Integrins are conformationally flexible cell surface receptors that survey the extracellular environment for their cognate ligands. Interactions with ligands are thought to be linked to global structural rearrangements involving transitions between bent, extended-closed and extended-open forms. Thus far, structural details are lacking for integrins in the extended conformations due to extensive flexibility between the headpiece and legs in this conformation. Here we present single-particle electron cryomicroscopy structures of human αvβ8 integrin in the extended-closed conformation, which has been considered to be a low-affinity intermediate. Our structures show the headpiece rotating about a flexible αv knee, suggesting a ligand surveillance mechanism for integrins in their extended-closed form. Our model predicts that the extended conformation is mainly stabilized by an interface formed between flexible loops in the upper and lower domains of the αv leg. Confirming these findings with the αvβ3 integrin suggests that our model of stabilizing the extended-closed conformation is generalizable to other integrins.

Integrin αvβ8 binds with exquisite specificity to latent transforming growth factor-β (L-TGF-β). This binding is essential for activating L-TGF-β presented by a variety of cell types. Inhibiting αvβ8-mediated TGF-β activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvβ8 ectodomain and its intact natural ligand, L-TGF-β, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvβ8 binding specificity and TGF-β activation. Our studies reveal a mechanism of TGF-β activation where mature TGF-β signals within the confines of L-TGF-β and the release and diffusion of TGF-β are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvβ8-mediated L-TGF-β activation.

Small airway fibrosis is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Chronic inflammatory cells accumulate around small airways in COPD and are thought to play a major role in small airway fibrosis. Mice deficient in α/β T cells have recently been shown to be protected from both experimental airway inflammation and fibrosis. In these models, CD4+Th17 cells and secretion of IL-17A are increased. However, a pathogenic role for IL-17 in specifically mediating fibrosis around airways has not been demonstrated. Here a role for IL-17A in airway fibrosis was demonstrated using mice deficient in the IL-17 receptor A (il17ra) Il17ra-deficient mice were protected from both airway inflammation and fibrosis in two different models of airway fibrosis that employ COPD-relevant stimuli. In these models, CD4+ Th17 are a major source of IL-17A with other expressing cell types including γδ T cells, type 3 innate lymphoid cells, polymorphonuclear cells, and CD8+ T cells. Antibody neutralization of IL-17RA or IL-17A confirmed that IL-17A was the relevant pathogenic IL-17 isoform and IL-17RA was the relevant receptor in airway inflammation and fibrosis. These results demonstrate that the IL-17A/IL-17 RA axis is crucial to murine airway fibrosis. These findings suggest that IL-17 might be targeted to prevent the progression of airway fibrosis in COPD.

Tobacco smoke-induced accelerated cell senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cell senescence is accompanied by the accumulation of damaged cellular components suggesting that in COPD, inhibition of autophagy may contribute to cell senescence. Here we look at whether autophagy contributes to cigarette smoke extract (CSE) - induced cell senescence of primary human bronchial epithelial cells (HBEC), and further evaluate p62 and ubiquitinated protein levels in lung homogenates from COPD patients. We demonstrate that CSE transiently induces activation of autophagy in HBEC, followed by accelerated cell senescence and concomitant accumulation of p62 and ubiquitinated proteins. Autophagy inhibition further enhanced accumulations of p62 and ubiquitinated proteins, resulting in increased senescence and senescence-associated secretory phenotype (SASP) with interleukin (IL)-8 secretion. Conversely, autophagy activation by Torin1, a mammalian target of rapamycin (mTOR inhibitor), suppressed accumulations of p62 and ubiquitinated proteins and inhibits cell senescence. Despite increased baseline activity, autophagy induction in response to CSE was significantly decreased in HBEC from COPD patients. Increased accumulations of p62 and ubiquitinated proteins were detected in lung homogenates from COPD patients. Insufficient autophagic clearance of damaged proteins, including ubiquitinated proteins, is involved in accelerated cell senescence in COPD, suggesting a novel protective role for autophagy in the tobacco smoke-induced senescence-associated lung disease, COPD.

TGF-β is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-β (L-TGF-β) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-β. Binding of L-TGF-β to integrin αvβ8 results in activation of TGF-β. We engineered and used αvβ8 antibodies optimized for blocking or detection, which - respectively - inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect β8 in human tumors. Inhibition of αvβ8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers - effects that are independent of PD-1/PD-L1. β8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-β, suggesting that tumor cell αvβ8 serves as a platform for activating cell-surface L-TGF-β presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to β8 inhibition with major increases in chemokine and tumor-eliminating genes. High β8 expression in tumor cells is seen in 20%-80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvβ8 is a PD-1/PD-L1-independent immunotherapeutic target.

