Cutaneous lupus is commonly present in patients with systemic lupus erythematosus (SLE). T cells have been strongly suspected to contribute to the pathology of cutaneous lupus, yet our understanding of the relevant T cell phenotypes and functions remains incomplete. Here, we present a detailed single-cell RNA sequencing profile of T and NK cell populations present within lesional and non-lesional skin biopsies of patients with cutaneous lupus. T cells across clusters from lesional and non-lesional skin biopsies expressed elevated levels of interferon-simulated genes (ISGs); however, compared to T cells from control skin, T cells from cutaneous lupus lesions did not show elevated expression profiles of activation, cytotoxicity, or exhaustion. Integrated analyses indicated that skin lymphocytes appeared less activated and lacked the expanded cytotoxic populations prominent in lupus nephritis kidney T/NK cells. Comparison of skin T cells from lupus and systemic sclerosis skin biopsies further revealed an elevated ISG signature specific to cells from lupus biopsies. Overall, these data represent the first detailed transcriptomic analysis of the T and NK cells in cutaneous lupus at the single cell level and have enabled a cross-tissue comparison that highlights stark differences in composition and activation of T/NK cells in distinct tissues in lupus.
Garrett S. Dunlap, Allison C. Billi, Xianying Xing, Feiyang Ma, Mitra P. Maz, Lam C. Tsoi, Rachael Wasikowski, Jeffrey B. Hodgin, Johann E. Gudjonsson, J. Michelle Kahlenberg, Deepak A. Rao
Spinocerebellar ataxia type1 (SCA1) is an adult-onset neurodegenerative disorder. As disease progresses motor neurons are affected, and their dysfunction contributes towards the inability to maintain proper respiratory function, a major driving force for premature death in SCA1. To investigate the isolated role of motor neurons in SCA1 we created a novel conditional SCA1 (cSCA1) mouse model. This model suppresses expression of the pathogenic SCA1 allele with a floxed stop cassette. cSCA1 mice crossed to a ubiquitous Cre line recapitulate all the major features of the original SCA1 mouse model, except they took twice as long to develop. We found that the cSCA1 mice produce less than half of the pathogenic protein compared to the unmodified SCA1 mice at 3 weeks of age. In contrast, restricted expression of the pathogenic SCA1 allele in motor neurons only leads to a decreased distance traveled of mice in the open field assay and did not affect body weight or survival. We conclude that a fifty percent or greater reduction of the mutant protein has a dramatic effect on disease onset and progression, and that expression of polyglutamine expanded ATXN1 at this level specifically in motor neurons is not sufficient to cause premature lethality.
James P. Orengo, Larissa Nitschke, Meike E. van der Heijden, Nicholas A. Ciaburri, Harry T. Orr, Huda Y. Zoghbi
The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not specifically assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created Rosa26-floxed-stop uracil phosphoribosyl-transferase (Uprt) expressing mice and inoculated 4-thiouracil (TU) to tag nascent RNA at selected time points. Cre-driven TU-tagged RNA was isolated from whole kidneys and demonstrated that volume depletion and ischemia induced different genetic programs. Even lineage related cell types expressed different genes in response to the two stressors. TU-tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa-26 locus, RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.
