Label-free single-cell phenotyping to determine tumor cell heterogeneity in pancreatic cancer in real time

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Abstract

Resistance to chemotherapy of pancreatic ductal adenocarcinoma (PDAC) is largely driven by intratumoral heterogeneity (ITH) due to tumor cell plasticity and clonal diversity. To develop alternative strategies to overcome this defined mechanism of resistance, tools to monitor and quantify ITH in a rapid and scalable fashion are needed urgently. Here, we employed label-free digital holographic microscopy (DHM) to characterize ITH in PDAC. We established a robust experimental and machine learning analysis pipeline to perform single-cell phenotyping based on DHM-derived phase images of PDAC cells in suspension. Importantly, we were able to detect dynamic changes in tumor cell differentiation and heterogeneity of distinct PDAC subtypes upon induction of epithelial-mesenchymal transition and under treatment-imposed pressure in murine and patient-derived model systems. This platform allowed us to assess phenotypic ITH in PDAC on a single-cell level in real time. Implementing this technology into the clinical workflow has the potential to fundamentally increase our understanding of tumor heterogeneity during evolution and treatment response.

Authors

Katja Wittenzellner, Manuel Lengl, Stefan Röhrl, Carlo Maurer, Christian Klenk, Aristeidis Papargyriou, Laura Schmidleitner, Nicole Kabella, Akul Shastri, David E. Fresacher, Farid Harb, Nawal Hafez, Stefanie Bärthel, Daniele Lucarelli, Carmen Escorial-Iriarte, Felix Orben, Rupert Öllinger, Ellen Emken, Lisa Fricke, Joanna Madej, Patrick Wustrow, I. Ekin Demir, Helmut Friess, Tobias Lahmer, Roland M. Schmid, Roland Rad, Günter Schneider, Bernhard Kuster, Dieter Saur, Oliver Hayden, Klaus Diepold, Maximilian Reichert

A CARD9 deficiency mouse model recapitulates human chronic CNS candidiasis identifying defective monocytic cell responses in immunopathogenesis

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Abstract

Human Caspase Recruitment Domain Containing Protein 9 (CARD9) deficiency predisposes to invasive fungal disease, particularly by Candida spp. CARD9 deficiency causes chronic central nervous system (CNS) candidiasis. Currently, no animal model recapitulates the chronicity of disease, precluding a better understanding of immunopathogenesis. We established a knock-in mouse homozygous for the recurring p.Y91H mutation (Y91HKI) and, in parallel to Card9-/– mice, titrated the intravenous fungal inoculum to the CARD9 genotype to develop a model of chronic invasive candidiasis. Strikingly, CARD9-deficient mice had predominantly CNS involvement, with neurological symptoms appearing late during infection and progressive brain fungal burden in the absence of fulminant sepsis, reflecting the human syndrome. Mononuclear cell aggregation at fungal lesions in the brain correlated with increased MHCII+Ly6C+ monocyte numbers at day 1 after infection in WT and Y91HKI mice, but not in Card9-/– mice. At day 4 after infection, neutrophils and additional Ly6C+ monocytes were recruited to the CARD9-deficient brain. As in humans, Y91HKI mutant mice demonstrated cerebral multinucleated giant cells and granulomata. Subtle immunologic differences between the hypomorphic (p.Y91H) and null mice were noted, perhaps explaining some of the variability seen in humans. Our work established a disease-recapitulating animal model to specifically decipher chronic CNS candidiasis due to CARD9 deficiency.

Authors

Marija Landekic, Isabelle Angers, Yongbiao Li, Marie-Christine Guiot, Marc-André Déry, Annie Beauchamp, Lucie Roussel, Annie Boisvert, Wen Bo Zhou, Christina Gavino, Julia Luo, Stéphane Bernier, Makayla Kazimerczak-Brunet, Yichun Sun, Brendan Snarr, Michail S. Lionakis, Robert T. Wheeler, Irah L. King, Salman T. Qureshi, Maziar Divangahi, Donald C. Vinh

Involvement of impaired carnitine-induced fatty acid oxidation in experimental and human diabetic kidney disease

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Abstract

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. Kidney tubular cells have a high energy demand, dependent on fatty acid oxidation (FAO). Although carnitine is indispensable for FAO, the pathological role of carnitine deficiency in DKD is not fully understood. We showed here that ectopic lipid accumulation owing to impaired FAO increased in patients with DKD and inversely correlated with kidney function. Organic cation/carnitine transporter 2–deficient (OCTN2-deficient) mice exhibited systemic carnitine deficiency with increased renal lipid accumulation. Cell death and inflammation were induced in OCTN2-deficient, but not wild-type, tubular cells exposed to high salt and high glucose. Compared with Spontaneously Diabetic Torii (SDT) fatty rats, uninephrectomized SDT fatty rats fed with 0.3% NaCl showed higher lipid accumulation and increased urinary albumin excretion with kidney dysfunction and tubulointerstitial injury, all of which were ameliorated by l-carnitine supplementation via stimulating FAO and mitochondrial biogenesis. In our single-center randomized control trial with patients undergoing peritoneal dialysis, l-carnitine supplementation preserved residual renal function and increased urine volume, the latter of which was correlated with improvement of tubular injury. The present study demonstrates the pathological role of impairment of carnitine-induced FAO in DKD, suggesting that l-carnitine supplementation is a potent therapeutic strategy for this devastating disorder.

