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Abstract
Secondary lymphedema is characterized by fibrosis and impaired lymphatic function. Although TGF-β is a key regulator of fibrosis in this disease, the cellular mechanisms regulating this process remain unknown. Epithelial–mesenchymal transition (EMT), a mechanism by which TGF-β induces fibrosis in other skin diseases, is characterized by loss of epithelial cell markers and cellular polarity, upregulation of fibrotic gene expression, and gain of migratory capacity. Using clinical lymphedema biopsy specimens and animal models, we show that keratinocytes in the basal layer of the epidermis undergo EMT in lymphedematous skin, migrate into the dermis, and contribute to dermal fibrosis. In vitro studies using cultured primary human keratinocytes treated with lymphatic fluid from the affected limbs of patients with secondary lymphedema resulted in a TGF-β–mediated increased expression of EMT markers. We show for the first time that EMT is activated by TGF-β in secondary lymphedema and that this process plays an important role in regulating skin fibrosis in this disease.
Authors
Hyeung Ju Park, Jinyeon Shin, Ananta Sarker, Mark G. Klang, Elyn Riedel, Michelle Coriddi, Joseph H. Dayan, Sarit Pal, Babak J. Mehrara, Raghu P. Kataru
Abstract
Background & Aims Liver cirrhosis is characterized by chronic inflammation and fibrosis, with Th17 cells playing a crucial role in its progression. Recent evidence suggests that dietary salt influences immune diseases by modulating Th17 differentiation. This study assessed the impact of dietary salt on Th17-driven inflammation in patients with compensated cirrhosis and explored its effects on liver injury in mouse models. Methods A non-drug, open-label, non-randomized study involved 37 patients with compensated cirrhosis, who were given personalized guidelines to reduce salt intake over three months. Changes in Th17-driven inflammation and liver function markers were assessed at baseline and after salt restriction. In parallel, the impact of a high-salt diet on hepatic CD4+ T cells was analyzed in mouse models of acute liver injury and fibrosis. Results High salt intake was associated with Th17-mediated inflammation and correlated with markers of impaired liver function in these patients. Importantly, moderating salt intake through a personalized nutritional intervention was sufficient to reduce CD4+ T cell- mediated inflammation. Furthermore, analysis of RNA-seq data revealed enrichment of salt-induced Th17 gene signatures in both liver tissue and peripheral cells from patients with liver disease. Similarly, mice fed a high salt diet showed hepatic enrichment of Th17 cells and exacerbated liver fibrosis upon injury. Mechanistic studies revealed that high sodium conditions activated NF-κB and induced IL-6 production in hepatocytes, which may promote Th17 responses. Conclusion Dietary salt exacerbates Th17-driven inflammation and contributes to cirrhosis progression. Salt reduction may represent a viable therapeutic approach to manage inflammation in compensated cirrhosis.
Authors
Amalia Tzoumpa, Beatriz Lozano-Ruiz, Yin Huang, Joanna Picó, Alba Moratalla, María Teresa Pomares, Iván Herrera, Juanjo Lozano, María Rodríguez-Soler, Cayetano Miralles, Pablo Bellot, Paula Piñero, Fabián Tarín, Pedro Zapater, Sonia Pascual, José Manuel González-Navajas
Abstract
Heterozygosity for missense mutations in one of 3 seemingly redundant calmodulin (CALM)-encoding genes can cause life-threatening arrhythmias, suggesting that small fractions of mutant CALM protein suffice to cause a severe phenotype. However, the exact molar ratios of wildtype to mutant CALM protein in calmodulinopathy hearts remain unknown. The aim of the present study was to quantitate mutant versus wildtype CALM transcript and protein levels in hearts of knock-in mice harboring the p.N98S mutation in the Calm1 gene. We found that the transcripts from the mutant Calm1 allele were the least abundantly expressed Calm transcripts in both hetero- and homozygous mutant hearts, while mutant hearts accumulate high levels of N98S-CALM protein in a Calm1N98S allele dosage-dependent manner, exceeding those of wildtype CALM protein. We further show that the severity of the electrophysiological phenotype incrementally increases with the graded increase in the mutant-to-wildtype CALM protein expression ratio seen in homozygous versus heterozygous mutant mice. We finally show a decrease in N98S-CALM protein degradation, suggesting that mutant CALM stabilization contributed to its enrichment in the heart. Our results support what we believe to be a novel mechanism by which a mutation in a single Calm gene can give rise to a severe phenotype.
