The genomics of primary prostate cancer differ from those of metastatic castration-resistant prostate cancer (mCRPC). We studied genomic aberrations in primary prostate cancer biopsies from patients who developed mCRPC, also studying matching, same-patient, diagnostic, and mCRPC biopsies following treatment. We profiled 470 treatment-naive prostate cancer diagnostic biopsies and, for 61 cases, mCRPC biopsies, using targeted and low-pass whole-genome sequencing (n = 52). Descriptive statistics were used to summarize mutation and copy number profile. Prevalence was compared using Fisher’s exact test. Survival correlations were studied using log-rank test. TP53 (27%) and PTEN (12%) and DDR gene defects (BRCA2 7%; CDK12 5%; ATM 4%) were commonly detected. TP53, BRCA2, and CDK12 mutations were markedly more common than described in the TCGA cohort. Patients with RB1 loss in the primary tumor had a worse prognosis. Among 61 men with matched hormone-naive and mCRPC biopsies, differences were identified in AR, TP53, RB1, and PI3K/AKT mutational status between same-patient samples. In conclusion, the genomics of diagnostic prostatic biopsies acquired from men who develop mCRPC differ from those of the nonlethal primary prostatic cancers. RB1/TP53/AR aberrations are enriched in later stages, but the prevalence of DDR defects in diagnostic samples is similar to mCRPC.
Joaquin Mateo, George Seed, Claudia Bertan, Pasquale Rescigno, David Dolling, Ines Figueiredo, Susana Miranda, Daniel Nava Rodrigues, Bora Gurel, Matthew Clarke, Mark Atkin, Rob Chandler, Carlo Messina, Semini Sumanasuriya, Diletta Bianchini, Maialen Barrero, Antonella Petermolo, Zafeiris Zafeirou, Mariane Fontes, Raquel Perez-Lopez, Nina Tunariu, Ben Fulton, Robert Jones, Ursula McGovern, Christy Ralph, Mohini Varughese, Omi Parikh, Suneil Jain, Tony Elliott, Shahneen Sandhu, Nuria Porta, Emma Hall, Wei Yuan, Suzanne Carreira, Johann S. de Bono
Deficits in social interaction (SI) are a core symptom of autism spectrum disorders (ASDs); 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 ASDs. 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 signaling reduced BLA-NAc glutamatergic activity and that pharmacological 2-AG augmentation via administration of JZL184, a monoacylglycerol lipase inhibitor, blocked SI deficits associated with in vivo BLA-NAc stimulation. Additionally, optogenetic inhibition of the BLA-NAc circuit markedly increased SI in the Shank3B–/– mouse, an ASD model with substantial SI impairment, without affecting SI in WT 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 feed-forward inhibition of NAc neurons in Shank3B–/– mice. These data reveal circuit-level and neuromodulatory mechanisms regulating social function relevant to ASDs 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
Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing 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 hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable 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 NADPH oxidase 2 (NOX2)/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 for preventing 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 of obtaining fresh tissues.METHODS This observational study evaluated 6 individuals with HIV (n = 4 with viral suppression using antiretroviral [ART] therapy; n = 2 with rebound viremia after stopping ART), who provided serial blood samples before death and their bodies for rapid autopsy. HIV reservoirs were characterized by digital droplet PCR, single-genome amplification, and sequencing of full-length (FL) envelope HIV. Phylogeographic methods were used to reconstruct HIV spread, and generalized linear models were tested for viral factors associated with dispersal.RESULTS Across participants, HIV DNA levels varied from approximately 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 (a) the emergence of large, identical, intact HIV RNA populations in blood after cessation of therapy, which repopulated tissues throughout the body; (b) that multiple sites acted as hubs for HIV dissemination but that blood and lymphoid tissues were the main source; (c) that viral exchanges occurred within brain areas and across the blood-brain barrier; and (d) that migration was associated with low HIV divergence between sites and greater diversity at the recipient site.CONCLUSION HIV reservoirs persisted in all deep tissues, and blood was the main source of dispersal. This may explain why eliminating HIV susceptibility in circulating T cells via bone marrow transplants allowed some individuals with HIV to experience therapy-free remission, even though deeper tissue reservoirs were not targeted.TRIAL REGISTRATION Not applicable.FUNDING NIH grants P01 AI31385, P30 AI036214, AI131971-01, AI120009AI036214, HD094646, AI027763, AI134295, and 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
