Regulatory T cells (Tregs) are immune regulatory T cells that are vital for controlling inflammation. The role of Tregs in inflammatory diseases namely psoriatic arthritis (PsA) is still poorly understood. The underlying reason being a lack of robust unbiased analysis to test the immune regulatory phenotype of human Tregs. Here, we propose that checkpoint receptors can identify functional Tregs in PsA. Using unbiased BD Rhapsody single-cell analysis, we have analyzed the expression pattern of checkpoint receptors in Tregs and found that PsA Tregs are enriched in the expression of CTLA4, TIGIT, PD-1, and GITR while TIM3 was downregulated. Furthermore, PD-1+ Tregs in PsA had an increased type 1 phenotype and expressed the protease asparaginyl endopeptidase. By harnessing the PD-1 signaling pathway and inhibiting asparaginyl endopeptidase, PsA Treg function was significantly enhanced in in vitro suppressor assays. Next, we interrogated the cell interaction pathways of Tregs in PsA and found a diminished crosstalk with circulating osteoclast precursors through the CD244-CD48 coreceptor pathways. Therapeutically, PsA Treg function could be enhanced by modulating PD1 and osteoclast interactions. Our study suggests that unconventional immune cell crosstalk with Tregs is severely diminished in PsA.

Stargardt disease is an inherited retinopathy affecting approximately 1:8000 individuals. It is characterised by biallelic variants in ABCA4 which encodes a vital protein for the recycling of retinaldehydes in the retina. Despite its prevalence and impact, there are currently no treatments available for this condition. Furthermore, 35% of STGD1 cases remain genetically unsolved. To investigate the cellular and molecular characteristics associated with STGD1, we generated iPSCs from two monoallelic unresolved (PT1 & PT2), late-onset STGD1 cases with the heterozygous complex allele - c.[5461-10 T > C;5603 A > T]. Both patient iPSCs and those from a biallelic affected control (AC) carrying -c.4892 T > C and c.4539+2001G > A, were differentiated to retinal organoids, which developed all key retinal neurons and photoreceptors with outer segments positive for ABCA4 expression. We observed patient-specific disruption to lamination with OPN1MW/LW+ cone photoreceptor retention in the retinal organoid centre during differentiation. Photoreceptor retention was more severe in the AC case affecting both cones and rods, suggesting a genotype/phenotype correlation. scRNA-Seq suggests retention may be due to the induction of stress-related pathways in photoreceptors. Whole genome sequencing successfully identified the missing alleles in both cases; PT1 reported c.-5603A > T in homozygous state and PT2 uncovered a rare hypomorph - c.-4685T > C. Furthermore, retinal organoids were able to recapitulate the retina-specific splicing defect in PT1 as shown by long-read RNA-seq data. Collectively, these results highlight the suitability of retinal organoids in STGD1 modelling. Their ability to display genotype-phenotype correlations enhances their utility as a platform for therapeutic development.

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy.

Purpose (the aim of the study): Understanding the aetiology of post-traumatic osteoarthritis (PTOA) is imperative to define the early initiating events and identify effective diagnostics for subsequent treatment; animal PTOA models are routinely used to recapitulate the pathological changes observed in human PTOA including destabilisation of the medial meniscus (DMM) and non-surgical mechanically induced rupture of the anterior cruciate ligament (ACL). MicroRNAs (miRNAs), small non-coding RNAs that control gene expression are regulated in the early phases of OA. In this study, we aimed to (i) characterise the expression profile of mRNA and miRNAs following mechanically induced ACL rupture to identify miRNAs, and their downstream mRNA targets, that are regulated in the early phases of PTOA disease progression and (ii) compare the transcriptome signature with that following DMM to assess commonality in gene mechano-regulation within these two models of PTOA.

Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation, presence of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development, and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids. Formation of the retina during development involves the coordinated action of retinal progenitor cells and their differentiated cell types, which is key for producing a functioning eye. Here the authors provide a detailed atlas of human retinal development, combining scRNA-seq and spatial transcriptomics, and identify key genetic factors that mediate retinal progenitor cell proliferation and differentiation.

Immune-mediated inflammatory diseases (IMIDs) are typically characterised by relapsing and remitting flares of inflammation. However, the unpredictability of disease flares impedes their study. Addressing this critical knowledge gap, we use the experimental medicine approach of immunomodulatory drug withdrawal in rheumatoid arthritis (RA) remission to synchronise flare processes allowing detailed characterisation. Exploratory mass cytometry analyses reveal three circulating cellular subsets heralding the onset of arthritis flare – CD45RO + PD1 hi CD4 + and CD8 + T cells, and CD27 + CD86 + CD21 - B cells – further characterised by single-cell sequencing. Distinct lymphocyte subsets including cytotoxic and exhausted CD4 + memory T cells, memory CD8 + CXCR5 + T cells, and IGHA1 + plasma cells are primed for activation in flare patients. Regulatory memory CD4 + T cells (Treg cells) increase at flare onset, but with dysfunctional regulatory marker expression compared to drug-free remission. Significant clonal expansion is observed in T cells, but not B cells, after drug cessation; this is widespread throughout memory CD8 + T cell subsets but limited to the granzyme-expressing cytotoxic subset within CD4 + memory T cells. Based on our observations, we suggest a model of immune dysregulation for understanding RA flare, with potential for further translational research towards novel avenues for its treatment and prevention.

Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here, skeletally mature C57Bl6 mice were subjected to either rapid-onset non-surgical mechanical rupture of the anterior cruciate ligament (ACL) or to surgical destabilisation of the medial meniscus (DMM). Transcriptome profiling of micro-dissected cartilage at day 7 or day 42 following ACL or DMM procedure, respectively, showed that the two models were comparable and highly correlative. Gene ontology (GO) enrichment analysis identified similarly enriched pathways that were overrepresented by anabolic terms. To address the transcriptome changes more completely in the ACL model, we also performed small RNA sequencing, describing the first microRNA profile of this model. miR-199-5p was amongst the most abundant, yet differentially expressed, microRNAs, and its inhibition in primary human chondrocytes led to a transcriptome response that was comparable to that observed in both human 'OA damaged vs intact cartilage' and murine DMM cartilage datasets. We also experimentally verified CELSR1, GIT1, ECE1 and SOS2 as novel miR-199-5p targets. Together, these data support the use of the ACL rupture model as a non-invasive companion to the DMM model.

BackgroundTracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs and excess mortality. The underlying mechanisms facilitating adverse respiratory outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses. MethodsTracheal aspirates, tracheal cytology brushings and nasal swabs were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome. ResultsChildren followed up serially from the time of tracheostomy up to 3 months postprocedure (n=9) were studied. A further cohort of children with a long-term tracheostomy were also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Long-term tracheostomy was associated with airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. Reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter. ConclusionsLong-term childhood tracheostomy is associated with a inflammatory tracheal phenotype characterised by neutrophilic inflammation and the ongoing presence of potential respiratory pathogens. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients.

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human fetal and adult RPE-choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE-choroid tissue. Single-cell comparative transcriptional analysis between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these findings need to be validated by additional techniques in a larger number of AMD patients and controls.

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.

Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.

Neurodegeneration in Parkinson’s disease (PD) precedes diagnosis by years. Early neurodegeneration may be reflected in RNA levels and measurable as a biomarker. Here, we present the largest quantification of whole blood linear and circular RNAs (circRNA) in early-stage PD, using RNA sequencing data from two cohorts (PPMI = 259 PD, 161 Controls; ICICLE-PD = 48 PD, 48 Controls). We identified a replicable increase in TMEM252 and LMNB1 gene expression in PD. We identified novel differences in the expression of circRNAs from ESYT2 , BMS1P1 and CCDC9 , and replicated trends of previously reported circRNAs. Overall, using circRNA as a diagnostic biomarker in Parkinson’s did not show any clear improvement over linear RNA. More interestingly, we observed a general reduction in circRNA expression in both PD cohorts, accompanied by an increase in RNASEL expression. This imbalance implicates the activation of an innate antiviral immune response and suggests a previously unknown aspect of circRNA regulation in PD.

Innate lymphoid cells (ILCs) play a key role in tissue-mediated immunity and can be controlled by coreceptor signaling. Here, we define a subset of ILCs that are Tbet + NK1.1 − and are present within the tumor microenvironment (TME). We show programmed death-1 receptor (PD-1) expression on ILCs within TME is found in Tbet + NK1.1 − ILCs. PD-1 significantly controlled the proliferation and function of Tbet + NK1.1 − ILCs in multiple murine and human tumors. We found tumor-derived lactate enhanced PD-1 expression on Tbet + NK1.1 − ILCs within the TME, which resulted in dampened the mammalian target of rapamycin (mTOR) signaling along with increased fatty acid uptake. In line with these metabolic changes, PD-1-deficient Tbet + NK1.1 − ILCs expressed significantly increased IFNγ and granzyme B and K. Furthermore, PD-1-deficient Tbet + NK1.1 − ILCs contributed toward diminished tumor growth in an experimental murine model of melanoma. These data demonstrate that PD-1 can regulate antitumor responses of Tbet + NK1.1 − ILCs within the TME.

Objectives: The objective of this study was to investigate the expression of genes in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), both at the mild cognitive impairment (MCI) and dementia stages, to improve our under-standing of disease pathophysiology and investigate the potential for diagnostic and prognostic biomarkers based on mRNA expression.Design: Cross-sectional observational study. Setting: University research center.Participants: People with MCI with Lewy bodies (MCI-LB, n=55), MCI-AD (n=19), DLB (n=38), AD (n=24) and a cognitively unimpaired comparison group (n=28).Measurements: Ribonucleic acid sequencing of whole blood. Differentially expressed genes (DEGs) were identified and gene set enrichment analysis was carried out.Results: Compared with the cognitively unimpaired group, there were 22 DEGs in MCI-LB/DLB and 61 DEGs in MCI-AD/AD. DEGS were also identified when comparing the two disease groups. Expression of ANP32A was associated with more rapid cognitive decline in MCI-AD/AD. Gene set enrich-ment analysis identified downregulation in gene sets including MYC targets and oxidative phosphorylation in MCI-LB/DLB; upregulation of immune and inflammatory responses in MCI-AD/AD; and upregulation of interferon -a and -g responses in MCI-AD/AD compared with MCI-LB/DLB.Conclusion: This study identified multiple DEGs in MCI-LB/DLB and MCI-AD/AD. One of these DEGs, ANP32A, may be a prognostic marker in AD. Genes related to mitochon-drial function were downregulated in MCI-LB/DLB. Previously reported

We previously discovered that deletion of c-Rel in the Eμ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that c-rel -/- Eµ-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- Eµ-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- Eµ-Myc in vivo, while having no effect on wild type Eμ-Myc cells. This data reveals an NF-κB regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.

Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5–10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10–95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs play a role in the pathophysiology of hypertension.