Glioblastoma is the most aggressive type of brain tumor with poor therapeutic response and prognosis. Passage of systemically delivered pharmacological agents into the brain is largely blocked by the blood-brain-barrier (BBB), an organotypic specialization of brain endothelial cells (EC). Tumor vessels in GBM are abnormal and more permeable, but the heterogeneity of BBB breakdown in different parts of the tumor vasculature and at the tumor’s invasive front is largely unknown. Here, through single-cell RNA sequencing (scRNA-seq) of freshly isolated ECs from human glioblastoma and paired tumor peripheral tissues, we have constructed a molecular atlas of human brain ECs providing unprecedented molecular insight into the heterogeneity of the human BBB and its molecular alteration in glioblastoma. We identified 5 distinct EC phenotypes representing different states of EC activation and BBB impairment, and associated with different anatomical locations within and around the tumor. This unique data resource provides key information for designing rational therapeutic regimens and optimizing drug delivery.
Yuan Xie, Liqun He, Roberta Lugano, Yanyu Zhang, Haiyan Cao, Qiyuan He, Min Chao, Boxuan Liu, Qingze Cao, Jianhao Wang, Yang Jiao, Yaqin Hu, Liying Han, Yong Zhang, Hua Huang, Lene Uhrbom, Christer Betsholtz, Liang Wang, Anna Dimberg, Lei Zhang
Historically, naive cells have been considered inconsequential to HIV persistence. Here, we compared the contribution of naive and memory cells to the reservoir of individuals with a spectrum of reservoir sizes and variable immunological control. We performed proviral sequencing of approximately 6000 proviruses from cellular subsets of 5 elite controllers (ECs) off antiretroviral therapy (ART) and 5 chronic progressors (CPs) on ART.The levels of naive infection were barely detectable in ECs and approximately 300-fold lower compared to CPs. Moreover, the ratio of infected naive to memory cells was significantly lower in ECs. Overall naive infection level increased as reservoir size increased such that naive cells were a major contributor to the intact reservoir of CPs, whose reservoirs were generally very diverse. In contrast, the reservoirs of ECs were dominated by proviral clones. Critically, the fraction of proviral clones increased with cell differentiation, with naive infection predicting reservoir diversity. Longitudinal sequencing revealed that the reservoir of ECs was less dynamic compared to CPs. Naive cells play a critical role in HIV persistence. Their infection level predicts reservoir size and diversity. Moreover, the diminishing diversity of the reservoir as cellular subsets mature suggests that naive T cells repopulate the memory compartment and that direct infection of naive T cells occurs in vivo.
Marilia Rita Pinzone, Sam Weissman, Alexander O. Pasternak, Ryan Zurakowski, Stephen Migueles, Una O'Doherty
The mitochondrial enzyme acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the detoxification of acetaldehyde and endogenous lipid aldehydes. Approximately 40% of East Asians, accounting for 8% of the human population, carry the E504K mutation in ALDH2 that leads to accumulation of toxic reactive aldehydes and increases the risk for cardiovascular disease (CVD), cancer and Alzheimer’s, among other diseases. However, the role of ALDH2 in acute kidney injury (AKI) remains poorly defined and is therefore the subject of the present study using various cellular and organismal sources. In murine models in which AKI was induced by either the contrast agent Iohexol or renal ischemia/reperfusion, knockout and activation/overexpression of ALDH2 was associated with increased and decreased renal injury, respectively. In murine renal tubular epithelial cells (RTECs), ALDH2 upregulated Beclin-1 expression, promoted autophagy activation and eliminated reactive oxygen species (ROS). In vivo and in vitro, both 3-MA and Beclin-1 siRNAs inhibited autophagy and abolished ALDH2 mediated renoprotection. In mice with Iohexol induced AKI, ALDH2 knockdown in RTECs using AAV-shRNA impaired autophagy activation and aggravated renal injury. In human renal proximal tubular epithelial HK-2 cells exposed to Iohexol, ALDH2 activation potentiated autophagy and attenuated apoptosis. In mice with AKI induced by renal ischemia ischemia/reperfusion, ALDH2 overexpression or pretreatment regulated autophagy mitigating apoptosis of RTECs and renal injury. Our data collectively substantiate a critical role of ALDH2 in AKI via autophagy activation involving the Beclin-1 pathway.
Tonghui Xu, Jialin Guo, Maozeng Wei, Jiali Wang, Kehui Yang, Chang Pan, Jiaojiao Pang, Li Xue, Qiu-huan Yuan, Mengyang Xue, Jian Zhang, Wentao Sang, Tangxing Jiang, Yuguo Chen, Feng Xu
Functional loss of MYO5B induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced epithelial-specific MYO5B knockout (VilCreERT2;Myo5bflox/flox) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations both in control and MYO5B knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor, dibenzazepine (DBZ), restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.
Izumi Kaji, Joseph T. Roland, Sudiksha Rathan-Kumar, Amy C. Engevik, Andreanna Burman, Anna E. Goldstein, Masahiko Watanabe, James R. Goldenring
Gain-of-function polymorphisms in the transcription factor IRF5 are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduces disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils does not reduce disease severity. B cell receptor and TLR7 signaling synergize to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increases B cell-intrinsic IL-6 and TNF-α production, both key requirements for germinal center responses, with IL-6 and TNF-α production in vitro and in vivo being substantially lower with loss of one allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation is reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression is dynamically regulated in vivo with increased expression in germinal center B cells compared to non-germinal center B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells is required to promote disease in murine lupus.
