The functional integrity of Treg cells is interwoven with cellular metabolism; however, the mechanisms governing Treg cell metabolic programs remain elusive. Here, we identified that the deubiquitinase USP47 inhibited RNA m6A reader YTHDF1-mediated c-Myc translation to maintain Treg cell metabolic and functional homeostasis. USP47 positively correlated with the tumor-infiltrating Treg cell signature in colorectal cancer and gastric cancer patient samples. USP47 ablation compromised Treg cell homeostasis and function in vivo, resulting in the development of inflammatory disorders, and boosted antitumor immune responses. USP47 deficiency in Treg cells triggered the accumulation of the c-Myc protein and in turn exacerbated hyperglycolysis. Mechanistically, USP47 prevented YTHDF1 ubiquitination to attenuate the association of YTHDF1 with translation initiation machinery, thereby decreasing m6A-based c-Myc translation efficiency. Our findings reveal that USP47 directs m6A-dependent metabolic programs to orchestrate Treg cell homeostasis and suggest novel approaches for selective immune modulation in cancer and autoimmune diseases by targeting USP47.
Aiting Wang, Haiyan Huang, Jian-Hong Shi, Xiaoyan Yu, Rui Ding, Yuerong Zhang, Qiaoqiao Han, Zhi-Yu Ni, Xia Li, Ren Zhao, Qiang Zou
Herpes zoster (HZ) is a substantial problem for people with decreased cell-mediated immunity, including older adults. The first vaccine approved for HZ prevention, the zoster vaccine live (ZVL), which provided limited and short-lived protection, has been supplanted by the superior recombinant zoster vaccine (RZV), which provides robust and durable protection. To understand the mechanisms underlying the differential immunologic characteristics of the two vaccines, we used T cell receptor beta sequencing and peptide-MHC class II tetramer staining to analyze gE-specific CD4+ T cell clonotypes in RZV and ZVL recipients. Compared to ZVL, RZV expanded more gE-specific CD4+ clonotypes with greater breadth and higher frequency of public clonotypes. RZV recruited a higher proportion of clonotypes from the naïve than from memory cells, while ZVL recruited equally from memory and naïve compartments. Compared to memory-, naïve-derived clonotypes were more likely to last ≥ 5 years post-immunization. Moreover, the frequency of tetramer+ persistent clones correlated with the frequency of tetramer+ naïve CD4+ T cells pre-vaccination. We conclude that the ability of RZV to recruit naive CD4+ T cells into the response may contribute to the durability of its effect. The abundance, breadth, and the frequency of public clonotypes may further add to its protective effect.
Kerry J. Laing, Emily S. Ford, Michael J. Johnson, Myron J. Levin, David M. Koelle, Adriana Weinberg
The endothelium plays a critical role in the host response to infection, and has been a focus of investigation in sepsis. While it is appreciated that intravascular thrombus formation, severe inflammation, and loss of endothelial integrity impair tissue oxygenation during sepsis, the precise molecular mechanisms that lead to endothelial injury remain poorly understood. We demonstrate herein that endothelial ADAM10 is essential for the pathogenesis of Staphylococcus aureus sepsis, contributing to a-toxin (Hla)-mediated microvascular thrombus formation and lethality. As ADAM10 is essential for endothelial development and homeostasis, we examined whether other major human sepsis pathogens also rely on ADAM10-dependent pathways in pathogenesis. Mice harboring an endothelial-specific knockout of ADAM10 are protected against lethal Pseudomonas aeruginosa and Streptococcus pneumoniae sepsis, yet remain fully susceptible to Group B Streptococci and Candida albicans sepsis. These studies illustrate a previously unknown role for ADAM10 in sepsis-associated endothelial injury, and suggest that understanding pathogen-specific divergent host pathways in sepsis may enable more precise targeting of disease.
Danielle N. Alfano, Mark J. Miller, Juliane Bubeck Wardenburg
Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying pro-tumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform to identify potential therapeutic targets to disrupt the contribution of pro-tumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC, and inform the development of therapeutics that target ECM stiffness in EAC.
