Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.
Simon P. Jochems, Karin de Ruiter, Carla Solórzano, Astrid Voskamp, Elena Mitsi, Elissavet Nikolaou, Beatriz F. Carniel, Sherin Pojar, Esther L. German, Jesús Reiné, Alessandra Soares-Schanoski, Helen Hill, Rachel Robinson, Angela D. Hyder-Wright, Caroline M. Weight, Pascal F. Durrenberger, Robert S. Heyderman, Stephen B. Gordon, Hermelijn H. Smits, Britta C. Urban, Jamie Rylance, Andrea M. Collins, Mark D. Wilkie, Lepa Lazarova, Samuel C. Leong, Maria Yazdanbakhsh, Daniela M. Ferreira
The stimulator of interferon genes (STING) signaling pathway is a critical link between innate and adaptive immunity, and induces anti-tumor immune responses. STING is expressed in vasculatures, but its role in tumor angiogenesis has not been elucidated. Here we investigated STING-induced tumor vascular remodeling and the potential of STING-based combination immunotherapy. Endothelial STING expression was correlated with enhanced T-cell infiltration and prolonged survival in human colon and breast cancer. Intratumoral STING activation with STING agonists (cGAMP or RR-CDA) normalized tumor vasculatures in implanted and spontaneous cancers, but not in STING-deficient mice. These were mediated by upregulation of type I/II interferon genes and vascular stabilizing genes (e.g., Angpt1, Pdgfrb, and Col4a). STING in non-hematopoietic cells is as important as STING in hematopoietic cells to induce a maximal therapeutic efficacy of exogenous STING agonist. Vascular normalizing effects of STING agonists were dependent on type I interferon signaling and CD8+ T cells. Notably, STING-based immunotherapy was maximally effective when combined with VEGFR2 blockade and/or immune checkpoint blockade (αPD-1 or αCTLA-4), leading to complete regression of immunotherapy-resistant tumors. Our data show that intratumoral STING activation can normalize tumor vasculature and the tumor microenvironment, providing a rationale for combining STING-based immunotherapy and anti-angiogenic therapy.
Hannah Yang, Won Suk Lee, So Jung Kong, Chang Gon Kim, Joo Hoon Kim, Sei Kyung Chang, Sewha Kim, Gwangil Kim, Hong Jae Chon, Chan Kim
3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in three genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5β, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5β. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism.
Pu Wang, Feng Yan, Zhijun Li, Yanbao Yu, Scott E. Parnell, Yue Xiong
Genetic susceptibility to type 2 diabetes is primarily due to β-cell dysfunction. However, a genetic study to directly interrogate β-cell function ex vivo has never been previously performed. We isolated 233,447 islets from 483 Diversity Outbred (DO) mice maintained on a Western-style diet, and measured insulin secretion in response to a variety of secretagogues. Insulin secretion from DO islets ranged >1,000-fold even though none of the mice were diabetic. The insulin secretory response to each secretagogue had a unique genetic architecture; some of the loci were specific for one condition, whereas others overlapped. Human loci that are syntenic to many of the insulin secretion QTL from mouse are associated with diabetes-related SNPs in human genome-wide association studies. We report on three genes, Ptpn18, Hunk and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These three genes encode a non-receptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Krϋppel-type zinc-finger transcription factor, respectively. Our results demonstrate that genetic variation in insulin secretion that can lead to type 2 diabetes is discoverable in non-diabetic individuals.
Mark P. Keller, Mary E. Rabaglia, Kathryn L. Schueler, Donnie S. Stapleton, Daniel M. Gatti, Matthew Vincent, Kelly A. Mitok, Ziyue Wang, Takanao Ishimura, Shane P. Simonett, Christopher H. Emfinger, Rahul Das, Tim Beck, Christina Kendziorski, Karl W. Broman, Brian S. Yandell, Gary A. Churchill, Alan D. Attie
Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1–6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased β-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.
Daisuke Yasuda, Daiki Kobayashi, Noriyuki Akahoshi, Takayo Ohto-Nakanishi, Kazuaki Yoshioka, Yoh Takuwa, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii
Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates interferon γ (IFNγ)-JAK-STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration and cancer immunity. IFNγ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased TH1-type chemokines and tumor-infiltrating lymphocytes (TILs) and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and TILs, enabling tumors to evade anti-tumor immunity and to resist anti-PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its co-factor, ascorbate/vitamin C, increased chemokine and TILs, leading to enhanced anti-tumor immunity and anti-PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFNγ-JAK-STAT-TET signaling pathway that mediates tumor response to anti-PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy and patient response to anti-PD-1/PD-L1 therapy, and stimulating TET activity as an adjuvant immunotherapy of solid tumors.
