INFECTION AND IMMUNITY, Feb. 2008, p. 671–677 0019-9567/08/$08.00⫹0 doi:10.1128/IAI.01079-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 2
Modulation of Pulmonary Dendritic Cell Function during Mycobacterial Infection䌤 Mursalin M. Anis,1,2 Scott A. Fulton,2 Scott M. Reba,2 Yi Liu,1 Clifford V. Harding,1# and W. Henry Boom2,3#* Department of Pathology,1 Division of Infectious Diseases,2 and Tuberculosis Research Unit,3 Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio Received 3 August 2007/Returned for modification 11 September 2007/Accepted 13 November 2007
We have previously reported that during mycobacterial infection, naı¨ve CD4ⴙ T-cell activation is enhanced in the lungs. We investigated the role of chemokine receptor CCR7 and its ligands in the ability of CD11cⴙ lung dendritic cells (DCs) to activate naı¨ve CD4ⴙ T cells during pulmonary infection with Mycobacterium bovis bacillus Calmette-Gue´rin (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. CD11cⴙ lung cells from BCG-infected mice activated ovalbumin (OVA)-specific naı¨ve CD4ⴙ T cells more than CD11cⴙ lung cells from uninfected mice. Interestingly, during peak mycobacterial infection, CD11chi MHChi lung DCs had slightly decreased chemotaxis toward the CCR7 ligand CCL21 and less efficiency in activating naive CD4ⴙ 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 naı¨ve CD4ⴙ T cells in situ. conditions by facilitating encounters among stromal cells, T cells, B cells, and DCs (1, 20, 21). CCR7 may have a restricted role in immune responses to airborne pathogens. Mice lacking the expression of CCL19 and CCL21-ser (plt/plt) have a delayed immune response that is still protective during influenza virus infection (24). Although CCR7⫺/⫺ mice can control virulent mycobacterial infection, the roles of CCL19 and CCL21 in pulmonary mycobacterial infection have not been evaluated (14, 35). CCR7 ligands within SLOs facilitate the interactions between naı¨ve T cells and mature DCs (20). Therefore, we examined how pulmonary BCG infection affects lung DCs and the expression of CCR7 ligands in the lungs in order to gain insight into naı¨ve CD4⫹ T-cell activation in the lung during chronic lung inflammation. Using an attenuated strain of mycobacteria, Mycobacterium bovis BCG, we report that BCG infection induced the pulmonary expression of CCL19 along with the increased activation of DCs in the lungs. The migration of lung DCs toward CCL21 and the activation of naı¨ve CD4⫹ T cells by lung DCs were slightly less efficient during peak infection than in late infection. All together, our findings suggest that the pulmonary expression of the naı¨ve T-cell chemoattractant CCL19 and the reduced CCR7-mediated migration of activated lung DCs lead to the retention of mature DCs in the lungs that can activate naı¨ve CD4⫹ T cells in situ during pulmonary mycobacterial infection.
During pulmonary mycobacterial infection, the migration of dendritic cells (DCs) and the dissemination of mycobacteria to draining lymph nodes are thought to be important for a successful cell-mediated immune response (6, 22, 36). Upon the engagement of Toll-like receptors (TLRs) on DCs by mycobacterium-derived, pathogen-associated molecular patterns, DCs undergo a coordinated maturation program that upregulates expression of the chemokine receptor CCR7. Chemokine receptor CCR7 expression is essential for the migration of DCs to the lymph nodes, where they coordinate adaptive immune responses following TLR stimulation (9, 17, 23, 33, 34). CCR7 and its ligands, CCL19 (Epstein-Barr virus-induced molecule 1 ligand chemokine) and CCL21 (secondary lymphoid chemokine), are important for both the initiation and regulation of adaptive immunity, as they direct the migration of mature DCs, naı¨ve CD4⫹ T cells, and central memory T cells to secondary lymphoid organs (SLOs) (31, 32). There are two isoforms of CCL21: CCL21-ser and CCL21-leu. CCL19 and CCL21-ser are produced by stromal cells within the T-cell zones of SLOs. CCL21-ser is also expressed in high endothelial venules, while CCL21-leu is expressed only in the lymphatic endothelium. Upon maturation, DCs also express CCL19 (26). Both CCL19 and CCL21 are involved in the organization of lymphoid structures under normal and chronic inflammatory
* Corresponding author. Mailing address: Division of Infectious Diseases, Biomedical Research Building, 1031, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4984. Phone: (216) 368-4844. Fax: (216) 368-2034. E-mail:
[email protected]. # W. Henry Boom and Clifford V. Harding share joint senior authorship. 䌤 Published ahead of print on 26 November 2007.
