Întegrins, matrix metalloproteases (MMPs), and the cytokine TGF-β have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-β exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-β. Because the latent domain of TGF-β1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-β (SLC) to the cell surface where TGF-β activation could be locally controlled. Here, we show that SLC binds to αvβ8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP–dependent release of active TGF-β, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell–matrix interactions.

Squamous metaplasia (SM) is common in smokers and is associated with airway obstruction in chronic obstructive pulmonary disease (COPD). A major mechanism of airway obstruction in COPD is thickening of the small airway walls. We asked whether SM actively contributes to airway wall thickening through alteration of epithelial-mesenchymal interactions in COPD. Using immunohistochemical staining, airway morphometry, and fibroblast culture of lung samples from COPD patients; genome-wide analysis of an in vitro model of SM; and in vitro modeling of human airway epithelial-mesenchymal interactions, we provide evidence that SM, through the increased secretion of IL-1β, induces a fibrotic response in adjacent airway fibroblasts. We identify a pivotal role for integrin-mediated TGF-β activation in amplifying SM and driving IL-1β-dependent profibrotic mesenchymal responses. Finally, we show that SM correlates with increased severity of COPD and that fibroblast expression of the integrin α v β 8 , which is the major mediator of airway fibroblast TGF-β activation, correlated with disease severity and small airway wall thickening in COPD. Our findings have identified TGF-β as a potential therapeutic target for COPD.

cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity.

The airway is a primary portal of entry for noxious environmental stimuli that can trigger airway remodeling, which contributes significantly to airway obstruction in chronic obstructive pulmonary disease (COPD) and chronic asthma. Important pathologic components of airway remodeling include fibrosis and abnormal innate and adaptive immune responses. The positioning of fibroblasts in interstitial spaces suggests that they could participate in both fibrosis and chemokine regulation of the trafficking of immune cells such as dendritic cells, which are crucial antigen-presenting cells. However, physiological evidence for this dual role for fibroblasts is lacking. Here, in two physiologically relevant models -conditional deletion in mouse fibroblasts of the TGF-β-activating integrin αvβ8 and neutralization of αvβ8 in human COPD fibroblasts -we have elucidated a mechanism whereby lung fibroblast chemokine secretion directs dendritic cell trafficking, in a manner that is critically dependent on αvβ8-mediated activation of TGF-β by fibroblasts. Our data therefore indicate that fibroblasts have a crucial role in regulating both fibrotic and immune responses in the lung.

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.

Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor–β (TGF-β) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-β is expressed in a latent form that requires activation. The integrin αvβ8 (encoded by the itgb8 gene) is a receptor for latent TGF-β and is essential for its activation. Expression of integrin αvβ8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvβ8 (B5) inhibited TGF-β activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-β activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvβ8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvβ8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-β pathway to treat fibroinflammatory airway diseases.

Human DNA-PK activates a STING-independent DNA sensing pathway Recognition of foreign nucleic acids is critical for antiviral defense. Detection of DNA is mediated by the cGAS-STING pathway, which activates a potent type I interferon response. This pathway is broadly required for antiviral defense across cell types and species, and its relevance in context of infection, cancer, and autoimmunity has been thoroughly established. However, we have discovered an additional, STING-independent DNA sensing pathway (SIDSP) in human cells. Using STING KO human cell lines, we find that the induction of interferon is indeed abrogated at early timepoints, but surprisingly, at later timepoints, we observe a robust DNAinduced interferon response. Here we identify DNA-PK as the sensor for the SIDSP and demonstrate that its kinase activity is required for the antiviral response. We show that a heat shock protein HSPA8/HSC70 is phosphorylated after DNA stimulation and acts as a marker for the SIDSP. Finally, we explore how DNA viruses antagonize both the cGAS-STING pathway and the SIDSP. Our work highlights the importance of nucleic acid sensing for both host and virus and has implications for modulating DNA sensing in order to improve therapies for cancer or autoimmunity.

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three ␣v integrins, ␣v␤1, ␣v␤3, and ␣v␤6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human ␤8 cDNA and expression of ␣v␤8 at the cell surface. The involvement of ␣v␤8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the ␣v␤8 heterodimer or the ␣v chain. Similar results were obtained with a chimeric ␣v␤8 including the ␤6 cytodomain (␣v␤8/6), showing that the ␤6 cytodomain can substitute efficiently for the corresponding region of ␤8. In contrast, virus binding to ␣v␤6 including the ␤8 cytodomain (␣v␤6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the ␣v␤6 chimera was recognized poorly by antibodies specific for the ectodomain of ␣v␤6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the ␤6 cytodomain is important for maintaining ␣v␤6 in a conformation required for productive infection by FMDV.Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease, a severe vesicular disease of cloven-hoofed animals including domesticated ruminants and pigs. The virus exists as seven serotypes, which are members of the genus Aphthovirus of the family Picornaviridae (35). The virion consists of an 8.5-kb strand of RNA enclosed within an icosahedral capsid formed from 60 copies each of four proteins, VP1 to VP4 (1).Two classes of cell surface receptors that mediate FMDV infection have been identified (30). Theses are the integrins (7, 31, 33) and heparan sulfate (HS) proteoglycans (HSPGs) (29). The ability to use HSPGs as receptors appears to be restricted to strains of FMDV that have been multiply passaged through cultured cell lines (4,5,22,41,52,58), and presently there is no convincing evidence of a role for HS in cell entry by field viruses. Instead, field viruses are dependent on integrin receptors to initiate infection in vitro, and integrins are believed to be the receptors used in the infected animal. Recently, two independent studies have shown that certain strains of FMDV can infect cultured cells via an entry pathway that is independent of both integrins and cellular HS, implying the existence of a third, as yet unidentified receptor family (4, 65).Integrins are a family of integral membrane receptors with distinct ligand-binding specificities and tissue distributions. They contribute to a variety of cellular functions, including cell-cell and cell-matrix adhesion, and exist in alternative lowand high-affinity states, enab...