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*Gene*GEO Profiles
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*Pulmonary Fibrosis
The Journal of Immunology, 2006, 177: 4436-4443.
Copyright © 2006 by The American Association of Immunologists, Inc.

Regulatory Role of {gamma}{delta} T Cells in the Recruitment of CD4+ and CD8+ T Cells to Lung and Subsequent Pulmonary Fibrosis1

Philip L. Simonian*, Christina L. Roark{dagger}, Fernando Diaz del Valle*, Brent E. Palmer*, Ivor S. Douglas*, Koichi Ikuta{ddagger}, Willi K. Born{dagger}, Rebecca L. O’Brien{dagger} and Andrew P. Fontenot2,*,{dagger}

* Department of Medicine, University of Colorado at Denver and Health Sciences Center, Denver, CO 80262; {dagger} Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206; and {ddagger} Laboratory of Biological Protection, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mechanisms by which T cells accumulate in the lungs of patients with pulmonary fibrosis are poorly understood. Because the lung is continually exposed to microbial agents from the environment, we repeatedly exposed C57BL/6 mice to the ubiquitous microorganism, Bacillus subtilis, to determine whether chronic exposure to an inhaled microorganism could lead to T cell accumulation in the lungs and subsequent pulmonary fibrosis. C57BL/6 mice repeatedly treated with B. subtilis for 4 consecutive weeks developed a 33-fold increase in the number of CD4+ T cells and a 354-fold increase in {gamma}{delta} T cells in the lung. The {gamma}{delta} T cells consisted almost entirely of V{gamma}6/V{delta}1+ cells, a murine subset bearing an invariant TCR the function of which is still unknown. Treatment of C57BL/6 mice with heat-killed vs live B. subtilis resulted in a 2-fold increase in the number of CD4+ T cells in the lung but no expansion of {gamma}{delta} T cells indicating that {gamma}{delta} cells accumulate in response to live microorganisms. In addition, mice treated with heat-killed B. subtilis developed significantly increased pulmonary fibrosis compared with mice treated with the live microorganism. Mice deficient in V{gamma}6/V{delta}1+ T cells when treated with B. subtilis had a 231-fold increase in lung CD4+ T cells and significantly increased collagen deposition compared with wild-type C57BL/6 mice, consistent with an immunoregulatory role for the V{gamma}6/V{delta}1 T cell subset. These findings indicate that chronic inhalation of B. subtilis can result in T cell accumulation in the lung and fibrosis, constituting a new model of immune-mediated pulmonary fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pulmonary fibrosis is characterized by the accumulation of excessive numbers of fibroblasts, deposition of extracellular matrix proteins, and distortion of normal tissue architecture with accompanying injury and cellular inflammation (1, 2, 3). This inflammatory infiltrate consists not only of neutrophils, macrophages, and eosinophils but also of T cells and plasma cells (2). The mechanisms by which lung injury and cellular inflammation result in pulmonary fibrosis are not completely understood. One hypothesis suggests that tissue injury is followed by cycles of inflammation and repair (4). If the tissue injury is minimal or self-limiting, repair will proceed with restoration of normal structure and function, but when injury is more extensive or repetitive, tissue repair may result in scarring and/or fibrosis (4). Inciting agents causing lung injury, inflammation, and pulmonary fibrosis are not well established except for certain therapeutic agents such as bleomycin (5). With the bleomycin-induced lung fibrosis model, studies have shown that type 1 cytokines (e.g., IFN-{gamma}, IL-2, and TNF-{alpha}) protect against the development of fibrotic lung disease whereas type 2 cytokines such as IL-4, IL-5, and IL-13 promote collagen deposition in the lungs by stimulating fibroblast secretion of extracellular matrix proteins, types I and III collagen, and fibronectin (4, 6, 7). Although this model has provided insight into the pathogenesis of pulmonary fibrosis, there is evidence to suggest that T cells are not required for bleomycin-induced fibrotic lung disease (8, 9).

Because T cells are present in the lungs of patients with pulmonary fibrosis due to a variety of causes, these cells may play a pivotal role in the fibrotic process. CD4+ T cells are abundant in the lungs of patients with granulomatous lung diseases such as sarcoidosis and berylliosis and are thought to be central to the pathogenesis of these disorders, but their role in the development of pulmonary fibrosis is not established (10, 11). CD8+ T cells have been associated with a decline in lung function in patients with fibrotic lung disease secondary to scleroderma (12). {gamma}{delta} T cells have also been found in the lungs of patients with hypersensitivity pneumonitis and sarcoidosis (13, 14, 15) and may be important for maintenance of mucosal immunity against microbial pathogens (16, 17). Although it is suggested that persistent or chronic exposure to inhaled Ags in genetically susceptible individuals results in the accumulation of immune cells in the lung, cytokine secretion, and pulmonary fibrosis, there is very little direct evidence that supports this hypothesis (18). The exception is the inflammatory lung condition known as hypersensitivity pneumonitis.

