IL-6 in the infarcted heart is preferentially formed by fibroblasts and is modulated by purinergic signaling

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Abstract

Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 and how its production is regulated is largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. IL-6, measured in various cell types in post MI hearts by qPCR, RNAscope and at protein level, was preferentially formed by fibroblasts (CFs). scRNAseq in infarcted mouse and human hearts confirmed this finding. Adenosine stimulated fibroblast IL-6 formation via A2bR in a Gq-dependent manner. CFs highly expressed Adora2b, rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans Adora2B was also mainly expressed by fibroblasts (scRNAseq). Global IL-6 formation was assessed in isolated hearts in mice lacking CD73 on T-cells (CD4CD73-/-) a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4CD73-/- mice, suggesting adenosine-mediated modulation. Together this demonstrates that post-MI IL-6 is mainly derived from activated CFs and is controlled by T-cell derived adenosine. Purinergic metabolic cooperation between CFs and T-cells is a novel mechanism with therapeutic potential which modulates IL6 formation by the heart.

Authors

Christina Alter, Anne Sophie Henseler, Christoph Owenier, Julia Hesse, Zhaoping Ding, Tobias Lautwein, Jasmin Bahr, Sikander Hayat, Rafael Kramann, Eva Kostenis, Jürgen Scheller, Jürgen Schrader

Kras oncogene ablation prevents resistance in advanced lung adenocarcinoma

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Abstract

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited anti-tumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS signaling pathways. Instead, sotorasib resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.

Authors

Marina Salmón, Ruth Álvarez-Díaz, Coral Fustero-Torre, Oksana Brehey, Carmen G. Lechuga, Manuel Sanclemente, Fernando Fernández-García, Alejandra López-García, María Carmen Martín-Guijarro, Sandra Rodríguez-Perales, Emily Bousquet-Mur, Lucía Morales-Cacho, Francisca Mulero, Fátima Al-Shahrour, Lola Martínez, Orlando Domínguez, Eduardo Caleiras, Sagrario Ortega, Carmen Guerra, Monica Musteanu, Matthias Drosten, Mariano Barbacid

Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction

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Abstract

CRISPR-Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ leading to decreased plasmid stability secondary to induction of the cytidine deaminase APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies of inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR-Cas9 therapeutic approaches.

Authors

Mrinal K. Sarkar, Ranjitha Uppala, Chang Zeng, Allison C. Billi, Lam C. Tsoi, Austin Kidder, Xianying Xing, Bethany E. Perez White, Shuai Shao, Olesya Plazyo, Sirisha Sirobhushanam, Enze Xing, Yanyun Jiang, Katherine A. Gallagher, John J. Voorhees, J. Michelle Kahlenberg, Johann E. Gudjonsson

RIP140 deficiency enhances cardiac fuel metabolism and protects mice from heart failure

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Abstract

During development of heart failure, capacity for cardiomyocyte fatty acid oxidation (FAO) and ATP production is progressively diminished contributing to pathologic cardiac hypertrophy and contractile dysfunction. Receptor interacting protein 140 (RIP140; Nrip1) has been shown to function as a transcriptional co-repressor of oxidative metabolism. We found that mice with striated muscle deficiency of RIP140 (strNrip1-/-) exhibit increased expression of a broad array of genes involved in mitochondrial energy metabolism and contractile function in heart and skeletal muscle. strNrip1-/- mice were resistant to the development of pressure overload-induced cardiac hypertrophy, and cardiomyocyte-specific RIP140 deficient (csNrip1-/-) mice were defended against development of heart failure caused by pressure overload combined with myocardial infarction. Genomic enhancers activated by RIP140 deficiency in cardiomyocytes were enriched in binding motifs for transcriptional regulators of mitochondrial function (estrogen-related receptor) and cardiac contractile proteins (myocyte enhancer factor 2). Consistent with a role in the control of cardiac fatty acid oxidation, loss of RIP140 in heart resulted in augmented triacylglyceride turnover and FA utilization. We conclude that RIP140 functions as a suppressor of a transcriptional regulatory network that controls cardiac fuel metabolism and contractile function, representing a potential therapeutic target for heart failure.

