Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

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Abstract

Radiation therapy is an effective cancer treatment, although damage to healthy tissues is common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damage in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type-specific reference DNA methylation atlases. We found that cell-type-specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damage was reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from patients with breast cancer undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation, indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type-specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.

Authors

Megan E. McNamara, Netanel Loyfer, Amber J. Kiliti, Marcel O. Schmidt, Sapir Shabi-Porat, Sidharth S. Jain, Sarah Martinez Roth, A. Patrick McDeed IV, Nesreen Shahrour, Elizabeth Ballew, Yun-Tien Lin, Heng-Hong Li, Anne Deslattes Mays, Sonali Rudra, Anna T. Riegel, Keith Unger, Tommy Kaplan, Anton Wellstein

Calcineurin regulates aldosterone production via dephosphorylation of NFATC4

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Abstract

The mineralocorticoid aldosterone, secreted by the adrenal zona glomerulosa (ZG), is critical for life, maintaining ion homeostasis and blood pressure. Therapeutic inhibition of protein phosphatase 3 (calcineurin, Cn) results in inappropriately low plasma aldosterone levels despite concomitant hyperkalemia and hyperreninemia. We tested the hypothesis that Cn participates in the signal transduction pathway regulating aldosterone synthesis. Inhibition of Cn with tacrolimus abolished the potassium-stimulated (K+-stimulated) expression of aldosterone synthase, encoded by CYP11B2, in the NCI-H295R human adrenocortical cell line as well as ex vivo in mouse and human adrenal tissue. ZG-specific deletion of the regulatory Cn subunit CnB1 diminished Cyp11b2 expression in vivo and disrupted K+-mediated aldosterone synthesis. Phosphoproteomics analysis identified nuclear factor of activated T cells, cytoplasmic 4 (NFATC4), as a target for Cn-mediated dephosphorylation. Deletion of NFATC4 impaired K+-dependent stimulation of CYP11B2 expression and aldosterone production while expression of a constitutively active form of NFATC4 increased expression of CYP11B2 in NCI-H295R cells. Chromatin immunoprecipitation revealed NFATC4 directly regulated CYP11B2 expression. Thus, Cn controls aldosterone production via the Cn/NFATC4 pathway. Inhibition of Cn/NFATC4 signaling may explain low plasma aldosterone levels and hyperkalemia in patients treated with tacrolimus, and the Cn/NFATC4 pathway may provide novel molecular targets to treat primary aldosteronism.

Authors

Mesut Berber, Sining Leng, Agnieszka Wengi, Denise V. Winter, Alex Odermatt, Felix Beuschlein, Johannes Loffing, David T. Breault, David Penton

Osteoclast-derived IGF1 induces RANKL production in osteocytes and contributes to pagetic lesion formation

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Abstract

We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of patients with Paget disease (PD) or targeted to the OCL lineage in MVNP-transgenic mice (MVNP mice) increases IGF1 production in osteoclasts (OCL-IGF1) and leads to development of PD OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of MVNP mice fully blocked development of PDLs. In this study, we examined whether osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of MVNP mice expressed less sclerostin, and had increased RANKL expression compared with OCys in bones from WT mice or normal patients. To test whether increased OCL-IGF1 is sufficient to induce PDLs and PD phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to determine whether increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce PDLs and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD OCLs, PDLs, and OCys, with decreased sclerostin and increased RANKL, similar to MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD OCLs and PDLs.

Authors

Kazuaki Miyagawa, Hirofumi Tenshin, Patrick L. Mulcrone, Jesus Delgado-Calle, Mark A. Subler, Jolene J. Windle, John M. Chirgwin, G. David Roodman, Noriyoshi Kurihara