Authors

Sakuya Ito, Kensei Taguchi, Goh Kodama, Saori Kubo, Tomofumi Moriyama, Yuya Yamashita, Yunosuke Yokota, Yosuke Nakayama, Yusuke Kaida, Masami Shinohara, Kyoko Tashiro, Keisuke Ohta, Sho-ichi Yamagishi, Kei Fukami

Macrophage-derived Spp1 promotes intramuscular fat in dystrophic muscle

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Abstract

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder involving cycles of muscle degeneration and regeneration, leading to accumulation of intramuscular fibrosis and fat. Ablation of Osteopontin/Spp1 in a murine model of DMD (mdx) improves the dystrophic phenotype, but the source of Spp1 and its impact on target cells in dystrophic muscles remain unknown. In dystrophic muscles, macrophages are the predominate infiltrating leukocyte and express high levels of Spp1. We used macrophage-specific ablation combined with single-cell transcriptional profiling to uncover the impact of macrophage-derived Spp1 on cell-cell interactions in mdx muscles. Ablation of macrophage-specific Spp1 (cKO) correlated with reduction of 2 PDGFRa+ stromal cell populations, expressing Lifr+ and Procr+. Sorting and transcriptional profiling of these populations confirmed that they are enriched in adipogenesis genes and are highly related to fibroadipogenic precursors (FAPS). These adipogenic stromal cells (ASC) displayed more adipogenic potential in vitro compared with FAPS, likely due to a more differentiated state. Reduction of ASCs correlated with reduced intramuscular diaphragmatic fat and improved diaphragm function. These data suggest a role for myeloid-derived Spp1 in the differentiation of stromal cells towards an adipogenic fate, leading to accumulation of intramuscular fat in dystrophic muscles.

Authors

Philip K. Farahat, Chino Kumagai-Cresse, Raquel L. Aragón, Feiyang Ma, Justin K. Amakor, Alejandro Espinoza, Irina Kramerova, Robert J. Jimenez, Bradley M. Smith, Jesus Perez, Rachelle H. Crosbie, Apoorva H. Nagendra, Jackie McCourt-Towner, Gerald Coulis, Oluwatayo F. Ikotun, April D. Pyle, Matteo Pellegrini, Elizabeth M. McNally, S. Armando Villalta, Melissa J. Spencer

Identification of bacteriophage DNA in human umbilical cord blood

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Abstract

Bacteriophages, viruses that parasitize bacteria, are abundant in the human microbiome and may influence human health, in part, through their interactions with bacterial hosts. Whether endogenous bacteriophages or their products are vertically transmitted from mother to fetus during human pregnancy is not known. Here, we searched for bacteriophage sequences from five bacteriophage databases (474,031 total sequences) in cell-free DNA (cfDNA) of paired maternal and umbilical cord blood samples from two independent cohorts. First, we sequenced cfDNA from 10 pairs of maternal and cord blood samples, including four pairs affected by preeclampsia. We validated our findings in a previously published dataset of 62 paired maternal and cord blood samples, including 43 pairs from preterm or chorioamnionitis-affected deliveries. We identified 94 and 596 bacteriophage sequences in maternal and cord blood cfDNA samples from the first and second cohort, respectively. We identified 58 phage sequences across maternal-infant dyads and 581 phage sequences that were unique to a single sample. We did not identify any phage sequences consistently associated with preeclampsia, preterm, or chorioamnionitis-affected samples. This study demonstrated the presence of bacteriophage DNA in human cord blood at birth, providing evidence that the human fetus is exposed to bacteriophage DNA in utero.

Authors

Jennifer A. Sequoia, Naomi L. Haddock, Paw Mar Gay, Layla J. Barkal, Purnima Narasimhan, Nadine Martinez, Virginia D. Winn, Paul L. Bollyky

Opposing roles for myeloid and smooth muscle cell STING in pulmonary hypertension

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Abstract

There is an emerging role for stimulator of interferon genes (STING) signaling in pulmonary hypertension (PH) development. Related to this, prior research has demonstrated the relevance of immune checkpoint protein programmed death ligand 1 (PD-L1) expression by immunoregulatory myeloid cells in PH. However, there remains a need to elucidate the cell-specific role of STING expression, and the STING/PD-L1 signaling axis in PH, before readily available disease-modifying therapies can be applied for patients with the disease. Here, through generation of bone marrow chimeric mice, we show that STING–/– mice receiving WT bone marrow were protected against PH secondary to chronic hypoxia. We further demonstrate a cellular dichotomous role for STING in PH development, with STING expression by smooth muscle cells contributing to PH and its activation on myeloid cells being pivotal in severe disease prevention. Finally, we provide evidence that a STING/PD-L1 axis modulates disease severity, suggesting the potential for future therapeutic applications. Overall, these data provide evidence of STING’s involvement in PH in a cell-specific manner, establishing the biologic plausibility of developing cell-targeted STING-related therapies for PH.