Authors
Wen-Chin Tsai, Chiu-Fen Yang, Shu-Yu Lin, Suh-Yuen Liang, Wei-Chung Tsai, Shuai Guo, Xiaochun Li, Susan Ofner, Kai-Chien Yang, Tzu-Ching Meng, Peng-Sheng Chen, Michael Rubart
Abstract
Multidrug-resistant (MDR) bacterial pneumonias pose a critical threat to global public health. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is a leading cause of nosocomial-associated pneumonia, and an effective vaccine could protect vulnerable populations, including the elderly, immunocompromised, and those with chronic respiratory diseases. Highly heterogeneous outer membrane vesicles (OMVs), shed from Gram-negative bacteria, are studded with immunogenic lipids, proteins, and virulence factors. To overcome limitations in OMV stability and consistency, we described a believed to be novel vaccine platform that combines immunogenic OMVs with precision nanotechnology—creating a bacterial cellular nanoparticle vaccine candidate (CNP), termed Pa-STING-CNP, which incorporates an adjuvanted core that activates the STING (stimulator of interferon genes) pathway. In this design, OMVs are coated onto the surface of self-adjuvanted STING nanocores. Pa-STING CNP vaccination induced substantial antigen presenting cell recruitment and activation in draining lymph nodes, robust anti-Pseudomonas antibody responses, and provided protection against lethal challenge with the hypervirulent clinical P. aeruginosa isolate PA14. Antibody responses mediated this protection and provided passive immunity against the heterologous P. aeruginosa strain PA01. These findings provided evidence that nanotechnology can be used to create a highly efficacious vaccine platform against high priority MDR pathogens such as P. aeruginosa.
Authors
Elisabet Bjånes, Nishta Krishnan, Truman Koh, Anh T.P. Ngo, Jason Cole, Joshua Olson, Ingrid Cornax, Chih-Ho Chen, Natalie Chavarria, Samira Dahesh, Shawn M. Hannah, Alexandra Stream, Jiaqi Amber Zhang, Hervé Besançon, Daniel Sun, Siri Yendluri, Sydney Morrill, Jiarong Zhou, Animesh Mohapatra, Ronnie H. Fang, Victor Nizet
Abstract
More than one third of patients with glioblastoma experience tumour progression during adjuvant therapy. In this study, we performed a high-throughput drug repurposing screen of FDA-approved agents capable of crossing the blood-brain barrier that to find agents to counteract acquired or inherent glioma cell resistance to temozolomide-associated cytotoxicity. We identified the cholesterol processing inhibitor, lomitapide, as a potential chemosensitizer in glioblastoma. In vitro treatment of temozolomide-resistant glioblastoma cells with lomitapide resulted in decreased intracellular ubiquinone levels and sensitized cells to temozolomide-induced ferroptosis. Concomitant treatment with lomitapide and temozolomide (TMZ) prolonged survival and delayed tumour recurrence in a mouse glioblastoma model, compared to treatment with TMZ alone. Our data identified lomitapide as a potential adjunct for treatment of temozolomide-resistant glioblastoma.
Authors
Alyona Ivanova, Taylor M. Wilson, Kimia Ghannad-Zadeh, Esmond Tse, Robert Flick, Megan Wu, Sunit Das
Abstract
Parathyroid hormone (PTH) regulates serum calcium and phosphate through its actions in bone and the kidney and is used to increase bone in osteoporosis treatment. In bone, PTH targets osteoblasts and osteocytes to regulate bone remodeling but also bone marrow stromal cells (BMSCs), regulating their differentiation in the osteoblast and/or the adipocyte lineages. PTH exerts its action through the PTH/PTH-related peptide (PTHrP) receptor (PTH1R), a G protein-coupled receptor (GPCR), activating adenylyl cyclase and phospholipase C (PLC). Although the effects of cAMP and PKA are well characterized, little is known about the effects of PLC activation or on the cross-talk between PTH signaling and other pathways. Here, bulk RNA-seq of PTH-treated murine BMSC line (W-20) revealed significant changes in the Hippo pathway. PTH stabilized YAP, a key target of Hippo, by decreasing YAP/LATS1 interaction, YAPS127 phosphorylation and YAP ubiquitination, leading to YAP nuclear translocation and expression of YAP target genes. Similar events occurred in osteocyte cell lines. This occurred via an increase in Src kinase activity: we identified YAPY428 as a key tyrosine residue phosphorylated by Src in response to PTH. Preventing YAP428 phosphorylation led to YAP instability, blocking both osteogenic and adipogenic differentiation of W-20 cells. These results demonstrate active crosstalk between the PTH/PTHrP and the Hippo signaling pathways and reveal that PTH signaling utilizes the PLC-Ca2+-Src tyrosine kinase signaling cascade to influence YAP stability, antagonizing Hippo signaling and favoring stromal cell differentiation. Thus, PTH signaling counteracts the effects of Hippo signaling in BMSCs to favor their differentiation.
