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Abstract
Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by peripheral airways inflammation and emphysema. Emerging evidence indicates a contribution of both innate and adaptive immune cells to the development of COPD. Transcription factor T-bet modulates the function of immune cells and therefore might be involved in the pathogenesis of COPD. To elucidate the role for T-bet in elastase-induced emphysema, pathological phenotypes were compared between wild-type and T-bet−/− mice. T-bet−/− mice demonstrated enhanced emphysema development on histological analyses, with higher values of mean linear intercept and dynamic compliance relative to wild-type mice. The number of neutrophils in BAL fluids, lung IL-6 and IL-17 expression, and the proportion of CD4+ T cells positive for IL-17 or retinoic acid receptor–related orphan receptor-γt were higher in T-bet−/− mice than in wild-type mice. Although T-bet downregulates cytokine expression in bone marrow–derived macrophages and MH-S cells, a murine alveolar cell line, depending on the surrounding environment, IL-6 expression in alveolar macrophages isolated from elastase-treated mice was not dependent on T-bet. Coculture of bone marrow–derived macrophages and CD4+ T cells revealed that T-bet regulation of IL-17 expression was dependent on CD4+ T cells. Neutralizing antibodies against IL-6R or IL-17 ameliorated the development of emphysema in T-bet−/− mice. In conclusion, we demonstrate that T-bet ameliorates elastase-induced emphysema formation by modulating the host immune response in the lungs.
Chronic obstructive pulmonary disease (COPD) is a progressive lung disease associated with an aberrant immune response to noxious gases and particles. The pathological hallmarks of COPD include destruction of the alveolar wall (emphysema) and inflammation of the peripheral airways, leading to airflow obstruction that is not fully reversible. Although the main cause of COPD is tobacco smoking in developed countries, only 20% of smokers actually develope COPD (1), suggesting a role of host factors predisposing to its development and progression. Understanding the molecular mechanisms regarding host factors, however, remains elusive.
In COPD, infiltration of neutrophils, macrophages, and T cells into the airways and the alveolar walls is observed (2), which may persist even after smoking cessation (3). Accumulating evidence demonstrates that macrophages play a key role in the development of COPD by producing inflammatory mediators (4). Besides, activation of the adaptive immunity, as shown by the infiltration of type 1 cytotoxic T, T-helper (Th) 1 and Th17 cells have been observed (5, 6). Inflammatory cytokines and chemokines produced by these cells mediate chronic inflammation, which is closely associated with the development of COPD (4). The role of cytokines in COPD has been extensively studied by using genetically modified animals. In mice lacking either IL-6 (7) or IL-17A (8), elastase-induced emphysema formation was attenuated, highlighting a contribution of these cytokines to the development of emphysema.
T-bet was originally identified as a T cell transcription factor promoting Th1 cell differentiation (9). T-bet is now recognized to play a pivotal role in dendritic cells as well (10), whereas its role in macrophages remains largely unknown. With the induction of Th1 response through the expression of IFN-γ, T-bet concomitantly suppresses Th2 (11) and Th17 (12) responses. In mice deficient in T-bet, therefore, blunted Th1 response with augmented Th2 (13) or Th17 (12) response has been demonstrated. Although the involvement of T-bet in diseases with dysregulated immune responses has been demonstrated (11, 13, 14), its role in COPD remains unknown.
In the current study, by using T-bet−/− mice, we investigated the role of T-bet in the pathogenesis of elastase-induced emphysema. We demonstrate T-bet attenuates emphysema development through orchestrating both innate and adaptive immune responses in the lungs.
T-bet−/− and wild-type BALB/c mice were obtained from The Jackson Laboratory and Charles River Breeding Laboratories, respectively. Female mice (8–12 wk old) were inoculated with 3.75 U of porcine pancreatic elastase (PPE; Sigma Aldrich) in 50 μl of saline intratracheally. All animal studies were approved by the University of Tsukuba Institutional Review Board.
Mice were killed on Day 21. Lung paraffin sections were stained with hematoxylin and eosin. The mean linear intercept (Lm) was quantified as described previously (15).
On Day 21, anesthetized mice were connected to a mechanical ventilator (FinePointe; Buxco) to measure dynamic compliance.
