Ehret Kasemo, Totta

Hackenberg, Stephan

Hagen, Rudolf

Meyer, Till

Scherzad, Agmal

English

KITopen

Klinik und Poliklinik für Hals-, Nasen- und Ohrenkrankheiten,plastische und ästhetische OperationenDirektor: Prof. Dr. Dr. h.c. R. HagenFunctional Effects of Carbon Black Nanoparticles on Primary Airway Epithelial Cells in vitroTotta Ehret Kasemo1, Till Meyer1, Agmal Scherzad1,Rudolf Hagen1, Stephan Hackenberg21) Poliklinik für Hals-, Nasen- und Ohrenkrankheiten, plastische und ästhetische Operationen, Universitätsklinikum Würzburg 2) Klinik für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie, RWTH Universitätsklinikum Aachen, Pauwelsstr. 30 52074 Aachen214.02.2023Ehret Kasemo, TottaSources Air qualityClimate effects CharacterizationDecision findingAbatement strategies and policiesHealth effects and mechanismsEpidemiologyUFP research initiative in Germany314.02.2023Ehret Kasemo, Totta„…close existing knowledgegaps, to support riskanalysis of ultrafineparticles (UFP) “ (free translation)• Measurement techniques• Chemical characterization• Epidemiology• Molecular mechanisms• ToxicologyUFP research initiative in Germany414.02.2023Ehret Kasemo, TottaCollaborating partnersProject partnersProf. Ralf Zimmermann514.02.2023Ehret Kasemo, TottaDetermine risks: toolkitFor mechanisms, (sensible) simplification is necessaryPerson: www.clipground.com; organs: media.istockphoto.comHealth effects and mechanismsThe nose is the entry point for aerosols614.02.2023Ehret Kasemo, TottaNose drawing: apkpure.com nose drawing tutorialsWhat happens to particles in the nose?Transport through the nose714.02.2023Ehret Kasemo, TottaHuman drawing: fineartamerica.com/featured/anatomy-nasal-cavity-granger.html, dog: www.cbc.ca, potoroo: Wikipedia (German), guinea pig: four-paws.orgNose structures: Zwicker, David, et al. "Physical and geometric constraints shape the labyrinth-like nasal cavity." Proceedings of the National Academy of Sciences 115.12 (2018): 2936-2941.Which model options are available (and which make sense)?Cellular level: what must we mimick?814.02.2023Ehret Kasemo, TottaCell types sketch: Mygind, Niels, and Ronald Dahl. "Anatomy, physiology and function of the nasal cavities in health and disease." Advanced drug deliveryreviews 29.1-2 (1998): 3-12. Tight junctions: Kojima, Takashi, et al. "Regulation of tight junctions in upper airway epithelium." BioMed research international 2013 (2013).H&E staining, human nasal mucosa. Image: Renate Bausch/Hieke ‚OberwinklerMucin polymers. Image by Katharina Ribbeck lab.Freeze-fracture image of tight junctionsin native nasal tissue by Kojima et al.Model selection should be based on the objectives of the projectHuman mucosal model of the upper airways9Ehret Kasemo, Totta 14.02.20235µm500µmMUC5ACGoblet cellsKinociliaTight junctionsCell types sketch: Mygind, Niels, and Ronald Dahl. "Anatomy, physiology and function of the nasal cavities in health and disease." Advanced drug deliveryreviews 29.1-2 (1998): 3-12.Our upper airway mucosa model1014.02.20231. PET filter with 0.4 µm pores (collagen-coated)2. Primary epithelial cells3. Primary fibroblasts4. Air interface with mucus  ALI5. Liquid interfaceLower image from K. Ickrath’sDoctoral Thesis1.2.3.5.4.Human biopsies used to establish model from primary cellsEhret Kasemo, TottaUltrafine particle exposures11BOTTOM RIGHT confocal and SEM: Cheng, Crystal, et al. "Toxicity and imaging of multi-walled carbon nanotubes in human macrophage cells." Biomaterials 30.25 (2009): 4152-4160. ALL OTHER: Jurkiewicz, Karolina, Mirosława Pawlyta, and Andrzej Burian. "Structure of carbon materials explored by localtransmission electron microscopy and global powder diffraction probes." C 4.4 (2018): 68.Submerged exposure1214.02.20231. Submerged exposure2. Nebulizer / Cloud system (Vitrocell) 3. Aerosol exposure (Vitrocell)Example: Multi-walled carbon nanotubes (MWCNT NM-401)Ehret Kasemo, TottaApprox. 