Authors

Tsunehisa Yamamoto, Santosh K. Maurya, Elizabeth Pruzinsky, Kirill Batmanov, Yang Xiao, Sarah M. Sulon, Tomoya Sakamoto, Yang Wang, Ling Lai, Kendra S. McDaid, Swapnil V. Shewale, Teresa C. Leone, Timothy R. Koves, Deborah M. Muoio, Pieterjan Dierickx, Mitchell A. Lazar, E. Douglas Lewandowski, Daniel P. Kelly

Persistent fasting lipogenesis links impaired ketogenesis with citrate synthesis in humans with non-alcoholic fatty liver

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Abstract

BACKGROUND. Hepatic de novo lipogenesis (DNL) and β-oxidation are tightly coordinated, and their dysregulation is thought to contribute to the pathogenesis of non-alcoholic fatty liver (NAFL). Fasting normally relaxes DNL-mediated inhibition of hepatic β-oxidation, dramatically increasing ketogenesis and decreasing reliance on the TCA cycle. Thus, we tested whether aberrant oxidative metabolism in fasting NAFL subjects is related to the inability to halt fasting DNL. METHODS. Forty consecutive non-diabetic individuals with and without a history of NAFL were recruited for this observational study. After phenotyping, subjects fasted for 24-hr, and hepatic metabolism was interrogated using a combination of 2H2O and 13C tracers, magnetic resonance spectroscopy, and high-resolution mass spectrometry. RESULTS. Within a subset of subjects, DNL was detectable after a 24-hr fast and was more prominent in those with NAFL, though it was poorly correlated with steatosis. However, fasting DNL negatively correlated with hepatic β-oxidation and ketogenesis and positively correlated with citrate synthesis. Subjects with NAFL but undetectable fasting DNL (25th percentile) were comparatively normal. However, those with the highest fasting DNL (75th percentile) were intransigent to the effects of fasting on the concentration of insulin, NEFA, and ketones. Additionally, they sustained glycogenolysis and spared the loss of oxaloacetate to gluconeogenesis in favor of citrate synthesis, which correlated with DNL and diminished ketogenesis. CONCLUSION. Metabolic flux analysis in fasted subjects indicates that shared metabolic mechanisms link the dysregulations of hepatic DNL, ketogenesis, and the TCA cycle in NAFL. TRIAL REGISTRATION. Data obtained during the enrollment/non-intervention phase of Effect of Vitamin E on Non-Alcoholic Fatty Liver Disease; ClinicalTrials.gov NCT02690792.

Authors

Xiaorong Fu, Justin A. Fletcher, Stanisław Deja, Melissa Inigo-Vollmer, Shawn C. Burgess, Jeffrey D. Browning

Intrinsic RIG-I restrains STAT5 activation to modulate anti-tumor activity of CD8⁺ T cells

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Abstract

Anti-tumor activity of CD8+ T cells is potentially restrained by a variety of negative regulatory pathways that are triggered in tumor microenvironment, yet exact mechanisms remain incompletely defined. Here we report that intrinsic RIG-I in CD8+ T cells represents such a factor, as evidenced by observations that tumor-restricting effect of endogenous or adoptively transferred CD8+ T cells was enhanced by intrinsic Rig-I deficiency or inhibition, with the increased accumulation, survival, and cytotoxicity of tumor-infiltrating CD8+ T cells. Mechanistically, T cell activation-induced RIG-I upregulation restrained STAT5 activation via competitively sequestering HSP90. In accordance, the frequency of RIG-I+ tumor-infiltrating CD8+ T cells in human colon cancer positively correlated with attenuated survival and effector signatures of CD8+ T cells as well as poor prognosis. Collectively, these results implicate RIG-I as a potentially druggable factor for improving CD8+ T cells-based tumor immunotherapy.

Authors

Xinyi Jiang, Jian Lin, Chengfang Shangguan, Xiaoyao Wang, Bin Xiang, Juan Chen, Hezhou Guo, Wu Zhang, Jun Zhang, Yan Shi, Jiang Zhu, Hui Yang

RB1-deficient prostate tumor growth and metastasis are vulnerable to ferroptosis induction via the E2F/ACSL4 axis

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Abstract

Inactivation of the RB1 tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies against RB1-deficient cancers, however, remain elusive. Here we showed that RB1-loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression of ACSL4 and enriching ACSL4-dependent arachidonic acid-containing phospholipids, which are key components of ferroptosis execution. ACSL4 appeared to be a direct E2F target gene and was critical to RB1 loss-induced sensitization to ferroptosis. Importantly, using cell line-derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blocked RB1-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis, and also suggest a promising approach for the treatment of RB1-deficient malignancies.

