Bridging therapy-induced phenotypes and genetic immune dysregulation to study interleukin-2-induced immunotoxicology.

Interleukin-2 (IL-2) holds promise for the treatment of cancer and autoimmune diseases, but its high-dose usage is associated with systemic immunotoxicity. Differential IL-2 receptor (IL-2R) regulation might impact function of cells upon IL-2 stimulation, possibly inducing cellular changes similar to patients with hypomorphic IL2RB mutations, presenting with multiorgan autoimmunity. Here, we show that sustained high-dose IL-2 stimulation of human lymphocytes drastically reduces IL-2Rß surface expression especially on T cells, resulting in impaired IL-2R signaling which correlates with high IL-2Rα baseline expression. IL-2R signaling in NK cells is maintained. CD4+ T cells, especially regulatory T cells are more broadly affected than CD8+ T cells, consistent with lineage-specific differences in IL-2 responsiveness. Given the resemblance of cellular characteristics of high-dose IL-2-stimulated cells and cells from patients with IL-2Rß defects, impact of continuous IL-2 stimulation on IL-2R signaling should be considered in the onset of clinical adverse events during IL-2 therapy.

Homeostatic T helper 17 cell responses triggered by complex microbiota are maintained in ex vivo intestinal tissue slices.

Segmented filamentous bacteria (SFB) are members of the commensal intestinal microbiome. They are known to contribute to the postnatal maturation of the gut immune system, but also to augment inflammatory conditions in chronic diseases such as Crohn's disease. Living primary tissue slices are ultrathin multicellular sections of the intestine and provide a unique opportunity to analyze tissue-specific immune responses ex vivo. This study aimed to investigate whether supplementation of the gut flora with SFB promotes T helper 17 (Th17) cell responses in primary intestinal tissue slices ex vivo. Primary tissue slices were prepared from the small intestine of healthy Taconic mice with SFB-positive and SFB-negative microbiomes and stimulated with anti-CD3/CD28 or Concanavalin A. SFB-positive and -negative mice exhibited distinct microbiome compositions and Th17 cell frequencies in the intestine and complex microbiota including SFB induced up to 15-fold increase in Th17 cell-associated mediators, serum amyloid A (SAA), and immunoglobulin A (IgA) responses ex vivo. This phenotype could be transmitted by co-housing of mice. Our findings highlight that changes in the gut microbiome can be observed in primary intestinal tissue slices ex vivo. This makes the system very attractive for disease modeling and assessment of new therapies.

IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-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.

Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Age-dependent inflammatory response is altered in an ex vivo model of bacterial pneumonia.

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-1ß, 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.

Beyond chemicals: Opportunities and challenges of integrating non-chemical stressors in adverse outcome pathways.

The adverse outcome pathways (AOPs) were developed to accelerate evidence-based chemical risk assessment by leveraging data from new approach methodologies. Thanks to their stressor-agnostic approach, AOPs were seen as instrumental in other fields. Here, we present AOPs that report non-chemical stressors along with the challenges encountered for their development. Challenges regarding AOPs linked to nanomaterials include non-specific molecular initiating events, limited understanding of nanomaterial biodistribution, and needs for adaptations of the in silico modeling and testing systems. Development of AOPs for radiation face challenges in how to incorporate ionizing events type, dose rate, energy deposition, and how to account for targeting multiple macromolecules. AOPs for COVID-19 required the inclusion of SARS-CoV-2-specific replicative steps to capture the essential events driving the disease. Developing AOPs to evaluate efficacy and toxicity of cell therapies necessitates addressing the cellular nature and the therapeutic function of the stressor. Finally, addressing toxicity of emerging biological stressors like microbial pesticides can learn from COVID-19 AOPs. We further discuss that the adaptations needed to expand AOP applicability beyond chemicals are mainly at the molecular and cellular levels while downstream key events at tissue or organ level, such as inflammation, are shared by many AOPs initiated by various stressors. In conclusion, although it is challenging to integrate non-chemical stressors within AOPs, this expands opportunities to account for real-world scenarios, to identify vulnerable individuals, and to bridge knowledge on mechanisms of adversity.

