Modulation of pulmonary dendritic cell function during mycobacterial infection

Abstract

We have previously reported that during mycobacterial infection, naïve CD4(+) T-cell activation is enhanced in the lungs. We investigated the role of chemokine receptor CCR7 and its ligands in the ability of CD11c(+) lung dendritic cells (DCs) to activate naïve CD4(+) T cells during pulmonary infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection resulted in the accumulation and maturation in the lungs of DCs that persisted as the mycobacterial burden declined. Lung DCs from infected mice expressed more major histocompatibility complex class II (MHC-II) than those from uninfected mice. CCR7 expression levels on lung DCs were comparable among uninfected and infected mice. The gene expression of the CCR7 ligand CCL19 progressively increased throughout BCG infection, and its expression was MyD88 dependent. CD11c(+) lung cells from BCG-infected mice activated ovalbumin (OVA)-specific naïve CD4(+) T cells more than CD11c(+) lung cells from uninfected mice. Interestingly, during peak mycobacterial infection, CD11c(hi) MHC(hi) lung DCs had slightly decreased chemotaxis toward the CCR7 ligand CCL21 and less efficiency in activating naive CD4(+) T cells than DCs from mice during late-stage infection, when few bacilli are found in the lung. These findings suggest that during BCG infection, the inflammation and sustained expression of CCL19 result in the recruitment, activation, and retention in the lung of DCs that can activate naïve CD4(+) T cells in situ.

FIG. 1.

Accumulation and maturation of DCs in the lung during BCG infection. (A) Lungs from BALB/c mice at 1 day, 4 to 6 weeks, and 12 to 14 weeks after aerosolized BCG infection were processed, and aliquots were plated to enumerate lung CFU. Mean numbers of CFU ± standard deviations (SD) from a single BCG infection of three mice per time point are shown. Similar bacterial burdens were measured in three separate experiments. Sorted CD11c⁺ lung cells from uninfected mice, mice infected for 4 to 6 weeks, and mice infected for 12 to 14 weeks were stained for MHC-II (I-A^d), CD11b, and CD11c. (B) Maturation of myeloid (CD11b^hi CD11c^hi) DCs was assessed by gating on these cells and by examining the geometric mean fluorescent intensity (MFI) of surface MHC-II staining. Uninf., uninfected. (C) After gating on live cells was done, the number of mature (CD11c^hi MHC^hi) lung DCs was calculated by multiplying the percentage of CD11c^hi MHC^hi cells by the total number of live cells, as assessed by trypan blue exclusion. Data in panels B and C are means ± SD of four independent experiments with three to four mice per experiment; #, ##, *, **, P < 0.02 compared to uninfected mice.

FIG. 2.

CCR7 mRNA and surface protein expression levels on lung DCs are not increased during peak and late stages of BCG infection. (A) CD11c⁺ lung cells were sorted from uninfected mice (Uninf.) and infected mice either 4 to 6 weeks or 12 to 14 weeks after BCG infection. Total RNA was extracted from the CD11c⁺ cells and used to perform qRT-PCR for CCR7 gene expression relative to GAPDH. Results are representative of two independent experiments (three mice per group). (B) Sorted CD11c⁺ lung cells were stained for CCR7 (clone 4B12), CD11b, and CD11c and gated on live cells. CCR7 expression on DCs (CD11c^hi CD11b^hi) and macrophages (CD11c^hi CD11b⁻) are shown. Representative plots are from three to five experiments. max, maximum.

FIG. 3.

BCG infection upregulates expression of CCL19 mRNA in lung in a MyD88-dependent manner. RNA was extracted from the right lower lobe of lungs from uninfected and infected mice. qRT-PCR was done using 1 μg of total RNA, and transcript levels were normalized to GAPDH. (A) BALB/c mice were left uninfected (Uninf.) or were infected with aerosolized BCG. At the indicated times postinfection, the induction of CCL19 and CCL21 in the lungs was measured by qRT-PCR (*, P = 0.01 between the two infected groups at 4 to 6 weeks and 12 to 14 weeks). (B) C57BL/6 mice and MyD88^−/− mice were infected with aerosolized BCG. Four to six weeks postinfection, the right lower lung lobes were harvested and used to quantitate CCL19 induction over that of uninfected mice (#, P = 0.04 between wild-type [wt] and MyD88^−/− mice). For all qRT-PCR data, gene expression levels of individual mice from each group were used to calculate mean ± standard deviation (three to five mice per group). The data shown are representative of two independent experiments.

FIG. 4.

BCG infection modulates CCR7-mediated chemotaxis of lung DCs. (A) CD11c⁺ lung cells (4 × 10⁵) were placed on 8-μm polycarbonate inserts and allowed to transmigrate toward CCL21 (100 ng/ml) for 5 h at 37°C. Migrated cells were collected from the bottom chamber and stained for CD11b, CD11c, and MHC-II (I-A^d). The number of transmigrating cells was calculated using the following formula: (total number of reference beads acquired) × (% of transmigrating cells)/(% of beads acquired). uninf., uninfected. (B) Data shown in panel A are expressed as a chemotaxis index normalized to the percentage of CD11c^hi MHC^hi mature lung DCs from the input population, as described in Materials and Methods. Chemotaxis assay data are representative of three similar experiments; #, P = 0.03 (three or four mice per group).

FIG. 5.

BCG infection alters the ability of CD11c⁺ lung cells to present peptide to naïve CD4⁺ T cells in vitro. CD11c⁺ lung cells from uninfected mice and mice infected with BCG for 4 to 6 weeks or 12 to 14 weeks were pulsed with the OVA_323-339 peptide (1 μM) for 90 min at 37°C. Cells were washed with culture medium and incubated with FAC-sorted naïve (CD44^low CD62L^hi) CD4⁺ T cells (5 × 10⁴) from DO11.10 mice. After 48 h, culture supernatants were tested for IL-2 production by enzyme-linked immunosorbent assay. (A) Data are expressed as increased numbers of CD11c⁺ lung cells added. Wells without naïve OVA-specific T cells had undetectable levels of IL-2 (data not shown). (B) Percentage of CD11c^hi MHC^hi cells present within the CD11c⁺ lung cells from the three groups of mice was multiplied by the number of CD11c⁺ lung cells added per well to obtain the number of CD11c^hi MHC^hi cells added to each well. Data are representative of two independent experiments.