Chronic Methadone Use Alters the CD8+ T Cell Phenotype In Vivo and Modulates Its Responsiveness Ex Vivo to Opioid Receptor and TCR Stimuli.

Endogenous opioid peptides are released at sites of injury, and their cognate G protein-coupled opioid receptors (ORs) are expressed on immune cells. Although drugs of misuse appropriate ORs, conflicting reports indicate immunostimulatory and immunosuppressive activity, in that opioid users have elevated infection risk, opioids activate innate immune cells, and opioids attenuate inflammation in murine T cell-mediated autoimmunity models. The i.v. use of drugs transmits bloodborne pathogens, particularly viruses, making the study of CD8+ T cells timely. From a cohort of nonuser controls and methadone users, we demonstrate, via t-Stochastic Neighbor Embedding and k-means cluster analysis of surface marker expression, that chronic opioid use alters human CD8+ T cell subset balance, with notable decreases in T effector memory RA+ cells. Studying global CD8+ T cell populations, there were no differences in expression of OR and several markers of functionality, demonstrating the need for finer analysis. Purified CD8+ T cells from controls respond to opioids ex vivo by increasing cytoplasmic calcium, a novel finding for OR signal transduction, likely because of cell lineage. CD8+ T cells from controls exposed to µ-OR agonists ex vivo decrease expression of activation markers CD69 and CD25, although the same markers are elevated in µ-OR-treated cells from methadone users. In contrast to control cells, T cell subsets from methadone users show decreased expression of CD69 and CD25 in response to TCR stimulus. Overall, these results indicate a direct, selective role for opioids in CD8+ T cell immune regulation via their ability to modulate cell responses through the opioid receptors and TCRs.

Insights From Dynamic Neuro-Immune Imaging on Murine Immune Responses to CNS Damage.

Evolving technologies and increasing understanding of human physiology over the past century have afforded our ability to intervene on human diseases using implantable bio-materials. These bio-electronic devices present a unique challenge through the creation of an interface between the native tissue and implantable bio-materials: the generation of host immune response surrounding such devices. While recent developments in cancer immunology seek to stimulate the immune system against cancer, successful long-term application of implantable bio-material devices need to durably minimize reactive immune processes at involved anatomical sites. Peripheral immune system response has been studied extensively for implanted bio-materials at various body sites. Examples include tooth composites (Gitalis et al., 2019), inguinal hernia repair (Heymann et al., 2019), and cardiac stents and pacemaker leads (Slee et al., 2016). Studies have also been extended to less well-studied immune reactivity in response to CNS neural-electronic implant devices. Recent technological advances in 2-Photon Laser Scanning Microscopy (2P-LSM) have allowed novel insights into in vivo immune response in a variety of tissue microenvironments. While imaging of peripheral tissues has provided an abundance of data with regards to immune cell dynamics, central nervous system (CNS) imaging is comparatively complicated by tissue accessibility and manipulation. Despite these challenges, the results of dynamic intravital neuro-immune imaging thus far have provided foundational insights into basic CNS biology. Utilizing a combination of intravital and ex vivo 2P-LSM, we have observed novel pathways allowing immune cells, stromal cells, cancer cells and proteins to communicate between the CNS parenchyma and peripheral vasculature. Similar to what has been reported in the intestinal tract, we have visualized myeloid cells extend dendritic processes across the blood brain barrier (BBB) into pial blood vessels. Furthermore, transient vessel leaks seen during systemic inflammation provide opportunities for cellular protein to be exchanged between the periphery and CNS. These insights provide new, visual information regarding immune surveillance and antigen presentation within the CNS. Furthermore, when combining intravital 2P-LSM and microfluidic devices complexed with mathematical modeling, we are gaining new insights into the intravascular behavior of circulating immune cells. This new knowledge into the basic mechanisms by which cells migrate to and interact with the CNS provide important considerations for the design of neuro-electronic biomaterials that have the potential to connect the peripheral-neural microenvironments into a unique, artificial interface.

Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis.

Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1ß, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.

Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular Smooth Muscle Cells Is Required for Optimal Peripheral Perfusion Recovery.

