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Abstract Background Aging is associated with an increased incidence and mortality of Pseudomonas aeruginosa-induced pneumonias. This might be partly due to age-dependent increases in inflammatory mediators, referred to as inflamm-aging and a decline in immune functions, known as immunosenescence. Still, the impact of dysregulated immune responses on lung infection during aging is poorly understood. Here, we aimed to mimic inflamm-aging using ex vivo precision-cut lung slices (PCLS) and neutrophils – as important effector cells of innate immunity – from young and old mice and investigated the influence of aging on inflammation upon infection with P. aeruginosa bacteria. Methods Murine PCLS were infected with the P. aeruginosa standard lab strain PAO1 and a clinical P. aeruginosa isolate D61. After infection, whole-transcriptome analysis of the tissue as well as cytokine expression in supernatants and tissue lysates were performed. Responses of isolated neutrophils towards the bacteria were investigated by quantifying neutrophil extracellular trap (NET) formation, cytokine secretion, and analyzing expression of surface activation markers using flow cytometry. Results Inflamm-aging was observed by transcriptome analysis, showing an enrichment of biological processes related to inflammation, innate immune response, and chemotaxis in uninfected PCLS of old compared with young mice. Upon P. aeruginosa infection, the age-dependent pro-inflammatory response was even further promoted as shown by increased production of cytokines and chemokines such as IL-1β, IL-6, CXCL1, TNF-α, and IL-17A. In neutrophil cultures, aging did not influence NET formation or cytokine secretion during P. aeruginosa infection. However, expression of receptors associated with inflammatory responses such as complement, adhesion, phagocytosis, and degranulation was lower in neutrophils stimulated with bacteria from old mice as compared to young animals. Conclusions By using PCLS and neutrophils from young and old mice as immunocompetent ex vivo test systems, we could mimic dysregulated immune responses upon aging on levels of gene expression, cytokine production, and receptor expression. The results furthermore reflect the exacerbation of inflammation upon P. aeruginosa lung infection as a result of inflamm-aging in old age.

Abstract Background The coordinated transcriptional regulation of activated T-cells is based on a complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by impulse and sustained patterns over the course. Here, we analyze the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation. Results Here, we identify and verify general biomarker signatures robustly evaluating T-cell activation in a time-resolved manner. We identify time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. The gene signatures include central transcriptional regulators of T-cell activation, representing successive waves as well as sustained patterns of induction. They cover sustained repressed, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. In addition, intermediate and late response activation signatures in CAR T-cell infusion products are correlated to immune effector cell-associated neurotoxicity syndrome. Conclusion This study is the first to describe temporally resolved gene expression patterns across T-cell populations. These biomarker signatures are a valuable source, e.g., monitoring transcriptional changes during T-cell activation with a reasonable number of genes, annotating T-cell states in single-cell transcriptome studies, or assessing dysregulated functions of human T-cell immunity.

AbstractDrug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Abstract Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract

Recently, Tummino et al. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.

Abstract Background Aberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone. Methods Precision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants. Results In rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied. Conclusions This study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.

Abstract Objective Human precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA isolation from PCLS to enable miRNA readouts. Results The modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human PCLS model.

Abstract To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 µg/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.

Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner.

Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.

Abstract Background Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. Methods Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. Results In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-β, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. Conclusions In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo.

