Docetaxel (DTX) combined with cisplatin and 5-FU has been used as induction chemotherapy for head and neck squamous cell carcinoma (HNSCC). However, the development of acquired resistance remains a major obstacle to treatment response. Tumor-associated macrophages are associated with chemotherapeutic resistance. In the present study, increased infiltration of macrophages into the tumor microenvironment was significantly associated with shorter overall survival and increased resistance to chemotherapeutic drugs, particularly DTX in HNSCC patients. Macrophage co-culture induced expression of intercellular adhesion molecule 1 (ICAM1), which promotes stemness and the formation of polyploid giant cancer cells, thereby reducing the efficacy of DTX. Both genetic silencing and pharmacological inhibition of ICAM1 sensitized HNSCC to DTX. Macrophage secretion of IL-1β was found to induce tumor expression of ICAM1. IL-1β neutralization and IL-1 receptor blockade reversed DTX resistance induced by macrophage co-culture. IL-1β activated superoxide dismutase 2 and inhibited catalase, thereby modulating intracellular levels of reactive oxygen species (ROS) and inducing ICAM1 expression. Arsenic trioxide (ATO) reduced macrophage infiltration into the TME and impaired IL-1β secretion by macrophages. The combinatorial use of ATO enhanced the in vivo efficacy of DTX in a mouse model, which may provide a revolutionary approach to overcoming acquired therapeutic resistance in HNSCC.
Ching-Yun Hsieh, Ching-Chan Lin, Yu-Wen Huang, Jong-Hang Chen, Yung-An Tsou, Ling-Chu Chang, Chi-Chen Fan, Chen-Yuan Lin, Wei-Chao Chang
Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and characterize the transcriptional profiles and metabolic pathways of pro-angiogenic microglia in a mouse model of oxygen-induced proliferative retinopathy (OIR). Using transcriptional single-cell sorting, we comprehensively map all microglia populations in retinas of room air (RA) and OIR mice. We unveil several unique types of PR-associated microglia (PRAM) and identify markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hyper-metabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes and pro-angiogenic insulin-like growth factor 1. Immunohistochemical staining shows these PRAMs were spatially located within or around neovascular (NV) tufts. These unique microglia-types have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis-related diseases.
Zhiping Liu, Huidong Shi, Jiean Xu, Qiuhua Yang, Qian Ma, Xiaoxiao Mao, Zhimin Xu, Yaqi Zhou, Qingen Da, Yongfeng Cai, David J.R. Fulton, Zheng Dong, Akrit Sodhi, Ruth B. Caldwell, Yuqing Huo
We describe affected members of a two-generation family segregating a Stargardt disease-like phenotype caused by a two base pair deletion-insertion, c.1014_1015delGAinsCT;p(Trp338_Asn339delinsCysTyr), in BEST1. The variant was identified by whole exome sequencing and its pathogenicity was verified through chloride channel recording using wild-type (WT) and transfected mutant HEK293 cells. Clinical examination of both patients revealed a similar phenotype at two different disease stages that were attributable to difference in their age at presentation. Hyperautofluorescent flecks along the arcades were observed in the proband, while the affected mother exhibited more advanced retinal pigment epithelium (RPE) loss in the central macula. Full-field electroretinogram testing was unremarkable in the daughter, however, moderate attenuation of generalized cone function was detected in the mother. Electro-oculogram testing in the daughter was consistent with widespread dysfunction of the RPE characteristic of Best disease. Whole-cell patch clamp recordings revealed statistically significant decrease in chloride conductance of the mutant compared to WT cells. This report broadens the clinical spectrum of BEST1-associated retinopathy in the form of a mother and daughter with BEST1 genotype phenocopying Stargardt disease.
