The kidney contains a population of resident macrophages from birth that expands as it grows and forms a contiguous network throughout the tissue. Kidney resident macrophages (KRMs) are important in homeostasis and the response to acute kidney injury (AKI). While the kidney contains many microenvironments, it is unknown whether KRMs are a heterogeneous population differentiated by function and location. We combined single-cell RNA sequencing (scRNAseq), spatial transcriptomics, flow cytometry, and immunofluorescence imaging to localize, characterize, and validate KRM populations during quiescence and following 19 minutes of bilateral ischemic kidney injury. scRNAseq and spatial transcriptomics revealed seven distinct KRM subpopulations, which are organized into zones corresponding to regions of the nephron. Each subpopulation was identifiable by a unique transcriptomic signature suggesting distinct functions. Specific protein markers were identified for two clusters allowing analysis by flow cytometry or immunofluorescence imaging. Following injury, the original localization of each subpopulation is lost, either from changing locations or transcriptomic signatures. The original spatial distribution of KRMs is not fully restored for at least 28 days post-injury. The change in KRM localization confirms a long hypothesized dysregulation of the local immune system following acute injury and may explain the increased risk for chronic kidney disease.
Matthew D. Cheung, Elise N. Erman, Kyle H. Moore, Jeremie M.P. Lever, Zhang Li, Jennifer R. LaFontaine, Gelare Ghajar-Rahimi, Shanrun Liu, Zhengqin Yang, Rafay Karim, Bradley K. Yoder, Anupam Agarwal, James F. George
Histopathology, the standard method to assess bone marrow in hematologic malignancies such as myeloproliferative neoplasms (MPNs), suffers from notable limitations in both research and clinical settings. Bone marrow biopsies in patients fail to detect disease heterogeneity; may yield a non-diagnostic sample; and cannot be repeated frequently in clinical oncology. Endpoint histopathology precludes monitoring disease progression and response to therapy in the same mouse over time, missing likely variations among mice. To overcome these shortcomings, we used magnetic resonance imaging (MRI) to measure changes in cellularity, macromolecular constituents, and fat versus hematopoietic cells in bone marrow using diffusion-weighted imaging, magnetization transfer, and chemical shift encoded fat imaging. Combining metrics from these imaging parameters revealed dynamic alterations in bone marrow following myeloablative radiation and transplantation. In a mouse MPLW515L bone marrow transplant model of MPN, MRI detected effects of a JAK2 inhibitor, ruxolitinib, within five days of initiating treatment and identified differing kinetics of treatment responses in sub-regions of the tibia. Histopathology validated MRI results for bone marrow composition and heterogeneity. Anatomic MRI scans also showed reductions in spleen volume during treatment. These findings establish an innovative, clinically translatable MRI approach to quantify spatial and temporal changes in bone marrow in MPN.
Tanner H. Robison, Manisha Solipuram, Kevin Heist, Ghoncheh Amouzandeh, Winston Y. Lee, Brock A. Humphries, Johanna M. Buschhaus, Avinash Bevoor, Anne Zhang, Kathryn E. Luker, Kristen Pettit, Moshe Talpaz, Dariya Malyarenko, Thomas L. Chenevert, Brian D. Ross, Gary D. Luker
The epidermis is the outermost layer of skin. Here, we use targeted lipid profiling to characterize the biogeographic alterations of human epidermal lipids across 12 anatomically distinct body sites, and use single-cell RNA sequencing to compare keratinocyte gene expression at acral and non-acral surfaces. We demonstrate that acral skin has low expression of EOS acyl-ceramides and the genes involved in their synthesis, as well as low expression of genes involved in filaggrin and keratin citrullination (PADI1 and PADI3) and corneodesmosome degradation, changes consistent with increased corneocyte retention. Several overarching principles governing epidermal lipid expression were also noted. For example, there is a strong negative correlation between the expression of 18-carbon and 22-carbon sphingoid base ceramides. Disease-specific alterations in epidermal lipid gene expression and their corresponding alterations to the epidermal lipidome were characterized. Lipid biomarkers with diagnostic utility for inflammatory and precancerous conditions were identified, and a two-analyte diagnostic model of psoriasis was constructed using a step-forward algorithm. Finally, gene co-expression analysis revealed a strong connection between lipid and immune gene expression. This work highlights mechanisms by which the epidermis is uniquely adapted for the specific environmental insults encountered at different body surfaces, and how inflammation-associated alterations in gene expression affect the epidermal lipidome.
