Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection, with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly, SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Archetypal Analysis for neuronal clique detection in low-rate calcium fluorescence imaging.

Archetypal analysis (AA) is a versatile data analysis method to cluster distinct features within a data set. Here, we demonstrate a framework showing the power of AA to spatio-temporally resolve events in calcium imaging, an imaging modality commonly used in neurobiology and neuroscience to capture neuronal communication patterns. After validation of our AA-based approach on synthetic data sets, we were able to characterize neuronal communication patterns in recorded calcium waves. Clinical relevance- Transient calcium events play an essential role in brain cell communication, growth, and network formation, as well as in neurodegeneration. To reliably interpret calcium events from personalized medicine data, where patterns may differ from patient to patient, appropriate image processing and signal analysis methods need to be developed for optimal network characterization.

Multi-curvature micropatterns unveil distinct calcium and mitochondrial dynamics in neuronal networks.

Tangential curvatures are a key geometric feature of tissue folds in the human cerebral cortex. In the brain, these smoother and firmer bends are called gyri and sulci and form distinctive curved tissue patterns imposing a mechanical stimulus on neuronal networks. This stimulus is hypothesized to be essential for proper brain cell function but lacks in most standard neuronal cell assays. A variety of soft lithographic micropatterning techniques can be used to integrate round geometries in cell assays. Most microfabricated patterns, however, focus only on a small set of defined curvatures. In contrast, curvatures in the brain span a wide physical range, leaving it unknown which precise role distinct curvatures may play on neuronal cell signaling. Here we report a hydrogel-based multi-curvature design consisting of over twenty bands of distinct parallel curvature ranges to precisely engineer neuronal networks' growth and signaling under patterns of arcs. Monitoring calcium and mitochondrial dynamics in primary rodent neurons grown over two weeks in the multi-curvature patterns, we found that static calcium signaling was locally attenuated under higher curvatures (k > 0.01 µm-1). In contrast, to randomize growth, transient calcium signaling showed higher synchronicity when neurons formed networks in confined multi-curvature patterns. Additionally, we found that mitochondria showed lower motility under high curvatures (k > 0.01 µm-1) than under lower curvatures (k < 0.01 µm-1). Our results demonstrate how sensitive neuronal cell function may be linked and controlled through specific curved geometric features. Furthermore, the hydrogel-based multi-curvature design possesses high compatibility with various surfaces, allowing a flexible integration of geometric features into next-generation neuro devices, cell assays, tissue engineering, and implants.

Low-cost calcium fluorometry for long-term nanoparticle studies in living cells.

Calcium fluorometry is critical to determine cell homeostasis or to reveal communication patterns in neuronal networks. Recently, characterizing calcium signalling in neurons related to interactions with nanomaterials has become of interest due to its therapeutic potential. However, imaging of neuronal cell activity under stable physiological conditions can be either very expensive or limited in its long-term capability. Here, we present a low-cost, portable imaging system for long-term, fast-scale calcium fluorometry in neurons. Using the imaging system, we revealed temperature-dependent changes in long-term calcium signalling in kidney cells and primary cortical neurons. Furthermore, we introduce fast-scale monitoring of synchronous calcium activity in neuronal cultures in response to nanomaterials. Through graph network analysis, we found that calcium dynamics in neurons are temperature-dependent when exposed to chitosan-coated nanoparticles. These results give new insights into nanomaterial-interaction in living cultures and tissues based on calcium fluorometry and graph network analysis.

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function.

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices.

Controlling the pro-inflammatory function of 6-sulfo LacNAc (slan) dendritic cells with dimethylfumarate.