BAL was performed and analyzed as described in the data supplement.
Cytokine and T-bet mRNA expression was analyzed by real-time RT-PCR, as described in the data supplement. Primer sequences are also shown in the data supplement.
Lung lymphocyte subsets and T cell expression of intracellular IL-17 and transcription factors were determined by flow cytometry, as described in the data supplement.
BAL fluids were cultured in wells of a 12-well plate at 37°C for 60 minutes. Nonadherent cells were removed by washing wells with PBS.
Anti–IL-6R antibody (Ab; 2 mg; Chugai Pharmaceutical) was injected intraperitoneally into T-bet−/− mice before elastase instillation. Thereafter, 0.5 mg Ab was given on Days 8 and 15. Anti–IL-17 Ab (100 μg; clone TC11-18H10.1; Biolegend) was injected intraperitoneally into T-bet−/− mice on Days 0, 8, and 15. Preimmune IgG was used as a control.
Bone marrow (BM)–derived macrophages were harvested as described previously (16), transferred to 12-well plates at a cell density of 4 × 105 cells/well, and cultured overnight. In polarization experiment, cells were cultured with LPS (100 ng/ml; Santa Cruz) plus IFN-γ (50 ng/ml; Peprotech), IL-4 (20 ng/ml; Peprotech), or medium alone.
Nuclear protein was extracted and analyzed by Western blotting, as described in the data supplement.
The siRNA targeting T-bet and the control siRNA were obtained from Invitrogen. MH-S, a murine alveolar macrophage cell line, cells were transfected with 20 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) and cultured for 48 hours. Culture medium was replaced with medium with or without elastase and RNA was harvested 24 hours later.
A 2.5-kb, full-length cDNA encoding the murine T-bet (17) was subcloned into pcDNA3.1 (Invitrogen). MH-S cells were transduced with 1.0 μg of construct using Lipofectamine LTX (Invitrogen) and cultured for 48 hours before harvesting RNA.
CD4+ T cells from splenic cells and BM-derived macrophages were cocultured as described in the data supplement.
Results are represented as means (±SEM). Comparisons of data were performed using one-way ANOVA followed by post hoc tests or two-tailed t test. P values less than 0.05 were considered statistically significant.
To clarify the role of T-bet in the development of elastase-induced emphysema, Balb/c wild-type and T-bet−/− mice were inoculated intratracheally with either saline or elastase. After 21 days, histological, morphological, and functional analyses of the lungs were performed to evaluate the severity of emphysema. Saline administration induced no evident histological change in either wild-type or T-bet−/− mice (Figure 1A). Elastase treatment induced alveolar wall destruction and air space enlargements in wild-type mice (Figure 1A). In T-bet−/− mice, elastase-induced emphysema formation was more severe compared with wild-type mice (Figure 1A). Accordingly, Lm values in elastase-instilled wild-type mice (51.23 ± 0.63 μm) were significantly higher than those in saline-treated wild-type mice (42.45 ± 1.94 μm) (Figure 1B). The values of Lm in elastase-treated T-bet−/− mice (72.67 ± 7.87 μm) were significantly higher than those in elastase-treated wild-type mice (Figure 1B).
Figure 1. Elastase-induced emphysema development is aggravated in T-bet−/− mice. Wild-type (WT) and T-bet−/− mice were inoculated intratracheally with either saline or elastase (porcine pancreatic elastase [PPE]). The development of emphysema was analyzed 21 days later. (A) Representative photomicrographs of the lungs. Scale bar: 200 μm. (B) Mean linear intercept (Lm) values of alveoli. (C) Dynamic lung compliance (Cdyn). Data represent mean ± SEM of four mice in each group. *P < 0.05.
[More] [Minimize]Next, to functionally assess the degree of emphysema, dynamic compliance (Cdyn) was examined. There was no significant difference in Cdyn values between saline-administered wild-type (0.031 ± 0.0010 ml/cm H2O) and T-bet−/− (0.030 ± 0.0013 ml/cm H2O) mice (Figure 1C). In wild-type mice, although elastase treatment slightly increased Cdyn values (0.034 ± 0.0013 ml/cm H2O), they were not significantly different from those of saline-instilled mice. In elastase-inoculated T-bet−/− mice, however, Cdyn values (0.043 ± 0.0026 ml/cm H2O) were significantly higher than those in saline-treated T-bet−/− mice and those in elastase-treated wild-type mice (Figure 1C). Collectively, these results demonstrate that T-bet plays a regulatory role in the development of elastase-induced emphysema.