The adverse outcome pathway (AOP) framework was developed to help predict whether chemicals have toxic effects on humans. Structuring available information in an accessible database can reduce animal testing. AOPs usually capture the path from the interaction of a stressor, usually a chemical, with the human body to an adverse outcome, e.g., a disease symptom. The concept of AOPs has now been expanded to include non-chemical stressors such as nanomaterials, radiation, viruses, cells used to treat patients, and microorganisms employed as pesticides. We use discuss how these stressors need to be accommodated within the framework and point out that pathways initiated by these stressors share downstream events like inflammation with chemical stressors. By integrating non-chemical stressors into the framework, real-world scenarios where people may be exposed to different stressor types can be considered, vulnerable individuals can be identified, and knowledge on toxic effects can be compounded.

A time-resolved meta-analysis of consensus gene expression profiles during human T-cell activation.

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.

Drug-induced phospholipidosis is not correlated with the inhibition of SARS-CoV-2 - inhibition of SARS-CoV-2 is cell line-specific.

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.

Bacterial vesicles block viral replication in macrophages via TLR4-TRIF-axis.

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.

Mice inflammatory responses to inhaled aerosolized LPS: effects of various forms of human alpha1-antitrypsin.

Rodent models of lipopolysaccharide (LPS)-induced pulmonary inflammation are used for anti-inflammatory drug testing. We aimed to characterize mice responses to aerosolized LPS alone or with intraperitoneal (i.p.) delivery of alpha1-antitrypsin (AAT). Balb/c mice were exposed to clean air or aerosolized LPS (0.21 mg/mL) for 10 min per day, for 3 d. One hour after each challenge, animals were treated i.p. with saline or with (4 mg/kg body weight) one of the AAT preparations: native (AAT), oxidized (oxAAT), recombinant (recAAT), or peptide of AAT (C-36). Experiments were terminated 6 h after the last dose of AATs. Transcriptome data of mice lungs exposed to clean air versus LPS revealed 656 differentially expressed genes and 155 significant gene ontology terms, including neutrophil migration and toll-like receptor signaling pathways. Concordantly, mice inhaling LPS showed higher bronchoalveolar lavage fluid neutrophil counts and levels of myeloperoxidase, inducible nitric oxide synthase, IL-1ß, TNFα, KC, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Plasma inflammatory markers did not increase. After i.p. application of AATs, about 1% to 2% of proteins reached the lungs but, except for GM-CSF, none of the proteins significantly influenced inflammatory markers. All AATs and C-36 significantly inhibited LPS-induced GM-CSF release. Surprisingly, only oxAAT decreased the expression of several LPS-induced inflammatory genes, such as Cxcl3, Cd14, Il1b, Nfkb1, and Nfkb2, in lung tissues. According to lung transcriptome data, oxAAT mostly affected genes related to transcriptional regulation while native AAT or recAAT affected genes of inflammatory pathways. Hence, we present a feasible mice model of local lung inflammation induced via aerosolized LPS that can be useful for systemic drug testing.

Cell Type-Specific Anti-Viral Effects of Novel SARS-CoV-2 Main Protease Inhibitors.

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.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

Precision cut intestinal slices, a novel model of acute food allergic reactions.

BACKGROUND: Food allergy affects up to 10% of the pediatric population. Despite ongoing efforts, treatment options remain limited. Novel models of food allergy are needed to study response patterns downstream of IgE-crosslinking and evaluate drugs modifying acute events. Here, we report a novel human ex vivo model that displays acute, allergen-specific, IgE-mediated smooth muscle contractions using precision cut intestinal slices (PCIS). METHODS: PCIS were generated using gut tissue samples from children who underwent clinically indicated surgery. Viability and metabolic activity were assessed from 0 to 24 h. Distribution of relevant cell subsets was confirmed using single nucleus RNA sequencing. PCIS were passively sensitized using plasma from peanut allergic donors or peanut-sensitized non-allergic donors, and exposed to various stimuli including serotonin, histamine, FcɛRI-crosslinker, and food allergens. Smooth muscle contractions and mediator release functioned as readouts. A novel program designed to measure contractions was developed to quantify responses. The ability to demonstrate the impact of antihistamines and immunomodulation from peanut oral immunotherapy (OIT) was assessed. RESULTS: PCIS viability was maintained for 24 h. Cellular distribution confirmed the presence of key cell subsets including mast cells. The video analysis tool reliably quantified responses to different stimulatory conditions. Smooth muscle contractions were allergen-specific and reflected the clinical phenotype of the plasma donor. Tryptase measurement confirmed IgE-dependent mast cell-derived mediator release. Antihistamines suppressed histamine-induced contraction and plasma from successful peanut OIT suppressed peanut-specific PCIS contraction. CONCLUSION: PCIS represent a novel human tissue-based model to study acute, IgE-mediated food allergy and pharmaceutical impacts on allergic responses in the gut.

