Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa-induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with lipopolysaccharides of P. aeruginosa, and is responsible for P. aeruginosa-induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1-dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa-induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa-induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilator-associated lung injury and fibrosis in P. aeruginosa-induced pneumonia.
Pei-Shan Sung, Yu-Chun Peng, Shao-Ping Yang, Cheng-Hsun Chiu, Shie-Liang Hsieh
BACKGROUND. Metabolomic profiling in individuals with chronic kidney disease (CKD) has the potential to identify novel biomarkers and provide insight into disease pathogenesis. Methods: We examined the association between blood metabolites and CKD progression, defined as the subsequent development of end-stage renal disease (ESRD) or estimated glomerular filtrate rate (eGFR) halving, in 1773 participants of the Chronic Renal Insufficiency Cohort (CRIC) study, 962 participants of the African American Study of Kidney Disease and Hypertension (AASK), and 5305 participants of the Atherosclerosis Risk in Communities (ARIC) study. RESULTS. In CRIC, more than half of measured metabolites were associated with CKD progression in minimally adjusted Cox proportional hazards models, but the number and strength of associations were markedly attenuated by serial adjustment for covariates, particularly eGFR. Ten metabolites were significantly associated with CKD progression in fully-adjusted models in CRIC; three of these metabolites were also significant in fully-adjusted models in AASK and ARIC, highlighting potential markers of glomerular filtration (pseudouridine), histamine metabolism (methylimidazoleacetate), and azotemia (homocitrulline). Our findings also nominate N-acetylserine as a potential marker of kidney tubular function, with significant associations with CKD progression observed in CRIC and ARIC. CONCLUSION. Together, our findings demonstrate the application of metabolomics to identify potential biomarkers and causal pathways in CKD progression. TRIAL REGISTRATION. Not applicable FUNDING. This study was supported by the NIH (U01 DK106981, U01 DK106982, U01 DK085689, R01 DK108803, R01 DK124399)
Donghai Wen, Zihe Zheng, Aditya Surapaneni, Bing Yu, Linda Zhou, Wen Zhou, Dawei Xie, Haochang Shou, Julian Avila-Pacheco, Sahir Kalim, Jiang He, Chi-yuan Hsu, Afshin Parsa, Panduranga Rao, James Sondheimer, Raymond Townsend, Sushrut S. Waikar, Casey M. Rebholz, Michelle R. Denburg, Paul L. Kimmel, Ramachandran S. Vasan, Clary B. Clish, Josef Coresh, Harold I. Feldman, Morgan E. Grams, Eugene P. Rhee
BACKGROUND. Increased reinfection rates with SARS-CoV-2 have recently been reported, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. In this study, we used the Johns Hopkins SARS-CoV-2 genomic surveillance data to evaluate the frequency of sequencing validated, confirmed and inferred reinfections between March 2020 and July 2022. METHODS. Patients who had two or more positive SARS-CoV-2 tests in our system with samples sequenced as a part of our surveillance efforts were identified as the cohort for our study. SARS-CoV-2 genomes of patients’ initial and later samples were compared. RESULTS. A total of 755 patients (920 samples) had a positive test at least 90 days after the initial test with a median time between tests of 377 days. Sequencing was attempted on 231 samples and was successful in 127. Successful sequencing spiked during the Omicron surge and showed higher median days from initial infection compared to failed sequences. A total of 122 (98%) patients showed evidence of reinfection, 45 of which had sequence validated reinfection and 77 had inferred reinfections (later sequence showed a clade that was not circulating when the patient was initially infected). Of 45 sequence validated reinfections, 43 (96%) were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%) were vaccinated prior to the second infection, 6 (13%) were Immunosuppressed, and only 2 (4%) were hospitalized. CONCLUSIONS. Sequence validated reinfections increased with the Omicron variant but were generally associated with mild infections.
