1Apoptotic cell-directed resolution of lung inflammation requires myeloid αv integrin-mediated induction of regulatory T lymphocytes Ailiang Zhang*, Adam Lacy-Hulbert†, Stephen Anderton*, Christopher Haslett*, John Savill*¶* Medical Research Council Centre for Inflammation Research University of Edinburgh, Queen's Medical Research Institute Edinburgh BioQuarter, 47 Little France Crescent Edinburgh, EH16 4TJ, UK † Benaroya Research Institute at Virginia Mason 1201 Ninth Avenue Seattle, WA 98101, USA ¶ Address Correspondence to John Savill Queen's Medical Research Institute, Edinburgh BioQuarter, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK E-mail: John.email@example.com This paper includes 29 text pages and 6 figures. Short Running title: Apoptotic cells resolve lung inflammation via αv and Tregs This work was supported by a grant from the UK Medical Research Council to J.S. and C.H. (G0802069); and by a grant from NIH to A.L-H. (U19 AI125378). Disclosures: The authors declare they have no conflicting interests.
2Abstract Intratracheal instillation of apoptotic cells enhances resolution of experimental lung inflammation by incompletely understood mechanisms. We report that this intervention induces functional regulatory T lymphocytes (Tregs) in mouse lung experimentally inflamed by intratracheal administration of lipopolysaccharide (LPS). Selective depletion demonstrated that Tregs were necessary for maximal apoptotic cell-directed enhancement of resolution and adoptive transfer of additional Tregs was sufficient to promote resolution without administering apoptotic cells. After intratracheal instillation labelled apoptotic cells were observed in the majority of CD11c+CD103+ myeloid dendritic cells (DCs) migrating to mediastinal draining lymph nodes and bearing migratory and immunoregulatory markers including increased CCR7 and β8 integrin (ITGB8) expression. In mice deleted for αv integrin in the myeloid line so as to reduce phagocytosis of dying cells by CD103+DCs, exogenous apoptotic cells failed to induce TGF-β1 expression or Treg accumulation and also failed to enhance resolution of LPS-induced lung inflammation. We conclude that in murine lung, myeloid phagocytes encountering apoptotic cells can deploy αv integrin-mediated mechanisms to induce Tregs and enhance resolution of acute inflammation. (word count=169)
3Introduction Although acute inflammation often resolves, persistent inflammation is a common and frequently intractable clinical problem.1-3 Nevertheless a growing body of data indicate that inflammation may normally be kept in check by safe immunosuppressive clearance of cells dying by apoptosis.4-10 We and others have employed in vitro and invivo approaches to demonstrate that myeloid phagocytes (both macrophages and immature dendritic cells, [DCs]) employ various molecular complexes assembled by phagocyte cell surface αv integrin to bind cells dying by apoptosis11-16 and trigger anti-inflammatory responses such as elaboration of active TGF-β1 and induction of CD4+CD25+FoxP3+ regulatory T lymphocytes (Tregs) by CD103+ DCs.11, 17-20 A substantial body of data implicates αvβ3 and αvβ5 integrins in phagocytosis of apoptotic cells by macrophages and DCs respectively, while myeloid αvβ8 integrin is required for efficient production of active TGF-β1 from the surface-bound latent form, a key immunosuppressive consequence of phagocytic clearance of cells dying by apoptosis.11-20 However, whether such αv-dependent mechanisms can be deployed in the acutely injured lung to promote resolution of inflammation has been unknown. Inhalation of bacterial endotoxins such as lipopolysaccharide (LPS) can cause lung inflammation and is implicated in a range of lung diseases affecting humans, such as various occupational dust disorders,21 and animals, for example equine heaves.22 Therefore, many have sought to dissect mechanisms of lung inflammation and its resolution by study of self-limited experimental lung inflammation induced by intratracheal (i.t.) administration of LPS. In this clinically-relevant model there is circumstantial evidence that clearance of cells dying by apoptosis is indeed important in directing resolution of acute lung inflammation. Huynh and colleagues10 demonstrated that i.t. administration of apoptotic cells enhanced resolution of LPS-induced lung inflammation in a TGF-β1-dependent manner. Although TGF-β1 is
4now widely recognized as a key stimulus inducing Tregs,23 the role of such lymphocytes was not dissected by these investigators. Nevertheless, although D’Alessio and colleagues24 did not examine effects of exogenous apoptotic cells, they did elegantly employ loss-of-function and gain-of-function approaches to demonstrate that Tregs are crucial for resolution of LPS-induced lung inflammation and are associated with increased TGF-β1 production and enhanced clearance of leucocytes by apoptosis and subsequent phagocytosis. Nevertheless, it was not known whether induction of Tregs is required for exogenous apoptotic cells to direct enhanced resolution of LPS-driven acute lung inflammation. In this study we demonstrate that intratracheal administration of exogenous apoptotic cells not only enhances resolution of LPS-induced lung inflammation, but also induces functional Tregs in the lung capable of suppressing T cell proliferation in mixed cell culture. By selective inducible deletion of Tregs,25 we confirmed that Tregs are necessary for maximalenhancement by administered apoptotic cells of resolution of lung inflammation. We also deployed adoptive transfer to demonstrate that Tregs are sufficient for enhanced resolution, being able to substitute for exogenous apoptotic cells in promoting resolution of LPS-induced lung inflammation in wild type mice. In view of strong evidence in the gut that Tregs are induced in draining lymph nodes by immunoregulatory CD103+ myeloid dendritic cells that have migrated from the gut wall having taken up cells dying by apoptosis at that site,26-32 we tracked the fate of labelled exogenous apoptotic cells administered intratracheally. We found that the majority of CD11c+CD103+ lung DCs migrating to draining mediastinal lymph nodes had ingested exogenous apoptotic cells and had acquired a migratory immunoregulatory phenotype expressing CCR7 and β8 integrin (ITGB8). Furthermore, in view of evidence that myeloid αv integrin is crucial for the induction of Tregs byimmunoregulatory CD103+DCs that have ingested apoptotic cells,11, 18, 20, 32 we studied mice selectively deleted for αv in the myeloid line, which is thought in myeloid DCs to inhibit