responses in L. pneumophila (10^7/mouse)-infected C57Bl/ 6 animals, three groups of animals will be studied as follows: 1) Rat anti-murine CXCL5/LIX antibodies (75 ug/75 ul/mouse) intratracheally [i.t.] followed by i.t. L. pneumophila 2) Isotype-matched control antibodies (75 ug/75 ul/mouse) i.t. followed by i.t. L. pneumophila 3) Saline (75 ul/mouse) i.t. followed by i.t. L. pneumophila The following parameters will be determined using the above groups: (i) Lung leukocyte and neutrophil influx: In this set of experiments, we will determine neutrophil recruitment at early time-point (6 h) and late time-point (24-72 h) using BAL cell counts, differentials, MPO activity and histology after L. pneumophila infection. (ii) Bacterial clearance: We will examine L. pneumophila CFUs in the lung, blood and spleen between 6-72 h after infection. We will use this time-frame for leukocyte influx and bacterial clearance because 1) L. pneumophila-induced neutrophil accumulation is significant within this frame [Figure 6]; and 2) we [Figures 9 and 11] and others (21) have observed substantial bacterial CFU in the lungs and bacterial dissemination in blood and spleen after i.t L. pneumophila infection. Progress/future experiments: Our preliminary results demonstrated that CXCL5 is important for host defense against L. pneumophila. However, KC, MIP-2 and CXCL5 are important for host defense against L. pneumophila. We are in the process of repeating those experiments. Sub-aim 2. To determine the role of CXCL5/LIX in neutrophil accumulation and bacterial clearance in L. pneumophila and E. coli pneumonia using CXCL5/LIX gene-deficient mice Experimental protocol: CXCL5/LIX, but not KC or MIP-2, is required for host defense against L. pneumophila whereas CXCL5/LIX, KC and MIP-2 are required for host defense against E. coli [Figure 12]. To examine the contribution of CXCL5/LIX to innate responses against Gram-negative bacterial pathogens more conclusively, we will use L. pneumophila (10^8/mouse for survival and 10^7/mouse for other parameters) with the following groups: 1. CXCL5/LIX-/- mice inoculated with i.t. L. pneumophila 2. CXCL5/LIX+/+ mice (littermate controls) inoculated with i.t. L. pneumophila 3. CXCL5/LIX-/- mice inoculated with i.t. E. coli 4. CXCL5/LIX+/+ mice (littermate controls) inoculated with i.t. E. coli 5. CXCL5/LIX-/- mice inoculated with i.t. saline 6. CXCL5/LIX+/+ mice inoculated with i.t. saline We will assess the following parameters: (i) Survival: Animals will be administrated with 108/bacteria and survival will be monitored up to 15 d. (ii) Lung leukocyte and neutrophil influx: In this set of experiments, we will determine neutrophil accumulation at early time-point (6 h) and late time-point (24-72 h) using BAL cell counts, differentials, MPO activity and histology after bacterial inoculation (10^7/mouse). (iii) Cytokine and chemokine responses: We will determine cytokine (TNF-a and IL-6) and chemokine (KC, MIP-2 and CXCL5/LIX) levels between 6 and 72 h at the mRNA (in lungs) and protein levels (in BALF and lung homogenates) after bacterial challenge (10^7/mouse). (iv) Bacterial clearance: We will examine L. pneumophila and E. coli CFU in lung, blood and spleen between 6-72 h after infection (10^7/mouse). Progress/future experiments: We have successfully generated CXCL5/LIX-gene disrupted mice. These mice are in a random hybrid/chimeric background (C57Bl/6 X129 sv). Our ICAUC protocol has recently been approved. We will investigate the role of CXCL5/LIX at the time of initial neutrophil influx (6 h) and at later time points (24-72 h). We will also assess the effects of CXCL5/LIX on bacterial clearance using lung, blood and spleen between 6-72 h since we observed substantial bacterial dissemination to spleen after i.t. L. pneumophila infection (10^7/mouse) during this time period.