Modeling SARS-CoV-2 and influenza infections and antiviral treatments in human lung epithelial tissue equivalents.

There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens.

Enhancement of Neuroglial Extracellular Matrix Formation and Physiological Activity of Dopaminergic Neural Cocultures by Macromolecular Crowding.

The neuroglial extracellular matrix (ECM) provides critical support and physiological cues for the proper growth, differentiation, and function of neuronal cells in the brain. However, in most in vitro settings that study neural physiology, cells are grown as monolayers on stiff surfaces that maximize adhesion and proliferation, and, therefore, they lack the physiological cues that ECM in native neuronal tissues provides. Macromolecular crowding (MMC) is a biophysical phenomenon based on the principle of excluded volume that can be harnessed to induce native ECM deposition by cells in culture. Here, we show that MMC using two species of Ficoll with vitamin C supplementation significantly boosts deposition of relevant brain ECM by cultured human astrocytes. Dopaminergic neurons cocultured on this astrocyte-ECM bed prepared under MMC treatment showed longer and denser neuronal extensions, a higher number of pre ad post synaptic contacts, and increased physiological activity, as evidenced by higher frequency calcium oscillation, compared to standard coculture conditions. When the pharmacological activity of various compounds was tested on MMC-treated cocultures, their responses were enhanced, and for apomorphine, a D2-receptor agonist, it was inverted in comparison to control cell culture conditions, thus emulating responses observed in in vivo settings. These results indicate that macromolecular crowding can harness the ECM-building potential of human astrocytes in vitro forming an ultra-flat 3D microenvironment that makes neural cultures more physiological and pharmacological relevant.

Neuroinflammation associated with ultrasound-mediated permeabilization of the blood-brain barrier.

The blood-brain barrier (BBB) continues to represent one of the most significant challenges for successful drug-based treatments of neurological disease. Mechanical modulation of the BBB using focused ultrasound (FUS) and microbubbles (MBs) has shown considerable promise in enhancing the delivery of therapeutics to the brain, but questions remain regarding possible long-term effects of such forced disruption. This review examines the evidence for inflammation associated with ultrasound-induced BBB disruption and potential strategies for managing such inflammatory effects to improve both the efficacy and safety of therapeutic ultrasound in neurological applications.

Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform.

The COVID-19 pandemic has highlighted the need for human respiratory tract-based assay platforms for efficient discovery and development of antivirals and disease-modulating therapeutics. Physiologically relevant tissue models of the lower respiratory tract (LRT), including the respiratory bronchioles and the alveolar sacs, are of high interest because they are the primary site of severe SARS-CoV-2 infection and are most affected during the terminal stage of COVID-19. Current epithelial lung models used to study respiratory viral infections include lung epithelial cells at the air-liquid interface (ALI) with fibroblasts and endothelial cells, but such models do not have a perfusable microvascular network to investigate both viral infectivity and viral infection-induced thrombotic events. Using a high throughput, 64-chip microfluidic plate-based platform, we have developed two novel vascularized, LRT multi-chip models for the alveoli and the small airway. Both models include a perfusable microvascular network consisting of human primary microvascular endothelial cells, fibroblasts and pericytes. The established biofabrication protocols also enable the formation of differentiated lung epithelial layers at the ALI on top of the vascularized tissue bed. We validated the physiologically relevant cellular composition, architecture and perfusion of the vascularized lung tissue models using fluorescence microscopy, flow cytometry, and electrical resistance measurements. These vascularized, perfusable microfluidic lung tissue on high throughput assay platforms will enable the development of respiratory viral infection and disease models for research investigation and drug discovery.

Operationalizing the Use of Biofabricated Tissue Models as Preclinical Screening Platforms for Drug Discovery and Development.

A wide range of complex in vitro models (CIVMs) are being developed for scientific research and preclinical drug efficacy and safety testing. The hope is that these CIVMs will mimic human physiology and pathology and predict clinical responses more accurately than the current cellular models. The integration of these CIVMs into the drug discovery and development pipeline requires rigorous scientific validation, including cellular, morphological, and functional characterization; benchmarking of clinical biomarkers; and operationalization as robust and reproducible screening platforms. It will be critical to establish the degree of physiological complexity that is needed in each CIVM to accurately reproduce native-like homeostasis and disease phenotypes, as well as clinical pharmacological responses. Choosing which CIVM to use at each stage of the drug discovery and development pipeline will be driven by a fit-for-purpose approach, based on the specific disease pathomechanism to model and screening throughput needed. Among the different CIVMs, biofabricated tissue equivalents are emerging as robust and versatile cellular assay platforms. Biofabrication technologies, including bioprinting approaches with hydrogels and biomaterials, have enabled the production of tissues with a range of physiological complexity and controlled spatial arrangements in multiwell plate platforms, which make them amenable for medium-throughput screening. However, operationalization of such 3D biofabricated models using existing automation screening platforms comes with a unique set of challenges. These challenges will be discussed in this perspective, including examples and thoughts coming from a laboratory dedicated to designing and developing assays for automated screening.

