Hearing loss is a significant public health concern, affecting over 250 million people worldwide. Both genetic and environmental etiologies are linked to hearing loss, but in many cases the underlying cellular pathophysiology is not well understood, highlighting the importance of further discovery. We found that inactivation of the gene, Tmtc4 (transmembrane and tetratricopeptide repeat 4), which was broadly expressed in the mouse cochlea, caused acquired hearing loss in mice. Our data showed Tmtc4 enriched in the endoplasmic reticulum, and that it functioned by regulating Ca2+ dynamics and the unfolded protein response (UPR). Given this genetic linkage of the UPR to hearing loss, we demonstrated a direct link between the more common noise-induced hearing loss (NIHL) and the UPR. These experiments suggested a novel approach to treatment. We demonstrated that the small-molecule UPR and stress response modulator ISRIB (Integrated Stress Response Inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout of Tmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing-loss disorders.
Jiang Li, Omar Akil, Stephanie L. Rouse, Conor W. McLaughlin, Ian R. Matthews, Lawrence R. Lustig, Dylan K. Chan, Elliott H. Sherr
Despite the success of T cell checkpoint blockade against melanoma, many “cold” tumors such as prostate cancer remain unresponsive. We find that hypoxic zones are prevalent across pre-clinical prostate cancer and resist T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We show that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperate to cure more than 80% of TRAMP-C2 prostate tumors. Immunofluorescence imaging shows that TH-302 drives an influx of T cells into hypoxic zones, which are then amplified by checkpoint blockade. Further, combination therapy reduces myeloid-derived suppressor cell density by more than 50%, and causes a persistent defect in the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which are completely resistant to checkpoint blockade, show minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors. Survival of Pb-Cre4, Ptenpc−/−Smad4pc−/− mice with highly aggressive prostate adenocarcinoma is also significantly extended by the combination of hypoxia-prodrug and checkpoint blockade. This combination of hypoxia disruption and T cell checkpoint blockade may render some of the most therapeutically resistant cancers sensitive to immunotherapy.
Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey R. Ager, Courtney Nicholas, Ashvin R. Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael A. Curran
Lysyl-tRNA synthetase (KRS) functions canonically in cytosolic translational processes. However, KRS is highly expressed in colon cancer, and localizes to distinct cellular compartments upon phosphorylations (i.e., the plasma membranes after T52-phosphorylation and the nucleus after S207-phosphorylation), leading to probably alternative non-canonical functions. It is unknown how other subcellular KRSs crosstalk with environmental cues during cancer progression. Here, we demonstrate that the KRS-dependent metastatic behavior of colon cancer spheroids within three-dimensional gels requires communication between cellular molecules and extracellular soluble factors and neighboring cells. Membranous and nuclear KRS were found to participate in invasive cell dissemination of colon cancer spheroids in three dimensional gels. Cancer spheroids secreted GAS6 via a KRS-dependent mechanism and caused the M2 polarization of macrophages, which activated the neighboring cells via secretion of FGF2/GROα/M-CSF to promote cancer dissemination under environmental remodeling via fibroblast-mediated laminins production. Analyses of tissues from clinical colon cancer patients and Krs–/+ animal models for cancer metastasis supported the roles of KRS, GAS6, and M2 macrophages in KRS-dependent positive feedback between tumors and environmental factors. Altogether, KRS in colon cancer cells remodels the microenvironment to promote metastasis, which can thus be therapeutically targeted at these bidirectional KRS-dependent communications of cancer spheroids with environmental cues.
Seo Hee Nam, Doyeun Kim, Doohyung Lee, Hye-Mi Lee, Dae-Geun Song, Jae Woo Jung, Ji Eon Kim, Hye-Jin Kim, Nam Hoon Kwon, Eun-Kyeong Jo, Sunghoon Kim, Jung Weon Lee
SMAD4 is the only common SMAD (co-SMAD) in transforming growth factor (TGF)-β signaling that usually impedes immune cell activation in the tumor microenvironment. However, here we demonstrated that selective deletion of Smad4 in natural killer (NK) cells actually led to dramatically reduced tumor cell rejection and augmented tumor cell metastases, reduced murine cytomegalovirus clearance, as well as impeded NK cell homeostasis and maturation. This was associated with a downregulation of granzyme B (Gzmb), Kit and Prdm1 in Smad4-deficient NK cells. We further unveiled the mechanism by which SMAD4 promoted Gzmb expression. Gzmb was identified as a direct target of a transcriptional complex formed by SMAD4 and JUNB. A JUNB binding site distinct from that for SMAD4 in the proximal Gzmb promoter was required for transcriptional activation by the SMAD4/JUNB complex. In a Tgfbr2 and Smad4 NK cell-specific double conditional knockout model, SMAD4-mediated events were found to be independent of canonical TGF-β signaling. Our study identifies and mechanistically characterizes unusual functions and pathways for SMAD4 in governing innate immune responses to cancer and viral infection, as well as NK cell development.