70nm wide, 4µm longAgglomerates in sampleKirsten, Rasmussen., et al. "Multi-Walled Carbon Nanotubes, NM-400, NM-401, NM-402, NM-403: Characterisation and Physico-Chemical Properties." (2014).Cloud exposure1314.02.2023Ehret Kasemo, Totta1. Submerged exposure2. Nebulizer / Cloud system (customized Vitrocell)3. Aerosol exposure (Vitrocell)Printex90 TEM: Shen, Y et al.: Particle and fibre toxicology, 15. BSU and particle sketch: Pawlyta, Miroslawa, Jean-Noël Rouzaud, and Stanislaw Duber. "Raman microspectroscopy characterization of carbon blacks: Spectral analysis and structural information." Carbon 84 (2015): 479-490.Example: Carbon black / Printex90 Basic structural unitsAerosol exposure1414.02.2023Ehret Kasemo, Totta1. Submerged exposure2. Nebulizer / Cloud system3. Aerosol exposure (Vitrocell) –collaboration Zimmermann/ di Bucchianico/ Delaval (Helmholtz German Research Center for Environmental Health)Generated Black carbon ~40% elemental carbon, 60% organic carbon compounds~45 nm diameterApproximated dose: 1.5 ± 0.2 ng/cm2Copy of drawing by Leonardo da Vinci fromwww.britannica.com/summary/Leonardo-da-Vincis-AchievementsDoes the model work?5µmMucosal model functionality - data1614.02.20231. Intact barrier – experiment with UFP (unpublished)2. Beating kinocilia (Lodes et al. (Hackenberg), 2020 + unpublished data)3. Mucus production (Lodes et al. (Hackenberg), 2020 + unpublished data)4. Responsive to toxins (unpublished)Ehret Kasemo, Totta1. Intact barrier – TEER data1714.02.2023Ehret Kasemo, Totta1. Intact barrier – potential effect of UFP1814.02.2023Transepithelial Electrical Resistance (TEER)Carbon Black (µg/cm2)0,01 2 4 10 150.05 1 8TEER (Ohm*cm2)ALI co-cultureNeg. ctrlPos. ctrlUFPEhret Kasemo, Totta2. Beating kinocilia19Ehret Kasemo, Totta 14.02.2023Ciliary beat frequency and heat-maps of beating kinocilia: Dissertation by Nina Lodes, 2019 (co-supervised by S. Hackenberg)Cell line HBEC3-KT Primary bronchial cells Primary nasal cells2. Beating kinocilia20Ehret Kasemo, Totta 14.02.2023Video: Nina Lodes / David Kessie in Steinke Lab, Fraunhofer ISC2. Beating kinocilia21Ehret Kasemo, Totta 14.02.2023Video: Nina Lodes / David Kessie in Steinke Lab, Fraunhofer ISC3. Mucus production22Ehret Kasemo, Totta 14.02.2023Table I adjusted from from Ali, Mahmoud S., and Jeffrey P. Pearson. "Upper airway mucin gene expression: a review." The Laryngoscope 117.5 (2007): 932-938.3. Mucus productionNow mucus functionality can be investigated23Ehret Kasemo, Totta 14.02.2023MUC5ACDAPICytokeratin18Mucin 5BTable I adjusted from from Ali, Mahmoud S., and Jeffrey P. Pearson. "Upper airway mucin gene expression: a review." The Laryngoscope 117.5 (2007): 932-938.Proteome by F. Neiersand J-M. Heydel, Center for Taste and Feeding Behaviour, Dijon, France in collaboration with S. Hackenberg and M. SteinkeGenes detected in mucussecreted from in vitro mucosal modelMUC1MUC4MUC5ACMUC5B4. Responsive to toner particles24Ehret Kasemo, Totta 14.02.2023Pos. ctrl.                 890.89Neg. ctrl.Toner particles (µg/cm2)8.92550751000Metabolic activity by MTT-TestCellviabilityrelative tountreated(%) Data by Nursen Tekin and Dr. Till Meyer, HNO WürzburgThe in vitro model isresponsive to toxicsubstances after ALI exposureParticle size by TEM: ~5-10µm4. Responsiveness to UFP25Ehret Kasemo, Totta 14.02.2023Carbon black (µg/cm2)Model UFP induces no cytotoxicity after 24h (MTT)ALI co-cultureEpithelialmonocultureFibroblast monocultureGrowth conditionViability(% ofneg. ctrl.)