C. Paul Morris, Raghda E. Eldesouki, Amary Fall, David C. Gaston, Julie M. Norton, Nicholas D. Gallagher, Chun Huai Luo, Omar Abdullah, Eili Y. Klein, Heba H. Mostafa
Mineralocorticoid receptor (MR) antagonists (MRAs) slow cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known anti-inflammatory drugs, remain standard-of-care for DMD, despite substantial side effects. Exact mechanisms underlying MR signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare to glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for one week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80Hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits anti-inflammatory properties without altering leukocyte distribution within skeletal muscles while prednisolone suppresses quadriceps cytokines, but increases diaphragm cytokines and pathology. Anti-inflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for DMD patients.
Zachary M. Howard, Chetan K. Gomatam, Charles P. Rabolli, Jeovanna Lowe, Arden B. Piepho, Shyam S. Bansal, Federica Accornero, Jill A. Rafael-Fortney
Collateral lethality occurs when loss of a gene/protein renders cancer cells dependent on its remaining paralog. Combining genome-scale CRISPR/Cas9 loss-of-function screens coupled with RNA-sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase Vaccinia-Related Kinase 1 (VRK1) for their survival in vivo. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout (KO) sensitized cells to VRK1 loss, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 loss. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas, and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced Barrier-to-Autointegration Factor (BAF) phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas.
Jonathan So, Nathaniel W. Mabe, Bernhard Englinger, Kin-Hoe Chow, Sydney M. Moyer, Smitha Yerrum, Maria C. Trissal, Joana Graca Marques, Jason J. Kwon, Brian H. Shim, Sangita Pal, Eshini Panditharatna, Thomas W. Quinn, Daniel A. Schaefer, Daeun Jeong, David L. Mayhew, Justin Hwang, Rameen Beroukhim, Keith L. Ligon, Kimberly Stegmaier, Mariella G. Filbin, William C. Hahn
The origin and mechanisms of autoantigen generation in systemic lupus erythematosus (SLE) are poorly understood. Here, we identified SLE neutrophils activated in vivo by interferon as a prominent source of Ro52/TRIM21 (hereafter Ro52), a critical autoantigen historically thought to be primarily generated by keratinocytes in SLE. Different to mononuclear cells and keratinocytes, SLE neutrophils are enriched in several unique Ro52 species containing a core sequence encoded by exon-4 (Ro52Ex4) in TRIM21. Ro52Ex4 is the main target of anti-Ro52 antibodies and is found in two Ro52 variants (Ro52α and a novel isoform termed Ro52γ) upregulated in SLE neutrophils. Further analysis of Ro52γ revealed a new subset of autoantibodies against a unique C-terminal domain (Ro52γCT) generated from a frameshift due to the lack of exon-6 in Ro52γ. Antibodies to Ro52Ex4 and Ro52γCT distinguish SLE patient subsets characterized by distinct clinical, laboratory, treatment and transcriptional profiles, which are not discerned by the “classical” anti-Ro52 antibodies. Together, these studies uncover interferon-activated neutrophils as a key source of unique immunogenic forms of Ro52 in SLE. Moreover, the finding of Ro52Ex4 and Ro52γCT as core targets of anti-Ro52 antibodies focus interest on Ro52γ as the potential isoform toward which immunological tolerance is initially lost in SLE.
Eduardo Gomez-Bañuelos, M. Javad Wahadat, Jessica Li, Merlin Paz, Brendan Antiochos, Alessandra Ida Celia, Victoria Andrade, Dylan P. Ferris, Daniel W. Goldman, Erika Darrah, Michelle Petri, Felipe Andrade
Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic PAM-Altering SNP (PAS), we developed an allele-specific CRISPR-Cas9 strategy to permanently inactivate mutant HTT through nonsense- mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant- specific CRISPR-Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR-Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR-Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR-Cas9 strategy inactivate mutant HTT through NMD. In addition, GUIDE-seq analysis and subsequent validation experiments supported high levels of on-target gene specificity. Together, our data demonstrated a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR-Cas9 and supporting its therapeutic potential in HD.