The incidence of human papillomavirus–positive (HPV+) head and neck squamous cell carcinoma (HNSCC) has surpassed that of cervical cancer and is projected to increase rapidly until 2060. The coevolution of HPV with transforming epithelial cells leads to the shutdown of host immune detection. Targeting proximal viral nucleic acid–sensing machinery is an evolutionarily conserved strategy among viruses to enable immune evasion. However, E7 from the dominant HPV subtype 16 in HNSCC shares low homology with HPV18 E7, which was shown to inhibit the STING DNA-sensing pathway. The mechanisms by which HPV16 suppresses STING remain unknown. Recently, we characterized the role of the STING/type I interferon (IFN-I) pathway in maintaining immunogenicity of HNSCC in mouse models. Here we extended those findings into the clinical domain using tissue microarrays and machine learning–enhanced profiling of STING signatures with immune subsets. We additionally showed that HPV16 E7 uses mechanisms distinct from those used by HPV18 E7 to antagonize the STING pathway. We identified NLRX1 as a critical intermediary partner to facilitate HPV16 E7–potentiated STING turnover. The depletion of NLRX1 resulted in significantly improved IFN-I–dependent T cell infiltration profiles and tumor control. Overall, we discovered a unique HPV16 viral strategy to thwart host innate immune detection that can be further exploited to restore cancer immunogenicity.
Xiaobo Luo, Christopher R. Donnelly, Wang Gong, Blake R. Heath, Yuning Hao, Lorenza A. Donnelly, Toktam Moghbeli, Yee Sun Tan, Xin Lin, Emily Bellile, Benjamin A. Kansy, Thomas E. Carey, J. Chad Brenner, Lei Cheng, Peter J. Polverini, Meredith A. Morgan, Haitao Wen, Mark E. Prince, Robert L. Ferris, Yuying Xie, Simon Young, Gregory T. Wolf, Qianming Chen, Yu L. Lei
BACKGROUND Undifferentiated systemic autoinflammatory diseases (USAIDs) 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 underwent screening for the presence of an interferon signature using a standardized type-I IFN-response-gene score (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 8 patients with pulmonary alveolar proteinosis (PAP) and recurrent macrophage activation syndrome (MAS). Among patients with panniculitis and progressive cytopenias, 2 patients were compound heterozygous for potentially novel LRBA mutations, 4 patients harbored potentially novel splice variants in IKBKG (which encodes NF-κB essential modulator [NEMO]), and 6 patients had de novo frameshift mutations in SAMD9L. Of additional 12 patients with elevated IRG-S and CANDLE-, SAVI- or Aicardi-Goutières syndrome–like (AGS-like) phenotypes, 5 patients carried mutations in either SAMHD1, TREX1, PSMB8, or PSMG2. Two patients had anti-MDA5 autoantibody–positive juvenile dermatomyositis, and 7 could not be classified. Patients with LRBA, IKBKG, 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 deleted exon 5–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.TRIAL REGISTRATION ClinicalTrials.gov NCT02974595.FUNDING The Intramural Research Program of the NIH, NIAID, NIAMS, and the Clinical Center.
Adriana A. de Jesus, Yangfeng 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, Maria 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’Neill, 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, Gisella Seminario, Andrew Shulman, Vidya Sivaraman, Mary Beth Son, Yuriy Stepanovskiy, 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
Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (SDM) of arginine, a posttranslational modification involved in oncogenesis and embryonic development. However, the role and mechanisms by which PRMT5 modulates Th cell polarization and autoimmune disease have not yet been elucidated. Here, we found that PRMT5 promoted SREBP1 SDM and the induction of cholesterol biosynthetic pathway enzymes that produce retinoid-related orphan receptor (ROR) agonists that activate RORγt. Specific loss of PRMT5 in the CD4+ Th cell compartment suppressed Th17 differentiation and protected mice from developing experimental autoimmune encephalomyelitis (EAE). We also found that PRMT5 controlled thymic and peripheral homeostasis in the CD4+ Th cell life cycle and invariant NK (iNK) T cell development and CD8+ T cell maintenance. This work demonstrates that PRMT5 expression in recently activated T cells is necessary for the cholesterol biosynthesis metabolic gene expression program that generates RORγt agonistic activity and promotes Th17 differentiation and EAE. These results point to Th PRMT5 and its downstream cholesterol biosynthesis pathway as promising therapeutic targets in Th17-mediated diseases.