… but monocultures are sensitive4. Responsiveness to UFP26Ehret Kasemo, Totta 14.02.2023Carbon Black (µg/cm2)Model UFP induces no genotoxicity after 24h (Comet)0,01 2 4 10 15Donor IDNeg. ctrlOlive TailMoment20100Positive controls5µmMucosal model suitablity2714.02.2023Ehret Kasemo, TottaParameter Model suitable? MethodsBarrier integrity Yes TEER, FD4, ultrastructure, IFAMucociliary clearance Yes Ciliary beating and frequency analysisMucus present Yes Histology, antibody detection, proteins, qPCR, rheology…Toxicity Yes LDH, MTT, Comet assay…Inflammation Molecular analysisor co-cultureELISA, Dotblot, qPCR, Western blot, FACS…Cell composition Yes Histology, antibody detection, ultrastructure, RNA-seqMorphology Yes Histology, ultrastructure, ciliary beatingheat-mapsRegeneration Most likely TEER, histology, mucus assays…Teaser: organoids28Ehret Kasemo, Totta 14.02.2023Images and cell cultures: Lili Szabo (Kai Kretzschmar Lab, Würzburg)P0, day 6, donor 1 P2, day 9, donor 2 Stem cellsExciting development29Ehret Kasemo, Totta 14.02.2023• Barrier damage without toxicity –new approaches to riskassessment?• Mucus-particle interactions• Mucus rheology• What is normal? Population variancePopulation/donor variationSlime illustration: www.seriouslynatural.org/. Viscoelasticity illustration: Picout, David R., and Simon B. Ross-Murphy. "Rheology of biopolymer solutions and gels." TheScientificWorldJOURNAL 3 (2003): 105-121. Population: www.dreamstime.comMucus proterties and particleinteractions?Thank you for your attentionEhret Kasemo, TottaCollaborationsFraunhofer ISC, Würzburg• Maria Steinke• Sofia DembskiUniversity of Burgundy Franche-Comté, Center for Taste and Feeding Behaviour, Dijon, France• Fabien Neier• Jean-Marie HeydelMSNZ for Cancer Research Würzburg• Kai Kretzschmar• Stephan Hackenberg• Agmal Scherzad• Till Meyer• Totta Ehret KasemoWithin BayUFPFriedrich-Alexander-Universität Erlangen-Nürnberg• Simone Schmitz-SpankeHelmholtz Zentrum München – German Research Center for Environmental Health• Ralf Zimmermann• Sebastiano di Bucchianico• Mathilde Delaval3314.02.2023MWCNT 4013414.02.2023MWCNT 4015µm3514.02.2023Ehret Kasemo, TottaEndpoints byusage in non-animal models3614.02.2023MWCNT 4013714.02.2023Carbon black / Printex90Projects – internal list to discuss3814.02.2023Ehret Kasemo, Totta Ultrafine particles in co-culture ALI model of upper airway mucosa Carbon black/Printex90 with nebulizer, inhouse Aerosols with Vitrocell system (collab. Helmholz München) Particle characterization (collab. Sofia Dembski, Fraunhofer ISC) Mucus Optimize model (e.g. induce goblet cells with IL-13, establish positive controls for expulsion (mechanical, LPS, ….. Co-supervised by Maria S.) Mucus proteome (SH + MS collab. France) Mucus rheology (upcoming - collab. Maria Steinke, Fraunhofer ISC) Organoid pre-cultures More replicates from each donor  quality improvement (collab. Kai Kretzschmar, Würzburg Biopsy VS brush based is an option (so far not explored) Microbiology Fungi and bacteria in voice prostheses So far a side project towards understanding challenges with the functional impact of biofilms on voice prostheses (map economic and healthgains of improvements as another side project?) Population variance – „how different are people?“  New project to initiate? Possible grant topic, synergies with almost all otherprojects using ALI-models Analyze different aspects of nasal physiology to gain better understanding for what is normal and what is disease-related or related to harmfulsubstance exposures RNA abundance („gene expression“) i.e. RNA-seq Mucus characterization Comet+LDH responses to (toxic?) substances (establish lab standard, e.g. UFPs (Printex and ZnO), LPS, …. Viruses with Maria?) Functional impact of (toxic?) substances  TEER, mucus production, ciliary beating (re-establish this protocol) Statistical analysis with generalized linear models (GLMs) or similar  collab London (ask Alice Balard? Or ask David Zwicker)

Functional Effects of Carbon Black Nanoparticles on Primary Airway Epithelial Cells in vitro

https://publikationen.bibliothek.kit.edu/1000158383/150667036

Functional Effects of Carbon Black Nanoparticles on Primary Airway Epithelial Cells in vitro

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