Masha Kolesnikova, Jin Kyun Oh, Jiali Wang, Winston Lee, Jana Zernant, Pei-Yin Su, Angela H. Kim, Laura A. Jenny, Tingting Yang, Rando Allikmets, Stephen H. Tsang
Deficiency of glycogen debranching enzyme in glycogen storage disease type III (GSD III) results in excessive glycogen accumulation in multiple tissues, primarily the liver, heart, and skeletal muscle. We recently reported that an adeno-associated virus (AAV) vector expressing a bacterial debranching enzyme (Pullulanase) driven by the ubiquitous CMV enhancer/chicken β-actin (CB) promoter cleared glycogen in major affected tissues of infant GSD IIIa mice. In this study, we developed a novel dual promoter consisting of a liver-specific promoter (LSP) and the CB promoter for gene therapy in adult GSD IIIa mice. Ten-week treatment with an AAV vector containing the LSP-CB dual promoter in adult GSD IIIa mice significantly increased Pullulanase expression and reduced glycogen contents in the liver (-60%), heart (-76%), and skeletal muscle (-63%), accompanied by the reversal of liver fibrosis, improved muscle function, and significant decrease in plasma biomarkers alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Compared to the CB promoter, the dual promoter effectively decreased Pullulanase-induced cytotoxic T lymphocyte responses and enabled persistent therapeutic gene expression in adult GSD IIIa mice. Future studies are needed to determine the long-term durability of the dual promoter mediated expression of Pullulanase in adult GSD IIIa mice and in large animal models.
Jeong-A Lim, Priya S. Kishnani, Baodong Sun
Identifying host factors that contribute to pneumonia incidence and severity are of utmost importance to guiding the development of more effective therapies. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor known to promote vascular injury and inflammation, but it is unknown whether and how LOX-1 functions in the lung. Here, we provide evidence of substantial accumulation of LOX-1 in the lungs of ARDS patients and in mice with pneumonia. Unlike previously described injurious contributions of LOX-1, we found that LOX-1 is uniquely protective in the pulmonary airspaces, limiting proteinaceous edema and inflammation. We also identified alveolar macrophages and recruited neutrophils as two prominent sites of LOX-1 expression in the lungs, whereby macrophages are capable of further induction during pneumonia and neutrophils exhibit a rapid, but heterogenous elevation of LOX-1 in the infected lung. Blockade of LOX-1 led to dysregulated immune signaling in alveolar macrophages, marked by alterations in activation markers and a concomitant elevation of inflammatory gene networks. However, bone marrow chimeras also suggested a prominent role for neutrophils in LOX-1-mediated lung protection, further supported by LOX-1+ neutrophils exhibiting transcriptional changes consistent with reparative processes. Taken together, this work establishes LOX-1 as a tissue-protective factor in the lungs during pneumonia, possibly mediated by its influence on immune signaling in alveolar macrophages (AMs) and LOX-1+ airspace neutrophils.
Filiz T. Korkmaz, Anukul T. Shenoy, Elise Symer, Lillia A. Baird, Christine V. Odom, Emad Arafa, Ernest L. Dimbo, Elim Na, William Molina-Arocho, Matthew Brudner, Theodore J. Standiford, Jawahar L. Mehta, Tatsuya Sawamura, Matthew R. Jones, Joseph P. Mizgerd, Katrina T. Traber, Lee J. Quinton
BACKGROUND. A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS. We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS. We reported an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed significant difference in response to standard of care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDOs cultures. CONCLUSION. While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION. Not applicable. FUNDING. This work was supported by the Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, CA265050, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, a University of Minnesota-Mayo Clinic Partnership grant, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).
Tara L. Hogenson, Hao Xie, William J. Phillips, Merih D. Toruner, Jenny J. Li, Isaac P. Horn, Devin J. Kennedy, Luciana L. Almada, David L. Marks, Ryan M. Carr, Murat Toruner, Ashley N. Sigafoos, Amanda N. Koenig-Kappes, Rachel L.O. Olson, Ezequiel J. Tolosa, Cheng Zhang, Hu Li, Jason D. Doles, Jonathan Bleeker, Michael T. Barrett, James H. Boyum, Benjamin R. Kipp, Amit Mahipal, Joleen M. Hubbard, Temperance J. Scheffler Hanson, Gloria M. Petersen, Surendra Dasari, Ann L. Oberg, Mark J. Truty, Rondell P. Graham, Michael J. Levy, Mojun Zhu, Daniel D. Billadeau, Alex A. Adjei, Nelson Dusetti, Juan L. Iovanna, Tanios S. Bekaii-Saab, Wen Wee Ma, Martin E. Fernandez-Zapico
Rest has long been considered beneficial to patient healing, yet remarkably there are no evidence-based experimental models determining how it benefits disease outcomes. Here, we create a novel experimental rest model in mice that briefly extends the morning rest period. We found, in two different major cardiovascular disease conditions (cardiac hypertrophy, myocardial infarction), that imposing a short, extended period of morning rest each day limits cardiac remodeling, as compared to controls. Mechanistically, rest mitigates autonomic-mediated hemodynamic stress on the cardiovascular system, relaxes myofilament contractility, attenuates cardiac remodeling genes, consistent with the benefits on cardiac structure and function. These same rest-responsive gene pathways underlie the pathophysiology of many major human cardiovascular conditions, as demonstrated by interrogating open-source transcriptomic data, and thus patients with other conditions may also benefit from a morning rest period in a similar manner. Our findings implicate rest as a key driver of physiology, creating an entirely new field – as broad and important as diet, sleep, or exercise – and provide a strong rationale for investigation of rest-based therapy for major clinical diseases.