T cell activation is insufficient to drive SIV disease progression

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Abstract

Resolution of T cell activation and inflammation is a key determinant of the lack of SIV disease progression in African green monkeys (AGMs). Although frequently considered together, T cell activation occurs in response to viral stimulation of acquired immunity, while inflammation reflects innate immune responses to mucosal injury. We dissociated T cell activation from inflammation through regulatory T cell (Treg) depletion with Ontak (interleukin-2 coupled with diphtheria toxin) during early SIV infection of AGMs. This intervention abolished control of T cell immune activation beyond the transition from acute to chronic infection. Ontak had no effect on gut barrier integrity, microbial translocation, inflammation, and hypercoagulation, despite increasing T cell activation. Ontak administration increased macrophage counts yet decreased their activation. Persistent T cell activation influenced SIV pathogenesis, shifting the ramp-up in viral replication to earlier time points, prolonging the high levels of replication, and delaying CD4+ T cell restoration yet without any clinical or biological sign of disease progression in Treg-depleted AGMs. Thus, by inducing T cell activation without damaging mucosal barrier integrity, we showed that systemic T cell activation per se is not sufficient to drive disease progression, which suggests that control of systemic inflammation (likely through maintenance of gut integrity) is the key determinant of lack of disease progression in natural hosts of SIVs.

Authors

Cristian Apetrei, Thaidra Gaufin, Egidio Brocca-Cofano, Ranjit Sivanandham, Paola Sette, Tianyu He, Sindhuja Sivanandham, Natalie Martinez Sosa, Kathryn J. Martin, Kevin D. Raehtz, Adam J. Kleinman, Audrey Valentine, Noah Krampe, Rajeev Gautam, Andrew A. Lackner, Alan L. Landay, Ruy M. Ribeiro, Ivona Pandrea

Individual transcriptomic response to strength training for patients with myotonic dystrophy type 1

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Abstract

Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by a CTG expansion resulting in significant transcriptomic dysregulation that leads to muscle weakness and wasting. While strength training is clinically beneficial in DM1, molecular effects had not been studied. To determine whether training rescued transcriptomic defects, RNA-Seq was performed on vastus lateralis samples from 9 male patients with DM1 before and after a 12-week strength-training program and 6 male controls who did not undergo training. Differential gene expression and alternative splicing analysis were correlated with the one-repetition maximum strength evaluation method (leg extension, leg press, hip abduction, and squat). While training program–induced improvements in splicing were similar among most individuals, rescued splicing events varied considerably between individuals. Gene expression improvements were highly varied between individuals, and the percentage of differentially expressed genes rescued after training were strongly correlated with strength improvements. Evaluating transcriptome changes individually revealed responses to the training not evident from grouped analysis, likely due to disease heterogeneity and individual exercise response differences. Our analyses indicate that transcriptomic changes are associated with clinical outcomes in patients with DM1 undergoing training and that these changes are often specific to the individual and should be analyzed accordingly.

Authors

Emily E. Davey, Cécilia Légaré, Lori Planco, Sharon Shaughnessy, Claudia D. Lennon, Marie-Pier Roussel, Hannah K. Shorrock, Man Hung, John Douglas Cleary, Elise Duchesne, J. Andrew Berglund

Endothelial-derived FABP4 constitutes the majority of basal circulating hormone and regulates lipolysis-driven insulin secretion

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Abstract

Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with obesity and metabolic pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with Fabp4 deletion in cells known to express the gene — adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) — to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was not significantly reduced in Adipo-KO mice, whereas Endo-KO mice showed ~87% reduction versus WT controls. In contrast, Adipo-KO mice exhibited ~62% decreased induction of FABP4 responses to lipolysis, while Endo-KO mice showed only mildly decreased induction, indicating that adipocytes are the main source of increases in FABP4 during lipolysis. We did not detect any myeloid contribution to circulating FABP4. Surprisingly, despite the nearly intact induction of FABP4, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis.

Authors

Karen E. Inouye, Kacey J. Prentice, Alexandra Lee, Zeqiu B. Wang, Carla Dominguez-Gonzalez, Mu Xian Chen, Jillian K. Riveros, M. Furkan Burak, Grace Y. Lee, Gökhan S. Hotamışlıgil

Targeting a xenobiotic transporter to ameliorate vincristine-induced sensory neuropathy