Yan-ping Xu, Lei Lv, Ying Liu, Matthew D. Smith, Wen-Cai Li, Xian-ming Tan, Meng Cheng, Zhijun Li, Michael Bovino, Jeffrey Aubé, Yue Xiong
Prostate cancer (PC) is initially dependent on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to suppress AR activity serve as the primary intervention for advanced disease. However, supraphysiological androgen (SPA) concentrations can produce paradoxical responses leading to PC growth inhibition. We sought to discern the mechanisms by which SPA inhibits PC and to determine if molecular context associates with anti-tumor activity. SPA produced an AR-mediated, dose-dependent induction of DNA double-strand breaks (DSBs), G0/G1 cell cycle arrest and cellular senescence. SPA repressed genes involved in DNA repair and delayed the restoration of damaged DNA which was augmented by PARP1 inhibition. SPA-induced DSBs were accentuated in BRCA2-deficient PCs, and combining SPA with PARP or DNA-PKcs inhibition further repressed growth. Next-generation sequencing was performed on biospecimens from PC patients receiving SPA as part of ongoing Phase II clinical trials. Patients with mutations in genes mediating homology-directed DNA repair were more likely to exhibit clinical responses to SPA. These results provide a mechanistic rationale for directing SPA therapy to PCs with AR amplification or DNA repair deficiency, and for combining SPA therapy with PARP inhibition.
Payel Chatterjee, Michael T. Schweizer, Jared M. Lucas, Ilsa Coleman, Michael D. Nyquist, Sander B. Frank, Robin Tharakan, Elahe Mostaghel, Jun Luo, Colin C. Pritchard, Hung-Ming Lam, Eva Corey, Emmanuel S. Antonarakis, Samuel R. Denmeade, Peter S. Nelson
Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.
Jenny E. Kanter, Baohai Shao, Farah Kramer, Shelley Barnhart, Masami Shimizu-Albergine, Tomas Vaisar, Mark J. Graham, Rosanne M. Crooke, Clarence R. Manuel, Rebecca A. Haeusler, Daniel Mar, Karol Bomsztyk, John E. Hokanson, Gregory L. Kinney, Janet K. Snell-Bergeon, Jay W. Heinecke, Karin E. Bornfeldt
Persistent, unresolved inflammation in the liver represents a key trigger for hepatic injury and fibrosis in various liver diseases and is controlled by classically activated pro-inflammatory macrophages, while restorative macrophages of the liver are capable of reversing inflammation once the injury trigger ceases. Here we have identified a novel role for neutrophils as key contributors to resolving the inflammatory response in the liver. Using two models of liver inflammatory resolution, we found that mice undergoing neutrophil depletion during the resolution phase exhibited unresolved hepatic inflammation, activation of the fibrogenic machinery and early fibrosis. These findings were associated with an impairment of the phenotypic switch of pro-inflammatory macrophages into a restorative stage after removal of the cause of injury and an increased NLRP3 / miR-223 ratio. Mice with a deletion of the granulocyte specific miR-223 gene showed a similarly impaired resolution profile that could be reversed by restoring miR-223 levels using a miR-223 3p mimic or infusing neutrophils from wildtype animals. Collectively, our findings reveal a novel role for neutrophils in the liver as resolving effector cells that induce pro-inflammatory macrophages into a restorative phenotype, potentially via miR-223.
Carolina Jimenez Calvente, Masahiko Tameda, Casey D. Johnson, Hana del Pilar, Yun Chin Lin, Nektaria Andronikou, Xavier De Mollerat Du Jeu, Cristina Llorente, Josh Boyer, Ariel E. Feldstein
Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRβ+ CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11-deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRβ was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRβ expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using five CAF subpopulations (one murine, four human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, integrin α11 pro-invasive activity relies on its ability to interact with PDGFRβ in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a pro-invasive matricellular protein. Pharmacological inhibition of PDGFRβ and JNK impaired tumor cell invasion induced by integrin α11-positive CAFs. Collectively, our study uncovers an integrin α11-positive subset of pro-tumoral CAFs that exploits PDGFRβ/JNK signalling axis to promote tumor invasiveness in BC.
Irina Primac, Erik Maquoi, Silvia Blacher, Ritva Heljasvaara, Jan Van Deun, Hilde Y. H. Smeland, Annalisa Canale, Thomas Louis, Linda Stuhr, Nor Eddine Sounni, Didier Cataldo, Taina Pihlajaniemi, Christel Pequeux, Olivier De Wever, Donald Gullberg, Agnès Noel
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