MATERIALS AND METHODS Mice. Eight- to 10-week-old female BALB/c and C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). MyD88⫺/⫺ mice were obtained from Osamu Takeuchi and Shizua Akira (Osaka University, Osaka, Japan) and backcrossed in a C57BL6 background for seven generations. Strain DO11.10 T-cell receptor (TCR) transgenic mice that express TCRs specific for
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the ovalbumin peptide comprising amino acids 323 to 339 (OVA323-339) presented in the context of I-Ad (25) were obtained from Alan Levine (Case Western Reserve University, Cleveland, OH) and bred on site. Mice were housed in specific-pathogen-free conditions. All studies were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University. Aerosolized BCG infection. Mice were exposed to aerosolized M. bovis BCG in an inhalation exposure system (Glas-Col, Terre Haute, IN) as previously described (16). Uninfected mice served as controls. Tissue isolation. Tissues were harvested and processed as previously described (16). Briefly, mice were anesthetized with a lethal dose of tribromoethanol (240 mg/kg). The abdominal cavity was incised, the spleen harvested, and the mouse exsanguinated. The trachea was cannulated, and bronchoalveolar lavage fluid was collected by aspirating it three times with 1 ml of phosphate-buffered saline (PBS). Lungs were perfused with 10 ml of PBS and harvested. Then draining mediastinal lymph nodes were harvested. Single cells were resuspended in complete medium (Dulbecco modified Eagle medium, 10% fetal bovine serum [FBS], 0.05 mM 2-mercaptoethanol, 2 mM HEPES, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 100 U/ml penicillin, and 0.1 mg/ml streptomycin). Lungs were minced and digested with 125 units/ml of type IV collagenase and 30 units/ml DNase for 90 min at 37°C. Lung tissue aggregates were drawn through an 18-gauge needle three times before being pressed through a 40-m nylon filter. Red blood cells were lysed, and lung tissue was resuspended in RPMI medium. Serial dilutions of lung suspension were plated on 7H10 plates to determine the number of bacterial CFU. Lung cells were then positively sorted for CD11c⫹ cells using N418 microbeads (Miltenyi Biotec, Auburn, CA). Mediastinal lymph nodes were pressed through a 70-m nylon filter using the plunger of a 1-ml syringe and resuspended in RPMI medium. Cell staining. Single-cell suspensions of tissues and sorted cells were counted, and viability was assessed by trypan blue exclusion. A total of 5 ⫻ 105 to 1 ⫻ 106 viable cells were preincubated in a 1% bovine serum albumin (BSA)-PBS solution of FcBlock (BD Pharmingen, San Jose, CA) for 15 min at 4°C. The cells were then stained with anti-CCR7 (4B12), anti-CD11c, anti-CD11b (eBioscience, San Diego, CA), and anti-I-Ad (BD Pharmingen; catalog number 553546). Cells were incubated for 30 min at 37°C according to the manufacturer’s staining protocol (eBioscience). Cells were washed once with 1% BSA, and pellets were resuspended in 1% paraformaldehyde in PBS. Stained samples were acquired using a BD LSR II flow cytometer. Flow cytometry results were analyzed with FlowJo software (Tree Star, Inc.). CD11cⴙ lung cell chemotaxis assay. Lung cells were sorted with magnetic CD11c (N418) beads (Miltenyi Biotec) as described above. In 24-well transwell plates with 8-m-pore-size polycarbonate inserts (Corning Costar Corp., Cambridge, MA), a total of 4 ⫻ 105 viable CD11c⫹ lung cells were added to inserts, and 600 l of chemotaxis medium (0.1% BSA in serum-free HL-1 medium) containing 100 ng/ml of CCL21 (R&D) was added to the lower chambers. After 5 h of incubation at 37°C, cells from the lower chambers were collected and pelleted. Migrated cells were stained with anti-CD11c, anti-CD11b, and antiI-Ad and counted along with 10-m reference beads (Polysciences, Warrington, PA) in a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA). The chemotaxis index was calculated by dividing the fraction of mature lung DCs from the input population that transmigrated to CCL21 by the fraction of mature DCs from the input population that migrated to medium alone. RNA isolation and quantitative real-time PCR (qRT-PCR). The right lower lobes of murine lungs were excised and flash frozen on dry ice. Lung tissues were stored at ⫺80°C until RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA) by following the manufacturer’s protocol. Freshly bead-sorted CD11c⫹ lung cells were lysed, and RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Total RNA yield was determined by use of a spectrophotometer, and 1 g of total RNA was used in a reverse transcriptase reaction (SuperScript First Strand synthesis system; Invitrogen, Carlsbad, CA) to convert RNA to single-stranded cDNA. One-tenth of the resulting cDNA template was used for real-time PCR analysis with Sybr green and the Bio-Rad iCycler fluorescence detection system (Bio-Rad Laboratories, Hercules, CA). Gene amplification was done at 95°C for 15 s, 59°C for 15 s, and 72°C for 30 s for 40 cycles. A standard curve for each gene was generated by the serial dilution of the amplified product standard of the known starting concentration. The following primers were used: CCR7 sense, 5⬘ TGT ACG TCA GTA TCA CCA GC 3⬘; CCR7 antisense, 5⬘ TTT TCC AGG TGT GCT TCT GC 3⬘; CCL19 sense, 5⬘ TGT GGC CTG CCT CAG ATT AT 3⬘; CCL19 antisense, 5⬘ AGT CTT CCG CAT CAT TAG CAC 3⬘; CCL21 sense, 5⬘ TCC AAG GGC TGC AAG AGA 3⬘; CCL21 antisense, 5⬘ TGA AGT TCG TGG GGG ATC T 3⬘; IL-10 sense, 5⬘ ATT TGA ATT CCC TGG GTG AGA AG 3⬘; IL-10 antisense 5⬘ CAC AGG GGA GAA ATC GAT GAC A 3⬘; GAPDH sense, 5⬘ CCA
