Resource and Technical Advance
Abstract
Geleophysic Dysplasia-1 (GD1) is an autosomal recessive disorder caused by ADAMTSL2 variants. It is characterized by distinctive facial features, limited joint mobility, short stature with brachydactyly, and the potential for life-threatening cardiovascular and respiratory complications. The clinical spectrum spans from perinatal lethality to milder phenotypes in adult survivors, manifesting a clinical heterogeneity. The Adamtsl2–/– mouse model dies perinatally and hinders further functional investigation. In this study, we developed and characterized cellular and mouse models, which were designed to replicate the genetic profile of a patient who is compound heterozygous for two ADAMTSL2 variants, namely p.R61H and p.A165T. The impairment of ADAMTSL2 secretion was observed in both variants, but notably, p.A165T exhibited a more severe impact. We conducted a thorough analysis of mice carrying different allelic combinations, including knockout, p.R61H, and p.A165T variants. This examination revealed a wide spectrum of phenotypic severity, spanning from lethality in knockout homozygotes to mild growth impairment observed in adult p.R61H homozygotes. While they survived, the homozygous and hemizygous p.A165T mice displayed severe respiratory and cardiac dysfunction. The respiratory dysfunction mainly affects the expiration phase without significant fibrosis in the lungs. Evidence of microscopic post-obstructive pneumonia was found in some hemizygous and homozygous p.A165T. Echocardiograms and MRI studies revealed a significant systolic dysfunction, accompanied by a reduction in the size of the aortic root. Histological examinations further confirmed the presence of hypertrophic cardiomyopathy with myocyte hypertrophy. In addition, evidence of elevated proteoglycan staining in the myocardium, chondroid metaplasia, along with patchy mild interstitial fibrosis within the myocardium was seen in hemizygous and homozygous p.A165T. In conclusion, our study revealed a significant correlation between the degree of impaired ADAMTSL2 secretion and the severity of the observed phenotype in GD1. The surviving mouse models we developed have provided valuable insights into the pathogenesis of GD and hold promise as valuable tools for informing and guiding future therapeutic interventions aimed at managing this disorder effectively.
Authors
Vladimir Camarena, Monique M. Williams, Alejo A. Morales, Mohammad F. Zafeer, Okan V. Kilic, Ali Kamiar, Clemer Abad, Monica A. Rasmussen, Laurence M. Briski, LéShon Peart, Guney Bademci, Deborah S. Barbouth, Sarah Smithson, Gaofeng Wang, Lina A. Shehadeh, Katherina Walz, Mustafa Tekin
Abstract
Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/−) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/− mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifts cell fate more toward the myeloid lineage but compromises lymphoid lineage development by reducing genes required for lymphoid and B-cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.
Authors
Lana Vukadin, Bohye Park, Mostafa Mohamed, Huashi Li, Amr Elkholy, Alex Torrelli-Diljohn, Jung-Hyun Kim, Kyuho Jeong, James M. Murphy, Caitlin A. Harvey, Sophia Dunlap, Leah Gehrs, Hanna Lee, Hyung-Gyoon Kim, Jay Prakash Sah, Seth N. Lee, Denise Stanford, Robert A. Barrington, Jeremy B. Foote, Anna G. Sorace, Robert S. Welner, Blake E. Hildreth III, Ssang-Taek Steve Lim, Eun-Young Erin Ahn
Abstract
With antimicrobial resistance (AMR) emerging as a major threat to global health, monoclonal antibodies (MAbs) have become a promising means to combat difficult-to-treat AMR infections. Unfortunately, in contrast with standard antimicrobials, for which there are well-validated clinical laboratory methodologies to determine whether an infecting pathogen is susceptible or resistant to a specific antimicrobial drug, no assays have been described that can inform clinical investigators or clinicians regarding the clinical efficacy of a MAb against a specific pathogenic strain. Using Acinetobacter baumannii as a model organism, we established and validated 2 facile clinical susceptibility assays, which used flow cytometry and latex bead agglutination, to determine susceptibility (predicting in vivo efficacy) or resistance (predicting in vivo failure) of 1 newly established and 3 previously described anti–A. baumannii MAbs. These simple assays exhibited impressive sensitivity, specificity, and reproducibility, with clear susceptibility breakpoints that predicted the in vivo outcomes in our preclinical model with excellent fidelity. These MAb susceptibility assays have the potential to enable and facilitate clinical development and deployment of MAbs that generally target the surface of microbes.
