Dexmedetomidine Alleviates Gut-Vascular Barrier Damage and Distant Hepatic Injury Following Intestinal Ischemia/Reperfusion Injury in Mice.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) challenge often results in gut barrier dysfunction and induces distant organ injury. Dexmedetomidine has been shown to protect intestinal epithelial barrier against I/R attack. The present study aims to investigate the degree to which intestinal I/R attack will contribute to gut-vascular barrier (GVB) damage, and to examine the ability of dexmedetomidine to minimize GVB and liver injuries in mice. METHODS: In vivo, intestinal ischemic challenge was induced in mice by clamping the superior mesenteric artery for 45 minutes. After clamping, the mice were subjected to reperfusion for either 2, 4, 6, or 12 hours. Intraperitoneal injection of dexmedetomidine 15, 20, or 25 µg·kg-1 was performed intermittently at the phase of reperfusion. For the in vitro experiments, the challenge of oxygen-glucose deprivation/reoxygenation (OGD/R) was established in cultured vascular endothelial cells, and dexmedetomidine (1 nM) was used to treat the cells for 24 hours. Moreover, in vivo and in vitro, SKL2001 (a specific agonist of ß-catenin) or XAV939 (a specific inhibitor of ß-catenin) was applied to determine the role of ß-catenin in the impacts provided by dexmedetomidine. RESULTS: The attack of intestinal I/R induced GVB damage. The greatest level of damage was observed at 4 hours after intestinal reperfusion. There was a significant increase in plasmalemma vesicle-associated protein-1 (PV1, a specific biomarker for endothelial permeability) expression (5.477 ± 0.718 vs 1.000 ± 0.149; P < .001), and increased translocation of intestinal macromolecules and bacteria to blood and liver tissues was detected (all P < .001). Liver damages were observed. There were significant increases in histopathological scores, serum parameters, and inflammatory factors (all P < .001). Dexmedetomidine 20 µg·kg-1 reduced PV1 expression (0.466 ± 0.072 vs 1.000 ± 0.098; P < .001) and subsequent liver damages (all P < .01). In vitro, dexmedetomidine significantly improved vascular endothelial cell survival (79.387 ± 6.447% vs 50.535 ± 1.766%; P < .001) and increased the productions of tight junction protein and adherent junction protein (all P < .01) following OGD/R. Importantly, in cultured cells and in mice, ß-catenin expression significantly decreased (both P < .001) following challenge. Dexmedetomidine or SKL2001 upregulated ß-catenin expression and produced protective effects (all P < .01). However, XAV939 completely eliminated the protective effects of dexmedetomidine on GVB (all P < .001). CONCLUSIONS: The disruption of GVB occurred following intestinal I/R. Dexmedetomidine alleviated I/R-induced GVB impairment and subsequent liver damage.

Rewiring T-cell responses to soluble factors with chimeric antigen receptors.

Chimeric antigen receptor (CAR)-expressing T cells targeting surface-bound tumor antigens have yielded promising clinical outcomes, with two CD19 CAR-T cell therapies recently receiving FDA approval for the treatment of B-cell malignancies. The adoption of CARs for the recognition of soluble ligands, a distinct class of biomarkers in physiology and disease, could considerably broaden the utility of CARs in disease treatment. In this study, we demonstrate that CAR-T cells can be engineered to respond robustly to diverse soluble ligands, including the CD19 ectodomain, GFP variants, and transforming growth factor beta (TGF-ß). We additionally show that CAR signaling in response to soluble ligands relies on ligand-mediated CAR dimerization and that CAR responsiveness to soluble ligands can be fine-tuned by adjusting the mechanical coupling between the CAR's ligand-binding and signaling domains. Our results support a role for mechanotransduction in CAR signaling and demonstrate an approach for systematically engineering immune-cell responses to soluble, extracellular ligands.

Bacterial effector NleL promotes enterohemorrhagic E. coli-induced attaching and effacing lesions by ubiquitylating and inactivating JNK.

As a major diarrheagenic human pathogen, enterohemorrhagic Escherichia coli (EHEC) produce attaching and effacing (A/E) lesions, characterized by the formation of actin pedestals, on mammalian cells. A bacterial T3SS effector NleL from EHEC O157:H7 was recently shown to be a HECT-like E3 ligase in vitro, but its biological functions and host targets remain elusive. Here, we report that NleL is required to effectively promote EHEC-induced A/E lesions and bacterial infection. Furthermore, human c-Jun NH2-terminal kinases (JNKs) were identified as primary substrates of NleL. NleL-induced JNK ubiquitylation, particularly mono-ubiquitylation at the Lys 68 residue of JNK, impairs JNK's interaction with an upstream kinase MKK7, thus disrupting JNK phosphorylation and activation. This subsequently suppresses the transcriptional activity of activator protein-1 (AP-1), which modulates the formation of the EHEC-induced actin pedestals. Moreover, JNK knockdown or inhibition in host cells complements NleL deficiency in EHEC infection. Thus, we demonstrate that the effector protein NleL enhances the ability of EHEC to infect host cells by targeting host JNK, and elucidate an inhibitory role of ubiquitylation in regulating JNK phosphorylation.

