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Abstract
Classical dynamins are large GTPases regulating membrane and cytoskeleton dynamics and are linked to different pathological conditions ranging from neuromuscular diseases to encephalopathy and cancer. Dominant DNM2 (dynamin 2) mutations lead to either mild adult onset or severe neonatal centronuclear myopathy (ADCNM). Our objectives were to better understand the pathomechanism of severe ADCNM and test a potential therapy. Here, we created the Dnm2SL/+ mouse line harboring the common S619L mutation found in patients with severe ADCNM and impairing the conformational switch regulating dynamin self-assembly and membrane remodeling. The Dnm2SL/+ mouse faithfully reproduces severe ADCNM hallmarks with early impaired muscle function and force together with myofibers hypotrophy. It revealed swollen mitochondria lacking cristae as the main ultrastructural defect and potential cause of the disease. Patient analysis confirmed this structural hallmark. In addition, DNM2 reduction with antisense oligonucleotides after disease onset efficiently reverted locomotor and force defects after only 3 weeks of treatment. Most histological defects including mitochondria alteration were partially or fully rescued. Overall, this study highlights an efficient approach to revert the severe form of dynamin-related centronuclear myopathy. These data also reveal that the dynamin conformational switch is key for muscle function and should be targeted for future therapeutic developments.
Authors
Xènia Massana Muñoz, Christine Kretz, Roberto Silva-Rojas, Julien Ochala, Alexia Menuet, Norma B. Romero, Belinda S. Cowling, Jocelyn Laporte
Abstract
Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4 T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a non-cytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.
Authors
Sungtae Park, Brad Griesenauer, Hua Jiang, Djamilatou Adom, Pegah Mehrpouya-Bahrami, Srishti Chakravorty, Majid Kazemian, Tanbeena Imam, Rajneesh Srivastava, Tristan A. Hayes, Julian Pardo, Sarath Chandra Janga, Sophie Paczesny, Mark H. Kaplan, Matthew R. Olson
Abstract
Background: Control of the tuberculosis (TB) pandemic remains hindered, in part, by a lack of simple and accurate measures of treatment efficacy. Current gold standard markers rely on sputum-based assays that are slow and challenging to implement. Previous work identified urinary N1, N12-diacetylspermine (DiAcSpm), neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 as potential biomarkers of active pulmonary TB (ATB). Here, we evaluated their ability to serve as biomarkers of TB treatment response and mycobacterial load. Methods: We analyzed urine samples prospectively collected from two cohorts with ATB: 34 participants from African countries treated with first line TB therapy (HRZE) and followed for one year, and 35 participants from Haiti treated with either HRZE or an experimental drug followed for 14 days. Blinded samples were analyzed by untargeted high-performance liquid chromatography-coupled-time of flight-mass spectrometry. Results: Urinary levels of all seven molecules exhibited significant decreases by week 26 of successful treatment (p=0.01-p<0.0001), and positive correlations with sputum mycobacterial load (p<0.0001). Urinary levels of DiAcSpm exhibited significant decreases in participants treated with HRZE as early as 14 days (p<0.0001) but were unchanged in participants receiving ineffective therapy (p=0.14). Conclusion: Reductions in urinary DiAcSpm, neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 correlated with successful anti-TB treatment and sputum mycobacterial load. Levels of DiAcSpm exhibited reductions capable of differentiating treatment success from failure as early as two weeks after the initiation of chemotherapy, commending its further development as a potentially simple, non-invasive biomarker of treatment response and bacterial load. Funding: This work was supported by the Clinical and Translational Science Center at Weill Cornell College of Medicine (NIH/NCATS 1 UL1 TR002384-02 and KL2TR000458), the Department of Defense (PR170782), the National Institute of Allergy and Infectious Disease grants (NIAID T32AI007613-16, K24 AI098627 and K23 AI131913), the NIH Fogarty International Center grants (R24 TW007988 and TW010062), the National Institute of Health grant (R01 GM135926 ), the Abby and Howard P. Milstein Program in Chemical Biology and Translational Medicine, and the Tuberculosis Research Units Networks (TBRU-N, AI111143).
