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. 2003 Dec;112(12):1862-70.
doi: 10.1172/JCI18125.

Chemokine-mediated recruitment of NK cells is a critical host defense mechanism in invasive aspergillosis

Brad E Morrison  1 , Stacy J Park, Jill M Mooney, Borna Mehrad
Affiliations

Chemokine-mediated recruitment of NK cells is a critical host defense mechanism in invasive aspergillosis

Brad E Morrison et al. J Clin Invest. 2003 Dec.

Abstract

Invasive aspergillosis is a severe pneumonia that is usually fatal despite currently available therapy. The disease disproportionately afflicts immunocompromised patients, indicating the critical importance of the immune status of the host in this infection, but the defense mechanisms against this pathogen remain incompletely understood. In the current study, we hypothesized that the chemokine ligand monocyte chemotactic protein-1, also designated CC chemokine ligand-2 (MCP-1/CCL2) is necessary for effective host defense against invasive aspergillosis in immunocompromised hosts. We found a rapid and marked induction of MCP-1/CCL2 in the lungs of neutropenic mice with invasive aspergillosis. Neutralizing MCP-1/CCL2 resulted in twofold greater mortality and greater than threefold increase in pathogen burden in the lungs. Neutralization of MCP-1/CCL2 also resulted in reduced recruitment of NK cells to the lungs at early time points, but did not affect the number of other leukocyte effector cells in the lungs. Ab-mediated depletion of NK cells similarly resulted in impaired defenses against the infection, resulting in a greater than twofold increase in mortality and impaired clearance of the pathogen from the lungs. These data establish MCP-1/CCL2-mediated recruitment of NK cells to the lungs as a critical early host defense mechanism in invasive aspergillosis and demonstrate NK cells to be an important and previously unrecognized effector cell in this infection.