Lindsay M. Webb, Shouvonik Sengupta, Claudia Edell, Zayda L. Piedra-Quintero, Stephanie A. Amici, Janiret Narvaez Miranda, Makenzie Bevins, Austin Kennemer, Georgios Laliotis, Philip N. Tsichlis, Mireia Guerau-de-Arellano
Currently, the incidence of HIV infection exceeds the death rate from HIV, and as a result, the prevalence of individuals living with the infection continues to increase. A critical limitation preventing the development of curative strategies is the lack of knowledge regarding mechanisms that allow HIV-infected cells to persist in individuals during combination antiviral therapy (ART). In this issue of the JCI, Chaillon and coworkers assessed HIV-infected cells from various anatomic compartments obtained through a rapid autopsy program of individuals undergoing long-term ART. This study, made possible with strong community collaboration, provides new insights on the potential locations of reservoirs of HIV-infected cells that persist during therapy.
The cardiomyopathy of Duchenne muscular dystrophy (DMD) is an important cause of morbidity and mortality in affected males with this dreaded muscle disease. Previous studies have implicated changes in expression and subcellular localization of connexin-43 (Cx43), the major ventricular gap junction protein, in DMD cardiomyopathy. In this issue of the JCI, Himelman et al. explore how hypophosphorylation of Cx43 at a triplet of serine residues (S325/S328/S330) in the regulatory C-terminus contributes to multiple features of the cardiomyopathy phenotype. Using a mouse model of DMD cardiomyopathy in which phosphomimetic glutamic acids are substituted for serines at these residues in Cx43, Himelman et al. observed reduced gap junction remodeling and lateralization of Cx43 immunosignals, protection against isoproterenol-induced arrhythmias, and improved Ca2+ homeostasis. This study contributes to the understanding of pathologic Cx43 remodeling and encourages further research into developing strategic interventions to mitigate cardiac dysfunction and arrhythmias in DMD patients.
Robin M. Shaw, Jeffrey E. Saffitz
Loss of androgen receptor (AR) signaling dependence occurs in approximately 15%–20% of advanced treatment-resistant prostate cancers, and this may manifest clinically as transformation from a prostate adenocarcinoma histology to a castration-resistant neuroendocrine prostate cancer (CRPC-NE). The diagnosis of CRPC-NE currently relies on a metastatic tumor biopsy, which is invasive for patients and sometimes challenging to diagnose due to morphologic heterogeneity. By studying whole-exome sequencing and whole-genome bisulfite sequencing of cell free DNA (cfDNA) and of matched metastatic tumor biopsies from patients with metastatic prostate adenocarcinoma and CRPC-NE, we identified CRPC-NE features detectable in the circulation. Overall, there was markedly higher concordance between cfDNA and biopsy tissue genomic alterations in patients with CRPC-NE compared with castration-resistant adenocarcinoma, supporting greater intraindividual genomic consistency across metastases. Allele-specific copy number and serial sampling analyses allowed for the detection and tracking of clonal and subclonal tumor cell populations. cfDNA methylation was indicative of circulating tumor content fraction, reflective of methylation patterns observed in biopsy tissues, and was capable of detecting CRPC-NE–associated epigenetic changes (e.g., hypermethylation of ASXL3 and SPDEF; hypomethylation of INSM1 and CDH2). A targeted set combining genomic (TP53, RB1, CYLD, AR) and epigenomic (hypo- and hypermethylation of 20 differential sites) alterations applied to ctDNA was capable of identifying patients with CRPC-NE.
