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Chronic respiratory infections with the gram-negative bacterium

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.

Introduction: COL4A3 degradation is significantly increased in severe, exacerbating type 2 asthmatics and correlates with increased chymase positive mast cells (mouse model of asthma). However, whether chymase is sufficient/necessary for COL4A3 degradation and if this mode of action is responsive to therapeutic intervention, is unknown. We hypothesized, that mast cell chymase is necessary for COL4A3 degradation and that treatment with anti-IgE reduces COL4A3 degradation. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in female 6-8 week old Balb/C mice, which were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28) and Chymase was inhibited with Fulacimstat (i.t.). Allergic, uncontrolled asthmatics (n=19, Omalizumab treatment), were monitored over six months. Treatment response was set as Asthma Control Test (ACT) improvement of g3 points. Serum levels of C4Ma3 were determined at baseline, 3 and 6 months. Results: Chymase inhibition prevented OVA-challenge induced C4Ma3 increase. Recombinant chymase alone did not induce elevated C4Ma3 level. Ten out of 19 patients had ACT score increases of g3 points after treatment. Responders mean level of circulating C4Ma3 was 8.87 ng/mL (confidence interval (CI): 7.02-10.72ng/mL), which decreased to 6.57 ng/mL (CI: 4.95-8.1ng/mL, pl0.01) after six months treatment. Non-responders did not show any change in serum levels of after treatment. Conclusion: COL4A3 degradation depends on mast cell chymase in vivo and monitoring serum C4Ma3, may afford a novel avenue to stratify and monitor anti-IgE therapy in severe, allergic asthma.

Introduction: Remodeling of the airway wall is a hallmark feature of asthma. Under physiological conditions, a finely tuned balance maintains a functional state of the extracellular matrix. This balance is disrupted in asthma. COL4A3 is reduced 18-fold in lung tissue from asthmatics, however, the mechanism leading to the diminished levels of COL4A3 has remained elusive. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in the ALLIANCE cohort study participants (Fuchs et al. 2018). Pediatric controls/cases: 34/134; Adult control/cases 31/149. C4Ma3 data was correlated with clinical data, serum cytokine profiles (27-Bioplex, Biorad). Female 6-8 week old Balb/C mice were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28). Murine precision cut lung slices (PCLS) were used to elucidate the role of sensitisation for COL4A3 degradation. Results: Increased C4Ma3 correlated with asthma exacerbation (pl0.01) and elevated levels of Th2/Th9 cytokines in the ALLIANCE cohort. Serum levels of C4Ma3 were elevated in OVA challenged mice (pl0.0001) and correlated with lung tissue levels of mast cell chymase (pl0.01), but not with neutrophils or matrix-metallo-proteinase 9. C4MA3 was elevated in PCLS of allergic, HDM challenged mice (pl0.05) with no further increase after PolyIC. Conclusion: COL4A3 degradation (circulating C4Ma3), is elevated in severe, exacerbating type 2 asthmatics. C4Ma3 levels correlate with chymase positive mast cells in a mouse model of asthma exacerbation. These data highlight that the degradation of COL4A3 is a central part of the pathology of asthma.

Background Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. Methods We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. Findings We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. Interpretation Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. Funding NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

IntroductionHuman respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%–40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required.MethodsIt is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl−) channel modulators to investigate their role during the HRSV life-cycle.ResultsWe demonstrate the requirement for TMEM16A, a calcium-activated Cl−channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01.DiscussionThese findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.

BackgroundAsthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3).ObjectiveTo delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response.MethodsThe serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test.ResultsSerum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5).ConclusionC4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

Chronic P. aeruginosa infections of the respiratory tract pose a major health care problem. The bacterial flagellum represents a tightly regulated virulence factor involved in biofilm formation as well as immune recognition of P. aeruginosa. Aim of this study was to delineate the role of flagellin using precision-cut lung slices (PCLS) as an organotypic, immunocompetent model of the human lung. PCLS were prepared from tumour-free lung tissue and infected with wildtype (wt) or flagellin mutant (∆fliC) PA14 strains. Persistent infection of the tissue over 24h was achieved by treatment with subinhibitory concentrations of tobramycin. Bacterial load was determined and lung tissue viability as well as specific cytokine responses were measured to assess the immune response. While the initial attachment of P. aeruginosa to the lung tissue was enhanced by the presence of the flagellum, both wt as well as ∆fliC strains were able to colonize the tissue over 24h. Analysis of cytokine profiles revealed a strong upregulation of pro-inflammatory mediators such as IL-8 (6-fold), IL-6 (6-fold), TNF-α (20-fold), GM-CSF (25-fold), IL-1β (52-fold), and MIP-3α (19-fold) after 24h of infection with wt PA14 compared to non-infected control PCLS. Importantly, infection with ∆fliC PA14 induced 2- to 4-fold lower levels of the respective molecules than the wt strain. Taken together, we show here that wt as well as flagellar mutant P. aeruginosa strains can infect human lung tissue ex vivo whereby flagellin acts as a potent immune stimulus, which could have implications for both immune evasion and biofilm formation of P. aeruginosa. Future studies will look at the transformation of acute towards persistent infection.