INFECT. IMMUN. GGT TGT CTC CTG CGA CT 3⬘; and GAPDH antisense, 5⬘ ATA CCA GGA AAT GAG CTT GAC AAA GT 3⬘. Quantification was determined based on a standard curve of known concentrations for each gene and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously described (29). CD11cⴙ lung cell culture. CD11c⫹ bead-sorted lung cells, enriched for DCs, were cultured at 1 ⫻ 106/ml in HL-1 medium (Cambrex Laboratories, Walkersville, MD) supplemented with 1% FBS for 18 h at 37°C. Culture supernatants were harvested and spun down to eliminate cells. Supernatants were frozen at ⫺20°C. Pelleted cells were stained as described above to examine CCR7 surface expression. DO11.10 T-cell isolation. Splenocytes from 9- to 14-week-old DO11.10 mice were isolated, and red blood cells were lysed in hypotonic lysis buffer (10 mM Tris-HCl and 0.83% ammonium chloride). Splenocytes were used to obtain untouched CD4⫹ T cells using the CD4⫹ T-cell negative selection kit (Miltenyi Biotec) according to the manufacturer’s instruction. The resulting CD4⫹ T cells were subsequently stained with anti-CD62L and anti-CD44 monoclonal antibodies and subjected to fluorescence-activated cell (FAC) sorting by gating on naı¨ve (CD62Lhi CD44low) T cells using a BD Aria cell sorter. FAC-sorted naı¨ve CD4⫹ T cells were then used in antigen presentation assays. FAC-sorted CD4⫹ T cells were ⬎98% CD44low CD62Lhi, and 65 to 75% of these naı¨ve CD4⫹ T cells were OVA-specific CD4⫹ T cells, as determined by staining with TCR-clonotypic KJ 1-26 antibody (Invitrogen). Antigen presentation assay. Sorted CD11c⫹ lung cells from BALB/c mice were incubated with different concentrations of the OVA323-339 peptide at 37°C for 90 min. Cells were washed and plated along with FAC-sorted naı¨ve (CD44low CD62Lhi) CD4⫹ T cells (5 ⫻ 104) from spleens of DO11.10 mice. After 48 h of incubation at 37°C, supernatants were harvested, and interleukin 2 (IL-2) levels were measured by enzyme-linked immunosorbent assay. Briefly, Immulon microtiter plates (Thermo Fisher Scientific, Waltham, MA) were precoated overnight at 4°C with anti-IL-2 capture antibody (eBioscience; 14-7022) at 1 g/ml. Plates were washed with PBS-Tween and blocked with 10% FBS-PBS for 1 h at 37°C. Plates were incubated at 37°C for 2 h with IL-2-containing supernatants. After being washed, plates were incubated at room temperature with biotinconjugated anti-IL-2 detection antibody (eBioscience; 13-7021) (1 g/ml). Plates were washed and incubated with streptavidin-alkaline phosphatase at room temperature for 30 min. Substrate was added, and the plates were read after 20 to 30 min at 405 nm. Statistical analysis. All statistical analyses were performed using Student’s t test. A P value of ⬍0.05 was considered statistically significant.
RESULTS BCG infection results in the accumulation and maturation of lung DCs beyond peak bacterial burden in the lungs. BALB/c mice were infected by aerosol with BCG. The BCG burden in the lungs peaked 4 to 6 weeks after infection and declined to ⬍100 bacilli/lung by 12 to 14 weeks (Fig. 1A). To determine the effect of BCG infection on the number and maturity of DCs in the lungs, the mice were divided into three groups: uninfected mice, mice infected for 4 to 6 weeks, and mice infected for 12 to 14 weeks. The mice were sacrificed, and their lungs harvested. Lung cells were stained for CD11b, CD11c, CD80, and I-Ad to measure the maturation state of myeloid DCs (CD11bhi CD11chi) as described by GonzalezJuarrero et al. (11). The expression of MHC-II on myeloid DCs increased six- to eightfold during infection and remained elevated for up to 12 to 14 weeks postinfection compared to that in uninfected mice (Fig. 1B). The expression of CD80 was not affected during early infection but was increased slightly (less than twofold) during late infection (data not shown), as observed by others (11). BCG infection also led to six- to eightfold increases in the numbers of mature lung DCs (CD11chi MHChi) in infected mice compared to those in uninfected mice (Fig. 1C). Interestingly, the maturation and accumulation of mature DCs in the lung persisted up to 12 to 14 weeks after infection, when the lung bacterial burden had decreased to below the limit of
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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-Ad), CD11b, and CD11c. (B) Maturation of myeloid (CD11bhi CD11chi) 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 (CD11chi MHChi) lung DCs was calculated by multiplying the percentage of CD11chi MHChi 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; #, ##, ⴱ, ⴱⴱ, P ⬍ 0.02 compared to uninfected mice.