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Abstract

Vincristine is a widely used chemotherapeutic drug for the treatment of multiple malignant diseases that causes a dose-limiting peripheral neurotoxicity. There is no clinically effective preventative treatment for vincristine-induced sensory peripheral neurotoxicity (VIPN), and mechanistic details of this side effect remain poorly understood. We hypothesized that VIPN is dependent on transporter-mediated vincristine accumulation in dorsal root ganglion neurons. Using a xenobiotic transporter screen, we identified OATP1B3 as a neuronal transporter regulating the uptake of vincristine. In addition, genetic or pharmacological inhibition of the murine orthologue transporter OATP1B2 protected mice from various hallmarks of VIPN — including mechanical allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes and neuronal morphology — without negatively affecting plasma levels or antitumor effects of vincristine. Finally, we identified α-tocopherol from an untargeted metabolomics analysis as a circulating endogenous biomarker of neuronal OATP1B2 function, and it could serve as a companion diagnostic to guide dose selection of OATP1B-type transport modulators given in combination with vincristine to prevent VIPN. Collectively, our findings shed light on the fundamental basis of VIPN and provide a rationale for the clinical development of transporter inhibitors to prevent this debilitating side effect.

Authors

Yang Li, Thomas Drabison, Mahesh Nepal, Richard H. Ho, Alix F. Leblanc, Alice A. Gibson, Yan Jin, Wenjian Yang, Kevin M. Huang, Muhammad Erfan Uddin, Mingqing Chen, Duncan F. DiGiacomo, Xihui Chen, Sobia Razzaq, Jeffrey R. Tonniges, Dana M. McTigue, Alice S. Mims, Maryam B. Lustberg, Yijia Wang, Amanda B. Hummon, William E. Evans, Sharyn D. Baker, Guido Cavaletti, Alex Sparreboom, Shuiying Hu

Pharmacological inhibition of TAK1 prevents and induces regression of experimental organ fibrosis

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Abstract

Multiorgan fibrosis in systemic sclerosis (SSc) accounts for substantial mortality and lacks effective therapies. Lying at the crossroad of TGF-β and TLR signaling, TGF-β–activated kinase 1 (TAK1) might have a pathogenic role in SSc. We therefore sought to evaluate the TAK1 signaling axis in patients with SSc and to investigate pharmacological TAK1 blockade using a potentially novel drug-like selective TAK1 inhibitor, HS-276. Inhibiting TAK1 abrogated TGF-β1 stimulation of collagen synthesis and myofibroblasts differentiation in healthy skin fibroblasts, and it ameliorated constitutive activation of SSc skin fibroblasts. Moreover, treatment with HS-276 prevented dermal and pulmonary fibrosis and reduced the expression of profibrotic mediators in bleomycin-treated mice. Importantly, initiating HS-276 treatment even after fibrosis was already established prevented its progression in affected organs. Together, these findings implicate TAK1 in the pathogenesis of SSc and identify targeted TAK1 inhibition using a small molecule as a potential strategy for the treatment of SSc and other fibrotic diseases.

Authors

Swarna Bale, Priyanka Verma, Bharath Yalavarthi, Scott Arthur Scarneo, Philip Hughes, M. Asif Amin, Pei-Suen Tsou, Dinesh Khanna, Timothy A.J. Haystead, Swati Bhattacharyya, John Varga

Multimodal single-cell analysis of nonrandom heteroplasmy distribution in human retinal mitochondrial disease

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Abstract

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multisystem disease. The wide range of manifestations observed in patients with mitochondrial disease results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify nonrandom distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA-Seq, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profiled the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and from healthy control donors. Utilizing the retina as a model for complex multilineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the WT allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the nonrandom nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

Authors

Nathaniel K. Mullin, Andrew P. Voigt, Miles J. Flamme-Wiese, Xiuying Liu, Megan J. Riker, Katayoun Varzavand, Edwin M. Stone, Budd A. Tucker, Robert F. Mullins

Age-related decline in hippocampal tyrosine phosphatase PTPRO is a mechanistic factor in chemotherapy-related cognitive impairment

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Abstract

Chemotherapy-related cognitive impairment (CRCI) or “chemo brain” is a devastating neurotoxic sequela of cancer-related treatments, especially for the elderly individuals. Here we show that PTPRO, a tyrosine phosphatase, is highly enriched in the hippocampus, and its level is tightly associated with neurocognitive function but declined significantly during aging. To understand the protective role of PTPRO in CRCI, a mouse model was generated by treating Ptpro–/– female mice with doxorubicin (DOX) because Ptpro–/– female mice are more vulnerable to DOX, showing cognitive impairments and neurodegeneration. By analyzing PTPRO substrates that are neurocognition-associated tyrosine kinases, we found that SRC and EPHA4 are highly phosphorylated/activated in the hippocampi of Ptpro–/– female mice, with increased sensitivity to DOX-induced CRCI. On the other hand, restoration of PTPRO in the hippocampal CA3 region significantly ameliorate CRCI in Ptpro–/– female mice. In addition, we found that the plant alkaloid berberine (BBR) is capable of ameliorating CRCI in aged female mice by upregulating hippocampal PTPRO. Mechanistically, BBR upregulates PTPRO by downregulating miR-25-3p, which directly targeted PTPRO. These findings collectively demonstrate the protective role of hippocampal PTPRO against CRCI.

Authors

Zhimeng Yao, Hongmei Dong, Jianlin Zhu, Liang Du, Yichen Luo, Qing Liu, Shixin Liu, Yusheng Lin, Lu Wang, Shuhong Wang, Wei Wei, Keke Zhang, Qingjun Huang, Xiaojun Yu, Weijiang Zhao, Haiyun Xu, Xiaofu Qiu, Yunlong Pan, Xingxu Huang, Sai-Ching Jim Yeung, Dianzheng Zhang, Hao Zhang

Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation

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Abstract

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C–expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H– but not K8-K393R–induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C–mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

Authors

Jingyuan Sun, Pei Li, Honglian Gui, Laure Rittié, David B. Lombard, Katrin Rietscher, Thomas M. Magin, Qing Xie, Li Liu, M. Bishr Omary

Alveolar repair following LPS-induced injury requires cell-ECM interactions

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Abstract

During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat AT1 epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administered E. coli lipopolysaccharide (LPS) by intratracheal injection to mice with a postdevelopmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells coexpressing AT2 epithelial, AT1 epithelial, and mixed intermediate cell state markers, with few mature type 1 cells. AT2 cells deficient in β1 showed persistently increased proliferation after injury, which was blocked by inhibiting NF-κB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature AT1 epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.

Authors

Jennifer M.S. Sucre, Fabian Bock, Nicholas M. Negretti, John T. Benjamin, Peter M. Gulleman, Xinyu Dong, Kimberly T. Ferguson, Christopher S. Jetter, Wei Han, Yang Liu, Seunghyi Kook, Jason J. Gokey, Susan H. Guttentag, Jonathan A. Kropski, Timothy S. Blackwell, Roy Zent, Erin J. Plosa

LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3

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Abstract

The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unraveled a potentially novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2, and LoVo) with either knockdown or overexpression of LIN28B, we identified claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and posttranscriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a potentially novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B/CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by posttranscriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.

Authors

Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Alice E. Shin, Nozomu Sakai, Hisahiro Matsubara, Masayuki Otsuka, Peter A. Sims, Christopher J. Lengner, Anil K. Rustgi

Understanding the treatment benefit of hyperimmune anti-influenza intravenous immunoglobulin (Flu-IVIG) for severe human influenza

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Abstract

BACKGROUND Antibody-based therapies for respiratory viruses are of increasing importance. The INSIGHT 006 trial administered anti-influenza hyperimmune intravenous immunoglobulin (Flu-IVIG) to patients hospitalized with influenza. Flu-IVIG treatment improved outcomes in patients with influenza B but showed no benefit for influenza A.METHODS To probe potential mechanisms of Flu-IVIG utility, sera collected from patients hospitalized with influenza A or B viruses (IAV or IBV) were analyzed for antibody isotype/subclass and Fcγ receptor (FcγR) binding by ELISA, bead-based multiplex, and NK cell activation assays.RESULTS Influenza-specific FcγR-binding antibodies were elevated in Flu-IVIG–infused IBV- and IAV-infected patients. In IBV-infected participants (n = 62), increased IgG3 and FcγR binding were associated with more favorable outcomes. Flu-IVIG therapy also improved the odds of a more favorable outcome in patients with low levels of anti-IBV Fc-functional antibody. Higher FcγR-binding antibody was associated with less favorable outcomes in IAV-infected patients (n = 50), and Flu-IVIG worsened the odds of a favorable outcome in participants with low levels of anti-IAV Fc-functional antibody.CONCLUSION These detailed serological analyses provide insights into antibody features and mechanisms required for a successful humoral response against influenza, suggesting that IBV-specific, but not IAV-specific, antibodies with Fc-mediated functions may assist in improving influenza outcome. This work will inform development of improved influenza immunotherapies.TRIAL REGISTRATION ClinicalTrials.gov NCT02287467.FUNDING Funding for this research was provided by subcontract 13XS134 under Leidos Biomedical Research Prime Contract HHSN261200800001E and HHSN261201500003I, NCI/NIAID.

Authors

Hillary A. Vanderven, Deborah N. Wentworth, Win Min Han, Heidi Peck, Ian G. Barr, Richard T. Davey Jr., John H. Beigel, Dominic E. Dwyer, Mamta K. Jain, Brian Angus, Christian T. Brandt, Analia Mykietiuk, Matthew G. Law, James D. Neaton, Stephen J. Kent, for the INSIGHT FLU-IVIG Study Group

P2Y₂ receptor decreases blood pressure by inhibiting ENaC

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Abstract

Stimulating the Gq-coupled P2Y2 receptor (P2ry2) lowers blood pressure. Global knockout of P2ry2 increases blood pressure. Vascular and renal mechanisms are believed to participate in P2ry2 effects on blood pressure. To isolate the role of the kidneys in P2ry2 effects on blood pressure and to reveal the molecular and cellular mechanisms of this action, we test here the necessity of the P2ry2 and the sufficiency of Gq-dependent signaling in renal principal cells to the regulation of the epithelial Na+ channel (ENaC), sodium excretion, and blood pressure. Activating P2ry2 in littermate controls but not principal cell–specific P2ry2-knockout mice decreased the activity of ENaC in renal tubules. Moreover, deletion of P2ry2 in principal cells abolished increases in sodium excretion in response to stimulation of P2ry2 and compromised the normal ability to excrete a sodium load. Consequently, principal cell–specific knockout of P2ry2 prevented decreases in blood pressure in response to P2ry2 stimulation in the deoxycorticosterone acetate–salt (DOCA-salt) model of hypertension. In wild-type littermate controls, such stimulation decreased blood pressure in this model of hypertension by promoting a natriuresis. Pharmacogenetic activation of Gq exclusively in principal cells using targeted expression of Gq–designer receptors exclusively activated by designer drugs and clozapine N-oxide decreased the activity of ENaC in renal tubules, promoting a natriuresis that lowered elevated blood pressure in the DOCA-salt model of hypertension. These findings demonstrate that the kidneys play a major role in decreasing blood pressure in response to P2ry2 activation and that inhibition of ENaC activity in response to P2ry2-mediated Gq signaling lowered blood pressure by increasing renal sodium excretion.

Authors

Antonio G. Soares, Jorge Contreras, Elena Mironova, Crystal R. Archer, James D. Stockand, Tarek Mohamed Abd El-Aziz

Early ART reduces viral seeding and innate immunity in liver and lungs of SIV-infected macaques

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Abstract

Identifying immune cells and anatomical tissues that contribute to the establishment of viral reservoirs is of central importance in HIV-1 cure research. Herein, we used rhesus macaques (RMs) infected with SIVmac251 to analyze viral seeding in the liver and lungs of either untreated or early antiretroviral therapy–treated (ART-treated) RMs. Consistent with viral replication and sensing, transcriptomic analyses showed higher levels of inflammation, pyroptosis, and chemokine genes as well as of interferon-stimulating gene (ISG) transcripts, in the absence of ART. Our results highlighted the infiltration of monocyte-derived macrophages (HLA-DR+CD11b+CD14+CD16+) in inflamed liver and lung tissues associated with the expression of CD183 and CX3CR1 but also with markers of tissue-resident macrophages (CD206+ and LYVE+). Sorting of myeloid cell subsets demonstrated that CD14+CD206–, CD14+CD206+, and CD14–CD206+ cell populations were infected, in the liver and lungs, in SIVmac251-infected RMs. Of importance, early ART drastically reduced viral seeding consistent with the absence of ISG detection but also of genes related to inflammation and tissue damage. Viral DNA was only detected in CD206+HLA-DR+CD11b+ cells in ART-treated RMs. The observation of pulmonary and hepatic viral rebound after ART interruption reinforces the importance of early ART implementation to limit viral seeding and inflammatory reactions.

