CCR7 deficiency leads to leukocyte activation and increased clearance in response to pulmonary Pseudomonas aeruginosa infection

Abstract

CCR7 is a chemokine receptor expressed on the surfaces of T cells, B cells, and mature dendritic cells that controls cell migration in response to the cognate ligands CCL19 and CCL21. CCR7 is critical for the generation of an adaptive T cell response. However, the roles of CCR7 in the host defense against pulmonary infection and innate immunity are not well understood. We investigated the role of CCR7 in the host defense against acute pulmonary infection with Pseudomonas aeruginosa. We intranasally infected C57BL/6 mice with P. aeruginosa and characterized the expression of CCR7 ligands and the surface expression of CCR7 on pulmonary leukocytes. In response to infection, expression of CCL19 and expression of CCL21 were oppositely regulated, and myeloid dendritic cells upregulated CCR7 expression. We further examined the effects of CCR7 deficiency on the inflammatory response to P. aeruginosa infection. We infected Ccr7(-/-) and wild-type mice with P. aeruginosa and characterized the accumulation of pulmonary leukocytes, production of proinflammatory mediators, neutrophil activation, and bacterial clearance. CCR7 deficiency led to an accumulation of myeloid dendritic cells and T cells in the lung in response to infection. CCR7 deficiency resulted in higher expression of CD80 and CD86 on dendritic cells; increased production of interleukin-12/23p40 (IL-12/23p40), gamma interferon (IFN-gamma), and IL-1 alpha; increased neutrophil respiratory burst; and, ultimately, increased clearance of acute P. aeruginosa infection. In conclusion, our results suggest that CCR7 deficiency results in a heightened proinflammatory environment in response to acute pulmonary P. aeruginosa infection and contributes to more efficient clearance.

FIG. 1.

Characterization of CCL19 and CCL21 expression in the lung in response to P. aeruginosa infection and CCR7 deficiency. (A) Total RNA was isolated from lung homogenates of infected and uninfected Ccr7^−/− and WT mice. CCL19 and CCL21 transcripts were assayed by quantitative RT-PCR and normalized to Rpl32. (B) CCL19 and CCL21 proteins were assayed by sandwich ELISA. Total protein was isolated from lung homogenates of infected and uninfected Ccr7^−/− and WT mice. Data are presented as means ± SEM (n = 5 to 12 mice/group). *, **, and ***, P < 0.05, P < 0.01, and P < 0.001, respectively.

FIG. 2.

Enumeration of pulmonary leukocyte populations. Lung cells were harvested from uninfected and infected Ccr7^−/− and WT mice. Leukocytes were isolated and stained with the following antibodies: FITC-conjugated B220, PerCP-Cy5.5-conjugated CD3e, PE-conjugated CD8a, FITC-conjugated CD4, FITC-conjugated CD11c, and PerCP-Cy5.5-conjugated CD11b. The numbers of myeloid dendritic cell populations (A) and lymphocyte populations (B) isolated from lung digests were enumerated by multiplying percentages obtained by flow cytometry by absolute counts. Data are presented as means ± SEM (n = 4 to 12 mice/group). *, **, and ***, P < 0.05, P < 0.01, and P < 0.001, respectively.

FIG. 3.

CD11c⁺ dendritic cells from Ccr7^−/− mice infected with P. aeruginosa express higher levels of costimulatory molecules CD80 and CD86 than those from WT mice. (A) Lungs from Ccr7^−/− and WT mice were digested at 16 h after infection with P. aeruginosa. Lung leukocytes were isolated and stained with the following Abs: FITC-conjugated CD11c, PerCP-Cy5.5-conjugated CD11b, and either allophycocyanin-conjugated CD80 or allophycocyanin-conjugated CD86. Myeloid dendritic cells (mDCs) were identified by high CD11c expression, low autofluorescence in FL2, and FSC and SSC properties. CD11b mDCs and IE mDCs were divided on the basis of high CD11b expression. The mean fluorescence intensities (MFI) of cells expressing high levels of CD80 or CD86 from each population are presented as means ± SEM (n = 5 mice/group). * and **, P < 0.05 and P < 0.01, respectively, compared to WT. (B) Representative histograms show expression profiles of costimulatory molecules gated on total mDCs. Heavy black lines represent Ccr7^−/−, dotted lines represent WT, and gray shaded histograms represent pooled mDCs from infected Ccr7^−/− and WT mice stained with the appropriate APC-conjugated isotype control. (C) The percentage of each population of mDCs expressing high levels of CD80 or CD86 is represented as open circles. Horizontal lines represent the mean for each group.

FIG. 4.

Ccr7^−/− mice have increased levels of IL-12/23p40, IFN-γ, and IL-1α in the BALF following P. aeruginosa infection compared to WT mice. Bronchoalveolar lavage was performed at 16 h after infection on Ccr7^−/− mice and WT mice and on uninfected control mice. IL-12/23p40 in BALF was assayed by sandwich ELISA. IFN-γ, IL-1α, TNF-α, MCP-1, and GM-CSF were assayed by a Luminex bead-based assay. Data are presented as means ± SEM (n = 5 to 10 mice/group) *, **, and ***, P < 0.05, P < 0.01, and P < 0.001, respectively, compared to WT.

FIG. 5.

Neutrophils recovered after P. aeruginosa infection from the BALF of Ccr7^−/− mice have increased respiratory burst compared to those from WT mice. (A) Ccr7^−/− and WT mice were lavaged at 16 h after infection with P. aeruginosa. Cells recovered from BALF were incubated with dihydrorhodamine-123, and respiratory burst was assayed by measuring conversion of dihydrorhodamine-123 to fluorescent rhodamine-123 by flow cytometry. Neutrophils and macrophages were identified according to FSC and SSC characteristics. Data presented as means ± SEM (n = 6 mice/group). **, P < 0.01 compared to WT. (B) Respiratory burst of peripheral blood neutrophils and macrophages from Ccr7^−/− and WT mice in response to PMA stimulation. Data are presented as means ± SEM (n = 4 mice/group). (C) Representative histograms showing intensity of rhodamine-123 staining of cells recovered from the BALF of infected mice. Heavy black lines, Ccr7^−/−; dotted lines, WT; shaded gray histograms, pooled cells recovered from BALF of infected Ccr7^−/− and WT mice without dihydrorhodamine-123 staining.

FIG. 6.

CCR7 deficiency leads to increased pulmonary clearance of acute P. aeruginosa respiratory infection. Ccr7^−/− and WT mice were intranasally infected with 1 × 10⁷ CFU of P. aeruginosa, and bacterial CFU in the lungs were assessed at 0, 4, and 16 h postinfection. Data are presented as means ± SEM (0 h, n = 4 mice/group; 4 h, n = 3 to 5 mice/group; 16 h, n = 9 to 12 mice/group). *, P < 0.05.