BACKROUND: The fumaric acid ester (FAE) dimethylfumarate (DMF) is a small molecule immunomodulator successfully used for the treatment of psoriasis and multiple sclerosis (MS). DMF is thought to inhibit pathogenic immune responses with Th17/Th1T cells, and IL-23/IL-12 producing dendritic cells (DCs). 6-sulfo LacNAc expressing dendritic cells (slanDCs) are a human pro-inflammatory cell type found frequently among the infiltrating leukocytes in skin lesions of psoriasis and brain lesions of MS. OBJECTIVE: To explore the influence of DMF on functional properties and cell signaling pathways of slanDCs. METHODS: In the context of slanDCs we studied the role of DMF in modulating cell migration, phenotypic maturation, cytokine production, cell signaling and T cell stimulation. RESULTS: Initially, we observed the reduction of slanDCs numbers in psoriasis skin lesions of FAE treated patients. Studying whether DMF controls the migratory capacity of slanDCs to chemotactic factors expressed in psoriasis we observed an inhibition of the CX3CL1 and C5a depedent cell migration. DMF also attenuated the rapid spontaneous phenotypic maturation of slanDCs, as judged by a reduced CD80, CD86, CD83 and HLA-DR expression. In addition, we observed a DMF-dependent decrease of IL-23, IL-12, TNF-α and IL-10 secretion, and noticed a reduced capacity to stimulate Th17/Th1 responses. DMF targeted in slanDCs different intracellular cell signaling pathways including NFκB, STAT1 and HO-1. CONCLUSIONS: With this study we identify a frequent pro-inflammatory cell type found in psoriasis and MS as a relevant target for the therapeutic immunomodulatory effects of DMF.

The phosphodiesterase 4 inhibitor apremilast inhibits Th1 but promotes Th17 responses induced by 6-sulfo LacNAc (slan) dendritic cells.

BACKGROUND: The phosphodiesterase 4 (PDE4) inhibitor apremilast increases cellular cAMP levels and has proven effective in the treatment of psoriasis and psoriasis arthritis. We recently described 6-sulfo LacNAc dendritic cells (slanDCs) as immature DCs in blood and as a subset of inflammatory dermal DCs in psoriasis with a pronounced capacity to produce proinflammatory cytokines and to program Th17/Th1 T cell responses. OBJECTIVE: The aim of this study was to investigate possible immune regulatory effects of the PDE4 inhibitor apremilast on slanDCs. METHODS: In vitro studies were performed analyzing the effects of apremilast on the proinflammatory function of slanDCs and their capacity to induce Th1/Th17-biased T cell responses. RESULTS: Increasing cAMP levels in slanDCs by PDE4 inhibition strongly reduced production of IL-12 and TNF-α. In line with these findings, co-culture experiments with apremilast-pulsed slanDCs and allogeneic T cells either from psoriasis patients or healthy controls, revealed a significant reduction of IFN-γ production and expression of the transcription factor T-bet. In parallel, production of IL-23 and IL-1ß by slanDCs was increased and co-cultured T cells revealed a largely augmented IL-17 production and an upregulated RORyt expression. CONCLUSIONS: We here demonstrate anti-inflammatory as well as Th17-promoting effects of apremilast when studying blood precursors of human inflammatory dermal dendritic cells. In the concert of the broad anti-inflammatory effects of apremilast on keratinocytes, fibroblasts and endothelial cells, the dual effect on slan+ inflammatory dermal DCs should be taken into account and may constrain therapeutic responses.

Modulating motility of intracellular vesicles in cortical neurons with nanomagnetic forces on-chip.

Vesicle transport is a major underlying mechanism of cell communication. Inhibiting vesicle transport in brain cells results in blockage of neuronal signals, even in intact neuronal networks. Modulating intracellular vesicle transport can have a huge impact on the development of new neurotherapeutic concepts, but only if we can specifically interfere with intracellular transport patterns. Here, we propose to modulate motion of intracellular lipid vesicles in rat cortical neurons based on exogenously bioconjugated and cell internalized superparamagnetic iron oxide nanoparticles (SPIONs) within microengineered magnetic gradients on-chip. Upon application of 6-126 pN on intracellular vesicles in neuronal cells, we explored how the magnetic force stimulus impacts the motion pattern of vesicles at various intracellular locations without modulating the entire cell morphology. Altering vesicle dynamics was quantified using, mean square displacement, a caging diameter and the total traveled distance. We observed a de-acceleration of intercellular vesicle motility, while applying nanomagnetic forces to cultured neurons with SPIONs, which can be explained by a decrease in motility due to opposing magnetic force direction. Ultimately, using nanomagnetic forces inside neurons may permit us to stop the mis-sorting of intracellular organelles, proteins and cell signals, which have been associated with cellular dysfunction. Furthermore, nanomagnetic force applications will allow us to wirelessly guide axons and dendrites by exogenously using permanent magnetic field gradients.