Youwei Wang, Jianhong Chu, Ping Yi, Wenjuan Dong, Jennifer N. Saultz, Yufeng Wang, Hongwei Wang, Steven D. Scoville, Jianying Zhang, Lai-Chu Wu, Youcai Deng, Xiaoming He, Bethany L. Mundy-Bosse, Aharon G. Freud, Li-Shu Wang, Michael A. Caligiuri, Jianhua Yu
The mechanisms that drive T cell aging are not understood. We report children and adult telomerase mutation carriers with short telomere length (TL) develop a T cell immunodeficiency that can manifest in the absence of bone marrow failure and causes life-threatening opportunistic infections. Mutation carriers shared T cell aging phenotypes seen in adults five decades older including depleted naïve T cells, increased apoptosis, and restricted T cell repertoire. T cell receptor excision circles (TRECs) were also undetectable or low, suggesting newborn screening may identify individuals with germline telomere maintenance defects. Telomerase null mice with short TL showed defects throughout T cell development including increased apoptosis of stimulated thymocytes, their intra-thymic precursors, in addition to depleted hematopoietic reserves. When we examined the transcriptional programs of T cells from telomerase mutation carriers, we found they diverged from older adults with normal TL. Short telomere T cells up-regulated DNA damage and intrinsic apoptosis pathways, while older adult T cells up-regulated extrinsic apoptosis pathways and PD-1 expression. T cells from mice with short TL also showed an active DNA damage response, in contrast to old wild-type mice, despite their shared propensity to apoptosis. Our data suggest there are telomere length-dependent and telomere length-independent mechanisms that differentially contribute to distinct molecular programs of T cell apoptosis with aging.
Christa L. Wagner, Vidya Sagar Hanumanthu, C. Conover Talbot Jr., Roshini S. Abraham, David Hamm, Dustin L. Gable, Christopher G. Kanakry, Carolyn D. Applegate, Janet Siliciano, J. Brooks Jackson, Stephen V. Desiderio, Jonathan K. Alder, Leo Luznik, Mary Armanios
Preeclampsia remains a clinical challenge due to its poorly understood pathogenesis. A prevailing notion is that increased placental production of soluble fms-like tyrosine kinase-1 (sFlt-1) causes the maternal syndrome by inhibiting proangiogenic placental growth factor (PlGF) and VEGF. However, the significance of PlGF suppression in preeclampsia is uncertain. To test whether preeclampsia results from the imbalance of angiogenic factors reflected by an abnormal sFlt-1:PlGF ratio, we studied PlGF knockout (KO; Pgf–/–) mice and noted that while sFlt-1 was significantly elevated in pregnancy, the mice did not develop signs or sequelae of preeclampsia. Notably, PlGF KO mice had morphologically distinct placentas, showing an accumulation of junctional zone glycogen. We next considered the role of placental PlGF in an established model of preeclampsia (pregnant catechol-O-methyltransferase (COMT)-deficient mice) by generating mice with deletions in both the Pgf and Comt genes. Deletion of placental PlGF in the context of COMT loss resulted in a reduction in maternal blood pressure and increased placental glycogen, indicating that loss of PlGF might be protective against the development of preeclampsia. These results identify a role for PlGF in placental development and support a complex model for the pathogenesis of preeclampsia beyond an angiogenic factor imbalance.