To examine the severity of pulmonary inflammation induced by elastase, BAL was performed 24 hours and 3 and 7 days after elastase inoculation. In addition, lung expression of chemokines for neutrophils and lymphocytes were examined by real-time RT-PCR. In saline-instilled mice, although the count of each leukocyte subset was higher in T-bet−/− mice than in wild-type mice, statistical significance was observed only in total cell count at 24 hours (Figure 2A). Elastase treatment led to a significant increase in the number of inflammatory cells in BAL fluid. The number of BAL neutrophils at 24 hours was significantly higher in T-bet−/− mice than in wild-type mice (Figure 2A). In line with this result, lung macrophage inflammatory protein 2 (MIP-2) mRNA expression after elastase instillation was significantly higher in T-bet−/− mice than in wild-type mice (Figure 2D). The number of BAL lymphocytes on Day 3 was slightly, but significantly, higher in T-bet−/− mice than in wild-type mice (Figure 2B), whereas expression of chemokines for Th cells after elastase exposure was not different between the two genotypes (Figure 2E). These results indicate that T-bet decreases elastase-induced recruitment of neutrophils and lymphocytes into the lungs.
Figure 2. Elastase-induced pulmonary inflammation is enhanced in T-bet −/− mice. (A–C) Numbers of total cells, macrophages, lymphocytes, and neutrophils in BAL fluids of WT and T-bet−/− mice 24 hours (A), 3 days (B), and 7 days (C) after the intratracheal inoculation of elastase (PPE) or saline. Data represent mean ± SEM of five mice in each group. (D and E) The mRNA expression of CXCL1 and macrophage inflammatory protein 2 (MIP-2) (D) and CXCL9, CXCL10, CXCL11, and CCL20 (E) in the lungs of WT and T-bet−/− mice as analyzed by real-time RT-PCR. Mice were killed 24 hours (D) and 5 days (E) after inoculation with saline or elastase. The y-axis of each graph represents the relative expression of the respective genes calculated using the ΔΔCT method and normalized against GAPDH mRNA. Data represent mean ± SEM of five mice in each group. *P < 0.05. CT = threshold cycle.
[More] [Minimize]To investigate the molecular basis underlying the enhanced pulmonary inflammation observed in T-bet−/− mice, we analyzed mRNA expression of cytokines in the lungs. The optimal time points for the analysis of each cytokine were determined by a preliminary study. Although elastase administration increased IL-6 mRNA expression in both genotypes at 24 hours, it was significantly higher in T-bet−/− mice than in wild-type mice (Figure 3A). Because T-bet is expressed only in immune cells (18), the cellular source responsible for the increased lung IL-6 expression in T-bet−/− mice was considered to be the innate immune cells. Expression of both TNF-α and IL-1β mRNA was not induced by elastase and was not different between the two genotypes (Figure 3A). Previous reports have demonstrated that Th17 response plays a critical role in the pathogenesis of elastase-induced emphysema (8). Therefore, we next assessed the expression of Th cytokines in the lungs 5 days after elastase administration. In saline-inoculated mice, IFN-γ expression was significantly lower in T-bet−/− mice than in wild-type mice (Figure 3B). After elastase inoculation, IFN-γ expression decreased in wild-type mice and was not different between the two genotypes (Figure 3B). IL-4 and IL-5 expression was not different between saline-inoculated mice of the two genotypes. After elastase treatment, expression of these cytokines decreased and was not different between the two genotypes (Figure 3B). IL-10 expression was not different between saline- and elastase-treated mice or between wild-type and T-bet−/− mice. In saline-exposed mice, IL-17 expression was significantly higher in T-bet−/− mice than in wild-type mice. In accordance with a previous report (8), after elastase inoculation, IL-17 expression was upregulated in both genotypes, and was significantly higher in T-bet−/− mice than in wild-type mice (Figure 3B).