Hypersensitivity pneumonitis is an environmental lung disease that results from the repeated inhalation of various aerosolized Ags including mammalian and avian proteins, fungi, and thermophilic bacteria (19). The classic example of hypersensitivity pneumonitis is farmer’s lung, which is caused by inhalation of the thermophilic actinomycete, Saccharopolyspora rectivirgula. The hallmark of hypersensitivity pneumonitis in both humans and animal models is the influx of large numbers of T cells to the lung (19); with repeated exposure, pulmonary fibrosis ensues (20). Similar to other pulmonary fibrotic diseases, patients who develop fibrosis associated with hypersensitivity pneumonitis have increased mortality (20). Because Bacillus subtilis is a common soil organism and based on a case report of a family who developed hypersensitivity pneumonitis after exposure to this ubiquitous microorganism (21), we repeatedly exposed C57BL/6 mice to B. subtilis to test the hypothesis that chronic exposure to an inhaled microorganism results in not only the accumulation of T cells in the lung but also pulmonary fibrosis. In this report, we show that C57BL/6 mice repetitively exposed to B. subtilis accumulate CD4+, CD8+, and {gamma}{delta} T cells expressing V{gamma}6/V{delta}1 TCR in their lungs and develop pulmonary fibrosis. In the absence of {gamma}{delta} T cells, both CD4+ and CD8+ T cells are further expanded, and increased collagen deposition was seen, suggesting that this subset of {gamma}{delta} T cells may regulate the expansion of {alpha}beta-expressing T cells and thus pulmonary fibrosis. These findings constitute a new model of immune-mediated pulmonary fibrosis, which should allow the delineation of the role of various T cell subsets in the generation of fibrotic lung disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Treatment of mice

Female C57BL/6 mice 6–8 wk old (The Jackson Laboratory) and V{gamma}4/6 knockout (KO)3 mice bred on a C57BL/6 background (22) were treated with 30 µl (5 million CFU) of B. subtilis (ATCC 21332) or sterile PBS on 3 consecutive days each week for up to 16 consecutive weeks by nasal inhalation. B. subtilis was prepared by growing the microorganism to log phase in tryptic soy broth at 37°C with constant agitation at 125 rpm. The B. subtilis culture was centrifuged and resuspended in sterile PBS before administration to mice by nasal inhalation. To determine the number of CFU given to the mice, serial dilutions of B. subtilis were grown on tryptic soy agar plates. Mice were lightly anesthetized with isofluorane to allow inhalation of either B. subtilis or sterile PBS. A Limulus amebocyte assay (Sigma-Aldrich) was performed each week to confirm that the B. subtilis preparations and sterile PBS contained <20 µg of endotoxin/5 million CFU. These studies were approved by the Animal Care and Use Committee at the University of Colorado at Denver and Health Sciences Center.

Preparation of mononuclear cells from lung homogenates

Mice at each time point were sacrificed 24 h after the last treatment with B. subtilis. The chest cavity was opened using sterile surgical dissection, and the inferior vena cava and abdominal aorta were clamped. The left atrium was opened by incision, and the right ventricle was infused with at least 2 ml of sterile PBS to remove any residual blood in the pulmonary vasculature. The heart and lungs were removed en bloc, and the heart, thymus, and lymph nodes were dissected away from the lungs. The right lung was removed, snap-frozen in liquid nitrogen, and stored at –80°C for collagen quantification. The left lung was cut into small pieces and placed in RPMI 1640 containing 5% FBS, collagenase (Sigma-Aldrich), and DNase (Boehringer Mannheim). After 30 min of collagenase digestion in a 37°C water bath, lungs were further disrupted by aspiration through an 18-gauge needle. The collagenase-digested lungs were layered on top of Ficoll (Accurate Chemical & Scientific) and centrifuged at 1200 rpm for 30 min at room temperature. The interface between the medium and Ficoll was removed and washed twice with RPMI 1640 containing 5% FBS.

Flow cytometry and immunofluorescence analysis

T lymphocytes isolated from the lungs of C57BL/6 mice and V{gamma}4/6 KO mice treated with either B. subtilis or sterile PBS were stained with mAbs directed against CD3, CD4, and CD8 (BD Biosciences). {gamma}{delta} T cells were identified by staining with anti-C{delta} mAb (23), and anti-CD3 mAb. {gamma}{delta} T cells that express the V{gamma}6+ TCR were identified using a mAb, 17D1, that recognizes the V{gamma}6+ TCR after staining with anti-C{delta} mAb (24). Briefly, T lymphocytes isolated from the lungs of C57BL/6 mice treated with either B. subtilis or sterile PBS were incubated with anti-C{delta} mAb for 30 min at 4°C in the dark. After washing with PBS containing 1% BSA, T lymphocytes were incubated with mAbs directed against the various V{gamma} TCRs (V{gamma}1, 4, 5, 6, and 7) for 30 min at 4°C in the dark as previously described (24, 25, 26, 27, 28, 29). Streptavidin-PE was used as a second-step reagent for TCR staining. The lymphocyte population was identified using forward and 90-degree light scatter patterns, and fluorescence intensity was analyzed using a FACSCalibur cytometer (BD Immunocytometry Systems) (30, 31).