Authors

Ann T. Pham, Shiza Virk, Aline C. Oliveira, Matthew D. Alves, Chunhua Fu, Yutao Zhang, Jimena Alvarez-Castanon, Brian B. Lee, Keira L. Lee, Radwan Mashina, Katherine E. Ray, Patrick Donabedian, Elnaz Ebrahimi, Harsh Patel, Reeha Patel, Duncan Lewis, Zhiguang Huo, Harry Karmouty-Quintana, Li Chen, Lei Jin, Andrew J. Bryant

Pan-H7 influenza human antibody virus neutralization depends on avidity and steric hindrance

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Abstract

H7N9 avian influenza virus is a zoonotic influenza virus of public health concern, with a 39% mortality rate in humans. H7N9-specific prevention or treatments for humans have not been approved. We previously isolated a human monoclonal antibody (mAb) designated H7-235 that broadly reacts to diverse H7 viruses and neutralizes H7N9 viruses in vitro. Here, we report the crystal structure of H7 HA1 bound to the fragment antigen-binding region (Fab) of recombinant H7-235 (rH7-235). The crystal structure revealed that rH7-235 recognizes residues near but outside of the receptor binding site (RBS). Nevertheless, the rH7-235 IgG potently inhibits hemagglutination mediated by H7N9 viruses due to avidity effect and Fc steric hindrance. This mAb prophylactically protects mice against weight loss and death caused by challenge with lethal H7N9 viruses in vivo. rH7-235 mAb neutralizing activity alone is sufficient for protection when used at a high dose in a prophylactic setting. This study provides insights into mechanisms of viral neutralization by protective, broadly reactive anti-H7 antibodies, informing the rational design of therapeutics and vaccines against H7N9 influenza virus.

Authors

Iuliia M. Gilchuk, Jinhui Dong, Ryan P. Irving, Cameron D. Buchman, Erica Armstrong, Hannah L. Turner, Sheng Li, Andrew B. Ward, Robert H. Carnahan, James E. Crowe Jr.

Allergen induces pulmonary neuroendocrine cell hyperplasia in a model of asthma

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Abstract

Asthma is characterized by exacerbated response to triggers such as allergen. While pulmonary neuroendocrine cells (PNECs), a rare population of airway epithelial cells, are essential for amplifying allergen-induced asthma response, how PNECs are regulated to achieve this role remains poorly understood. Here we show that in the adult mouse airway, inactivation of achaete-scute-like protein 1 gene in PNECs led to loss of these cells. Intriguingly, exposure of these mutants to house dust mites (HDM), a common allergen, led to reappearance of PNECs. Similarly, exposure of wild-type mice to HDM led to PNEC hyperplasia, a result of proliferation of existing PNECs and transdifferentiation from club cells. Single-cell RNA-Seq experiments revealed PNEC heterogeneity, including the emergence of an allergen-induced PNEC subtype. Notch signaling was downregulated in HDM-treated airway, and treatment with Notch agonist prevented PNEC hyperplasia. These findings together suggest that HDM-induced PNEC hyperplasia may contribute to exacerbated asthma response.

Authors

Estelle Kim, Brian K. Wells, Hannah Indralingam, Yujuan Su, Jamie Verheyden, Xin Sun

An amphiregulin reporter mouse enables transcriptional and clonal expansion analysis of reparative lung Tregs

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Abstract

Regulatory T cells (Tregs) are known to play critical roles in tissue repair via provision of growth factors, such as amphiregulin (Areg). Areg-producing Tregs have previously been difficult to study because of an inability to isolate live Areg-producing cells. In this report, we created a reporter mouse to detect Areg expression in live cells (AregThy1.1). We employed influenza A and bleomycin models of lung damage to sort Areg-producing and non-Areg-producing Tregs for transcriptomic analyses. Single-cell RNA-Seq revealed distinct subpopulations of Tregs and allowed transcriptomic comparisons of damage-induced populations. Single-cell TCR sequencing showed that Treg clonal expansion was biased toward Areg-producing Tregs and largely occurred within damage-induced subgroups. Gene module analysis revealed functional divergence of Tregs into immunosuppression-oriented and tissue repair–oriented groups, leading to identification of candidate receptors for induction of repair activity in Tregs. We tested these using an ex vivo assay for Treg-mediated tissue repair, identifying 4-1BB agonism as a mechanism for reparative activity induction. Overall, we demonstrate that the AregThy1.1 mouse is a promising tool for investigating tissue repair activity in leukocytes.

