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Type-1 IFN primed monocytes in pathogenesis of idiopathic pulmonary fibrosis
ABSTRACT Idiopathic pulmonary fibrosis (IPF) is the most severe form of lung fibrosis. It is progressive, and has an extremely poor outcome and limited treatment options. The disease exclusively affects the lungs, and thus less attention has been focused on blood-borne immune cells. which could be a more effective therapeutic target than lung-based cells. Here, we questioned if circulating monocytes, which has been shown to be increased in IPF, bore abnormalities that might contribute to its pathogenesis. We found that levels of circulating monocytes correlated directly with the extent of fibrosis in the lungs, and increased further during acute clinical deterioration. Monocytes in IPF were phenotypically distinct, displaying increased expression of CD64, a type 1 IFN gene expression signature and a greater magnitude of type 1 IFN response when stimulated. These abnormalities were accompanied by markedly raised CSF-1 levels in the serum, prolonged survival of monocytes ex vivo , and increased numbers of monocytes in lung tissue. Our study defines the key monocytic abnormalities in IPF, proposing type 1 IFN-primed monocytes as a potential driver of an aberrant repair response and fibrosis. It provides a rationale for targeting monocytes and identifies monocytic CD64 as a potential specific therapeutic target for IPF.
Isolation of human fetal intestinal cells for single-cell RNA sequencing.
The intestine has a large number of cell types. Thus, digestion of pure and viable populations is necessary for downstream techniques including single-cell RNA sequencing. We outline a protocol to isolate both epithelial and non-epithelial cells from human fetal samples at high viability, which was used to produce a full thickness atlas of intestinal cells across human development. This protocol can also be adapted to adult endoscopy and surgical specimens. For details on the use of this protocol, please refer to Fawkner-Corbett et al. (2021).
Tracking in situ checkpoint inhibitor-bound target T cells in patients with checkpoint-induced colitis.
The success of checkpoint inhibitors (CPIs) for cancer has been tempered by immune-related adverse effects including colitis. CPI-induced colitis is hallmarked by expansion of resident mucosal IFNγ cytotoxic CD8+ T cells, but how these arise is unclear. Here, we track CPI-bound T cells in intestinal tissue using multimodal single-cell and subcellular spatial transcriptomics (ST). Target occupancy was increased in inflamed tissue, with drug-bound T cells located in distinct microdomains distinguished by specific intercellular signaling and transcriptional gradients. CPI-bound cells were largely CD4+ T cells, including enrichment in CPI-bound peripheral helper, follicular helper, and regulatory T cells. IFNγ CD8+ T cells emerged from both tissue-resident memory (TRM) and peripheral populations, displayed more restricted target occupancy profiles, and co-localized with damaged epithelial microdomains lacking effective regulatory cues. Our multimodal analysis identifies causal pathways and constitutes a resource to inform novel preventive strategies.
Heterozygous COL17A1 variants are a frequent cause of amelogenesis imperfecta.
BACKGROUND: Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised. METHODS: Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth. RESULTS: Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting. CONCLUSION: These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care.
A systems approach evaluating the impact of SARS-CoV-2 variant of concern mutations on CD8+ T cell responses.
T cell recognition of SARS-CoV-2 antigens after vaccination and/or natural infection has played a central role in resolving SARS-CoV-2 infections and generating adaptive immune memory. However, the clinical impact of SARS-CoV-2-specific T cell responses is variable and the mechanisms underlying T cell interaction with target antigens are not fully understood. This is especially true given the virus' rapid evolution, which leads to new variants with immune escape capacity. In this study, we used the Omicron variant as a model organism and took a systems approach to evaluate the impact of mutations on CD8+ T cell immunogenicity. We computed an immunogenicity potential score for each SARS-CoV-2 peptide antigen from the ancestral strain and Omicron, capturing both antigen presentation and T cell recognition probabilities. By comparing ancestral vs. Omicron immunogenicity scores, we reveal a divergent and heterogeneous landscape of impact for CD8+ T cell recognition of mutated targets in Omicron variants. While T cell recognition of Omicron peptides is broadly preserved, we observed mutated peptides with deteriorated immunogenicity that may assist breakthrough infection in some individuals. We then combined our scoring scheme with an in silico mutagenesis, to characterise the position- and residue-specific theoretical mutational impact on immunogenicity. While we predict many escape trajectories from the theoretical landscape of substitutions, our study suggests that Omicron mutations in T cell epitopes did not develop under cell-mediated pressure. Our study provides a generalisable platform for fostering a deeper understanding of existing and novel variant impact on antigen-specific vaccine- and/or infection-induced T cell immunity.
