Charles L. Hardy

The roles of activin A and its binding protein, follistatin, in inflammation and tissue repair.

Activin A, a member of the transforming growth factor-β superfamily of cytokines, is a critical controller of inflammation, immunity and fibrosis. It is rapidly released into the blood following a lipopolysaccharide challenge in experimental animals, through activation of the Toll-like receptor 4 signalling pathway. Blocking activin action by pre-treatment with its binding protein, follistatin, modifies the inflammatory cytokine cascade, and reduces the severity of the subsequent inflammatory response and mortality. Likewise, high serum levels of activin A are predictive of death in patients with septicaemia. However, activin A has complex immunomodulatory actions. It is produced by inflammatory macrophages, but can regulate either pro- or anti-inflammatory responses in these cells, depending on their prior activation status. Activin A is also produced by Th2 cells, and stimulates antibody production by B cells and the development of regulatory T cells. Production of activin A during inflammatory responses stimulates fibrosis and tissue remodelling, and follistatin inhibits these actions of activin A. The modulation of activin by follistatin may represent an important therapeutic target for the modulation and amelioration of inflammatory and fibrotic disorders.

Promising particle-based vaccines in cancer therapy

Immunotherapy and preventative cancer vaccines offer the hope of controlling cancer in humans with few of the undesirable side effects associated with current chemotherapy-based methods. Particulate vaccines are effectively taken up by dendritic cells, inducing both T-cell and antibody responses. Virus-like particles (VLPs) have shown preventive efficacy against cervical cancer. Herein we review a range of leading particle-based vaccine approaches: VLPs, immunostimulating complexes, liposomes, synthetic nanoparticles and microparticles (both biocompatible and biodegradable, such as polylactide-co-glycolides and poly[D,L-lactic-co-glycolic] acid). Immune efficacy, regulatory and safety issues, as well the application of immunotherapeutics to immunosuppressed patients with high levels of Tregs are also discussed. We argue that developmental issues (cost and intellectual property lifespan) and the lack of reliable preclinical animal models, rather than the lack of innovative vaccine approaches, currently present a major obstacle to rapid and effective vaccine development.

Glucocorticoid receptor deficient thymic and peripheral T cells develop normally in adult mice

The involvement of glucocorticoid receptor (GR) signaling in T cell development is highly controversial, with several studies for and against. We have previously demonstrated that GR–/– mice, which usually die at birth because of impaired lung development, exhibit normal T cell development, at least in embryonic mice and in fetal thymus organ cultures. To directly investigate the role of GR signaling in adult T cell development, we analyzed the few GR–/– mice that occasionally survive birth, and irradiated mice reconstituted with GR–/– fetal liver precursors. All thymic and peripheral T cells, as well as other leukocyte lineages, developed and were maintained at normal levels. Anti‐CD3‐induced cell death of thymocytes in vitro, T cell repertoire heterogeneity and T cell proliferation in response to anti‐CD3 stimulation were normal in the absence of GR signaling. Finally, we show that metyrapone, an inhibitor of glucocorticoid synthesis (commonly used to demonstrate a role for glucocorticoids in T cell development), impaired thymocyte development regardless of GR genotype indicating that this reagent inhibits thymocyte development in a glucocorticoid‐independent fashion. These data demonstrate that GR signaling is not required for either normal T cell development or peripheral maintenance in embryonic or adult mice.

Differential Uptake of Nanoparticles and Microparticles by Pulmonary APC Subsets Induces Discrete Immunological Imprints

There is increasing interest in the use of engineered particles for biomedical applications, although questions exist about their proinflammatory properties and potential adverse health effects. Lung macrophages and dendritic cells (DC) are key regulators of pulmonary immunity, but little is known about their uptake of different sized particles or the nature of the induced immunological imprint. We investigated comparatively the immunological imprints of inert nontoxic polystyrene nanoparticles 50 nm in diameter (PS50G) and 500 nm in diameter (PS500G). Following intratracheal instillation into naive mice, PS50G were preferentially taken up by alveolar and nonalveolar macrophages, B cells, and CD11b+ and CD103+ DC in the lung, but exclusively by DC in the draining lymph node (LN). Negligible particle uptake occurred in the draining LN 2 h postinstillation, indicating that particle translocation does not occur via lymphatic drainage. PS50G but not PS500G significantly increased airway levels of mediators that drive DC migration/maturation and DC costimulatory molecule expression. Both particles decreased frequencies of stimulatory CD11b+MHC class IIhi allergen-laden DC in the draining LN, with PS50G having the more pronounced effect. These distinctive particle imprints differentially modulated induction of acute allergic airway inflammation, with PS50G but not PS500G significantly inhibiting adaptive allergen-specific immunity. Our data show that nanoparticles are taken up preferentially by lung APC stimulate cytokine/chemokine production and pulmonary DC maturation and translocate to the lung-draining LN via cell-associated transport. Collectively, these distinctive particle imprints differentially modulate development of subsequent lung immune responses. These findings support the development of lung-specific particulate vaccines, drug delivery systems, and immunomodulators.

