Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent red blood cells (sRBC). Macrophages are also essential in defense against microbial threats, but pathologic states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype following intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, as K. pneumoniae mutant lacking siderophore function recapitulated findings observed with wildtype strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and interferon-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulates the STAT1 pathway with implications in severe infection.
Tolani F. Olonisakin, Tomeka L. Suber, Shekina Gonzalez-Ferrer, Zeyu Xiong, Hernán F. Peñaloza, Rick van der Geest, Yuting Xiong, David O. Osei-Hwedieh, Jesus Tejero, Matthew R. Rosengart, Wendy M. Mars, Daria Van Tyne, Andreas Perlegas, Samuel Brashears, Daniel B. Kim-Shapiro, Mark T. Gladwin, Michael A. Bachman, Eldad A. Hod, Claudette St. Croix, Yulia Y. Tyurina, Valerian E. Kagan, Rama K. Mallampalli, Anuradha Ray, Prabir Ray, Janet S. Lee
RNA binding protein Apobec1 Complementation Factor (A1CF) regulates posttranscriptional ApoB mRNA editing but the range of RNA targets and long-term impact of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf transgenic (A1cf +/Tg), A1cf+/Tg Apobec1–/– and A1cf –/– mice fed chow or high fat/high fructose diets using RNA-Seq, RNA-CLIP Seq and tissue microarrays from human hepatocellular cancer (HCC). A1cf +/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf +/Tg mice developed spontaneous fibrosis, dysplasia and HCC, which was accelerated on a high fat/fructose diet and independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), proliferation (Kif20a, Mcm2, Mcm4, Mcm6) with a subset of mRNAs (including Sox4, Sox9, Cdh1) identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative and inflammatory pathways leading to HCC.
Valerie Blanc, Jesse D. Riordan, Saeed Soleymanjahi, Joseph Nadeau, ILKe Nalbantoglu, Yan Xie, Elizabeth A. Molitor, Blair B. Madison, Elizabeth M. Brunt, Jason C. Mills, Deborah C. Rubin, Irene O.L. Ng, Yeonjung Ha, Lewis R. Roberts, Nicholas O. Davidson
CD4+ T cells interactions with B cells play a critical role in the pathogenesis of systemic autoimmune diseases such as systemic lupus and chronic graft-versus-host disease (cGVHD). Extrafollicular CD44hiCD62LloPSGL1loCD4+ (PSGL1loCD4+) T cells are associated with the pathogenesis of lupus and cGVHD, but their causal role has not been established. With murine and humanized MHC–/–HLA-A2+DR4+ murine models of cGVHD, we show that both murine and human PSGL1loCD4+ T cells from GVHD target tissues have features of B cell helpers with upregulated-expression of PD1 and ICOS and production of IL-21. They reside in non-lymphoid tissues without circulating in the blood and have features of tissue-resident memory T cells with upregulated-expression of CD69. Murine PSGL1loCD4+ T cells from GVHD target tissues augmented B cell differentiation into plasma cells and production of autoantibodies via their PD1 interaction with PD-L2 on B cells. Human PSGL1loCD4+ T cells were apposed with memory B cells in the liver tissues of humanized mice and cGVHD patients. Human PSGL1loCD4+ T cells from humanized GVHD target tissues also augmented autologous memory B cell differentiation into plasma cells and antibody production in PD1/PD-L2-dependent manner. Further preclinical studies targeting tissue-resident T cells to treat antibody-mediated features of autoimmune diseases are warranted.
Xiaohui Kong, Deye Zeng, Xiwei Wu, Bixin Wang, Shijie Yang, Qingxiao Song, Yongping Zhu, Martha Salas, Hanjun Qin, Ubaydah Nasri, Karen M. Haas, Arthur D. Riggs, Ryotaro Nakamura, Paul J. Martin, Aimin Huang, Defu Zeng
Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kD (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45/myosin IIA-containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a-dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8+ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55/G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.
Xiaochao Tan, Lei Shi, Priyam Banerjee, Xin Liu, Hou-Fu Guo, Jiang Yu, Neus Bota-Rabassedas, B. Leticia Rodriguez, Don L. Gibbons, William K. Russell, Chad J. Creighton, Jonathan M. Kurie
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the relationship between anti-spike ectodomain (ECD), anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two in vitro assays using convalescent plasma samples from 68 COVID-19 patients. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titer. The probability of a VN titer ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2,814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers ≥1:160, all of which had anti-RBD titer ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.
