Research Article
Abstract
Early initiation of antiretroviral therapy (ART) in perinatally HIV-infected children significantly limits the establishment of the viral reservoir. However, the long-term impact of this intervention remains unclear. We measured the frequency of inducible, translation-competent, and replication-competent proviruses in samples from 62 children who initiated ART early and remained virally suppressed for up to 9.9 years. Only a small fraction of HIV genomes produced HIV transcripts, viral proteins, or infectious virions. Accordingly, replication-competent virus was detected in only 11% of the participants. Despite the predominance of naive cells in pediatric blood, most proviruses were detected in memory CD4+ T cells, especially central memory cells. Longitudinal analysis revealed a biphasic decay in HIV DNA: an initial decline followed by long-term stability, which was associated with extensive expansions of infected T cell clones. In contrast, inducible proviruses declined continuously and became undetectable in most children after 5 years. Near full-length sequencing of 1,305 HIV genomes revealed a dramatic reduction in genetically intact proviruses, from pre-ART to after 7 years of ART. Together, these findings suggest that the intact viral reservoir rapidly decays in early-treated children, offering critical insights for pediatric HIV cure strategies.
Authors
Marta Massanella, Caroline Dufour, Amélie Pagliuzza, Audrée Lemieux, Corentin Richard, Jintanat Ananworanich, Louise Leyre, Thidarat Jupimai, Supranee Buranapraditkun, Rapisa Nantanee, Julie L. Mitchell, Panadda Sawangsinth, Mark de Souza, Piyarat Suntarattiwong, Suparat Kanjanavanit, Pope Kosalaraksa, Thitiporn Borkird, Witaya Petdachai, Kulkanya Chokephaibulkit, Lydie Trautmann, Rémi Fromentin, Thanyawee Puthanakit, Nicolas Chomont, on behalf of the HIV Netherlands Australia Thailand Research Collaboration 209 and 194 (HIVNAT209 and HIVNAT194) study groups
Abstract
Typ515 (W515) mutations in the protein MPL are one of the key driver mutations promoting BCR-ABL-negative myeloproliferative neoplasms (MPNs), but, to our knowledge, their effects on hematopoietic stem cells (HSCs) and MPN-related hematological abnormalities have not been studied in physiological contexts. Here, we established a MplW514L knock-in mouse model, which largely mimics human MPLW515L mutation during hematopoiesis. The mutant mice developed an essential thrombocythemia–like (ET-like) MPN phenotype, displaying excess megakaryopoiesis and thrombocytosis and progressive myelofibrosis. Mechanistically, we observed that the MplW514L-conditioned HSC compartment had a unique disease-initiating capacity; however, it did not exhibit a obvious advantage of competitive repopulation over the WT control. Notably, single-cell analysis and flow cytometry profiles support that MplW514L expression led to a significant expansion of megakaryocyte-biased stem cell fate within the HSC pool. Finally, JAK2 inhibitor treatment phenotypically alleviated the ET signs but failed to eliminate the disease-initiating HSCs. These findings underscore the etiology of physiological expression of the MPLW515L mutation in HSCs and also provide a valuable in vivo model to evaluate potential therapeutic options for patients with MPLW515L-positive MPN.
Authors
Shujing Zhang, Jingjing Liu, Yuan Li, Yi Wang, Lingling Wang, Miaomiao Xu, Yanxia Li, Ge Dong, Shanshan Wang, Yanmei Li, Zhigang Cai, Baobing Zhao
Abstract
BORCS5 encodes a subunit of the BLOC-One-Related Complex (BORC), which is known to promote anterograde movement and fusion of lysosomes. We identified 16 individuals from 9 families with bi-allelic BORCS5 variants, revealing a spectrum of neurodevelopmental and neurodegenerative phenotypes. Carriers of homozygous protein-truncating variants (PTVs), resulting in complete loss of BORCS5, presented with prenatally lethal arthrogryposis multiplex congenita, brain malformations, and neuropathological evidence of neuroaxonal dystrophy. Individuals with missense or splice-site variants presented differently, with microcephaly, developmental epileptic encephalopathy, optic atrophy, spasticity, and progressive movement disorders. In this group, brain MRI showed diffuse hypomyelination, corpus callosum abnormalities, and progressive global cerebral atrophy, consistent with neurodegeneration. Borcs5 KO in zebrafish resulted in microcephaly, motor deficits, and increased seizure susceptibility, mirroring the patients’ clinical presentation. At the cellular level, only BORCS5 PTVs, but not missense variants, led to perinuclear lysosomal clustering and impaired lysosomal axonal trafficking in induced pluripotent stem cell–derived forebrain neurons. However, PTVs and missense variants were associated with reduced lysosomal proteolysis and activity of lysosomal hydrolases glucocerebrosidase and cathepsin B, indicating lysosomal dysfunction. Our study reveals a role for BORCS5 in modulation of lysosomal function, in addition to its known role in lysosome movement and fusion, possibly underlying the diverse clinical manifestations in individuals with BORCS5-related disorders.
Authors
Niccolò E. Mencacci, Georgia Minakaki, Reza Maroofian, Raffaella De Pace, Adeline Paimboeuf, Tiago Branco Fonseca, Tatiana Abramova, Patrick Shannon, David Chitayat, Francesca Magrinelli, Wesley J. Peng, Diptaman Chatterjee, Sara H. Eldessouky, Julia Baptista, Tamas Marton, Julie Vogt, Juan Dario Ortigoza-Escobar, Loreto Martorell, Marta Gómez-Chiari, Ingrid M. Wentzensen, Erik-Jan Kamsteeg, Maha S. Zaki, Annarita Scardamaglia, Giovanni Zifarelli, Zuhair Nasser Al-Hassnan, Elka Miller, Shiri Shinar, Lova S. Matsa, Sri Hari Chandan Appikonda, Ghada A. Otaify, Khalid Al-Thihli, Almundher Al-Maawali, Michael Schwake, Mariasavina Severino, Henry Houlden, Shunmoogum A. Patten, Juan S. Bonifacino, Kailash P. Bhatia, Dimitri Krainc
Abstract
Cellular and molecular heterogeneity in the liver has been increasingly recognized to drive liver fibrosis progression, but the particular events that occur initially in response to liver injury and trigger immune cell recruitment remain unclear. Here, we identify epigenetically aberrant liver sinusoidal endothelial cells (LSECs) as key players in this process. Mechanistically, the epigenetic readers like bromodomain-containing protein 4–dependent (BRD4-dependent) super enhancers (SEs) activate proinflammatory genes, including promyelocytic leukemia (PML). The PML protein, in turn, binds BRD4 and amplifies proinflammatory angiocrine signaling through phase separation–dependent SE activation via PML/BRD4 condensate formation. In mouse models, LSEC-specific depletion of the PML/BRD4 complex mitigates liver inflammation and fibrosis. Single-cell RNA-seq reveals that epigenetically aberrant LSECs exhibit a reprogrammed proinflammatory angiocrine landscape in mouse fibrotic livers. TIMP1+ LSECs promote the recruitment of CD63+ monocyte–derived macrophages (MoMFs) during liver fibrosis progression. Thereby, PML/BRD4 in LSECs governs inflammatory immune cell recruitment in liver fibrosis. Pharmacological BRD4 inhibition or epigenetic PML-SE repression alleviates liver inflammation and fibrosis. In conclusion, PML/BRD4-mediated SE activation via phase separation drives proinflammatory angiocrine signaling in LSECs, initiating the inflammatory cascade and subsequent immune cell recruitment during liver fibrosis.
Authors
Can Gan, Enjiang Lai, Yang Tai, Shuai Chen, Chong Zhao, Wenting Dai, Zhu Yang, Bei Li, Tian Lan, Yang Xiao, Yangkun Guo, Jiaxin Chen, Bo Wei, Zhaodi Che, Sheng Cao, Mengfei Liu, Frank Tacke, Chengwei Tang, Vijay H. Shah, Haopeng Yu, Fei Wang, Zhiyin Huang, Jinhang Gao
Abstract
Immunotherapies achieve durable responses in several cancers but show limited efficacy in refractory hepatocellular carcinoma (HCC). The mechanisms by which hepatoma cells evade immune recognition and limit immune checkpoint blockade (ICB) efficacy are incompletely defined. Here, we identified tumor-intrinsic tescalcin (TESC) as a previously unrecognized phagocytic checkpoint that contributes to immune evasion and ICB resistance in HCC. Mechanistically, H3K4 methylation drove TESC expression in hepatoma cells, facilitating cytosolic Ca2+ buffering and attenuating endoplasmic reticulum (ER) stress–induced calreticulin (CALR) plasma membrane exposure, an essential “eat-me” signal. Consequently, this process abrogated membrane CALR-directed phagocytosis by antigen-presenting cells (APCs), including macrophages and DCs, thereby impairing antigen presentation and subsequent T cell activation. Clinically, we found that elevated H3K4me3-TESC signaling was a promising prognostic biomarker for a poor ICB response in HCC. Importantly, in vivo disruption of this axis restored APC phagocytic function and enhanced the antitumor effects of ICB therapy. Therefore, targeting TESC-driven immune escape and its underlying epigenetic regulation may restore APC function and offer a precise therapeutic strategy to enhance immunotherapeutic efficacy in HCC.
Authors
Jiong-Liang Wang, Jun-Cheng Wang, Yangxun Pan, Minrui He, Zhikai Zheng, Hao Zou, Tianqing Wu, Yuhan Zhang, Zili Hu, Yizhen Fu, Wei Peng, Zhenyun Yang, Li Xu, Yao-Jun Zhang, Min-Shan Chen, Dandan Hu, Jinbin Chen, Ming Zhao, Dong-Ping Chen, Zhong-Guo Zhou
Abstract
Metabolic dysfunction–associated steatotic liver disease (MASLD) and metabolic dysfunction–associated steatohepatitis (MASH) are leading causes of cirrhosis and hepatocellular carcinoma. Defects in autophagy contribute to the development of MASLD; however, the role of Unc-51–like autophagy-activating kinase 1 (ULK1) in the pathophysiology of MASLD remains unclear. Herein, we show that ULK1, a serine/threonine kinase and core autophagy protein, is significantly repressed in human MASH livers, and that hepatocyte-specific loss of ULK1 promotes, unexpectedly, hepatic steatosis and progression to liver fibrosis, without affecting basal autophagy flux. Phospho-proteomics identified the transcriptional coactivator NCOA3 as a downstream phospho-target of ULK1. Mechanistically, ULK1 phosphorylates NCOA3 to repress its transcriptional activity and restrain the CREB/CBP-mediated de novo lipogenic program. Accordingly, a phosphorylation-deficient NCOA3 mutant drives CREB/CBP-mediated lipogenesis, whereas genetic or pharmacological NCOA3 inhibition prevents steatosis, hepatic inflammation, and profibrotic signaling. Hence, ULK1-mediated NCOA3 phosphorylation is a fundamental and druggable checkpoint against the entire MASLD spectrum.
Authors
Young Do Koo, Romilia Tatiana Castillo, Asha Sukumaran Nair, Michael Garneau, Chad Gochee, Zachary V. Campbell, Tashya Shreyas Vakil, Jua Ha, Alex Marti, Jamie Soto, Debajyoti Das, Nuria Martinez-Lopez, Shipra Sharma, Yennifer Delgado, Callie Phung, Immy A. Ashley, Edmund D. Kapelczak, Rashel Jacobo, Eric T. Weatherford, Dao-Fu Dai, Jihane N. Benhammou, Andrea G. Marshall, Antentor Hinton Jr., Ling Yang, Renata O. Pereira, Tara TeSlaa, Mehdi Bouhaddou, Rajat Singh, E. Dale Abel
Abstract
The Western diets (WD), high in saturated fats such as palmitic acid (PA), promotes enteric neurodegeneration and motility disorders. Using murine models, in vitro systems, and human myenteric ganglia, we investigated whether a WD and PA drive iron-dependent ferroptotic injury in the enteric nervous system (ENS). Mice were fed a control diet (CD) or a WD for 12 weeks, with or without systemic AAV9-MaCPNS2 delivery of Nfe2l2 to enteric neurons. Colonic motility was assessed by a bead expulsion assay. We assessed ferroptosis using convergent readouts including iron dysregulation (transferrin receptor 1 [TfR1], ferritin heavy chain 1 [FTH1], labile and mitochondrial iron [Fe2+]), lipid peroxidation (C11-BODIPY and 4-hydroxynonenal [4-HNE]), glutathione peroxidase 4 (GPX4) suppression, and pharmacologic inhibition by ferrostatin 1 (Fer-1) in primary enteric neurons, murine myenteric plexuses, and human networks of myenteric ganglia (nhMPG). WD-fed mice exhibited delayed colonic transit, increased TfR1 and FTH1, and vulnerability of nNOS neurons; these changes were reversed by nuclear factor erythroid 2–related factor 2; (Nfe2l2, also known as Nrf2) overexpression. RNA-seq of PA-treated immortalized murine fetal enteric neurons (IM-FENs) revealed disrupted neurotransmitter signaling, reduced mitochondrial and antioxidant programs, and increased iron import and lipid peroxidation signatures. PA increased labile Fe2+, mitochondrial ROS, membrane depolarization, Ca2+ dysregulation, 4-HNE, and mitoferrin 2 (Mfrn2), whereas Fer-1 preserved mitochondrial integrity, viability, and ENS function. In human nhMPG, PA induced enteric neuronal iron loading and ferroptosis, supporting the translational relevance to diet-associated enteric neuropathy.
Authors
Arun Balasubramaniam, Dmitrii Pavlov, Yunpeng Du, Jeremy Reeves, Alan Harzman, Yunshan Liu, Francesca Cingolani, Xinxu Yuan, Jay M. Patel, Simon Musyoka Mwangi, Peijian He, C. Michael Hart, Wenhui Hu, Fievos L. Christofi, Shanthi Srinivasan
Abstract
Mucociliary clearance (MCC) is an innate defense mechanism that normally keeps airways clean but is dysfunctional in cystic fibrosis (CF) and other muco-obstructive pulmonary diseases. Previously we discovered that activating adenyl cyclase in combination with a cholinergic agonist increased MCC velocity (MCCV) synergistically in ex vivo WT and CF ferret and WT piglets. For what we believe is the first time, we show in vivo synergistic MCC using FDA approved β-adrenergic and cholinergic drugs delivered to the apical surface of WT and CF rats and a CF sheep model. Also, a single dose of the combined drugs is tolerated by humans. As for mechanisms, via ex vivo experiments, we show the combined agonists increased net fluid secretion mainly by stimulating gland secretion and by inhibiting surface absorption, consequently increasing airway surface liquid depth. They also increased net base secretion and increased ciliary beat frequency. Additional ex vivo and in vitro experiments show that the combined agonists had additive effects when combined with highly effective CF transmembrane conductance regulator modulator therapy. The synergistic increase in MCCV induced by this combination of agonists offers therapeutic potential for treating muco-obstructive pulmonary diseases, including CF.
Authors
Nam Soo Joo, Susan E. Birket, Johnathan D. Keith, Juan P. Ianowski, Xiaojie Luan, Jacquelyn Spano, Jennifer B. Bollyky, Marissa N. Dobry, Juan R. Sabater, Ryan W. Williams, John F. Engelhardt, Jeffrey J. Wine, Carlos E. Milla
Abstract
CD48 is a surface molecule with immunoregulatory functions. Following our initial report of a patient with a de novo heterozygous variant at amino acid S220 in the CD48 gene, we describe a second, unrelated patient with similar features of immune dysregulation and a missense change affecting the same residue. To further elucidate the specific pathogenic mechanisms of the identified variants, we reviewed patient records, analyzed patient-derived cells, and employed complementary in vitro and in vivo model systems, including transfected cell lines and CD48-deficient mice. We demonstrate that the variants are associated with altered distribution of CD48, characterized by diminished CD48 surface expression, intracellular retention, and activation of ER stress signaling. Patient T cells displayed increased susceptibility to apoptosis, reduced antiviral responses, and enhanced inflammation. Both patients exhibited T cell lymphopenia, a restricted T cell receptor repertoire diversity, and oligoclonal expansions consistent with antigen-driven selection. In parallel, virally infected CD48-deficient mice recapitulate key aspects of the human phenotype, including delayed antiviral immune responses, impaired viral clearance, and pronounced inflammation. We conclude that identified variants compromise CD48 cell surface localization, impair T cell survival and function, and predispose to inflammation, thereby highlighting the role of CD48 in immune regulation and the prevention of excessive inflammation.
Authors
Samantha Milanesi, Tiziana Lorenzini, Tommaso Marchetti, Diana Tintor, Raquel Planas, Ola Sabet, Lars Malmström, Sudip Acharya, Carson D. Williams, Zoe E. Manning, Jack H. Roser, Angelica C. Ehler, Michael Huber, Seraina Prader, Stefano Vavassori, Cullen M. Dutmer, Jordan K. Abbott, Jana Pachlopnik Schmid
Abstract
Metabolic signals critically shape innate immune responses. Through pharmacological screening of metabolic pathways, we identified aspartate metabolism as a key regulator of cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling. Genetically or aminooxyacetic acid–mediated (AOA-mediated) pharmacologically reducing aspartate levels markedly potentiated the cGAS-STING pathway, leading to stronger upregulation of type I interferons and interferon-stimulated genes. Mechanistically, disruption of de novo pyrimidine synthesis, a major downstream pathway of aspartate, induced mtDNA replication stress and increased mtDNA double-strand breaks, promoting mtDNA release into the cytosol. Cytosolic mtDNA synergized with cGAS-STING agonists to upregulate Z-DNA binding protein 1 (ZBP1), which recruits RIPK1/3 to sustain IRF3 phosphorylation, forming a positive feedback loop that amplifies innate immune signaling. In immunocompetent mouse models, AOA enhanced the antitumor efficacy of STING agonists, chemotherapy, or radiotherapy, whereas aspartate supplementation abrogated these effects. Consistently, aspartate levels negatively correlated with antitumor immunity in colorectal cancer patient samples. Together, our study identifies aspartate–pyrimidine metabolism as a critical metabolic checkpoint that licenses STING signaling by enabling mtDNA stress to cooperate with agonist stimulation, driving type I interferon–dependent ZBP1 induction and feed-forward amplification of STING signaling, thus offering a promising strategy to enhance antitumor immunity.
Authors
Yuheng Liao, Hanze Wang, Hengxin Liu, Xi Chen, Renqiang Sun, Xie Li, Zhen Yang, Chenying Liu, Wei Wu, Ziqian He, Yuzheng Zhao, Ying Mao, Dan Ye, Hui Yang
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