Authors
Qianjing Xia, Myung Hee Lee, Kathleen F. Walsh, Kathrine McAulay, James M. Bean, Daniel W. Fitzgerald, Kathryn M. Dupnik, Warren D. Johnson, Jean W. Pape, Kyu Y. Rhee, Flonza Isa
Abstract
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies to the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological enzyme-linked immunosorbent assay (ELISA) using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 RT-PCR confirmed SARS-CoV-2 hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies to the two different viral proteins showed a moderate correlation. Antibodies to N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.6% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD binding antibodies exhibited neutralizing capacity, but only 74% of N-protein positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein binding antibodies does not always correlate with presence of S-RBD neutralizing antibodies, and cautions against the extensive use of N-protein based serology testing for determination of potential COVID-19 immunity.
Authors
Kathleen M. McAndrews, Dara P. Dowlatshahi, Jianli Dai, Lisa M. Becker, Janine Hensel, Laura M. Snowden, Jennifer M. Leveille, Michael R. Brunner, Kylie Holden, Nikolas S. Hopkins, Alexandria Harris, Jerusha J. Kumpati, Michael A. Whitt, J. Jack Lee, Luis Ostrosky-Zeichner, Ramesha Papanna, Valerie LeBleu, James Allison, Raghu Kalluri
Abstract
Angiogenesis is essential for cardiac functional recovery after myocardial infarction (MI). HSPA12B is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) plays an important role in tumor angiogenesis. This study investigated the cooperative role of HSPA12B and YAP in angiogenesis post-MI. Silencing of either HSPA12B or YAP impairs hypoxia-promoted endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppresses YAP expression and nuclear translocation following hypoxia. Knockdown of YAP attenuates hypoxia-stimulated HSPA12B nuclear translocation and abrogates HSPA12B-promoted endothelial cell angiogenesis. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. ChIP assay shows that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain4 (TEAD4) and a co-activator in YAP-associated angiogenesis. In vivo studies using the MI model show that endothelial specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impairs angiogenesis and exacerbates cardiac dysfunction, when compared with wild type (WT) mice. MI increased YAP expression and nuclear translocation in WT hearts, but not in eHspa12b-/- hearts. HSPA12B expression and nuclear translocation were up-regulated in WT MI hearts, but not in eYap-/- MI myocardium. Our data demonstrated that endothelial HSPA12B is a novel target and co-activator for YAP/TEAD4 and cooperates with YAP to regulate endothelial angiogenesis post-MI.
Authors
Min Fan, Kun Yang, Xiaohui Wang, Yana Wang, Fei Tu, Tuanzhu Ha, Li Liu, David L. Williams, Chuanfu Li
Abstract
Age-associated systemic, chronic inflammation is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can partially rescue age-related thymic involution. Given the findings that TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected naturally young TECs can lead to middle-aged thymus regrowth, we attempted to extend these two findings by combining them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these two-types of FREFs directly into the aged murine thymus. We found significant regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and auto-reactive T cell-mediated inflammation in old mice. Therefore, this strategy has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to significantly, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in the elderly.
Authors
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
Abstract
Abstract: S-Nitroso-L-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase (sGC)-independent effects are stereoselective - that is, not recapitulated by S-nitroso-D-cysteine (D-CSNO) – and are inhibited by chemical cogeners. However, candidate L-CSNO receptors have never been identified. Here, we have used two complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy, hydrogen deuterium exchange and, in Kv1.1/Kv1.2/Kvβ2 overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv 1.1 prevented this effect. In intact animals, L-CSNO injection at level of the carotid body (CB) dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO cogener S-methyl-L-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.
Authors
Benjamin Gaston, Laura A. Smith, Jürgen Bosch, James M. Seckler, Diana L. Kunze, Janna Kiselar, Nadzeya Marozkina, Craig Hodges, Patrick Wintrode, Kellen McGee, Tatiana Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis
Abstract
New strategies are needed to enhance the efficacy of anti-programmed cell death protein (PD-1) antibody (Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen primed T cells to macrophage conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma specific killing. Genetic or chemical PPT1 inhibition resulted an M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells (MDSCs) in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1) pathway activation and the secretion of interferon β (IFN-β) in macrophages which was a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for a melanoma clinical trial testing this new immunotherapy combination and may also be effective in other cancers.