Total and differential cell counts

Total and differential cell counts were performed on collagenase-digested lung before purification of mononuclear cells over Ficoll. Epithelial cells were not included in the total cell count while RBC were removed by RBC lysis before total and differential cell determination. Single-cell suspensions of total lung cells were transferred to a glass slide using a cytocentrifuge apparatus and stained with Wright and Giemsa. Differential cell counts were performed by counting at least 100 cells. To calculate the absolute number of T and B cells, the percentage of cells that express CD3 and B220 in the lymphocyte gate by flow cytometry was multiplied by the absolute lymphocyte count determined by differential cell count. Absolute cell numbers of CD4+, CD8+, and {gamma}{delta} T cells were then calculated by multiplying the absolute CD3 count by the percentage of CD3+ cells that expressed CD4+, CD8+, or {gamma}{delta} T cells, respectively, in the lymphocyte gate identified by flow cytometry as described above.

Histology

C57BL/6 and V{gamma}4/6 KO mice were sacrificed 24 h after their last exposure to B. subtilis. The lungs were removed and infused with 10% formalin, embedded in paraffin and stained with H&E, Masson trichrome, or Sirius Red staining (Accurate Chemical & Scientific) following the manufacturer’s instructions.

Collagen quantification

The collagen content of the right lung from C57BL/6 and V{gamma}4/6 KO mice treated with either B. subtilis or sterile PBS was determined using Sirius Red staining (6). Sirius Red stain allows quantification of type I–V mammalian collagen. This reagent was chosen because it has been shown to accurately reflect tissue collagen content when compared with hydroxyproline assays (32). Each lung sample was thawed at 4°C in sterile PBS supplemented with protease inhibitors (Sigma-Aldrich), homogenized in 5 ml of 0.5 M acetic acid containing 1 mg of pepsin/10 mg of tissue, and incubated for 24 h at 4°C with constant stirring. After centrifugation at high speed for 10 min, 100 µl of each supernatant were mixed with 1 ml of Sirius Red dye reagent, allowed to incubate at room temperature for 30 min, and then centrifuged at high speed for 10 min. After the supernatant containing excess Sirius Red dye reagent was aspirated, the pellet containing the complex of soluble collagen and Sirius Red dye reagent was resuspended in 0.5 M NaOH, and the OD was measured using a spectrophotometer. The collagen content in micrograms was calculated from a standard curve generated using known concentrations of collagen per the manufacturer’s instructions.

Statistical analysis

A Mann-Whitney U analysis was used to determine whether there was a significant differences between the treatment groups at each time point (Prism 4; GraphPad Software). p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
C57BL/6 mice repeatedly exposed to B. subtilis develop peribronchovascular mononuclear infiltrates

C57BL/6 mice were exposed to B. subtilis by nasal inhalation on 3 consecutive days each week for up to 16 consecutive weeks to determine whether chronic inhalational exposure to the microbial agent, B. subtilis, could result in the accumulation of mononuclear cells in the lungs. As seen in Fig. 1, C57BL/6 mice (n = 20) repeatedly treated with B. subtilis develop a peribronchovascular infiltrate of mononuclear cells. Conversely, no peribronchial, perivascular, or parenchymal cellular infiltrates were seen in the lungs of PBS-treated control mice at any time point.


Figure 1
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  		FIGURE 1. Accumulation of mononuclear cells in a peribronchovascular distribution after exposure to B. subtilis. Representative H&E staining of lungs from C57BL/6 mice after repeated treatment with B. subtilis (5 million CFU) for 3 consecutive days each week for 1, 4, 8, 12, and 16 consecutive weeks is shown. A total of 20 mice with at least 3 mice from each time point treated with B. subtilis were examined by H&E. Staining was performed on the lungs from at least two separate mice treated with B. subtilis at each time point. All B. subtilis-treated mice at each time point develop peribronchovascular mononuclear infiltrates. Control mice were treated for 1, 4, 8, 12, and 16 consecutive weeks with sterile PBS. None of the control mice developed peribronchovascular mononuclear infiltrates. A representative example of H&E staining of lungs from a PBS-treated control mice treated for 16 consecutive weeks is shown (x10). A total of 14 control mice were examined by H&E with at least 2 mice from each time point.

 
To determine the cellular composition of the peribronchovascular infiltrates of mice treated with B. subtilis over time, total and differential cell counts were performed on collagenase-digested lungs before purification of mononuclear cells over a Ficoll gradient. As seen in Fig. 2, there was a significant and progressive increase in the total number of cells infiltrating the lungs of mice treated with B. subtilis that peaked after 4 wk of treatment compared with PBS-treated controls. Additionally, there was a steady increase in the number of lymphocytes and macrophages in the lungs of B. subtilis-treated mice over the first 4 wk of treatment which remained elevated throughout the entire course of exposure (Fig. 2A). For example, a 54-fold increase in the number of lymphocytes in the B. subtilis-treated mice (1.68 ± 0.39 x 106 cells; mean ± SD) compared with PBS-treated control animals (0.031 ± 0.0059 x 106 cells) after 4 wk of treatment was seen. Neutrophil counts also increased during the first 4 wk of treatment but steadily declined on repeated exposure (Fig. 2A). PBS-treated control mice never developed an increase in the total number of cells consistent with the lack of inflammation as seen on H&E (Figs. 1 and 2B). Lungs of PBS-treated control mice contained mostly macrophages with very few lymphocytes and virtually no neutrophils (Fig. 2B).


