Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

Cancer-Associated Fibroblasts Regulate Kinase Activity in Mesothelioma Cell Lines via Paracrine Signaling and Thereby Dictate Cell Faith and Behavior.

BACKGROUND: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro. METHODS: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients.

Olfactory receptors impact pathophysiological processes of lung diseases in bronchial epithelial cells.

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are limited. Bronchial epithelial cells are key in the pathogenesis by releasing the central proinflammatory cytokine interleukine-8 (IL-8). Olfactory receptors (ORs) are expressed in various cell types. This study examined the drug target potential of ORs by investigating their impact on associated pathophysiological processes in lung epithelial cells. METHODS: Experiments were performed in the A549 cell line and in primary human bronchial epithelial cells. OR expression was investigated using RT-PCR, Western blot, and immunocytochemical staining. OR-mediated effects were analyzed by measuring 1) intracellular calcium concentration via calcium imaging, 2) cAMP concentration by luminescence-based assays, 3) wound healing by scratch assays, 4) proliferation by MTS-based assays, 5) cellular vitality by Annexin V/PI-based FACS staining, and 6) the secretion of IL-8 in culture supernatants by ELISA. RESULTS: By screening 100 potential OR agonists, we identified two, Brahmanol and Cinnamaldehyde, that increased intracellular calcium concentrations. The mRNA and proteins of the corresponding receptors OR2AT4 and OR2J3 were detected. Stimulation of OR2J3 with Cinnamaldehyde reduced 1) IL-8 in the absence and presence of bacterial and viral pathogen-associated molecular patterns (PAMPs), 2) proliferation, and 3) wound healing but increased cAMP. In contrast, stimulation of OR2AT4 by Brahmanol increased wound healing but did not affect cAMP and proliferation. Both ORs did not influence cell vitality. CONCLUSION: ORs might be promising drug target candidates for lung diseases with non-type 2 inflammation. Their stimulation might reduce inflammation or prevent tissue remodeling by promoting wound healing.

Treatment with Helicobacter pylori-derived VacA attenuates allergic airway disease.

Background: Asthma is an incurable heterogeneous disease with variations in clinical and underlying immunological phenotype. New approaches could help to support existing therapy concepts. Neonatal infection of mice with Helicobacter pylori or administration of H. pylori-derived extracts or molecules after birth have been shown to prevent the development of allergic airway disease later in life. This study evaluated the potential therapeutic efficacy of H. pylori vacuolating cytotoxin A (VacA) in allergic airway inflammation and investigated the underlying immunological mechanisms for its actions. Methods: Murine models of allergic airway diseases, and murine and human in vitro models were used. Results: In both an acute model and a therapeutic house dust mite model of allergic airway disease, treatment with H. pylori-derived VacA reduced several asthma hallmarks, including airway hyperresponsiveness, inflammation and goblet cell metaplasia. Flow cytometry and ELISA analyses revealed induction of tolerogenic dendritic cells (DC) and FoxP3 positive regulatory T cells (Tregs), and a shift in the composition of allergen-specific immunoglobulins. Depletion of Tregs during treatment with VacA reversed treatment-mediated suppression of allergic airway disease. Human monocyte derived DCs (moDC) that were exposed to VacA induced Tregs in co-cultured naïve autologous T cells, replicating key observations made in vivo. Conclusion: H. pylori-derived VacA suppressed allergic airway inflammation via induction of Tregs in both allergic airway disease models. These data suggest that the immunomodulatory activity of VacA could potentially be exploited for the prevention and treatment of allergic airway disease.

Regulatory T cell enhancement in adults with cystic fibrosis receiving Elexacaftor/Tezacaftor/Ivacaftor therapy.

Introduction: Cystic fibrosis (CF), especially CF lung disease, is characterized by chronic infection, immune dysfunction including impairment of regulatory T cells (Tregs) and an exaggerated inflammatory response. CF transmembrane conductance regulator (CFTR) modulators have shown to improve clinical outcomes in people with CF (PwCF) with a wide range of CFTR mutations. However, it remains unclear whether CFTR modulator therapy also affects CF-associated inflammation. We aimed to examine the effect of elexacaftor/tezacaftor/ivacaftor therapy on lymphocyte subsets and systemic cytokines in PwCF. Methods: Peripheral blood mononuclear cells and plasma were collected before and at three and six months after the initiation of elexacaftor/tezacaftor/ivacaftor therapy; lymphocyte subsets and systemic cytokines were determined using flow cytometry. Results: Elexacaftor/tezacaftor/ivacaftor treatment was initiated in 77 PwCF and improved percent predicted FEV1 by 12.5 points (p<0.001) at 3 months. During elexacaftor/tezacaftor/ivacaftor therapy, percentages of Tregs were enhanced (+18.7%, p<0.001), with an increased proportion of Tregs expressing CD39 as a marker of stability (+14.4%, p<0.001). Treg enhancement was more pronounced in PwCF clearing Pseudomonas aeruginosa infection. Only minor, non-significant shifts were observed among Th1-, Th2- and Th17-expressing effector T helper cells. These results were stable at 3- and 6-month follow-up. Cytokine measurements showed a significant decrease in interleukin-6 levels during treatment with elexacaftor/tezacaftor/ivacaftor (-50.2%, p<0.001). Conclusion: Treatment with elexacaftor/tezacaftor/ivacaftor was associated with an increased percentage of Tregs, especially in PwCF clearing Pseudomonas aeruginosa infection. Targeting Treg homeostasis is a therapeutic option for PwCF with persistent Treg impairment.

Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro.

Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff's reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.