detection (i.e., ⬍100 CFU/lung). Thus, BCG infection resulted not only in the maturation of lung DCs but also in greater retention of activated lung DCs 6 to 8 weeks beyond the peak of the mycobacterial growth. BCG infection does not increase surface expression of CCR7 on lung DCs during peak and late stages of infection. The maturation of DCs is accompanied by a coordinated switch in their chemokine receptor expression that may explain the increased retention of DCs in the lungs during BCG infection. Upon maturation, tissue DCs upregulate CCR7 expression and migrate to SLOs (34). To determine if the maturation of lung DCs during mycobacterial infection resulted in the increased expression of CCR7, lungs were harvested from uninfected mice and mice that had been infected with aerosolized BCG 4 to 6 weeks (peak infection group) or 12 to 14 weeks earlier (late-infection group). Lung cells from these different groups of mice were sorted with CD11c beads to enrich for lung DCs. Total RNA was extracted from a total of 2 ⫻ 106 to 3 ⫻ 106 sorted CD11c⫹ cells from the three groups of mice and used to measure CCR7 mRNA transcript levels by qRT-PCR. BCG infection induced 1.5- to 2-fold increases (P ⬎ 0.05) in CCR7 gene expression in CD11c⫹ lung cells compared to that in uninfected mice (Fig. 2A). In addition, CCR7 protein expression levels on lung macrophages (CD11chi CD11b⫺) and myeloid lung DCs (CD11chi CD11bhi) were assessed using flow cytometry (Fig. 2B). Lung macrophages from the three groups of mice did not express detectable levels of CCR7. Although lung DCs expressed low levels of CCR7, a significant difference in CCR7 surface expression levels between lung DCs from infected and uninfected mice was not detected. The surface expression of CCR7 was also determined by staining with CCL19-Fc ligand (eBioscience); however, no evident shift in mean fluorescent intensity was noted among the three groups with this method (data not shown). Thus, the accumulation of DCs in the lung during infection was associated with an increased amount of CCR7 mRNA in the lung, as measured by qRT-PCR, without causing significant changes in CCR7 protein expression on individual lung DCs or lung macrophages as determined by flow cytometry.
BCG infection upregulates expression of CCL19 in lung in a MyD88-dependent manner. Like the expression of other chemokine receptors, CCR7 surface expression is modulated by its cognate ligands CCL19 and CCL21 via ligand-induced downregulation (4, 27). To determine if the lack of upregulation of CCR7 surface protein expression during BCG infection could be due to the expression of CCL19 and CCL21 in infected lungs, total RNA from the lungs of uninfected and BCGinfected mice was isolated and CCL19 and CCL21 mRNA levels were measured by qRT-PCR. BCG infection induced CCL19, but not CCL21, gene expression in the lungs (Fig. 3A). Mice infected 12 to 14 weeks earlier expressed higher levels of CCL19 mRNA than mice infected 4 to 6 weeks earlier (P ⫽ 0.01). Increased CCL19 expression in the lung during BCG infection may have inhibited the upregulation of CCR7 on DCs. CCL19 expression is also increased in the lungs of mice infected with virulent Mycobacterium tuberculosis that grows to greater numbers (106 CFU/mouse lung) during late infection (14, 35). Thus, it was surprising that BCG infection upregulated CCL19 mRNA expression during late infection (12 to 14 weeks postinfection), when the lung bacterial burden was reduced to fewer than 102 lung CFU/mouse (Fig. 1A). Recent studies have shown that MyD88 along with other TLR adaptors and cytokines regulate the CCL19 promoter (30). To determine if the recognition of whole mycobacteria or mycobacterial products by TLRs had a role in the increased expression of CCL19 mRNA in BCG-infected mice, wild-type C57Bl/6 and MyD88⫺/⫺ mice were infected with BCG. MyD88⫺/⫺ mice are deficient in the recognition of bacteria and bacterial products through multiple TLRs, including TLR2 and TLR9, that have a role in mycobacterial infection (3). BCG-induced CCL19 mRNA expression was MyD88 dependent (Fig. 3B). BCG infection modulates CCR7-mediated chemotaxis of lung DCs. The lack of CCR7 upregulation on the surface of lung DCs and the enhanced expression of CCL19 in the lungs of infected mice suggested that the CCR7-mediated migration of lung DCs to draining lymph nodes might be affected by BCG infection. We used transwell assays to examine the ability of
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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 g 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 (ⴱ, 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.