Authors
Sara Monaci, Mengrui Wu, Hiroyuki Okada, Kedkanya Mesil, Byeong-Rak Keum, Maisa Monseff Rodrigues da Silva, Clifford J. Rosen, Francesca Gori, Roland Baron
Abstract
Malignancies increase the risk for thrombosis and metastasis dependent on complex interactions of innate immune cells, platelets, and the coagulation system. Immunosuppressive functions of platelets and macrophage-derived coagulation factors in the tumor microenvironment (TME) drive tumor growth. Here we show that patients with malignancies and tumor-bearing mice have increased levels of coagulation factor (F) X expressing circulating monocytes engaged in platelet aggregate formation. This interaction and resulting thrombin generation on platelets interferes with monocyte differentiation and antigen uptake of antigen-presenting cells (APCs). Myeloid cell-specific deletion of FX or abrogated FXa signaling via protease activated receptor 2 (PAR2) averts the suppressive activity of platelets on tumor cell debris uptake and promotes the immune stimulatory activity of APCs in the TME. Myeloid cell FXa-PAR2 signaling deficiency specifically enhances activation of the cGAS-STING-IFN-I pathway with a resulting expansion of antigen experienced progenitor exhausted CD8+ T cells. Pharmacological blockade of FXa with direct oral anticoagulants expands T cell priming-competent immune cells in the TME and synergizes with the reactivation of exhausted CD8+ T cells by immune checkpoint inhibitors for improved anti-tumor responses. These data provide mechanistic insights into the emerging clinical evidence demonstrating the translational potential of FXa inhibition to synergize with immunotherapy.
Authors
Petra Wilgenbus, Jennifer Pott, Sven Pagel, Claudius Witzler, Jennifer Royce, Federico Marini, Sabine Reyda, Thati Madhusudhan, Thomas Kindler, Anne Hausen, Matthias M. Gaida, Hartmut Weiler, Wolfram Ruf, Claudine Graf
Abstract
RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL) and frequently co-occurs with chromosome 13q deletion (del(13q)). This combination is associated with poor prognosis in CLL, suggesting these lesions increase CLL aggressiveness. While del(13q) in murine B cells (Mdr mice), but not expression of Sf3b1-K700E, drives the initiation of CLL, we hypothesize that SF3B1 mutation accelerates CLL progression. In this study, we crossed mice with a B-cell-specific Sf3b1-K700E allele with Mdr mice to determine the impact of Sf3b1 mutation on CLL progression. We found that the co-occurrence of these two lesions in murine B cells caused acceleration of CLL. We showed that Sf3b1-K700E impacted alternative RNA splicing of Nfatc1 and activated mTOR signaling and the MYC pathway, contributing to CLL acceleration. Moreover, concurrent inhibition of RNA splicing and mTOR pathways led to cell death in vitro and in vivo in murine CLL cells with SF3B1 mutation and del(13q). Our results thus suggest that SF3B1 mutation contributes to the aggressiveness of CLL by activating the mTOR pathway through alternative splicing of Nfatc1, providing a rationale for targeting mTOR and RNA splicing in the subset of CLL patients with both SF3B1 mutations and del(13q).
