Biology subjects

Young, E. E.

Publications and source records attributed to Young, E. E..

5 recordsLinked to original sources

bioRxiv2024

Diminished Immune Cell Adhesion in Hypoimmune ICAM-1 Knockout Pluripotent Stem Cells

Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g., T cell) immune responses, but have largely not addressed the innate immune cells (e.g., monocytes, neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies, we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding, thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. HighlightsO_LIAntibody blocking of ICAM-1 on human pluripotent stem cell-derived cells inhibits immune cell adhesion C_LIO_LICRISPR/Cas9 knock-out of ICAM-1 ablates surface and secreted ICAM-1 protein and inhibits immune adhesion C_LIO_LIICAM-1 knock-out results in decreased T cell proliferative responses to human pluripotent stem cell-derived grafts in vitro, and resistance to immune-mediated graft loss in vivo C_LIO_LIAddition of ICAM-1 knock-out to first generation MHC knock-out human pluripotent stem cells confers protection against immune adhesion C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/597791v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@890b56org.highwire.dtl.DTLVardef@147449borg.highwire.dtl.DTLVardef@1e94f08org.highwire.dtl.DTLVardef@43c55c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC - pluripotent stem cell-derived endothelial cells; KO - knock-out; dSMAC - distal supramolecular activation complex; pSMAC - peripheral supramolecular activation complex; cSMAC - central supramolecular activation complex. C_FIG

immunology↗

bioRxiv2023

Identification of arginine-vasopressin receptor 1a (Avpr1a/AVPR1A) as a novel candidate gene for chronic visceral pain.

Chronic abdominal pain in the absence of ongoing disease is the hallmark of disorders of gut-brain interaction (DGBIs), including irritable bowel syndrome (IBS). While the etiology of DGBIs remains poorly understood, there is evidence that both genetic and environmental factors play a role. In this study, we report the identification and validation of Avpr1a as a novel candidate gene for visceral hypersensitivity (VH), a primary peripheral mechanism underlying abdominal pain in DGBI/IBS. Comparing two C57BL/6 (BL/6) substrains (C57BL/6NTac and C57BL/6J) revealed differential susceptibility to the development of chronic VH following intrarectal zymosan (ZYM) instillation, a validated preclinical model for post-inflammatory IBS. Using whole genome sequencing, we identified a SNP differentiating the two strains in the 5 intergenic region upstream of Avpr1a, encoding the protein arginine-vasopressin receptor 1A (AVPR1A). We used behavioral, histological, and molecular approaches to identify distal colon- specific gene expression differences and neuronal hyperresponsiveness covarying with Avpr1a genotype and VH susceptibility. While the two BL/6 substrains did not differ across other gastrointestinal (GI) phenotypes (e.g., GI motility), VH-susceptible BL/6NTac mice had higher colonic Avpr1a mRNA and protein expression. Moreover, neurons of the enteric nervous system were hyperresponsive to the AVPR1A agonist AVP, suggesting a role for enteric neurons in the pathology underlying VH. These results parallel our findings that patients colonic Avpr1a mRNA expression was higher in patients with higher pain ratings. Taken together, these findings implicate differential regulation of Avpr1a as a novel mechanism of VH-susceptibility as well as a potential therapeutic target specific to VH. SummaryA combination of approaches, from genomic analysis to functional analyses, confirm Avpr1a as a high priority candidate gene for visceral pain.

genetics↗

bioRxiv2021

Comprehensive Phenotyping of Cutaneous Afferents Reveals Rapid-Onset Alterations in Nociceptor Response Properties Following Spinal Cord Injury

Spinal cord injury (SCI) is a complex syndrome that has profound effects on patient well-being, including the development of medically-resistant chronic pain. The mechanisms underlying SCI pain have been the subject of thorough investigation but remain poorly understood. While the majority of the research has focused on changes occurring within and surrounding the site of injury in the spinal cord, there is now a consensus that alterations within the peripheral nervous system, namely sensitization of nociceptors, contribute to the development and maintenance of chronic SCI pain. Here we demonstrate that thoracic spinal contusion injury results in the emergence of autotomy and spasticity, both indicators of spontaneous pain, in areas below the level of the injury within 24 hr of SCI. These behaviors were associated with hindpaw edema and elevated cutaneous calcitonin gene-related peptide (CGRP) concentration. Electrophysiological recordings using an ex vivo skin/nerve/DRG/spinal cord preparation demonstrated that SCI increased mechanical and thermal sensitivity, as well as the incidence of spontaneous activity (SA) and afterdischarge (AD), in below-level C-fiber nociceptors 24 hr following injury. Interestingly, the distribution of nociceptors that exhibit SA and AD are not identical, and the development of SA was observed more frequently in nociceptors with low thermal thresholds, while AD was found more frequently in nociceptors with high thermal thresholds. These results demonstrate that SCI causes the rapid-onset of peripheral inflammation-like processes that sensitize nociceptors, which may contribute to the early emergence and persistence of chronic SCI pain.

neuroscience↗

bioRxiv2019

TIMP-1 attenuates the development of cutaneous inflammation-induced hypersensitivity

Unresolved inflammation is a significant predictor for developing chronic pain, and targeting the mechanisms underlying inflammation offers opportunities for therapeutic intervention. During inflammation, matrix metalloproteinase (MMP) activity contributes to tissue remodeling and inflammatory signaling through proteolytic maturation of cytokines. MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs) 1-4. TIMP-1 and -2 have known roles in pain, but only in the context of MMP inhibition. However, TIMP-1 also has receptor-mediated cell signaling functions that are not well understood. Here, we examined how TIMP-1-dependent cell signaling impacted inflammatory hypersensitivity and ongoing pain. We found that hindpaw injection of complete Freunds adjuvant (CFA) increased keratinocyte-derived TIMP-1 that peaked 3 days following inflammation, when mechanical hypersensitivity began to emerge in WT mice. These data suggest that TIMP-1 expression inhibits the development of inflammatory hypersensitivity. To examine this possibility, we injected TIMP-1 knockout (T1KO) mice with a diluted CFA mixture to examine how subtle cutaneous inflammation affected behavioral hypersensitivity. T1KO mice exhibited rapid onset thermal and mechanical hypersensitivity at the site of inflammation that was absent or attenuated in WT controls. We also found that T1KO mice exhibited hypersensitivity in adjacent tissues innervated by different sets of afferents, and skin contralateral to the site of inflammation. Replacement of recombinant murine (rm)TIMP-1 alleviated hypersensitivity when administered at the site and time of inflammation. To examine the MMP-dependent and -independent mechanisms of rmTIMP-1, T1KO mice were administered full-length rmTIMP-1, the N-terminal region (TIMP-1(N)) with MMP-inhibitory properties, or the C-terminal region (TIMP-1(C)) that retains receptor signaling function. Each of the peptides prevented inflammatory hypersensitivity, suggesting that rmTIMP-1 acts through mechanisms that also include receptor-mediated cell signaling. We also found that hypersensitivity was neither due to genotype-specific differences in MMP-9 activity or expression, nor to differences in cytokine expression. Finally, to evaluate the potential clinical utility of TIMP-1, we administered rmTIMP-1 to WT mice and found that rmTIMP-1 prevented clonidine-induced conditioned place preference (e.g., ongoing pain) and inflammatory mechanical hypersensitivity. Collectively, our data suggest a novel role for TIMP-1 in the attenuation of inflammatory pain that occurs through previously uncharacterized cell signaling mechanisms.

neuroscience↗

bioRxiv2018

Genetic mapping in Diversity Outbred mice identifies a Trpa1 variant influencing late phase formalin response

Identification of genetic variants that influence susceptibility to chronic pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with chronic pain, we measured late phase response to formalin injection in 275 male and female Diversity Outbred (DO) mice genotyped for over 70 thousand SNPs. One quantitative trait locus (QTL) reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity inflammatory QTL 4; MGI:5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of DO founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the seven other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, Trpa1/Trpv1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.

bioinformatics↗