Our data from physiologically relevant models collectively provide functional and mechanistic in vivo evidence for a central role of the fibroblast in regulating pathologic innate and adaptive immunity. Specifically, conditional deletion of the TGF-β–activating integrin αvβ8 selectively in adult fibroblasts reduced IL-1β–induced and allergen-induced airway remodeling and secretion of CCL2 and CCL20. This cascade of events was associated with decreased DC numbers in the draining lymph node and subsequently muted adaptive immunity. We extended these studies to human disease by demonstrating that enhancement of αvβ8-mediated TGF-β activation, CCL20 expression, and DC migration is a feature of COPD fibroblasts. To further support our hypothesis that enhanced integrin β8–dependent DC trafficking in human airways may facilitate the abnormal persistent innate and adaptive immune response that characterizes human COPD, we found that increased DC accumulation in COPD airways correlates with both the stage of COPD and β8 expression.
The most likely explanation for reduction in the number of migratory DCs in the lung and lymph node that we observe with fibroblast Itgb8
deletion is altered chemokine secretion from fibroblasts. We determined that CCL2 and CCL20 are secreted at high levels by IL-1β–stimulated lung fibroblasts — 1,000 and 30 times, respectively, the levels reported to be released by cultured human airway epithelial cells stimulated with the same dose of IL-1β, on a per-cell basis, suggesting a dominant role for fibroblasts as a source of these chemokines (58
). Our data demonstrating αvβ8-dependent CCL2 and CCL20 expression levels in lung homogenates and BAL suggest that the respective roles for CCL2 and CCL20 may differ in different disease models, with CCL2 and CCL20 playing larger roles in allergic asthma and COPD, respectively.
Fibroblasts have been hypothesized to modulate immune responses (60
), but clear in vivo demonstration of this function, and the mechanisms underlying this modulation, are lacking. Previous studies have shown that fibroblastic reticular cells, which are fibroblast-like cells of lymphoid organs, secrete chemokines and thus are thought to act as “organizers” of lymphoid tissues (61
). Cytokines are released by fibroblasts in patterns that distinguish fibroblasts from different anatomic sites and disease states (33
). These studies validate and extend recent in vitro studies suggesting that cultured skin fibroblasts stimulated with TNF/IL-1 can promote DC migration or increase the maturation of LPS-stimulated DCs by a complex feedback mechanism involving IL-1β secreted from DCs (62
). Our data now demonstrate that in physiologic models, fibroblast Itgb8
is required for lung DC migration. We could not detect a role for Itgb8
on fibroblasts in lung DC maturation.
The integrins αvβ6 and αvβ8 account for the majority of TGF-β activation in murine genetic models (49
). However, fibroblast deletion of Itgb8
appeared to account for the majority of protection against airway remodeling in chronic asthma and Ad-IL-1β models. In addition, Ad-IL-1β did not increase the expression of Itgb6
, and a neutralizing anti-αvβ6 antibody had no effect on Ad-IL-1β–mediated airway remodeling. These findings support genetic studies suggesting that Itgb6
is not an asthma or a COPD target, as mice deficient in Itgb6
develop airway hyperresponsiveness and emphysema (64
). The striking role for Itgb8
in promoting airway remodeling and lack of a role for Itgb6
may be due to differences in cell type distribution or differences in mechanisms of TGF-β activation. Integrin αvβ8 is expressed in the lung by epithelial cells and fibroblasts, while αvβ6 is expressed exclusively by epithelial cells (66
). Integrin αvβ8 binds to latent TGF-β, and following the recruitment of a transmembrane metalloprotease, MMP14, active TGF-β is proteolytically released as a paracrine factor (48
). In contrast, the integrin αvβ6 binds to latent TGF-β only on epithelial cells and alters the conformation of the LAP, and can only present active TGF-β to adjacent cells (67
). Therefore, it appears that localized epithelial activation of TGF-β is insufficient, while fibroblast activation and paracrine secretion of TGF-β are required for airway remodeling.
