Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We investigated a male infant with neonatal skin/eye/mouth (SEM) HSV1 disease who had complete recovery after acyclovir but developed HSV1 encephalitis at 1 year of age. Immune work up showed an anergic peripheral blood monocyte cytokine (PBMC) response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IRF7 and UNC93B1. PBMC single cell RNA sequencing done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and THP-1 showed that both variants individually suppressed TLR3-driven IRF3 promoter activity and type I interferon response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I interferon response upon HSV1 challenge. This study reports an infant with recurrent HSV1 disease complicated by encephalitis associated with deleterious variants in IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent severe HSV.
Megan H. Tucker, Wei Yu, Heather L. Menden, Sheng Xia, Carl F. Schreck, Margaret I. Gibson, Daniel A. Louiselle, Tomi Pastinen, Nikita Raje, Venkatesh Sampath
HSV-2 coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well-defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2 infected and bystander 2D10 cells. Bulk and single-cell RNA sequencing studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms including upregulation of MALAT1 to release epigenetic silencing.
Carl A. Pierce, Lip Nam Loh, Holly R. Steach, Natalia Cheshenko, Paula Preston-Hurlburt, Fengrui Zhang, Stephanie Stransky, Leah Kravets, Simone Sidoli, William M. Philbrick, Michel N. Nassar, Smita Krishnaswamy, Kevan C. Herold, Betsy C. Herold
Renal osteodystrophy (ROD) is a disorder of bone metabolism that affects virtually all patients with chronic kidney disease (CKD), and is associated with adverse clinical outcomes including fractures, cardiovascular events and death. In the present study, we showed that hepatocyte nuclear factor 4 alpha (HNF4α), a transcription factor mostly expressed in the liver, is also expressed in bone, and that osseous HNF4α expression was dramatically reduced in patients and mice with ROD. Osteoblast-specific deletion of Hnf4α resulted in impaired osteogenesis in cells and mice. Using multi-omics analyses of bones and cells lacking or overexpressing Hnf4α1 and Hnf4α2, we showed that HNF4α2 is the main osseous Hnf4α isoform that regulates osteogenesis, cell metabolism, and cell death. As a result, osteoblast-specific overexpression of Hnf4α2 prevented bone loss in mice with CKD. Our results showed that HNF4α2 is a transcriptional regulator of osteogenesis, implicated in the development of ROD.
Marta Martinez-Calle, Guillaume Courbon, Bridget Hunt-Tobey, Connor Francis, Jadeah J. Spindler, Xueyan Wang, Luciene M. dos Reis, Carolina Steller Wagner Martins, Isidro B. Salusky, Hartmut H. Malluche, Thomas L. Nickolas, Rosa M.A. Moyses, Aline Martin, Valentin David
Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis (CH) of indeterminate potential (CHIP) is frequent in ageing, involves expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, risk factors that contribute to CHIP-associated CH are poorly understood. Obesity induces a pro-inflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data from 47,466 individuals with validated CHIP in UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1 and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and a pro-inflammatory state can potentiate progression of CHIP to more significant hematologic neoplasia. Calcium channel blocker, nifedipine or SKF-96365, either alone or in combination with metformin, MCC950 or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in obese individuals.
Santhosh Kumar Pasupuleti, Baskar Ramdas, Sarah S. Burns, Lakshmi Reddy Palam, Rahul Kanumuri, Ramesh Kumar, Taruni R. Pandhiri, Utpal Dave, Nanda Kumar Yellapu, Xinyu Zhou, Chi Zhang, George E. Sandusky, Zhi Yu, Michael C. Honigberg, Alexander G. Bick, Gabriel K. Griffin, Abhishek Niroula, Benjamin L. Ebert, Sophie Paczesny, Pradeep Natarajan, Reuben Kapur
Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) responding to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs of the brain (CVOs) focus on mechanosensitive membrane proteins. The present study demonstrated that an intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuronal-specific knockout (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1-cKO also blunted mannitol-indued AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.
Xin Jin, Jian Xie, Chia-Wei Yeh, Jen-Chi Chen, Chih-Jen Cheng, Cheng-Chang Lien, Chou-Long Huang
Neuropathic pain remains poorly managed by current therapies highlighting the need to improve our knowledge of chronic pain mechanisms. In neuropathic pain models, dorsal root ganglia (DRG) nociceptive neurons transfer miR-21 packaged in extracellular vesicles to macrophages that promote pro-inflammatory phenotype and contribute to allodynia. Here we show that miR-21 conditional deletion in DRG neurons was coupled with lack of up-regulation of CCL2 chemokine after nerve injury and reduced accumulation of CCR2-expressing macrophages, which showed TGFB-related pathway activation and acquired M2-like anti-nociceptive phenotype. Indeed, neuropathic allodynia was attenuated in cKO and restored by a TGFB receptor inhibitor (SB431542) administration. Since TGFBR2 and TGFB1 are known miR-21 targets, we suggest that miR-21 transfer from injured neurons to macrophages maintains a pro-inflammatory phenotype via suppression of such an anti-inflammatory pathway. These data support miR-21 inhibition as a possible approach to maintain polarization of DRG macrophages at M2-like state and attenuate neuropathic pain.
Lynda Zeboudj, George Sideris-Lampretsas, Rita Silva, Sabeha Al-Mudaris, Francesca Picco, Sarah Fox, David Chambers, Marzia Malcangio
The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Further, our analysis identified two important milestones: 1) a diploid stage, where iATC cells were diploids with inflammatory phenotypes, and 2) an aneuploid stage, where mATCs gained aneuploid genomes and mesenchymal phenotypes producing excessive collagens and collagen-interacting receptors. In parallel, cancer-associated-fibroblasts showed strong interactions among mesenchymal cell-types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for ATC.