Alexander A. Merleev, Stephanie T. Le, Claire Alexanian, Atrin Toussi, Yixuan Xie, Alina I. Marusina, Steven M. Watkins, Forum Patel, Allison C. Billi, Julie Wiedemann, Yoshihiro Izumiya, Ashish Kumar, Ranjitha Uppala, J. Michelle Kahlenberg, Fu-Tong Liu, Iannis E. Adamopoulos, Elizabeth A. Wang, Chelsea Ma, Michelle Y. Cheng, Halani Xiong, Amanda Kirane, Guillaume Luxardi, Bogi Andersen, Lam C. Tsoi, Carlito B. Lebrilla, Johann E. Gudjonsson, Emanual Maverakis
Despite being a leading cause of advanced liver disease, alcohol-associated liver disease (ALD) has no effective medical therapies. The circulating proteome, which comprises proteins secreted by different cells and tissues in the context of normal physiological function or in the setting of disease and illness, represents an attractive target for uncovering novel biology related to the pathogenesis of ALD. In this work, we used the aptamer-based SomaScan proteomics platform to quantify the relative concentration of over 1300 proteins in a well-characterized cohort of patients with the spectrum of ALD. We found a distinct circulating proteomic signature that correlated with ALD severity, including over 600 proteins that differed significantly between ALD stages, many of which have not previously been associated with ALD to our knowledge. Notably, certain proteins that were markedly dysregulated in patients with alcohol-associated hepatitis were also altered, to a lesser degree, in patients with subclinical ALD and may represent early biomarkers for disease progression. Taken together, our work highlights the vast and distinct changes in the circulating proteome across the wide spectrum of ALD, identifies potentially novel biomarkers and therapeutic targets, and provides a proteomic resource atlas for ALD researchers and clinicians.
Jay Luther, Augustin G.L. Vannier, Esperance A. Schaefer, Russell P. Goodman
The androgen receptor (AR) is a master transcription factor that regulates prostate cancer (PC) development and progression. Inhibition of AR signaling by androgen deprivation is the first-line therapy with initial efficacy for advanced and recurrent PC. Paradoxically, supraphysiological levels of testosterone (SPT) also inhibit PC progression. However, as with any therapy, not all patients show a therapeutic benefit, and responses differ widely in magnitude and duration. In this study, we evaluated whether differences in the AR cistrome before treatment can distinguish between SPT-responding (R) and -nonresponding (NR) tumors. We provide the first preclinical evidence to our knowledge that SPT-R tumors exhibit a distinct AR cistrome when compared with SPT-NR tumors, indicating a differential biological role of the AR. We applied an integrated analysis of ChIP-Seq and RNA-Seq to the pretreatment tumors and identified an SPT-R signature that distinguishes R and NR tumors. Because transcriptomes of SPT-treated clinical specimens are not available, we interrogated available castration-resistant PC (CRPC) transcriptomes and showed that the SPT-R signature is associated with improved survival and has the potential to identify patients who would respond to SPT. These findings provide an opportunity to identify the subset of patients with CRPC who would benefit from SPT therapy.
Xintao Qiu, Lisha G. Brown, Jennifer L. Conner, Holly M. Nguyen, Nadia Boufaied, Sarah Abou Alaiwi, Ji-Heui Seo, Talal El Zarif, Connor Bell, Edward O’Connor, Brian Hanratty, Mark Pomerantz, Matthew L. Freedman, Myles Brown, Michael C. Haffner, Peter S. Nelson, Felix Y. Feng, David P. Labbé, Henry W. Long, Eva Corey
Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PV). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2 mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell-type-distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF-predisposition genes, suggesting combinatorial risk for AF-genesis. Our data further reveals that PV and LA Pitx2 mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF-predisposition in the PV of humans.