Millions of asthmatics suffer from virus induced exacerbation mainly caused by rhinovirus (RV). Insufficient virus elimination and inadequate immune response are assumed to be responsible for exacerbation. Therefore, in this study the impact of an asthmatic background on the anti-viral and pro-inflammatory immune response was investigated in precision-cut lung slices (PCLS) of HDM-sensitized mice. Balb/c mice were sensitized and challenged with HDM or saline i.n. for 28 days. Then PCLS were sliced and infected with RV1b (105 TCID50/mL) or UV-inactivated RV1b for 48 h. HDM sensitization resulted in a TH-2/TH-17 dominated allergic immune response with significantly higher secretion of IL-4, IL-5, IL-10, IL-17A and IP-10 which was maintained ex vivo. RV1b infection significantly upregulated anti-viral cytokines such as IFN-β (954±311 vs. 566±111 ng/mg protein) and IP-10 (61±17 vs. 40±6 pg/mg protein) in healthy vs. asthmatic tissue, respectively. Furthermore, RV1b exacerbated IL-6 (40±13 vs. 102±16 ng/mg protein) and IL-4 (823±201 vs. 1374±340 pg/mg protein) secretion in asthmatic tissue ex vivo in comparison to the corresponding UV-control but remained unchanged in healthy tissue. Rupintrivir treatment significantly reduced cytokine secretion of IFN-α, IP-10, IL-4, IL-6 and MCP-1 in asthmatic tissue but had only an impact on MCP-1 levels in healthy tissue. The in vivo established asthmatic disease background by HDM-challenge maintained in cell culture, leads to a significantly dysregulated immune response to virus infection ex vivo. Further studies are required to reveal pathways involved in the mechanisms of virus-induced exacerbation of asthma.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), and interleukin 1 alpha (IL-1alpha) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.

Millions of asthmatic patients suffer from virus induced exacerbation mainly caused by human rhinovirus (HRV). Insufficient virus elimination and inadequate immune response are assumed to be responsible for exacerbation. Therefore, in this study the impact of an asthmatic background on the anti-viral and pro-inflammatory immune response was investigated in precision-cut lung slices (PCLS) of HDM sensitized mice. Balb/c mice were sensitized and challenged with HDM or saline i.n. for 28 days. PCLS were prepared and infected with HRV1B (105 TCID50/mL) or UV-inactivated HRV1B for 48 h and cytokine secretion was assessed by MSD. HDM sensitization resulted in a TH-2 dominated allergic immune response present in lung tissue ex vivo as shown by significant induction of IL-4, IL-5, IL-10, IL-17A and IP-10. HRV1B infection of sham-sensitized PCLS resulted in induction of an anti-viral and pro-inflammatory immune response as indicated by a significantly higher secretion of IFN-γ (480 %), IP-10 (1625 %), IL-17A (1300%) or TNF-α (200 %). Contrary to this, HDM-sensitized PCLS showed a significantly attenuated secretion of IFN-γ (206%), IP-10 (133 %), IL-17A (140 %) demonstrating an impaired immune response to HRV1B. Interestingly, IL-4 was further induced in HRV1B infected PCLS of HDM-sensitized mice. The asthmatic disease background induced by in vivo HDM challenge was maintained ex vivo and lead to an inadequate immune response to virus infection. In future studies we will investigate pathways involved in the mechanisms of virus-induced exacerbation of asthma.

Background Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures. Results We established an optimized protocol for RNA isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis. Conclusions In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications.