Cristine J. Reitz, Mina Rasouli, Faisal J. Alibhai, Tarak Nath Khatua, W. Glen Pyle, Tami A. Martino
BRD4 is a bromodomain extra-terminal domain (BET) family member and functions primarily as a chromatin reader regulating genes involved in cell fate decisions. Here we bred Brd4f/fOx40-Cre mice in which Brd4 was conditionally deleted in OX40-expressing cells to examine the role of BRD4 in regulating immune responses. We found that the Brd4f/fOx40-Cre mice developed profound alopecia and dermatitis while other organs and tissues were not affected. Surprisingly, lineage-tracing experiments using the Rosa26f/f-Yfp mice identified a subset of hair follicle stem cells (HFSCs) that constitutively express OX40 and deletion of Brd4 specifically in such HFSCs resulted in cell death and a complete loss of skin hair growth. We also found that death of HFSCs triggered massive activation of the intra-dermal γδ T cells, which induced epidermal hyperplasia and dermatitis by producing the inflammatory cytokine IL-17. Interestingly, deletion of Brd4 in Foxp3+ Tregs, which also constitutively express OX40, compromised their suppressive functions and this in turn contributed to the enhanced activation of γδ T cells as well as the severity of dermatitis and hair follicle destruction. Thus, our data demonstrate an unexpected role of BRD4 in regulating skin follicle stem cells and skin inflammation.
Mou Wen, Yuanlin Ying, Xiang Xiao, Preston R. Arnold, Guangchuan Wang, Xiufeng Chu, Rafik M. Ghobrial, Xian C. Li
Pancreatic ductal adenocarcinoma (PDA) remains resistant to immune therapies, largely due to robustly fibrotic and immunosuppressive tumor microenvironments. It has been postulated that excessive accumulation of immunosuppressive myeloid cells influences immunotherapy resistance and recent studies targeting macrophages in combination with checkpoint blockade have demonstrated promising preclinical results. Yet, our understanding of tumor-associated macrophage (TAM) function, complexity, and diversity in PDA remains limited. Here, analysis reveals significant macrophage heterogeneity, with bone marrow-derived monocytes serving as the primary source for immunosuppressive TAMs. These cells also serve as a primary source of TNF-α, which suppresses expression of the alarmin IL33 in carcinoma cells. Deletion of Ccr2 in genetically engineered mice decreases monocyte recruitment resulting in profoundly decreased TNF-α and increased IL33 expression, decreased metastasis, and increased survival. Moreover, intervention studies targeting CCR2 with a new orthosteric inhibitor (CCX598) renders PDA susceptible to checkpoint blockade resulting in reduced metastatic burden and increased survival. Our data indicate that this shift in anti-tumor immunity is influenced by increased levels of IL-33, which increases dendritic cell and cytotoxic T cell activity. These data demonstrate that interventions to disrupt infiltration of immunosuppressive macrophages, or their signaling, have the potential to overcome barriers to effective immunotherapeutics for PDA.
Ajay Dixit, Aaron L. Sarver, Jon Zettervall, Huocong Huang, Kexin Zheng, Rolf A. Brekken, Paolo Provenzano
Hevin/Sparcl1 is an astrocyte-secreted protein and regulates synapse formation. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared to wild-type mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than wild-type mice from neuropathic pain after nerve injury. Intrathecal injection of wild-type hevin was sufficient to induce persistent mechanical allodynia in naïve mice. In hevin-null mice with nerve injury, AAV-mediated re-expression of hevin in GFAP-expressing spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin potentiated NMDA currents mediated by the GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing antibody against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal antibody may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.
Gang Chen, Jing Xu, Hao Luo, Xin Luo, Sandeep K. Singh, Juan J. Ramirez, Michael L. James, Joseph P. Mathew, Miles Berger, Cagla Eroglu, Ru-Rong Ji
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