Research Article
Abstract
T cells have a remarkable capacity to clonally expand, a process that is intricately linked to their effector activities. As vigorously proliferating T cell also incur substantial DNA lesions, how the dividing T cells safeguard their genomic integrity to allow the generation of T effector cells remains largely unknown. Here we report the identification of the apurinic/apyrimidinic endonuclease-1 (Apex1) as an indispensable molecule for the induction of cytopathic T effectors in mouse models. We demonstrate that conditional deletion of Apex1 in T cells resulted in a remarkable accumulation of baseless DNA sites in the genome of proliferating T cells, which further led to genomic instability and apoptotic cell death. Consequently, Apex1-deleted T cells failed to acquire any effector features after activation and failed to mediate autoimmune diseases and allergic tissue damages. Detailed mutational analyses pinpointed the importance of its endonuclease domain in the generation of T effector cells. We provide further evidence that inhibiting the base repair activities of Apex1 with chemical inhibitors similarly abrogated the induction of autoimmune diseases. Collectively, our study suggests that Apex1 serves as a gatekeeper for the generation of cytopathic T cells and that therapeutically targeting Apex1 may have important clinical implications in the treatment of autoimmune diseases.
Authors
Xiang Xiao, Yong Du, Si Sun, Xiaojun Su, Junji Xing, Guangchuan Wang, Steven M. Elzein, Dawei Zou, Laurie J. Minze, Zhuyun Mao, Rafik M. Ghobrial, Ashton A. Connor, Wenhao Chen, Zhiqiang Zhang, Xian C. Li
Abstract
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. While there is no curative treatment, the immune system’s involvement with autoimmune T cells that recognize the protein α-synuclein (α-syn) in a subset of individuals suggests new areas for therapeutic strategies. As not all patients with PD have T cells specific for α-syn, we explored additional autoantigenic targets of T cells in PD. We generated 15-mer peptides spanning several PD-related proteins implicated in PD pathology, including glucosylceramidase β 1 (GBA), superoxide dismutase 1 (SOD1), PTEN induced kinase 1 (PINK1), Parkin RBR E3 ubiquitin protein ligase (parkin), oxoglutarate dehydrogenase (OGDH), and leucine rich repeat kinase 2 (LRRK2). Cytokine production (IFN-γ, IL-5, IL-10) against these proteins was measured using a fluorospot assay and PBMCs from patients with PD and age-matched healthy controls. We identified PINK1, a regulator of mitochondrial stability, as an autoantigen targeted by T cells, as well as its unique epitopes, and their HLA restriction. The PINK1-specific T cell reactivity revealed sex-based differences, as it was predominantly found in male patients with PD, which may contribute to the heterogeneity of PD. Identifying and characterizing PINK1 and other autoinflammatory targets may lead to antigen-specific diagnostics, progression markers, and/or novel therapeutic strategies for PD.
Authors
Gregory P. Williams, Antoine Freuchet, Tanner Michaelis, April Frazier, Ngan K. Tran, João Rodrigues Lima-Junior, Elizabeth J. Phillips, Simon A. Mallal, Irene Litvan, Jennifer G. Goldman, Roy N. Alcalay, John Sidney, David Sulzer, Alessandro Sette, Cecilia S. Lindestam Arlehamn
Abstract
Nerve growth factor (NGF) monoclonal antibodies inhibit chronic pain, yet failed to gain approval due to worsened joint damage in osteoarthritis patients. We report that neuropilin-1 (NRP1) is a coreceptor for NGF and tropomyosin-related kinase A (TrkA) pain signaling. NRP1 was coexpressed with TrkA in human and mouse nociceptors. NRP1 inhibitors suppressed NGF-stimulated excitation of human and mouse nociceptors and NGF-evoked nociception in mice. NRP1 knockdown inhibited NGF/TrkA signaling, whereas NRP1 overexpression enhanced signaling. NGF bound NRP1 with high affinity and interacted with and chaperoned TrkA from the biosynthetic pathway to the plasma membrane and endosomes, enhancing TrkA signaling. Molecular modeling suggested that the C-terminal R/KXXR/K NGF motif interacts with the extracellular “b” NRP1 domain within a plasma membrane NGF/TrkA/NRP1 of 2:2:2 stoichiometry. G α interacting protein C-terminus 1 (GIPC1), which scaffolds NRP1 and TrkA to myosin VI, colocalized in nociceptors with NRP1/TrkA. GIPC1 knockdown abrogated NGF-evoked excitation of nociceptors and pain-like behavior. Thus, NRP1 is a nociceptor-enriched coreceptor that facilitates NGF/TrkA pain signaling. NRP binds NGF and chaperones TrkA to the plasma membrane and signaling endosomes via the GIPC1 adaptor. NRP1 and GIPC1 antagonism in nociceptors offers a long-awaited nonopioid alternative to systemic antibody NGF sequestration for the treatment of chronic pain.