Authors

Lucas F. Loffredo, Katherine A. Kaiser, Adam Kornberg, Samhita Rao, Kenia de los Santos-Alexis, Arnold Han, Nicholas Arpaia

Glycoprotein NMB mediates bidirectional GSC-TAM interactions to promote tumor progression

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Abstract

Glioblastoma (GBM) is a lethal brain tumor containing a subpopulation of GBM stem cells (GSCs) that interaction with surrounding cells, including infiltrating tumor-associated macrophages and microglia (TAMs). While GSCs and TAMs are in close proximity and likely interact to coordinate tumor growth, a limited number of mechanisms have been identified that support their communication. Here, we identified glycoprotein NMB (GPNMB) as a key factor mediating a unique bidirectional interaction between GSCs and TAMs in GBM. Specifically, GSCs educated macrophages and microglia to preferentially express GPNMB in the GBM tumor microenvironment. As a result, TAM-secreted GPNMB interacted with its receptor CD44 on GSCs to promote their glycolytic and self-renewal abilities via activating the PYK2/RSK2 signaling axis. Disrupting GPNMB-mediated GSC-TAM interplay suppressed tumor progression and self-renewal in GBM mouse models. Our study found a protumor function of GPNMB-mediated GSC-TAM bidirectional communication and supports GPNMB as a promising therapeutic target for GBM.

Authors

Yang Liu, Lizhi Pang, Fatima Khan, Junyan Wu, Fei Zhou, Craig Horbinski, Shideng Bao, Jennifer S. Yu, Justin D. Lathia, Peiwen Chen

MTOR signaling regulates the development of airway mucous cell metaplasia associated with severe asthma

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Abstract

In asthma, airway epithelial remodeling is characterized by aberrant goblet cell metaplastic differentiation accompanied by epithelial cell hyperplasia and hypertrophy. These pathologic features in severe asthma indicate a loss of control of proliferation, cell size, differentiation, and migration. MTOR is a highly conserved pathway that regulates protein synthesis, cell size, and proliferation. We hypothesized that the balance between MTOR and autophagy regulates mucous cell metaplasia. Airways from individuals with severe asthma showed increased MTOR signaling by RPS6 phosphorylation, which was reproduced using an IL-13–activated model of primary human airway epithelial cells (hAEC). MTOR inhibition by rapamycin led to a decrease of IL-13–mediated cell hypertrophy, hyperplasia, and MUC5AC mucous metaplasia. BrdU labeling during IL-13–induced mucous metaplasia confirmed that MTOR was associated with increased basal-to-apical hAEC migration. MTOR activation by genetic deletion of Tsc2 in cultured mouse AECs increased IL-13–mediated hyperplasia, hypertrophy, and mucous metaplasia. Transcriptomic analysis of IL-13–stimulated hAEC identified MTOR-dependent expression of genes associated with epithelial migration and cytoskeletal organization. In summary, these findings point to IL-13–dependent and –independent roles of MTOR signaling in the development of pathogenic epithelial changes contributing to airway obstruction in severe asthma.

Authors

Katrina M. Kudrna, Luis F. Vilches, Evan M. Eilers, Shailendra K. Maurya, Steven L. Brody, Amjad Horani, Kristina L. Bailey, Todd A. Wyatt, John D. Dickinson

Stearoyl-CoA desaturase inhibition normalizes brain lipid saturation, α-synuclein homeostasis, and motor function in mutant Gba1-Parkinson mice

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Abstract

Loss-of-function mutations in the GBA1 gene are a prevalent risk factor for Parkinson’s disease (PD). Defining features are Lewy bodies that can be rich in α-synuclein (αS), vesicle membranes, and other lipid membranes, coupled with striatal dopamine loss and progressive motor dysfunction. Of these, lipid abnormalities are the least understood. An altered lipid metabolism in PD patient-derived neurons — carrying mutations in either GBA1, encoding for glucocerebrosidase (GCase), or αS — shifted the physiological αS tetramer/monomer (T:M) equilibrium, resulting in PD phenotypes. We previously reported inhibition of stearoyl-CoA desaturase (SCD), the rate-limiting enzyme for fatty acid desaturation, stabilized αS tetramers and improved motor deficits in αS mice. Here we show that mutant GBA1-PD cultured neurons have increased SCD products (monounsaturated fatty acids [MUFAS]) and reduced αS T:M ratios that were improved by inhibiting SCD. Oral treatment of symptomatic L444P and E326K Gba1 mutant mice with 5b also improved the αS T:M homeostasis and dopaminergic striatal integrity. Moreover, SCD inhibition normalized GCase maturation and dampened lysosomal and lipid-rich clustering, key features of neuropathology in GBA-PD. In conclusion, this study supports that brain MUFA metabolism links GBA1 genotype and WT αS homeostasis to downstream neuronal and behavioral impairments, identifying SCD as a therapeutic target for GBA-PD.

Authors

Silke Nuber, Harrison Hsiang, Esra’a Keewan, Tim E. Moors, Sydney J. Reitz, Anupama Tiwari, Gary P.H. Ho, Elena Su, Wolf Hahn, Marie-Alexandre Adom, Riddhima Pathak, Matthew Blizzard, Sangjune Kim, Han Seok Ko, Xiaoqun Zhang, Per Svenningsson, Dennis J. Selkoe, Saranna Fanning

Polyfunctional T follicular helper cells drive checkpoint-inhibitor diabetes and are targeted by JAK inhibitor therapy

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Abstract

Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy, but their use is limited by the development of autoimmunity in healthy tissues as a side effect of treatment. Such immune-related adverse events (IrAE) contribute to hospitalizations, cancer treatment interruption, and even premature death. ICI-induced autoimmune diabetes mellitus (ICI-T1DM) is a life-threatening IrAE that presents with rapid pancreatic β-islet cell destruction leading to hyperglycemia and life-long insulin dependence. While prior reports have focused on CD8+ T cells, the role for CD4+ T cells in ICI-T1DM is less understood. We identify expansion of CD4+ T follicular helper (Tfh) cells expressing IL-21 and IFN-γ as a hallmark of ICI-T1DM. Furthermore, we show that both IL-21 and IFN-γ are critical cytokines for autoimmune attack in ICI-T1DM. Because IL-21 and IFN-γ both signal through JAK/STAT pathways, we reasoned that JAK inhibitors (JAKi) may protect against ICI-T1DM. Indeed, JAKi provide robust in vivo protection against ICI-T1DM in a mouse model that is associated with decreased islet-infiltrating Tfh cells. Moreover, JAKi therapy impaired Tfh cell differentiation in patients with ICI-T1DM. These studies highlight CD4+ Tfh cells as underrecognized but critical mediators of ICI-T1DM that may be targeted with JAKi to prevent this grave IrAE.

Authors

Nicole L. Huang, Jessica G. Ortega, Kyleigh Kimbrell, Joah Lee, Lauren N. Scott, Esther M. Peluso, Sarah J. Wang, Ellie Y. Kao, Kristy Kim, Jarod Olay, Jaden N. Nguyen, Zoe Quandt, Trevor E. Angell, Maureen A. Su, Melissa G. Lechner

Metagenomic detection of central nervous system infections missedby conventional testing

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Abstract

Community-acquired infectious meningoencephalitis is associated with high rates of mortality and morbidity, compounded by limited access to diagnostic resources. The current study assessed acute central nervous system (CNS) infections in patients with meningoencephalitis enrolled in a hospital-based diagnostic surveillance study in São Paulo, Brazil. Cerebrospinal fluid (CSF) was collected from 600 patients between March 2018 and November 2019 and initially screened for a broad range of pathogens according to a local diagnostic algorithm. Standard microbiological and molecular diagnostic methods were applied. Metagenomic sequencing was used as a complementary approach to investigating etiology in instances where no pathogen was initially identified. Standard testing identified infectious etiologies in 292 patients (48.6%), with 227 (77.7%) confirmed as viral infections, predominantly caused by enteroviruses (n = 144) and herpesviruses (n = 40). Nonviral agents were identified in 65 patients (22.3%). Metagenomic sequencing (mNGS) of 277 of 308 undiagnosed patients revealed several additional potential etiologies, including Parvovirus B19, Toxoplasma gondii, Picobirnavirus, other enterovirus species and Vesivirus, the latter being associated with CNS infection for the first time. These findings underscore the complexity of CNS infections and highlight the potential of metagenomics to improve diagnostic accuracy, inform treatment strategies, and support efforts to address future pandemics.

Authors

Noely Evangelista Ferreira, Michael G. Berg, Antonio C. da Costa, Mary A. Rodgers, Esper G. Kallas, Cassia G. Terrasani Silveira, Mateus Vailant Thomazella, Ana Carolina Soares de Oliveira, Layla Honorato, Heuder G.O. Paião, Renan Barros Domingues, Carlos Senne, Marina F. Côrtes, Tania R. Tozetto-Mendoza, Hélio R. Gomes, Maria Laura Mariano Matos, Geovani de Oliveira Ribeiro, Steven S. Witkin, Gavin A. Cloherty, Maria Cassia Mendes-Correa

Human pegivirus alters brain and blood immune and transcriptomic profiles of patients with Parkinson’s disease

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Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder with both genetic and environmental factors contributing to pathogenesis. Viral infections are potential environmental triggers that influence PD pathology. Using ViroFind, an unbiased platform for whole virome sequencing, along with quantitative PCR (qPCR), we identified human pegivirus (HPgV) in 5 of 10 (50%) of PD brains, confirmed by IHC in 2 of 2 cases, suggesting an association with PD. All 14 age- and sex-matched controls were HPgV negative. HPgV-brain positive patients with PD showed increased neuropathology by Braak stage and Complexin-2 levels, while those positive in the blood had higher IGF-1 and lower pS65-ubiquitin, supporting disruption in metabolism or mitophagy in response to HPgV. RNA-Seq revealed altered immune signaling in HPgV-infected PD samples, including consistent suppression of IL-4 signaling in both the brain and blood. Longitudinal analysis of blood samples showed a genotype-dependent viral response, with HPgV titers correlating directly with IL-4 signaling in a LRRK2 genotype–dependent manner. YWHAB was a key hub gene in the LRRK2 genotypic response, which exhibited an altered relationship with immune-related factors, including NFKB1, ITPR2, and LRRK2 itself, in patients with PD who are positive for HPgV. These results suggest a role for HPgV in shaping PD pathology and highlight the complex interplay between viral infection, immunity, and neuropathogenesis.