Authors
Bo Zhang, Prajish Iyer, Meiling Jin, Elisa ten Hacken, Zachary Cartun, Kevyn L. Hart, Mike Fernandez, Kristen Stevenson, Laura Rassenti, Emanuela M. Ghia, Thomas J. Kipps, Donna Neuberg, Ruben Carrasco, Wing Chan, Joo Y. Song, Yu Hu, Catherine Wu, Lili Wang
Abstract
Psoriasis is a chronic autoimmune skin disease characterized by abnormal keratinocyte proliferation and immune dysregulation. Altered lipid metabolism has been implicated in its pathogenesis, but the underlying mechanisms remain unclear. In this study, we generated an keratinocyte-specific Sprouty RTK signaling antagonist 1 (SPRY1) knockout (Spry1ΔEpi) mouse model, which exhibits psoriasis-like symptoms. Using both psoriasis patient samples and Spry1ΔEpi mice, we investigated the role of diacylglycerol acyltransferase 2 (DGAT2) in psoriasis. Our results show that DGAT2 expression is reduced, and glycerides metabolism is disrupted in psoriatic lesions in both psoriasis patients and Spry1ΔEpi mice. Lipidomic analysis reveals significant alterations in glycerides, glycerophospholipids, sphingolipids, and fatty acids in Spry1ΔEpi mice. At the cellular level, DGAT2 downregulation and lipid dysregulation enhance Toll-like receptor 3 (TLR3)-mediated inflammatory signaling in keratinocytes. Furthermore, increased DGAT2 secretion from keratinocytes promotes CD8⁺ T cell activation, proliferation and survival, amplifying psoriatic inflammation. These findings highlight the role of DGAT2 and lipid metabolism in the pathogenesis of psoriasis and reveal their interaction with immune responses in psoriasis.
Authors
Ying-Ying Li, Li-Ran Ye, Ying-Zhe Cui, Fan Xu, Xi-Bei Chen, Feng-Fei Zhang, Yi Lu, Yu-Xin Zheng, Xiao-Yong Man
Abstract
Hematopoietic stem cell transplantation (HCT) is a potentially life-saving therapy but can lead to lung injury due to chemoradiation toxicity, infection, and immune dysregulation. We previously showed that bronchoalveolar lavage (BAL) transcriptomes representing pulmonary inflammation and cellular injury can phenotype post-HCT lung injury and predict mortality. To test whether peripheral blood might be a suitable surrogate for BAL, we compared 210 paired BAL and blood transcriptomes obtained from 166 pediatric HCT patients at 27 hospitals. BAL and blood RNA abundance showed minimal correlation at the level of individual genes, gene set enrichment scores, imputed cell fractions, and T- and B-cell receptor clonotypes. Instead, we identified significant site-specific transcriptional programs. In BAL, pathways related to immunity, hypoxia, and epithelial mesenchymal transition were tightly co-expressed and linked to mortality. In contrast, in blood, expression of endothelial injury, DNA repair, and cellular metabolism pathways was associated with mortality. Integration of paired BAL and blood transcriptomes dichotomized patients into two groups with significantly different rates of hypoxia and clinical outcomes within 1 week of BAL. These findings reveal a compartmentalized injury response, where BAL and blood transcriptomes provide distinct but complementary insights into local and systemic mechanisms of post-HCT lung injury.
Authors
Emma M. Pearce, Erica Evans, Madeline Y. Mayday, Gustavo Reyes, Miriam R. Simon, Jacob Blum, Hanna Kim, Jessica Mu, Peter J. Shaw, Courtney M. Rowan, Jeffery J. Auletta, Paul L. Martin, Caitlin Hurley, Erin M. Kreml, Muna Qayed, Hisham Abdel-Azim, Amy K. Keating, Geoffrey D.E. Cuvelier, Janet R. Hume, James S. Killinger, Kamar Godder, Rabi Hanna, Christine N. Duncan, Troy C. Quigg, Paul Castillo, Nahal R. Lalefar, Julie C. Fitzgerald, Kris M. Mahadeo, Prakash Satwani, Theodore B. Moore, Benjamin Hanisch, Aly Abdel-Mageed, Dereck B. Davis, Michelle P. Hudspeth, Greg A. Yanik, Michael A. Pulsipher, Christopher C. Dvorak, Joseph L. DeRisi, Matt S. Zinter
Chronic integrated stress response causes dysregulated cholesterol synthesis in white matter disease
Abstract
Maladaptive integrated stress response (ISR) activation is observed in human diseases of the brain. Genetic mutations of eIF2B, a critical mediator of protein synthesis, cause chronic pathway activation resulting in a leukodystrophy but the precise mechanism is unknown. We generated N208Y eIF2Bα mice and found that this metabolite binding mutation leads to destabilization of eIF2Bα, a systemic ISR, and neonatal lethality. 2BAct, an eIF2B activator, rescued lethality and significantly extended the lifespan of this severe model, underscoring its therapeutic potential in pediatric disease. Continuous treatment was required for survival, as withdrawal led to ISR induction in all tissues and rapid deterioration, thereby providing a model to assess the impact of the ISR in vivo by tuning drug availability. Single nuclei RNA-sequencing of the CNS identified astrocytes, oligodendrocytes, and ependymal cells as the cell types most susceptible to eIF2B dysfunction and revealed dysfunctional maturation of oligodendrocytes. Moreover, ISR activation decreased cholesterol biosynthesis, a process critical for myelin formation and maintenance. As such, persistent ISR engagement may contribute to pathology in other demyelinating diseases.