Figure 2
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  		FIGURE 2. Total and differential cell counts from homogenized lungs after exposure to either B. subtilis or sterile PBS. A, C57BL/6 mice treated with B. subtilis by nasal inhalation for 3 consecutive days each week for up to 16 consecutive weeks developed increased numbers of macrophages, lymphocytes, and neutrophils over the first 4 wk of treatment. Macrophage and lymphocyte numbers remained elevated, whereas total cell count and neutrophil numbers declined with continued exposure to B. subtilis. Data represent the mean ± SD of five mice at each time point. B, C57BL/6 mice treated with sterile PBS by nasal inhalation for 3 consecutive days each week for up to 16 consecutive weeks. Data represent the mean ± SD of three mice at each time point. There was a statistically significant difference (p < 0.05) between B. subtilis- and PBS-treated mice at each time point after the first week of treatment for total cell number and lymphocyte, macrophage, and neutrophil counts.

 
Peribronchovascular mononuclear infiltrates contain large numbers of CD4+, CD8+, and {gamma}{delta} T cells

Flow cytometry was performed to characterize the T cell subsets isolated from the lungs of mice chronically exposed to B. subtilis and PBS. In the representative density plots shown in Fig. 3A, 22, 9, and 67% of the CD3+ T cells expressed CD4, CD8, and {gamma}{delta} T cell phenotypes, respectively, after 4 wk of exposure to B. subtilis. As seen in Fig. 3B, there was a 33-fold increase in the number of CD4+ T cells and a 17-fold increase in CD8+ T cells in the lungs of mice exposed to B. subtilis compared with control animals at 4 wk. Interestingly, there was an even larger expansion of {gamma}{delta} T cells (354-fold; Fig. 3B). The number of CD4+, CD8+, and {gamma}{delta} T cells remained elevated with continued exposure to B. subtilis (Fig. 3B).


Figure 3
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  		FIGURE 3. Expansion of CD4+, CD8+, and {gamma}{delta} T cells after repeated exposure to live B. subtilis. A, Representative density plots after 4 wk of treatment with B. subtilis, with the percentage of CD3+ T cells expressing CD4, CD8, and {gamma}{delta}, respectively, shown in the upper right quadrant of each density plot. B, C57BL/6 mice treated with B. subtilis ({blacksquare}) by nasal inhalation for 3 consecutive days each week for up to 16 consecutive weeks developed increased numbers of CD4+, CD8+, and {gamma}{delta} T cells over the first 3 wk of treatment and remained elevated with continued treatment with B. subtilis. PBS-treated control mice (•) did not have increased numbers of T cell subsets in their lungs. Data represent the mean ± SD of five mice treated with B. subtilis and three mice treated with PBS at each time point. *, Statistically significant difference (p < 0.05) between B. subtilis- and PBS-treated mice at each time point after the first week of treatment for CD4+, CD8+, and {gamma}{delta} T cells. C, Representative density plot displaying the percentage of {gamma}{delta} T cells expressing the V{gamma}6 TCR. C57BL/6 mice treated with B. subtilis by nasal inhalation for 3 consecutive days each week for up to 16 consecutive weeks developed increased numbers of {gamma}{delta} T cells expressing the V{gamma}6 TCR.

 
The vast majority of {gamma}{delta} T cells expressed the normally rare V{gamma}6+ TCR (Fig. 3C). Junctional region nucleotide sequencing of the TCR GV2 (33) showed that the TCR expressed by these cells was primarily the V{gamma}6/V{delta}1+ canonical receptor (data not shown), which has been previously described (34). The lung V{gamma} TCR repertoire from PBS-treated mice was difficult to analyze due to the scarcity of {gamma}{delta} T cells in the lungs of control animals, which is consistent with published reports (35, 36).

NK T cells were also slightly expanded in the lungs of mice treated with B. subtilis compared with control animals and remained elevated with continued exposure to B. subtilis (data not shown). In addition, the number of B cells was increased in lung (24-fold) after 4 wk of treatment with B. subtilis compared with control animals (data not shown).

Chronic exposure to B. subtilis results in pulmonary fibrosis

To determine whether chronic exposure to inhaled B. subtilis results in collagen deposition, lungs from C57BL/6 mice were assessed by Masson trichrome staining. As seen in Fig. 4A, C57BL/6 mice treated with B. subtilis developed pulmonary fibrosis with collagen deposition in a peribronchovascular distribution, which is the identical location of the mononuclear infiltrates seen in Fig. 1. Minimal, if any, Masson trichrome staining was detected in PBS-treated control mice (Fig. 4B).