Eric Salazar, Suresh V. Kuchipudi, Paul A. Christensen, Todd Eagar, Xin Yi, Picheng Zhao, Zhicheng Jin, S. Wesley Long, Randall J. Olsen, Jian Chen, Brian Castillo, Christopher Leveque, Dalton Towers, Jason J. Lavinder, Jimmy Gollihar, Jose A. Cardona, Gregory C. Ippolito, Ruth H. Nissly, Ian Bird, Denver Greenawalt, Randall M. Rossi, Abhinay Gontu, Sreenidhi Srinivasan, Indira Poojary, Isabella M. Cattadori, Peter Hudson, Nicole M. Josleyn, Laura Prugar, Kathleen E. Huie, Andrew S. Herbert, David W. Bernard, John M. Dye, Vivek Kapur, James M. Musser
Homeostasis of bone metabolism is regulated by the central nervous system and mood disorders such as anxiety are associated with bone metabolism abnormalities, yet our understanding of the central neural circuits regulating bone metabolism is limited. Here, we demonstrate that chronic stress in crewmembers resulted in decreased bone density and elevated anxiety in an isolated habitat mimicking a space station. We then used a mouse model to demonstrate that GABAergic neural circuitry in the ventromedial hypothalamus (VMH) mediates chronic stress-induced bone loss. We show that GABAergic inputs in the VMHdm arise from a specific group of somatostatin neurons in the posterior region of bed nucleus of the stria terminalis (BNST), which is indispensable for stress-induced bone loss and is able to trigger bone loss in the absence of stressors. In addition, the sympathetic system and glutamatergic neurons in nucleus tractus solitaries (NTS) were employed to regulate stress-induced bone loss. Our study has therefore identified the central neural mechanism by which chronic stress induced mood disorders, such as anxiety, influence bone metabolism.
Fan Yang, Yunhui Liu, Shanping Chen, Zhongquan Dai, Dazhi Yang, Dashuang Gao, Jie Shao, Yuyao Wang, Ting Wang, Zhijian Zhang, Lu Zhang, William W. Lu, Yinghui Li, Liping Wang
Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from sickle cell disease (SCD) patients induces fetal hemoglobin (HbF) without detectable toxicity as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited HSPCs in four non-human primates. We utilized a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer or control locus (AAVS1) targeted cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Non-homologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we find that edited HSCs can persist for at least 101 weeks post-transplant, and biallelic edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.
Selami Demirci, Jing Zeng, Yuxuan Wu, Naoya Uchida, Anne H. Shen, Danilo Pellin, Jackson Gamer, Morgan Yapundich, Claire Drysdale, Jasmine Bonanno, Aylin C. Bonifacino, Allen Krouse, Nathaniel Seth Linde, Theresa Engels, Robert E. Donahue, Juan J. Haro-Mora, Alexis Leonard, Tina Nassehi, Kevin Luk, Shaina N. Porter, Cicera R. Lazzarotto, Shengdar Q. Tsai, Mitchell Weiss, Shondra M. Pruett-Miller, Scot A. Wolfe, Daniel E. Bauer, John F. Tisdale
Chronic viral infections are often established by the exploitation of immune regulatory mechanisms that result in non-functional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase (SphK) 2 generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2’s role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.
Caleb J. Studstill, Curtis J. Pritzl, Young-Jin Seo, Dae Young Kim, Chuan Xia, Jennifer J. Wolf, Ravi Nistala, Madhuvanthi Vijayan, Yong-Bin Cho, Kyung Won Kang, Sang-Myeong Lee, Bumsuk Hahm
Regulatory T (Treg) cells require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Treg cells persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg cell lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator Uhrf1 is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet non-uniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg cell lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg cell genome for both lineage establishment and stability of identity and suppressive function.
Kathryn A. Helmin, Luisa Morales-Nebreda, Manuel A. Torres Acosta, Kishore R. Anekalla, Shang-Yang Chen, Hiam Abdala-Valencia, Yuliya Politanska, Paul Cheresh, Mahzad Akbarpour, Elizabeth M. Steinert, Samuel E. Weinberg, Benjamin D. Singer
Germ cell tumors (GCTs) are the most common cancer in men between the ages of 15-40. While most patients are cured, those with disease arising in the mediastinum have distinctly poor outcomes. One in every 17 patients with primary mediastinal non-seminomatous GCTs develop an incurable hematologic malignancy and prior data intriguingly suggests a clonal relationship exists between hematologic malignancies and GCTs in these cases. To date however, the precise clonal relationship between GCTs and the diverse additional somatic malignancies arising in such individuals has not been determined. Here, we traced the clonal evolution and characterized the genetic features of each neoplasm from a cohort of fifteen patients with GCTs and associated hematologic malignancies. We discovered that GCTs and hematologic malignancies developing in such individuals evolved from a common shared precursor, nearly all of which harbored allelically imbalanced TP53 and/or RAS pathway mutations. Hematologic malignancies arising in this setting genetically resembled mediastinal GCTs rather than de novo myeloid neoplasms. Our findings argue that this scenario represents a unique clinical syndrome, distinct from de novo GCTs or hematologic malignancies, initiated by an ancestral precursor which gives rise to the parallel evolution of GCTs and blood cancers in these patients.
Justin Taylor, Mark T.A. Donoghue, Caleb Ho, Kseniya Petrova-Drus, Hikmat A. Al-Ahmadie, Samuel A. Funt, Yanming Zhang, Umut Aypar, Pavitra Nagesh Rao, Shweta S. Chavan, Michael Haddadin, Roni Tamari, Sergio Giralt, Martin S. Tallman, Raajit K. Rampal, Priscilla Baez, Rajya Kappagantula, Satyajit Kosuri, Ahmet Dogan, Satish K. Tickoo, Victor E. Reuter, George J. Bosl, Christine A. Iacobuzio-Donahue, David B. Solit, Barry S. Taylor, Darren R. Feldman, Omar Abdel-Wahab
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