lung DCs from infected and uninfected mice to migrate in response to the CCR7 ligand CCL21, which has affinity for CCR7 similar to that of CCL19 but which causes less downregulation of CCR7 (4, 27). As shown in Fig. 4A, CD11chi MHChi mature DCs from BCG-infected lungs migrated toward CCL21 in greater numbers than lung DCs from uninfected mice. Interestingly, lung DCs from mice infected 12 to 14 weeks earlier migrated even more than lung DCs from mice infected for 4 to 6 weeks (P ⫽ 0.04), even though both lung DC populations expressed similar levels of CCR7 and MHC-II (Fig. 1B and 2B). To compare the chemotactic responses of individual mature lung DCs (CD11chi MHChi) among the three groups of mice, the data in Fig. 4A were normalized to a chemotaxis index as described in Materials and Methods (Fig. 4B). Mature lung DCs isolated during peak BCG infection had a lower chemotaxis index than mature lung DCs from mice infected 12 to 14 weeks earlier (P ⫽ 0.03). Thus, during peak BCG infection (4 to 6 weeks), the CCR7-mediated chemotactic responses of 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 (CD11chi CD11bhi) and macrophages (CD11chi CD11b⫺) are shown. Representative plots are from three to five experiments. max, maximum.
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FIG. 4. BCG infection modulates CCR7-mediated chemotaxis of lung DCs. (A) CD11c⫹ lung cells (4 ⫻ 105) were placed on 8-m 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-Ad). 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 CD11chi MHChi 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).
mature lung DCs were slightly blunted on an individual cell basis compared to those of uninfected mice, even though the accumulation of mature DCs in the lung led to greater numbers of DCs that could migrate in the assay. Thus, the increased number of mature DCs in infected lungs was not due to an intrinsic inability of these cells to migrate during peak infection but rather to a relative change in migratory functions that may have resulted in greater retention of mature DCs in the lung. BCG infection alters the ability of CD11cⴙ lung cells to present peptide to naı¨ve CD4ⴙ T cells in vitro. The increased number of mature DCs in BCG-infected lungs along with the expression of the naı¨ve T-cell chemoattractant CCL19 suggested that naı¨ve CD4⫹ T-cell–DC interactions could occur in infected lungs and result in naı¨ve CD4⫹ T-cell activation. To measure the antigen-presenting cell (APC) function of lung DCs and their ability to activate naı¨ve CD4⫹ T cells, CD11c⫹ lung cells from BCG-infected and uninfected mice were pulsed with the OVA323-339 peptide and incubated with FAC-sorted naı¨ve (CD44low CD62Lhi) CD4⫹ T cells specific for OVA from DO11.10 TCR transgenic mice. Naı¨ve T-cell activation was assessed by measuring IL-2 production. CD11c⫹ lung cells from BCG infected mice caused greater naı¨ve CD4⫹ T-cell activation (Fig. 5A). In addition, T-cell activation was greater when using CD11c⫹ lung cells from mice infected for 12 to 14 weeks than when using CD11c⫹ lung cells from uninfected
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FIG. 5. BCG infection alters the ability of CD11c⫹ lung cells to present peptide to naı¨ve 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 OVA323-339 peptide (1 M) for 90 min at 37°C. Cells were washed with culture medium and incubated with FAC-sorted naı¨ve (CD44low CD62Lhi) 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 naı¨ve OVA-specific T cells had undetectable levels of IL-2 (data not shown). (B) Percentage of CD11chi MHChi 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 CD11chi MHChi cells added to each well. Data are representative of two independent experiments.
