and G

and G.A.D also share support from the National Institute of Allergy and Infectious Diseases (NIH R21 AI142050) and the Cystic Fibrosis Foundation (DUNCAN18I0). to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding. 0.05; Mann-Whitney test. Reprinted with permission from Duncan, G.; Kim, N.; Colon-Cortes, Y.; Pamiparib Rodriguez, J.; Mazur, M.; Birket, S.; Rowe, S.; West, N.; Livraghi-Butrico, A.; Boucher, R.; Hanes, J.; Aslanidi, G.; and Suk, J. An adeno-associated viral vector capable of penetrating the mucus barrier to inhaled gene therapy ( Molecular TherapyMethods & Clinical Development 2018, 9:296-304. Copyright 2018, The American Society of Gene and Cell Therapy [286]. 6.2. Viral Particle Tracking, HostCVirus Interactions, and Specific Barrier Component Contributions Viral transit through the mucus gel and subsequent PCL is a necessary component of all respiratory infections (see Section 4), and therefore evaluating the diffusion of viral particles through mucus represents an important aspect of viral pathogenesis. Individual virions can be tracked in real time by directly labelling viral particles with reactive, lipophilic, or intercalating dyes [287]. Quantum dots, a type of semiconductor nanoparticles, can Pamiparib also be used to label virions [288] without significantly impacting infectivity [289]. Once labeled, particles can be imaged directly [290] in mucus or engineered surrogates [273]. Trajectories of virion movement can be imaged, as shown in Figure 3B, to measure diffusion and mucus penetration [272,286]. As opposed to muco-inert particles used to study microrheology, viral particles often exhibit adhesive interactions with airway mucus components [286]. The measured pore sizes of airway mucus (~200C500 nm) would imply rapid diffusion of viral particles through the mucus layer based on viral particle size [259,266]. However, adhesive Pamiparib interactions between viral surface glycoprotein domains have been shown to significantly reduce viral diffusion through airway mucus [257,291]. For example, particle tracking microrheology studies using fluorescently-labelled adeno-associated virus revealed that diffusion of the 20 nm virions through CF sputum was substantially slower compared to 100 nm nanoparticles, which are significantly larger [292]. Importantly, viral particle tracking can be done with any mucus source, including directly on ALI systems. Evidence of viral adhesion can then be further investigated outside the context of 3D model systems using surface plasmon resonance [293], optical tweezers and atomic force microscopy [294], or a quartz crystal microbalance [295]. However, to date there have been few attempts at direct tracking of viral particles in mucus gel or on ALI systems [286]. Finally, engineered mucus hydrogels and genetic ablation of mucin expression in ALI or organoid systems represent potentially powerful tools to study the contributions of specific barrier components to infection. Engineered mucus can be produced in large Pamiparib volumes and can be tuned to desired parameters [273,296,297,298] such as variable cross-linking concentration [296] or mucin gels p35 composed of only MUC5B or MUC5AC [273,297]. As with ex vivo mucus, these surrogate mucin gels could then be applied to in vitro systems to explore infection phenotypes. However, difficulty in mimicking both bulk and microrheological properties of native mucus combined with the genetic tractability of in vitro culture systems (see Section 2) highlights the utility in creating modified mucus gels through altered gene expression within the context of in vitro human ASL. Similarly, the contribution of tethered mucins as well as other host factors in the ASL can be dissected at baseline and during viral infection. For instance, CRISPR/Cas9-mediated depletion of the tethered mucin MUC18 from ALI cultures suggests a general pro-inflammatory role [40]. Pamiparib Koh et al. demonstrated that ablation of the SAM-pointed domain containing ETS transcription factor (SPDEF) from ALI cultures prevented MUC5AC induction and subsequent MCC impairment after stimulation with interleukin 13 [42]. Still, more work remains to dissect the contribution that individual mucins and other respiratory factors make towards a functional ASL barrier which protects from viral infection. Additionally, the extent to which individual host factors influence viral pathogenesis in both healthy and diseased human airways still needs to be addressed. 7. Conclusions and Future Perspectives Understanding mucosal.