Acetylsalicylic Acid and Salicylic Acid Inhibit SARS-CoV-2 Replication in Precision-Cut Lung Slices.

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.

Nintedanib modulates type III collagen turnover in viable precision-cut lung slices from bleomycin-treated rats and patients with pulmonary fibrosis.

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.

Infection of Human Precision-Cut Lung Slices with the Influenza Virus.

Viral infections are common causes of asthma exacerbations. To model these processes ex vivo, human precision-cut lung slices (PCLSs) can be used. Here we describe the infection of human PCLSs with the human influenza virus. We then provide methods to quantify the virus and reveal its localization within infected PCLSs and study consequences of infection, including cell death and production of cytokines, chemokine, and mucus. We also describe the stimulation of PCLSs with immune mediators such as pro-inflammatory tumor necrosis factor α (TNF-α). These models are useful to investigate mechanisms of virally induced asthma exacerbations and study modes of action and efficacy of antiviral and/or anti-inflammatory drugs.

Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2.

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.

The Challenge of Long-Term Cultivation of Human Precision-Cut Lung Slices.

Human precision-cut lung slices (PCLS) have proven to be an invaluable tool for numerous toxicologic, pharmacologic, and immunologic studies. Although a cultivation period of <1 week is sufficient for most studies, modeling of complex disease mechanisms and investigating effects of long-term exposure to certain substances require cultivation periods that are much longer. So far, data regarding tissue integrity of long-term cultivated PCLS are incomplete. More than 1500 human PCLS from 16 different donors were cultivated under standardized, serum-free conditions for up to 28 days and the viability, tissue integrity, and the transcriptome was assessed in great detail. Even though viability of PCLS was well preserved during long-term cultivation, a continuous loss of cells was observed. Although the bronchial epithelium was well preserved throughout cultivation, the alveolar integrity was preserved for about 2 weeks, and the vasculatory system experienced significant loss of integrity within the first week. Furthermore, ciliary beat in the small airways gradually decreased after 1 week. Interestingly, keratinizing squamous metaplasia of the alveolar epithelium with significantly increasing manifestation were found over time. Transcriptome analysis revealed a significantly increased immune response and significantly decreased metabolic activity within the first 24 hours after PCLS generation. Overall, this study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of PCLS.

Cigarette smoke alters inflammatory genes and the extracellular matrix - investigations on viable sections of peripheral human lungs.

Chronic obstructive pulmonary disease (COPD) is a complex chronic respiratory disorder often caused by cigarette smoke. Cigarette smoke contains hundreds of toxic substances. In our study, we wanted to identify initial mechanisms of cigarette smoke induced changes in the distal lung. Viable slices of human lungs were exposed 24 h to cigarette smoke condensate, and the dose-response profile was analyzed. Non-toxic condensate concentrations and lipopolysaccharide were used for further experiments. COPD-related protein and gene expression was measured. Cigarette smoke condensate did not induce pro-inflammatory cytokines and most inflammation-associated genes. In contrast, lipopolysaccharide significantly induced IL-1α, IL-1ß, TNF-α and IL-8 (proteins) and IL1B, IL6, and TNF (genes). Interestingly, cigarette smoke condensate induced metabolism- and extracellular matrix-associated proteins and genes, which were not influenced by lipopolysaccharide. Also, a significant regulation of CYP1A1 and CYP1B1, as well as MMP9 and MMP9/TIMP1 ratio, was observed which resembles typical findings in COPD. In conclusion, our data show that cigarette smoke and lipopolysaccharide induce significant responses in human lung tissue ex vivo, giving first hints that COPD starts early in smoking history.

A modified protocol for successful miRNA profiling in human precision-cut lung slices (PCLS).

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.