Lina Lu, Jennifer Rui Wang, Ying C. Henderson, Shanshan Bai, Jie Yang, Min Hu, Cheng-Kai Shiau, Timothy Y. Pan, Yuanqing Yan, Tuan M. Tran, Jianzhuo Li, Rachel Kieser, Xiao Zhao, Jiping Wang, Roza Nurieva, Michelle D. Williams, Maria E. Cabanillas, Ramona Dadu, Naifa Busaidy, Mark Zafereo, Nicholas Navin, Stephen Y. Lai, Ruli Gao
Germline or somatic loss-of-function mutations of fumarate hydratase (FH) predispose patients to an aggressive form of renal cell carcinoma (RCC). Since other than tumor resection, there is no effective therapy for metastatic FH-deficient RCC, an accurate method for early diagnosis is needed. Although MRI or CT scans are offered, they cannot differentiate FH-deficient tumors from other RCCs. Therefore, finding noninvasive plasma biomarkers suitable for rapid diagnosis, screening and surveillance would improve clinical outcomes. Taking advantage of the robust metabolic rewiring that occurs in FH-deficient cells, we performed plasma metabolomics analysis and identified two tumor-derived metabolites, succinyl-adenosine and succinic-cysteine, as outstanding plasma biomarkers for early diagnosis (receiver operating characteristic area under curve (ROCAUC) = 0.98). These two molecules reliably reflected the FH mutation status and tumor mass. We further identified the enzymatic cooperativity by which these biomarkers are produced within the tumor microenvironment. Longitudinal monitoring of patients demonstrated that these circulating biomarkers can be used for reporting on treatment efficacy and identifying recurrent or metastatic tumors.
Liang Zheng, Zi-Ran Zhu, Tal Sneh, Weituo Zhang, Zao-Yu Wang, Guang-Yu Wu, Wei He, Hong-Gang Qi, Hang Wang, Xiao-Yu Wu, Jonatan Fernández-García, Ifat Abramovich, Yun-Ze Xu, Jin Zhang, Eyal Gottlieb
Many hepatocellular carcinoma (HCC) patients do not respond to the first-line immune checkpoint inhibitor treatment. Immunization with effective cancer vaccines is an attractive alternative approach to immunotherapy. However, its efficacy remains insufficiently evaluated in preclinical studies. Here, we investigated HCC-associated self/tumor antigen, α-fetoprotein (AFP) based vaccine immunization for treating AFP (+) HCC mouse models. We found that AFP immunization effectively induced AFP-specific CD8+ T cells in vivo. However, these CD8+ T cells expressed exhaustion markers, including PD1, LAG3, and Tim3. Furthermore, the AFP vaccine effectively prevented c-MYC/Mcl1 HCC initiation when administrated before tumor formation, while it was ineffective against full-blown c-MYC/Mcl1 tumors. Similarly, anti-PD1 and anti-PD-L1 monotherapy showed no efficacy in this murine HCC model. In striking contrast, AFP immunization combined with anti-PD-L1 treatment triggered significant inhibition of HCC progression in most liver tumor nodules, while combining with anti-PD1 induced slower tumor progression. Mechanistically, we demonstrated that HCC intrinsic PD-L1 expression was the primary target of anti-PD-L1 in this combination therapy. Notably, the combination therapy had a similar therapeutic effect in the cMet/β-Catenin mouse HCC model. These findings suggest that combining the AFP vaccine and immune checkpoint inhibitors may be effective for AFP (+) HCC treatment.
Xinjun Lu, Shanshan Deng, Jiejie Xu, Benjamin L. Green, Honghua Zhang, Guofei Cui, Yi Zhou, Yi Zhang, Hongwei Xu, Fapeng Zhang, Rui Mao, Sheng Zhong, Thorsten Cramer, Matthias Evert, Diego F. Calvisi, Yukai He, Chao Liu, Xin Chen
Excessive Erythrocytosis (EE) is a major hallmark of patients suffering from chronic mountain sickness (CMS, Monge’s disease) and is responsible for major morbidity and even mortality in early adulthood. We took advantage of unique populations, one living at high altitude (Peru) showing EE, while another population, at the same altitude and region, shows no evidence of EE (non-CMS). Through RNA-seq, we identified and validated the function of a group of long non-coding RNA (lncRNAs) that regulate erythropoiesis in Monge’s disease but not in the non-CMS population. Among these lncRNAs is HIKER (Hypoxia Induced Kinase-mediated Erythropoietic Regulator)/LINC02228 which we showed plays a critical role in erythropoiesis in CMS cells. Under hypoxia, HIKER modulated CSNK2B (the regulatory subunit of Casein kinase 2). A down-regulation of HIKER down-regulated CSNK2B, remarkably reducing erythropoiesis (<70% reduction of BFUs); furthermore, an up-regulation of CSNK2B on the background of HIKER down-regulation rescued erythropoiesis defects. Pharmacologic inhibition of CSNK2B drastically reduced erythroid colonies (50-75% reduction in BFU colonies) and knock-down of CSNK2B in zebrafish lead to a defect in hemoglobinization (<97% morphants show reduction in hemoglobin levels). We conclude that HIKER regulates erythropoiesis in Monge’s disease and acts through at least one specific target, CSNK2B, a casein kinase.
Priti Azad, Dan Zhou, Hung-Chi Tu, Francisco C. Villafuerte, David Traver, Tariq M. Rana, Gabriel G. Haddad
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