BACKGROUND: Limb ischemia resulting from peripheral vascular disease is a common cause of morbidity. Vessel occlusion limits blood flow, creating a hypoxic environment that damages distal tissue, requiring therapeutic revascularization. Hypoxia-inducible factors (HIFs) are key transcriptional regulators of hypoxic vascular responses, including angiogenesis and arteriogenesis. Despite vascular smooth muscle cells' (VSMCs') importance in vessel integrity, little is known about their functional responses to hypoxia in peripheral vascular disease. This study investigated the role of VSMC HIF in mediating peripheral ischemic responses. METHODS AND RESULTS: We used ArntSMKO mice with smooth muscle-specific deletion of aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1ß), required for HIF transcriptional activity, in a femoral artery ligation model of peripheral vascular disease. ArntSMKO mice exhibit impaired perfusion recovery despite normal collateral vessel dilation and angiogenic capillary responses. Decreased blood flow manifests in extensive tissue damage and hypoxia in ligated limbs of ArntSMKO mice. Furthermore, loss of aryl hydrocarbon receptor nuclear translocator changes the proliferation, migration, and transcriptional profile of cultured VSMCs. ArntSMKO mice display disrupted VSMC morphologic features and wrapping around arterioles and increased vascular permeability linked to decreased local blood flow. CONCLUSIONS: Our data demonstrate that traditional vascular remodeling responses are insufficient to provide robust peripheral tissue reperfusion in ArntSMKO mice. In all, this study highlights HIF responses to hypoxia in arteriole VSMCs critical for the phenotypic and functional stability of vessels that aid in the recovery of blood flow in ischemic peripheral tissues.

Biomimetic post-capillary venule expansions for leukocyte adhesion studies.

Leukocyte adhesion and extravasation are maximal near the transition from capillary to post-capillary venule, and are strongly influenced by a confluence of scale-dependent physical effects. Mimicking the scale of physiological vessels using in vitro microfluidic systems allows the capture of these effects on leukocyte adhesion assays, but imposes practical limits on reproducibility and reliable quantification. Here we present a microfluidic platform that provides multiple (54-512) technical replicates within a 15-minute sample collection time, coupled with an automated computer vision analysis pipeline that captures leukocyte adhesion probabilities as a function of shear and extensional stresses. We report that in post-capillary channels of physiological scale, efficient leukocyte adhesion requires erythrocytes forcing leukocytes against the wall, a phenomenon that is promoted by the transitional flow in post-capillary venule expansions and dependent on the adhesion molecule ICAM-1.

Live-cell visualization of gasdermin D-driven pyroptotic cell death.

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.

Spatiotemporal ablation of CXCR2 on oligodendrocyte lineage cells: Role in myelin repair.

BACKGROUND: Residual CXCR2 expression on CNS cells in Cxcr2 (+/-) →Cxcr2 (-/-) chimeric animals slowed remyelination after both experimental autoimmune encephalomyelitis and cuprizone-induced demyelination. METHODS: We generated Cxcr2 (fl/-) :PLPCre-ER(T) mice enabling an inducible, conditional deletion of Cxcr2 on oligodendrocyte lineage cells of the CNS. Cxcr2 (fl/-) :PLPCre-ER(T) mice were evaluated in 2 demyelination/remyelination models: cuprizone-feeding and in vitro lysophosphatidylcholine (LPC) treatment of cerebellar slice cultures. RESULTS: Cxcr2 (fl/-) :PLPCre-ER(T)(+) (termed Cxcr2-cKO) mice showed better myelin repair 4 days after LPC-induced demyelination of cerebellar slice cultures. Cxcr2-cKOs also displayed enhanced hippocampal remyelination after a 2-week recovery from 6-week cuprizone feeding. CONCLUSION: Using 2 independent demyelination/remyelination models, our data document enhanced myelin repair in Cxcr2-cKO mice, consistent with the data obtained from radiation chimerism studies of germline CXCR2. Further experiments are appropriate to explore how CXCR2 function in the oligodendrocyte lineage accelerates myelin repair.

Gelation chemistries for the encapsulation of nanoparticles in composite gel microparticles for lung imaging and drug delivery.