IT-Ad-IL-1β increased active TGF-β in the BAL fluid, which was blocked by 61% with simultaneous injection of Ad-Cre. This incomplete blockade was seen despite efficient reduction of Itgb8
mRNA by Ad-Cre. Because suitable reagents are not available to assess mouse αvβ8 protein, we cannot exclude that residual αvβ8 protein remains after Ad-Cre–mediated deletion. Alternatively, other mechanisms may contribute to TGF-β activation within the alveolar lining fluid. For instance, previous literature suggests that additional integrins (i.e., αvβ3 and αvβ5) mediate activation of TGF-β in cultured fibroblasts under specific circumstances (68-72). However, αvβ3 and αvβ5 bind poorly to latent TGF-β relative to αvβ8 and αvβ6 (48
), and mice with deficiency of αvβ3, αvβ5, or αvβ3 and αvβ5 combined develop normally and have no evidence of TGF-β insufficiency (74
). Finally, in our models, αvβ8 appears to account for the majority if not all of TGF-β effects, since Itgb8fko
mice have a protective effect equal to if not a greater than a pan–TGF-β blocking antibody from IL-1β–mediated inflammation and fibrosis. Therefore, neither αvβ3, αvβ5, nor αvβ6 compensate for loss of αvβ8 in airway remodeling, in vivo.
Fibroblasts can be derived by epithelial-mesenchymal transition, from circulating fibrocytes, or from resident fibroblasts (75
). We have not formally addressed the source of the fibroblasts in airway remodeling in this study. However, resident fibroblasts are likely a major source of fibroblasts in airway remodeling, because we saw no obvious epithelial or hematopoietic cell recombination in Col-Cre-ER(T) mice. Furthermore, primary cultures of lung fibroblasts, which are derived from resident fibroblasts, possess all the functional characteristics to explain the airway remodeling findings in vivo; they increase expression of αvβ8 in response to IL-1β, support αvβ8-mediated TGF-β activation, and secrete CCL2, CCL20, and collagen in an αvβ8-dependent manner.
Airway fibroblasts interact not only with immune cells, but also with adjacent airway epithelial cells to maintain homeostasis. Airway epithelial cells have recently been implicated in modulating adaptive immune responses through TLR4-dependent pathways. This recent study has shown that TLR4 expressed by airway epithelial cells is required for LPS- or allergen-induced pathology, including increases in lung DC numbers, maturation, migration to MLN, and CCL2 and CCL20 expression (76
). The intriguing similarity of these findings to the findings that we present here support the hypothesis that excessive pattern recognition receptor engagement plays a significant role in airway remodeling in COPD and chronic asthma by caspase-1–mediated increased bioactive IL-1β secretion by airway epithelial cells or macrophages; this leads to the induction of αvβ8 expression and subsequent TGF-β activation by adjacent fibroblasts, which affects fibroblast chemokine secretion, DC migration, and adaptive immunity (Figure ). However, we cannot exclude that other cytokines also influence αvβ8 expression, or that IL-1β influences αvβ8 activity (i.e., through affecting integrin activation state, or localization or activity of MMP14).
Model for fibroblast αvβ8–directed DC migration in airway remodeling.
IT delivery of active IL-1β by the adenoviral method bypasses the requirement for IL-1β activation by the inflammasome. We have exploited the Ad-IL-1β model to mechanistically dissect the pathway that is downstream of IL-1β activation and common to the pathogenic environmental stimuli that lead to airway remodeling in COPD and chronic asthma (12
). The Ad-IL-1β model has many physiologically relevant features, since it recapitulates most of the morphologic and immunologic characteristics of airway remodeling in COPD in humans. Specifically, there are increases in airway wall fibrosis and airway wall inflammation; and a COPD-like innate and adaptive immune response, with increased neutrophils, mDCs, and Th17 and Th1 cells and increased production of IL-17, CCL2 and CCL20 (20
The use of this system has provided insights into the proinflammatory role of Itgb8
and TGF-β in the lung and in COPD. TGF-β has recently been implicated as a driver of pathologic immunity through its critical role in the development of Th17 cells. This important subset of CD4+
T cells produces IL-17(A,F), a cytokine that has been implicated in the pathogenesis of asthma, COPD, psoriasis, arthritis, diabetes, multiple sclerosis, and inflammatory bowel disease (77
). Two recent studies demonstrate that the loss of αvβ8 on DCs, which are crucial APCs, protects against experimental autoimmune encephalitis through inhibition of Th17 cell differentiation (51
). In the fibroblast conditional deletion model, there is no evidence of Cre-mediated deletion of Itgb8
by purified lung DCs; instead, loss of Itgb8
on fibroblasts indirectly impairs DC function through decreased trafficking to lymph nodes. Thus, Itgb8
participates in proinflammatory responses through two distinct mechanisms in different conditional deletion models, one that indirectly and one that directly influences the functional capacity of DCs to prime a pathologic immune response. Our data suggest that inhibition of integrin αvβ8–mediated TGF-β activation by lung fibroblasts represents a novel therapeutic target for airway remodeling in both COPD and asthma.