Figure 3. Elastase-induced pulmonary IL-6 expression and T-helper (Th) 17 differentiation are downregulated by T-bet. (A–C) The mRNA expression of IL-6, TNF-α, and IL-1β (A), IFN-γ, IL-4, IL-5, IL-10, and IL-17 (B) in the lungs of WT and T-bet−/− mice, and IL-6 and IL-17 in the alveolar macrophages isolated by BAL from WT and T-bet−/− mice (C), as analyzed by real-time RT-PCR. Mice were killed 24 hours (A) and 5 days (B) after inoculation with saline or elastase. BAL was performed 24 hours after inoculation with saline or elastase. Data represent mean ± SEM of four mice in each group. (D–F) The ratio of CD4+ to CD8+ T cells (D), IL-17 production in CD4+ T cells (E), and T-bet (left), GATA-3 (center), and retinoic acid receptor–related orphan receptor (ROR)-γt (right) expression in CD4+ T cells (F) in the lungs of WT and T-bet−/− mice 5 days after the exposure to saline or elastase. Data represent mean ± SEM of six mice in each group. *P < 0.05.
[More] [Minimize]To identify the cellular source of enhanced lung IL-6 expression in T-bet−/− mice, alveolar macrophages were isolated by BAL and analyzed for IL-6 expression. Although IL-6 mRNA expression was significantly increased by elastase in both wild-type and T-bet−/− mice, no significant difference was observed between the two genotypes.
Furthermore, to assess the lymphocyte subsets and CD4+ T cell expression of IL-17 and transcription factors regulating Th cell differentiation, intracellular cytokine and transcription factor analysis was performed using flow cytometry. There was no significant difference in the CD4:CD8 ratio between saline- and elastase-treated mice or between the two genotypes (Figure 3D), suggesting that neither T-bet nor elastase exposure has a significant impact on the composition of CD4+ and CD8+ T cells in the lungs. The proportion of CD4+ T cells producing IL-17, as well as those positive for retinoic acid receptor-related orphan receptor (ROR)-γt after elastase inoculation, was significantly higher in T-bet−/− mice relative to wild-type mice (Figures 3E and 3F). The fraction of CD4+ T cells positive for T-bet or GATA-3 was not different between saline- and elastase-instilled mice (Figure 3F). The expression of GATA-3 in CD4+ T cells was not different between the two genotypes (Figure 3D). Taken together, these results suggest T-bet decreases elastase-induced IL-6 expression in innate immune cells except for alveolar macrophages as well as subsequent IL-17 expression in CD4+ T cells.
Although T-bet is expressed and is induced by IFN-γ (19) and LPS (20) in macrophages, its role in macrophages has not been well understood. Pancreatic elastase directly induces proinflammatory responses in myeloid cells through TLR4 and NF-κB (21). To investigate the role of T-bet in elastase-induced cytokine expression in macrophages, BM-derived macrophages were generated from wild-type and T-bet−/− mice and their cytokine expression profile was compared. Although elastase exposure induced IL-6 expression in BM-derived macrophages from both genotypes, it was significantly higher in cells from T-bet−/− mice than in cells from wild-type mice (Figure 4A). TNF-α expression was not induced by elastase and not different between two genotypes (Figure 4A). IL-1β expression was upregulated by elastase in cells of both genotypes and was not different between the two genotypes (Figure 4A). These results demonstrate that, whereas IL-6 and IL-1β are induced by elastase, only IL-6 expression is attenuated by T-bet in BM-derived macrophages.
Figure 4. T-bet attenuates cytokine expression depending on the surrounding environment in bone marrow–derived macrophages (BMDMs). (A) The mRNA expression of IL-6, TNF-α, and IL-1β in BMDMs generated from WT and T-bet−/− mice cultured in the absence or the presence of elastase (PPE) for 24 hours, as analyzed by real-time RT-PCR. (B) The mRNA expression of IL-6, TNF-α, IL-1β, transforming growth factor (TGF)-β1 and arginase in BMDMs generated from WT and T-bet−/− mice cultured in medium alone (cont) or in medium with LPS + IFN-γ (M1) or with IL-4 (M2). The presence or absence of elastase (PPE) for 24 hours, as analyzed by real-time RT-PCR. The y-axis of each graph represents the relative expression of the respective genes calculated using the ΔΔCT method and normalized against GAPDH mRNA. Data represent mean ± SEM of four mice in each group. *P < 0.05.