mice or mice infected 4 to 6 weeks earlier (P ⫽ 0.01 and P ⫽ 0.02, respectively, for the highest number of CD11c⫹ lung cells added). Interestingly, the number of mature CD11chi MHChi lung DCs in mice infected for 4 to 6 weeks was comparable to the number in mice infected 12 to 14 weeks earlier (Fig. 1B). Thus, to normalize T-cell responses to APC numbers between the two groups of infected mice, the data in Fig. 5A were expressed to show the number of mature (CD11chi MHChi) lung DCs added to the in vitro T-cell activation assay. During peak BCG infection (4 to 6 weeks), mature lung DCs were able to activate naı¨ve CD4⫹ T cells but were slightly less efficient than lung DCs from mice infected 12 to 14 weeks earlier (Fig. 5B). This decrease in APC function was not associated with a decrease in the expression of CD80, which is expressed on more than 80 to 85% of all CD11c⫹ lung cells (data not shown). The same patterns of T-cell activation were observed when the data in Fig. 5A were normalized for the total number of myeloid (CD11bhi CD11chi) DCs added to the T-cell assay (data not shown). These results suggest that BCG infection renders the lung permissive for naı¨ve CD4⫹ T-cell activation in situ. Increased numbers and the retention of mature lung DCs during infection result in enhanced activation of naı¨ve CD4⫹ T
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cells. At the same time, the in vivo environment during peak infection renders the lung DCs slightly less effective for naı¨ve CD4⫹ T-cell activation than DCs from the late stage of infection (Fig. 5B). DISCUSSION We have previously used adoptive-transfer techniques to demonstrate that naı¨ve CD4⫹ T cells are activated in the lung during ongoing M. bovis BCG infection (2). These studies led us to focus on the role of lung DCs in priming naı¨ve CD4⫹ T cells in the lungs in situ (2). Immune responses ordinarily involve the activation of naı¨ve T cells in the organized microenvironment of SLOs (13, 19). Recently, investigations have demonstrated that mycobacterial and influenza virus infections lead to the development of neolymphoid structures in the lung that could serve as potential sites for the priming of naı¨ve T cells (14, 24, 35). We report here that pulmonary mycobacterial infection results in the accumulation and maturation in the lung of DCs that persist beyond peak bacterial burden for up to 12 to 14 weeks postinfection. The accumulation of mature DCs in infected lungs was associated with a mild reduction in the CCR7-mediated chemotaxis of mature lung DCs during peak infection (4 to 6 weeks postinfection), while surface expression levels of CCR7 remained similar between infected and uninfected mice. The reduction in the CCR7-mediated migration of mature lung DCs suggests that DCs are retained in the lungs during BCG infection. Mycobacterial infection induced the expression of the naı¨ve T-cell chemoattractant CCL19 in the lungs through a MyD88-dependent pathway. OVA-specific naı¨ve CD4⫹ T cells were readily activated by CD11c⫹ lung cells from BCG-infected mice. Lung DCs during peak infection (4 to 6 weeks postinfection) were slightly less efficient, on a per cell basis, in activating naı¨ve CD4⫹ T cells than lung DCs during the late stage of infection (12 to 14 weeks postinfection), despite similar levels of MHC-II on lung DCs at both stages of infection. The stimulation of TLRs by mycobacterial products leads to the maturation of DCs (9, 28). BCG infection caused the accumulation, maturation, and retention of lung DCs even as the lung mycobacterial burden declined. Although BCG infection induced a higher level of surface expression of MHC-II on myeloid lung DCs, CCR7 surface expression was minimally influenced. Moreover, the CCR7-mediated chemotaxis of mature lung DCs was reduced when the bacterial burden peaked in the lungs. A reduction in CCR7-mediated chemotaxis may result in the decreased migration of mature lung DCs to draining lymph nodes and increased retention in the lungs. Naı¨ve CD4⫹ T cells are present in both lymphoid and nonlymphoid organs such as the lungs (8). The formation of bronchus-associated lymphoid tissue (BALT) during pulmonary mycobacterial infection may facilitate encounters between such naı¨ve T cells and lung DCs (14, 35). The expression of the CCR7 ligands CCL19 and CCL21 is associated with the maturation and organization of neolymphoid follicles (10). Pulmonary BCG infection induced the expression of CCL19 in the lung. However, CCR7 signaling is not essential, since mice deficient in CCR7 or its ligands, CCL19 and CCL21-ser (plt/ plt), develop BALT during mycobacterial and influenza infections (14, 24). Recently, it has been demonstrated that the
formation of BALT in mice and the T-cell responses elicited in such animals are directly affected by the lack of CCR7-mediated migration of regulatory T cells (15). Although the mechanism of BALT formation is still not clearly understood, increased activation and retention of DCs in the lungs along with increased expression of CCL19 may render productive interactions between naı¨ve CD4⫹ T cells and lung DCs more likely during BCG infection. We had previously shown that lung inflammation during peak BCG infection caused enhanced pulmonary antigen-specific CD4⫹ T-cell responses in vivo (2). The enhanced CD4⫹ T-cell responses in infected lungs could have occurred due to naı¨ve T-cell priming in situ, secondary to increased naı¨ve T-cell recruitment to the lung and the retention of mature DCs in the lung. In the present study, OVA-specific naı¨ve CD4⫹ T-cell activation by lung DCs was slightly less during peak infection (4 to 6 weeks) than in late infection (12 to 14 weeks) and was not associated with a change in CD80 costimulatory molecule expression. This may represent the modulation of DC function during peak mycobacterial infection that subsides as the infection is controlled. Schreiber et al. have also reported that T cells that are unable to migrate to lymph nodes