Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested to play a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD), however a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypo-phosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knock-in mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or non-phosphorylatable alanines (mdxS3A). The mdxS3E but not mdxS3A mice were resistant to Cx43 remodeling with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NOX2/reactive oxygen species (ROS) production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43-remodeling and suggest that targeting Cx43 may be a therapeutic strategy to prevent heart dysfunction and arrhythmias in DMD patients.
Eric Himelman, Mauricio A. Lillo, Julie Nouet, J. Patrick Gonzalez, Qingshi Zhao, Lai-Hua Xie, Hong Li, Tong Liu, Xander H.T. Wehrens, Paul D. Lampe, Glenn I. Fishman, Natalia Shirokova, Jorge E. Contreras, Diego Fraidenraich
Background. Understanding HIV dynamics across the human body is important for cure efforts. This goal has been hampered by technical difficulties and the challenge to obtain fresh tissues. Methods. This observational study evaluated 6 persons with HIV (4 virally suppressed with antiretroviral therapy and 2 with rebound viremia after stopping therapy) who provided blood serially before death and their bodies for rapid autopsy. HIV reservoirs were characterized by digital droplet PCR and single genome amplification and sequencing of full-length (FL) envelope HIV. Phylogeographic methods reconstructed HIV spread and generalized linear models tested for viral factors associated with dispersal. Results. Across participants, HIV DNA levels varied from ~0 to 659 copies/106 cells (IQR:22.9-126.5). A total of 605 intact FL env sequences were recovered in antemortem blood cells and across 28 tissues (IQR:5-9). Sequence analysis showed: 1) emergence of large, identical, intact HIV RNA populations in blood after stopping therapy, which repopulated tissues throughout the body, 2) multiple sites acted as hubs for HIV dissemination but blood and lymphoid tissues were the main source, and 3) viral exchanges occurred within brain areas and across the blood brain barrier, and 4) migration was associated with low HIV divergence between sites and higher diversity at the recipient site. Conclusion. HIV reservoirs persist in all deep tissues, and blood is the main source of dispersal. This may explain why eliminating HIV susceptibility in circulating T cells via bone marrow transplants allowed some people with HIV to have therapy free remission, even though deeper tissue reservoirs were not targeted. Trial registration. Not applicable. Funding. National Institute of Health Grants (P01 AI31385, P30 AI036214, AI131971-01, AI120009AI036214,HD094646, AI027763, AI134295, AI68636).
Antoine Chaillon, Sara Gianella, Simon Dellicour, Stephen A. Rawlings, Timothy E. Schlub, Michelli Faria De Oliveira, Caroline Ignacio, Magali Porrachia, Bram Vrancken, Davey M. Smith
Bruton tyrosine kinase (BTK) is present in a wide variety of cells and may thus have important non-B cell functions. Here we explored the function of this kinase in macrophages with studies of its regulation of the NLRP3 inflammasome. We found that bone marrow-derived macrophages (BMDMs) from BTK-deficient mice or monocytes from X-linked agammaglobulinemia patients exhibit increased NLRP3 inflammasome activity; this was also the case with BMDMs exposed to low doses of BTK inhibitor such as ibrutinib and monocytes from chronic lymphocytic leukemia patients being treated with ibrutinib. In mechanistic studies, we found that BTK binds to NLRP3 during the priming phase of inflammasome activation and in doing so inhibits LPS/nigericin-induced assembly of the NLRP3 inflammasome during the activation phase of inflammasome activation. This inhibitory effect was caused by BTK inhibition of PP2A-mediated dephosphorylation of Ser5 in the pyrin domain of NLRP3. Finally, we showed that BTK-deficient mice are subject to severe experimental colitis and such colitis is normalized by administration of anti-IL-β or an inhibitor of IL-1β signaling, anakinra. Together, these studies strongly suggest that BTK functions as a physiologic inhibitor of NLRP3 inflammasome activation; they thereby explain the fact that XLA patients are prone to develop Crohn’s disease.