[More] [Minimize]To further investigate the role of T-bet in regulating macrophage phenotype, the gene expression profiles of BM-derived macrophages polarized toward M1 and M2 phenotypes were compared. Under the M1 polarizing condition (LPS + IFN-γ), IL-6, TNF-α, and IL-1β mRNA expression was significantly induced, whereas transforming growth factor (TGF)-β1 mRNA expression was significantly decreased compared with control cells (Figure 4B). On the other hand, under the M2 polarizing condition (IL-4), TGF-β and Arginase mRNA expression was significantly increased, whereas expression of M1 cytokines was not changed in BM-derived macrophages from wild-type mice (Figure 4B). There was no significant difference in any cytokine expression, except for TNF-α expression under the M1 polarizing condition, between wild-type and T-bet−/− mice under each culture condition. These results indicate that T-bet does not regulate cytokine expression upon typical M1 and M2 stimulation conditions, except for TNF-α under the M1 condition, in BM-derived macrophages.
In BM-derived macrophages, T-bet differentially regulates cytokine expression depending on the environment. To further delineate the role for T-bet in macrophages, cytokine expression was analyzed in murine alveolar macrophage cell line MH-S in which T-bet expression level was genetically modified. First, to evaluate the effect of elastase exposure on the activation of T-bet, T-bet nuclear protein levels were examined. T-bet nuclear expression was significantly higher in elastase-exposed cells than in control cells at 24 hours (Figure 5A), suggesting that an activation of T-bet is involved in the elastase-induced inflammatory response. Next, T-bet expression was suppressed by transiently transfecting siRNA. The effect of siRNA was confirmed by real-time RT-PCR (Figure 5B). Knockdown of T-bet expression significantly increased mRNA expression of IL-6, IL-1β, TNF-α, and IL-12p40 relative to those in control cells in the absence of elastase (Figure 5B). Although elastase exposure increased expression of these cytokines in both control and T-bet siRNA transfected cells, each expression level was significantly higher in T-bet siRNA transfected cells than in control cells (Figure 5B). IL-23p19 expression was increased only in T-bet siRNA transfected cells in the presence of elastase (Figure 5B). IL-10 expression was lower, whereas TGF-β expression level was higher, in T-bet siRNA transfected cells than in control cells in the presence of elastase (Figure 5B). To further examine the relationship between T-bet and cytokine expression, the effect of T-bet cDNA transfection was analyzed. Overexpression of T-bet was confirmed by real-time RT-PCR (Figure 5C). In contrast to knockdown experiments, T-bet overexpression led to a significant decrease in mRNA expression of IL-6, IL-1β, TNF-α, and IL-12p40 (Figure 5C). IL-23p19, IL-10, and TGF-β expression is not changed by T-bet overexpression (Figure 5C). Collectively, these results demonstrate that elastase-induced inflammatory cytokine expression is negatively correlated with T-bet expression level in MH-S cells.
Figure 5. T-bet decreases elastase-induced cytokine expression in MH-S cells. (A) Western blotting of T-bet nuclear protein levels in murine alveolar macrophage cell line MH-S cultured in the absence (PPE−) or presence (PPE+) of 5 U/ml elastase for 0, 6, and 24 hours. Densitometric analysis of Western blots was performed and T-bet signal normalized to lamin B signal are presented as mean ± SEM of three samples in each group. (B and C) The mRNA expression of T-bet, IL-6, IL-1β, TNF-α, IL-12p40, IL-23p19, IL-10, and TGF-β1 in MH-S transfected with either control (NC) or T-bet siRNA (B), or with either empty or T-bet cDNA construct (C), was analyzed by real-time RT-PCR. In an siRNA experiment, cells were cultured in the presence (PPE 5) or absence (PPE 0) of 5 U/ml elastase for 24 hours. The y-axis of each graph represents the relative expression of the respective genes calculated using ΔΔCT method and normalized against GAPDH mRNA. Data represent mean ± SEM of three or four samples in each group. *P < 0.05.