Authors
Matthew J. Slarve, Neven Bowler, Elizabeth Burk, Jun Yan, Ulrike Carlino-MacDonald, Thomas A. Russo, Brian M. Luna, Brad Spellberg
Abstract
Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for four weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transients measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype.
Authors
Kalina J. Rossler, Willem J. De Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge
Abstract
Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This library was validated using seven genetic mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2–/– and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn–/– IgD+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.
Authors
Elze Rackaityte, Irina Proekt, Haleigh S. Miller, Akshaya Ramesh, Jeremy F. Brooks, Andrew F. Kung, Caleigh Mandel-Brehm, David J.L. Yu, Colin R. Zamecnik, Rebecca Bair, Sara E. Vazquez, Sara Sunshine, Clare L. Abram, Clifford A. Lowell, Gabrielle Rizzuto, Michael R. Wilson, Julie Zikherman, Mark S. Anderson, Joseph L. DeRisi
Abstract
Adipose tissue macrophage (ATM) infiltration is associated with adipose tissue dysfunction and insulin resistance in mice and humans. Recent single cell data highlight increased ATM heterogeneity in obesity, but do not provide a spatial context for ATM phenotype dynamics. We integrated single cell RNA-sequencing, spatial transcriptomics, and imaging of murine adipose tissue in a time course of diet-induced obesity. Overall, proinflammatory immune cells were predominant in early obesity, while non-resident antiinflammatory ATMs predominated in chronic obesity. A subset of these antiinflammatory ATMs were transcriptomically intermediate between monocytes and mature lipid-associated macrophages (LAM) and were consistent with a LAM precursor (pre-LAM). Pre-LAMs were spatially associated with early obesity crown-like structures (CLS), which indicate adipose tissue dysfunction. Spatial data showed colocalization of ligand-receptor transcripts related to lipid signaling among monocytes, pre-LAMs, and LAMs, including Apoe, Lrp1, Lpl and App. Pre-LAM expression of these ligands in early obesity suggested signaling to LAMs in the CLS microenvironment. Our results refine understanding of ATM diversity and provide insight into the dynamics of the LAM lineage during development of metabolic disease.
Authors
Cooper M. Stansbury, Gabrielle A. Dotson, Harrison Pugh, Alnawaz Rehemtulla, Indika Rajapakse, Lindsey A. Muir
Abstract
Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.
Authors
Samantha Slight-Webb, Kevin Thomas, Miles Smith, Catriona A. Wagner, Susan Macwana, Aleksandra Bylinska, Michele Donato, Mai Dvorak, Sarah E. Chang, Alex Kuo, Peggie Cheung, Laurynas Kalesinskas, Ananthakrishnan Ganesan, Denis Dermadi, Carla J. Guthridge, Wade DeJager, Christian Wright, Mariko H. Foecke, Joan T. Merrill, Eliza Chakravarty, Cristina Arriens, Holden T. Maecker, Purvesh Khatri, Paul J. Utz, Judith A. James, Joel M. Guthridge
Abstract
IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with unclear pathogenesis. We performed single-cell RNA-seq and surface proteome analyses on 61,379 PBMCs from 9 treatment-naïve IgG4-RD patients and 7 age- and sex-matched healthy controls. Integrative analyses were performed for altered gene expression in IgG4-RD, and flow cytometry and immunofluorescence were used for validation. We observed expansion of plasmablasts with enhanced protein processing and activation, which correlated with number of involved organs in IgG4-RD. Increased proportions of CD4+ cytotoxic T lymphocytes (CTLs), CD8+ CTLs-GNLY (granulysin) and γδT cells with enhanced chemotaxis and cytotoxicity but with suppressed inhibitory receptors characterize IgG4-RD. Prominent infiltration of lymphocytes with distinct compositions were found in different organs of IgG4-RD patients. Transcription factors (TFs) including PRDM1/XBP1 and RUNX3 were upregulated in IgG4-RD, promoting the differentiation of plasmablasts and CTLs, respectively. Monocytes in IgG4-RD have stronger expression of genes related to cell adhesion and chemotaxis, which may give rise to profibrotic macrophages in lesions. The gene activation pattern in peripheral immune cells indicated activation of multiple interaction pathways between cell types, in part through chemokines or growth factors and their receptors. Specific upregulation of TFs and expansion of plasmablasts and CTLs may be involved in the pathogenesis of IgG4-RD, and each of these populations are candidate targets for therapeutic interventions in this disease.