Liming Mao, Atsushi Kitani, Eitaro Hiejima, Kim Montgomery-Recht, Wenchang Zhou, Ivan Fuss, Adrian Wiestner, Warren Strober
Cancer–related anemia is present in over 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and red blood cell (RBC) transfusion-independence in AML patients treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor, enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRIPSR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71+ progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.
Ritika Dutta, Tian Yi Zhang, Thomas Köhnke, Daniel Thomas, Miles Linde, Eric Gars, Melissa Stafford, Satinder Kaur, Yusuke Nakauchi, Raymond Yin, Armon Azizi, Anupama Narla, Ravindra Majeti
BACKGROUND. Residual C-peptide is detected in many people for years following the diagnosis of type 1 diabetes; however, the physiologic significance of low levels of detectable C-peptide is not known. METHODS. We studied sixty-three adults with type 1 diabetes classified by peak mixed-meal tolerance test (MMTT) C-peptide as negative (<0.007; n =15), low (0.017–0.200; n =16), intermediate (>0.200–0.400; n =15), or high (>0.400 pmol/mL; n =17). We compared the groups’ glycemia from continuous glucose monitoring (CGM), β-cell secretory responses from a glucose-potentiated arginine (GPA) test, insulin sensitivity from a hyperinsulinemia euglycemic (EU) clamp, and glucose counterregulatory responses from a subsequent hypoglycemic (HYPO) clamp. RESULTS. Low and intermediate MMTT C-peptide groups did not exhibit β-cell secretory responses to hyperglycemia, whereas the high C-peptide group showed increases in both C-peptide and proinsulin (P ≤0.01). All groups with detectable MMTT C-peptide demonstrated acute C-peptide and proinsulin responses to arginine that were positively correlated with peak MMTT C-peptide (P <0.0001 for both analytes). During the EU-HYPO clamp, C-peptide levels were proportionately suppressed in the low, intermediate, and high C-peptide compared to the negative group (P ≤0.0001), whereas glucagon increased from EU to HYPO only in the high C-peptide group compared to negative (P =0.01). CGM demonstrated lower mean glucose and more time-in-range for the high C-peptide group. CONCLUSION. These results indicate that in adults with type 1 diabetes, β-cell responsiveness to hyperglycemia and α-cell responsiveness to hypoglycemia are only observed at high levels of residual C-peptide that likely contribute to glycemic control.
Michael R. Rickels, Carmella Evans-Molina, Henry T. Bahnson, Alyssa Ylescupidez, Kristen J. Nadeau, Wei Hao, Mark A. Clements, Jennifer L. Sherr, Richard E. Pratley, Tamara S. Hannon, Viral N. Shah, Kellee M. Miller, Carla J. Greenbaum
Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive bone marrow failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of two genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor Microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress signaling pathways, including the SMAD2/3, p38MAPK, NF-kB and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.
Alessia Oppezzo, Julie Bourseguin, Emilie Renaud, Patrycja Pawlikowska, Filippo Rosselli
Oncogenic KRAS is a major driver in lung adenocarcinoma (LUAD) that has yet to be therapeutically conquered. Here we report that the SLC7A11/glutathione axis displays metabolic synthetic lethality with oncogenic KRAS. Through metabolomics approaches, we found that mutationally activated KRAS strikingly increased the intracellular cystine level and glutathione biosynthesis. SLC7A11, a cystine/glutamate antiporter conferring specificity for cystine uptake, was overexpressed in patients with KRAS-mutant LUAD and showed positive association with tumor progression. Furthermore, SLC7A11 inhibition either by genetic depletion or pharmacological inhibition by sulfasalazine resulted in selective killing across a panel of KRAS-mutant cancer cells in vitro and tumor growth inhibition in vivo, suggesting the functionality and specificity of SLC7A11 as a therapeutic target. Importantly, we further identified a potent SLC7A11 inhibitor, HG106 that markedly decreased cystine uptake and intracellular glutathione biosynthesis. Furthermore, HG106 exhibited selective cytotoxicity towards KRAS-mutant cells by increasing oxidative stress- and endoplasmic reticulum stress-mediated cell apoptosis. Of note, treatment of KRAS-mutant LUAD with HG106 in several lung cancer preclinical mouse models led to marked tumor suppression and prolonged mouse survival. Overall, our findings reveal that KRAS-mutant LUAD cells are vulnerable to SLC7A11 inhibition, providing promising therapeutic approaches to the treatment of this currently incurable disease.