The formation of 10-40 µm composite gel microparticles (CGMPs) comprised of ∼100 nm drug containing nanoparticles (NPs) in a poly(ethylene glycol) (PEG) gel matrix is described. The CGMP particles enable targeting to the lung by filtration from the venous circulation. UV radical polymerization and Michael addition polymerization reactions are compared as approaches to form the PEG matrix. A fluorescent dye in the solid core of the NP was used to investigate the effect of reaction chemistry on the integrity of encapsulated species. When formed via UV radical polymerization, the fluorescence signal from the NPs indicated degradation of the encapsulated species by radical attack. The degradation decreased fluorescence by 90% over 15 min of UV exposure. When formed via Michael addition polymerization, the fluorescence was maintained. Emulsion processing using controlled shear stress enabled control of droplet size with narrow polydispersity. To allow for emulsion processing, the gelation rate was delayed by adjusting the solution pH. At a pH = 5.4, the gelation occurred at 3.5 h. The modulus of the gels was tuned over the range of 5 to 50 kPa by changing the polymer concentration between 20 and 70 vol %. NP aggregation during polymerization, driven by depletion forces, was controlled by the reaction kinetics. The ester bonds in the gel network enabled CGMP degradation. The gel modulus decreased by 50% over 27 days, followed by complete gel degradation after 55 days. This permits ultimate clearance of the CGMPs from the lungs. The demonstration of uniform delivery of 15.8 ± 2.6 µm CGMPs to the lungs of mice, with no deposition in other organs, is shown, and indicates the ability to concentrate therapeutics in the lung while avoiding off-target toxic exposure.

Dissociable functional networks of the human dentate nucleus.

The cerebellar dentate nucleus has been reported to project to motor and prefrontal cortical regions in nonhuman primates from 2 anatomically distinct areas. However, despite a wealth of human neuroimaging data implicating the cerebellum in motor and cognitive behaviors, evidence of dissociable motor and cognitive networks comprising the human dentate is lacking. To investigate the existence of these 2 networks in the human brain, we used resting-state functional connectivity magnetic resonance imaging. The resting-state fMRI signal was extracted from regions of interest in the dorsal and ventral dentate nucleus. We report a "motor" network involving the dorsal dentate, anterior regions of the cerebellum, and the precentral gyrus, and a "cognitive" network involving the ventral dentate, Crus I, and prefrontal cortex. The existence of these 2 distinct networks supports the notion that cerebellar involvement in cognitive tasks is above and beyond that associated with motor response components.

An "off-the-shelf" capillary microfluidic device that enables tuning of the droplet breakup regime at constant flow rates.

The fabrication of glass capillary microfluidic devices is technically challenging, often hampering use of the design. We describe a new technique, based on commercially available components, for assembling flow focusing capillary devices that can readily be taken apart and cleaned between uses. This design strategy allows for generation of both water-in-oil and oil-in-water emulsions in the same device after an ethanol rinse. The modularity of the device enables the adjustment of the tip separation between the two inner capillaries during droplet generation, which enables tuning of the age of the interface. Time-dependent surfactant diffusion to the interface changes the interfacial tension, thus providing an approach for adjusting the capillary number in addition to the usual method of changing flow rates. This design enables the tuning of the mode of breakup and the droplet size.

Aerosol delivery of nanoparticles in uniform mannitol carriers formulated by ultrasonic spray freeze drying.

PURPOSE: While most examples of nanoparticle therapeutics have involved parenteral or IV administration, pulmonary delivery is an attractive alternative, especially to target and treat local infections and diseases of the lungs. We describe a successful dry powder formulation which is capable of delivering nanoparticles to the lungs with good aerosolization properties, high loadings of nanoparticles, and limited irreversible aggregation. METHODS: Aerosolizable mannitol carrier particles that encapsulate nanoparticles with dense PEG coatings were prepared by a combination of ultrasonic atomization and spray freeze drying. This process was contrasted to particle formation by conventional spray drying. RESULTS: Spray freeze drying a solution of nanoparticles and mannitol (2 wt% solids) resulted in particles with an average diameter of 21 ± 1.7 µm, regardless of the fraction of nanoparticles loaded (0-50% of total solids). Spray freeze dried (SFD) powders with a 50% nanoparticle loading had a fine particle fraction (FPF) of 60%. After formulation in a mannitol matrix, nanoparticles redispersed in water to < 1 µm with hand agitation and to < 250 nm with the aid of sonication. Powder production by spray drying was less successful, with low powder yields and extensive, irreversible aggregation of nanoparticles evident upon rehydration. CONCLUSIONS: This study reveals the unique advantages of processing by ultrasonic spray freeze drying to produce aerosol dry powders with controlled properties for the delivery of therapeutic nanoparticles to the lungs.