Authors

Julien A. Clain, Henintsoa Rabezanahary, Gina Racine, Steven Boutrais, Calaiselvy Soundaramourty, Charles Joly Beauparlant, Mohammad-Ali Jenabian, Arnaud Droit, Petronela Ancuta, Ouafa Zghidi-Abouzid, Jérôme Estaquier

An integrated single-cell analysis of human adrenal cortex development

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Abstract

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress.

Authors

Ignacio del Valle, Matthew D. Young, Gerda Kildisiute, Olumide K. Ogunbiyi, Federica Buonocore, Ian C. Simcock, Eleonora Khabirova, Berta Crespo, Nadjeda Moreno, Tony Brooks, Paola Niola, Katherine Swarbrick, Jenifer P. Suntharalingham, Sinead M. McGlacken-Byrne, Owen J. Arthurs, Sam Behjati, John C. Achermann

Leishmania major drives host phagocyte death and cell-to-cell transfer depending on intracellular pathogen proliferation rate

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Abstract

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major–infected (L. major–infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

Authors

Iris Baars, Moritz Jaedtka, Leon-Alexander Dewitz, Yan Fu, Tobias Franz, Juliane Mohr, Patricia Gintschel, Hannes Berlin, Angelina Degen, Sandra Freier, Stefan Rygol, Burkhart Schraven, Sascha Kahlfuß, Ger van Zandbergen, Andreas J. Müller

Platelets exacerbate cardiovascular inflammation in a murine model of Kawasaki disease vasculitis

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Abstract

Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Increased platelet counts and activation are observed during the course of KD, and elevated platelet counts are associated with higher risks of developing intravenous immunoglobulin resistance and coronary artery aneurysms. However, the role of platelets in KD pathogenesis remains unclear. Here, we analyzed transcriptomics data generated from the whole blood of patients with KD and discovered changes in the expression of platelet-related genes during acute KD. In the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, LCWE injection increased platelet counts and the formation of monocyte-platelet aggregates (MPAs), upregulated the concentration of soluble P-selectin, and increased circulating thrombopoietin and interleukin 6 (IL-6). Furthermore, platelet counts correlated with the severity of cardiovascular inflammation. Genetic depletion of platelets (Mpl–/– mice) or treatment with an anti-CD42b antibody significantly reduced LCWE-induced cardiovascular lesions. Furthermore, in the mouse model, platelets promoted vascular inflammation via the formation of MPAs, which likely amplified IL-1B production. Altogether, our results indicate that platelet activation exacerbates the development of cardiovascular lesions in a murine model of KD vasculitis. These findings enhance our understanding of KD vasculitis pathogenesis and highlight MPAs, which are known to enhance IL-1B production, as a potential therapeutic target for this disorder.

Authors

Begüm Kocatürk, Youngho Lee, Nobuyuki Nosaka, Masanori Abe, Daisy Martinon, Malcolm E. Lane, Debbie Moreira, Shuang Chen, Michael C. Fishbein, Rebecca A. Porritt, Bernardo S. Franklin, Magali Noval Rivas, Moshe Arditi

Influenza A–induced cystic fibrosis transmembrane conductance regulator dysfunction increases susceptibility to Streptococcus pneumoniae

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Abstract

Influenza A virus (IAV) infection is commonly complicated by secondary bacterial infections that lead to increased morbidity and mortality. Our recent work demonstrates that IAV disrupts airway homeostasis, leading to airway pathophysiology resembling cystic fibrosis disease through diminished cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we use human airway organotypic cultures to investigate how IAV alters the airway microenvironment to increase susceptibility to secondary infection with Streptococcus pneumoniae (Spn). We observed that IAV-induced CFTR dysfunction and airway surface liquid acidification is central to increasing susceptibility to Spn. Additionally, we observed that IAV induced profound transcriptional changes in the airway epithelium and proteomic changes in the airway surface liquid in both CFTR-dependent and -independent manners. These changes correspond to multiple diminished host defense pathways and altered airway epithelial function. Collectively, these findings highlight both the importance of CFTR function during infectious challenge and demonstrate a central role for the lung epithelium in secondary bacterial infections following IAV.

