During cutaneous tick attachment, the feeding cavity becomes a site of transmission for tick salivary compounds and tick-borne pathogens. However, the immunological consequences of tick feeding on human skin remain unclear. Here, we assessed human skin and blood samples upon tick bite and developed a human skin explant model mimicking Ixodes ricinus bite and tick-borne pathogen infection. Following tick attachment, we observed rapidly occurring patterns of immunomodulation including increase in neutrophils and cutaneous B and T cells. T cells up-regulated tissue-residency markers, while lymphocytic cytokine production was impaired. In early stages of Borrelia burgdorferi model infections, we detected strain-specific immune responses and close spatial relationships of macrophages and spirochetes. Pre-incubation of spirochetes with tick salivary gland extracts hampered accumulation of immune cells and increased spirochete loads. Collectively, we showed that tick feeding exerts profound changes on the skin immune network, which interfere with the primary response against tick-borne pathogens.
Johanna Strobl, Verena Muendler, Sophie Müller, Anna Gindl, Sara Berent, Anna-Margarita Schötta, Lisa Kleissl, Clement Staud, Anna Redl, Luisa Unterluggauer, Ana Elena Aguilar González, Sophie Therese Weninger, Denise Atzmüller, Romana Klasinc, Gerold Stanek, Mateusz Markowicz, Hannes Stockinger, Georg Stary
Fusion oncoproteins are the initiating event in the pathogenesis of many pediatric AML. The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem/progenitor cells (CB HSPCs) in an endothelial cell (EC) co-culture system, that recapitulates the transcriptome, morphology and immunophenotype of C/G AML and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, FOLR1, and demonstrated their pre-clinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.
Quy Le, Brandon Hadland, Jenny L. Smith, Amanda Leonti, Benjamin J. Huang, Rhonda Ries, Tiffany A. Hylkema, Sommer Castro, Thao T. Tang, Cyd N. McKay, LaKeisha Perkins, Laura Pardo, Jay Sarthy, Amy K. Beckman, Robin Williams, Rhonda Idemmili, Scott Furlan, Takashi Ishida, Lindsey Call, Shivani Srivastava, Anisha M. Loeb, Filippo Milano, Suzan Imren, Shelli M. Morris, Fiona Pakiam, James M. Olson, Michael R. Loken, Lisa Eidenschink Brodersen, Stanley R. Riddell, Katherine Tarlock, Irwin D. Bernstein, Keith R. Loeb, Soheil Meshinchi
Astrocytes are highly heterogenic in their phenotype and function, which contribute to CNS disease, repair and aging; however, the molecular mechanism of their functional states remains largely unknown. Here we show that activation of sirtuin 1 (SIRT1), a protein deacetylase, plays an important role in the detrimental actions of reactive astrocytes, whereas its inactivation endorsed these cells with anti-inflammatory functions that inhibited the production of proinflammatory mediators by myeloid cells/microglia and promoted the differentiation of oligodendrocyte progenitor cells. Mice with astrocyte-specific Sirt1 knockout had suppressed progression of experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammatory demyelinating diseases. Ongoing EAE was also suppressed when Sirt1 expression in astrocytes was diminished by CRISPR/Cas vector, resulting in reduced demyelination, decreased numbers of T cells, and increased rate of IL-10-producing macrophages/microglia in the CNS, whereas peripheral immune response remained unaffected. Mechanistically, Sirt1-/- astrocytes expressed a range of nuclear factor erythroid-derived 2-like 2 (Nfe2l2) target genes, and Nfe2l2 deficiency shifted the beneficial action of Sirt1-/- astrocytes to a detrimental one. These findings identify a novel approach for switching functional state of reactive astrocytes and facilitate the development of astrocyte-targeting therapies for inflammatory neurodegenerative diseases such as multiple sclerosis.