Ricardo Cruz-Acuña, Secunda W. Kariuki, Kensuke Sugiura, Spyros Karaiskos, Eleanor M. Plaster, Claudia Loebel, Gizem Efe, Tatiana A. Karakasheva, Joel T. Gabre, Jianhua Hu, Jason A. Burdick, Anil K. Rustgi
Idiopathic Pulmonary Fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins, and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin (KRT) 8 genetic variants are associated with IPF. Krt8-/- mice are protected from fibrosis and accumulation of the transitional state. Keratin (K) 8 regulates expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promote accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs are highly senescent, basaloid, and do not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induce and are maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolve, whereas in human IPF, transitional AECs evolve into an aberrant basaloid state which persists with progressive fibrosis.
Fa Wang, Christopher Ting, Kent A. Riemondy, Michael T. Douglas, Kendall M. Foster, Nisha Patel, Norihito Kaku, Alexander E. Linsalata, Jean Nemzek, Brian M. Varisco, Erez Cohen, Jasmine A. Wilson, David W.H. Riches, Elizabeth F. Redente, Diana M. Toivola, Xiaofeng Zhou, Bethany B. Moore, Pierre A. Coulombe, M. Bishir Omary, Rachel L. Zemans
BACKGROUND. Macrophage activation syndrome (MAS) is a life-threatening complication of Still’s disease (SD) characterized by overt immune cell activation and cytokine storm. We aimed to further understand the immunologic landscape of SD and MAS. METHOD. We profiled peripheral blood mononuclear cells (PBMC) from healthy controls and patients with SD with or without MAS using bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq). We validated and expanded the findings by mass cytometry, flow cytometry and in vitro studies. RESULTS. Bulk RNA-seq of PBMC from patients with SD-associated MAS revealed strong expression of genes associated with type I interferon (IFN-I) signaling and cell proliferation, in addition to the expected IFN-γ signal, compared to healthy controls and SD patients without MAS. scRNA-seq analysis of > 65,000 total PBMC confirmed IFN-I and IFN-γ signatures and localized the cell proliferation signature to cycling CD38+HLA-DR+ cells within CD4+ T cell, CD8+ T cell and NK cell populations. CD38+HLA-DR+ lymphocytes exhibited prominent IFN-g production, glycolysis, and mTOR signaling. Cell-cell interaction modeling suggested a network linking CD38+HLA-DR+ lymphocytes with monocytes through IFN-γ signaling. Notably, the expansion of CD38+HLA-DR+ lymphocytes in MAS was greater than in other systemic inflammatory conditions in children. In vitro stimulation of PBMC demonstrated that IFN-I and IL-15 – both elevated in MAS patients – synergistically augmented the generation of CD38+HLA-DR+ lymphocytes, while Janus kinase inhibition mitigated this response. CONCLUSION. MAS associated with SD is characterized by overproduction of IFN-I, which may act in synergy with IL-15 to generate CD38+HLA-DR+ cycling lymphocytes that produce IFN-γ.
Zhengping Huang, Kailey E. Brodeur, Liang Chen, Yan Du, Holly Wobma, Evan E. Hsu, Meng Liu, Joyce C. Chang, Margaret H. Chang, Janet Chou, Megan Day-Lewis, Fatma Dedeoglu, Olha Halyabar, James A. Lederer, Tianwang Li, Mindy S. Lo, Meiping Lu, Esra Meidan, Jane W. Newburger, Adrienne G. Randolph, Mary Beth F. Son, Robert P. Sundel, Maria L. Taylor, Huaxiang Wu, Qing Zhou, Scott W. Canna, Kevin Wei, Lauren A. Henderson, Peter A. Nigrovic, Pui Y. Lee
Aberrant androgen receptor (AR) signalling drives prostate cancer (PC) and is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signalling and are refractory to the current repertoire of AR-targeting therapies, which together drives disease progression. There is an unmet clinical need therefore to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of co-regulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signalling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesised that DNA-PKcs is critical for AR-V function. Using a novel proximity biotinylation approach, we demonstrate that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing, and via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicates that targeting DNA-PKcs attenuates AR-V signalling and provides evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V positive PC patients.