Figure 4
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  		FIGURE 4. C57BL/6 mice repeatedly exposed to B. subtilis develop lung fibrosis. Masson trichrome staining shows increased collagen deposition in a peribronchovascular distribution in C57BL/6 mice treated with B. subtilis for 16 consecutive weeks (A) as compared with PBS-treated control mice (B). Arrow, peribronchovascular collagen deposition as stained by Masson trichrome. Arrowhead, continued presence of the peribronchovascular mononuclear infiltrates (x10). Masson trichrome staining of lungs from PBS-treated control mice showed very little collagen deposition after 16 wk of treatment (x10). Sirius Red staining also showed increased collagen deposition in a peribronchovascular distribution in C57BL/6 mice treated with B. subtilis for 16 consecutive weeks (C) as compared with PBS-treated control mice (D) (x10). In PBS-treated control mice, collagen deposition was only in the subepithelial and subendothelial basement membrane of large airways and pulmonary arteries after 16 wk of treatment (x10). Sirius Red staining also showed collagen deposition in the alveolar walls of C57BL/6 mice treated with B. subtilis for 16 consecutive weeks (E) and its absence in PBS-treated control mice (F) (x20). Masson trichrome and Sirius Red staining were performed on three B. subtilis- and two PBS-treated mice at each time point (1, 2, 3, 4, 8, 12, and 16 wk of consecutive treatment). Masson trichrome and Sirius Red sections were chosen based on the most representative area of staining when mice at each time point were collectively viewed. G, Quantification of collagen content using Sirius Red stain by colorimetric assay in the lungs of C57BL/6 mice treated with either B. subtilis or sterile PBS is shown. A statistically significant increase in collagen deposition in the lungs of C57BL/6 mice treated with B. subtilis compared with PBS-treated control mice was detected from 3–16 wk. *, p < 0.05. Data represent the mean ± SD of five mice treated with B. subtilis and three mice treated with PBS at each time point.

 
Sirius Red is an anionic dye with sulfonic acid side chain groups that react with the basic amino acid side chain groups of collagen, allowing detection and quantification of types I through V collagen (6). Sections of lung were stained with Sirius Red and visualized by phase contrast light microscopy. As seen in Fig. 4, C and D, and similar to Masson trichrome staining (Fig. 4, A and B), Sirius Red staining demonstrated increased peribronchovascular collagen deposition in the lungs of B. subtilis-treated mice compared with PBS-treated control mice. Higher magnification also showed increased collagen deposition in the alveolar walls of mice treated with B. subtilis compared with PBS-treated control mice (Fig. 4, E and F), suggesting that repeated inhalation of B. subtilis results in both peribronchovascular and interstitial fibrosis. Quantitative collagen content was assessed by Sirius Red colorimetric assay. As shown in Fig. 4G, collagen content was significantly increased (>3-fold) in B. subtilis-treated mice compared with control animals at 3, 4, 8, 12, and 16 wk of treatment with B. subtilis (p < 0.05).

Exposure to heat-killed B. subtilis results in peribronchovascular mononuclear infiltrates and increased pulmonary fibrosis

C57BL/6 mice repetitively treated with B. subtilis developed increased numbers of neutrophils in their lungs during the first 4 wk of treatment which steadily declined. Conversely, lymphocytes remain elevated with continued exposure to B. subtilis (Fig. 2), suggesting a transition from an acute infectious phase to a chronic cell-mediated hypersensitivity phase. To determine whether the accumulation of mononuclear cells in the lungs was due to infection or cell-mediated hypersensitivity, we exposed C57BL/6 mice to heat-killed B. subtilis. As seen in Fig. 5A, mice repeatedly treated for 4 consecutive weeks with heat-killed B. subtilis developed diffuse mononuclear infiltrates similar to treatment with the live microorganism. Mice exposed to heat-killed B. subtilis had increased numbers of both CD4+ (p < 0.05; Fig. 5B) and CD8+ (p < 0.05; Fig. 5C) T cells with decreased numbers of lung neutrophils compared with mice treated with live B. subtilis for 4 wk (see Fig. 2B; data not shown), consistent with a cell-mediated hypersensitivity reaction to the microorganism. Interestingly, the large expansion of {gamma}{delta} T cells expressing the V{gamma}6/V{delta}1+ TCR present upon exposure to the live microorganism was not detected in the lungs of mice treated with heat-killed B. subtilis (Fig. 5D). These findings suggest that only the live microorganism triggers expansion of {gamma}{delta} T cells.


Figure 5
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  		FIGURE 5. Exposure of C57BL/6 mice to heat-killed B. subtilis resulted in peribronchovascular mononuclear infiltrates. H&E staining of lungs from C57BL/6 mice showed a peribronchovascular distribution of mononuclear cells after repeated treatment with heat-killed B. subtilis (5 million CFU) for 3 consecutive days each week for 4 consecutive weeks (x10) (A). The mononuclear infiltrates were composed of greater numbers of CD4+ (B) and CD8+ (C) T cells compared with mice exposed to the live microorganism. The use of the heated-killed B. subtilis eliminated the large {gamma}{delta} T cell expansion that was seen with the live microorganism (D). Data represent data collected from at least two separate experiments.

 
C57BL/6 mice treated with heat-killed B. subtilis also developed pulmonary fibrosis with collagen deposition in a peribronchovascular distribution (Fig. 6A). In addition, a significantly greater amount of collagen was detected in mice exposed to heat-killed vs live microorganisms (p < 0.05; Fig. 6B). These findings suggest that in the absence of an immunoregulatory {gamma}{delta} T cell population, increased CD4+ T cells and, to a lesser degree, CD8+ T cells contribute to the more robust fibrotic process. In addition, the recruitment of CD4+ and CD8+ T cells to a peribronchovascular distribution and the development of fibrosis in the setting of exposure to heat-killed microorganisms strengthen the contention that these effects are not due to infection but to the generation of an immune response directed against unknown T cell epitopes within the microorganism.