Jeffrey D. Steimle, Francisco J. Grisanti Canozo, Minjun Park, Zachary A. Kadow, Md. Abul Hassan Samee, James F. Martin
Inflammasomes are a class of innate immune signaling platforms that activate in response to an array of cellular damage and pathogens. Inflammasomes promote inflammation under many circumstances to enhance immunity against pathogens and inflammatory responses through their effector cytokines, IL-1β and IL-18. Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune conditions influenced by inflammasomes. Despite work investigating inflammasomes during EAE, little remains known concerning the role of inflammasomes in the central nervous system (CNS) during the disease. Here, we used multiple genetically modified mouse models to monitor activated inflammasomes in situ based on oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC) in the spinal cord. Using inflammasome reporter mice, we found heightened inflammasome activation in astrocytes after the disease peak. In contrast, microglia and CNS-infiltrated myeloid cells had few activated inflammasomes in the CNS during EAE. Astrocyte inflammasome activation during EAE was dependent on absent in melanoma 2 (AIM2), but low IL-1β release and no significant signs of cell death were found. Thus, the AIM2 inflammasome activation in astrocytes may have a distinct role from traditional inflammasome-mediated inflammation.
William E. Barclay, Nupur Aggarwal, M. Elizabeth Deerhake, Makoto Inoue, Toshiaki Nonaka, Kengo Nozaki, Nathan A. Luzum, Edward A. Miao, Mari L. Shinohara
Severe COVID-19 disease is associated with dysregulation of the myeloid compartment during acute infection. Survivors frequently experience long-lasting sequelae but little is known about the eventual persistence of this immune alteration. Herein, we evaluated Toll-like receptor-induced cytokine responses in a cohort of mild to critical patients during acute or convalescent phases (n=97). In the acute phase, we observed impaired cytokine production by monocytes in the most severe patients. This capacity was globally restored in convalescent patients. Yet, we observed increased responsiveness to TLR1/2 ligation in patients that recovered from severe disease, indicating that these cells display distinct functional properties at the different stages of the disease. We identified a specific transcriptomic and epigenomic state in monocytes from acute severe patients that can account for their functional refractoriness. The molecular profile of monocytes from recovering patients was distinct and characterized by increased chromatin accessibility at AP1 and MAF loci. These results demonstrate that severe COVID-19 infection has a profound impact on the differentiation status and function of circulating monocytes both during the acute and the convalescent phases in a completely distinct manner. This could have important implications for our understanding of short and long-term COVID19-related morbidity.
Elisa Brauns, Abdulkader Azouz, David Grimaldi, Hanxi Xiao, Séverine Thomas, Muriel Nguyen, Véronique Olislagers, Ines Vu Duc, Carmen Orte Cano, Véronique Del Marmol, Pieter Pannus, Frédérick Libert, Sven Saussez, Nicolas Dauby, Jishnu Das, Arnaud Marchant, Stanislas Goriely
Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell–derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.
Kristine M. Abo, Julio Sainz de Aja, Jonathan Lindstrom-Vautrin, Konstantinos-Dionysios Alysandratos, Alexsia Richards, Carolina Garcia-de-Alba, Jessie Huang, Olivia T. Hix, Rhiannon B. Werder, Esther Bullitt, Anne Hinds, Isaac Falconer, Carlos Villacorta-Martin, Rudolf Jaenisch, Carla F. Kim, Darrell N. Kotton, Andrew A. Wilson
Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing-based method by RNA-Seq on a single skin biopsy for concomitant identification of the cutaneous virome and underlying IEI. Skin biopsies were obtained from normal and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 different viruses in a viral genome catalog. In nine families studied, the patients carried pathogenic variants in six human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping which assisted in identification of candidate genes in consanguineous families. This automated, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspicion of IEI and viral dermatosis.
Amir Hossein Saeidian, Leila Youssefian, Charles Y. Huang, Fahimeh Palizban, Mahtab Naji, Zahra Saffarian, Hamidreza Mahmoudi, Azadeh Goodarzi, Soheila Sotoudeh, Fatemeh Vahidnezhad, Maliheh Amani, Narjes Tavakoli, Ali Ajami, Samaneh Mozafarpoor, Mehrdad Teimoorian, Saeed Dorgaleleh, Sima Shokri, Mohammad Shenagari, Nima Abedi, Sirous Zeinali, Paolo Fortina, Vivien Béziat, Emmanuelle Jouanguy, Jean-Laurent Casanova, Jouni Uitto, Hassan Vahidnezhad
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