are recruited to the lungs during pulmonary mycobacterial infection, where they become activated and acquire effector functions after a delay (35). Perhaps the delayed acquisition of effector function by T cells in that study reflects the mycobacterium-induced modulation of lung DC function. The modulation of the CCR7-mediated migration of DCs may occur at multiple levels. Ligand-induced receptor downregulation is a common mechanism used by chemokine receptors, although CCR7 is more resistant to this form of regulation than other chemokine receptors (33). Once bound by its ligand, CCR7 is endocytosed and recycled back, while bound ligand dissociates and is degraded (27). The expression of CCL19 in the lungs of BCG-infected mice indicated that this mode of receptor downregulation might play a role. However, the reduction in chemotaxis during peak infection was not associated with reduced surface expression of CCR7. In addition, mice had higher expression of CCL19 in the lungs 12 to 14 weeks after BCG infection, yet mature lung DCs from these same mice were more responsive to chemokine-mediated chemotaxis than lung DCs from mice infected for 4 to 6 weeks. Another possible mechanism for the decreased chemotaxis of lung DCs from mice infected for 4 to 6 weeks may involve the CCX-CKR decoy receptor that efficiently scavenges CCL19, CCL21, and CCL25 (7). CCX-CKR does not couple to downstream signaling components; instead, it acts as a chemokine sink. BCG infection may modulate the expression of CCXCKR and in an in vitro transwell assay could limit the amount of CCL21 available to bind and signal through CCR7. In summary, BCG infection caused the accumulation and maturation of DCs in the lungs. Surprisingly, the chemotaxis of mature lung DCs was reduced during peak bacterial burden. Our results do not exclude the possibility that acute mycobacterial infection increases the migration of lung DCs to the lymph node, as others have seen with bone-marrow-derived DCs (5). Mycobacteria have been found inside lymph node DCs, but the origin of these DCs has not been determined (12). The ability of infection to impede the migration of DCs is not exclusive to mycobacterial infection. Legge and Braciale
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have shown that lung DCs during pulmonary virus infection become refractory to migration even as inflammation persists and viral replication continues to occur (18). Therefore, the modulation seen in mature lung DC chemotaxis during mycobacterial infection may involve a common set of pathways that recognize pathogen-associated molecular patterns from different pathogens. Additional studies will determine how DCs in the lung become refractory to CCR7-mediated migration and thus become available to activate naı¨ve CD4⫹ T cells in the lungs during mycobacterial infection. ACKNOWLEDGMENTS We thank Nancy Nagy for mouse husbandry. Roxana Rojas provided guidance and suggestions for assays examining the chemotaxis of DCs. This work was supported by National Institutes of Health grants AI27243 and HL55967 to W. Henry Boom; AI35726, AI069085, and AI34343 to Clifford V. Harding; T32 GM07250 to Mursalin M. Anis; and American Lung Association grant RG-8407-N to Scott A. Fulton. REFERENCES 1. Aloisi, F., and R. Pujol-Borrell. 2006. Lymphoid neogenesis in chronic inflammatory diseases. Nat. Rev. Immunol. 6:205–217. 2. Anis, M. M., S. A. Fulton, S. M. Reba, C. V. Harding, and W. H. Boom. 2007. Modulation of naive CD4⫹ T-cell responses to an airway antigen during pulmonary mycobacterial infection. Infect. Immun. 75:2260–2268. 3. Bafica, A., C. A. Scanga, C. G. Feng, C. Leifer, A. Cheever, and A. Sher. 2005. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J. Exp. Med. 202:1715– 1724. 4. Bardi, G., M. Lipp, M. Baggiolini, and P. Loetscher. 2001. The T cell chemokine receptor CCR7 is internalized on stimulation with ELC, but not with SLC. Eur. J. Immunol. 31:3291–3297. 5. Bhatt, K., S. P. Hickman, and P. Salgame. 2004. Cutting edge: a new approach to modeling early lung immunity in murine tuberculosis. J. Immunol. 172:2748–2751. 6. Chackerian, A. A., J. M. Alt, T. V. Perera, C. C. Dascher, and S. M. Behar. 2002. Dissemination of Mycobacterium tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity. Infect. Immun. 70: 4501–4509. 7. Comerford, I., S. Milasta, V. Morrow, G. Milligan, and R. Nibbs. 2006. The chemokine receptor CCX-CKR mediates effective scavenging of CCL19 in vitro. Eur. J. Immunol. 36:1904–1916. 8. Cose, S., C. Brammer, K. M. Khanna, D. Masopust, and L. Lefrancois. 2006. Evidence that a significant number of naive T cells enter non-lymphoid organs as part of a normal migratory pathway. Eur. J. Immunol. 36:1423– 1433. 9. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188:373–386. 10. Fukuyama, S., T. Nagatake, D. Y. Kim, K. Takamura, E. J. Park, T. Kaisho, N. Tanaka, Y. Kurono, and H. Kiyono. 2006. Cutting edge: uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis. J. Immunol. 177:4276– 4280. 11. Gonzalez-Juarrero, M., T. S. Shim, A. Kipnis, A. P. Junqueira-Kipnis, and I. M. Orme. 2003. Dynamics of macrophage cell populations during murine pulmonary tuberculosis. J. Immunol. 171:3128–3135. 12. Humphreys, I. R., G. R. Stewart, D. J. Turner, J. Patel, D. Karamanou, R. J. Snelgrove, and D. B. Young. 2006. A role for dendritic cells in the dissemination of mycobacterial infection. Microbes Infect. 8:1339–1346. 13. Jenkins, M. K., A. Khoruts, E. Ingulli, D. L. Mueller, S. J. McSorley, R. L. Reinhardt, A. Itano, and K. A. Pape. 2001. In vivo activation of antigenspecific CD4 T cells. Annu. Rev. Immunol. 19:23–45. 14. Kahnert, A., U. E. Hopken, M. Stein, S. Bandermann, M. Lipp, and S. H. Kaufmann. 2007. Mycobacterium tuberculosis triggers formation of lymphoid structure in murine lungs. J. Infect. Dis. 195:46–54. 15. Kocks, J. R., A. C. Davalos-Misslitz, G. Hintzen, L. Ohl, and R. Forster. 2007. Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue. J. Exp. Med. 204:723–734.