Tian Huai Shen, Jacob Stauber, Katherine Xu, Alexandra Jacunski, Neal Paragas, Miriam Callahan, Run Banlengchit, Abraham D. Levitman, Beatriz Desanti de Oliveira, Andrew Beenken, Madeleine S. Grau, Edwin Mathieu, Qingyin Zhang, Yuanji Li, Tejashree Gopal, Nathaniel Askanase, Siddarth Arumugam, Sumit Mohan, Pamela I. Good, Jacob S. Stevens, Fangming Lin, Samuel K. Sia, Chyuan-Sheng Lin, Vivette D'Agati, Krzysztof Kiryluk, Nicholas P. Tatonetti, Jonathan Barasch
Mechanisms governing entry and exit of immune cells into, and out of, inflamed joints, remain poorly understood. We sought herein to identify the key molecular pathways regulating such migration. Using murine models of inflammation in conjunction with mice expressing a photoconvertible fluorescent protein we characterized the migration of cells from joints to draining lymph nodes (LN) and performed RNA-seq analysis on isolated cells, identifying genes associated with migration and retention. We further refined the gene list to those specific for joint inflammation. RNA-seq data revealed pathways and genes previously highlighted as characteristic of RA in patient studies, validating the methodology. Focusing on gene regulatory pathways associated with cell migration, adhesion and movement, we identified genes involved in the retention of immune cells in the inflamed joint, namely JAM-A, and identified a role for such molecules in T cell differentiation in vivo.Thus, using a combination of novel cell tracking approaches and murine models of inflammatory arthritis we have identified genes, pathways and anatomically specific tissue signatures regulating cell migration in a variety of inflamed sites. This unique skin and joint specific dataset will be an invaluable resource for the identification of novel therapeutic targets for arthritis and other inflammatory disorders.
Catriona T. Prendergast, Robert A. Benson, Hannah E. Scales, Caio S. Bonilha, John J. Cole, Iain McInnes, James M. Brewer, Paul Garside
Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there is a lack of methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic datasets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal datasets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk and single-cell RNA sequencing time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways as well as impaired type I interferon (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS-CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets.
Xinge Wang, Mark A. Sanborn, Yang Dai, Jalees Rehman
Remodeling of injured sympathetic nerves on the heart after myocardial infarction (MI) contributes to adverse outcomes such as sudden arrhythmic death, yet the underlying structural mechanisms are poorly understood. We sought to examine microstructural changes on the heart after MI and to directly link these changes with electrical dysfunction. We developed a high-resolution pipeline for anatomically precise alignment of electrical maps with structural myofiber and nerve-fiber maps created by customized computer vision algorithms. Using this integrative approach in a mouse model, we identified distinct structure-function correlates to objectively delineate the infarct border zone, a known source of arrhythmias after MI. During tyramine-induced sympathetic nerve activation, we demonstrated regional patterns of altered electrical conduction aligned directly with altered neuroeffector junction distribution, pointing to potential neural substrates for cardiac arrhythmia. This study establishes a synergistic framework for examining structure-function relationships after MI with microscopic precision that has potential to advance understanding of arrhythmogenic mechanisms.
Ching Zhu, Pradeep S. Rajendran, Peter Hanna, Igor R. Efimov, Guy Salama, Charless C. Fowlkes, Kalyanam Shivkumar
Hundreds of genetic variants in KCNQ2 encoding the voltage-gated potassium channel KV7.2 are associated with early onset epilepsy and/or developmental disability, but the functional consequences of most variants are unknown. Absent functional annotation for KCNQ2 variants hinders identification of individuals who may benefit from emerging precision therapies. We employed automated patch clamp recording to assess at an unprecedented scale the functional and pharmacological properties of 79 missense and 2 inframe deletion KCNQ2 variants. Among the variants we studied were 18 known pathogenic variants, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. We analyzed electrophysiological data recorded from 9,480 cells. The functional properties of 18 known pathogenic variants largely matched previously published results and validated automated patch clamp for this purpose. Unlike rare population variants, most disease-associated KCNQ2 variants exhibited prominent loss-of-function with dominant-negative effects, providing strong evidence in support of pathogenicity. All variants responded to retigabine, although there were substantial differences in maximal responses. Our study demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine, a proposed precision therapy for KCNQ2 developmental and epileptic encephalopathy.
Carlos G. Vanoye, Reshma R. Desai, Zhigang Ji, Sneha Adusumilli, Nirvani Jairam, Nora Ghabra, Nishtha Joshi, Eryn Fitch, Katherine L. Helbig, Dianalee McKnight, Amanda S. Lindy, Fanggeng Zou, Ingo Helbig, Edward C. Cooper, Alfred L. George Jr.
