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2019

Amariuta T, Luo Y, Knevel R, Okada Y, Raychaudhuri S. Advances in genetics toward identifying pathogenic cell states of rheumatoid arthritis. Immunol Rev. 2019;
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Rheumatoid arthritis (RA) risk has a large genetic component (~60%) that is still not fully understood. This has hampered the design of effective treatments that could promise lifelong remission. RA is a polygenic disease with 106 known genome-wide significant associated loci and thousands of small effect causal variants. Our current understanding of RA risk has suggested cell-type-specific contexts for causal variants, implicating CD4 + effector memory T cells, as well as monocytes, B cells and stromal fibroblasts. While these cellular states and categories are still mechanistically broad, future studies may identify causal cell subpopulations. These efforts are propelled by advances in single cell profiling. Identification of causal cell subpopulations may accelerate therapeutic intervention to achieve lifelong remission.
Luo, Suliman, Asgari, Amariuta, Baglaenko, Martinez-Bonet, Ishigaki, Gutierrez-Arcelus, Calderon, Lecca, Leon, Jimenez, Yataco, Contreras, Galea, Becerra, Nejentsev, Nigrovic, Moody D, Murray, Raychaudhuri. Early progression to active tuberculosis is a highly heritable trait driven by 3q23 in Peruvians. Nature Communications. 2019;10(3765).
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Of the 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5–15% will develop active tuberculosis (TB). Approximately half will progress to active TB within the first 18 months after infection, presumably because they fail to mount an effective initial immune response. Here, in a genome-wide genetic study of early TB progression, we genotype 4002 active TB cases and their household contacts in Peru. We quantify genetic heritability (h2ghg2) of early TB progression to be 21.2% (standard error 0.08). This suggests TB progression has a strong genetic basis, and is comparable to traits with well-established genetic bases. We identify a novel association between early TB progression and variants located in a putative enhancer region on chromosome 3q23 (rs73226617, OR = 1.18; P = 3.93 × 10−8). With in silico and in vitro analyses we identify rs73226617 or rs148722713 as the likely functional variant and ATP1B3 as a potential causal target gene with monocyte specific function.
Arazi, Rao D, Berthier C, Davidson, Liu Y, Hoover P, Chicoine, Eisenhaure T, Jonsson A, Li, Lieb D, Zhang, Slowikowski, Browne E, Norma, Sutherby, Steelman, Smilek D, Tosta, Apruzzese, Massarotti, Dall’Era, Park, Kamen D, Furie R, Payan-Schober, Pendergraft W, McInnes E, Buyon J, Petri M, Putterman, Kalunian K, Woodle E, Lederer J, Hildeman D, Nusbaum C, Raychaudhuri, Kretzler, Anolik J, Brenner, Wofsy, Hacohen, Diamond, network AMPS. The immune cell landscape in kidneys of patients with lupus nephritis. Nature Immunology. 2019;20(7):902–914.
Publisher's Version
Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.
Der, Suryawanshi, Morozov, Kustagi, Goilav, Ranabathou, Izmirly, Clancy, Belmont H, Koenigsberg, Mockrzycki, Rominieki, Graham J, Rocca J, Bornkamp, Jordan, Schulte, Wu, Pullman, Slowikowski, Raychaudhuri, Guthridge, James, Buyon, Tuschl, Putterman, Consortium AMPRASLE (AMP R. Tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways. Nature Immunology. 2019;20(7):915–927.
Publisher's Version
The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.
Croft A, Campos, Jansen, Turner J, Marshall, Attar, Savary, Wehmeyer, Naylor A, Kemble, Begum, Durholz, Perlman, Barone, McGettrick H, Fearon D, Wei, Raychaudhuri, Korsunsky, Brenner, Coles, Sansom S, Filer, Buckley C. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature. 2019;570:246–251.
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The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune mediated inflammatory diseases (IMIDs). However it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue driven processes observed in IMIDs such as inflammation and damage. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of FAPα+ synovial cells suppressed both inflammation and bone erosions in murine models of resolving and persistent arthritis. Single cell transcriptional analysis identified two distinct fibroblast subsets: FAPα+ THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+ THY1- destructive fibroblasts restricted to the synovial lining. When adoptively transferred into the joint, FAP α+ THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation whereas transfer of FAP α+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell based therapies aimed at modulating inflammation and tissue damage.
Fine R, Pers T, Amariuta T, Raychaudhuri S, Hirschhorn J. Benchmarker: An Unbiased, Association-Data-Driven Strategy to Evaluate Gene Prioritization Algorithms. American Journal of Human Genetics. 2019;104(6):1025–1039.
Publisher's Version
Genome-wide association studies (GWASs) are valuable for understanding human biology, but associated loci typically contain multiple associated variants and genes. Thus, algorithms that prioritize likely causal genes and variants for a given phenotype can provide biological interpretations of association data. However, a critical, currently missing capability is to objectively compare performance of such algorithms. Typical comparisons rely on “gold standard” genes harboring causal coding variants, but such gold standards may be biased and incomplete. To address this issue, we developed Benchmarker, an unbiased, data-driven benchmarking method that compares performance of similarity-based prioritization strategies to each other (and to random chance) by leave-one-chromosome-out cross-validation with stratified linkage disequilibrium (LD) score regression. We first applied Benchmarker to 20 well-powered GWASs and compared gene prioritization based on strategies employing three different data sources, including annotated gene sets and gene expression; genes prioritized based on gene sets had higher per-SNP heritability than those prioritized based on gene expression. Additionally, in a direct comparison of three methods, DEPICT and MAGMA outperformed NetWAS. We also evaluated combinations of methods; our results indicated that combining data sources and algorithms can help prioritize higher-quality genes for follow-up. Benchmarker provides an unbiased approach to evaluate any similarity-based method that provides genome-wide prioritization of genes, variants, or gene sets and can determine the best such method for any particular GWAS. Our method addresses an important unmet need for rigorous tool assessment and can assist in mapping genetic associations to causal function.
Kuo, Ding, Cohn I, Zhang, Wei, Rao D, Rozo, Sohki U, Shanaj, Oliver D, Echeverria A, DiCarlo E, Brenner, Bykerk V, Goodman S, Raychaudhuri, Ratsch, Ivashkiv L, Donlin L. Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defin. Science Translational Medicine. 2019;11(491):eaau8587.
Publisher's Version
Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF ⁺ inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor–dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF ⁺ inflammatory macrophages; however, in some cases, medication redirected them into a state that is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions.
Stanaway IB, Hall TO, Rosenthal EA, Palmer M, Naranbhai V, Knevel R, Namjou‐Khales B, Carroll RJ, Kiryluk K, Gordon AS, Linder J, Howell KM, Mapes BM, Lin FT, Joo YY, Hayes MG, Gharavi AG, Pendergrass SA, Ritchie MD, , Croteau‐Chonka DC, Raychaudhuri S, Weiss ST, Lebo M, Amr SS, Carrell D, Larson EB, Chute CG, Rasmussen‐Torvik LJ, Roy‐Puckelwartz MJ, Sleiman P, Hakonarson H, Li R, Karlson EW, Peterson JF, Kullp IJ, Chisholm R, Denny JC, Jarvik GP, Network T, Crosslin DR. The eMERGE genotype set of 83,717 subjects imputed to ~40 million variants genome wide and association with the herpes zoster medical record phenotype. Genetic Epidemiology. 2019;43(1):63–81.
Publisher's Version

