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Abstract
Recent studies have revealed that, beyond their classical role in platelet production, megakaryocytes (MKs) express immune-related genes and exert important immunoregulatory functions. However, it remains unclear whether these functions arise from a single versatile population or from distinct specialized subtypes, and how such subtypes influence infection and inflammation. Here, we identified three specialized immune MK subtypes (imm-MKs)—macrophage-like MKs (Mac-MKs), neutrophil-like MKs (Neu-MKs), and antigen-presenting MKs (APC-MKs)—each defined by distinct transcriptional programs and regulatory networks, with comparable heterogeneity observed in human MKs. Developmental analyses showed that MK immune-related programs increased with maturation and that immune MK subtypes exhibited distinct tissue- and stage-dependent patterns. Functionally, MK subtypes exhibited phase-specific responses during bacterial pneumonia: early infection preferentially induced Mac-MKs and Neu-MKs, which contributed to pulmonary inflammation, whereas during the post-peak acute-to-early-recovery stage, APC-MKs supported a Treg-associated regulatory program that contributed to pulmonary inflammatory control. This MK–Treg axis uncovers a previously unrecognized mechanism of hematopoietic–immune crosstalk. Collectively, our study delineates organ- and stage-specific immune specialization of MKs and identifies immune MK subtypes as dynamic contributors to phase-specific inflammatory and Treg-linked regulatory programs during development and infection.
Authors
Huizhen He, Yezi Ma, Yifei Cai, xiaoyuan chen, Tianran Cheng, Ziqi Huo, Sibei Guo, Meijuan Xia, Dan Feng, Minmin Li, Jingjing Zhao, Nananan Zhao, Pei Su, Wen Zhou, Fei Wang, Cuicui Liu, Hongtao Wang, Jiaxi Zhou
Receptor-tuned Interleukin-2 Mutein Decouples Treg Expansion from ILC2-driven Cutaneous Inflammation
Abstract
Low-dose interleukin-2 (IL-2) and IL-2 muteins are being developed to expand regulatory T cells (Tregs) for autoimmune disease therapy, but injection site reactions (ISRs) remain frequent and poorly understood. Here we show IL2Mut24, a murine surrogate of IL-2 receptor α (IL-2Rα, CD25)-biased IL-2 mutein, enhances Treg expansion yet paradoxically exacerbates cutaneous inflammation compared to wild-type IL-2. Using immunodeficient mice, antibody blockade and adoptive transfer, we identify group 2 innate lymphoid cells (ILC2s) as key drivers of IL-2-induced skin inflammation, defining a CD25-dependent innate activation axis that constrains immune tolerance. In cynomolgus monkeys, the CD25-biased IL-2 mutein efavaleukin alfa promotes greater peripheral Treg expansion than aldesleukin (recombinant human IL-2) but is accompanied by transient increases in IL-5 and dose-dependent ISRs, indicating conservation of this innate inflammatory program across species. To improve the therapeutic window, we engineer receptor clamps by linking IL2Mut24 to antibodies against CD25 to restrict IL-2 access to CD25. This receptor-tuned IL-2 preserves Treg selectivity, suppresses ISRs, and outperforms IL2Mut24 in experimental autoimmune encephalomyelitis by restraining Th17 responses. These findings reveal a conserved innate mechanism underlying IL-2–associated toxicity and establish receptor-tuning as a strategy to improve the safety and efficacy of IL-2–based immunotherapy.
Authors
Anupama Sahoo, Cody Moorman, Mina Tsenkova, Yi Jing, Alexis Valdovinos, Xin Luo, Shiping Lu, Songyu Wang, Renee R. Hukkanen, Madeline Fort, Helen S.H. Tang, Ronya Primack, Shweta Mandavalli, Weiwen Deng
Abstract
Atrial fibrillation (AF) is the most common sustained human cardiac arrhythmia and linked to a drastic increase in stroke and heart failure risk. While sequence variations in the PITX2 non-coding region are the strongest genetic signature of AF risk, the direct role of PITX2 in AF remains a topic of debate. Here, we generated a mouse model (Pitx2Pro41Ser) of a human PITX2 coding variant linked to increased AF risk in the Finnish population. The Pitx2Pro41Ser mice exhibit near-complete penetrance of pacing-induced AF, and transcriptional profiling indicates that Pitx2Pro41Ser is a loss-of-function mutation. In vivo cleavage under targets and tagmentation (CUT&Tag) reveals that PITX2 acts as a transcriptional repressor in developing left atrial cardiomyocytes independent of DNA methylation. Ectopic Pitx2 expression in postnatal right atrial cardiomyocytes via adeno-associated virus (AAV) delivery or genetic overexpression represses right atrial genes and induces a left atrial transcriptome, revealing unexpected plasticity of postnatal atrial cardiomyocytes. Strikingly, delivery of Pitx2 AAV into Pitx2Pro41Ser mice rescues AF inducibility uncovering a direct link between PITX2 activity and AF susceptibility.
Authors
Jeffrey D. Steimle, Yue Yuan, Shaohai Fang, Vaibhav Deshmukh, Christine Rodriguez, Fansen Meng, Taotao Tan, Md. Abul Hassan Samee, Yun Huang, Na Li, James F. Martin
Abstract
Chronic kidney disease (CKD) disrupts mineral homeostasis, leading to impaired skeletal mineralization and cardiovascular pathology, yet the mechanism linking these processes remains undefined. Here we identify glycerol-3-phosphate acyltransferase 2 (GPAT2) as a regulator that couples free fatty acid (FFA) partitioning in osteoblasts to systemic phosphate balance. In mouse and human CKD, elevated osteoblast GPAT2 routes FFA away from mitochondrial oxidation, limiting phosphate incorporation into bone, and toward lysophosphatidic acid synthesis, increasing production of the phosphaturic hormone fibroblast growth factor 23 (FGF23). By contrast, osteoblast-specific Gpat2 deletion restores osteoblast FFA oxidation and skeletal phosphate incorporation, lowers circulating phosphate and FGF23, and attenuates vascular calcification and cardiac hypertrophy in CKD. Further, a bone-targeted GPAT inhibitor recapitulates most of these beneficial effects. These findings establish a fundamental role for osteoblast lipid metabolism in mineral homeostasis and identify GPAT2 in bone as a promising therapeutic target for both skeletal and cardiovascular complications of CKD.
Authors
Petra Simic, Han Xie, Wen Zhou, Yu Fan, Renata C. Pereira, Fangcong Dong, Jason D. Roh, Isidro B. Salusky, Charandeep Singh, Russell P. Goodman, Ashok Khatri, Eugene P. Rhee
Abstract
Authors
Tony Yao, Rita E. Chen, Melissa Yamada, J. Russell Moore, Marissa Jimenez, Tammy Huang, Lynn Cornelius, George Ansstas, Naresha Saligrama, David Y. Chen
Abstract
Cyclin-dependent kinase like 5 (CDKL5) is a serine-threonine kinase enriched in the mammalian brain whose loss of function causes a severe developmental and epileptic encephalopathy named CDKL5 Deficiency Disorder. We previously showed that CDKL5 phosphorylates the microtubule-associated protein MAP1S, but how this regulates microtubule-dependent functions is not well understood. To address this question, we generated MAP1S phosphomutant mice in which the CDKL5 phosphorylation sites S786 and S812 were mutated to alanine (MAP1S S786/812A; MAP1S SA). Using a microtubule cosedimentation assay, we found that dynein binding to microtubules was reduced in MAP1S SA and CDKL5 knockout (KO) brain lysates, and time-lapse imaging showed impaired dynein motility in dendrites from both genotypes. MAP1S SA mice also exhibited reduced AMPA receptor transport, dendritic spine density, and excitatory synapses, accompanied by anxiety-like behavior and motor, social, and memory deficits relevant to CDD. Mechanistically, MAP1S SA and CDKL5 KO neurons showed increased microtubule stability and reduced tubulin tyrosination, consistent with excessive MAP1S-mediated stabilization. Restoring tubulin tyrosination by expressing tubulin-tyrosine ligase rescued dynein transport defects. Together, these findings identify MAP1S phosphorylation as a critical regulator of microtubule dynamics and dynein-dependent transport.
Authors
André T. Lopes, Ondine Janiv, Suzanne Claxton, Sila K. Ultanir
Abstract
Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare condition causing nephrotic syndrome, neuropathy, and other manifestations. SPLIS is caused by mutations in SGPL1, which encodes sphingosine-1-phosphate lyase (SPL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme needed to degrade the bioactive sphingolipid sphingosine-1-phosphate (S1P). Supplementation with the PLP precursor pyridoxine benefits some individuals with PLP-dependent enzymopathies. We investigated whether pyridoxine has therapeutic activity in SPLIS. Neurological improvement, plasma S1P normalization, and increased SPL activity in patient-derived fibroblasts were observed after pyridoxine supplementation in a patient with R222Q-variant SPLIS. Additionally, PLP dose-dependently augmented recombinant R222Q-variant SPL activity. To further explore pyridoxine’s effects, gene editing was employed to create an R222Q-variant SPLIS mouse model. SPLR222Q mice fed pyridoxine-enriched chow lacked obvious phenotypes. However, SPL inactivation, S1P accumulation, proteinuria, and glomerulosclerosis developed in SPLR222Q but not WT mice fed chow with reduced pyridoxine. Ultrastructural analysis and super-resolution microscopy showed podocyte loss and foot process effacement. Transcriptional profiling revealed patterns of cytokine upregulation and extracellular matrix remodeling. Inhibiting S1P production or RhoA/ROCK signaling prevented nephrosis in SPLR222Q mice fed chow lacking pyridoxine. Our findings establish a SPLIS mouse model that recapitulates R222Q-variant SPLIS, demonstrates its responsiveness to pyridoxine, and implicates a S1P/RhoA/ROCK pathway in its pathophysiology. Running Title: Cofactor supplementation in R222Q-variant SPLIS
Authors
Ranjha Khan, Maria L. Allende, Ehtesham Khalid, Joanna Y. Lee, Everett Stone, Max Rodnick-Smith, Audrey Izuhara, Vadym Buncha, Georgina Gyarmati, Janos Peti-Peterdi, Ranya Al-Khaledy, Jeffrey B. Hodgin, Gizachew Tassew, Babak Oskouian, Alina Chen, Yu-Tang Chang, Rachel Zhang, Richard L. Proia, Julie D. Saba
Abstract
Metabolic dysfunction-associated steatohepatitis (MASH) is rising globally despite recent therapeutic advances, highlighting the need to identify new targetable pathways. While dysregulated lipid and amino acid metabolism are established features of MASH, the interplay between these two metabolic pathways remains unexplored. Here, metabolomics of livers from humans and mice with MASH uncovered depletion of lipidated amino acids, where these two distinctive pathways converge. Notably, hepatic levels of N-oleoyl-leucine (C18:1-Leu) were inversely correlated with the severity of MASH-fibrosis. The C18:1-Leu-regulating enzyme, peptidase M20 domain containing 1 (PM20D1), was suppressed in MASH due to attenuated de novo transcription, and stable-isotope tracing confirmed impaired hepatic biosynthesis of C18:1-Leu in MASH. Hepatocyte-specific PM20D1 ablation lowered hepatic C18:1-Leu and exacerbated MASH, whereas hepatocyte-specific PM20D1 overexpression restored C18:1-Leu and ameliorated both MASH progression and established disease. Exogenous administration of C18:1-Leu similarly ameliorated MASH-fibrosis. In silico modeling, transcriptomics, metabolic flux analyses and hepatocyte-specific in vivo manipulations revealed that C18:1-Leu binds and activates peroxisome proliferator-activated receptor alpha to suppress C-C motif chemokine ligand 2, concurrently enhancing fatty acid β-oxidation and attenuating monocyte recruitment to reduce MASH-fibrosis. These findings highlight C18:1-Leu deficiency as a driver and a therapeutic target in MASH-fibrosis, warranting further clinical evaluation.
Authors
Sumit Kumar Anand, Sandeep Das, Fabio Arias, Koral S.E. Richard, Sumati Rohilla, Alia Ghrayeb, M. Peyton McKinney, Lu Wang, Lin Tan, Jibin Ding, Dhananjay Kumar, Nilesh Pandey, Jennifer Lee, Ying Zhao, Suman Mohajan, Gurranna Male, Kelley Nunez, Alexandra C. Finney, Brenna H. Pearson-Gallion, Reethika Gade, Jemiah Maxie, Yu Liu, Harpreet Kaur, Rajan Pandit, Cyrine Ben Dhaou, Joseph Eniafe, Xiaolu Zhang, Zhipeng Liu, Andrew D. Yurochko, Minerva T. Garcia-Barrio, Babak Razani, Fei Chang, Francisco J. Schopfer, Xin Huang, Bishuang Cai, Eyal Gottlieb, Bo Wen, Duxin Sun, Wanqing Liu, Paul T. Thevenot, Ari J. Cohen, Jifeng Zhang, Y. Eugene Chen, A. Wayne Orr, Nirav Dhanesha, Arif Yurdagul Jr., Oren Rom
Abstract
Receptor activator of NFκB ligand (RANKL) is important for bone metabolism, but also modulates immune processes. We showed that mast cells (MCs) are involved in RANKL regulation, but the importance of MC-derived RANKL in skin inflammation has not yet been investigated. In contact hypersensitivity (CHS), the absence of MC-derived RANKL led to reduced skin inflammation due to impaired leukocyte infiltration and blood lymphopenia. Surprisingly, we observed a massive hyperplasia of the distant inguinal lymph nodes in the absence of MC-RANKL. Using adoptive transfers, flow cytometry and whole-mount 3D imaging, we demonstrated that this was not caused by structural maladaptation, but rather by the inability of lymphocytes to exit in a timely manner. Importantly, RANKL deletion in skin MCs only replicated the effect of LN hyperplasia and blood lymphopenia. Moreover, MCs were involved in serum sphingosine-1-phosphate (S1P) regulation during sensitization and challenge. Intravascular administration of S1P restored timely lymphocyte egress, demonstrating a MC-induced organ-spanning RANKL-S1P axis. Consequently, peripheral skin MC-derived RANKL is essential for the timely lymphocyte egress from distant LNs, which may have important implications for the targeted treatment of inflammatory skin diseases.
Authors
Konstantinos Katsoulis-Dimitriou, Waqar Umer, Laura Knop, Tanja Schickschneit, Aaron Hoffmann, Ali El-Bizri, Lea M. Schmitter, Kathleen Baumgart, Nouria Jantz-Naeem, Vladyslava Dovhan, Charlotte Heidelbach, Lars Philipsen, Andreas J. Müller, Stephan Fricke, Sascha Kahlfuß, Thomas Schüler, Jan Dudeck, Anne Dudeck
Abstract
Oil-producing sebaceous glands (SGs), attached to the upper/ middle portion of hair follicles, are indispensable for maintaining skin hydration, and their dysfunction leads to dry skin. However, the molecular mechanisms underlying regulation of SG stem cells remain largely unknown. We identified transcription factor KROX20 as a marker of SG stem cells that sustains their stemness throughout SG morphogenesis and homeostasis. We developed an inducible mouse model in which ablation of KROX20-positive cells causes SG loss and rapidly and robustly induces dry, flaky, alopecia symptoms following induction. This model, termed Xeroflacia (Xero = xerosis, fla = flaky, cia = alopecia), provides a tool for studying the biology of dry skin. Furthermore, we found that KROX20 directly regulates Notch1 transcription to orchestrate the balance between SG stem cell self-renewal and differentiation. Small molecule drug modulation of Notch1 signaling to activate or inhibit the pathway enabled us to regulate SG differentiation to maintain skin oil levels, providing a proof-of-principle that other signaling pathways downstream of Krox20 could be potential therapeutic targets for sebaceous gland-related diseases.
Authors
Yumeng Zhang, Pernelle Pulh, Michelle F. Pan, Yi He, Juanzhu Yan, Annie Li, Renée M. McKay, Lu Q. Le
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Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)