Himisha Beltran, Alessandro Romanel, Vincenza Conteduca, Nicola Casiraghi, Michael Sigouros, Gian Marco Franceschini, Francesco Orlando, Tarcisio Fedrizzi, Sheng-Yu Ku, Emma Dann, Alicia Alonso, Juan Miguel Mosquera, Andrea Sboner, Jenny Xiang, Olivier Elemento, David M. Nanus, Scott T. Tagawa, Matteo Benelli, Francesca Demichelis
BACKGROUND In retinitis pigmentosa (RP), rod photoreceptors degenerate from 1 of many mutations, after which cones are compromised by oxidative stress. N-acetylcysteine (NAC) reduces oxidative damage and increases cone function/survival in RP models. We tested the safety, tolerability, and visual function effects of oral NAC in RP patients.METHODS Subjects (n = 10 per cohort) received 600 mg (cohort 1), 1200 mg (cohort 2), or 1800 mg (cohort 3) NAC bid for 12 weeks and then tid for 12 weeks. Best-corrected visual acuity (BCVA), macular sensitivity, ellipsoid zone (EZ) width, and aqueous NAC were measured. Linear mixed-effects models were used to estimate the rates of changes during the treatment period.RESULTS There were 9 drug-related gastrointestinal adverse events that resolved spontaneously or with dose reduction (maximum tolerated dose 1800 mg bid). During the 24-week treatment period, mean BCVA significantly improved at 0.4 (95% CI: 0.2–0.6, P < 0.001), 0.5 (95% CI: 0.3–0.7, P < 0.001), and 0.2 (95% CI: 0.02–0.4, P = 0.03) letters/month in cohorts 1, 2, and 3, respectively. There was no significant improvement in mean sensitivity over time in cohorts 1 and 2, but there was in cohort 3 (0.15 dB/month, 95% CI: 0.04–0.26). There was no significant change in mean EZ width in any cohort.CONCLUSION Oral NAC is safe and well tolerated in patients with moderately advanced RP and may improve suboptimally functioning macular cones. A randomized, placebo-controlled trial is needed to determine if oral NAC can provide long-term stabilization and/or improvement in visual function in patients with RP.TRIAL REGISTRATION NCT03063021.FUNDING Mr. and Mrs. Robert Wallace, Mr. and Mrs. Jonathan Wallace, Rami and Eitan Armon, Marc Sumerlin, Cassandra Hanley, and Nacuity Pharmaceuticals, Inc.
Peter A. Campochiaro, Mustafa Iftikhar, Gulnar Hafiz, Anam Akhlaq, Grace Tsai, Dagmar Wehling, Lili Lu, G. Michael Wall, Mandeep S. Singh, Xiangrong Kong
Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM 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 2 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, p38 MAPK, NF-κB, 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
Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing–based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER Tnffl/– mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-β–activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1creER Tak1fl/fl– mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor–based approach against GVHD-induced neurotoxicity.
Nimitha R. Mathew, Janaki M. Vinnakota, Petya Apostolova, Daniel Erny, Shaimaa Hamarsheh, Geoffroy Andrieux, Jung-Seok Kim, Kathrin Hanke, Tobias Goldmann, Louise Chappell-Maor, Nadia El-Khawanky, Gabriele Ihorst, Dominik Schmidt, Justus Duyster, Jürgen Finke, Thomas Blank, Melanie Boerries, Bruce R. Blazar, Steffen Jung, Marco Prinz, Robert Zeiser
BACKGROUND Cerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.METHODS Using multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or CM–), iRBC sequestration (Seqhi, Seqlo, Seq0) and HIV status (HIV+ or HIV–).RESULTS We identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV– children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ > CM–, P = 0.004) and sequestration level (Seqhi > Seqlo, P = 0.010). HIV coinfection significantly increased T cell numbers (P = 0.017) and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.CONCLUSION Within the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.FUNDING This research was supported by the Intramural Research Program of the National Institutes of Health.
Brittany A. Riggle, Monica Manglani, Dragan Maric, Kory R. Johnson, Myoung-Hwa Lee, Osorio Lopes Abath Neto, Terrie E. Taylor, Karl B. Seydel, Avindra Nath, Louis H. Miller, Dorian B. McGavern, Susan K. Pierce
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. Using a chicken egg OVA–expressing S. aureus strain to analyze OVA-specific T cell responses, we demonstrated that primary skin infection was 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 DCs, attributable to S. aureus α-toxin (Hla) expression. Gene- 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 affecting 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 M. Kwiecinski, Juliane Bubeck Wardenburg
Cantu syndrome (CS) is a complex disorder caused by gain-of-function (GoF) mutations in ABCC9 and KCNJ8, which encode the SUR2 and Kir6.1 subunits, respectively, of vascular smooth muscle (VSM) KATP channels. CS includes dilated vasculature, marked cardiac hypertrophy, and other cardiovascular abnormalities. There is currently no targeted therapy, and it is unknown whether cardiovascular features can be reversed once manifest. Using combined transgenic and pharmacological approaches in a knockin mouse model of CS, we have shown that reversal of vascular and cardiac phenotypes can be achieved by genetic downregulation of KATP channel activity specifically in VSM, and by chronic administration of the clinically used KATP channel inhibitor, glibenclamide. These findings demonstrate that VSM KATP channel GoF underlies CS cardiac enlargement and that CS-associated abnormalities are reversible, and provide evidence of in vivo efficacy of glibenclamide as a therapeutic agent in CS.
Conor McClenaghan, Yan Huang, Zihan Yan, Theresa M. Harter, Carmen M. Halabi, Rod Chalk, Attila Kovacs, Gijs van Haaften, Maria S. Remedi, Colin G. Nichols
Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls blood phosphate levels by increasing renal phosphate excretion and reducing 1,25-dihydroxyvitamin D3 [1,25(OH)2D] production. Disorders of FGF23 homeostasis are associated with significant morbidity and mortality, but a fundamental understanding of what regulates FGF23 production is lacking. Because the kidney is the major end organ of FGF23 action, we hypothesized that it releases a factor that regulates FGF23 synthesis. Using aptamer-based proteomics and liquid chromatography–mass spectrometry–based (LC-MS–based) metabolomics, we profiled more than 1600 molecules in renal venous plasma obtained from human subjects. Renal vein glycerol-3-phosphate (G-3-P) had the strongest correlation with circulating FGF23. In mice, exogenous G-3-P stimulated bone and bone marrow FGF23 production through local G-3-P acyltransferase–mediated (GPAT-mediated) lysophosphatidic acid (LPA) synthesis. Further, the stimulatory effect of G-3-P and LPA on FGF23 required LPA receptor 1 (LPAR1). Acute kidney injury (AKI), which increases FGF23 levels, rapidly increased circulating G-3-P in humans and mice, and the effect of AKI on FGF23 was abrogated by GPAT inhibition or Lpar1 deletion. Together, our findings establish a role for kidney-derived G-3-P in mineral metabolism and outline potential targets to modulate FGF23 production during kidney injury.
Petra Simic, Wondong Kim, Wen Zhou, Kerry A. Pierce, Wenhan Chang, David B. Sykes, Najihah B. Aziz, Sammy Elmariah, Debby Ngo, Paola Divieti Pajevic, Nicolas Govea, Bryan R. Kestenbaum, Ian H. de Boer, Zhiqiang Cheng, Marta Christov, Jerold Chun, David E. Leaf, Sushrut S. Waikar, Andrew M. Tager, Robert E. Gerszten, Ravi I. Thadhani, Clary B. Clish, Harald Jüppner, Marc N. Wein, Eugene P. Rhee
Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway–modulating therapeutics.
Jeremy M. Sullivan, William W. Motley, Janel O. Johnson, William H. Aisenberg, Katherine L. Marshall, Katy E.S. Barwick, Lingling Kong, Jennifer S. Huh, Pamela C. Saavedra-Rivera, Meriel M. McEntagart, Marie-Helene Marion, Lucy A. Hicklin, Hamid Modarres, Emma L. Baple, Mohamed H. Farah, Aamir R. Zuberi, Cathleen M. Lutz, Rachelle Gaudet, Bryan J. Traynor, Andrew H. Crosby, Charlotte J. Sumner
The ATP-sensitive K+ channel (KATP) is formed by the association of four inwardly rectifying K+ channel (Kir6.x) pore subunits with four sulphonylurea receptor (SUR) regulatory subunits. Kir6.x or SUR mutations result in KATP channelopathies, which reflect the physiological roles of these channels, including but not limited to insulin secretion, cardiac protection, and blood flow regulation. In this issue of the JCI, McClenaghan et al. explored one of the channelopathies, namely Cantu syndrome (CS), which is a result of one kind of KATP channel mutation. Using a knockin mouse model, the authors demonstrated that gain-of-function KATP mutations in vascular smooth muscle resulted in cardiac remodeling. Moreover, they were able to reverse the cardiovascular phenotypes by administering the KATP channel blocker glibenclamide. These results exemplify how genetic mutations can have an impact on developmental trajectories, and provide a therapeutic approach to mitigate cardiac hypertrophy in cases of CS.