Authors
Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, Ravi K. Amaravadi
Abstract
Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found two peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on the Tim-4 (T-cell immunoglobulin and mucin domain containing 4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FIP200 resulted in Tim-4+ TAM loss via ROS-mediated apoptosis, and elevated T cell-immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated CRIg (complement receptor of the Immunoglobulin superfamily) positive macrophages were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat ovarian cancer patients with peritoneal metastasis.
Authors
Houjun Xia, Shasha Li, Xiong Li, Weichao Wang, Yingjie Bian, Shuang Wei, Sara Grove, Weimin Wang, Linda Vatan, J. Rebecca Liu, Karen McLean, Ramandeep Rattan, Adnan R. Munkarah, Jun-Lin Guan, Ilona Kryczek, Weiping Zou
Abstract
Electroconvulsive therapy is highly effective in neuropsychiatric disorders by unknown mechanisms. Microglial toxicity plays key role in neuroinflammatory and degenerative diseases, where there is critical shortage in therapies. This study examined the effects of electroconvulsive seizures (ECS) on chronic neuroinflammation and microglial neurotoxicity.Electric brain stimulation inducing full tonic-clonic seizures during chronic relapsing-progressive experimental autoimmune encephalomyelitis (EAE) reduced spinal immune cell infiltration, reduced myelin and axonal loss, and prevented clinical deterioration. Using the transfer EAE model we examined the effect of ECS on systemic immune response in donor mice versus ECS effect on CNS innate immune activity in recipient mice. ECS did not affect encephalitogenicity of systemic T cells, but targeted the CNS directly to inhibit T-cell induced neuroinflammation. In vivo and ex-vivo assays indicated that ECS suppressed microglial neurotoxicity, by reducing iNOS expression, nitric oxide and reactive oxygen species (ROS) production, and by reducing CNS oxidative stress. Microglia from ECS treated EAE mice expressed less T cell stimulatory and chemoattractant factors. Our finding indicate that Electroconvulsive therapy targets the CNS innate immune system to reduce neuroinflammation by attenuating microglial neurotoxicity. These findings signify a novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric and neurodegenerative diseases.
Authors
Smadar Goldfarb, Nina Fainstein, Tamir Ben-Hur
Abstract
Background Tuberculosis (TB) kills more people than any other infection and new diagnostic tests to identify active cases are urgently required. We aimed to discover and verify novel markers for TB in non-depleted plasma. Methods We applied an optimised quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation hyphenated with high-resolution mass spectrometry (q3D LC-MS) to study non-depleted plasma of 11 patients with active TB compared to 10 healthy control donors. Prioritised candidates were verified in an independent UK-based (n=118) and a South African cohorts (n=203). Results We generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We further analysed the predominantly low molecular weight sub-proteome; identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤1%, q-value ≤0.05). Biological network analysis showed regulation of new pathways involving lipid and organophosphate ester transport. Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in two independent cohorts. These proteins were elevated in both TB and other respiratory diseases (ORD). Receiver-operating-characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP and SAA1) exhibited discriminatory power in distinguishing between TB and ORD (AUC =0.81). Conclusions We report the most comprehensive TB plasma proteome to date, identifying numerous novel markers with verification in two independent cohorts, which led to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test. Funding Colombia: Colciencias. UK: Medical Research Council, Innovate UK, National Institute for Health Research, Academy of Medical Sciences. Peru: Program for Advanced Research Capacities for AIDS. South Africa: Wellcome Centre for Infectious Diseases Research.
Authors
Diana J. Garay-Baquero, Cory H. White, Naomi F. Walker, Marc Tebruegge, Hannah F. Schiff, Cesar Ugarte-Gil, Stephen Morris-Jones, Ben G. Marshall, Antigoni Manousopoulou, John H. Adamson, Andres F. Vallejo, Magdalena K. Bielecka, Robert J. Wilkinson, Liku B. Tezera, Christopher H. Woelk, Spiros D. Garbis, Paul Elkington
Abstract
Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1 thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further reported that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system indicating NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation dependent functions of key proteins involved in the DDR.