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

Interleukin-13 is a key cytokine of asthma and elevated in asthmatics resulting in airway hyperresponsiveness (AHR). AHR is a hallmark of allergic asthma defined as exaggerated bronchoconstriction in response to contractile stimuli. Research for the development of new drugs is mainly based on appropriate in vivo and in vitro models. There is a need for translational models with improved predictivity for human. For comparison of different species we used precision-cut lung slices (PCLS) and assessed IL-13 induced hyperreactivity in PCLS of mice, rats, and humans. PCLS were prepared from Balb/c mice, Brown Norway rats, and humans. IL-13 receptor was stained in the airways of mouse, rat and human PCLS by immunohistochemistry. Airways of all species were pre-incubated with 100 ng/mL IL-13. Subsequently, bronchoconstriction was induced by addition of methacholine (MCh) and visualized by videomicroscopy. IL-13 receptor was present in epithelial cells and smooth muscle cells in PCLS of all species. Methacholine-induced bronchoconstriction in mouse exhibited an EC50 of 80 nM and decreased by pre-incubation with IL-13 to 50 nM, in rat from 220 nM to 170 nM and human from 180 nM to 47 nM MCh. In general, pre-incubation of PCLS in the presence of IL-13 resulted in all species in stronger bronchoconstriction at maximum methacholine concentration. Maximal constriction of initial airway area resulted in mouse in Cmax 61% by control and decreased in IL-13 pre-incubated tissue to 80%, in rat by 49% to 69% and human by 85% to 94% compared to untreated tissue. This study shows that IL-13 receptor is similar distributed in epithelial cells and smooth muscle cells of all three species. IL-13 induced airway hyperreactivity in all tested species with different methacholine sensitivity. In future studies, PCLS will be used for pre-clinical studies to valuate the antagonist efficacy.

Question: Human risk assessment of chemicals is currently based on the evaluation of in vivo animal studies. The use of high animal numbers and the suffering of animals in experiments such as repeated dose toxicity studies (RDT) demand for alternative test approaches. One alternative approach currently used in human risk assessment is read-across (RAX). In a RAX approach toxicity data of one or several tested source compound(s) are used to predict the toxicity of ""similar"" non-tested target compound(s). Similar compounds shall share structural- and physico-chemical proper-ties as well as a similar mode of action. This study was designed to evaluate the use of data from ex vivo experiments such as rator human precision-cut lung slices (PCLS). Three RAX-categories were tested, namely vicinal halogenides, naphthalene derivatives and vinyl esters. Each RAX category was selected based on shared structural properties and similar toxicological effects in RDT studies extracted from the FhG RepDose database (www.fraunhofer-repdose.de). Methods: Exposure of rat and human PCLS was performed on three days for three hours daily. The cytotoxicity of chemicals was assessed by LDH and WST-1 assay. Ex vivo IC50 values were calculated by sigmoidal curve fitting. These values were correlated to the LD50 values. Results: At least one substance in each category showed cyto-toxic effects in dose dependent manner. Linear regression analysis of public available in vivo LD50 values to the obtained ex vivo IC50values showed good linear correlation. Vicinal halogenides and naphthalene derivatives were less toxic in human lung sections compared to rat sections, where as the vinyl esters showed the comparable cytotoxicity in both species. Thus only one substance group showed similar cytotoxicity in both tested species, whereas two other groups reveal interspecies and metabolism diversity based on cytotoxicity endpoint. Conclusion: This first evaluation of rat and human and PCLs show that ex vivo chemical testing in human lung sections result in a promising approach for toxicity screening as no human in vivo reference data exist.

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host cell proteins for viral entry into human lung cells. One of them, the protease TMPRSS2, is required to activate the viral spike protein (S). Even though two inhibitors, camostat and nafamostat, are known to inhibit TMPRSS2 and block cell entry of SARS-CoV-2, finding further potent therapeutic options is still an important task. In this study, we report that a late-stage drug candidate, otamixaban, inhibits SARS-CoV-2 cell entry. We show that otamixaban suppresses TMPRSS2 activity and SARS-CoV-2 infection of a human lung cell line, although with lower potency than camostat or nafamostat. In contrast, otamixaban inhibits SARS-CoV-2 infection of precision cut lung slices with the same potency as camostat. Furthermore, we report that otamixaban's potency can be significantly enhanced by (sub-) nanomolar nafamostat or camostat supplementation. Dominant molecular TMPRSS2-otamixaban interactions are assessed by extensive 109 μs of atomistic molecular dynamics simulations. Our findings suggest that combinations of otamixaban with supplemental camostat or nafamostat are a promising option for the treatment of COVID-19., SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host proteins for viral entry into human lung cells and is blocked by otamixaban in combination with a covalent protease inhibitor.

Background Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. Methods Printex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. Results All particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 μg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. Conclusions Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells. Electronic supplementary material The online version of this article (doi:10.1186/s12989-017-0189-1) contains supplementary material, which is available to authorized users.