[More] [Minimize]Previous reports have shown that T-bet inhibits IL-17 production from CD4+ T cells (12). IL-6 and TGF-β play a crucial role in Th17 differentiation through the upregulation of ROR-γt. The role of macrophages in T-bet modulation of Th17 response remains elusive. To assess the contribution of T-bet in macrophages and lymphocytes, separately, to the development of Th17 response, coculture experiments were performed. Naive splenic CD4+ T cells isolated from either wild-type or T-bet−/− mice were cultured with or without BM-derived macrophages generated from either wild-type or T-bet−/− mice. The concentration of IL-17 in the culture supernatant was significantly lower in the media without BM-derived macrophages (Figure 6), suggesting a pivotal role for the interaction between these cells to induce IL-17. The production of IL-17 was dependent on the genotype of CD4+ T cells, but not on that of macrophages, and was significantly higher in the media of CD4+ T cells from T-bet−/− mice than in those from wild-type mice (Figure 6). These results demonstrate that, under a coculture setting, induction of Th17 response is downregulated by T-bet. Although macrophages are necessary to elicit Th17 response, T-bet regulation of IL-17 expression is mediated by lymphocytes, but not by macrophages.
Figure 6. T-bet in CD4+ T cells, but not in macrophages, downregulates Th17 expression. Concentration of IL-17 in the coculture media of BMDMs generated from WT or T-bet−/− mice and splenic CD4+ cells isolated from WT or T-bet−/− mice. Experiments were performed using 3 biological replicates for each group and repeated twice with similar results. Data represent mean ± SEM of three replicates in each group. *P < 0.05.
[More] [Minimize]To clarify whether enhanced IL-6 and IL-17 expression is inducing the phenotype observed in elastase-treated T-bet−/− mice, either anti–IL-6R or anti–IL-17 neutralizing antibodies were given to T-bet−/− mice. Both of these antibodies significantly attenuated emphysema formation compared with control mice. The values of Lm in IL-6R Ab–treated mice (54.0 ± 6.12 μm) and IL-17 Ab–treated mice (56.7 ± 2.73 μm) were significantly lower than those in control mice (62.5 ± 3.36 μm and 63.1 ± 2.89 μm, respectively) (Figure 7). These results demonstrate that both IL-6 and IL-17 are required to drive emphysema development in T-bet−/− mice.
Figure 7. Anti–IL-6R and anti–IL-17 neutralizing antibodies ameliorate emphysema development in T-bet−/− mice. Anti–IL-6R, anti–IL-17, or their respective control antibodies were administered to T-bet−/− mice inoculated with elastase. After 21 days, the development of emphysema was analyzed. Representative photomicrographs of the lungs and Lm values of alveoli of control (cont) and anti–IL-6R (anti–IL6R) antibody–treated mice (A) and control and anti–IL-17 (anti–IL17) antibody–treated mice (B). Scale bars: 200 μm. Data represent mean ± SEM of four to eight mice in each group. *P < 0.05.
[More] [Minimize]COPD is a progressive lung disease associated with an aberrant innate (22) and adaptive (6) immune response to noxious particles and gases within the lung (2). Dysregulation of the functional phenotype of immune cells, therefore, might be associated with the susceptibility to and/or the severity of COPD. In this study, we demonstrate a novel role for T-bet in attenuating the development of elastase-induced emphysema in mice. T-bet decreases elastase-induced IL-6 expression in the acute phase as well as the subsequent Th17 response in the lungs. Although T-bet does not attenuate IL-6 expression in alveolar macrophages of elastase-instilled mice, it does downregulate elastase-induced expression of IL-6 and other cytokines, depending on the surrounding environment, in BM-derived macrophages and MH-S cells. Both IL-6 and IL-17 play a crucial role in driving emphysema formation.