Jun Wan Shin, Eun Pyo Hong, Seri S. Park, Doo Eun Choi, Ihn Sik Seong, Madelynn N. Whittaker, Benjamin P. Kleinstiver, Richard Z. Chen, Jong-Min Lee
BACKGROUND. Apolipoprotein CIII is a regulator of triglyceride (TG) metabolism, and due to its association with risk of cardiovascular disease, is an emergent target for pharmacological intervention. The impact of substantially lowering apoC-III on lipoprotein metabolism is not clear. METHODS. We investigated the kinetics of apolipoproteins B48 and B100 in chylomicrons, VLDL1, VLDL2, IDL and LDL in subjects heterozygous for a loss-of-function (LOF) mutation in the APOC3 gene. Studies were conducted in the post-prandial state to provide a more comprehensive view of the influence of this protein on TG transport. RESULTS. Compared to non-LOF subjects, a genetically-determined decrease in apoC-III resulted in marked acceleration of lipolysis of triglyceride-rich lipoproteins (TRL), increased removal of VLDL remnants from the bloodstream, and a substantial decrease in circulating levels of VLDL1, VLDL2 and IDL particles. Production rates for apolipoprotein B48-containing chylomicrons and apoB100-containing VLDL1 and VLDL2 were not different between LOF carriers and non-carriers. Likewise, the rate of production of LDL was not affected by the lower apoC-III level, nor was the concentration of LDL-apoB100 or its clearance rate. CONCLUSION. These findings indicate that apoC-III lowering will have a marked effect on TRL and remnant metabolism, with possibly significant consequences for cardiovascular disease prevention. TRIAL REGISTRATIONS. Clinical Trials NCT04209816 and NCT01445730 FUNDING. This project was funded by grants from Swedish Heart-Lung Foundation, Swedish Research Council, ALF grant from the Sahlgrenska University Hospital, Novo Nordisk Foundation, Sigrid Juselius Foundation, Helsinki University Hospital Government Research funds, Finnish Heart Foundation, and Finnish Diabetes Research Foundation.
Marja-Riitta Taskinen, Elias Björnson, Niina Matikainen, Sanni Söderlund, Joel Rämo, Mari-Mia Ainola, Antti Hakkarainen, Carina Sihlbom, Annika Thorsell, Linda Andersson, Per-Olof Bergh, Marcus Henricsson, Stefano Romeo, Martin Adiels, Samuli Ripatti, Markku Laakso, Chris J. Packard, Jan Borén
Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in DM1 patients resemble the appearance of an accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from DM1 patients and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which related to the accumulation of cellular senescence. The data from transcriptome analyses were corroborated in human myoblasts and blood samples as well as in mouse and Drosophila models of the disease. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts, whereas DM1 Drosophila model showed reduced longevity and impaired locomotor activity. Moreover, functional studies highlighted the impact of BMI1 and downstream p16INK4A/RB and ARF/p53/p21CIP pathways in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds, Quercetin, Dasatinib, or Navitoclax, reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in Drosophila in vivo. Our results identified the accumulation of senescence as part of DM1 pathophysiology and therefore, demonstrated the efficacy of senolytic compounds in the pre-clinical setting.
Mikel García-Puga, Ander Saenz-Antoñanzas, Gorka Gerenu, Alex Arrieta, Roberto Fernandez-Torron, Miren Zulaica, Amets Saenz, Joseba Elizazu, Gisela Nogales-Gadea, Shahinaz M. Gadalla, Marcos J. Araúzo-Bravo, Adolfo Lopez de Munain, Ander Matheu
The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kD exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2-low MM had a better response to PIs and a longer overall survival than patients with ISG20L2-high MM. Biotinylated-bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance (SPR) assay further confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2-high MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.
Yan Yang, Yuhan Gao, Jingcao Huang, Zhuang Yang, Hongmei Luo, Fangfang Wang, Juan Xu, Yushan Cui, Hong Ding, Zhimei Lin, Xinyu Zhai, Ying Qu, Li Zhang, Ting Liu, Lingqun Ye, Ting Niu, Yuhuan Zheng
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