Alex Pellerin, Kei Yasuda, Abraham Cohen-Bucay, Vanessa Sandra, Prachi Shukla, Barry K. Horne Jr, Kerstin Nundel, Gregory A. Viglianti, Yao Xie, Ulf Klein, Ying Tan, Ramon G. Bonegio, Ian R. Rifkin
Inherited retinal diseases such as retinitis pigmentosa (RP) can be caused by thousands of different mutations, a small number of which have been successfully treated with gene replacement. However, this approach has yet to scale and may not be feasible in many cases, highlighting the need for interventions that could benefit more patients. Here, we found that microglial phagocytosis is upregulated during cone degeneration in RP, suggesting that expression of “don’t eat me” signals such as CD47 might confer protection to cones. To test this, we delivered an adeno-associated viral (AAV) vector expressing CD47 on cones, which promoted cone survival in three mouse models of RP and preserved visual function. Cone rescue with CD47 required a known interacting protein, signal regulatory protein alpha (SIRPα), but not an alternative interacting protein, thrombospondin-1 (TSP1). Despite the correlation between increased microglial phagocytosis and cone death, microglia were dispensable for the pro-survival activity of CD47, suggesting that CD47 interacts with SIRPα on non-microglial cells to alleviate degeneration. These findings establish augmentation of CD47-SIRPα signaling as a potential treatment strategy for RP and possibly other forms of neurodegeneration.
Sean K. Wang, Yunlu Xue, Constance L. Cepko
Previously we reported heightened expression of human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of PA hypertension (PAH) patients. Furthermore, recombinant HERV-K dUTPase increased IL6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG, and proinflammatory molecules IL6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response (MYD)-88 to increase IL6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to pJNK-pSMAD3 required for SNAIL, and pSTAT1 for IL6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor (ATF)-2 to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated pro-inflammatory molecules.
Shoichiro Otsuki, Toshie Saito, Shalina Taylor, Dan Li, Jan-Renier Moonen, David P. Marciano, Rebecca L. Harper, Aiqin Cao, Lingli Wang, Maria E. Ariza, Marlene Rabinovitch
Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab, a mAb that blocks IL-13 and IL-4 signaling, had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, hyaluronan synthase 1 (Has1) was the most downregulated gene and accumulation of the hyaluronan polysaccharide was decreased in the lung. In patients with COVID-19, hyaluronan was increased in the lungs and plasma. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator. Finally, hyaluronan was directly induced in the lungs of mice by administration of IL-13, indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.
Alexandra N. Donlan, Tara E. Sutherland, Chelsea Marie, Saskia Preissner, Benjamin T. Bradley, Rebecca M. Carpenter, Jeffrey M. Sturek, Jennie Z. Ma, G. Brett Moreau, Jeffrey R. Donowitz, Gregory A. Buck, Myrna G. Serrano, Stacey L. Burgess, Mayuresh M. Abhyankar, Cameron Mura, Philip E. Bourne, Robert Preissner, Mary K. Young, Genevieve R. Lyons, Johanna J. Loomba, Sarah J. Ratcliffe, Melinda D. Poulter, Amy J. Mathers, Anthony J. Day, Barbara J Mann, Judith E. Allen, William A. Petri Jr.
Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry-based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch-repair deficient (dMMR)-CRC tissue sample carrying 3,967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of non-mutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.
Tomomi Hirama, Serina Tokita, Munehide Nakatsugawa, Kenji Murata, Yasuhito Nannya, Kazuhiko Matsuo, Hidetoshi Inoko, Yoshihiko Hirohashi, Shinichi Hashimoto, Seishi Ogawa, Ichiro Takemasa, Noriyuki Sato, Fumitake Hata, Takayuki Kanaseki, Toshihiko Torigoe
BACKGROUND. Wolfram syndrome is a rare endoplasmic reticulum disorder characterized by insulin-dependent diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Although there is currently no treatment to delay, halt, or reverse the progression of Wolfram syndrome, preclinical studies in cell and rodent models suggest that therapeutic strategies targeting endoplasmic reticulum calcium homeostasis, including dantrolene sodium, may be beneficial. METHODS. Based on the results from preclinical studies on dantrolene sodium and ongoing longitudinal studies, our group put together the first-ever clinical trial in pediatric and adult patients with Wolfram syndrome. An open-label phase 1b/2a trial design was chosen. The primary objective of the study was to assess the safety and tolerability of dantrolene sodium in adult and pediatric patients with Wolfram syndrome. Secondary objectives were to evaluate the efficacy of dantrolene sodium on residual pancreatic beta-cell functions, visual acuity, quality of life measures related to vision, and neurological functions. RESULTS. The results indicate that dantrolene sodium is well tolerated by patients with Wolfram syndrome. Overall, β-cell functions were not significantly improved by dantrolene, but there was a significant correlation between baseline β-cell functions and the change in β-cell responsiveness (R2, p=0.004) after 6 months of dantrolene therapy. Other outcome measures, including visual acuity and neurological functions, were not improved by dantrolene sodium treatment within 6 months. As previously reported, markers of inflammatory cytokines and oxidative stress, such as IFNγ, IL-1β, TNFα, and isoprostane, were elevated in subjects with Wolfram syndrome. CONCLUSION. This study justifies further investigation into using dantrolene sodium and other small molecules targeting the endoplasmic reticulum for the treatment of Wolfram syndrome. TRIAL REGISTRATION. ClinicalTrials.gov Identifier NCT02829268
Damien Abreu, Stephen I. Stone, Toni S. Pearson, Robert C. Bucelli, Ashley N. Simpson, Stacy Hurst, Cris M. Brown, Kelly Kries, Chinyere Onwumere, Hongjie Gu, James Hoekel, Lawrence Tychsen, Gregory P. Van Stavern, Neil H. White, Bess A. Marshall, Tamara Hershey, Fumihiko Urano
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