An inverse correlation between T-bet and IL-6 expression has been demonstrated (12, 23). In T-bet−/− mice, increase in lung IL-6 expression is demonstrated in a murine model of Mycobacterium avium complex disease (24) and asthma (25). In the current study, T-bet deficiency upregulates elastase-induced lung IL-6 expression in the acute phase. Although alveolar macrophages have been shown as the primary source of lung IL-6 in response to various stimuli (26), elastase-induced IL-6 expression is not dependent on T-bet in alveolar macrophages. In recent years, lung macrophages have been recognized to comprise a heterogeneous population, including monocyte-derived and interstitial macrophages, with a distinct functional phenotype (27). In cigarette smoke–induced emphysema, CX3CR1+ interstitial mononuclear phagocytes produce IL-6 and TNF-α and promote emphysema formation (28). Hence, comprehensive analyses of the function of T-bet in each subpopulation of macrophages and other innate immune cells are required to identify the cellular source responsible for enhanced IL-6 expression observed in T-bet−/− mice.
Macrophages acquire a distinct functional phenotype through the activation of the transcription factors according to their tissue microenvironment. M1 macrophages are induced by the activation of STAT1, STAT5, IRF5, and NF-κB, produce proinflammatory cytokines, such as IL-6, IL-1β, TNF-α, IL-12, and IL-23 and low levels of IL-10, and are associated with the development of Th1 and Th17 immunity (29). On the other hand, M2 macrophages are induced by C/EBPβ, IRF4, and IL-6, produce antiinflammatory cytokines, and are involved in the differentiation toward Th2 (30). M1 and M2 are considered to represent two extremes of activation status, and other subsets remain largely unknown. In the current study, we demonstrate a novel role for T-bet in regulating the function of macrophages. In BM-derived macrophages, T-bet decreases TNF-α expression under M1-polarizing conditions without affecting other typical M1 and M2 cytokine expression. On the other hand, T-bet differentially regulates elastase-induced M1-like response in BM-derived macrophages and MH-S cells. These results suggest that T-bet may play a role in regulating M1 cytokine expression, depending on the cell type and the surrounding environment. Investigating the interaction of T-bet with other polarization-related transcription factors may be helpful in understanding the molecular mechanisms of T-bet regulation of macrophage function.
Although T-bet decreases elastase-induced IL-6 expression in both BM-derived macrophages and MH-S cells in vitro, elastase-induced IL-6 expression in alveolar macrophages is not dependent on T-bet ex vivo. Upon elastase inoculation, inflammatory mediators are produced from immune cells and structural cells. Furthermore, lung extracellular matrix is degraded to generate matrikines, which attract immune cells and further enhance inflammatory response (31). These differences in the surrounding environment as well as in the cell type are considered to explain the discrepancy as to the role of T-bet in regulating IL-6 expression in these cells.
Several reports have shown the involvement of Th17 response in the pathogenesis of elastase-induced emphysema and COPD. In either IL-17A– (8) or IL-23 (29) –deficient mice, elastase-induced emphysema formation is attenuated. The expression of Th17-related cytokines is increased in the bronchus in COPD (6). Our study demonstrates that the elastase-induced pulmonary Th17 response is upregulated in T-bet−/− mice. Lineage-specific transcription factors promote the differentiation of naive CD4+ T cells and concomitantly inhibit their differentiation toward other lineages. T-bet promotes Th1 differentiation, while suppressing the polarization toward Th2 and Th17. In T-bet−/− mice, therefore, an exaggerated Th17 response with its significant impact on disease phenotype has been demonstrated in various inflammatory diseases (12, 13, 15). Mechanistic studies have revealed that T-bet interacts with the transcription factor, Runx1, to block its transactivation of the Rorc, which encodes the transcription factor, ROR-γt (12). Furthermore, genome-wide analysis demonstrated that T-bet inhibits Th17 lineage commitment by suppressing IFN regulatory factor-4 (32). These results suggest that T-bet directly modulates the polarization mechanisms in CD4+ T cells to attenuate their differentiation toward Th17.
Recently, a novel macrophage population with a unique phenotype (including IL-12+, IL-1βhigh, IL-6+, TNF-α+, NOS2+, and IL-10high) is demonstrated to induce a Th17 response in Mycobacterium infection (33). However, the role of macrophages in the development of Th17 differentiation largely remains elusive. In our coculture study, IL-17 production is dependent on T-bet expression in CD4+ T cells, but not in macrophages, supporting the notion that T-bet directly attenuates CD4+ T cell polarization to Th17. Further studies are required to examine whether T-bet suppresses Th17 differentiation by modulating the production of IL-6 and other cytokines from macrophage and other innate immune cells.