Authors
Chloe J. Peach, Raquel Tonello, Elisa Damo, Kimberly Gomez, Aida Calderon-Rivera, Renato Bruni, Harsh Bansia, Laura Maile, Ana-Maria Manu, Hyunggu Hahn, Alex R.B. Thomsen, Brian L. Schmidt, Steve Davidson, Amedee des Georges, Rajesh Khanna, Nigel W. Bunnett
Abstract
Red blood cells (RBCs), traditionally recognized for their role in transporting oxygen, play a pivotal role in the body’s immune response by expressing TLR9 and scavenging excess host cell-free DNA. DNA capture by RBCs leads to accelerated RBC clearance and triggers inflammation. Whether RBCs can also acquire microbial DNA during infections is unknown. Murine RBCs acquire microbial DNA in vitro, and bacterial DNA–induced (bDNA-induced) macrophage activation was augmented by WT, but not Tlr9-deleted, RBCs. In a mouse model of polymicrobial sepsis, RBC-bound bDNA was elevated in WT mice but not in erythroid Tlr9–deleted mice. Plasma cytokine analysis in these mice revealed distinct sepsis clusters characterized by persistent hypothermia and hyperinflammation in the most severely affected mice. RBC Tlr9 deletion attenuated plasma and tissue IL-6 production in the most severely affected group. Parallel findings in humans confirmed that RBCs from patients with sepsis harbored more bDNA than did RBCs from healthy individuals. Further analysis through 16S sequencing of RBC-bound DNA illustrated distinct microbial communities, with RBC-bound DNA composition correlating with plasma IL-6 in patients with sepsis. Collectively, these findings unveil RBCs as overlooked reservoirs and couriers of microbial DNA, capable of influencing host inflammatory responses in sepsis.
Authors
L.K. Metthew Lam, Nathan J. Klingensmith, Layal Sayegh, Emily Oatman, Joshua S. Jose, Christopher V. Cosgriff, Kaitlyn A. Eckart, John McGinnis, Piyush Ranjan, Matthew Lanza, Nadir Yehya, Nuala J. Meyer, Robert P. Dickson, Nilam S. Mangalmurti
Abstract
Antagonists — such as Ziconotide and Gabapentin — of the CaV2.2 (N-type) calcium channels are used clinically as analgesics for chronic pain. However, their use is limited by narrow therapeutic windows, difficult dosing routes (Ziconotide), misuse, and overdoses (Gabapentin), as well as a litany of adverse effects. Expansion of novel pain therapeutics may emerge from mechanism-based interrogation of CaV2.2. Here, we report the identification of C2230, an aryloxy-hydroxypropylamine, as a CaV2.2 blocker. C2230 trapped and stabilized inactivated CaV2.2 in a slow-recovering state and accelerated the open-state inactivation of the channel, conferring an advantageous use-dependent inhibition profile. C2230 inhibited CaV2.2 during high-frequency stimulation, while sparing other voltage-gated ion channels. C2230 inhibited CaV2.2 in dorsal root and trigeminal ganglia neurons from rats, marmosets, and humans in a G-protein-coupled-receptor–independent manner. Further, C2230 reduced evoked excitatory postsynaptic currents and excitatory neurotransmitter release in the spinal cord, leading to relief of neuropathic, orofacial, and osteoarthritic pain-like behaviors via 3 different routes of administration. C2230 also decreased fiber photometry-based calcium responses in the parabrachial nucleus, mitigated aversive behavioral responses to mechanical stimuli after neuropathic injury, and preserved protective pain responses, all without affecting motor or cardiovascular function. Finally, site-directed mutation analysis demonstrated that C2230 binds differently than other known CaV2.2 blockers, making it a promising lead compound for analgesic development.