Regulatory T cells (T_regs) that promote tumor immune evasion are enriched in certain tumors and correlate with poor prognosis. However, mechanisms for T_reg enrichment remain incompletely understood. We described a mechanism for T_reg enrichment in mouse and human tumors mediated by the αvβ8 integrin. Tumor cell αvβ8 bound to latent transforming growth factor-β (L-TGF-β) presented on the surface of T cells, resulting in TGF-β activation and immunosuppressive T_reg differentiation in vitro. In vivo, tumor cell αvβ8 expression correlated with T_reg enrichment, immunosuppressive T_reg gene expression, and increased tumor growth, which was reduced in mice by αvβ8 inhibition or T_reg depletion. Structural modeling and cell-based studies suggested a highly geometrically constrained complex forming between αvβ8-expressing tumor cells and L-TGF-β-expressing T cells, facilitating TGF-β activation, independent of release and diffusion, and providing limited access to TGF-β inhibitors. These findings suggest a highly localized tumor-specific mechanism for T_reg enrichment.

Cigarette smoke (CS)-induced accumulation of mitochondrial damage has been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Mitophagy plays a crucial role in eliminating damaged mitochondria, and is governed by the PINK1 (PTEN induced putative protein kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase) pathway. Although both increased PINK1 and reduced PRKN have been implicated in COPD pathogenesis in association with mitophagy, there are conflicting reports for the role of mitophagy in COPD progression. To clarify the involvement of PRKN-regulated mitophagy in COPD pathogenesis, prkn knockout (KO) mouse models were used. To illuminate how PINK1 and PRKN regulate mitophagy in relation to CS-induced mitochondrial damage and cellular senescence, overexpression and knockdown experiments were performed in airway epithelial cells (AEC). In comparison to wild-type mice, prkn KO mice demonstrated enhanced airway wall thickening with emphysematous changes following CS exposure. AEC in CS-exposed prkn KO mice showed accumulation of damaged mitochondria and increased oxidative modifications accompanied by accelerated cellular senescence. In vitro experiments showed PRKN overexpression was sufficient to induce mitophagy during CSE exposure even in the setting of reduced PINK1 protein levels, resulting in attenuation of mitochondrial ROS production and cellular senescence. Conversely PINK1 overexpression failed to recover impaired mitophagy caused by PRKN knockdown, indicating that PRKN protein levels can be the rate-limiting factor in PINK1-PRKN-mediated mitophagy during CSE exposure. These results suggest that PRKN levels may play a pivotal role in COPD pathogenesis by regulating mitophagy, suggesting that PRKN induction could mitigate the progression of COPD. Abbreviations: AD: Alzheimer disease; AEC: airway epithelial cells; BALF: bronchoalveolar lavage fluid; AKT: AKT serine/threonine kinase; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; COPD: chronic obstructive pulmonary disease; CS: cigarette smoke; CSE: CS extract; CXCL1: C-X-C motif chemokine ligand 1; CXCL8: C-X-C motif chemokine ligand 8; HBEC: human bronchial epithelial cells; 4-HNE: 4-hydroxynonenal; IL: interleukin; KO: knockout; LF: lung fibroblasts; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; OPTN: optineurin; PRKN: parkin RBR E3 ubiquitin protein ligase; PCD: programmed cell death; PFD: pirfenidone; PIK3C: phosphatidylinositol-4:5-bisphosphate 3-kinase catalytic subunit; PINK1: PTEN induced putative kinase 1; PTEN: phosphatase and tensin homolog; RA: rheumatoid arthritis; ROS: reactive oxygen species; SA-GLB1/β-Gal: senescence-associated-galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SNP: single nucleotide polymorphism; TNF: tumor necrosis factor.

TGF-β, essential for development and immunity, is expressed as a latent complex (L-TGF-β) non-covalently associated with its prodomain and presented on immune cell surfaces by covalent association with GARP. Binding to integrin αvβ8 activates L-TGF-β1/GARP. The dogma is that mature TGF-β must physically dissociate from L-TGF-β1 for signaling to occur. Our previous studies discovered that αvβ8-mediated TGF-β autocrine signaling can occur without TGF-β1 release from its latent form. Here, we show that mice engineered to express TGF-β1 that cannot release from L-TGF-β1 survive without early lethal tissue inflammation, unlike those with TGF-β1 deficiency. Combining cryogenic electron microscopy with cell-based assays, we reveal a dynamic allosteric mechanism of autocrine TGF-β1 signaling without release where αvβ8 binding redistributes the intrinsic flexibility of L-TGF-β1 to expose TGF-β1 to its receptors. Dynamic allostery explains the TGF-β3 latency/activation mechanism and why TGF-β3 functions distinctly from TGF-β1, suggesting that it broadly applies to other flexible cell surface receptor/ligand systems.