Transcallosal sensorimotor fiber tract structure-function relationships.

Recent studies have demonstrated neuroanatomically selective relationships among white matter tract microstructure, physiological function, and task performance. Such findings suggest that the microstructure of transcallosal motor fibers may reflect the capacity for interhemispheric inhibition between the primary motor cortices, although full characterization of the transcallosal inhibitory sensorimotor network is lacking. Thus, the goal of this study was to provide a comprehensive description of transcallosal fibers connecting homologous sensorimotor cortical regions and to identify the relationship(s) between fiber tract microstructure and interhemispheric inhibition during voluntary cortical activity. To this end, we assessed microstructure of fiber tracts connecting homologous sensorimotor regions of the cortex with diffusion tensor imaging. We also assessed interhemispheric inhibition by eliciting the ipsilateral silent period (iSP) within the same participants. We mapped mutually exclusive transcallosal connections between homologous sensorimotor regions and computed quantitative metrics of each fiber tract. Paralleling work in non-human primates, we found the densest interhemispheric sensorimotor connections to be between the medial motor areas. Additionally, we provide a midsagittal callosal atlas in normalized Montreal Neurological Institute (MNI) space for future studies to use when investigating callosal fiber tracts connecting primary and secondary sensorimotor cortices. Finally, we report a strong, positive relationship (r = 0.76) between strength of interhemispheric inhibition (iSP) and microstructure of interhemispheric fibers that is specific to tracts connecting the primary motor cortices. Thus, increased fiber microstructure in young adults predicts interhemispheric inhibitory capacity.

Microfluidic generation of droplets with a high loading of nanoparticles.

Microfluidic approaches for controlled generation of colloidal clusters, for example, via encapsulation of colloidal particles in droplets, have been used for the synthesis of functional materials including drug delivery carriers. Most of the studies, however, use a low concentration of an original colloidal suspension (<10 wt %). Here we demonstrate microfluidic approaches for directly making droplets with moderate (10-25 wt %) and high (>60 wt %) particle concentrations. Three types of microfluidic devices, PDMS flow-focusing, PDMS T-junction, and microcapillary devices, are investigated for direct encapsulation of a high concentration of polystyrene (PS) nanoparticles in droplets. In particular, it is shown that PDMS devices fabricated by soft lithography can generate droplets from a 25 wt % PS suspension, whereas microcapillary devices made from glass capillary tubes are able to produce droplets from a 67 wt % PS nanoparticle suspension. When the PS concentration is between 0.6 and 25 wt %, the size of the droplets is found to change with the oil-to-water flow rate ratio and is independent of the concentration of particles in the initial suspensions. Drop sizes from ~12 to 40 µm are made using flow rate ratios Q(oil)/Q(water) from 20 to 1, respectively, with either of the PDMS devices. However, clogging occurs in PDMS devices at high PS concentrations (>25 wt %) arising from interactions between the PS colloids and the surface of PDMS devices. Glass microcapillary devices, on the other hand, are resistant to clogging and can produce droplets continuously even when the concentration of PS nanoparticles reaches 67 wt %. We believe that our findings indicate useful approaches and guidelines for the controlled generation of emulsions filled with a high loading of nanoparticles, which are useful for drug delivery applications.

Resting state cortico-cerebellar functional connectivity networks: a comparison of anatomical and self-organizing map approaches.

The cerebellum plays a role in a wide variety of complex behaviors. In order to better understand the role of the cerebellum in human behavior, it is important to know how this structure interacts with cortical and other subcortical regions of the brain. To date, several studies have investigated the cerebellum using resting-state functional connectivity magnetic resonance imaging (fcMRI; Krienen and Buckner, 2009; O'Reilly et al., 2010; Buckner et al., 2011). However, none of this work has taken an anatomically-driven lobular approach. Furthermore, though detailed maps of cerebral cortex and cerebellum networks have been proposed using different network solutions based on the cerebral cortex (Buckner et al., 2011), it remains unknown whether or not an anatomical lobular breakdown best encompasses the networks of the cerebellum. Here, we used fcMRI to create an anatomically-driven connectivity atlas of the cerebellar lobules. Timecourses were extracted from the lobules of the right hemisphere and vermis. We found distinct networks for the individual lobules with a clear division into "motor" and "non-motor" regions. We also used a self-organizing map (SOM) algorithm to parcellate the cerebellum. This allowed us to investigate redundancy and independence of the anatomically identified cerebellar networks. We found that while anatomical boundaries in the anterior cerebellum provide functional subdivisions of a larger motor grouping defined using our SOM algorithm, in the posterior cerebellum, the lobules were made up of sub-regions associated with distinct functional networks. Together, our results indicate that the lobular boundaries of the human cerebellum are not necessarily indicative of functional boundaries, though anatomical divisions can be useful. Additionally, driving the analyses from the cerebellum is key to determining the complete picture of functional connectivity within the structure.

