Abstract Response to mechanical signals is an important mechanism by which T lymphocytes drive inflammation. A potential contributor in this process is Piezo1, a mechanosensitive cation channel which is abundantly expressed in T lymphocytes. Previously, our lab has shown that Piezo1 colocalizes with high affinity LFA-1 on activated human T lymphocytes during chemokine-activated crawling, suggesting a role for Piezo1 in the adhesion cascade. However, the mechanism by which Piezo1 contributes to the regulation of the adhesion cascade is unknown. We hypothesize that Piezo1 mediates T lymphocyte migration through direct contact with integrin to regulate its function. We investigate the role of Piezo1 in the expression, binding affinity, and turnover of LFA-1 and VLA-4. Co-immunoprecipitation revealed a preferential association of Piezo1 with the alpha subunit of integrins, specifically LFA-1, MAC-1, and VLA-4 in the active forms. Moreover, microfluidic devices were used to study the contribution of Piezo1 on T lymphocytes under laminar flow-induced shear stress. We found that activated T lymphocytes with CRISPR/Cas9-mediated Piezo1 ablation resulted in a reduced chemokine-induced crawling speed that correlates to the efficiency of Piezo1 gene ablation. Finally, in studies of lymph node homing where fluorescently labeled T lymphocytes were tracked in vivo, Piezo1 deficient cells exhibit increased ingress and decreased egress through the lymph node, suggesting an intricate role of Piezo1 on T lymphocyte migration. Taken together, our data support the hypothesis that Piezo1 modulates inflammatory processes through complex integrin interactions and mediates T lymphocyte migration from the lymph node to the site of inflammation. Case Postbaccalaureate Research Education Program grant NIH/NIGMS 5R25GM075207-15; St. Baldrick’s Foundation; Hyundai Hope-on-Wheels Program; The I’m Not Done Yet Foundation; and Pediatric Cancer Research Foundation

Novel therapies for the pediatric brain tumor medulloblastoma (MB) are hindered by limited knowledge of the roles played by host-derived cells at the local tumor site. Immune resistance in a murine model of MB (MM1) is associated with enhanced tumor expression of programmed death ligand-1 (PD-L1) in response to anti-tumor cytokine interferon-gamma (IFNγ) signaling. Disrupting this pathway via knockdown of cyclin dependent kinase 5 (CDK5), an essential transducer of the IFNγ signal, leads to an inflammatory tumor microenvironment (TME) and enhanced tumor rejection in vivo. In this study, we sought to detail the mechanism of CDK5-mediated PD-L1 regulation. In response to IFNγ, PD-L1 expression is thought to be regulated at the promoter level by competition between the transcriptional activator IRF1 and repressor IRF2. We hypothesized that CDK5-IFNγ signaling inhibits IRF2 activity, resulting in unleashed PD-L1 promoter activity. We assessed IRF1/IRF2-DNA interactions using chromatin immunoprecipitation (ChIP) and gene reporter assays, with complementary analysis of PD-L1 transcription and surface expression with IRF1/IRF2 depletion. ChIP-qPCR revealed a similar, IFNγ-induced enrichment of both IRF1 and IRF2 at the PD-L1 promoter. This is a surprising result, given the assumption that they compete for the same binding site and exert opposing effects. Additionally, inducible PD-L1 promoter activity was found to be decreased in tumors with IRF2 depletion. Together, these results suggest that IRF2 may be necessary for full activity of the PD-L1 promoter in MB. IRF1/IRF2 also bind to other cis-regulatory sites at genes that contribute to tumor immunogenicity, necessitating genome-wide characterization of their interactions.

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 m-OR agonists ex vivo decrease expression of activation markers CD69 and CD25, although the same markers are elevated in m-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.

Abstract Efficient recruitment of circulating immune cells to various tissues plays a critical role in homeostasis and immune surveillance, a process that serves as the basis of any successful cell-based immunotherapeutic strategies in cancer, particularly for solid tumors and cancers residing in body sites outside of blood vessels and sinusoid network. Clinical and experimental observations suggest that in vivo leukocyte adhesion and extravasation are maximal near the transition from capillary to post-capillary venule, through a multistep process that includes the intravascular capture, rolling, arrest, crawling of cells through interactions of adhesion molecules (selectins, integrins, chemokines/receptors, for example), eventually leading to transcellular or paracellular transmigration through intact endothelium. These cellular and molecular processes are strongly influenced by a confluence of scale-dependent physical effects. Mimicking the scale of physiologic vessels using in vitro microfluidic systems allows the capture and investigation of these effects on leukocyte adhesion assays, but imposes practical limits on reproducibility and reliable quantification. We have developed 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 not only shear stress imposed on leukocytes within the vessels as conventional wisdom dictates, but also of the extensional stresses imposed by the topology of post-capillary venules and the rheology of circulating leukocytes in these vessels. We identified that in post-capillary channels of physiologic 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 highly dependent on the adhesion molecule ICAM-1. These studies help identified a mechanosensory mechanism that determines the increased likelihood of leukocyte adhesion in post-capillary venules, and further suggests a significant role of mechanosensory channel(s) in influencing leukocyte integrin affinity for cellular capture to the vessel wall. Through a series of truncation mutants, we have narrowed down a small region of putative interacting domain between integrin and a candidate mechanosensory channel—PIEZO1—on leukocytes. These ongoing investigations offer new insights into immune cellular recruitment and new molecular targets to enhance leukocyte recruitment to peripheral tumor sites. Citation Format: Alex Y. Huang, Bryan L. Benson, Luis Correa, Lucy Li, Jay T. Myers, Umut A. Gurkan, Richard Ransohoff. Mechanosensory mechanisms and in vivo tissue topology contribute to rheology of circulating leukocytes resulting in efficient post-capillary vessel wall adhesion and recruitment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A195.

