The ability to treat severe viral infections is limited by our understanding of the mechanisms behind virus-induced immunopathology. While the role of type I interferons (IFNs) in early control of ...viral replication is clear, less is known about how IFNs can regulate the development of immunopathology and affect disease outcomes. Here, we report that absence of type I IFN receptor (IFNAR) is associated with extensive immunopathology following mucosal viral infection. This pathology occurred independent of viral load or type II immunity but required the presence of macrophages and IL-6. The depletion of macrophages and inhibition of IL-6 signaling significantly abrogated immunopathology. Tissue destruction was mediated by macrophage-derived matrix metalloproteinases (MMPs), as MMP inhibition by doxycycline and Ro 28-2653 reduced the severity of tissue pathology. Analysis of post-mortem COVID-19 patient lungs also displayed significant upregulation of the expression of MMPs and accumulation of macrophages. Overall, we demonstrate that IFNs inhibit macrophage-mediated MMP production to prevent virus-induced immunopathology and uncover MMPs as a therapeutic target towards viral infections.
Immunogenic cell death (ICD) can switch immunologically "cold" tumors "hot", making them sensitive to immune checkpoint inhibitor (ICI) therapy. Many therapeutic platforms combine multiple modalities ...such as oncolytic viruses (OVs) and low-dose chemotherapy to induce ICD and improve prognostic outcomes. We previously detailed many unique properties of oncolytic bovine herpesvirus type 1 (oBHV) that suggest widespread clinical utility. Here, we show for the first time, the ability of oBHV monotherapy to induce bona fide ICD and tumor-specific activation of circulating CD8
T cells in a syngeneic murine model of melanoma. The addition of low-dose mitomycin C (MMC) was necessary to fully synergize with ICI through early recruitment of CD8
T cells and reduced infiltration of highly suppressive PD-1
Tregs. Cytokine and gene expression analyses within treated tumors suggest that the addition of MMC to oBHV therapy shifts the immune response from predominantly anti-viral, as evidenced by a high level of interferon-stimulated genes, to one that stimulates myeloid cells, antigen presentation and adaptive processes. Collectively, these data provide mechanistic insights into how oBHV-mediated therapy modalities overcome immune suppressive tumor microenvironments to enable the efficacy of ICI therapy.
Abstract
Introduction
Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50–80% of asthma ...exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35–50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP).
Hypothesis
Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy.
Methods
Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA.
Results
Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES.
Conclusion
Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.
The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar ...GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2-4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3-dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.
The accumulation of macrophages in the airways and the pulmonary interstitium is a hallmark of cigarette smoke-associated inflammation. Notably, pulmonary macrophages are not a homogenous population ...but consist of several subpopulations. To date, the manner in which cigarette smoke exposure affects the relative composition and functional capacity of macrophage subpopulations has not been elucidated.
Using a whole-body cigarette smoke exposure system, we investigated the impact of cigarette smoke on macrophage subpopulations in C57BL/6 mice using flow cytometry-based approaches. Moreover, we used bromodeoxyuridine labelling plus
and
mice to assess the relative contribution of local proliferation and monocyte recruitment to macrophage accumulation. To assess the functional consequences of altered macrophage subpopulations, we used a model of concurrent bleomycin-induced lung injury and cigarette smoke exposure to examine tissue remodelling processes.
Cigarette smoke exposure altered the composition of pulmonary macrophages increasing CD11b
subpopulations including monocyte-derived alveolar macrophages (Mo-AM) as well as interstitial macrophages (IM)1, -2 and -3. The increase in CD11b
subpopulations was observed at multiple cigarette smoke exposure timepoints. Bromodeoxyuridine labelling and studies in
mice demonstrated that increased Mo-AM and IM3 turnover in the lungs of cigarette smoke-exposed mice was IL-1α dependent. Compositional changes in macrophage subpopulations were associated with impaired induction of fibrogenesis including decreased α-smooth muscle actin positive cells following intratracheal bleomycin treatment. Mechanistically,
and
assays demonstrated predominant macrophage M1 polarisation and reduced matrix metallopeptidase 9 activity in cigarette smoke-exposed mice.
Cigarette smoke exposure modified the composition of pulmonary macrophage by expanding CD11b
subpopulations. These compositional changes were associated with attenuated fibrogenesis, as well as predominant M1 polarisation and decreased fibrotic activity. Overall, these data suggest that cigarette smoke exposure altered the composition of pulmonary macrophage subpopulations contributing to impaired tissue remodelling.
Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) in the lung parenchyma. The abnormal ECM deposition slowly overtakes ...normal lung tissue, disturbing gas exchange and leading to respiratory failure and death. ECM cross-linking and subsequent stiffening is thought to be a major contributor of disease progression and also promotes the activation of transforming growth factor (TGF)-β1, one of the main profibrotic growth factors. Lysyl oxidase-like (LOXL) 1 belongs to the cross-linking enzyme family and has been shown to be up-regulated in active fibrotic regions of bleomycin-treated mice and patients with IPF. We demonstrate in this study that LOXL1-deficient mice are protected from experimental lung fibrosis induced by overexpression of TGF-β1 using adenoviral (Ad) gene transfer (AdTGF-β1). The lack of LOXL1 prevented accumulation of insoluble cross-linked collagen in the lungs, and therefore limited lung stiffness after AdTGF-β1. In addition, we applied mechanical stretch to lung slices from LOXL1
and LOXL1
mice treated with AdTGF-β1. Lung stiffness (Young's modulus) of LOXL1
lung slices was significantly lower compared with LOXL1
lung slices. Moreover, the release of activated TGF-β1 after mechanical stretch was significantly lower in LOXL1
mice compared with LOXL1
mice after AdTGF-β1. These data support the concept that cross-linking enzyme inhibition represents an interesting therapeutic target for drug development in IPF.
Background and Aims:
Idiopathic pulmonary fibrosis (IPF) is a common and severe form of pulmonary fibrosis. Nintedanib, a triple angiokinase inhibitor, is approved for treating IPF. Galectin 3 ...(Gal-3) activates a variety of profibrotic processes. Currently, the Gal-3 inhibitor TD139 is being tested in phase II clinical trials. Since this treatment is given ‘on top’ of nintedanib, it is important to estimate its effect on Gal-3 levels. Therefore, we evaluated the impact of nintedanib on Gal-3 expression using both in vitro and in vivo models, in addition to serum samples from patients with IPF.
Methods:
Gal-3 levels were evaluated in IPF and control tissue samples, primary human lung fibroblasts (HLFs) following nintedanib treatment (10–100 nM, quantitative polymerase chain reaction), and in a silica-induced fibrosis mouse model with/without nintedanib (0.021–0.21 mg/kg) by immunohistochemistry. In addition, Gal-3 levels were analyzed in serum samples from 41 patients with interstitial lung disease patients with/without nintedanib treatment by ELISA.
Results:
Nintedanib addition to HLFs resulted in significant elevations in Gal-3, phospho-signal transducer and activator of transcription 3 (pSTAT3), as well as IL-8 mRNA levels (p < 0.05). Gal-3 expression was higher in samples from IPF patients compared with non-IPF controls at the protein and mRNA levels (p < 0.05). In the in vivo mouse model, Gal-3 levels were increased following fibrosis induction and even further increased with the addition of nintedanib, mostly in macrophages (p < 0.05). Patients receiving nintedanib presented with higher Gal-3 serum levels compared with those who did not receive nintedanib (p < 0.05).
Conclusion:
Nintedanib elevates Gal-3 levels in both experimental models, along with patient samples. These findings highlight the possibility of using combined inhibition therapy for patients with IPF.
Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic ...acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems
Oncolytic virotherapies have shown excellent promise in a variety of cancers by promoting antitumor immunity. However, the effects of oncolytic virus-mediated type I interferon (IFN-I) production on ...antitumor immunity remain unclear. Recent reports have highlighted immunosuppressive functions of IFN-I in the context of checkpoint inhibitor and cell-based therapies. In this study, we demonstrate that oncolytic virus-induced IFN-I promotes the expression of PD-L1 in tumor cells and leukocytes in a IFN receptor (IFNAR)-dependent manner. Inhibition of IFN-I signaling using a monoclonal IFNAR antibody decreased IFN-I-induced PD-L1 expression and promoted tumor-specific T cell effector responses when combined with oncolytic virotherapy. Furthermore, IFNAR blockade improved therapeutic response to oncolytic virotherapy in a manner comparable with PD-L1 blockade. Our study highlights a critical immunosuppressive role of IFN-I on antitumor immunity and uses a combination strategy that improves the response to oncolytic virotherapy.
Display omitted
Oncolytic virotherapies have shown promise in cancer by promoting antitumor immunity. In this study, we demonstrate that oncolytic virus-induced IFNα/β promotes the expression of PD-L1 in tumor cells and leukocytes. Inhibition of IFNα/β signaling decreased promoted tumor-specific T cell effector responses and improved therapeutic response to oncolytic virotherapy.
Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences ...is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (
= 22), compared to those that had recovered from other mild respiratory infections (
= 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.
PDF