Authors
Cheng Tang, Kimberly Gomez, Yan Chen, Heather N. Allen, Sara Hestehave, Erick J. Rodríguez-Palma, Santiago Loya-Lopez, Aida Calderon-Rivera, Paz Duran, Tyler S. Nelson, Siva Rama Raju Kanumuri, Bijal Shah, Nihar R. Panigrahi, Samantha Perez-Miller, Morgan K. Schackmuth, Shivani Ruparel, Amol Patwardhan, Theodore J. Price, Paramjit S. Arora, Ravindra K. Sharma, Abhisheak Sharma, Jie Yu, Olga A. Korczeniewska, Rajesh Khanna
Abstract
Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte (CM) TTN expression, we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human CMs from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, whereas heterozygous mice showed an allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted binding sites of NK2 homeobox 5 (NKX2-5) and myocyte enhancer factor 2 (MEF2) within E1 abolished its transcriptional activity. In embryonic mice expressing E1 reporter gene constructs, we validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5– and MEF2-binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2-binding motif that was identified in a patient with unexplained dilated cardiomyopathy (DCM) showed reduced TTN expression. Together, these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.
Authors
Yuri Kim, Seong Won Kim, David Saul, Meraj Neyazi, Manuel Schmid, Hiroko Wakimoto, Neil Slaven, Joshua H. Lee, Olivia Layton, Lauren K. Wasson, Justin H. Letendre, Feng Xiao, Jourdan K. Ewoldt, Konstantinos Gkatzis, Peter Sommer, Bénédicte Gobert, Nicolas Wiest-Daesslé, Quentin McAfee, Nandita Singhal, Mingyue Lun, Joshua M. Gorham, Zolt Arany, Arun Sharma, Christopher N. Toepfer, Gavin Y. Oudit, William T. Pu, Diane E. Dickel, Len A. Pennacchio, Axel Visel, Christopher S. Chen, J.G. Seidman, Christine E. Seidman
Abstract
Group 2 innate lymphoid cells (ILC2s) play a pivotal role in the development of airway hyperreactivity (AHR). However, the regulatory mechanisms governing ILC2 function remain inadequately explored. This study uncovers V-domain Ig suppressor of T cell activation (VISTA) as an inhibitory immune checkpoint crucial for modulating ILC2-driven lung inflammation. VISTA is upregulated in activated pulmonary ILC2s and plays a key role in regulating lung inflammation, as VISTA-deficient ILC2s demonstrate increased proliferation and function, resulting in elevated type 2 cytokine production and exacerbation of AHR. Mechanistically, VISTA stimulation activates Forkhead box O1 (FOXO1), leading to modulation of ILC2 proliferation and function. The suppressive effects of FOXO1 on ILC2 effector function were confirmed using FOXO1 inhibitors and activators. Moreover, VISTA-deficient ILC2s exhibit enhanced fatty acid oxidation and oxidative phosphorylation to meet their high energy demands. Therapeutically, VISTA agonist treatment reduces ILC2 function both ex vivo and in vivo, significantly alleviating ILC2-driven AHR. Our murine findings were validated in human ILC2s, whose function was reduced ex vivo by a VISTA agonist, and in a humanized mouse model of ILC2-driven AHR. Our studies unravel VISTA as an immune checkpoint for ILC2 regulation via the FOXO1 pathway, presenting potential therapeutic strategies for allergic asthma by modulating ILC2 responses.
Authors
Mohammad Hossein Kazemi, Zahra Momeni-Varposhti, Xin Li, Benjamin P. Hurrell, Yoshihiro Sakano, Stephen Shen, Pedram Shafiei-Jahani, Kei Sakano, Omid Akbari
Abstract
Lysosomes are implicated in a wide spectrum of human diseases, including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration, and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in patients remains challenging. Here, we report the development of the ‘tagless LysoIP’ method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell–derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients’ lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.