Authors
Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abian, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, Jose Luis Neira, Juan L. Iovanna
Abstract
Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and skeletal abnormalities. Biallelic mutations in SBDS, which encodes a ribosome maturation factor, are found in 90% of SDS cases. Sbds-/- mice are embryonic lethal. Using CRISPR/Cas9 editing, we created sbds-deficient zebrafish strains. Sbds protein levels progressively decreased and became undetectable at 10 days post fertilization (dpf). Polysome analysis revealed decreased 80S ribosomes. Homozygous mutant fish developed normally until 15 dpf. Mutant fish subsequently have stunted growth and shows signs of atrophy in pancreas, liver, and intestine. In addition, neutropenia occurred by 5 dpf. Upregulation of tp53 mRNA did not occur until 10 dpf and inhibition of proliferation correlating with death by 21 dpf. Transcriptome analysis showed tp53 activation through upregulation of genes involved in cell cycle arrest, cdkn1a and ccng1, and apoptosis, puma and mdm2. However, elimination of Tp53 function did not prevent lethality. Because of growth retardation and atrophy of intestinal epithelia, we studied the effects of starvation on wildtype fish. Starved wildtype fish showed intestinal atrophy, zymogen granule loss, and tp53 upregulation – similar to the mutant phenotype. In addition, there was reduction in neutral lipid storage and ribosomal protein amount, similar to the mutant phenotype. Thus, loss of Sbds in zebrafish phenocopies much of the human disease and is associated with growth arrest and tissue atrophy, particularly of the gastrointestinal system, at the larval stage. A variety of stress responses, some associated with Tp53, contribute to pathophysiology of SDS.
Authors
Usua Oyarbide, Arish N. Shah, Wilmer Amaya-Mejia, Matthew Snyderman, Margaret Kell, Daniela Allende, Eliezer Calo, Jacek Topczewski, Seth Corey
Abstract
One of the most significant adverse post-burn responses is abnormal scar formation, such as keloids. Despite its prolificacy, the underlying pathophysiology of keloid development is unknown. We recently demonstrated that NLRP3 inflammasome, the master regulator of inflammatory and metabolic responses (e.g. aerobic glycolysis), is essential for physiological wound healing. Therefore, burn patients who develop keloids may exhibit altered immunometabolic responses at the site of injury, which interferes with normal healing and portends keloid development. Here, we confirmed keloid NLRP3 activation (caspase-1 (p<0.05), IL1β (p<0.05), IL18 (p<0.01)) and upregulation in Glut1 (p<0.001) and glycolytic enzymes. Burn skin similarly displayed enhanced glycolysis and Glut1 expression (p<0.01). However, Glut1 was significantly higher in keloid compared to non-keloid burn patients (>2 standard deviations above mean). Targeting aberrant glucose metabolism with shikonin, a pyruvate kinase M2 inhibitor, dampened NLRP3-mediated inflammation (caspase-1 (p<0.05), IL1β (p<0.01)) and improved healing in vivo. In summary, burn skin exhibited evidence of Warburg-like metabolism, similar to keloids. Targeting this altered metabolism could change the trajectory towards normal scarring, indicating the clinical possibility of shikonin for abnormal scar prevention.
Authors
Roohi Vinaik, Dalia Barayan, Christopher Auger, Abdikarim Abdullahi, Marc G. Jeschke
Abstract
Background: The complement system plays a key role in host defense but is activated by ischemia-reperfusion injury (IRI). Primary graft dysfunction (PGD) is a form of acute lung injury occurring predominantly due to IRI, which worsens survival after lung transplantation (LTx). Local complement activation is associated with acute lung injury, but whether it is more reflective of allograft injury compared to systemic activation remains unclear. We proposed that local complement activation would help identify those who develop PGD post-LTx. We also aimed to identify which complement activation pathways are associated with PGD. Methods: We performed a multicenter cohort study at the University of Pennsylvania and Washington University. Bronchoalveolar lavage (BAL) and plasma specimens were obtained from recipients within 24 h post-LTx. PGD was scored based on the consensus definition. Complement activation products and components of each arm of the complement cascade were measured using ELISA. Results: In both cohorts, sC4d and sC5b-9 levels were increased in BAL of subjects with PGD compared to those without PGD. Subjects with PGD also had higher C1q, C2, C4, and C4b, compared to subjects without PGD, suggesting classical and lectin pathway involvement. Ba levels were higher in subjects with PGD, suggesting alternative pathway activation. Among lectin pathway-specific components, MBL and FCN-3 had a moderate-to-strong correlation with the terminal complement complex in the BAL but not in the plasma. Conclusion: Complement activation fragments are detected in the BAL within 24 h post-LTx. Components of all three pathways are locally increased in subjects with PGD. Our findings create a precedent for investigating complement-targeted therapeutics to mitigate PGD. Funding: This research was supported by the National Institutes of Health (NIH), American Lung Association, Children’s Discovery Institute, the Robert Wood Johnson Foundation, the Cystic Fibrosis Foundation, the Barnes-Jewish Hospital Foundation, The Danish Hearth Foundation], The Danish Research Foundation of Independent Research, The Svend Andersen Research Foundation and the Novo Nordisk Research Foundation.
Authors
Hrishikesh S. Kulkarni, Kristy Ramphal, Lina Ma, Melanie Brown, Michelle L. Oyster, Kaitlyn Speckhart, Tsuyoshi Takahashi, Derek E. Byers, Mary K. Porteous, Laurel Kalman, Ramsey R. Hachem, Melanie Rushefski, Ja'Nia McPhatter, Marlene Cano, Daniel Kreisel, Masina Scavuzzo, Brigitte Mittler, Edward Cantu, Katrine Pilely, Peter Garred, Jason D. Christie, John Atkinson, Andrew E. Gelman, Joshua M. Diamond
Abstract
Decreased cardiac myosin-binding protein C (cMyBPC) expression due to inheritable mutations is thought to contribute to the hypertrophic cardiomyopathy (HCM) phenotype, suggesting increasing cMyBPC content is of therapeutic benefit. In vitro assays show cMyBPC N-terminal domains (NTDs) contain structural elements necessary and sufficient to modulate acto-myosin interactions, but it is unknown if they can regulate in vivo myocardial function. To test if NTDs can recapitulate the effects of full-length (FL) cMyBPC in rescuing cardiac function in a cMyBPC-null mouse model of HCM, we assessed the efficacy of AAV9 gene transfer of a cMyBPC NTD containing domains C0C2 and compared its therapeutic potential with AAV9-FL gene replacement. AAV9 vectors were administered systemically at neonatal day 1, when early-onset disease phenotypes begin to manifest. A comprehensive analysis of in vivo and in vitro function was performed following cMyBPC gene transfer. Our results show that a systemic injection of AAV9-C0C2 gene transfer significantly improved cardiac function (e.g. 52.24±1.69 ejection fraction in C0C2 treated group compared to 40.07±1.97 in control cMyBPC-/- group, p<0.05) and reduced the histopathologic signs of cardiomyopathy. Furthermore, C0C2 significantly slowed and normalized the accelerated cross-bridge kinetics (32.41% decrease of krel) found in cMyBPC-/- control myocardium. Results indicate that C0C2 can rescue biomechanical defects of cMyBPC deficiency and the NTD may be a target region for therapeutic myofilament kinetic manipulation.
Authors
Jiayang Li, Ranganath Mamidi, Chang Yoon Doh, Joshua B. Holmes, Nikhil Bharambe, Rajesh Ramachandran, Julian E. Stelzer
Abstract
Specialized pro-resolving mediators (SPMs) actively limit inflammation and expedite its resolution by modulating leukocyte recruitment and function. Here we profiled intramuscular lipid mediators via LC-MS based metabolipidomics following myofiber injury and investigated the potential role of SPMs in skeletal muscle inflammation and repair. Both pro-inflammatory eicosanoids and SPMs increased following myofiber damage induced by either intramuscular injection of barium chloride or synergist ablation-induced functional muscle overload. Daily systemic administration of the SPM resolvin D1 (RvD1) as an immunoresolvent limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokine expression, enhanced polymorphonuclear cell clearance, modulated the local muscle stem cell response, and polarized intramuscular macrophages to a more pro-regenerative subset. RvD1 had minimal direct impact on in-vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs can modulate both infiltrating myeloid and resident muscle cell populations. These data reveal the efficacy of immunoresolvents as a novel alternative to classical anti-inflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.