Authors

Erin Y. Earnhardt, Jennifer L. Tipper, Adonis D’Mello, Ming-Yuan Jian, Elijah S. Conway, James A. Mobley, Carlos J. Orihuela, Hervé Tettelin, Kevin S. Harrod

Mucosal transcriptomics highlight lncRNAs implicated in ulcerative colitis, Crohn’s disease, and celiac disease

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Abstract

Ulcerative colitis (UC), Crohn’s disease (CD), and celiac disease are prevalent intestinal inflammatory disorders with nonsatisfactory therapeutic interventions. Analyzing patient data-driven cohorts can highlight disease pathways and new targets for interventions. Long noncoding RNAs (lncRNAs) are attractive candidates, since they are readily targetable by RNA therapeutics, show relative cell-specific expression, and play key cellular functions. Uniformly analyzing gut mucosal transcriptomics from 696 subjects, we have highlighted lncRNA expression along the gastrointestinal (GI) tract, demonstrating that, in control samples, lncRNAs have a more location-specific expression in comparison with protein-coding genes. We defined dysregulation of lncRNAs in treatment-naive UC, CD, and celiac diseases using independent test and validation cohorts. Using the Predicting Response to Standardized Pediatric Colitis Therapy (PROTECT) inception UC cohort, we defined and prioritized lncRNA linked with UC severity and prospective outcomes, and we highlighted lncRNAs linked with gut microbes previously implicated in mucosal homeostasis. HNF1A-AS1 lncRNA was reduced in all 3 conditions and was further reduced in more severe UC form. Similarly, the reduction of HNF1A-AS1 ortholog in mice gut epithelia showed higher sensitivity to dextran sodium sulfate–induced colitis, which was coupled with alteration in the gut microbial community. These analyses highlight prioritized dysregulated lncRNAs that can guide future preclinical studies for testing them as potential targets.

Authors

Tzipi Braun, Katya E. Sosnovski, Amnon Amir, Marina BenShoshan, Kelli L. VanDussen, Rebekah Karns, Nina Levhar, Haya Abbas-Egbariya, Rotem Hadar, Gilat Efroni, David Castel, Camila Avivi, Michael J. Rosen, Anne M. Grifiths, Thomas D. Walters, David R. Mack, Brendan M. Boyle, Syed Asad Ali, Sean R. Moore, Melanie Schirmer, Ramnik J. Xavier, Subra Kugathasan, Anil G. Jegga, Batya Weiss, Chen Mayer, Iris Barshack, Shomron Ben-Horin, Igor Ulitsky, Anthony Beucher, Jorge Ferrer, Jeffrey S. Hyams, Lee A. Denson, Yael Haberman

Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells

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Abstract

Specific and efficient smooth muscle cell–targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLSP2A or CreERT2–P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2–P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

Authors

Yang Zhao, Guizhen Zhao, Ziyi Chang, Tianqing Zhu, Ying Zhao, Haocheng Lu, Chao Xue, Thomas L. Saunders, Yanhong Guo, Lin Chang, Y. Eugene Chen, Jifeng Zhang

CD84 is a regulator of the immunosuppressive microenvironment in multiple myeloma

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Abstract

Authors

Hadas Lewinsky, Emine G. Gunes, Keren David, Lihi Radomir, Matthias P. Kramer, Bianca Pellegrino, Michal Perpinial, Jing Chen, Ting-fang He, Anthony G. Mansour, Kun-Yu Teng, Supriyo Bhattacharya, Enrico Caserta, Estelle Troadec, Peter Lee, Mingye Feng, Jonathan Keats, Amrita Krishnan, Michael Rosenzweig, Jianhua Yu, Michael A. Caligiuri, Yosef Cohen, Olga Shevetz, Shirly Becker-Herman, Flavia Pichiorri, Steven Rosen, Idit Shachar