Authors

Barbara A. Hanson, Xin Dang, Pouya Jamshidi, Alicia Steffens, Kaleigh Copenhaver, Zachary S. Orban, Bernabe Bustos, Steven J. Lubbe, Rudolph J. Castellani, Igor J. Koralnik

NEXN regulates vascular smooth muscle cell phenotypic switching and neointimal hyperplasia

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Abstract

Vascular smooth muscle cells (VSMCs) exhibit substantial heterogeneity and plasticity, enabling them to switch between contractile and synthetic states, which is crucial for vascular remodeling. Nexilin (NEXN) has been identified as a high-confidence gene associated with dilated cardiomyopathy. Existing evidence indicates NEXN is involved in phenotypic switching of VSMCs. However, a comprehensive understanding of the cell-specific roles and precise mechanisms of NEXN in vascular remodeling remains elusive. Using integrative transcriptomics analysis and smooth muscle–specific lineage-tracing mice, we demonstrated NEXN was highly expressed in VSMCs, and the expression of NEXN was significantly reduced during the phenotypic transformation of VSMCs and intimal hyperplasia induced by vascular injury. VSMC-specific NEXN deficiency promoted the phenotypic transition of VSMCs and exacerbated neointimal hyperplasia in mice following vascular injury. Mechanistically, we found NEXN primarily mediated VSMC proliferation and phenotypic transition through endoplasmic reticulum (ER) stress and Krüppel-like factor 4 signaling. Inhibiting ER stress ameliorated VSMC phenotypic transition by reducing cell cycle activity and proliferation caused by NEXN deficiency. These findings indicate targeting NEXN could be explored as a promising therapeutic approach for proliferative arterial diseases.

Authors

Zexuan Lin, Chaojie Wang, Zhuohua Wen, Zhaohui Cai, Wenjie Guo, Xin Feng, Zengyan Huang, Rongjun Zou, Xiaoping Fan, Canzhao Liu, Hanyan Yang

ADAR1 expression is associated with cervical cancer progression and negatively regulates NK cell activity

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Abstract

ADAR1 edits double-stranded RNAs (dsRNAs) by deaminating adenosines into inosines, preventing aberrant activation of innate immunity by endogenous dsRNAs, which may resemble viral structures. Several tumors exploit ADAR1 to evade immune surveillance; indeed, its deletion reduces tumor viability and reshapes infiltrating leukocytes. Here we investigated the role of ADAR1 in immune evasion mechanisms during cervical cancer (CC) progression. Patients’ biopsy samples showed higher ADAR1 expression already in premalignant lesions (squamous intraepithelial lesions [SIL]) and a substantially reduced percentage of infiltrating CD7+ innate cells in in situ and invasive carcinomas compared with normal mucosa, with CD56+ NK cells showing phenotypic alterations that may have affected their functional responses. In CC-derived cell lines (SiHa, CaSki), ADAR1 silencing reduced cell proliferation, an effect further enhanced by exogenous IFN-β administration. It also induced proinflammatory gene expression, as demonstrated by RNA-Seq analysis, and conditioned supernatants collected from these cells activated several NK cell effector functions. NK cell infiltration and activation were also confirmed in organotypic 3D tissue models of SiHa cells knocked out for ADAR1. In conclusion, ADAR1 expression increased with CC progression and was accompanied by alterations in tumor-infiltrating NK cells, but its silencing in CC-derived cell lines potentiated antitumor NK cell activities. Thus, ADAR1 inhibition may represent a therapeutic perspective for CC and possibly other malignancies.

Authors

Valentina Tassinari, Marta Kaciulis, Stefano Petrai, Helena Stabile, Angelina Pernazza, Martina Leopizzi, Valeria Di Maio, Francesca Belleudi, Danilo Ranieri, Vanessa Mancini, Innocenza Palaia, Federica Tanzi, Ludovica Lospinoso Severini, Silvia Ruggeri, Maria Emanuela Greco, Giovanni Bernardini, Alessandra Zingoni, Marco Cippitelli, Cristina Cerboni, Alessandra Soriani