The Age of Cortical Neural Networks Affects Their Interactions with Magnetic Nanoparticles.

Despite increasing use of nanotechnology in neuroscience, the characterization of interactions between magnetic nanoparticles (MNPs) and primary cortical neural networks remains underdeveloped. In particular, how the age of primary neural networks affects MNP uptake and endocytosis is critical when considering MNP-based therapies for age-related diseases. Here, primary cortical neural networks are cultured up to 4 weeks and with CCL11/eotaxin, an age-inducing chemokine, to create aged neural networks. As the neural networks are aged, their association with membrane-bound starch-coated ferromagnetic nanoparticles (fMNPs) increases while their endocytic mechanisms are impaired, resulting in reduced internalization of chitosan-coated fMNPs. The age of the neurons also negates the neuroprotective effects of chitosan coatings on fMNPs, attributing to decreased intracellular trafficking and increased colocalization of MNPs with lysosomes. These findings demonstrate the importance of age and developmental stage of primary neural cells when developing in vitro models for fMNP therapeutics targeting age-related diseases.

Induction of Calcium Influx in Cortical Neural Networks by Nanomagnetic Forces.

Nanomagnetic force stimulation with ferromagnetic nanoparticles was found to trigger calcium influx in cortical neural networks without observable cytotoxicity. Stimulated neural networks showed an average of 20% increment in calcium fluorescence signals and a heightened frequency in calcium spiking. These effects were also confined spatially to areas with engineered high magnetic field gradients. Furthermore, blockage of N-type calcium channels inhibited the stimulatory effects of the nanomagnetic forces, suggesting the role of mechano-sensitive ion channels in mediating calcium influx.

Normal mast cell numbers in the tissues of AhR-deficient mice.

The transcription factor aryl hydrocarbon receptor (AhR) acts as an immunomodulatory molecule in several immune cell lineages. Recently, it has been implicated in development and maintenance of immune cells in barrier tissues such as skin and mucosa. To investigate its role on mast cell development and maintenance in skin, peritoneal exudate cells (PECs) and lymph nodes, we studied in depth their phenotype in AhR-deficient mice. Our findings do not provide any evidence for a suspected role of the AhR in mast cell homeostasis.

Increased Numbers of 6-sulfo LacNAc (slan) Dendritic Cells in Hand Transplant Recipients.

BACKGROUND: Vascularized composite allotransplantation (VCA) is a modern option for the treatment of functionally significant limb and tissue defects. In our study we aimed to characterize the morphological and histological features of the hand transplant recipient skin rejection. MATERIAL AND METHODS: We clinically evaluated the skin and mucous membranes and microscopically assessed biopsies taken from the patient's own skin and from the allogenic grafted limb (n=5). We also performed immunohistochemistry for presence of T lymphocytes (CD3, CD4, and CD8), B lymphocytes (CD20), macrophages (CD68), Langerhans cells (CD1a+), plasmacytoid dendritic cells (pDCs) (CD123+) and 6-sulfo LacNAc+ dendritic cells (slanDCs) (DD2+). RESULTS: Only scattered pDCs were present in both own and skin grafts. The number of LC in the epidermis was higher in graft skin in all cases and CD1a+ cells were also present in the dermis in transplanted skin in patients with grade 1 rejection. Most interestingly, we identified far increased numbers of dermal slanDCs in the grafted skin. SlanDCs have a high capacity to produce proinflammatory cytokines and have been described as inflammatory dermal dendritic cells in psoriasis and lupus erythematosus. CONCLUSIONS: It may be hypothesized that slanDCs identified in the skin after limb transplantation may support the local inflammatory skin reaction.

Engineering cortical neuron polarity with nanomagnets on a chip.