Kewen Hu, Kun Li, Jing Lv, Jie Feng, Jing Chen, Haigang Wu, Feixiong Cheng, Wenhao Jiang, Jieqiong Wang, Haixiang Pei, Paul J. Chiao, Zhenyu Cai, Yihua Chen, Mingyao Liu, Xiufeng Pang
Staphylococcus aureus remains a leading cause of human infection. These infections frequently recur when the skin is a primary site of infection, especially in infants and children. In contrast, invasive staphylococcal disease is less commonly associated with reinfection, suggesting that tissue-specific mechanisms govern the development of immunity. Knowledge of how S. aureus manipulates protective immunity has been hampered by a lack of antigen-specific models to interrogate the T cell response. Utilizing a chicken egg ovalbumin (OVA)-expressing S. aureus strain to analyze OVA-specific T cell responses, we demonstrated that primary skin infection is associated with impaired development of T cell memory. Conversely, invasive infection induced antigen-specific memory and protected against reinfection. This defect in adaptive immunity following skin infection was associated with a loss of dendritic cells, attributable to S. aureus α-toxin (Hla) expression. Genetic and immunization-based approaches to protect against Hla during skin infection restored the T cell response. Within the human population, exposure to α-toxin through skin infection may modulate the establishment of T cell-mediated immunity, adversely impacting long-term protection. These studies prompt consideration that vaccination targeting S. aureus may be most effective if delivered prior to initial contact with the organism.
Brandon Lee, Reuben Olaniyi, Jakub Kwiecinski, Juliane Bubeck Wardenburg
Deficits in social interaction (SI) are a core symptom of Autism Spectrum Disorders (ASD), however treatments for social deficits are notably lacking. Elucidating brain circuits and neuromodulatory signaling systems that regulate sociability could facilitate a deeper understanding of ASD pathophysiology and reveal novel treatments for ASD. Here we found that in vivo optogenetic activation of the basolateral amygdala-nucleus accumbens (BLA-NAc) glutamatergic circuit reduced SI and increased social avoidance in mice. Furthermore, we found that 2-arachidonoylglycerol (2-AG) endocannabinoid (eCB) signaling reduced BLA-NAc glutamatergic activity, and that pharmacological 2-AG augmentation via administration of JZL184 blocked SI deficits associated with in vivo BLA-NAc stimulation. Additionally, optogenetic inhibition of the BLA-NAc circuit significantly increased SI in the Shank3B-/-, an ASD model with substantial SI impairment, without affecting SI in wild-type mice. Finally, we demonstrated that JZL184 delivered systemically or directly to the NAc also normalized SI deficits in Shank3B-/-mice, while ex vivo JZL184 application corrected aberrant NAc excitatory and inhibitory neurotransmission and reduced BLA-NAc-elicited feedforward inhibition of NAc neurons in Shank3B-/- mice. These data reveal circuit-level and neuromodulatory mechanisms regulating social function relevant to ASD and suggest 2-AG augmentation could reduce social deficits via modulation of excitatory and inhibitory neurotransmission in the NAc.
