ROLE OF CXCL5 IN BACTERIAL PNEUMONIA

CXCL5 在细菌性肺炎中的作用

• 批准号: 7960598
• 负责人: Samithamby Jeyaseelan
• 金额: $ 6.89万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960598
• 关键词: Animals; Antibodies; Bacteria; Bacterial Pneumonia; Blood; CXCL5 gene; Cell Count; Computer Retrieval of Information on Scientific Projects Database; Escherichia coli; Funding; Future; Genes; Goals; Grant; Histology; Host Defense; Hybrids; Immune response; Infection; Infectious Diseases Research; Institution; Interleukin-6; Leukocytes; Lung; Messenger RNA; Monitor; Mus; Neutrophil Infiltration; Pneumonia; Process; Proteins; Protocols documentation; Rat-1; Relative (related person); Research; Research Personnel; Resources; Role; Saline; Source; Spleen; Time; United States National Institutes of Health; chemokine; cytokine; neutralizing antibody; neutrophil; pathogen; research study; response; time use

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目的目的是确定在肺炎乳杆菌感染期间，趋化因子，CXCL5在中性粒细胞募集中的相对重要性。我们将通过以下特定目标来实现我们的目标： 目的。评估CXCL5对肺炎乳杆菌中嗜中性粒细胞募集的贡献。假设：CXCL5对于肺炎乳杆菌感染后的肺中性粒细胞积累至关重要。 Sub-aim 1。使用特定的中和抗体，确定CXCL5/LIX对肺炎乳杆菌肺炎猪乳杆菌的贡献和细菌清除的贡献 实验方案：评估CXCL5/Lix对肺炎乳杆菌（10^7/小鼠）感染的C57BL/6动物中免疫反应的贡献，将研究三组​​动物： 1）大鼠抗杀菌CXCL5/Lix抗体（75 ug/75 ul/鼠标）在气管内[I.T。]，然后是I.T. L.肺炎 2）同型匹配的对照抗体（75 ug/75 ul/鼠标）I.T。然后是I.T. L.肺炎 3）盐水（75 ul/鼠标）I.T。然后是I.T. L.肺炎 以下参数将使用上述组确定： （i）肺白细胞和中性粒细胞流入：在这组实验中，我们将使用BAL细胞计数，差异，MPO活性和L. pneumophila感染后的早期时间点（6 h）和晚期时间点（6 h）和晚期（24-72 h）（24-72 h）募集。 （ii）细菌清除率：我们将检查感染后6-72小时之间肺，血液和脾脏中的肺炎乳杆菌CFU。 我们将使用此时间框架来进行白细胞的流入和细菌清除率，因为1）在此框架内，肺炎乳杆菌诱导的中性粒细胞的积累很重要[图6]； 2）我们[图9和11]等（21）观察到肺部和肺炎感染后血液和脾脏中的细菌CFU以及细菌传播。 进度/未来实验：我们的初步结果表明，CXCL5对于宿主防御肺炎乳杆菌很重要。但是，KC，MIP-2和CXCL5对于宿主防御肺炎乳杆菌很重要。我们正在重复这些实验。 Sub-aim 2。确定CXCL5/Lix在使用CXCL5/Lix基因缺乏小鼠中的肺炎乳杆菌和大肠杆菌肺炎中的中性粒细胞积累和细菌清除率中的作用 实验方案：针对肺炎乳杆菌的宿主防御需要CXCL5/LIX，而不是KC或MIP-2，而CXCL5/LIX，KC和MIP-2是针对大肠杆菌的宿主防御所必需的[图12]。为了更结论性地检查CXCL5/Lix对革兰氏阴性细菌病原体的先天反应的贡献，我们将使用以下组使用肺炎L. pneumophila（10^8/小鼠的生存和10^7/小鼠）的贡献。 1。CXCL5/Lix - / - 鼠标接种I.T. L.肺炎 2。CXCL5/Lix+/+小鼠（同窝控制）接种I.T. L.肺炎 3。CXCL5/Lix - / - 小鼠接种I.T.大肠杆菌 4。CXCL5/Lix+/+小鼠（同窝控制）接种I.T.大肠杆菌 5。CXCL5/Lix - / - 鼠标接种I.T.盐水 6。CXCL5/Lix+/+小鼠接种I.T.盐水 我们将评估以下参数： （i）生存：动物将用108/细菌施用，并将监测生存期限为15 d。 （ii）肺白细胞和嗜中性粒细胞流入：在这组实验中，我们将使用BAL细胞计数，差异，MPO活性和组织学后，在早期时间点（6 h）和晚期时间点（6 h）和晚期时间点（24-72 h）积累（10^7/小鼠）。 （iii）细胞因子和趋化因子反应：我们将确定细胞因子（TNF-A和IL-6）和趋化因子（KC，MIP-2和CXCL5/LIX）水平在mRNA（肺部）和蛋白质水平（BALF和蛋白质中）（在BALF和肺同源物中）的6至72 h之间。 （iv）细菌清除率：我们将检查感染后6-72小时之间的肺，血液和脾脏中的肺炎乳杆菌和大肠杆菌CFU（10^7/小鼠）。 进度/未来实验：我们成功地产生了CXCL5/Lix-Gene中断的小鼠。这些小鼠在随机的杂种/嵌合背景（C57BL/6 x129 SV）中。我们的ICAUC协议最近已获批准。我们将在初始中性粒细胞流入时（6小时）和以后的时间点（24-72 h）研究CXCL5/Lix的作用。我们还将评估CXCL5/Lix对使用肺，血液和脾脏在6-72 h之间的细菌清除率的影响，因为我们观察到I.T。在此期间，肺炎。肺炎杆菌感染（10^7/小鼠）。