Authors
James F. Markworth, Lemuel A. Brown, Eunice Lim, Carolyn Floyd, Jacqueline Larouche, Jesus A. Castor-Macias, Kristoffer B. Sugg, Dylan C. Sarver, Peter C. D. Macpherson, Carol S. Davis, Carlos A. Aguilar, Krishna Rao Maddipati, Susan V. Brooks
Abstract
Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and non-alcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor (GIPR) than the GLP-1 receptor (GLP-1R), corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIPR but show bias at the GLP-1R to favor cAMP generation over β-arrestin recruitment, coincident with a weaker ability to drive GLP-1R internalization compared with GLP-1. Experiments in primary islets reveal β-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIPR, combined with distinct signaling properties at the GLP-1R, together may account for the promising efficacy of this new investigational agent.
Authors
Francis S. Willard, Jonathan D. Douros, Maria B. N. Gabe, Aaron D. Showalter, David B. Wainscott, Todd M. Suter, Megan E. Capozzi, Wijnand J. C. van der Velden, Cynthia. Stutsman, Guemalli R. Cardona, Shweta Urva, Paul J. Emmerson, Jens J. Holst, David A. D'Alessio, Matthew P. Coghlan, Mette M. Rosenkilde, Jonathan E. Campbell, Kyle W. Sloop
Abstract
The angiopoietin-like protein ANGPTL8 (A8) is one of three ANGPTLs (A8, A3, A4) that coordinate changes in triglyceride (TG) delivery to tissues by inhibiting lipoprotein lipase (LPL), an enzyme that hydrolyzes TG. Previously we showed that A8, which is expressed in liver and adipose tissue, is required to redirect dietary TG from oxidative to storage tissues following food intake. Here we show that A8 from liver and adipose tissue have different roles in this process. Mice lacking hepatic A8 have no circulating A8, high intravascular LPL activity, low plasma TG levels, and evidence of decreased delivery of dietary lipids to adipose tissue. In contrast, mice lacking A8 in adipose tissue have higher postprandial TG levels and no alteration in fatty acid composition in adipocytes. Expression of A8, together with A4, in cultured cells reduced A4 secretion and A4-mediated LPL inhibition. Thus, hepatic A8 (with A3) acts in an endocrine fashion to inhibit intravascular LPL in oxidative tissues, whereas A8 in adipose tissue enhances LPL activity by autocrine/paracrine inhibition of A4. These combined actions of A8 ensure that TG stores are rapidly replenished and sufficient energy is available until the next meal.
Authors
Federico Oldoni, Haili Cheng, Serena Banfi, Viktoria Gusarova, Jonathan C. Cohen, Helen H. Hobbs
Abstract
Dysregulated sensing of self nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type-I interferons by innate cells. Here we show that complexes of self-DNA and autoantibodies (DNA-IC) contribute to elevated interferon levels via activation of the cGAS-STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp causes a delay in endolysosomal maturation and prolongs the transit time of ingested DNA-IC. Stalling in maturation-defective organelles facilitates leakage of DNA-IC into the cytosol, promoting activation of the TBK1-STING pathway. Genetic deletion of STING, STING and cGAS chemical inhibitors abolish interferon production and rescue systemic activation of interferon stimulated genes in vivo. These data unveil the contribution of cytosolic self-nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodelling to prevent innate activation.
Authors
Giulia Maria Piperno, Asma Naseem, Giulia Silvestrelli, Roberto Amadio, Nicoletta Caronni, Karla Evelia Cervantes Luevano, Nalan Liv, Judith Klumperman, Andrea Colliva, Hashim Ali, Francesca Graziano, Philippe Benaroch, Hans Haecker, Richard N. Hanna, Federica Benvenuti
