Tumor necrosis factor receptor (TNF)-associated factor 4 (TRAF4) is an important regulator of type 2 responses in the airway; however, the underlying cellular and molecular mechanisms remain elusive. Herein, we generated T cell-specific TRAF4-deficient (CD4cre-Traf4fl/fl) mice and investigated the role of TRAF4 in interleukin (IL)-33 receptor (ST2, suppression of tumorigenicity 2)-expressing memory Th2 cells (ST2+ mTh2) in IL-33-mediated type 2 airway inflammation. We found that in vitro polarized TRAF4-deficient (CD4cre-Traf4fl/fl) ST2+ mTh2 cells exhibited decreased IL-33-induced proliferation as compared with TRAF4-sufficient (Traf4fl/fl) cells. Moreover, CD4cre-Traf4fl/fl mice showed less ST2+ mTh2 cell proliferation and eosinophilic infiltration in the lungs than Traf4fl/fl mice in the preclinical models of IL-33-mediated type 2 airway inflammation. Mechanistically, we discovered that TRAF4 was required for the activation of AKT/mTOR and ERK1/2 signaling pathways as well as the expression of transcription factor Myc and nutrient transporters (Slc2a1, Slc7a1, and Slc7a5), signature genes involved in T cell growth and proliferation, in ST2+ mTh2 cells stimulated by IL-33. Taken together, the current study reveals a previously unappreciated role of TRAF4 in ST2+ mTh2 cells in IL-33-mediated type 2 pulmonary inflammation, opening up avenues for the development of new therapeutic strategies.
Jianxin Xiao, Xing Chen, Weiwei Liu, Wen Qian, Katarzyna Bulek, Lingzi Hong, William Miller-Little, Xiaoxia Li, Caini Liu
Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in approximately 10 million US women. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyoma that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant (mut)- vs wild-type (wt)-MED12 leiomyoma. Integration of cistromic and transcriptomic data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene, which was significantly upregulated in mut-MED12 leiomyoma when compared to adjacent myometrium and wt-MED12 leiomyoma. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyoma. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2-kynurenine-AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptor regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2-kynurenine-AHR pathway selectively in mut-MED12 leiomyoma promotes tumor growth and may inform the future development of targeted treatments and precision medicine.
Azna Zuberi, Yongchao Huang, Ariel J. Dotts, Helen Wei, John S. Coon V, Shimeng Liu, Takashi Iizuka, Olivia Wu, Olivia Sotos, Priyanka Saini, Debabrata Chakravarti, Thomas G. Boyer, Yang Dai, Serdar E. Bulun, Ping Yin
Cancer stem cells (CSCs) are responsible for tumor progression and recurrence. However, the mechanisms regulating hepatocellular carcinoma (HCC) stemness remain unclear. Applying a genome-scale CRISPR knockout screen, we identify that the H3K4 methyltransferase SETD1A and other members of Trithorax group proteins drive cancer stemness in HCC. SETD1A is positively correlated with poor clinical outcome in HCC patients. Combination of SETD1A and serum AFP significantly improves the accuracy of predicting HCC relapse. Mechanistically, SETD1A mediates transcriptional activation of various histone-modifying enzymes, facilitates deposition of H3K4me3 and H3K27me3 and activates oncogenic enhancers and super-enhancers, leading to activation of oncogenes and inactivation of tumor suppressor genes simultaneously in HCC CSCs. In addition, SETD1A cooperates with PABPC1 to regulate H3K4me3 modification on oncogenes. Our data pinpoint SETD1A as a key epigenetic regulator driving HCC stemness and progression, highlighting the potential of SETD1A as a candidate target for HCC intervention and therapy.
Jianxu Chen, Zhijie Xu, Hongbin Huang, Yao Tang, Hong Shan, Fei Xiao
IL-15 is under clinical investigation towards the goal of curing HIV infection, due to its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15-mediated latency reversal and NK function, by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T-cell responses. We show that ex vivo IL-15+HODHBt treatment markedly enhances HIV-specific granzyme B-releasing T-cell responses in PBMCs from ARV-suppressed donors. We also observed upregulation of antigen processing and presentation in CD4+ T-cells, and increased surface MHC-I. In ex vivo PBMCs, IL-15+HODHBt was sufficient to reduce intact proviruses in 1 of 3 ARV-suppressed donors. Our findings reveal the potential for 2nd-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.