The effects of working memory resource depletion and training on sensorimotor adaptation.

We have recently demonstrated that visuospatial working memory performance predicts the rate of motor skill learning, particularly during the early phase of visuomotor adaptation. Here, we follow up these correlational findings with direct manipulations of working memory resources to determine the impact on visuomotor adaptation, a form of motor learning. We conducted two separate experiments. In the first one, we used a resource depletion strategy to investigate whether the rate of early visuomotor adaptation would be negatively affected by fatigue of spatial working memory resources. In the second study, we employed a dual n-back task training paradigm that has been shown to result in transfer effects [1] over five weeks to determine whether training-related improvements would boost the rate of early visuomotor adaptation. The depletion of spatial working memory resources negatively affected the rate of early visuomotor adaptation. However, enhancing working memory capacity via training did not lead to improved rates of visuomotor adaptation, suggesting that working memory capacity may not be the factor limiting maximal rate of visuomotor adaptation in young adults. These findings are discussed from a resource limitation/capacity framework with respect to current views of motor learning.

A spatial explicit strategy reduces error but interferes with sensorimotor adaptation.

Although sensorimotor adaptation is typically thought of as an implicit form of learning, it has been shown that participants who gain explicit awareness of the nature of the perturbation during adaptation exhibit more learning than those who do not. With rare exceptions, however, explicit awareness is typically polled at the end of the study. Here, we provided participants with either an explicit spatial strategy or no instructions before learning. Early in learning, explicit instructions greatly reduced movement errors but also resulted in increased trial-to-trial variability and longer reaction times. Late in adaptation, performance was indistinguishable between the explicit and implicit groups, but the mechanisms underlying performance improvements remained fundamentally different, as revealed by catch trials. The progression of implicit recalibration in the explicit group was modulated by the use of an explicit strategy: these participants showed a lower level of recalibration as well as decreased aftereffects. This phenomenon may be due to the reduced magnitude of errors made to the target during adaptation or inhibition of implicit learning mechanisms by explicit processing.

Tetherless thermobiochemically actuated microgrippers.

We demonstrate mass-producible, tetherless microgrippers that can be remotely triggered by temperature and chemicals under biologically relevant conditions. The microgrippers use a self-contained actuation response, obviating the need for external tethers in operation. The grippers can be actuated en masse, even while spatially separated. We used the microgrippers to perform diverse functions, such as picking up a bead on a substrate and the removal of cells from tissue embedded at the end of a capillary (an in vitro biopsy).

Pick-and-place using chemically actuated microgrippers.

In this communication, we demonstrate the concept of single-use, chemically triggered, reversible tools in the form of mobile grippers that can be used to manipulate micro-objects. Both the closing and opening of the mobile grippers are triggered by chemicals, namely acetic acid (CH(3)COOH) and hydrogen peroxide (H(2)O(2)), respectively. The grippers close and open en masse based on chemically triggered, mechanical property changes within trilayer joints patterned within the gripper, and no external power is needed for operation. We describe the actuation of the gripper using a multilayer thin film model and demonstrate the utility of the gripper by picking-and-placing 200 microm diameter tubes and beads. Our pick-and-place microgripper is a first step toward the development of functional Micro Chemo-Mechanical Systems (MCMS), which are actuated by chemistry as opposed to electricity [as in Micro Electro-Mechanical Systems (MEMS)].

Self-loading lithographically structured microcontainers: 3D patterned, mobile microwells.

We demonstrate mass-producible, mobile, self-loading microcontainers that can be used to encapsulate both non-living and living objects, thus forming three-dimensionally patterned, mobile microwells.