Degradation of the Separase-cleaved Rec8, a Meiotic Cohesin Subunit, by the N-end Rule Pathway.

The Ate1 arginyltransferase (R-transferase) is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. Ate1 arginylates N-terminal Asp, Glu, or (oxidized) Cys. The resulting N-terminal Arg is recognized by ubiquitin ligases of the N-end rule pathway. In the yeastSaccharomyces cerevisiae, the separase-mediated cleavage of the Scc1/Rad21/Mcd1 cohesin subunit generates a C-terminal fragment that bears N-terminal Arg and is destroyed by the N-end rule pathway without a requirement for arginylation. In contrast, the separase-mediated cleavage of Rec8, the mammalian meiotic cohesin subunit, yields a fragment bearing N-terminal Glu, a substrate of the Ate1 R-transferase. Here we constructed and used a germ cell-confinedAte1(-/-)mouse strain to analyze the separase-generated C-terminal fragment of Rec8. We show that this fragment is a short-lived N-end rule substrate, that its degradation requires N-terminal arginylation, and that maleAte1(-/-)mice are nearly infertile, due to massive apoptotic death ofAte1(-/-)spermatocytes during the metaphase of meiosis I. These effects ofAte1ablation are inferred to be caused, at least in part, by the failure to destroy the C-terminal fragment of Rec8 in the absence of N-terminal arginylation.

Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors.

BACKGROUND: T cells expressing chimeric antigen receptors (CARs) have shown exciting promise in cancer therapy, particularly in the treatment of B-cell malignancies. However, optimization of CAR-T cell production remains a trial-and-error exercise due to a lack of phenotypic benchmarks that are clearly predictive of anti-tumor functionality. A close examination of the dynamic changes experienced by CAR-T cells upon stimulation can improve understanding of CAR-T-cell biology and identify potential points for optimization in the production of highly functional T cells. METHODS: Primary human T cells expressing a second-generation, anti-CD19 CAR were systematically examined for changes in phenotypic and functional responses to antigen exposure over time. Multi-color flow cytometry was performed to quantify dynamic changes in CAR-T cell viability, proliferation, as well as expression of various activation and exhaustion markers in response to varied antigen stimulation conditions. RESULTS: Stimulated CAR-T cells consistently bifurcate into two distinct subpopulations, only one of which (CAR(hi)/CD25(+)) exhibit anti-tumor functions. The use of central memory T cells as the starting population and the resilience-but not antigen density-of antigen-presenting cells used to expand CAR-T cells were identified as critical parameters that augment the production of functionally superior T cells. We further demonstrate that the CAR(hi)/CD25(+) subpopulation upregulates PD-1 but is resistant to PD-L1-induced dysfunction. CONCLUSIONS: CAR-T cells expanded ex vivo for adoptive T-cell therapy undergo dynamic phenotypic changes during the expansion process and result in two distinct populations with dramatically different functional capacities. Significant and sustained CD25 and CAR expression upregulation is predictive of robust anti-tumor functionality in antigen-stimulated T cells, despite their correlation with persistent PD-1 upregulation. The functionally superior subpopulation can be selectively augmented by careful calibration of antigen stimulation and the enrichment of central memory T-cell type.

GENERATION OF TOLEROGENIC DENDRITIC CELLS UNDER THE PERSISTENT INFLAMMATION STIMULATION.

ABSTRACT: Immunosuppression, commonly accompanied by persistent inflammation, is a key feature in the later phase of sepsis. However, the pathophysiological mechanisms underlying this phenomenon remain unclear. Dendritic cells (DCs), specifically tolerogenic DCs (tolDCs), play a crucial role in this process by regulating immune responses through inducing T cell anergy and releasing anti-inflammatory cytokines. Nevertheless, the existing cell models are inadequate for investigating tolDCs during the immunosuppressive phase of sepsis. Therefore, this study aimed to develop a novel in vitro model to generate tolDCs under chronic inflammatory conditions. We have successfully generated tolDCs by exposing them to sublethal lipopolysaccharide (LPS) for 72 h while preserving cell viability. Considering that IL-10-induced tolDCs (IL-10-tolDCs) are well-established models, we compared the immunological tolerance between LPS-tolDCs and IL-10-tolDCs. Our findings indicated that both LPS-tolDCs and IL-10-tolDCs exhibited reduced expression of maturation markers, whereas their levels of inhibitory markers were elevated. Furthermore, the immunoregulatory activities of LPS-tolDCs and IL-10-tolDCs were found to be comparable. These dysfunctions include impaired antigen presenting capacity and suppression of T cell activation, proliferation, and differentiation. Notably, compared with IL-10-tolDCs, LPS-tolDCs showed a reduced response in maturation and cytokine production upon stimulation, indicating their potential as a better model for research. Overall, in comparison with IL-10-tolDCs, our data suggest that the immunological dysfunctions shown in LPS-tolDCs could more effectively elucidate the increased susceptibility to secondary infections during sepsis. Consequently, LPS-tolDCs have emerged as promising therapeutic targets for ameliorating the immunosuppressed state in septic patients.