Authors
Karin Lin, Nina Ly, Rejani B. Kunjamma, Ngoc Vu, Bryan King, Holly M. Robb, Eric G. Mohler, Janani Sridar, Qi Hao, José Zavala-Solorio, Chunlian Zhang, Varahram Shahryari, Nick van Bruggen, Caitlin F. Connelly, Bryson D. Bennett, James J. Lee, Carmela Sidrauski
Abstract
X-linked Lymphoproliferative Syndromes (XLP), arising from mutations in SH2D1A or XIAP genes, are characterized by fulminant Epstein-Barr Virus (EBV) infection. Lymphomas occur frequently in XLP-1 and in other congenital conditions with heightened EBV susceptibility, but not in XLP-2. Why XLP-2 patients are apparently protected from EBV-driven lymphomagenesis remains a key open question. To gain insights, newly EBV-infected versus receptor-stimulated primary B-cells from XLP-2 patients or with XIAP CRISPR editing were compared to healthy controls. XIAP perturbation impeded outgrowth of newly EBV-infected B-cells, but not that of CD40 ligand and interleukin-21 stimulated B-cells. XLP-2 deficient B-cells showed significantly lower EBV transformation efficiency than healthy controls. Interestingly, EBV-immortalized lymphoblastoid cell proliferation was not impaired by XIAP knockout, implicating an XIAP role in early EBV B-cell transformation. Mechanistically, nascent EBV infection activated p53-mediated apoptosis signaling, which was counteracted by XIAP in control cells. With XIAP deficiency, EBV markedly elevated apoptosis rates over the first two weeks of infection. Interferon-gamma, whose levels are increased with severe XLP2 EBV infection, markedly increased newly EBV-infected B-cell apoptosis. These findings underscored XIAP's crucial role in support of the earliest stages of EBV-mediated B-cell immortalization and provide insights into the curious absence of EBV+ lymphoma in XLP-2 patients.
Authors
Yizhe Sun, Janet Chou, Kevin D. Dong, Steven P. Gygi, Benjamin E. Gewurz
Abstract
Autophagy is a catabolic quality control pathway that has been linked to neurodegenerative disease, atherosclerosis and ageing, and can be modified by nutrient availability in preclinical models. Consequently, there is immense public interest in stimulating autophagy in people. However, progress has been hampered by the lack of techniques to measure human autophagy. As a result, several key concepts in the field, including nutritional modulation of autophagy, have yet to be validated in humans. We conducted a single arm pre-post study in 42 healthy individuals, to assess whether an acute nutritional intervention could modify autophagy in humans. Two blood samples were collected per participant: after a 12 h overnight fast and 1 h post-consumption of a high protein meal. Autophagy turnover was assessed using a physiologically relevant measure of autophagic flux in peripheral blood mononuclear cells. A lysosomal inhibitor was added directly to whole blood, with the resulting build-up of autophagy marker LC3B-II designated as flux, and measured quantitatively via ELISA. Notably, consumption of a high protein meal had no impact on autophagy, with no differences between overnight fasting and postprandial autophagic flux. We observed sexual dimorphism in autophagy, with females having higher autophagic flux compared to males (p = 0.0031). Exploratory analyses revealed sex-specific correlations between autophagy, insulin and glucose signalling. Importantly, our findings show that an acute nutritional intervention (overnight fasting followed by consumption of a protein-rich meal) does not change autophagic flux in humans, highlighting the need to conduct further autophagy studies in humans.