Weifeng Zhang, Dan Xiao, Xing Li, Yuan Zhang, Javad Rasouli, Giacomo Casella, Alexandra Boehm, Daniel Hwang, Larissa L.W. Ishikawa, Rodolfo Thome, Bogoljub Ciric, Mark T. Curtis, Abdolmohamad Rostami, Guang-Xian Zhang
Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we showed that hyperoxia-induced experimental BPD in newborn mice led to life-long impairments in cerebrovascular structure and function, as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing non-human primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Altogether, our findings established a relationship between BPD and abnormal neurodevelopmental outcomes and identified molecular and cellular players of neonatal brain injury that persist throughout adulthood, that may be targeted for early intervention to aid this vulnerable patient population.
Marissa A. Lithopoulos, Xavier Toussay, Shumei Zhong, Liqun Xu, Shamimunisa B. Mustafa, Julie Ouellette, Moises Freitas-Andrade, Cesar C. Comin, Hayam A. Bassam, Adam N. Baker, Yiren Sun, Michael Wakem, Alvaro G. Moreira, Cynthia L. Blanco, Arul Vadivel, Catherine Tsilfidis, Steven R. Seidner, Ruth S. Slack, Diane C. Lagace, Jing Wang, Baptiste Lacoste, Bernard Thébaud
22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations; thymus hypoplasia, hypoparathyroidism and/or cardiac defects. Thymus hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymus hypoplasia in 22q11.2DS remain unknown. Comparing embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed similar proportions of mesenchymal-, epithelial- and hematopoietic- cell types as controls. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymus mesenchymal cells with normal ones restored tissue growth. Comparative single cell RNA sequencing of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 line. Transcripts impacted include extracellular matrix (ECM) proteins, consistent with increased collagen deposition seen in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymus tissue expansion for hypoplastic lobes. In colony forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, contrasting the normal growth of thymus epithelial cells. These findings suggest that mesenchymal cells are causal to the small embryonic thymuses in 22q11.2DS mouse models, correctable by substituting with normal mesenchyme.
Pratibha Bhalla, Qiumei Du, Ashwani Kumar, Chao Xing, Angela Moses, Igor Dozmorov, Christian A. Wysocki, Ondine B. Cleaver, Timothy J. Pirolli, Mary Louise Markert, M. Teresa de la Morena, Antonio Baldini, Nicolai S.C. van Oers
A fundamental issue in regenerative medicine is whether there exist endogenous regulatory mechanisms that limit the speed and efficiency of the repair process. We report the existence of a maturation checkpoint during muscle regeneration which pauses myofibers at a neonatal stage. This checkpoint is regulated by the mitochondrial protein mitofusin 2 (Mfn2), whose expression is activated in response to muscle injury. Mfn2 is required for growth and maturation of regenerating myofibers; in the absence of Mfn2, new myofibers arrested at a neonatal stage, characterized by centrally nucleated myofibers and loss of H3K27me3 repressive marks at the neonatal myosin heavy chain gene. A similar arrest at the neonatal stage was observed in infantile cases of human centronuclear myopathy. Mechanistically, Mfn2 upregulation suppressed expression of Hypoxia-induced Factor 1α (Hif1α), which is induced in the setting of muscle damage. Sustained Hif1α signaling blocked maturation of new myofibers at the neonatal-to-adult fate transition, revealing the existence of a checkpoint that delays muscle regeneration. Correspondingly, inhibition of Hif1α allowed myofibers to bypass the checkpoint, thereby accelerating the repair process. We conclude that skeletal muscle contains a regenerative checkpoint which regulates the speed of myofiber maturation in response to Mitofusin 2 and Hif1α activity.
Xun Wang, Yuemeng Jia, Jiawei Zhao, Nicholas P. Lesner, Cameron J. Menezes, Spencer D. Shelton, Siva Sai Krishna Venigalla, Jian Xu, Chunyu Cai, Prashant Mishra
Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation leads to downregulation of the DNA repair pathways. This leads to enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG leads to accumulation of extrachromosomal DNA, which activates the cGAS-STING pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier permeable PARP inhibitor, pamiparib, and the cell cycle checkpoint CHK1/2 inhibitor, AZD7762), and these combinations resulted in approximately 50% long-term survivors. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induce immune-mediated therapeutic efficacy in G34-mutant pHGG.