Improvement of gut-vascular barrier by terlipressin reduces bacterial translocation and remote organ injuries in gut-derived sepsis.

Gut-vascular barrier (GVB) serves as the last barrier to limit the migration of intestinal toxins into the blood circulation. The efficacy of terlipressin (a vasopressin V1 receptor agonist) in reducing GVB and multiple organ damage in gut-derived sepsis is unknown. In this study, we hypothesized that, besides other intestinal barriers, GVB play a key role in gut-derived sepsis and terlipressin improve GVB damage and then reduce bacterial translocation and organ injuries. In vivo, a cecal ligation and puncture mouse model was established. The mice were subjected to examine the damage of GVB determined by intestinal plasmalemma vesicle-associated protein-1(PV-1) and vascular endothelial-cadherin. And the intestinal permeability was assessed by translocation of intestinal bacteria and macromolecules. In vitro, transendothelial electrical resistance (TER) during interleukin (IL)-1ß stimulation was measured on endothelial cells with or without small interfering RNA targeting ß-catenin (si ß-catenin). Terlipressin significantly improved GVB damage and reduced translocation of intestinal macromolecules and bacteria by activating PI3K signaling. Of note, intestinal PV-1 expression was significantly correlated with translocation of macromolecules, and dramatic increase of macromolecules was observed in intestinal tissues whereas fewer macromolecules and bacteria were observed in blood, liver and lung following terlipressin treatment. In vitro, terlipressin restored TER during IL-1ß stimulation and si ß-catenin transfection blocked the changes delivered by terlipressin. Collectively, terlipressin alleviated GVB damage and subsequent bacterial translocation via blood vessels after sepsis challenge, resulting in reduced distant organ injuries and the responsible mechanisms may involve the activation of PI3K/ß-catenin pathway.

HMGB1 signaling-regulated endoplasmic reticulum stress mediates intestinal ischemia/reperfusion-induced acute renal damage.

BACKGROUND: Ischemia/reperfusion of the intestine often leads to distant organ injury, but the mechanism of intestinal ischemia/reperfusion-induced renal dysfunction is still not clear. The present study aimed to investigate the mechanisms of acute renal damage after intestinal ischemia/reperfusion challenge and explore the role of released high-mobility group box-1 in this process. METHODS: Intestinal ischemia/reperfusion was induced in male Sprague-Dawley rats by clamping the superior mesenteric artery for 1.5 hours. At different reperfusion time points, anti-high-mobility group box-1 neutralizing antibodies or ethyl pyruvate were administered to neutralize or inhibit circulating high-mobility group box-1, respectively. RESULTS: Significant kidney injury was observed after 6 hours of intestinal reperfusion, as indicated by increased serum levels of urea nitrogen and creatinine, increased expression of neutrophil gelatinase-associated lipocalin, interleukin-6, and MIP-2, and enhanced cell apoptosis, as indicated by cleaved caspase 3 levels in renal tissues. The levels of phosphorylated eIF2ɑ, activating transcription factor 4, and C/EBP-homologous protein (CHOP) were markedly elevated, indicating the activation of endoplasmic reticulum stress in the impaired kidney. High-mobility group box-1 translocated to cytoplasm in the intestine and serum concentrations of high-mobility group box-1 increased notably during the reperfusion phase. Both anti-high-mobility group box-1 antibodies and ethyl pyruvate treatment significantly reduced serum high-mobility group box-1 concentrations, attenuated endoplasmic reticulum stress in renal tissue and inhibited the development of renal damage. Moreover, the elevated expression of receptor for advanced glycation end products in the kidneys after intestinal ischemia/reperfusion was abrogated after high-mobility group box-1 inhibition. CONCLUSION: These results suggested that high-mobility group box-1 signaling regulated endoplasmic reticulum stress and promoted intestinal ischemia/reperfusion-induced acute kidney injury. High-mobility group box-1 neutralization/inhibition might serve as a pharmacological intervention strategy for these pathophysiological processes.