Authors

Mu-En Wang, Jiaqi Chen, Yi Lu, Alyssa R. Bawcom, Jinjin Wu, Jianhong Ou, John M. Asara, Andrew J. Armstrong, Qianben Wang, Lei Li, Yuzhuo Wang, Jiaoti Huang, Ming Chen

Aurora A kinase inhibition elevates PD-L1 expression and compromises its anti-tumor efficacy

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Abstract

Aurora A plays a critical role in G2/M transition and mitosis, making it an attractive target for cancer treatment. Aurora A inhibitors showed remarkable antitumor effects in preclinical studies, but unsatisfactory outcomes in clinical trials have greatly limited their development. In this study, the Aurora A inhibitor alisertib upregulated PD-L1 expression in a panel of tumor cells both in vitro and in vivo. The upregulation of the checkpoint protein PD-L1 reduced antitumor immunity in immune-competent mice, paradoxically inhibiting the antitumor effects of alisertib. Mechanistically, Aurora A directly bound to and phosphorylated cGAS, suppressing PD-L1 expression in tumor cells. Aurora A inhibition by alisertib activated the cGAS-STING-NF-κB pathway and promoted PD-L1 expression. Combining alisertib with anti-PD-L1 antibody improved antitumor immunity and enhanced antitumor effects of alisertib in immune-competent mice. Our results revealed the immunomodulatory functions of Aurora A inhibitors，which provide a plausible explanation for the poor clinical outcomes of Aurora A inhibitors in clinical settings and suggest a potential approach to improve their anti-tumor efficacy.

Authors

Xiaobo Wang, Jing Huang, Fenglin Liu, Qian Yu, Ruina Wang, Jiaqi Wang, Zewen Zhu, Juan Yu, Jun Hou, Joong Sup Shim, Wei Jiang, Zengxia Li, Yuanyuan Zhang, Yongjun Dang

Human IAPP is a contributor to painful diabetic peripheral neuropathy

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Abstract

Peripheral neuropathy is a frequent complication of type 2 diabetes mellitus (T2DM). We investigated whether human islet amyloid polypeptide (hIAPP), which forms pathogenic aggregates that damage pancreatic islet β-cells in T2DM, is involved in T2DM-associated peripheral neuropathy. In vitro, hIAPP incubation with sensory neurons reduced neurite outgrowth and increased levels of mitochondrial reactive oxygen species. Transgenic hIAPP mice that have elevated plasma hIAPP levels without hyperglycemia developed peripheral neuropathy as evidenced by pain-associated behavior and reduced intra-epidermal nerve fiber (IENF) density. Similarly, hIAPP Ob/Ob mice that have hyperglycaemia in combination with elevated plasma hIAPP levels had signs of neuropathy, although more aggravated.In wild-type mice, intraplantar and intravenous hIAPP injections induced long-lasting allodynia and decreased IENF density. Non-aggregating murine IAPP, mutated hIAPP (Pramlintide), or hIAPP with pharmacologically inhibited aggregation did not induce these effects. T2DM patients had reduced IENF density and more hIAPP oligomers in the skin compared to non-T2DM controls. Thus, we provide evidence that hIAPP aggregation is neurotoxic and mediates peripheral neuropathy in mice. The increased abundance of hIAPP aggregates in the skin of T2DM patients supports the notion that hIAPP is a potential contributor to T2DM neuropathy in humans.

Authors

Mohammed M.H. Albariqi, Sabine Versteeg, Elisabeth M. Brakkee, J. Henk Coert, Barend O.W. Elenbaas, Judith Prado, C. Erik Hack, Jo W.M. Höppener, Niels Eijkelkamp

Elevated prelimbic cortex-to-basolateral amygdala circuit activity mediates comorbid anxiety-like behaviors in chronic pain

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Abstract

Chronic pain could cause both hyperalgesia and anxiety symptoms. How the two components are encoded in the brain remains unclear. The prelimbic cortex (PrL), a critical brain region for both nociceptive and emotional modulations, serves as an ideal medium for comparing the encoding of the two components. We report that PrL neurons projecting to the basolateral amygdala (PrLBLA) and those projecting to the ventrolateral periaqueductal gray (PrLl/vlPAG) were segregated and displayed elevated and reduced neuronal activity, respectively, during pain chronicity. Consistently, optogenetic suppression of PrL→BLA circuit reversed anxiety-like behaviors whereas activation of PrL→l/vlPAG circuit attenuated hyperalgesia in mice with chronic pain. Moreover, mechanistic studies indicated that elevated TNF-α/TNFR1 signaling in PrL caused increased insertion of GluA1 receptors into PrLBLA neurons contributing to anxiety-like behaviors in mice with chronic pain. Together, these results provide insights into the circuit and molecular mechanisms in PrL for controlling pain-related hyperalgesia and anxiety-like behaviors.

Authors

Feng Gao, Jie Huang, Guo-Bin Huang, Qiang-Long You, Shan Yao, Shen-Ting Zhao, Jian Liu, Cui-Hong Wu, Gui-Fu Chen, Shi-Min Liu, Zongyan Yu, Yan-Ling Zhou, Yu-Ping Ning, Shenquan Liu, Bing-Jie Hu, Xiang-Dong Sun