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Figures

Figure 1
Figure 1
Lung MCP-1/CCL2 levels after intratracheal A. fumigatus challenge in immunocompetent and neutropenic animals. Both groups were inoculated intratracheally with conidia, and MCP-1/CCL2 was measured in lung homogenates using a specific ELISA. *P < 0.05 compared with immunocompetent animals at that time point. Data shown represent mean ± SEM; n = 6 for each group at each time point. Neutrophil-depleted, RB6-8C5 mAb-treated animals.
Figure 2
Figure 2
Outcome of invasive aspergillosis in neutropenic mice with or without neutralization of MCP-1/CCL2. (a) Survival study; n = 15 for each group. (b) Lung chitin content after intratracheal A. fumigatus challenge on day 3 after intratracheal inoculation with conidia. Data represent mean ± SEM of n = 6 for each group. *P < 0.05 compared with infected mice with polymorphonuclear (PMN) cell depletion. PMN depletion indicates mice treated with RB6-8C5 and PBS; PMN depletion/MCP-1 neutralization indicates mice treated with RB6-8C5 and anti–MCP-1/CCL2 mAb.
Figure 3
Figure 3
Effect of MCP-1/CCL2 neutralization on lung leukocyte subsets in transiently neutropenic mice after intratracheal challenge with A. fumigatus conidia. Cell populations were identified as CD45+ cells expressing the markers in parentheses. *P < 0.05 compared with infected mice with PMN depletion at the same time point. Data shown represent mean ± SEM; n = 4–6 for each group at each time point. Uninfected indicates normal uninfected mice; infected/PBS indicates mice treated with RB6-8C5 and PBS and challenged with intratracheal A. fumigatus conidia; infected/anti–MCP-1 indicates mice treated with RB6-8C5 and anti–MCP-1/CCL2 mAb and challenged with intratracheal A. fumigatus conidia.
Figure 4
Figure 4
Effect of MCP-1/CCL2 neutralization on NK cells in neutropenic mice on day 1 after intratracheal challenge with A. fumigatus conidia. Flow-cytometry data gated on CD45+ cells with lymphocyte light-scatter characteristics. Data are representative of two experiments, with a total of five animals per group. PMN depletion/PBS indicates animals treated with RB6-8C5 mAb and PBS and challenged with intratracheal A. fumigatus conidia; PMN depletion/anti–MCP-1 indicates animals treated with RB6-8C5 and anti–MCP-1/CCL2 mAb’s and challenged with intratracheal A. fumigatus conidia.
Figure 5
Figure 5
Role of MCP-1/CCL2 in in vivo recruitment of NK cells to the lungs in invasive aspergillosis. Cultured NK cells were left unlabeled or labeled with the vital cytoplasmic fluorochrome, CFSE, and transferred to neutrophil-depleted mice before intratracheal challenge with A. fumigatus conidia or vehicle. Infected mice were treated with PBS or neutralizing anti–MCP-1/CCL2 Ab, and lungs were examined for the presence of CFSE-labeled cells after 1 day. (a) A representative scatter diagram of total lung cells, demonstrating the gate used in b. (b) Representative scatter diagrams from each group, gated on lung CD3– NK1.1+ cells in a. The upper-right quadrants contain transferred CFSE+ NK cell populations used for statistical comparisons in c. (c) Mean ± SEM of the number of lung CFSE+ CD3– NK cells in each group (n = 3–5 per group). *P < 0.05 compared with uninfected mice given labeled cells; **P < 0.05 compared with infected mice with PMN depletion.
Figure 6
Figure 6
Role of CCR2 in in vivo recruitment of NK cells to the lungs in invasive aspergillosis. CFSE-labeled or unlabeled NK cells from WT or CCR2–/– animals were transferred to neutrophil-depleted mice before intratracheal challenge with A. fumigatus conidia. Infected mice were treated with PBS or neutralizing anti–MCP-1/CCL2 Ab. (a) Mean ± SEM of number of lung CFSE+ CD3– DX5+ NK cells (n = 3–5 mice per group). *P < 0.05 compared with corresponding recipients of WT NK cells. (b) Lung chitin content on day 3 of invasive aspergillosis after NK cell transfer. Mean ± SEM (n = 6 mice per infected groups; n = 2, uninfected group). *P < 0.05 compared with recipients of CCR2–/– NK cells. (c) Mean ± SEM of number of lung CFSE+ CD3– DX5+ NK cells in each group (n = 4–6 mice per group). Anti–MCP-1, mice treated with anti–MCP-1/CCL2 mAb. *P < 0.05 compared with WT recipients of WT NK cells; **P < 0.05 compared with each of the other groups.
Figure 7
Figure 7
Survival after intratracheal A. fumigatus challenge in neutropenic mice with or without NK cell depletion. (a) Survival study; n = 15 for each group. *P < 0.05. (b) Lung chitin content and MPO activity on day 3 after intratracheal inoculation with conidia in neutropenic mice. MPO activity is a surrogate for presence of neutrophils in the lung in this model. Data represent mean ± SEM; n = 6 for each group. PMN depletion indicates mice treated with RB6-8C5 and PBS; PMN and NK depletion indicates mice treated with RB6-8C5 and NK1.1.
Figure 8
Figure 8
Effect of NK cell depletion or MCP-1/CCL2 neutralization on lung histology in transiently neutropenic mice with invasive aspergillosis. Representative lung H&E and GMS stains 3 days after inoculation with A. fumigatus conidia are shown (original magnification ×100). H&E panels in all three groups show areas of intense inflammatory infiltration of leukocytes with some areas of associated necrosis. GMS stains show greater numbers of associated fungal elements in PMN depletion and anti–MCP-1 and PMN and NK depletion groups. PMN depletion indicates RB6-8C5 and PBS; PMN depletion and anti–MCP-1 indicates RB6-8C5–treated mice with MCP-1/CCL2 neutralization; PMN and NK depletion indicates mice treated with RB6-8C5 and NK1.1.
Figure 9
Figure 9
Lung chitin content 3 days after intratracheal challenge with A. fumigatus conidia in neutrophil-depleted mice. Data shown represent mean ± SEM of n = 6 per group. PMN/NK depletion PBS indicates mice treated with RB6-8C5, NK1.1, and PBS; PMN/NK depletion anti–MCP-1 indicates mice treated with RB6-8C5, NK1.1, and anti–MCP-1 mAb.
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