Engineering primary T cells with chimeric antigen receptors for rewired responses to soluble ligands.

The expression of synthetic receptors in primary T cells enables the programming of user-defined responses when designing T-cell therapies. Chimeric antigen receptors (CARs) are synthetic receptors that have demonstrated efficacy in cancer therapy by targeting immobilized antigens on the surface of malignant cells. Recently, we showed they can also rewire T-cell responses to soluble ligands. In contrast to other synthetic receptors, CARs are not only readily engineered by rational design, but also clinically translatable, with robust function in primary human T cells. This protocol discusses design principles for CARs responsive to soluble ligands and delineates steps for producing T cells expressing synthetic receptors. Functional assays for quantifying the ability of CAR T cells to sense and respond to soluble ligands are also presented. This protocol provides a framework for proficient immune-cell researchers to test novel T-cell therapies targeting soluble ligands in <2 weeks.

A role for the p53 pathway in the pathology of meningiomas with NF2 loss.

The neurofibromatosis 2 locus (NF2) is inactivated through mutation and loss of heterozygosity (LOH) in 40-65% of all sporadic meningiomas, while the role of the p53 tumor suppression pathway in meningioma initiation and progression is still unclear. This study aims to determine if a p53 codon 72 arginine-to-proline polymorphism, found to be correlated with cancer development and cancer patient survival in other tumors, is associated with sporadic meningioma initiation or progression. We investigated Pro72 incidence in a cohort of 92 sporadic meningiomas and analyzed its association with histological grade (WHO classification) and with NF2 LOH (determined using polymorphic microsatellite markers on 22q). The Pro72 allele was not found to be selected for in the cohort. However, in the subgroup of meningiomas with NF2 LOH and carrying Pro72, 50.0% had high grade tumors (WHO grades II and III) compared to only 14.3% of those without NF2 LOH (OR = 6.0, CI = 1.56-23.11, P = 0.012). The significant association occurred only when considering subgroups of meningiomas with or without NF2 LOH, suggesting that not including NF2 status when analyzing study cohorts may explain the variability seen in the literature where all meningiomas were grouped together. Our data suggests a role for the p53 pathway in the progression of meningiomas in which NF2 is inactivated, and highlights the importance of accounting for NF2 LOH in future studies of meningiomas and the p53 pathway.

TGF-ß-responsive CAR-T cells promote anti-tumor immune function.

A chimeric antigen receptor (CAR) that responds to transforming growth factor beta (TGF-ß) enables the engineering of T cells that convert this immunosuppressive cytokine into a potent T-cell stimulant. However, clinical translation of TGF-ß CAR-T cells for cancer therapy requires the ability to productively combine TGF-ß responsiveness with tumor-targeting specificity. Furthermore, the potential concern that contaminating, TGF-ß?producing regulatory T (Treg) cells may preferentially expand during TGF-ß CAR-T cell manufacturing and suppress effector T (Teff) cells demands careful evaluation. Here, we demonstrate that TGF-ß CAR-T cells significantly improve the anti-tumor efficacy of neighboring cytotoxic T cells. Furthermore, the introduction of TGF-ß CARs into mixed T-cell populations does not result in the preferential expansion of Treg cells, nor do TGF-ß CAR-Treg cells cause CAR-mediated suppression of Teff cells. These results support the utility of incorporating TGF-ß CARs in the development of adoptive T-cell therapy for cancer.

CARs: Synthetic Immunoreceptors for Cancer Therapy and Beyond.

Chimeric antigen receptors (CARs) are versatile synthetic receptors that provide T cells with engineered specificity. Clinical success in treating B-cell malignancies has demonstrated the therapeutic potential of CAR-T cells against cancer, and efforts are underway to expand the use of engineered T cells to the treatment of diverse medical conditions, including infections and autoimmune diseases. Here, we review current understanding of the molecular properties of CARs, how this knowledge informs the rational design and characterization of novel receptors, the successes and shortcomings of CAR-T cells in the clinic, and emerging solutions for the continued improvement of CAR-T cell therapy.

The heme-p53 interaction: Linking iron metabolism to p53 signaling and tumorigenesis.

Recently, we reported that heme binds to tumor suppressor p53 protein (TP53, best known as p53) and promotes its nuclear export and cytosolic degradation, whereas iron chelation stabilizes p53 protein and suppresses tumors in a p53-dependent manner. This not only provides mechanistic insights into tumorigenesis associated with iron excess, but also helps guide the administration of chemotherapy based on iron deprivation in the clinic.

Iron metabolism regulates p53 signaling through direct heme-p53 interaction and modulation of p53 localization, stability, and function.

Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy.