Resource and Technical Advance
Abstract
The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific FLS biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated transcriptomic and chromatin accessibility data from FLS to identify the transcription factors (TFs) and pathways. Networks were constructed by integration of chromatin accessibility and gene expression data. Analysis revealed joint-specific patterns of FLS phenotype, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an ‘activated’ state relative to the ‘resting’ state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.
Authors
Eunice Choi, Camilla R. L. Machado, Takaichi Okano, David L. Boyle, Wei Wang, Gary S. Firestein
Abstract
Applying advanced molecular profiling together with highly specific targeted therapies offers the possibility to better dissect the mechanisms underlying immune mediated inflammatory diseases such as systemic lupus erythematosus (SLE) in humans. Here we apply a combination of single cell RNA sequencing and T/B cell repertoire analysis to perform an in-depth characterization of molecular changes in the immune-signature upon CD19 CAR T cell-mediated depletion of B cells in SLE patients. The resulting datasets do not only confirm a selective CAR T cell-mediated reset of the B cell response, but simultaneously reveal consequent changes in the transcriptional signature of monocyte and T cell subsets that respond with a profound reduction in type 1 interferon signaling. Our current data thus provide evidence for a causal relationship between the B cell response and the increased interferon signature observed in SLE and additionally demonstrate the usefulness of combining targeted therapies and novel analytic approaches to decipher molecular mechanisms of immune-mediated inflammatory diseases in humans.
Authors
Artur Wilhelm, David Chambers, Fabian Müller, Aline Bozec, Ricardo Grieshaber-Bouyer, Thomas Winkler, Dimitrios Mougiakakos, Andreas Mackensen, Georg Schett, Gerhard Krönke
Abstract
Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned hIL-6 transgenic NSG mice (NSG+hIL6) reliably support the engraftment of malignant and pre-malignant human plasma cells including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and post-relapse myeloma, plasma cell leukemia, and AL amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells, developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single cell RNA sequencing showed non-malignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma engrafted mice given CAR T-cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results established NSG+hIL6 mice as an effective patient derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.
Authors
Zainul S. Hasanali, Alfred L. Garfall, Lisa Burzenski, Leonard D. Shultz, Yan Tang, Siddhant Kadu, Neil C. Sheppard, Wei Liu, Derek Dopkin, Dan T. Vogl, Adam D. Cohen, Adam J. Waxman, Sandra P. Susanibar-Adaniya, Martin Carroll, Edward A. Stadtmauer, David Allman
Abstract
Substantial evidence suggests a role for immunotherapy in treating Alzheimer’s disease (AD). While the precise pathophysiology of AD is incompletely understood, clinical trials of antibodies targeting aggregated forms of β amyloid (Aβ) have shown that reducing amyloid plaques can mitigate cognitive decline in patients with early-stage AD. Here, we describe what we believe to be a novel approach to target and degrade amyloid plaques by genetically engineering macrophages to express an Aβ-targeting chimeric antigen receptor (CAR-Ms). When injected intrahippocampally, first-generation CAR-Ms have limited persistence and fail to significantly reduce plaque load, which led us to engineer next-generation CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. Cytokine secreting “reinforced CAR-Ms” have greater survival in the brain niche and significantly reduce plaque load locally in vivo. These findings support CAR-Ms as a platform to rationally target, resorb, and degrade pathogenic material that accumulates with age, as exemplified by targeting Aβ in AD.
Authors
Alexander B. Kim, Qingli Xiao, Ping Yan, Qiuyun Pan, Gaurav Pandey, Susie Grathwohl, Ernesto Gonzales, Isabella Xu, Yoonho Cho, Hans Haecker, Slava Epelman, Abhinav Diwan, Jin-Moo Lee, Carl J. DeSelm
Abstract
Spine metastases can result in severe neurologic compromise and decreased overall survival. Despite treatment advances, local disease progression is frequent, highlighting the need for novel therapies. Tumor treating fields (TTFields) impair tumor cell replication and are influenced by properties of surrounding tissue. We hypothesize bone’s dielectric properties will enhance TTFields mediated suppression of tumor growth in spine metastasis models. Computational modeling of TTFields intensity was performed following surgical resection of a spinal metastasis and demonstrated enhanced TTFields intensity within the resected vertebral body. Additionally, luciferase-tagged human KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized bone grafts and exposed to TTFields. Following TTFields exposure, BLI signal decreased 10-80% of baseline while control cultures displayed 4.48-9.36 fold increase in signal. Lastly, TTFields were applied in an orthotopic murine model of spinal metastasis. After 21 days of treatment, control mice demonstrated a 5-fold increase in BLI signal compared to TTFields treated mice. TTFields similarly prevented tumor invasion into the spinal canal and development of neurologic symptoms. Our data suggest that TTFields can be leveraged as a local therapy within minimally-conductive bone of spine metastases. This provides the groundwork for future studies investigating TTFields for patients with treatment-refractory spine metastases.
Authors
Daniel Ledbetter, Romulo de Almeida, Xizi Wu, Ariel Naveh, Chirag B. Patel, Queena Gonzalez, Thomas H. Beckham, Robert North, Laurence Rhines, Jing Li, Amol Ghia, David Aten, Claudio Tatsui, Christopher Alvarez-Breckenridge
Abstract
Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement of cholesterol for activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and non-responsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells detects T cell activation and has potential to assess the success of ICI therapy.
Authors
Nicholas G. Ciavattone, Jenny Nan Guan, Alex Farfel, Jenelle Stauff, Timothy J. Desmond, Benjamin L. Viglianti, Peter J.H. Scott, Allen F. Brooks, Gary D. Luker
Abstract
Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet–based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.
Authors
Samantha Donsante, Alice Pievani, Biagio Palmisano, Melissa Finamore, Grazia Fazio, Alessandro Corsi, Andrea Biondi, Shunji Tomatsu, Rocco Piazza, Marta Serafini, Mara Riminucci
Abstract
Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the bone marrow. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-sequencing to characterize immune and tumor cell alterations in neuroblastoma bone marrow metastases by comparative analysis with patients without metastases. Our results revealed remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the bone marrow metastatic niche we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of regulatory T cells and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and identified prognostic markers in malignant cells that are associated with worse clinical outcomes in three independent neuroblastoma cohorts. Our results provide insights into the cellular, compositional and transcriptional shifts underlying neuroblastoma bone marrow metastases contributing to the development of new therapeutic strategies.
Authors
Shenglin Mei, Adele M. Alchahin, Bethel Tesfai Embaie, Ioana Maria Gavriliuc, Bronte Manouk Verhoeven, Ting Zhao, Xiangyun Li, Nathan Elias Jeffries, Adena Pepich, Hirak Sarkar, Thale Kristin Olsen, Malin Wickström, Jakob Stenman, Oscar Reina-Bedoya, Peter V. Kharchenko, Philip J. Saylor, John Inge Johnsen, David B. Sykes, Per Kogner, Ninib Baryawno
Abstract
The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis–specific SNPs were enriched in distal colon–related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.
Authors
John L. Johnson, Davit Sargsyan, Eric M. Neiman, Amy Hart, Aleksandar Stojmirovic, Roman Kosoy, Haritz Irizar, Mayte Suárez-Fariñas, Won-Min Song, Carmen Argmann, Stefan Avey, Liraz Shmuel-Galia, Tim Vierbuchen, Gerold Bongers, Yu Sun, Leonard Edelstein, Jacqueline Perrigoue, Jennifer E. Towne, Aisling O’Hara Hall, Katherine A. Fitzgerald, Kasper Hoebe
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
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