Authors
Sanjna Singh, Célia Fourrier, Kathryn J. Hattersley, Leanne K. Hein, Jemima Gore, Alexis Martin, Linh V.P. Dang, Barbara King, Rachael A. Protzman, Paul J. Trim, Leonie K. Heilbronn, Julien Bensalem, Timothy J. Sargeant
Abstract
Acute kidney injury (AKI) is characterized by a rapid decline in renal function. In severe or recurrent cases, AKI can progress to chronic kidney disease (CKD), marked by renal inflammation and fibrosis. Despite the severity of these outcomes, early-stage diagnostic tools and pharmacological interventions for AKI-to-CKD progression remain limited. In this study, we examined circular RNA (circRNA) expression profiles in mouse renal cortex tissues 14 days post-ischemia/reperfusion (I/R) injury using circRNA sequencing. The renal biopsy samples of patients after AKI exhibited reduced CircNSD1 expression, which was inversely associated with inflammation and fibrosis. Overexpression of CircNSD1 attenuated ferroptosis in vivo and in vitro, while slowing AKI-to-CKD progression. Mechanistically, CircNSD1 downregulated ACSL4 and SlC39A14 expression through histone H3 lysine 36 (H3K36) methylation, a critical pathway regulating ferroptosis after AKI or hypoxia/reoxygenation (H/R) injury. Furthermore, we identified that CircNSD1 encoded a NSD1-916aa peptide, which may functionally contribute to its observed effect. Collectively, these findings demonstrated that CircNSD1 may serve as a diagnostic and therapeutic target for early detection of AKI-to-CKD transition.
Authors
Li Gao, Junsheng Zhang, Chaoyi Chen, Sai Zhu, Xianglong Wei, Guiqin Tang, Sheng Wang, Yukai Wang, Xinran Liu, Ling Jiang, Yonggui Wu
Abstract
Metabolic health is influenced by adipose tissue, and obesity and lipodystrophy are characterized by inflammation and metabolic dysfunction. Whereas obesity and lipodystrophy treatments involve pharmacological approaches and lifestyle changes, these therapies require long-term, repeated dosing, and are not successful for all patients. Gene therapy with targets such as FGF21 and sTGFBR2 provides an alternative approach, specifically in lipodystrophy. Preclinical experiments in mice housed at 22°C are confounded by a mild cold stress not generally experienced by humans, which can negatively affect translation of metabolic therapeutics. In this study, we investigated effects of FGF21/sTGFBR2 combination gene therapy on obese and lipodystrophic mice, and how housing temperature influences therapeutic efficacy. In obese mice, FGF21/sTGFBR2 improved insulin resistance and hyperlipidemia more dramatically at warmer temperatures. In lipodystrophic mice on a high fat diet, combination therapy required adipose tissue to improve insulin resistance at 30°C, whereas FGF21 alone improved insulin resistance at 22°C. Transcriptomic analyses revealed that lipodystrophic mice had upregulated hepatic cell proliferation and fibrosis pathways, and that FGF21 promoted hepatic metabolism. Thus, metabolic dysfunction caused by lipodystrophy is improved by targeting FGF21 and TGFB signaling, but effectiveness in preclinical models may be dependent upon environmental temperature and presence of adipose tissue.
Authors
Jessica N. Maung, Yang Chen, Keegan S. Hoose, Rose E. Adler, Hadla Hariri, Mia J. Dickson, Taryn A. Hetrick, Gabriel A. Ferguson, Rebecca L. Schill, Hiroyuki Mori, Romina M. Uranga, Kenneth T. Lewis, Isabel D. K. Hermsmeyer, Donatella Gilio, Christopher de Solis, Amber Toliver, Noah Davidsohn, Elif A. Oral, Ormond A. MacDougald
Abstract
Uracil DNA glycosylase (UNG) excises uracil and 5-fluorouracil bases from DNA and is implicated in fluorodeoxyuridine (FdU) resistance. Here we explore the effects of inhibiting UNG activity, or depleting the UNG protein, in two mouse syngeneic models for colorectal cancer. Overexpressing the uracil DNA glycosylase inhibitor protein in mismatch repair (MMR)-deficient MC38 cells injected into C57/B6 mice delayed tumor growth and prolonged survival when combined with FdU. Combining UNG inhibition with FdU numerically increased CD4+ T lymphocytes and B cells compared to FdU or UNG inhibition alone, suggesting an immune component to the effects. In contrast, shRNA depletion of UNG in the absence of FdU treatment resulted in 70% of mice clearing their tumors, and a 3-fold increase in overall survival compared to FdU. Analysis of MC38 tumor-infiltrating immune cells showed UNG depletion increased monocyte and dendritic cell populations, with CD8+ T cells also numerically increased. shRNA depletion of UNG in MMR-proficient CT-26 cells injected into Balb/C mice produced minimal benefit; the addition of anti-PD-1 antibody synergized with UNG-depletion to increase survival. Cytotoxic T cell depletion abolished the benefits of UNG depletion in both models. These findings suggest UNG inhibition and/or depletion could enhance antitumor immune response in humans.