Targeting human plasmacytoid dendritic cells through BDCA2 prevents skin inflammation and fibrosis in a novel xenotransplant mouse model of scleroderma.
OBJECTIVES: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. METHODS: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression. RESULTS: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses. CONCLUSIONS: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc.
TNF-α Regulates Human Plasmacytoid Dendritic Cells by Suppressing IFN-α Production and Enhancing T Cell Activation.
Human plasmacytoid dendritic cells (pDCs) play a vital role in modulating immune responses. They can produce massive amounts of type I IFNs in response to nucleic acids via TLRs, but they are also known to possess weak Ag-presenting properties inducing CD4+ T cell activation. Previous studies showed a cross-regulation between TNF-α and IFN-α, but many questions remain about the effect of TNF-α in regulating human pDCs. In this study, we showed that TNF-α significantly inhibited the secretion of IFN-α and TNF-α of TLR-stimulated pDCs. Instead, exogenous TNF-α promoted pDC maturation by upregulating costimulatory molecules and chemokine receptors such as CD80, CD86, HLA-DR, and CCR7. Additionally, RNA sequencing analysis showed that TNF-α inhibited IFN-α and TNF-α production by downregulating IRF7 and NF-κB pathways, while it promoted Ag processing and presentation pathways as well as T cell activation and differentiation. Indeed, TNF-α-treated pDCs induced in vitro higher CD4+ T cell proliferation and activation, enhancing the production of Th1 and Th17 cytokines. In conclusion, TNF-α favors pDC maturation by switching their main role as IFN-α-producing cells to a more conventional dendritic cell phenotype. The functional status of pDCs might therefore be strongly influenced by their overall inflammatory environment, and TNF-α might regulate IFN-α-mediated aspects of a range of autoimmune and inflammatory diseases.
Evaluating performance of existing computational models in predicting CD8+ T cell pathogenic epitopes and cancer neoantigens
T cell recognition of a cognate peptide-MHC complex (pMHC) presented on the surface of infected or malignant cells, is of utmost importance for mediating robust and long-term immune responses. Accurate predictions of cognate pMHC targets for T Cell Receptors (TCR) would greatly facilitate identification of vaccine targets for both pathogenic diseases as well as personalized cancer immunotherapies. Predicting immunogenic peptides therefore has been at the centre of intensive research for the past decades but has proven challenging. Although numerous models have been proposed, performance of these models has not been systematically evaluated and their success rate in predicting epitopes in the context of human pathology, has not been measured and compared. In this study, we evaluated the performance of several publicly available models, in identifying immunogenic CD8+ T cell targets in the context of pathogens and cancers. We found that for predicting immunogenic peptides from an emerging virus such as SARS-CoV-2, none of the models perform substantially better than random or offer considerable improvement beyond HLA ligand prediction. We also observed suboptimal performance for predicting cancer neoantigens. Through investigation of potential factors associated with ill performance of models, we highlight several data- and model-associated issues. In particular, we observed that cross-HLA variation in the distribution of immunogenic and non-immunogenic peptides in training data of the models seem to substantially confound the predictions. We additionally compared key parameters associated with immunogenicity between pathogenic peptides and cancer neoantigens and observed evidence for differences in the thresholds of binding affinity and stability, which suggested the need to modulate different features in identifying immunogenic pathogen vs. cancer peptides. Overall, we demonstrate that accurate and reliable prediction of immunogenic CD8+ T cell targets remains unsolved, thus we hope our work will guide users and model developers regarding potential pitfalls and unsettled questions in existing immunogenicity predictors.
Functionally impaired plasmacytoid dendritic cells and non-haematopoietic sources of type I interferon characterize human autoimmunity.