Smokers with apparently “healthy” lungs suffer from more severe and frequent viral respiratory infections, but the mechanisms underlying this observation are still unclear. Epithelial cells and dendritic cells (DC) form the first line of defense against inhaled noxes such as smoke or viruses. We therefore aimed to obtain insight into how cigarette smoke affects DCs and epithelial cells and how this influences the response to viral infection. Female C57BL/6J mice were exposed to cigarette smoke (CS) for 1 h daily for 24 days and then challenged i.n. with the viral mimic and Toll-like receptor 3 (TLR3) ligand poly (I:C) after the last exposure. DC subpopulations were analyzed 24 h later in whole lung homogenates by flow cytometry. Calu-3 cells or human precision-cut lung slices (PCLS) cultured at air-liquid interface were exposed to CS or air and subsequently inoculated with influenza H1N1. At 48 h post infection cytokines were analyzed by multiplex technology. Cytotoxic effects were measured by release of lactate dehydrogenase (LDH) and confocal imaging. In Calu-3 cells the trans-epithelial electrical resistance (TEER) was assessed. Smoke exposure of mice increased numbers of inflammatory and plasmacytoid DCs in lung tissue. Additional poly (I:C) challenge further increased the population of inflammatory DCs and conventional DCs, especially CD11b+ cDCs. Smoke exposure led to a loss of the barrier function in Calu-3 cells, which was further exaggerated by additional influenza H1N1 infection. Influenza H1N1-induced secretion of antiviral cytokines (IFN-α2a, IFN-λ, interferon-γ-induced protein 10 [IP-10]), pro-inflammatory cytokine IL-6, as well as T cell-associated cytokines (e.g., I-TAC) were completely suppressed in both Calu-3 cells and human PCLS after smoke exposure. In summary, cigarette smoke exposure increased the number of inflammatory DCs in the lung and disrupted epithelial barrier functions, both of which was further enhanced by viral stimulation. Additionally, the antiviral immune response to influenza H1N1 was strongly suppressed by smoke. These data suggest that smoke impairs protective innate mechanisms in the lung, which could be responsible for the increased susceptibility to viral infections in “healthy” smokers.

Pulmonary infections constitute a substantial health problem worldwide. Here the authors show that phagocytes similar to primitive macrophages can be generated from human induced pluripotent stem cells, by the use of industry-compatible, stirred-tank bioreactors, and applied as a cell-based therapy to treat acute bacterial infections in mice.

Summary: Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model. : Schambach and colleagues show evidence that hematopoiesis in hiPSC-derived teratomas occurs by endothelial-to-hematopoietic transition in a highly polyclonal manner. This process was supported by the presence of HUVECs in the NSGS mouse, which provides human SCF, IL-3, and GM-CSF. The established hematopoiesis model can potentially be used for disease modeling and improvement of hematopoietic differentiation protocols. Keywords: hematopoiesis, teratoma, hiPSC, EHT, genetic barcoding, HUVECs, embryogenesis

Abstract Background Investigation of basic chronic inflammatory mechanisms and development of new therapeutics targeting the respiratory tract requires appropriate testing systems, including those to monitor long- persistence. Human precision-cut lung slices (PCLS) have been demonstrated to mimic the human respiratory tract and have potential of an alternative, ex-vivo system to replace or augment in-vitro testing and animal models. So far, most research on PCLS has been conducted for short cultivation periods (≤72 h), while analyses of slowly metabolized therapeutics require long-term survival of PCLS in culture. In the present study, we evaluated viability, physiology and structural integrity of PCLS cultured for up to 15 days. Methods PCLS were cultured for 15 days and various parameters were assessed at different time points. Results Structural integrity and viability of cultured PCLS remained constant for 15 days. Moreover, bronchoconstriction was inducible over the whole period of cultivation, though with decreased sensitivity (EC501d = 4 × 10−8 M vs. EC5015d = 4 × 10−6 M) and reduced maximum of initial airway area (1d = 0.5% vs. 15d = 18.7%). In contrast, even though still clearly inducible compared to medium control, LPS-induced TNF-α secretion decreased significantly from day 1 to day 15 of culture. Conclusions Overall, though long-term cultivation of PCLS need further investigation for cytokine secretion, possibly on a cellular level, PCLS are feasible for bronchoconstriction studies and toxicity assays.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