Oakleigh M. Folkes, Rita Báldi, Veronika Kondev, David J. Marcus, Nolan D. Hartley, Brandon D. Turner, Jade K. Ayers, Jordan J. Baechle, Maya P. Misra, Megan Altemus, Carrie A. Grueter, Brad A. Grueter, Sachin Patel
BACKGROUND. Undifferentiated systemic autoinflammatory diseases (USAID) present diagnostic and therapeutic challenges. Chronic interferon (IFN) signaling and cytokine dysregulation may identify diseases with available targeted treatments. METHODS. Sixty-six consecutively-referred USAID patients underwent standardized evaluation of Type-I IFN-response-gene-signature (IRG-S); cytokine profiling, and genetic evaluation by next-generation sequencing. RESULTS. Thirty-six USAID patients (55%) had elevated IRG-S. Neutrophilic panniculitis (40% vs 0%), basal ganglia calcifications (46% vs 0%), interstitial lung disease (47% vs 5%), and myositis (60% vs 10%) were more prevalent in patients with elevated IRG-S. Moderate IRG-S elevation and highly-elevated serum IL-18 distinguished eight patients with pulmonary alveolar proteinosis (PAP) and recurrent macrophage activation syndrome (MAS). Among patients with panniculitis and progressive cytopenias, two patients were compound heterozygous for novel LRBA mutations, four patients harbored novel splice variants in IKBKG/NEMO, and six patients had de novo frameshift mutations in SAMD9L. Of additional 12 patients with elevated IRG-S and CANDLE-, SAVI- or Aicardi-Goutières-Syndrome (AGS)-like phenotypes, five patients carried mutations in either SAMHD1, TREX1, PSMB8 or PSMG2. Two patients had anti-MDA5 autoantibody-positive juvenile dermatomyositis, and seven could not be classified. Patients with LRBA, IKBKG/NEMO and SAMD9L mutations showed a pattern of IRG elevation that suggests prominent NF-κB activation different from the canonical interferonopathies CANDLE, SAVI and AGS. CONCLUSIONS. In patients with elevated IRG-S, we identified characteristic clinical features and 3 additional autoinflammatory diseases: IL-18-mediated PAP and recurrent MAS (IL-18PAP-MAS), NEMO∆5-associated autoinflammatory syndrome (NEMO-NDAS), and SAMD9L-associated autoinflammatory disease (SAMD9L-SAAD). The IRG-S expands the diagnostic armamentarium in evaluating USAIDs and points to different pathways regulating IRG expression.
Adriana A. de Jesus, Yanfeng Hou, Stephen Brooks, Louise Malle, Angelique Biancotto, Yan Huang, Katherine R. Calvo, Bernadette Marrero, Susan Moir, Andrew J. Oler, Zuoming Deng, Gina A. Montealegre Sanchez, Amina Ahmed, Eric Allenspach, Bita Arabshahi, Edward Behrens, Susanne Benseler, Liliana Bezrodnik, Sharon Bout-Tabaku, AnneMarie C. Brescia, Diane Brown, Jon M. Burnham, María Soledad Caldirola, Ruy Carrasco, Alice Y. Chan, Rolando Cimaz, Paul Dancey, Jason Dare, Marietta DeGuzman, Victoria Dimitriades, Ian Ferguson, Polly Ferguson, Laura Finn, Marco Gattorno, Alexei A. Grom, Eric P. Hanson, Philip J. Hashkes, Christian M. Hedrich, Ronit Herzog, Gerd Horneff, Rita Jerath, Elizabeth Kessler, Hanna Kim, Daniel J. Kingsbury, Ronald M. Laxer, Pui Y. Lee, Min Ae Lee-Kirsch, Laura Lewandowski, Suzanne Li, Vibke Lilleby, Vafa Mammadova, Lakshmi N. Moorthy, Gulnara Nasrullayeva, Kathleen M. O’Neil, Karen Onel, Seza Ozen, Nancy Pan, Pascal Pillet, Daniela G.P. Piotto, Marilynn G. Punaro, Andreas Reiff, Adam Reinhardt, Lisa G. Rider, Rafael Rivas-Chacon, Tova Ronis, Angela Rösen-Wolff, Johannes Roth, Natasha Mckerran Ruth, Marite Rygg, Heinrike Schmeling, Grant Schulert, Christiaan Scott, Gisela Seminario, Andrew Shulman, Vidya Sivaraman, Mary Beth Son, Yuriy Stepanovskyy, Elizabeth Stringer, Sara Taber, Maria Teresa Terreri, Cynthia Tifft, Troy Torgerson, Laura Tosi, Annet Van Royen-Kerkhof, Theresa Wampler Muskardin, Scott W. Canna, Raphaela Goldbach-Mansky
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