The Electronic Medical Records and Genomics (eMERGE) network is a network of medical centers with electronic medical records linked to existing biorepository samples for genomic discovery and genomic medicine research. The network sought to unify the genetic results from 78 Illumina and Affymetrix genotype array batches from 12 contributing medical centers for joint association analysis of 83,717 human participants. In this report, we describe the imputation of eMERGE results and methods to create the unified imputed merged set of genome-wide variant genotype data. We imputed the data using the Michigan Imputation Server, which provides a missing single-nucleotide variant genotype imputation service using the minimac3 imputation algorithm with the Haplotype Reference Consortium genotype reference set. We describe the quality control and filtering steps used in the generation of this data set and suggest generalizable quality thresholds for imputation and phenotype association studies. To test the merged imputed genotype set, we replicated a previously reported chromosome 6 HLA-B herpes zoster (shingles) association and discovered a novel zoster-associated loci in an epigenetic binding site near the terminus of chromosome 3 (3p29).


 

2018

Davenport EE, Amariuta T, Gutierrez-Arcelus M, Slowikowski K, Westra HJ, Luo Y, Shen C, Rao DA, Zhang Y, Pearson S, Schack D, Beebe JS, Bing N, John S, Vincent MS, Zhang B, Raychaudhuri S. Discovering in vivo cytokine eQTL interactions from a lupus clinical trial. Genome Biology. 2018;19(1):168.
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BACKGROUND: 

Cytokines are critical to human disease and are attractive therapeutic targets given their widespread influence on gene regulation and transcription. Defining the downstream regulatory mechanisms influenced by cytokines is central to defining drug and disease mechanisms. One promising strategy is to use interactions between expression quantitative trait loci (eQTLs) and cytokine levels to define target genes and mechanisms.

RESULTS: 

In a clinical trial for anti-IL-6 in patients with systemic lupus erythematosus, we measure interferon (IFN) status, anti-IL-6 drug exposure, and whole blood genome-wide gene expression at three time points. We show that repeat transcriptomic measurements increases the number of cis eQTLs identified compared to using a single time point. We observe a statistically significant enrichment of in vivo eQTL interactions with IFN status and anti-IL-6 drug exposure and find many novel interactions that have not been previously described. Finally, we find transcription factor binding motifs interrupted by eQTL interaction SNPs, which point to key regulatory mediators of these environmental stimuli and therefore potential therapeutic targets for autoimmune diseases. In particular, genes with IFN interactions are enriched for ISRE binding site motifs, while those with anti-IL-6 interactions are enriched for IRF4 motifs.

CONCLUSIONS: 

This study highlights the potential to exploit clinical trial data to discover in vivo eQTL interactions with therapeutically relevant environmental variables.

Fonseka C, Rao DA, Teslovich NC, Hannes SK, Slowikowski K, Gurish MF, Donlin LT, Weinblatt ME, Massarotti EM, Coblyn JS, Helfgott SM, Todd DJ, Bykerk VP, Karlson EW, Ermann J, Lee YC, Brenner MB, Raychaudhuri S. Mixed Effects Association of Single Cells Identifies an Expanded Th1-Skewed Cytotoxic Effector CD4+ T Cell Subset in Rheumatoid Arthritis.. Science Translational Medicine. 2018;10(463).
Publisher's Version
High dimensional single-cell analyses have dramatically improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging due to technical and inter-individual variation. Here we present Mixed effects modeling of Associations of Single Cells (MASC), a novel reverse single cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounds and biological variation. Applying MASC to mass cytometry analyses of CD4+ T cells from blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+ T cells, identified as CD27- HLA-DR+ effector memory cells, in RA patients (OR = 1.7; p = 0.0011). The frequency of CD27- HLA-DR+ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27- HLA-DR+ cell frequency decreased in RA patients who respond to immunosuppressive therapy. Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained ~5-fold higher frequencies of CD27- HLA-DR+ cells, which comprised ~10% of synovial CD4+ T cells. We find that CD27- HLA-DR+ cells are abundant producers of IFN-γ and also express perforin and granzyme A at elevated levels. Thus MASC identified the expansion of a unique Th1 skewed effector T cell population with cytotoxic capacity in RA. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single cell data.