Authors
Chenyang Lu, Shasha Li, Pingying Qing, Qiuping Zhang, Xing Ji, Zhigang Tang, Chunyan Chen, Tong Wu, Yidan Hu, Yi Zhao, Xiaohui Zhang, Qi He, David A. Fox, Chunyu Tan, Yubin Luo, Yi Liu
Abstract
Darier, Hailey-Hailey, and Grover’s diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. To better understand the underlying mechanisms leading to disease in these conditions we performed RNA-seq on lesional skin samples from patients. The transcriptomic profiles of Darier, Hailey-Hailey, and Grover’s disease were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases. Analysis of enriched pathways showed a shared upregulation in keratinocyte differentiation, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grover’s disease. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in the acantholytic skin diseases. Further, upstream regulator analysis suggested that a decrease in SRF/MRTF activity was responsible for the downregulation of actin organization pathways. Staining for MRTFA in lesional skin samples showed a decrease in nuclear MRTFA in patient skin compared to normal skin. These findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grover’s disease, and identify decreases in actin organization pathways as a unique signature present in these conditions.
Authors
Quinn R. Roth-Carter, Hope E. Burks, Ziyou Ren, Jennifer L. Koetsier, Lam C. Tsoi, Paul W. Harms, Xianying Xing, Joseph Kirma, Robert M. Harmon, Lisa M. Godsel, Abbey L. Perl, Johann E. Gudjonsson, Kathleen J. Green
Abstract
Rationale: Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. Objectives: We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 total patients undergoing femoral (N=23) or carotid (N=26) endarterectomy using single-cell ribonucleic acid sequencing (scRNA-seq; N=13), flow cytometry (N=24), and immunohistochemistry (N=12). Findings: Comparative scRNA-seq of CD45 positive-selected leukocytes from femoral (N=9; 35265 cells) and carotid (N=4; 30655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised 2.5- to 4-fold higher proportions of myeloid cells in carotid plaques, whereas non-inflammatory foam cell-like macrophages and LYVE1-overexpressing resident-like macrophages comprised 3.5- to 9-fold higher proportions of myeloid cells in femoral plaque (p<0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus femoral plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively anti-inflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. Conclusion: Human femoral plaques exhibit distinct macrophage profiles and diminished CD8+ T cell populations compared with carotid plaques. Experimental models elucidating determinants of plaque site-specific cell polarization cues are warranted.
Authors
Joshua Slysz, Arjun Sinha, Matthew DeBerge, Shalini Singh, Harris Avgousti, Inhyeok Lee, Kristofor Glinton, Reina Nagasaka, Prarthana J. Dalal, Shaina J. Alexandria, Ching Man Wai, Ricardo Tellez, Mariavittoria Vescovo, Ashwin Sunderraj, Xinkun Wang, Matthew J. Schipma, Ryan K Sisk, Rishab Gulati, Jenifer Vallejo, Ryosuke Saigusa, Donald M. Lloyd-Jones, Jon Lomasney, Samuel E. Weinberg, Karen J. Ho, Klaus Ley, Chiara Giannarelli, Edward B. Thorp, Matthew J. Feinstein
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