Authors
Daniel Saarela, Pawel Lis, Sara Gomes, Raja S. Nirujogi, Wentao Dong, Eshaan Rawat, Sophie Glendinning, Karolina Zeneviciute, Enrico Bagnoli, Rotimi Fasimoye, Cindy Lin, Kwamina Nyame, Fanni A. Boros, Friederike Zunke, Frederic Lamoliatte, Sadik Elshani, Matthew Jaconelli, Judith J.M. Jans, Margriet A. Huisman, Christian Posern, Lena M. Westermann, Angela Schulz, Peter M. van Hasselt, Dario R. Alessi, Monther Abu-Remaileh, Esther M. Sammler
Abstract
Vitamin D regulates mineral homeostasis. The most biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), is synthesized by CYP27B1 from 25-dihydroxyvitamin D (25D) and is inactivated by CYP24A1. Human monogenic diseases and genome-wide association studies support a critical role for CYP24A1 in regulation of mineral homeostasis, but little is known about its tissue-specific effects. Here, we describe the responses of mice with inducible global deletion, kidney-specific, and intestine-specific deletion of Cyp24a1 to dietary calcium challenge and chronic kidney disease (CKD). Global and kidney-specific Cyp24a1 deletion caused similar syndromes of systemic vitamin D intoxication: elevated circulating 1,25D, 25D, and fibroblast growth factor 23 (FGF23), activation of vitamin D target genes in the kidney and intestine, hypercalcemia, and suppressed parathyroid hormone (PTH). In contrast, mice with intestine-specific Cyp24a1 deletion demonstrated activation of vitamin D target genes exclusively in the intestine, despite no changes in systemic vitamin D levels. In response to a high calcium diet, PTH was suppressed, despite normal serum calcium. In mice with CKD, intestinal Cyp24a1 deletion decreased PTH and FGF23 without precipitating hypercalcemia. These results implicate kidney CYP24A1 in systemic vitamin D regulation while independent local effects of intestinal CYP24A1 could be targeted to treat secondary hyperparathyroidism in CKD.
Authors
Michaela A.A. Fuchs, Alexander Grabner, Melody Shi, Susan L. Murray, Emily J. Burke, Nejla Latic, Venkataramana Thiriveedi, Jatin Roper, Shintaro Ide, Koki Abe, Hiroki Kitai, Tomokazu Souma, Myles Wolf
Abstract
PARP inhibitors (PARPi) have received regulatory approval for the treatment of several tumors, including prostate cancer (PCa), and demonstrate remarkable results in the treatment of castration-resistant prostate cancer (CRPC) patients characterized by defects in homologous recombination repair (HRR) genes. Preclinical studies showed that DNA repair genes (DRG) other than HRR genes may have therapeutic value in the context of PARPi. To this end, we performed multiple CRISPR/Cas9 screens in PCa cell lines using a custom sgRNA library targeting DRG combined with PARPi treatment. We identified DNA ligase 1 (LIG1), essential meiotic structure-specific endonuclease 1 (EME1), and Fanconi anemia core complex associated protein 24 (FAAP24) losses as PARPi sensitizers and assessed their frequencies from 3% to 6% among CRPC patients. We showed that concomitant inactivation of LIG1 and PARP induced replication stress and DNA double-strand breaks, ultimately leading to apoptosis. This synthetic lethality (SL) is conserved across multiple tumor types (e.g., lung, breast, and colorectal), and its applicability might be extended to LIG1-functional tumors through a pharmacological combinatorial approach. Importantly, the sensitivity of LIG1-deficient cells to PARPi was confirmed in vivo. Altogether, our results argue for the relevance of determining the status of LIG1 and potentially other non-HRR DRG for CRPC patient stratification and provide evidence to expand their therapeutic options.
Authors
Giulia Fracassi, Francesca Lorenzin, Francesco Orlando, Ubaldo Gioia, Giacomo D’Amato, Arnau S. Casaramona, Thomas Cantore, Davide Prandi, Frédéric R. Santer, Helmut Klocker, Fabrizio d’Adda di Fagagna, Joaquin Mateo, Francesca Demichelis
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ISSN: 0021-9738 (print), 1558-8238 (online)