Figure 6
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  		FIGURE 6. Development of lung fibrosis after exposure of C57BL/6 mice to heat-killed B. subtilis. A, Masson trichrome staining showed increased collagen deposition in a peribronchovascular distribution in C57BL/6 mice treated with heat-killed B. subtilis for 4 consecutive weeks. Arrow, peribronchovascular collagen deposition as stained by Masson trichrome (x10). B, Quantification of collagen content using Sirius Red by colorimetric assay in the lungs of individual C57BL/6 mice treated with heat-killed B. subtilis, live B. subtilis, or sterile PBS for 4 consecutive weeks is shown. Collagen content was significantly increased in the lungs of C57BL/6 mice treated with heat-killed B. subtilis compared with live B. subtilis and PBS-treated control mice. The median collagen content of the right lung for each condition is shown as a solid line. Data represent those data collected from at least two separate experiments.

 
Exposure of V{gamma}4/6 knockout mice to B. subtilis results in peribronchovascular mononuclear infiltrates, increased numbers of CD4+ and CD8+ T cells and accelerated pulmonary fibrosis

C57BL/6 mice repeatedly exposed to live B. subtilis developed expansions of {gamma}{delta} T cells that expressed the V{gamma}6/V{delta}1+ TCR, whereas mice treated with heat-killed B. subtilis developed increased pulmonary fibrosis and increasing numbers of CD4+ T cells in the absence of an expanded population of {gamma}{delta} T cells. To determine whether V{gamma}6+ T cells might attenuate the development of pulmonary fibrosis through a regulatory effect on CD4+ T cells, we exposed V{gamma}4/6 KO mice to live B. subtilis (5 million CFU) for up to 16 consecutive weeks. V{gamma}4/6 KO mice on a C57BL/6 background were used because V{gamma}6 KO mice are not currently available, and V{gamma}4+ {gamma}{delta} T cells were not expanded in the lungs of wild-type C57BL/6 mice treated with B. subtilis (Fig. 3C). As seen in Fig. 7A, V{gamma}4/6 KO mice treated with live B. subtilis developed mononuclear infiltrates in a peribronchovascular distribution after 1 wk which persisted over the 16 wk of treatment. These peribronchovascular infiltrates resolved when treatment with B. subtilis was discontinued (data not shown), suggesting that these infiltrates occurred due to repeated exposure to the microorganism. Mononuclear cell infiltration was not seen in the PBS-treated controls (Fig. 7B). Analysis of the peribronchial infiltrates by flow cytometry showed a much larger expansion of lung CD4+ and CD8+ T cells in V{gamma}4/6-deficient mice compared with wild-type C57BL/6 mice (Fig. 7C). For example, V{gamma}4/6 KO mice had a 7-fold increase in the numbers of CD4+ T cells (1.37 ± 0.35 x 106 cells) after 12 wk of B. subtilis exposure compared with wild-type C57BL/6 mice (0.19 ± 0.030 x 106 cells; p < 0.05). CD8+ T cells also increased during the first 12 wk of treatment but were no longer statistically significant after 16 wk compared with wild-type C57BL/6 mice exposed to live B. subtilis. The live B. subtilis microorganism was no longer present in the lung, spleen, or peripheral blood of both wild-type C57BL/6 and V{gamma}4/6-deficient mice at 8 h after the last dose of B. subtilis, confirming that the expansion of CD4+ and CD8+ T cells in the absence of V{gamma}6/V{delta}1+ T cells was not due to bacterial persistence in the lungs of mice deficient in V{gamma}6/V{delta}1+ T cells (data not shown).


Figure 7
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  		FIGURE 7. Repeated exposure of V{gamma}4/6 knockout mice to live B. subtilis resulted in peribronchovascular mononuclear infiltrates and increased numbers of CD4+ and CD8+ T cells. Representative H&E staining of lungs from V{gamma}4/6 KO mice after repeated exposure to live B. subtilis (A) and sterile PBS (B) for 16 consecutive weeks is shown. C, V{gamma}4/6 KO mice treated with B. subtilis by nasal inhalation for 3 consecutive days each week for up to 16 consecutive weeks developed increased numbers of CD4+ and CD8+ T cells beginning after 4 consecutive weeks of treatment with live B. subtilis (5 million CFU) compared with wild-type C57BL/6 mice. Data represent the mean ± SD of 5 mice for both V{gamma}4/6 KO and wild-type C57BL/6 mice treated with B. subtilis at each time point. *, p < 0.05.

 
Masson trichrome and Sirius Red staining showed striking collagen deposition in alveolar walls and peribronchovascular areas of the lung of V{gamma}4/6 KO mice compared with PBS-treated control animals (Fig. 8, A–D). Interestingly, collagen deposition was accelerated in these mice compared with wild-type C57BL/6 mice with a statistically significant increase in pulmonary fibrosis after 4, 8, and 12 wk of treatment (p < 0.05), which correlated with the increased numbers of CD4+ and CD8+ T cells in the lungs of V{gamma}4/6-deficient mice (Figs. 8E and 7C). After 16 wk of exposure to live B. subtilis, there was no longer a significant difference in collagen content in the lungs of V{gamma}4/6-defcient compared with wild-type C57BL/6 mice, suggesting the V{gamma}6/V{delta}1+ cells may retard albeit not prevent collagen deposition in the lung (Figs. 7C and 8E).