Dennis C. Copertino, Jr, Carissa S. Holmberg, Jared Weiler, Adam R. Ward, J. Natalie Howard, Callie Levinger, Alina P.S. Pang, Michael J. Corley, Friederike Dündar, Paul Zumbo, Doron Betel, Rajesh T. Gandhi, Deborah K. McMahon, Ronald J. Bosch, Noemi Linden, Bernard J. Macatangay, Joshua C. Cyktor, Joseph J. Eron, John W. Mellors, Colin Kovacs, Erica Benko, Alberto Bosque, R. Brad Jones
Given the resurgence of pertussis, several countries have introduced maternal tetanus, diphtheria, and acellular pertussis (aP) vaccination during pregnancy to protect young infants against severe pertussis. Although protective against the disease, the effect of maternal aP vaccination on bacterial colonization of the offspring is unknown. Here, we used a mouse model to demonstrate that maternal aP immunization, either before or during pregnancy, protects pups from lung colonization by Bordetella pertussis. However, it substantially prolongs nasal carriage by inhibiting the recruitment of IL-17-producing resident memory T cells and ensuing neutrophil influx in the nasal tissue upon B. pertussis infection, especially of those with pro-inflammatory and cytotoxic properties. Prolonged nasal carriage after aP vaccination is due to IL-4 signaling, as it is abolished in IL-4Ra-/- mice. The effect of maternal aP vaccination can be transferred transplacentally to the offspring or by breastfeeding and is long-lasting, as it persists into adulthood. Maternal aP vaccination may thus potentially augment the B. pertussis reservoir.
Violaine Dubois, Jonathan Chatagnon, Manon Depessemier, Camille Locht
Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine and spermine. These metabolites play critical roles in activation-induced T-cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eIF5A. Here, we demonstrated that the glutamine-polyamine-hypusine axis controls the expression of CD69, an important regulator of tissue resident memory T cells (TRM). Inhibition of this circuit augmented the development of TRM cells ex vivo and in vivo in the bone marrow, a well-established niche for TRM cells. Furthermore, blocking the polyamine-hypusine axis augmented CD69 expression and IFN-γ and TNF-α production in human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes, as well as in human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate TRM cells for therapeutic benefit.
Aya G. Elmarsafawi, Rebecca S. Hesterberg, Mario R. Fernandez, Chunying Yang, Lancia N.F. Darville, Min Liu, John M. Koomen, Otto Phanstiel IV, Reginald Atkins, John E. Mullinax, Shari A. Pilon-Thomas, Frederick L. Locke, Pearlie K. Epling-Burnette, John L. Cleveland
DNASE1L3, an enzyme highly expressed in dendritic cells, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of anti-tumor immunity, the foundation of modern anti-tumor immunotherapy. In this study, we identify DNASE1L3 as a new regulator of anti-tumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating dendritic cells, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired anti-tumor immunity, including a substantial reduction of cytotoxic T cells and a unique subset of dendritic cells. Consistently, Dnase1l3-deficient dendritic cells directly modulate cytotoxic T cells in vitro. Collectively, our study unveils a previously unknown link between DNASE1L3 and anti-tumor immunity, and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anti-cancer therapy.
Wenling Li, Hideki Nakano, Wei Fan, Yuanyuan Li, Payel Sil, Keiko Nakano, Fei Zhao, Peer W. Karmaus, Sara A. Grimm, Min Shi, Xin Xu, Ryushin Mizuta, Daisuke Kitamura, Yisong Wan, Michael B. Fessler, Donald N. Cook, Igor Shats, Xiaoling Li, Leping Li
Denosumab is an anti-RANKL antibody that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast number in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term but not short-term denosumab administration reduced OPG mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.
Qiang Fu, Nancy C. Bustamante-Gomez, Humberto Reyes-Pardo, Igor Gubrij, Diana Escalona-Vargas, Jeff D. Thostenson, Michela Palmieri, Joseph J. Goellner, Intawat Nookaew, C. Lowry Barnes, Jeffrey B. Stambough, Elena Ambrogini, Charles A. O'Brien
Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechano-protective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the novel integrin α8 (Itga8)-Cre mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within two weeks of knockout induction and in smaller arteries at later times. The vascular specificity of the Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA-sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS-STING pathway, were increased. A sizeable increase of SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring three days after knockout induction and before the pro-inflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.
Marycarmen Arévalo Martínez, Olivia Ritsvall, Joakim A. Bastrup, Selvi Celik, Gabriel Jakobsson, Fatima Daoud, Christopher Winqvist, Anders Aspberg, Catarina Rippe, Lars Maegdefessel, Alexandru Schiopu, Thomas A. Jepps, Johan Holmberg, Karl Swärd, Sebastian Albinsson
Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, bi-allelic variants in the highly conserved flightless-1 (FLII) gene were identified in three families with idiopathic early-onset dilated CM. We demonstrate that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, result in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provide insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identify Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM, and report bi-allelic variants as a novel genetic cause of pediatric CM.
Claudine W.B. Ruijmbeek, Filomena Housley, Hafiza Idrees, Michael P. Housley, Jenny Pestel, Leonie Keller, Jason Kuan Han Lai, Herma C. van der Linde, Rob Willemsen, Janett Piesker, Zuhair N. Al-Hassnan, Abdulrahman Almesned, Michiel Dalinghaus, Lisa M. van den Bersselaar, Marjon A. van Slegtenhorst, Federico Tessadori, Jeroen Bakkers, Tjakko J. van Ham, Didier Y.R. Stainier, Judith M.A. Verhagen, Sven Reischauer
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