Lung IL-17 expression was slightly increased in the absence of T-bet 5 days after saline instillation (Figure 3B). In alveolar macrophages, IL-17 expression was not dependent on T-bet (Figure 3C). Although the difference was not statistically significant, the number of BAL lymphocytes (Figure 2) and the proportion of IL-17–producing CD4+ T cells (Figure 3) were slightly higher in T-bet−/− mice, implyng that the absolute number of IL-17–producing CD4+ T cells was possibly increased in T-bet−/− mice. Therefore, although the exact mechanism remains elusive, CD4+ T cells are likely to contribute to the enhanced IL-17 expression in control T-bet−/− mice.
Neutralization studies reveal that both IL-6 and IL-17 play pivotal roles in the pathogenesis of elastase-induced emphysema. Alveolar wall destruction mediated by matrix metalloproteinases (MMPs) and other lytic enzymes leads to permanent airspace enlargement. Among all MMPs, MMP-12 and MMP-9 have been shown to play crucial roles in emphysema development. Mmp12−/− mice are resistant to cigarette smoke–induced emphysema (34). An MMP-9/MMP-12 inhibitor ameliorates cigarette smoke–induced emphysema (35). Several reports have shown the correlation between IL-17 and these MMPs. IL-17 induces MMP-12 expression in human bronchial epithelium (36) and MMP-9 activity in mouse airways (37). IL-17 signaling is required for cigarette smoke–induced MMP-12 induction and emphysema formation (36). Evidence also indicates the role of IL-6 in driving emphysema formation. Mice overexpressing IL-6 in the lungs show emphysema-like airspace enlargement (38). In gp130F/F mice, upregulated IL-6 develops emphysema, which is accompanied by an increased pulmonary apoptosis and an excessive proteinase activity (39). IL-6 recruits neutrophils into the lungs by, as observed in our study, inducing chemokine expression such as MIP-2 (40). Neutrophils might contribute to alveolar destruction by secreting serine proteinases and MMPs (4). Furthermore, IL-6 might affect the development of Th17 differentiation. Therefore, IL-6– and IL-17–dependent induction of proteinases and apoptosis are possibly involved in the mechanisms underlining the aggravated emphysema formation demonstrated in the absence of T-bet.
One of the limitations in our study is the use of elastase-induced emphysema as a model system for COPD. The mechanisms of proteolytic degradation of extracellular matrix is totally different between emphysema induced by elastase and that induced by smoking. In the elastase model, proteinase is delivered into the airways by a single instillation, whereas, in the smoking model, proteinases are persistently delivered by inflammatory cells. Future studies are required to analyze the role of T-bet in the smoking-induced emphysema model, which more closely reflects emphysema in COPD, to obtain more clinically relevant insights. Another limitation is that the molecular mechanisms linking the enhanced lung inflammation to the aggravated emphysema development in elastase-treated T-bet−/− mice remain unknown. Analyzing proteinase and antiproteinase activity is necessary for a greater understanding of the mechanisms of T-bet regulation of emphysema development.
In conclusion, we demonstrate a novel role for T-bet in the pathogenesis of elastase-induced emphysema in mice. T-bet ameliorates emphysema formation by suppressing an inflammatory response in the lungs. Therapy targeting T-bet might be effective against emphysema development by modulating both the innate and the adaptive immune systems, and has to be validated by future study. In addition, further studies are warranted to investigate the role for T-bet as a host factor influencing the progression of COPD.
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Supported in part by Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science 16K09571 (Y. Matsuno).
Author Contributions: Conception and design—S.H. and Y. Matsuno; data collection: S.H. and Y. Matsuno; analysis and interpretation—S.H., Y. Matsuno, Y.T., H.S., T.K., Y. Morishima, Y.I., K.Y., S.T., and N.H.; drafting and editing the manuscript—all authors.
This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1165/rcmb.2018-0109OC on April 9, 2019
Author disclosures are available with the text of this article at www.atsjournals.org.