Figure 8
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  		FIGURE 8. Repeated exposure of V{gamma}4/6 KO mice to live B. subtilis resulted in accelerated pulmonary fibrosis. Representative Masson trichrome staining showed increased collagen deposition in a peribronchovascular distribution in V{gamma}4/6 KO mice treated with live B. subtilis (A) compared with collagen deposition in the lungs of V{gamma}4/6 KO mice treated with sterile PBS for 16 consecutive weeks (B) (x10). Representative Sirius Red staining showed increased collagen deposition in a peribronchovascular distribution in V{gamma}4/6 KO mice treated with live B. subtilis (C) compared with collagen deposition in the lungs of V{gamma}4/6 KO mice treated with sterile PBS for 16 consecutive weeks (D) (x10). Masson trichrome (A and B) and Sirius Red (C and D) staining were performed on three B. subtilis- and two PBS-treated mice at each time point (1, 2, 3, 4, 8, 12, and 16 wk of consecutive treatment). Masson trichrome and Sirius Red sections were chosen based on the most representative area of staining when mice at each time point were collectively viewed. E, Quantification of collagen content by colorimetric assay using Sirius Red in the lungs of individual V{gamma}4/6 KO and wild-type C57BL/6 mice treated with live B. subtilis or sterile PBS for up to 16 consecutive weeks. Collagen content was significantly increased in the lungs of V{gamma}4/6 KO compared with wild-type C57BL/6 mice after 4, 8, and 12 wk of treatment with live B. subtilis. *, p < 0.05. Data represent the mean ± SD of five mice treated with B. subtilis and three mice treated with PBS at each time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we demonstrate that the repeated inhalation of the Gram-positive microorganism, B. subtilis, results in the development of peribronchovascular mononuclear infiltrates that contain large numbers of CD4+ and CD8+ T lymphocytes with a striking expansion of {gamma}{delta} T cells. Chronic inhalational exposure to the B. subtilis microorganism also led to collagen deposition in the alveolar walls and peribronchovascular areas of the lung, confirming that repeated inhalation of B. subtilis results in both the recruitment of large populations of T cells to the lung and pulmonary fibrosis. In the absence of {gamma}{delta} T cells, CD4+ and CD8+ T cell numbers are further increased, suggesting that {gamma}{delta} T cells limit the expansion of these T cell subsets in response to repeated exposure to this microorganism. The fact that CD4+ and CD8+ T cells are markedly increased in the lungs in response to inhalation of both live and heat-killed B. subtilis suggests that a cell-mediated immune response against the microorganism is responsible for the histopathologic changes rather than an infectious cause.

Although the mechanisms responsible for the development of pulmonary fibrosis are not well defined, there is evidence that collagen deposition in the lung is the result of dysregulated repair following alveolar epithelial injury (4). The source of alveolar injury that results in cellular inflammation and fibrosis in most cases, however, is unknown. In this model, the injury pattern of diffuse alveolar damage characterized by the presence of hyaline membranes, interstitial edema, and alveolar epithelial cell destruction was not seen even during the first few weeks of exposure, suggesting that alveolar injury may not be a prerequisite for the development of pulmonary fibrosis in response to chronic inhalation of B. subtilis. Although mice inhaled live B. subtilis, there was never a significant accumulation of neutrophils in the alveolar space or lung consolidation consistent with an acute infection but rather a progressive accumulation of mononuclear cells in the lung characteristic of a hypersensitivity reaction. Additionally, mice did not succumb to chronic inhalation of B. subtilis, unlike mice given a single treatment of Listeria monocytogenes, a known human pathogen and Gram-positive organism. Mice treated with just a single dose of 1000 CFU of L. monocytogenes died within 3–5 days of inhalation. Necropsy studies showed neutrophilic infiltration and lung consolidation with disseminated L. monocytogenes in the spleen, suggesting overwhelming lung and systemic infection. Because B. subtilis is a common microorganism found in soil, it seems possible that chronic inhalation of this and possibly other microorganisms may result in a chronic mononuclear inflammation and pulmonary fibrosis consistent with hypersensitivity pneumonitis and not an acute infectious pneumonia. Because epithelial injury most likely does not precede the development of pulmonary fibrosis in this model, it is plausible that a variety of inhaled allergens, environmental toxins, or microbial agents may cause cellular inflammation and pulmonary fibrosis without alveolar injury.

We show here that C57BL/6 mice treated with heat-killed B. subtilis developed both increased collagen deposition and larger expanded populations of CD4+ and CD8+ T cells. Furthermore, V{gamma}4/6-deficient mice treated with live B. subtilis had accelerated collagen deposition and increased numbers of CD4+ and CD8+ T cells. The results further implicate {alpha}beta T cells in the pathogenesis of pulmonary fibrosis. Neutrophils and macrophages may also be important for collagen deposition in the lungs in response to chronic inhalation of B. subtilis. Neutrophils, however, may be less important for progression of lung fibrosis given that the number of neutrophils declined as collagen deposition progressed in response to inhaled live B. subtilis. CD4+ T cells have been implicated in the initiation and perpetuation of several granulomatous lung diseases in humans (11). For example, in berylliosis, large populations of beryllium-specific CD4+ T cells are recruited to the lung in response to the persistent presence of the beryllium Ag (11, 30, 31, 37). These T cells recognize beryllium in an MHC II-restricted manner (38, 39, 40), and their recruitment to lung precedes the development of granulomatous inflammation (41). Although berylliosis is considered a progressive disease with one-third of affected subjects historically progressing to respiratory insufficiency (42), the specific role of beryllium-specific CD4+ T cells in the development of fibrosis is unclear. Therefore, defining the mechanisms by which CD4+ and CD8+ T cells promote collagen deposition in the lungs may result in a far better understanding of how pulmonary fibrosis develops in response to chronic exposure to an inhaled Ag.