Beth Adamson, Nicholas Brittain, Laura Walker, Ruaridh Duncan, Sara Luzzi, Pasquale Rescigno, Graham R. Smith, Suzanne McGill, Richard J.S. Burchmore, Elaine Willmore, Ian Hickson, Craig N. Robson, Denisa Bogdan, Juan M. Jimenez-Vacas, Alec Paschalis, Jonathan Welti, Wei Yuan, Stuart R. McCracken, Rakesh Heer, Adam Sharp, Johann de Bono, Luke Gaughan
Background: Proglucagon can be processed to Glucagon-Like Peptide-1 (GLP-1) within the islet but its contribution to islet function in humans remains unknown. We sought to understand whether ‘pancreatic’ GLP-1 alters islet function in humans and whether this is affected by type 2 diabetes.Methods: We therefore studied individuals with and without type 2 diabetes on 2 occasions in random order. On one occasion exendin 9-39, a competitive antagonist of the GLP-1 Receptor (GLP1R), was infused, while on the other saline was infused. The tracer dilution technique ([3-3H] glucose) was used to measure glucose turnover during fasting and during a hyperglycemic clamp.Results: Exendin 9-39 increased fasting glucose concentrations; fasting islet hormone concentrations were unchanged, but inappropriate for the higher fasting glucose observed. In people with type 2 diabetes fasting glucagon concentrations were markedly elevated and persisted despite hyperglycemia. This impaired suppression of endogenous glucose production by hyperglycemia. These data show that GLP1R blockade impairs islet function, implying that intra-islet GLP1R activation alters islet responses to glucose and does so to a greater degree in people with type 2 diabetes.
Andrew A. Welch, Rahele A. Farahani, Aoife M. Egan, Marcello C. Laurenti, Maya Zeini, Max Vella, Kent R. Bailey, Claudio Cobelli, Chiara Dalla Dalla Man, Aleksey Matveyenko, Adrian Vella
The BCL-2 inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B-cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL-2 mutations have been described, this likely only explains a subset of resistant cases. Using two complementary functional precision medicine techniques -- BH3-profiling and high throughput-kinase activity mapping -- we found that hyperphosphorylation of BCL-2 family proteins, including anti-apoptotic MCL-1 and BCL-2 and pro-apoptotic BAD and BAX, underlies functional mechanisms of both intrinsic and acquired resistance of venetoclax in CLL and DLBCL. Additionally, we provide evidence that anti-apoptotic BCL-2 family protein phosphorylation alters the apoptotic protein interactome, thereby changing the profile of functional dependence on these pro-survival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs re-wired these dependences, thus restoring sensitivity to venetoclax in a panel of venetoclax resistant lymphoid cell lines, resistant mouse model, and paired patient samples pre-venetoclax and at time of progression.
Stephen Jun Fei Chong, Fen Zhu, Olga Dashevsky, Rin Mizuno, Jolin X.H. Lai, Liam Hackett, Christine E. Ryan, Mary C. Collins, J. Bryan Iorgulescu, Romain Guièze, Johany Penailillo, Ruben Carrasco, Yeonjoo C. Hwang, Denise P. Muñoz, Mehdi Bouhaddou, Yaw Chyn Lim, Catherine J. Wu, John N. Allan, Richard R. Furman, Boon Cher Goh, Shazib Pervaiz, Jean-Philippe Coppé, Constantine S. Mitsiades, Matthew S. Davids
Monocytes and monocyte-derived macrophages (MDM) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here we show that PDGFB-driven GBM cells induce the expression of the potent pro-inflammatory cytokine IL-1β in MDM, which engages IL-1R1 in tumor cells, activates the NF-kB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1β/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1β/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, reduced exhausted CD8+ T cells, and thereby extends the survival of tumor-bearing mice. In contrast to IL-1β, IL-1α exhibits anti-tumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss of interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively-active NF-kB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1β could be considered as an effective therapy specifically for proneural GBM.
Zhihong Chen, Bruno Giotti, Milota Kaluzova, Montserrat Puigdelloses Vallcorba, Kavita Rawat, Gabrielle Price, Cameron J. Herting, Gonzalo Piñero, Simona Cristea, James L. Ross, James Ackley, Victor Maximov, Frank Szulzewsky, Wes Thomason, Mar Marquez-Ropero, Angelo Angione, Noah Nichols, Nadejda M. Tsankova, Franziska Michor, Dmitry M. Shayakhmetov, David H. Gutmann, Alexander M. Tsankov, Dolores Hambardzumyan
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