Authors
Eric S. Christenson, Brandon E. Smith, Thanh J. Nguyen, Alens Valentin, Soren Charmsaz, Nicole E Gross, Sarah M. Shin, Alexei Hernandez, Won Jin Ho, Srinivasan Yegnasubramanian, James T. Stivers
Abstract
Yellow Fever virus (YFV) infection is fatal in 5–10% of the 200,000 yearly cases. There is currently no available antiviral treatment. We showed previously that administration of 50 mg/kg of a YFV-specific neutralizing monoclonal antibody (nmAb) at 2 days post-infection (dpi), prior to the onset of severe disease, protected YFV-infected rhesus macaques from death. To further explore the clinical applicability of our nmAb MBL-YFV-01, we treated rhesus macaques with a lower dose (10 mg/kg) of this nmAb prophylactically or therapeutically at 3.5 dpi. We show that a single prophylactic or therapeutic intravenous dose of our nmAb protects rhesus macaques from death following challenge. A comprehensive analysis of 167 inflammatory cytokine and chemokines revealed that protection was associated with significantly reduced expression of 125 of these markers, including type I interferons, IL6, and CCL2. This study further expands the potential clinical use of our YFV-specific nmAb, which could be used during an outbreak for immediate prophylactic immunity or for patients with measurable serum viremia.
Authors
Lauren N. Rust, Michael J. Ricciardi, Savannah S. Lutz, Sofiya Yusova, Johan J. Louw, Aaron Yrizarry-Medina, Sreya Biswas, Miranda Fischer, Aaron Barber-Axthelm, Gavin Zilverberg, Lauren Bailey, Tonya Swanson, Rachael Tonelli, G.W. McElfresh, Brandon C. Rosen, Thomas B. Voigt, Christakis Panayiotou, Jack T. Mauter, Noor Ghosh, Jenna Meanor, Giovana Godoy, Michael Axthelm, Jeremy Smedley, Mark K. Slifka, Esper G. Kallas, Gabriela Webb, Robert Zweig, Caralyn S. Labriola, Benjamin N. Bimber, Jonah B. Sacha, David I. Watkins, Benjamin J. Burwitz
Abstract
The aryl hydrocarbon receptor (AhR) is proposed to mediate the frailty-promoting effects of the tryptophan metabolite kynurenine (Kyn), which increases with age in mice and humans. The goal of the current study was to test whether administration of pharmacological AhR inhibitors, BAY2416964 and CH-223191, could abrogate musculoskeletal decline in aging mice. Female C57BL/6 mice (18 months old) were treated with vehicle (VEH) or BAY2416964 (30 mg/kg) via daily oral gavage 5 days/week for 8 weeks. A second AhR antagonist, CH-223191, was administered to 16-month-old male and female C57BL/6 mice via intraperitoneal injections (3.3 mg/kg) 3 days/week for 12 weeks. While grip strength declined over time in VEH-treated mice, BAY2416964 preserved grip strength in part by improving integrity of neuromuscular junctions, an effect replicated during in vitro studies with siRNA against AhR. Cortical bone mass was also greater in BAY2416964- than VEH-treated mice. Similarly, CH-223191 treatment improved cortical bone and showed beneficial effects in skeletal muscle, including reducing oxidative stress as compared to VEH-treated animals. Transcriptomic and proteomic data from BAY2416964-treated mice supported a positive impact of BAY2416964 on molecular targets that affect neuromuscular junction function. Taken together, these data support AhR as a therapeutic target for improving musculoskeletal health during aging.