Subgroups of patients with severe asthma are insensitive to inhaled corticosteroids and require novel therapies on top of standard medical care. IL-13 is considered one of the key cytokines in the asthma pathogenesis, however, the effect of IL-13 was mostly studied in rodents. This study aimed to assess IL-13 effect in human lung tissue for the development of targeted therapy approaches such as inhibition of soluble IL-13 or its receptor IL-4Rα subunit. Precision-cut lung slices (PCLS) were prepared from lungs of rodents, non-human primates (NHP) and humans. Direct effect of IL-13 on human lung tissue was observed on inflammation, induction of mucin5AC, and airway constriction induced by methacholine and visualized by videomicroscopy. Anti-inflammatory treatment was evaluated by co-incubation of IL-13 with increasing concentrations of IL-13/IL-13 receptor inhibitors. IL-13 induced a two-fold increase in mucin5AC secretion in human bronchial tissue. Additionally, IL-13 induced release of proinflammatory cytokines eotaxin-3 and TARC in human PCLS. Anti-inflammatory treatment with four different inhibitors acting either on the IL-13 ligand itself (anti-IL-13 antibody, similar to Lebrikizumab) or the IL-4Rα chain of the IL-13/IL-4 receptor complex (anti-IL-4Rα #1, similar to AMG 317, and #2, similar to REGN668) and #3 PRS-060 (a novel anticalin directed against this receptor) could significantly attenuate IL-13 induced inflammation. Contrary to this, IL-13 did not induce airway hyperresponsiveness (AHR) in human and NHP PCLS, although it was effective in rodent PCLS. Overall, this study demonstrates that IL-13 stimulation induces production of mucus and biomarkers of allergic inflammation in human lung tissue ex-vivo but no airway hyperresponsiveness. The results of this study show a more distinct efficacy than known from animals models and a clear discrepancy in AHR induction. Moreover, it allows a translational approach in inhibitor profiling in human lung tissue.

The cellular serine protease TMPRSS2, a member of the type II transmembrane serine protease (TTSP) family, cleaves and activates the hemagglutinin of influenza A viruses (FLUAV) in cell culture and is essential for spread of diverse FLUAV in mice. Non-human primates (NHP), in particular rhesus and cynomolgus macaques, serve as animal models for influenza and experimental FLUAV infection of common marmosets has recently also been reported. However, it is currently unknown whether the NHP orthologues of human TMPRSS2 cleave and activate FLUAV hemagglutinin and contribute to viral spread in respiratory tissue. Here, we cloned and functionally analyzed the macaque and marmoset orthologues of human TMPRSS2. In addition, we analyzed the macaque orthologues of human TMPRSS4 and HAT, which also belong to the TTSP family. We found that all NHP orthologues of human TMPRSS2, TMPRSS4 and HAT cleave and activate HA upon directed expression and provide evidence that endogenous TMPRSS2 is expressed in the respiratory epithelium of rhesus macaques. Finally, we demonstrate that a serine protease inhibitor active against TMPRSS2 suppresses FLUAV spread in precision-cut lung slices of human, macaque and marmoset origin. These results indicate that FLUAV depends on serine protease activity for spread in diverse NHP and in humans. Moreover, our findings suggest that macaques and marmosets may serve as models to study FLUAV activation by TMPRSS2 in human patients.

Rhinovirus (RV) is the predominant virus causing respiratory tract infections. Bronchobini® is a low dose multi component, multi target preparation used to treat inflammatory respiratory diseases such as the common cold, described to ease severity of symptoms such as cough and viscous mucus production. The aim of the study was to assess the efficacy of Bronchobini® in RV infection and to elucidate its mode of action. Therefore, Bronchobini®’s ingredients (BRO) were assessed in an ex vivo model of RV infection using mouse precision-cut lung slices, an organotypic tissue capable to reflect the host immune response to RV infection. Cytokine profiles were assessed using enzyme-linked immunosorbent assay (ELISA) and mesoscale discovery (MSD). Gene expression analysis was performed using Affymetrix microarrays and ingenuity pathway analysis. BRO treatment resulted in the significant suppression of RV-induced antiviral and pro-inflammatory cytokine release. Transcriptome analysis revealed a multifactorial mode of action of BRO, with a strong inhibition of the RV-induced pro-inflammatory and antiviral host response mediated by nuclear factor kappa B (NFkB) and interferon signaling pathways. Interestingly, this was due to priming of these pathways in the absence of virus. Overall, BRO exerted its beneficial anti-inflammatory effect by priming the antiviral host response resulting in a reduced inflammatory response to RV infection, thereby balancing an otherwise excessive inflammatory response.

Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥10(3) µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1β, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.

The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30 ± 7.1%) than in guinea pig (79 ± 5.1%) PCLS. The transient receptor potential (TRP) channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.