{gamma}{delta} T cells represent a separate lymphocyte subset, whose primary function is not well understood (43, 44). However, this T cell subset has been shown to be important in certain lung diseases, such as asthma (45, 46). For example, V{gamma}1+ {gamma}{delta} T cells enhance airway hyperresponsiveness, whereas V{gamma}4+ {gamma}{delta} T cells suppress this response in a murine model of asthma (46 ). In the present study, we identified large expansions of {gamma}{delta} T cells that consisted almost entirely of V{gamma}6/V{delta}1+ cells in the lungs of mice repeatedly treated with live B. subtilis. V{gamma}6/V{delta}1-expressing {gamma}{delta} T cells normally reside in several tissues, including the female reproductive tract (34), tongue (34), and lung (47) but are quite rare in lymphoid and most other tissues (24). Responses of this TCR-invariant subset have been previously reported in other models, including the liver of Listeria-infected mice (48), testis during Listeria-induced orchitis (49), peritoneum of Escherichia coli-infected mice (50), kidneys of mice infected intrarenally with L. monocytogenes (51), kidneys of rats treated with adriamycin (52) or in which autoimmune (Heymann) nephritis has been induced (53), and in the brains of mice with experimental allergic encephalomyelitis (54). Perhaps most importantly, V{gamma}6/V{delta}1+ cells were found to be the predominantly responding {gamma}{delta} T cell type in the testes of mice with autoimmune orchitis (55), a disease model in which an autoimmune attack of the testes is induced by introducing testicular cells into the bloodstream, in the absence of any foreign agent or adjuvant. Thus, although the ligand for this TCR remains unknown, it is likely to be a host molecule the expression of which is induced during inflammation. Our report is the first to show a specific response of this {gamma}{delta} T cell subset in the course of a disease in the lung, a tissue in which these cells normally reside. Compared with prior studies, the response of these {gamma}{delta} T cells was unique in two ways: 1) the {gamma}{delta} T cell expansion was almost completely limited to the V{gamma}6/V{delta}1+ {gamma}{delta} T cell population; and 2) the expansion was so prominent that it exceeded ongoing responses by both CD4+ and CD8+ {alpha}beta T cells. Interestingly, our results also imply that live bacteria in this system are required to induce the response. Previous studies have also shown a preferential {gamma}{delta} T cell response when live vs dead microorganisms were used (56, 57, 58). How {gamma}{delta} T cells distinguish a live from a killed microbe remains an important unanswered question.

On the basis of the substantially enhanced numbers of responding CD4+ and CD8+ T cells evident in mice unable to produce V{gamma}6+ {gamma}{delta} T cells, our results suggest that the V{gamma}6 subset in this system is strongly inhibiting the overall immune response. This was not unexpected based on a previous report that these {gamma}{delta} T cells can produce substantial quantities of the immunosuppressive cytokine, TGF-beta (53). In addition, in Listeria-induced orchitis, the predominantly responding V{gamma}6+ {gamma}{delta} T cells appeared to reduce and retard inflammatory damage (59). In preliminary experiments, lung-derived CD4+ T cells showed greater proliferation and IFN-{gamma} expression in the absence of {gamma}{delta} T cells in response to heat-killed B. subtilis. It is also possible that {gamma}{delta} T cells not only regulate the expansion of CD4+ and CD8+ T cells by inhibiting their proliferation but also delay the development of pulmonary fibrosis by altering the cytokine secretion profile of {alpha}beta-expressing T cells in response to B. subtilis exposure.

In summary, we have shown the presence of large populations of CD4+, CD8+, and {gamma}{delta} T cells in the lungs of mice exposed to B. subtilis, with the subsequent development of lung fibrosis. In addition, fibrosis developed upon exposure to both live- and heat-killed microorganisms, suggesting a delayed type hypersensitivity reaction. An immunoregulatory role of V{gamma}6+ {gamma}{delta} T cells was also seen, with an increased recruitment of {alpha}beta-expressing T cells to the lung and accelerated lung fibrosis in the absence of this T cell subset. Taken together, this murine model of lung fibrosis will extend our current understanding of the immune mechanisms involved in the development of this fatal condition.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants HL62410 and ES011810 (to A.P.F.), AI40611 and HL65410 (to W.K.B.), and AI044920, EY015840, and AI063400 (to R.L.O.). P.L.S. and C.L.R. are supported by the Flight Attendants Medical Research Institute and the Arthritis Foundation Investigator Award, respectively. Back

2 Address correspondence and reprint requests to Dr. Andrew P. Fontenot, Division of Clinical Immunology (B164), University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: andrew.fontenot{at}uchsc.edu Back

3 Abbreviation used in this paper: KO, knockout. Back

Received for publication May 10, 2006. Accepted for publication July 19, 2006.


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