Jacqueline G. Parchem, Keizo Kanasaki, Megumi Kanasaki, Hikaru Sugimoto, Liang Xie, Yuki Hamano, Soo Bong Lee, Vincent H. Gattone, Samuel Parry, Jerome F. Strauss, Vesna D. Garovic, Thomas F. McElrath, Karen H. Lu, Baha M. Sibai, Valerie S. LeBleu, Peter Carmeliet, Raghu Kalluri
Enterovirus 71 (EV-A71) receptors that have been identified to date cannot fully explain the pathogenesis of EV-A71, which is an important global cause of hand-foot-and-mouth disease and life-threatening encephalitis. We identified an interferon-gamma (IFNγ)-inducible EV-A71 cellular entry factor, human tryptophanyl-tRNA synthetase (hWARS), using genome-wide RNAi library screening. The importance of hWARS in mediating virus entry and infectivity was confirmed by virus attachment, in vitro pull-down, antibody/antigen blocking, and CRISPR/Cas9. Upon IFNγ treatment, induced hyperexpression and plasma membrane translocation of hWARS were observed, which sensitized semi-permissive (human neuronal NT2)/non-permissive (mouse fibroblast L929) cells to EV-A71 infection. Our hWARS-transduced mouse infection model showed pathological changes similar to patients with severe EV-A71 infection. The expression of hWARS is also required for productive infection by other human enteroviruses, including the clinically important CV-A16 and EV-D68. This is the first report on the discovery of an entry factor, hWARS, which can be induced by IFNγ for EV-A71. Given that a high level of IFNγ was observed in patients with severe EV-A71 infection, our findings extend the knowledge of the pathogenicity of EV-A71 in relation to the expression of entry factor upon IFNγ stimulation and the therapeutic options for treating severe EV-A71-associated complications.
Man Lung Yeung, Lilong Jia, Cyril C.Y. Yip, Jasper F.W. Chan, Jade L.L. Teng, Kwok-Hung Chan, Jian-Piao Cai, Chaoyu Zhang, Anna J. Zhang, Wan-Man Wong, Kin-Hang Kok, Susanna K.P. Lau, Patrick C.Y. Woo, Janice Y.C. Lo, Dong-Yan Jin, Shin-Ru Shih, Kwok-Yung Yuen
Cardiac two pore domain potassium channels (K2P) exist in organisms from Drosophila to humans, however their role in cardiac function is not known. We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK related potassium channel (TREK-1 or Kcnk2), exhibit exaggerated pressure overload induced concentric hypertrophy and alterations in fetal gene expression, yet retain preserved systolic and diastolic cardiac function. While cardiomyocyte specific deletion of TREK-1 in response to in vivo pressure overload resulted in cardiac dysfunction, TREK-1 deletion in fibroblasts prevented deterioration in cardiac function. The absence of pressure overload induced dysfunction in TREK-1 KO mice was associated with diminished cardiac fibrosis and reduced activation of c-Jun N-terminal kinase activity (JNK) in cardiomyocytes and fibroblasts. These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload-induced cardiac dysfunction and serve as a conceptual basis for its inhibition for as a potential therapy.
Dennis M. Abraham, Teresa E. Lee, Lewis J. Watson, Lan Mao, Gurangad S. Chandok, Hong-Gang Wang, Stephan Frangakis, Geoffrey S. Pitt, Svati H. Shah, Matthew J. Wolf, Howard A. Rockman
Chronic allergic inflammatory diseases are a major cause of morbidity, allergic asthma alone affecting over 300 million people worldwide. Epidemiological studies demonstrate that environmental stimuli are associated with either promotion or prevention of disease. Major reductions in asthma prevalence are documented in European and US farming communities. Protection is associated with exposure of mothers during pregnancy to microbial breakdown products present in farm dusts and unprocessed foods, and enhancement of innate immune competence in the children. We sought to develop a scientific rationale for progressing these findings towards clinical application for primary disease prevention. Treatment of pregnant mice with a defined clinically-approved immune-modulator was shown to markedly reduce susceptibility of their offspring to development of the hallmark clinical features of allergic airway inflammatory disease. Mechanistically, offspring displayed enhanced dendritic cell-dependent airway mucosal immune surveillance function, which resulted in more efficient generation of mucosal-homing T-regulatory cells in response to local inflammatory challenge. We provide evidence that the principal target for maternal treatment effects was the fetal dendritic cell progenitor compartment, equipping the offspring for accelerated functional maturation of the airway mucosal dendritic cell network following birth. These data provide proof-of-concept supporting the rationale for development of transplacental immune reprogramming approaches for primary disease prevention.
Kyle T. Mincham, Naomi M. Scott, Jean-Francois Lauzon-Joset, Jonatan Leffler, Alexander N. Larcombe, Philip A. Stumbles, Sarah A. Robertson, Christian Pasquali, Patrick G. Holt, Deborah H. Strickland
It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding pre-clinical models with the same G+E combinations are useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including pro-apoptosis, metabolic dysregulation, and selective down-regulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease relevant phenotype, and provides rational strategies to identify actionable targets.
Ta-Chiang Liu, Justin T. Kern, Kelli L. VanDussen, Shanshan Xiong, Gerard E. Kaiko, Craig B. Wilen, Michael W. Rajala, Roberta Caruso, Michael J. Holtzman, Feng Gao, Dermot P.B. McGovern, Gabriel Nunez, Richard D. Head, Thaddeus S. Stappenbeck
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