This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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 antibodiesExperimental 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. pneumophila2) Isotype-matched control antibodies (75 ug/75 ul/mouse) i.t. followed by i.t. L. pneumophila3) Saline (75 ul/mouse) i.t. followed by i.t. L. pneumophilaThe 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 miceExperimental 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. pneumophila3. CXCL5/LIX-/- mice inoculated with i.t. E. coli4. CXCL5/LIX+/+ mice (littermate controls) inoculated with i.t. E. coli5. CXCL5/LIX-/- mice inoculated with i.t. saline6. CXCL5/LIX+/+ mice inoculated with i.t. salineWe 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.
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