Guiling Zhao, Aaron Kaplan, Maura Greiser, W. Jonathan Lederer
Mosquito-transmitted Plasmodium falciparum infection can cause human cerebral malaria (HCM) with high mortality rates. The abundance of infected red blood cells that accumulate in the cerebral vasculature of patients has led to the belief that these brain-sequestered cells solely cause pathogenesis. However, animal models suggest that CD8+ T cells migrate to and accumulate in the brain, directly contributing to experimental cerebral malaria (ECM) mortality. In this issue of the JCI, Riggle et al. explored the brain vasculature from 34 children who died from HCM or other causes and frequently found CD3+ CD8+ T cells in contact with endothelial cells. Further, the authors show that coinfection with HIV enhanced such CD3+ CD8+ T cell luminal distribution. These findings suggest that the mouse model for cerebral malaria may accurately reflect human disease pathology. This study sheds new light on the mechanisms behind blood-brain barrier breakdown in this complicated neurological disease and opens up alternative approaches for treatment.
Laurent Rénia, Georges E.R. Grau, Samuel C. Wassmer
Salt inducible kinases (SIKs) are key regulators of cellular metabolism and growth, but their role in cardiomyocyte plasticity and heart failure pathogenesis remains unknown. Here, we showed that loss of SIK1 kinase activity protected against adverse cardiac remodeling and heart failure pathogenesis in rodent models and human iPSC-derived cardiomyocytes. We found that SIK1 phosphorylated and stabilized histone deacetylase 7 (HDAC7) protein during cardiac stress, an event that is required for pathologic cardiomyocyte remodeling. Gain- and loss-of-function studies of HDAC7 in cultured cardiomyocytes implicated HDAC7 as a pro-hypertrophic signaling effector that can induce c-Myc expression, indicating a functional departure from the canonical MEF2 co-repressor function of class IIa HDACs. Taken together, our findings reveal what we believe to be a previously unrecognized role for a SIK1-HDAC7 axis in regulating cardiac stress responses and implicate this pathway as a potential target in human heart failure.
Austin Hsu, Qiming Duan, Sarah McMahon, Yu Huang, Sarah A.B. Wood, Nathanael S. Gray, Biao Wang, Benoit G. Bruneau, Saptarsi M. Haldar
De novo lipogenesis is tightly regulated by insulin and nutritional signals to maintain metabolic homeostasis; excessive lipogenesis induces lipotoxicity, leading to nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Genetic lipogenic programs have been extensively investigated, but epigenetic regulation of lipogenesis is poorly understood. Here, we identified Slug as an important epigenetic regulator of lipogenesis. Hepatic Slug levels were markedly upregulated in mice by either feeding or insulin treatment. In primary hepatocytes, insulin stimulation increased Slug expression, stability, and interactions with epigenetic enzyme lysine-specific demethylase-1 (Lsd1). Slug bound to the fatty acid synthase (Fasn) promoter where Slug-associated Lsd1 catalyzed H3K9 demethylation, thereby stimulating Fasn expression and lipogenesis. Ablation of Slug blunted insulin-stimulated lipogenesis; conversely, overexpression of Slug, but not a Lsd1 binding-defective Slug mutant, stimulated Fasn expression and lipogenesis. Lsd1 inhibitor treatment also blocked Slug-stimulated lipogenesis. Remarkably, hepatocyte-specific deletion of Slug inhibited the hepatic lipogenic program and protected against obesity-associated NAFLD, insulin resistance, and glucose intolerance in mice. Conversely, liver-restricted overexpression of Slug, but not the Lsd1 binding-defective Slug mutant, had the opposite effects. These results unveil an insulin/Slug/Lsd1/H3K9 demethylation lipogenic pathway that promotes NAFLD and type 2 diabetes.
Yan Liu, Haiyan Lin, Lin Jiang, Qingsen Shang, Lei Yin, Jiandie D. Lin, Wen-Shu Wu, Liangyou Rui
Given the numerous health benefits of exercise, understanding how exercise capacity is regulated is a question of paramount importance. Circulating interleukin-6 (IL-6) levels surge during exercise and IL-6 favors exercise capacity. However, neither the cellular origin of circulating IL-6 during exercise nor the means by which this cytokine enhances exercise capacity have been formally established yet. Here we show through genetic means that the majority of circulating IL-6 detectable during exercise originates from muscle and that to increase exercise capacity, IL-6 must signal in osteoblasts to favor osteoclast differentiation and the release of bioactive osteocalcin in the general circulation. This explains why mice lacking the IL-6 receptor only in osteoblasts exhibit a deficit in exercise capacity of similar severity to the one seen in mice lacking muscle-derived IL-6 (mIL-6), and why this deficit is correctable by osteocalcin but not by IL-6. Furthermore, in agreement with the notion that IL-6 acts through osteocalcin, we demonstrate that mIL-6 promotes nutrient uptake and catabolism into myofibers during exercise in an osteocalcin-dependent manner. Lastly, we show that the crosstalk between osteocalcin and IL-6 is conserved between rodents and humans. This study provides evidence that a muscle-bone-muscle endocrine axis is necessary to increase muscle function during exercise in rodents and humans.