Intra- and extracellular signaling play critical roles in cell polarity, ultimately leading to the development of functional cell-cell connections, tissues, and organs. In the brain, pathologically oriented neurons are often the cause for disordered circuits, severely impacting motor function, perception, and memory. Aside from control through gene expression and signaling pathways, it is known that nervous system development can be manipulated by mechanical stimuli (e.g., outgrowth of axons through externally applied forces). The inverse is true as well: intracellular molecular signals can be converted into forces to yield axonal outgrowth. The complete role played by mechanical signals in mediating single-cell polarity, however, remains currently unclear. Here we employ highly parallelized nanomagnets on a chip to exert local mechanical stimuli on cortical neurons, independently of the amount of superparamagnetic nanoparticles taken up by the cells. The chip-based approach was utilized to quantify the effect of nanoparticle-mediated forces on the intracellular cytoskeleton as visualized by the distribution of the microtubule-associated protein tau. While single cortical neurons prefer to assemble tau proteins following poly-L-lysine surface cues, an optimal force range of 4.5-70 pN by the nanomagnets initiated a tau distribution opposed to the pattern cue. In larger cell clusters (groups comprising six or more cells), nanoparticle-mediated forces induced tau repositioning in an observed range of 190-270 pN, and initiation of magnetic field-directed cell displacement was observed at forces above 300 pN. Our findings lay the groundwork for high-resolution mechanical encoding of neural networks in vitro, mechanically driven cell polarization in brain tissues, and neurotherapeutic approaches using functionalized superparamagnetic nanoparticles to potentially restore disordered neural circuits.

Research highlights: cell separation at the bench and beyond.

We highlight recent progress in applying micro- and nanotechnology enabled cell separations to life sciences and clinical use. Microfluidic systems operate on a scale that matches that of cells (10-100 µm) and therefore allow interfacing and separations that are sensitive at this scale. Given the corresponding dimensions, it is not surprising that a wide array of microfluidic cell separation technologies have been developed using hydrodynamic, electrical, magnetic and optical forces, and have been applied to a range of biological and clinical problems in sample preparation. Passive separation approaches have distinct advantages for point of care applications or when downstream cell-based therapies are envisioned. We highlight a recent approach that allows for passive hydrodynamic filtering of cells over almost two orders of magnitude in flow conditions, which allowed the researchers to interface with a standard manual pipettor, creating a "microfluidic pipette tip". In a second work, passive separation by size yields distinct populations of mesenchymal stem cells that can be used therapeutically. The researchers report on other biophysical separations that would be expected to refine these cell populations further for the most efficacious cell-based therapies. In an intriguing twist, we highlight a creative idea in which stem cell populations could potentially also be extracted from a patient with less invasive surgeries, performing the separation using magnetic nanoparticles in vivo without bulk tissue disruption. New cell separation technologies will continue to be demonstrated, however, a major research thrust appears to be now developing these technologies to address unique application niches in point-of-care sample preparation for research and diagnostics or cell-based therapies.

Flexible and stretchable micromagnet arrays for tunable biointerfacing.

A process to surface pattern polydimethylsiloxane (PDMS) with ferromagnetic structures of varying sizes (micrometer to millimeter) and thicknesses (>70 µm) is developed. Their flexibility and magnetic reach are utilized to confer dynamic, additive properties to a variety of substrates, such as coverslips and Eppendorf tubes. It is found that these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.

Research highlights: Microtechnologies for engineering the cellular environment.

In this issue we highlight recent microtechnology-enabled approaches to control the physical and biomolecular environment around cells: (1) developing micropatterned surfaces to quantify cell affinity choices between two adhesive patterns, (2) controlling topographical cues to align cells and improve reprogramming to a pluripotent state, and (3) controlling gradients of biomolecules to maintain pluripotency in embryonic stem cells. Quantitative readouts of cell-surface affinity in environments with several cues should open up avenues in tissue engineering where self-assembly of complex multi-cellular structures is possible by precisely engineering relative adhesive cues in three dimensional constructs. Methods of simple and local epigenetic modification of chromatin structure with microtopography and biomolecular gradients should also be of use in regenerative medicine, as well as in high-throughput quantitative analysis of external signals that impact and can be used to control cells. Overall, approaches to engineer the cellular environment will continue to be an area of further growth in the microfluidic and lab on a chip community, as the scale of the technologies seamlessly matches that of biological systems. However, because of regulations and other complexities with tissue engineered therapies, these micro-engineering approaches will likely first impact organ-on-a-chip technologies that are poised to improve drug discovery pipelines.

Advances in high-throughput single-cell microtechnologies.

Micro-scale biological tools that have allowed probing of individual cells--from the genetic, to proteomic, to phenotypic level--have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics.

Astrocyte-neuron co-culture on microchips based on the model of SOD mutation to mimic ALS.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.