Tools for noninvasive detection of bacterial pathogens are needed but are not currently available for clinical use. We have previously shown that para-aminobenzoic acid (PABA) rapidly accumulates in a wide range of pathogenic bacteria, motivating the development of related PET radiotracers. In this study, 11C-PABA PET imaging was used to accurately detect and monitor infections due to pyogenic bacteria in multiple clinically relevant animal models. 11C-PABA PET imaging selectively detected infections in muscle, intervertebral discs, and methicillin-resistant Staphylococcus aureus–infected orthopedic implants. In what we believe to be first-in-human studies in healthy participants, 11C-PABA was safe, well-tolerated, and had a favorable biodistribution, with low background activity in the lungs, muscles, and brain. 11C-PABA has the potential for clinical translation to detect and localize a broad range of bacteria.
Alvaro A. Ordonez, Matthew F.L. Parker, Robert J. Miller, Donika Plyku, Camilo A. Ruiz-Bedoya, Elizabeth W. Tucker, Justin M. Luu, Dustin A. Dikeman, Wojciech G. Lesniak, Daniel P. Holt, Robert F. Dannals, Lloyd S. Miller, Steven P. Rowe, David M. Wilson, Sanjay K. Jain
The discovery of the oncometabolite 2-hydroxyglutarate in isocitrate dehydrogenase (IDH)1-mutated tumor entities affirmed the role of metabolism in cancer. Yet, large databases with tissue metabolites that are modulated by IDH1 mutation remain an area of development. Here, we present an unprecedented and valuable resource for tissue metabolites in diffuse glioma and their modulations by IDH1 mutation, histology and tumor treatments in 101 tissue samples from 73 diffuse glioma patients (24 astrocytoma, 17 oligodendroglioma, 32 glioblastoma), investigated by NMR-based metabolomics and supported by RNA sequencing. We discovered comparison-specific metabolites and pathways modulated by IDH1 (“IDH1 mutation status cohort”) and tumor entity. The “Longitudinal investigation cohort” provides metabolic profiles of untreated and corresponding treated glioma samples at first progression. Most interestingly, univariate and multivariate cox regressions and Kaplan Meier analyses revealed that tissue metabolites correlate with progression-free and overall survival. Thus, this study introduces novel candidate prognostic and surrogate metabolite biomarkers for future prospective clinical studies aiming at further refining patient stratification in diffuse glioma. Furthermore, our data will facilitate the generation of so far unanticipated hypotheses for experimental studies to advance our molecular understanding of glioma biology.
Christoph Trautwein, Laimdota Zizmare, Irina Mäurer, Benjamin Bender, Björn Bayer, Ulrike Ernemann, Marcos Tatagiba, Stefan J. Grau, Bernd J. Pichler, Marco Skardelly, Ghazaleh Tabatabai
A hallmark of chronic bacterial infections is the long-term persistence of 1 or more pathogen species at the compromised site. Repeated detection of the same bacterial species can suggest that a single strain or lineage is continually present. However, infection with multiple strains of a given species, strain acquisition and loss, and changes in strain relative abundance can occur. Detecting strain-level changes and their effects on disease is challenging because most methods require labor-intensive isolate-by-isolate analyses, and thus, only a few cells from large infecting populations can be examined. Here, we present a population-level method for enumerating and measuring the relative abundance of strains called population multi-locus sequence typing (PopMLST). The method exploits PCR amplification of strain-identifying polymorphic loci, next-generation sequencing to measure allelic variants, and informatic methods to determine whether variants arise from sequencing errors or low-abundance strains. These features enable PopMLST to simultaneously interrogate hundreds of bacterial cells that are cultured en masse from patient samples or are present in DNA directly extracted from clinical specimens without ex vivo culture. This method could be used to detect epidemic or super-infecting strains, facilitate understanding of strain dynamics during chronic infections, and enable studies that link strain changes to clinical outcomes.
Sarah J. Morgan, Samantha L. Durfey, Sumedha Ravishankar, Peter Jorth, Wendy Ni, Duncan T. Skerrett, Moira L. Aitken, Edward F. McKone, Stephen J. Salipante, Matthew C. Radey, Pradeep K. Singh
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