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16. Kuchtey, J., S. A. Fulton, S. M. Reba, C. V. Harding, and W. H. Boom. 2006. Interferon-alphabeta mediates partial control of early pulmonary Mycobacterium bovis bacillus Calmette-Gue´rin infection. Immunology 118:39–49. 17. Lande, R., E. Giacomini, T. Grassi, M. E. Remoli, E. Iona, M. Miettinen, I. Julkunen, and E. M. Coccia. 2003. IFN-alpha beta released by Mycobacterium tuberculosis-infected human dendritic cells induces the expression of CXCL10: selective recruitment of NK and activated T cells. J. Immunol. 170:1174–1182. 18. Legge, K. L., and T. J. Braciale. 2003. Accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection. Immunity 18:265–277. 19. Luther, S. A., K. M. Ansel, and J. G. Cyster. 2003. Overlapping roles of CXCL13, interleukin 7 receptor alpha, and CCR7 ligands in lymph node development. J. Exp. Med. 197:1191–1198. 20. Luther, S. A., A. Bidgol, D. C. Hargreaves, A. Schmidt, Y. Xu, J. Paniyadi, M. Matloubian, and J. G. Cyster. 2002. Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J. Immunol. 169:424–433. 21. Marinkovic, T., A. Garin, Y. Yokota, Y. X. Fu, N. H. Ruddle, G. C. Furtado, and S. A. Lira. 2006. Interaction of mature CD3⫹CD4⫹ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid. J. Clin. Investig. 116:2622–2632. 22. Marino, S., S. Pawar, C. L. Fuller, T. A. Reinhart, J. L. Flynn, and D. E. Kirschner. 2004. Dendritic cell trafficking and antigen presentation in the human immune response to Mycobacterium tuberculosis. J. Immunol. 173: 494–506. 23. MartIn-Fontecha, A., S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp, A. Lanzavecchia, and F. Sallusto. 2003. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J. Exp. Med. 198:615–621. 24. Moyron-Quiroz, J. E., J. Rangel-Moreno, K. Kusser, L. Hartson, F. Sprague, S. Goodrich, D. L. Woodland, F. E. Lund, and T. D. Randall. 2004. Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity. Nat. Med. 10:927–934. 25. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4⫹CD8⫹TCRlo thymocytes in vivo. Science 250:1720–1723. 26. Ngo, V. N., H. L. Tang, and J. G. Cyster. 1998. Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells. J. Exp. Med. 188:181–191. 27. Otero, C., M. Groettrup, and D. F. Legler. 2006. Opposite fate of endocytosed CCR7 and its ligands: recycling versus degradation. J. Immunol. 177:2314–2323. 28. Pecora, N. D., A. J. Gehring, D. H. Canaday, W. H. Boom, and C. V. Harding. 2006. Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function. J. Immunol. 177:422–429. 29. Pennini, M. E., R. K. Pai, D. C. Schultz, W. H. Boom, and C. V. Harding. 2006. Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-gammainduced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling. J. Immunol. 176:4323–4330. 30. Pietila, T. E., V. Veckman, A. Lehtonen, R. Lin, J. Hiscott, and I. Julkunen. 2007. Multiple NF- and IFN regulatory factor family transcription factors regulate CCL19 gene expression in human monocyte-derived dendritic cells. J. Immunol. 178:253–261. 31. Randolph, G. J., V. Angeli, and M. A. Swartz. 2005. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat. Rev. Immunol. 5:617–628. 32. Randolph, G. J., G. Sanchez-Schmitz, and V. Angeli. 2005. Factors and signals that govern the migration of dendritic cells via lymphatics: recent advances. Springer Semin. Immunopathol. 26:273–287. 33. Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen, I. Julkunen, R. Forster, R. Burgstahler, M. Lipp, and A. Lanzavecchia. 1999. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur. J. Immunol. 29:1617–1625. 34. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Qin, and A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28:2760–2769. 35. Schreiber, T., S. Ehlers, S. Aly, A. Holscher, S. Hartmann, M. Lipp, J. B. Lowe, and C. Holscher. 2006. Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis. J. Immunol. 176: 1131–1140. 36. Tian, T., J. Woodworth, M. Skold, and S. M. Behar. 2005. In vivo depletion of CD11c⫹ cells delays the CD4⫹ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection. J. Immunol. 175:3268– 3272.