JAK inhibitors dampen activation of interferon-stimulated transcription of ACE2 isoforms in human airway epithelial cells.

SARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Here, we report on transcriptomes activated in primary airway cells by interferons and their suppression by Janus kinase (JAK) inhibitors. Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. ChIP-seq for activating histone marks and Pol II loading identified candidate enhancer elements controlling the ACE2 locus, including the intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE2 and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib, used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.

Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.

Activation of Interferon-Stimulated Transcriptomes and ACE2 Isoforms in Human Airway Epithelium Is Curbed by Janus Kinase Inhibitors.

SARS-CoV-2 infection of human airway epithelium activates genetic programs that lead to progressive hyperinflammation in COVID-19 patients. Here we report on genetic programs activated by interferons and the suppression by Janus kinase (JAK) inhibitors. The angiotensin-converting enzyme 2 (ACE2) is the receptor for SARS-CoV-2 and deciphering its regulation is paramount for understanding the cell tropism of SARS-CoV-2 infection. We identified candidate regulatory elements in the ACE2 locus in human primary airway cells and lung tissue. Activating histone and promoter marks and Pol II loading characterize the intronic dACE2 and define novel candidate enhancers distal to the genuine ACE2 promoter and within additional introns. dACE2, and to a lesser extent ACE2, RNA levels increased in primary cells treated with interferons and this induction was mitigated by JAK inhibitors that are used therapeutically in COVID-19 patients. Our analyses provide insight into ACE2 regulatory elements and highlight JAK inhibitors as suitable tools to suppress interferon-activated genetic programs in bronchial cells.

Regulation of the ACE2 locus in human airways cells.

The angiotensin-converting enzyme 2 (ACE2) receptor is the gateway for SARS-CoV-2 to airway epithelium1,2 and the strong inflammatory response after viral infection is a hallmark in COVID-19 patients. Deciphering the regulation of the ACE2 gene is paramount for understanding the cell tropism of SARS-CoV-2 infection. Here we identify candidate regulatory elements in the ACE2 locus in human primary airway cells and lung tissue. Activating histone and promoter marks and Pol II loading characterize the intronic dACE2 and define novel candidate enhancers distal to the genuine ACE2 promoter and within additional introns. dACE2, and to a lesser extent ACE2, RNA levels increased in primary bronchial cells treated with interferons and this induction was mitigated by Janus kinase (JAK) inhibitors that are used therapeutically in COVID-19 patients. Our analyses provide insight into regulatory elements governing the ACE2 locus and highlight that JAK inhibitors are suitable tools to suppress interferon-activated genetic programs in bronchial cells.

Contrast enhanced ultrasound imaging by nature-inspired ultrastable echogenic nanobubbles.

Advancement of ultrasound molecular imaging applications requires not only a reduction in size of the ultrasound contrast agents (UCAs) but also a significant improvement in the in vivo stability of the shell-stabilized gas bubble. The transition from first generation to second generation UCAs was marked by an advancement in stability as air was replaced by a hydrophobic gas, such as perfluoropropane and sulfur hexafluoride. Further improvement can be realized by focusing on how well the UCAs shell can retain the encapsulated gas under extreme mechanical deformations. Here we report the next generation of UCAs for which we engineered the shell structure to impart much better stability under repeated prolonged oscillation due to ultrasound, and large changes in shear and turbulence as it circulates within the body. By adapting an architecture with two layers of contrasting elastic properties similar to bacterial cell envelopes, our ultrastable nanobubbles (NBs) withstand continuous in vitro exposure to ultrasound with minimal signal decay and have a significant delay on the onset of in vivo signal decay in kidney, liver, and tumor. Development of ultrastable NBs can potentially expand the role of ultrasound in molecular imaging, theranostics, and drug delivery.

Ultrasound Contrast Agents and Delivery Systems in Cancer Detection and Therapy.

Ultrasound is the second most utilized imaging modality in the world because it is widely accessible, robust, and safe. Aside from its extensive use in diagnostic imaging, ultrasound has also been frequently utilized in therapeutic applications. Particularly, when combined with appropriate delivery systems, ultrasound provides a flexible platform for simultaneous real-time imaging and triggered release, enabling precise, on-demand drug delivery to target sites. This chapter will discuss the basics of ultrasound including its mechanism of action and how it can be used to trigger the release of encapsulated drug either through thermal or cavitation effects. Fundamentals of ultrasound contrast agents, how they enhance ultrasound signals, and how they can be modified to function as carriers for triggered and targeted release of drugs will also be discussed.

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease.

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease.

Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.