Biology subjects

Clancy, R.

Publications and source records attributed to Clancy, R..

6 recordsLinked to original sources

bioRxiv2025

Deep profiling of lupus nephritis kidneys reveals dynamic changes in myeloid cells associated with disease progression

ObjectivesLupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aim to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. MethodsSingle-cell RNA-sequencing (scRNA-seq) was used to profile dissociated kidney samples from 156 LN patients and 30 healthy individuals. Spatial transcriptomics (ST), utilizing a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, was applied to kidney samples acquired from 6 LN patients and 2 healthy controls. ResultsWe generated a full catalog of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the NIH chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4+ T, GZMK+CD8+ T, B and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are indicated to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. ConclusionsWe suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.

immunology↗

bioRxiv2024

Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

immunology↗

bioRxiv2023

Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

Resident macrophages and infiltrating monocytes in kidneys of patients with lupus nephritis are altered both in frequency and function relative to their counterparts in healthy kidneys. The extent to which mouse models might be useful in developing approaches to target these cells for treating lupus nephritis is poorly understood. Here, we studied four common lupus mouse models that share clinical, serologic, and histopathologic kidney changes with humans. Using single-cell profiling and multiplex spatial imaging to analyze the intrarenal myeloid compartment with the onset of clinical disease in these models, we identified monocyte and macrophage subsets that expand or contract in kidneys with clinical nephritis. A unique subset of classical monocytes expanded with the onset of disease and expressed genes such as CD9, Spp1, Ctsd, Cd63, Apoe, and Trem2 that were previously shown to be induced by tissue injury and play a role in inflammation, lipid metabolism and tissue repair in other organs. Resident macrophages transitioned from a pro-inflammatory to a similar injury-associated state with onset of disease. To test whether these findings in mouse models were also observed in humans, we re-analyzed monocytes and macrophages in a single-cell RNAseq dataset of kidney biopsies from 155 patients with lupus nephritis and 30 healthy donors, collected by the NIH AMP RA/SLE consortium. Human monocytes and macrophages showed conserved changes in gene expression programs associated with lupus nephritis disease indices, and localized to similar kidney microenvironments as in mice. By identifying myeloid subsets and disease-associated alterations in biological processes that are conserved across species, we provide a strong rationale for functional studies of these cells and pathways in mice to uncover mechanisms and find targets relevant to human lupus nephritis. One sentence summaryThis study characterizes intrarenal myeloid cells from four lupus mouse models and 155 patients with lupus nephritis using single-cell RNA-seq and imaging, and identifies novel infiltrating and resident myeloid subsets that are conserved between mouse and human lupus nephritis, thus providing a map and strong rationale for functional studies in mice with relevance to human disease.

immunology↗

bioRxiv2022

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

biophysics↗

bioRxiv2020

APOL1 Variant-Expressing Endothelial Cells Exhibit Autophagic Dysfunction and Mitochondrial Stress

Apolipoprotein L1 (APOL1) gene risk variants (RV) associate with renal and cardiovascular disease particularly in SLE. We hypothesized that in RV-carrying human umbilical vein endothelial cells (HUVECs) cytokine-induced APOL1 expression compromises mitochondrial respiration, lysosome integrity, and autophagic flux. HUVEC cultures of each APOL1 genotype were generated. APOL1 was expressed using IFN{gamma}; HUVEC mitochondrial function, lysosome integrity, and autophagic flux were measured. IFN{gamma} increased APOL1 expression across all genotypes 20-fold (p=0.001). Compared to the homozygous G0 (ancestral) allele (0RV), high risk (2RV) HUVECs showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosome compartment (p<0.001), and an accumulation of autophagosomes suggesting a defect in autophagic flux. Treatment of 0RV HUVECs with a non-selective lysosome inhibitor, hydroxychloroquine, produced autophagosome accumulations similar to the 2RV cells, thus implicating lysosome dysfunction in blocking autophagy. Compared to 0RV and 2RV HUVECs, 1 RV cells demonstrated an intermediate autophagy defect which was exacerbated by IFN{gamma}. Our findings implicate dysfunction of mitochondrial respiration, lysosome, and autophagy in APOL1 RV-mediated endothelial cytotoxicity. IFN{gamma} amplified this phenotype even in variant heterozygous cells-a potential underpin of the APOL1/inflammation interaction. This is the first description of APOL1 pathobiology in variant heterozygous cell cultures.

cell biology↗

bioRxiv2018

Insights from Comparison of the Renal and Skin Single Cell Transcriptomes in Lupus Nephritis

Lupus nephritis (LN) occurs in up to 50% of patients with systemic lupus erythematosus (SLE), and is a major contributor to mortality and morbidity. LN presents as a highly heterogeneous disease both in histopathology and response to therapy. The molecular and cellular processes leading to renal damage and to the heterogeneity of the disease are not well understood. To elucidate the processes underpinning the heterogeneity of LN, we applied singlecell RNA-sequencing (scRNA-seq) to renal biopsies from LN patients. Skin biopsies were evaluated as a source of biomarkers for monitoring kidney disease. Type-I interferon (IFN) response signatures were identified in tubular cells and keratinocytes, differentiating LN patients from healthy controls. Non-responders associated with higher IFN signatures in both tissue compartments. Moreover, non-response was also associated with a fibrotic signature in the tubular cells. Receptor-ligand interaction analysis indicated that the fibrotic process is likely mediated by FGF receptors with the initiating signal originating from infiltrating leukocytes. Differential expression analysis of tubular cells between proliferative and membranous LN pointed to several fibrosis-relevant pathways, which may offer insight into their histological differences. In summary, scRNA-seq was applied to LN to deconstruct its heterogeneity and provide novel targets for personalized approaches to therapy.

immunology↗