Subrata Chowdhury, Logan C Schulz, Biagio Palmisano, Parminder Singh, Julian Meyer Berger, Vijay K. Yadav, Paula Mera, Helga Ellingsgaard, Juan Hidalgo, Jens C. Brüning, Gerard Karsenty
Chimeric antigen receptor (CAR) T cell therapies can eliminate relapsed and refractory tumors, but the durability of anti-tumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, as well as influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and substituting this asparagine to phenylalanine (CD28-YMFM) promoted durable anti-tumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing towards Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and anti-tumor activity. This finding suggests modifications to the co-stimulatory domains of CAR-T cells can enable longer persistence and thereby improve anti-tumor response.
Sonia Guedan, Aviv Madar, Victoria Casado-Medrano, Carolyn E. Shaw, Anna Wing, Fang Liu, Regina M. Young, Carl H. June, Avery D. Posey Jr.
Chronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease. Despite numerous animal model studies, questions remain about local immune characteristics in human CP. We profiled pancreatic immune cell characteristics in control organ donors and CP patients that included hereditary and idiopathic CP undergoing total pancreatectomy with islet auto-transplantation. Flow cytometric analysis revealed a significant increase in the frequency of CD68+ macrophages in idiopathic CP. In contrast, hereditary CP showed a significant increase in CD3+ T cell frequency, which prompted us to investigate the T cell receptor β (TCRβ) repertoire in CP and controls. TCRβ-sequencing revealed a significant increase in TCRβ repertoire diversity and reduced clonality in both CP groups versus controls. Interestingly, we observed differences in Vβ-Jβ gene family usage between hereditary and idiopathic CP and a positive correlation of TCRβ rearrangements with disease severity scores. Immunophenotyping analyses in hereditary and idiopathic CP pancreata indicate differences in innate and adaptive immune responses, which highlights differences in immunopathogenic mechanism of disease among subtypes of CP. TCR repertoire analysis further suggests a role for specific T cell responses in hereditary versus idiopathic CP pathogenesis providing new insights into immune responses associated with human CP.
Bomi Lee, Julia Z. Adamska, Hong Namkoong, Melena D. Bellin, Joshua J. Wilhelm, Gregory L. Szot, David M. Louis, Mark M. Davis, Stephen Pandol, Aida Habtezion
Massive amounts of health care data can be gleaned from patient health records, pharmacies, clinical trials, insurance providers, regulatory bodies, hospitals and clinics, wearables and sensors, and more. This month in the JCI, a series of Reviews describes how large-scale data sets can provide valuable insights that help personalize the treatment of disease, diagnose and understand rare diseases, optimize clinical trial design, facilitate drug discovery and development, reduce health care costs, and more. This issue’s cover is a playful representation of the complex networks that visualize meaningful connections in large-scale data sets, such as multiomics analyses.
JCI This Month is a digest of the research, reviews, and other features published each month.
The healthcare industry generates massive amounts of data originating from a variety of sources, among them patient health records, pharmacies, clinical trials, insurance providers, regulatory bodies, hospitals and clinics, and wearable devices and sensors. Advances in data processing, analysis, and deep learning techniques have made it possible to leverage this wealth of healthcare data to optimize patient care. In this series, reviews discuss the potential for large-scale datasets to provide valuable insights that help personalize therapies, diagnose and understand rare diseases, optimize clinical trial design, facilitate drug discovery and development, reduce healthcare costs, and more. The reviews also discuss the limitations of existing analysis methods, asserting that analyses of large-scale datasets can complement traditional preclinical and clinical research. As healthcare data increases in volume and complexity, so does its potential to transform medical practice and research.