In order to respond to infection, injury and stress, T lymphocytes must successfully migrate from the bloodstream into inflamed tissue in a process called the leukocyte adhesion cascade. Of these steps, blockade of adhesion has shown clear clinical benefit in autoimmunity, and the step of intraluminal crawling is of great potential clinical importance. In order to coordinate adhesion and crawling while in circulation, leukocytes must respond appropriately to complex hemodynamic forces, including shear stresses and erythrocyte driven margination against the venule walls. Therefore, we hypothesized that the mechanosensitive calcium channel PIEZO1, which is preferentially expressed in T lymphocytes, mediates T lymphocyte responses to force during adhesion and crawling. To test this hypothesis, we genetically ablated PIEZO1 in primary T lymphocytes from healthy human donors using CRISPR/Cas9, and observed crawling ability in vitro. In this assay, PIEZO1 knockout cells exhibited decreased crawling and disrupted morphology. Moreover, we performed immunofluorescence of PIEZO1 on chemokine-activated crawling T lymphocytes, which demonstrated that PIEZO1 redistributes to the contact zone of crawling cells in a pattern reminiscent of a high-affinity LFA-1 focal zone. Additionally, PIEZO1 colocalizes specifically with high affinity LFA-1. Using co-immunoprecipitation indicated that PIEZO1 preferentially associates with the alpha integrin subunit of LFA-1 in its active form, through a conserved amphipathic eight amino acid motif. Taken together, our data suggest that PIEZO1 contributes to coordination of T lymphocyte crawling via interaction with integrins, modulating the affinity state and turnover of LFA-1.

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.

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-1β, 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.

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.

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.

Post capillary venules (PCVs) contribute a tiny fraction of the total vasculature, but are the predominant site of leukocyte adhesion and infiltration. Given the astronomical number of leukocytes and length of the vasculature, this restriction of adhesion to PCV requires tight and redundant regulation: adhesion molecule expression, chemokine presentation, and wall shear stress (WSS) from blood flow converge to promote this phenomenon in PCVs. To mimic WSS, parallel plate fluidic channels have become the standard of in vitro models to study leukocyte adhesion. These models match the WSS of in vivo PCVs, but vary in their height (h), the smallest dimension that most strongly dictates fluid forces. Surprisingly, in our own experiments and in literature, leukocyte adhesion decreases as h decreases towards the 25 micron diameter of in vivo PCVs, whereas computational studies predict more adhesion with smaller h. This discrepancy suggests that a biological response within leukocytes opposes passive physical forces, or that other mediating factors are at play in vivo. In the transition from capillary to PCV, leukocytes experience another fluid stress besides WSS: extensional stress, due to the cross-sectional area of the vessels increasing at bifurcations. We created biomimetic models of the capillary to PCV transition in which we independently vary extensional stress and WSS and assay leukocyte adhesion to adsorbed CXCL12 and ICAM-1. Here, extensional stress has a powerful influence on human peripheral blood mononuclear cell (PBMC) and Jurkat adhesion probability, with higher extensional stress strongly promoting leukocyte adhesion. We are currently investigating the roles of mechanosensitive channels PIEZO1 and TRPV2 in this effect.

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.

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.

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. Hum Brain Mapp, 2013. © 2011 Wiley Periodicals, Inc.

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.

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.

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.

27 Although sensorimotor adaptation is typically thought of as an implicit form of learning, 28 it has been shown that participants who gain explicit awareness of the nature of the perturbation 29 during adaptation exhibit more learning than those that do not. With rare exceptions, however, 30 explicit awareness is typically polled at the end of the study. Here, we provided participants with 31 either an explicit spatial strategy or no instructions prior to learning. Early in learning, explicit 32 instructions greatly reduced movement errors but also resulted in increased trial-to-trial 33 variability and longer reaction times. Late in adaptation, performance was indistinguishable 34 between the explicit and implicit groups, but the mechanisms underlying performance 35 improvements remained fundamentally different, as revealed by catch trials. The progression of 36 implicit recalibration in the explicit group was modulated by the use of an explicit strategy: these 37 participants showed a lower level of recalibration as well as decreased after-effects. This 38 phenomenon may be due to the reduced magnitude of errors made to the target during 39 adaptation, or inhibition of implicit learning mechanisms by explicit processing. 40 41

In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: a b s t r a c t 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.