Impact of the IL-15 superagonist N-803 on lymphatic reservoirs of HIV

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Abstract

BACKGROUND NK cell function is impaired in people with HIV (PWH), hindering their potential to reduce the lymphoid tissue (LT) reservoir. The IL-15 superagonist N-803 has been shown to enhance NK and T cell function and thus may reduce viral reservoirs.METHODS To determine the impact of N-803 on LTs, we conducted a clinical trial where 10 PWH on effective antiretroviral therapy (ART) were given 3 subcutaneous 6 mcg/kg doses of N-803. We obtained PBMCs and lymph node (LN) and gut biopsies at baseline and after the last N-803 dose.RESULTS We found a nonstatistically significant approximately 0.50 median log reduction in the frequency of viral RNA+ (vRNA+) and vDNA+ cells/g in the 6 participants with baseline and posttreatment LN biopsies. In the ileum, we observed reductions of vRNA+ cells in 8/10 participants and vDNA+ cells in all participants. We also found significant inverse correlations between NK cell proliferation and the frequency of vRNA+ cells and between NKG2A expression on NK cells and the frequency of vRNA+ cells.CONCLUSION Our findings suggest N-803 may reduce the HIV reservoir in LTs of PWH on ART, an effect likely mediated by enhanced NK cell function. Controlled studies assessing the impact of NK cell therapy on HIV LTs are needed.TRIAL REGISTRATION ClinicalTrials.gov NCT04808908.FUNDING NIH grants 5UM1AI126611, UL1TR002494, R01 AI147912, R35 CA283892, and UM1AI164561.

Authors

Joshua Rhein, Jeffrey G. Chipman, Gregory J. Beilman, Ross Cromarty, Kevin Escandón, Jodi Anderson, Garritt Wieking, Jarrett Reichel, Rodolfo Batres, Alexander Khoruts, Christopher M. Basting, Peter Hinderlie, Zachary B. Davis, Anne Eaton, Byron P. Vaughn, Elnaz Eilkhani, Jeffrey T. Safrit, Patrick Soon-Shiong, Jason V. Baker, Nichole R. Klatt, Steven G. Deeks, Jeffrey S. Miller, Timothy W. Schacker

Distinct cell types along thick ascending limb express pathways for monovalent and divalent cation transport

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Abstract

Kidney thick ascending limb (TAL) cells reabsorb sodium, potassium, calcium, and magnesium and contribute to urinary concentration. These cells are typically viewed as a single type that recycles potassium across the apical membrane and generates a lumen-positive transepithelial voltage driving calcium and magnesium reabsorption, though variability in potassium channel expression has been reported. Additionally, recent transcriptomic analyses suggest that different cell types exist along this segment, but classifications have varied and have not led to a new consensus model. We used immunolocalization, electrophysiology, and enriched single-nucleus RNA-Seq to identify TAL cell types in rats, mice, and humans. We identified 3 major TAL cell types defined by expression of potassium channels and claudins. One has apical potassium channels, has low basolateral potassium conductance, and is bordered by a monovalent cation-permeable claudin. A second lacks apical potassium channels, has high basolateral potassium conductance, and is bordered by calcium- and magnesium-permeable claudins. A third type also lacks apical potassium channels and has high basolateral potassium conductance, but these cells are ringed by monovalent cation-permeable claudins. The recognition of diverse cell types may resolve longstanding questions about how solute transport can be modulated selectively and how disruption of these cells leads to human disease.

Authors

Hasan Demirci, Jessica P. Bahena-Lopez, Alina Smorodchenko, Xiao-Tong Su, Jonathan W. Nelson, Chao-Ling Yang, Joshua N. Curry, Xin-Peng Duan, Wen-Hui Wang, Yuliya Sharkovska, Ruisheng Liu, Duygu Elif Yilmaz, Catarina Quintanova, Katie Emberley, Ben Emery, Nina Himmerkus, Markus Bleich, David H. Ellison, Sebastian Bachmann

High-affinity CD16A polymorphism associated with reduced risk ofsevere COVID-19

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Abstract

CD16A is an activating Fc receptor on NK cells that mediates antibody-dependent cellular cytotoxicity (ADCC), a key mechanism in antiviral immunity. However, the role of NK cell–mediated ADCC in SARS-CoV-2 infection remains unclear, particularly whether it limits viral spread and disease severity or contributes to the immunopathogenesis of COVID-19. We hypothesized that the high-affinity CD16AV176 polymorphism influences these outcomes. Using an in vitro reporter system, we demonstrated that CD16AV176 is a more potent and sensitive activator than the common CD16AF176 allele. To assess its clinical relevance, we analyzed 1,027 patients hospitalized with COVID-19 from the Immunophenotyping Assessment in a COVID-19 cohort (IMPACC), a comprehensive longitudinal dataset with extensive transcriptomic, proteomic, and clinical data. The high-affinity CD16AV176 allele was associated with a significantly reduced risk of ICU admission, mechanical ventilation, and severe disease trajectories. Lower anti–SARS-CoV-2 IgG titers were correlated to CD16AV176; however, there was no difference in viral load across CD16A genotypes. Proteomic analysis revealed that participants homozygous for CD16AV176 had lower levels of inflammatory mediators. These findings suggest that CD16AV176 enhances early NK cell–mediated immune responses, limiting severe respiratory complications in COVID-19. This study identifies a protective genetic factor against severe COVID-19, informing future host-directed therapeutic strategies.