Inert 50-nm Polystyrene Nanoparticles That Modify Pulmonary Dendritic Cell Function and Inhibit Allergic Airway Inflammation

Nanoparticles are being developed for diverse biomedical applications, but there is concern about their potential to promote inflammation, particularly in the lung. Although a variety of ambient, anthropogenic and man-made nanoparticles can promote lung inflammation, little is known about the long-term immunomodulatory effects of inert noninflammatory nanoparticles. We previously showed polystyrene 50-nm nanoparticles coated with the neutral amino acid glycine (PS50G nanoparticles) are not inflammatory and are taken up preferentially by dendritic cells (DCs) in the periphery. We tested the effects of such nanoparticles on pulmonary DC function and the development of acute allergic airway inflammation. Surprisingly, exposure to PS50G nanoparticles did not exacerbate but instead inhibited key features of allergic airway inflammation including lung airway and parenchymal inflammation, airway epithelial mucus production, and serum allergen-specific IgE and allergen-specific Th2 cytokines in the lung-draining lymph node (LN) after allergen challenge 1 mo later. PS50G nanoparticles themselves did not induce lung oxidative stress or cardiac or lung inflammation. Mechanistically, PS50G nanoparticles did not impair peripheral allergen sensitization but exerted their effect at the lung allergen challenge phase by inhibiting expansion of CD11c+MHCIIhi DCs in the lung and draining LN and allergen-laden CD11bhiMHCIIhi DCs in the lung after allergen challenge. PS50G nanoparticles further suppressed the ability of CD11bhi DCs in the draining LN of allergen-challenged mice to induce proliferation of OVA-specific CD4+ T cells. The discovery that a defined type of nanoparticle can inhibit, rather than promote, lung inflammation via modulation of DC function opens the door to the discovery of other nanoparticle types with exciting beneficial properties.

The activin A antagonist follistatin inhibits asthmatic airway remodelling.

Background Current pharmacotherapy is highly effective in the clinical management of the majority of patients with stable asthma, however severe asthma remains inadequately treated. Prevention of airway remodelling is a major unmet clinical need in the management of patients with chronic severe asthma and other inflammatory lung diseases. Accumulating evidence convincingly demonstrates that activin A, a member of the transforming growth factor (TGF)-β superfamily, is a key driver of airway inflammation, but its role in chronic asthmatic airway remodelling is ill-defined. Follistatin, an endogenously produced protein, binds activin A with high affinity and inhibits its bioactivity. The aim of this study was to test the potential of follistatin as a therapeutic agent to inhibit airway remodelling in an experimental model of chronic allergic airway inflammation. Methods BALB/c mice were systemically sensitised with ovalbumin (OVA), and challenged with OVA intranasally three times a week for 10 weeks. Follistatin was instilled intranasally during allergen challenge. Results Chronic allergen challenge induced mucus hypersecretion and subepithelial collagen deposition which persisted after cessation of challenge. Intranasal follistatin (0.05, 0.5, 5 µg) inhibited the airway remodelling and dose-dependently decreased airway activin A and TGF-β1, and allergen-specific T helper 2 cytokine production in the lung-draining lymph nodes. Follistatin also impaired the loss of TGF-β1 and activin RIB immunostaining in airway epithelium which occurred following chronic allergen challenge. Conclusions These data demonstrate that follistatin attenuates asthmatic airway remodelling. Our findings point to the potential of follistatin as a therapeutic for prevention of airway remodelling in asthma and other inflammatory lung diseases.

The effects of engineered nanoparticles on pulmonary immune homeostasis.