Autoimmune connective tissue diseases arise in a stepwise fashion from asymptomatic preclinical autoimmunity. Type I interferons have a crucial role in the progression to established autoimmune diseases. The cellular source and regulation in disease initiation of these cytokines is not clear, but plasmacytoid dendritic cells have been thought to contribute to excessive type I interferon production. Here, we show that in preclinical autoimmunity and established systemic lupus erythematosus, plasmacytoid dendritic cells are not effector cells, have lost capacity for Toll-like-receptor-mediated cytokine production and do not induce T cell activation, independent of disease activity and the blood interferon signature. In addition, plasmacytoid dendritic cells have a transcriptional signature indicative of cellular stress and senescence accompanied by increased telomere erosion. In preclinical autoimmunity, we show a marked enrichment of an interferon signature in the skin without infiltrating immune cells, but with interferon-κ production by keratinocytes. In conclusion, non-hematopoietic cellular sources, rather than plasmacytoid dendritic cells, are responsible for interferon production prior to clinical autoimmunity.
Translating Biomarkers of Cholangiocarcinoma for Theranosis: A Systematic Review.
Cholangiocarcinoma (CCA) is a rare disease with poor outcomes and limited research efforts into novel treatment options. A systematic review of CCA biomarkers was undertaken to identify promising biomarkers that may be used for theranosis (therapy and diagnosis). MEDLINE/EMBASE databases (1996-2019) were systematically searched using two strategies to identify biomarker studies of CCA. The PANTHER Go-Slim classification system and STRING network version 11.0 were used to interrogate the identified biomarkers. The TArget Selection Criteria for Theranosis (TASC-T) score was used to rank identified proteins as potential targetable biomarkers for theranosis. The following proteins scored the highest, CA9, CLDN18, TNC, MMP9, and EGFR, and they were evaluated in detail. None of these biomarkers had high sensitivity or specificity for CCA but have potential for theranosis. This review is unique in that it describes the process of selecting suitable markers for theranosis, which is also applicable to other diseases. This has highlighted existing validated markers of CCA that can be used for active tumor targeting for the future development of targeted theranostic delivery systems. It also emphasizes the relevance of bioinformatics in aiding the search for validated biomarkers that could be repurposed for theranosis.
Multi-Modal Characterization of Monocytes in Idiopathic Pulmonary Fibrosis Reveals a Primed Type I Interferon Immune Phenotype.
Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo. These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64hi monocytes and "transitional macrophages" with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF.
A Chromosome 7 Pericentric Inversion Defined at Single-Nucleotide Resolution Using Diagnostic Whole Genome Sequencing in a Patient with Hand-Foot-Genital Syndrome.
Next generation sequencing methodologies are facilitating the rapid characterisation of novel structural variants at nucleotide resolution. These approaches are particularly applicable to variants initially identified using alternative molecular methods. We report a child born with bilateral postaxial syndactyly of the feet and bilateral fifth finger clinodactyly. This was presumed to be an autosomal recessive syndrome, due to the family history of consanguinity. Karyotype analysis revealed a homozygous pericentric inversion of chromosome 7 (46,XX,inv(7)(p15q21)x2) which was confirmed to be heterozygous in both unaffected parents. Since the resolution of the karyotype was insufficient to identify any putatively causative gene, we undertook medium-coverage whole genome sequencing using paired-end reads, in order to elucidate the molecular breakpoints. In a two-step analysis, we first narrowed down the region by identifying discordant read-pairs, and then determined the precise molecular breakpoint by analysing the mapping locations of "soft-clipped" breakpoint-spanning reads. PCR and Sanger sequencing confirmed the identified breakpoints, both of which were located in intergenic regions. Significantly, the 7p15 breakpoint was located 523 kb upstream of HOXA13, the locus for hand-foot-genital syndrome. By inference from studies of HOXA locus control in the mouse, we suggest that the inversion has delocalised a HOXA13 enhancer to produce the phenotype observed in our patient. This study demonstrates how modern genetic diagnostic approach can characterise structural variants at nucleotide resolution and provide potential insights into functional regulation.
Deficiency of the myogenic factor MyoD causes a perinatally lethal fetal akinesia.
BACKGROUND: Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. METHODS: Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. RESULTS: Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. CONCLUSIONS: We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme.
Amelogenesis Imperfecta; Genes, Proteins, and Pathways.
Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the possibility of novel treatments and prevention strategies for AI.
m6aViewer: software for the detection, analysis, and visualization of N6-methyladenosine peaks from m6A-seq/ME-RIP sequencing data.
Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.