Authors
Kanglun Yu, Sagar Vyavahare, Dima W. Alhamad, Husam Bensreti, Ling Ruan, Anik Tuladhar, Caihong Dai, Joseph C. Shaver, Alok Tripathi, Kehong Ding, Rafal Pacholczyk, Marion A. Cooley, Roger Zhong, Maribeth H. Johnson, Jie Chen, Wendy B. Bollag, Carlos M. Isales, William D. Hill, Mark W. Hamrick, Sadanand Fulzele, Meghan E. McGee-Lawrence
Abstract
Tumor immunosuppression impacts survival and treatment efficacy. Tumor NOS2/COX2 coexpression strongly predicts poor outcome in ER– breast cancer by promoting metastasis, drug resistance, cancer stemness, and immune suppression. Herein, a spatially distinct NOS2/COX2 and CD3+CD8+PD1– T effector (TEff) cell landscape correlated with poor survival in ER– tumors. NOS2 was primarily expressed at the tumor margin, whereas COX2 together with B7H4 was associated with immune desert regions lacking TEff cells, where a higher ratio of tumor NOS2 or COX2 to TEff cells predicted poor survival. Also, PDL1/PD1, regulatory T cells (TReg) and IDO1 were primarily associated with stroma restricted TEff cells. Regardless of the survival outcome, CD4+ T cells and macrophages were primarily in stromal lymphoid aggregates. Finally, in a 4T1 model, COX2 inhibition led to increased CD8+ TEff/CD4+ TReg ratio and CD8+ TEff infiltration while Nos2 deficiency had no significant effect, thus reinforcing our observations that COX2 is an essential component of immunosuppression through CD8+ TEff cell exclusion from the tumor. Our study indicates that tumor NOS2/COX2 expression plays a central role in tumor immune evasion, suggesting that strategies combining clinically available NOS2/COX2 inhibitors with immune therapy could provide effective options for the treatment of aggressive and drug-resistant ER– breast tumors.
Authors
Lisa A. Ridnour, Robert Y.S. Cheng, William F. Heinz, Milind Pore, Ana L. Gonzalez, Elise L. Femino, Rebecca L. Moffat, Adelaide L. Wink, Fatima Imtiaz, Leandro L. Coutinho, Donna Butcher, Elijah F. Edmondson, M. Cristina Rangel, Stephen T.C. Wong, Stanley Lipkowitz, Sharon A. Glynn, Michael P. Vitek, Daniel W. McVicar, Xiaoxian Li, Stephen K. Anderson, Nazareno Paolocci, Stephen M. Hewitt, Stefan Ambs, Timothy R. Billiar, Jenny C. Chang, Stephen J. Lockett, David A. Wink
Abstract
Extracellular DNA (ecDNA) released from injured and dying cells powerfully induces injurious inflammation. In this study we define the role of ecDNA in systemic vasculitis affecting the kidney, using human kidney biopsies and murine models of myeloperoxidase anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (MPO-ANCA GN). Twice daily administration of intravenous DNase I (ivDNase I) in two models of anti-MPO GN reduced glomerular deposition of ecDNA, histological injury, leukocyte infiltration and NETosis. Comprehensive investigation into DNase I modes of action revealed that after exposure to MPO, DNase I reduced lymph node DC numbers and their activation status, resulting in decreased frequency of MPO-specific CD4 effector T cells (IFN-, and IL-17A producing), and reductions in dermal anti-MPO delayed type hypersensitivity responses. To overcome the translational obstacle of the short half-life of DNase I (<5 hours), we tested an adeno-associated viral vector encoding DNase I (vec-DNase I). The method of DNase I delivery was more effective, as in addition to the histological and anti-inflammatory changes described above, a single vector treatment also reduced circulating MPO-ANCA titers and albuminuria. These results indicate ecDNA is a potent driver of anti-MPO GN and that DNase I is a potential therapeutic that can be delivered using gene technology
Authors
Anne Cao Le, Virginie Oudin, Jonathan Dick, Maliha A. Alikhan, Timothy A. Gottschalk, Lu Lu, Kate E. Lawlor, Daniel Koo Yuk Cheong, Mawj Mandwie, Ian E. Alexander, A R. Kitching, Poh-Yi Gan, Grant J. Logan, Kim M. O'Sullivan