Abstract Engineered nanoparticles (ENP), which could be composed of inorganic metals, metal oxides, metalloids, organic biodegradable and inorganic biocompatible polymers, are being used as carriers for vaccine and drug delivery. There is also increasing interest in their application as delivery agents for the treatment of a variety of lung diseases. Although many studies have shown ENP can be effectively and safely used to enhance the delivery of drugs and vaccines in the periphery, there is concern that some ENP could promote inflammation, with unknown consequences for lung immune homeostasis. In this study, we review research on the effects of ENP on lung immunity, focusing on recent studies using diverse animal models of human lung disease. We summarize how the inflammatory and immune response to ENP is influenced by the diverse biophysical and chemical characteristics of the particles including composition, size and mode of delivery. We further discuss newly described unexpected beneficial properties of ENP administered into the lung, where biocompatible polystyrene or silver nanoparticles can by themselves decrease susceptibility to allergic airways inflammation. Increasing our understanding of the differential effects of diverse types of nanoparticles on pulmonary immune homeostasis, particularly previously underappreciated beneficial outcomes, supports rational ENP translation into novel therapeutics for prevention and/or treatment of inflammatory lung disorders.

The immunoregulatory and fibrotic roles of activin A in allergic asthma

Activin A, a member of the TGF‐β superfamily of cytokines, was originally identified as an inducer of follicle stimulating hormone release, but has since been ascribed roles in normal physiological processes, as an immunoregulatory cytokine and as a driver of fibrosis. In the last 10–15 years, it has also become abundantly clear that activin A plays an important role in the regulation of asthmatic inflammation and airway remodelling. This review provides a brief introduction to the activin A/TGF‐β superfamily, focussing on the regulation of receptors and signalling pathways. We examine the contradictory evidence for generalized pro‐ vs. anti‐inflammatory effects of activin A in inflammation, before appraising its role in asthmatic inflammation and airway remodelling specifically by evaluating data from both murine models and clinical studies. We identify key issues to be addressed, paving the way for safe exploitation of modulation of activin A function for treatment of allergic asthma and other inflammatory lung diseases.

Interleukin-13 Regulates Secretion of the Tumor Growth Factor–β Superfamily Cytokine Activin A in Allergic Airway Inflammation

Activin A is a member of the TGF-beta superfamily and plays a role in allergic inflammation and asthma pathogenesis. Recent evidence suggests that activin A regulates proinflammatory cytokine production and is regulated by inflammatory mediators. In a murine model of acute allergic airway inflammation, we observed previously that increased activin A concentrations in bronchoalveolar lavage (BAL) fluid coincide with Th2 cytokine production in lung-draining lymph nodes and pronounced mucus metaplasia in bronchial epithelium. We therefore hypothesized that IL-13, the key cytokine for mucus production, regulates activin A secretion into BAL fluid in experimental asthma. IL-13 increased BAL fluid activin A concentrations in naive mice and dose dependently induced activin A secretion from cultured human airway epithelium. A key role for IL-13 in the secretion of activin A into the BAL fluid during allergic airway inflammation was confirmed in IL-13-deficient mice. Eosinophils were not involved in this response because there was no difference in BAL fluid activin A concentrations between wild-type and eosinophil-deficient mice. Our data highlight an important role for IL-13 in the regulation of activin A intraepithelially and in BAL fluid in naive mice and during allergic airway inflammation. Given the immunomodulatory and fibrogenic effects of activin A, our findings suggest an important role for IL-13 regulation of activin A in asthma pathogenesis.

Increased Thymic B Cells but Maintenance of ThymicStructure, T Cell Differentiation and Negative Selectionin Lymphotoxin-α and TNF Gene-Targeted Mice

TNF, lymphotoxin (LT) and their receptors are expressed constitutively in the thymus. It remains unclear whether these cytokines play a role in normal thymic structure or function. We have investigated thymocyte differentiation, selection and thymic organogenesis in gene targeted mice lacking LTalpha, TNF, or both (TNF/LTalpha-/-). The thymus was normal in TNF/LTalpha-/- mice with regard to cell yields and stromal architecture. Detailed analysis of alphabeta and gammadelta T cell-lineage thymocyte subsets revealed no abnormalities, implying that neither TNF nor LT play an essential role in T cell differentiation or positive selection. The number and distribution of thymic CD11c+ dendritic cells was also normal in the absence of both TNF and LTalpha. A three-fold increase in B cell numbers was observed consistently in the TNF/LTalpha-/- thymus. This phenotype was due entirely to the LTalpha deficiency and associated with changes in the hemopoietic compartment, rather than the thymic stromal compartment of LTalpha-/- mice. Finally, specific Vbeta8+ T cell deletion within the thymus following intrathymic injection of staphylococcal enterotoxin B (SEB) was TNF/LT independent. Thus, despite the presence of these cytokines and their receptors in the normal thymus, there appears no essential role for either TNF or LT in development of organ structure or for those processes associated with T cell repertoire selection.