Authors

Anita E. Qualls, Tasha Tsao, Irene Lui, Shion A. Lim, Yapeng Su, Ernie Chen, Dylan Duchen, Holden T. Maecker, Seunghee Kim-Schulze, Ruth R. Montgomery, Florian Krammer, Charles R. Langelier, Ofer Levy, Lindsey R. Baden, Esther Melamed, Lauren I.R. Ehrlich, Grace A. McComsey, Rafick P. Sekaly, Charles B. Cairns, Elias K. Haddad, Albert C. Shaw, David A. Hafler, David B. Corry, Farrah Kheradmand, Mark A. Atkinson, Scott C. Brakenridge, Nelson I. Agudelo Higuita, Jordan P. Metcalf, Catherine L. Hough, William B. Messer, Bali Pulendran, Kari C. Nadeau, Mark M. Davis, Ana Fernandez-Sesma, Viviana Simon, Monica Kraft, Christian Bime, Carolyn S. Calfee, David J. Erle, Joanna Schaenmann, Al Ozonoff, Bjoern Peters, Steven H. Kleinstein, Alison D. Augustine, Joann Diray-Arce, Patrice M. Becker, Nadine Rouphael, IMPACC Network, Jason D. Goldman, Daniel R. Calabrese, James R. Heath, James A. Wells, Elaine F. Reed, Lewis L. Lanier, Harry Pickering, Oscar A. Aguilar

Myocardial pyruvate dehydrogenase kinase 4 drives sex-specific cardiac responses to endotoxemia

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Abstract

Males often experience worse cardiac outcomes than females in sepsis. This study identified pyruvate dehydrogenase kinase 4 (PDK4) as a key mediator of this disparity. PDK4 regulates glucose utilization by inhibiting pyruvate dehydrogenase (PDH) in mitochondria. In a mouse endotoxemia model, a sublethal dose of lipopolysaccharide (LPS, 5 mg/kg) significantly upregulated myocardial PDK4 and induced cardiac dysfunction in males but not females. Cardiac-specific PDK4 overexpression promoted this cardiac dysfunction in both sexes, whereas PDK4 knockout provided protection. In WT males, LPS reduced PDH activity and fatty acid oxidation (FAO) while increasing lactate levels, suggesting a shift toward glycolysis. These effects were exacerbated by PDK4 overexpression but attenuated by knockout. In females, metabolic changes were minimal, aside from reduced FAO in LPS-challenged females overexpressing PDK4. Additionally, a higher LPS dose (8 mg/kg) triggered cardiac dysfunction in females, accompanied by modest upregulation of PDK4, but without changes in PDH or lactate. Dichloroacetate (DCA), restraining PDK-mediated PDH inhibition, improved cardiac function in males but not females during endotoxemia. PDK4 overexpression also exacerbated cardiac mitochondrial damage, reduced mitophagy, and increased oxidative stress and inflammation during endotoxemia — effects that were prevented by PDK4 knockout. These findings suggest that PDK4 drives sex-specific cardiac responses in sepsis.

Authors

John Q. Yap, Azadeh Nikouee, Matthew Kim, Quan Cao, David J. Rademacher, Jessie E. Lau, Ananya Arora, Leila Y. Zou, Yuxiao Sun, Luke Szweda, Hesham Sadek, Sharon Elliot, Benjamin Roos, Marilyn K. Glassberg, Hong-Long Ji, Xiang Gao, Qunfeng Dong, Qun Sophia Zang

Cord blood proteomics identifies biomarkers of early-onset neonatal sepsis

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Abstract

BACKGROUND Symptoms of early-onset neonatal sepsis (EOS) in preterm infants are nonspecific and overlap with normal postnatal physiological adaptations and noninfectious pathologies. This clinical uncertainty and the lack of reliable EOS diagnostics results in liberal use of antibiotics in the first days to weeks of life, leading to increased risk of antibiotic-related morbidities in infants who do not have an invasive infection. METHODS To identify potential biomarkers for EOS in newborn infants, we used unlabeled tandem mass spectrometry proteomics to identify differentially abundant proteins in the umbilical cord blood of infants with and without culture-confirmed EOS. Proteins were then confirmed using immunoassay, and logistic regression and random forest models were built, including both biomarker concentration and clinical variables to predict EOS. RESULTS These data identified 5 proteins that were significantly upregulated in infants with EOS, 3 of which (serum amyloid A, C-reactive protein, and lipopolysaccharide-binding protein) were confirmed using a quantitative immunoassay. The random forest classifier for EOS was applied to a cohort of infants with culture-negative presumed sepsis. Most infants with presumed sepsis were classified as resembling infants in the control group, with low EOS biomarker concentrations.CONCLUSION These results suggest that cord blood biomarker screening may be useful for early stratification of EOS risk among neonates, improving targeted, evidence-based use of antibiotics early in life. FUNDING NIH, Gerber Foundation, Friends of Prentice, Thrasher Research Fund, Ann & Robert H. Lurie Children’s Hospital, and Stanley Manne Children’s Research Institute of Lurie Children’s.

Authors

Leena B. Mithal, Mark E. Becker, Ted Ling-Hu, Young Ah Goo, Sebastian Otero, Aspen Kremer, Surya Pandey, Nicola Lancki, Yawei Li, Yuan Luo, William Grobman, Denise Scholtens, Karen K. Mestan, Patrick C. Seed, Judd F. Hultquist