Santiago Haase, Kaushik Banerjee, Anzar A. Mujeeb, Carson S. Hartlage, Fernando M. Nunez, Felipe J. Nuñez, Mahmoud S. Alghamri, Padma Kadiyala, Stephen Carney, Marcus Barissi, Ayman W. Taher, Emily K. Brumley, Sarah Thompson, Justin T. Dreyer, Caitlin T. Alindogan, Maria B. Garcia-Fabiani, Andrea Comba, Sriram Venneti, Visweswaran Ravikumar, Carl Koschmann, Angel M. Carcaboso, Maria Vinci, Arvind Rao, Jennifer S. Yu, Pedro R. Lowenstein, Maria G. Castro
Cell surface receptors, ligands and adhesion molecules underlie development, circuit formation and synaptic function of the central nervous system and represent important therapeutic targets for many neuropathologies. The functional contributions of interactions between cell surface proteins of neurons and non-neuronal cells have not been fully addressed. Using an unbiased protein-protein interaction screen, we showed that the human immunomodulatory ligand, B7-1 (hB7-1), interacts with the p75 neurotrophin receptor (p75NTR), and that the B7-1:p75NTR interaction is a recent evolutionary adaptation present in humans and other primates, but absent in mice, rats, and other lower mammals. The surface of hB7-1 that engages p75NTR overlapped with the hB7-1 surface involved in CTLA-4/CD28 recognition, and these molecules directly competed for binding to p75NTR. Soluble or membrane bound hB7-1 altered dendritic morphology of cultured hippocampal neurons, with loss of the postsynaptic protein PSD95 in a p75NTR-dependent manner. Abatacept, an FDA-approved therapeutic (CTLA-4-hFc fusion) inhibited these processes. In vivo injection of hB7-1 into the murine subiculum, a hippocampal region affected in Alzheimer’s Disease, resulted in p75NTR-dependent pruning of dendritic spines. Collectively, we have reported the biochemical interaction between B7-1 and p75NTR, described biological effects on neuronal morphology, and identified a therapeutic opportunity for treatment of neuroinflammatory diseases.
Nicholas C. Morano, Roshelle S. Smith, Victor Danelon, Ryan Schreiner, Uttsav Patel, Natalia G. Herrera, Carla Smith, Steven M. Olson, Michelle K. Laerke, Alev Celikgil, Scott J. Garforth, Sarah C. Garrett-Thomson, Francis S. Lee, Barbara L. Hempstead, Steven C. Almo
A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under non-hemophilic conditions was maintained by programmed death (PD) ligand 1 (PD-L1)-expressing regulatory T cells (Treg) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Treg and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1+ Tregs and re-engaged removal of inhibitor-forming B cells. We demonstrated the existence of FVIII-specific Tregs also in humans and showed that such Tregs upregulated PD-L1 after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1-mediated B cell tolerance against FVIII operated in healthy individuals and in hemophilia A patients without inhibitors, and that ITI re-engaged this mechanism. These findings may impact monitoring of ITI success and treatment of hemophilia A patients.
Janine Becker-Gotot, Mirjam Meissner, Vadim Kotov, Blanca Jurado-Mestre, Andrea Maione, Andreas Pannek, Thilo Albert, Chrystel Flores, Frank A. Schildberg, Paul A. Gleeson, Birgit M. Reipert, Johannes Oldenburg, Christian Kurts
The stomach corpus epithelium is organized into anatomical units that consist of glands and pits and contain different specialized secretory cells. Acute and chronic injury of the corpus are associated with characteristic changes of cellular differentiation and proliferation. Processes that control cellular differentiation under homeostatic conditions and upon injury are not well understood. R-spondin 3 (Rspo3) is a Wnt signalling enhancer secreted by gastric stromal cells, which controls stem cell homeostasis in different organs. Here we investigated the function of Rspo3 in the corpus during homeostasis, acute injury, and H. pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland towards parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells – caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneraton was maintained long-term, promoing severe glandular hyperproliferation and the development of premalignant metaplasia.
Anne-Sophie Fischer, Stefanie Müllerke, Alexander Arnold, Julian Heuberger, Hilmar Berger, Manqiang Lin, Hans-Joachim Mollenkopf, Jonas Wizenty, David Horst, Frank Tacke, Michael Sigal
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