ABSTRACT
The redirection of T cells has emerged as an attractive therapeutic principle in B cell non-Hodgkin lymphoma (B-NHL). However, a detailed characterization of lymphoma-infiltrating T cells across B-NHL entities is missing. Here we present an in-depth T cell reference map of nodal B-NHL, based on cellular indexing of transcriptomes and epitopes, T cell receptor sequencing, flow cytometry and multiplexed immunofluorescence applied to 101 lymph nodes from patients with diffuse large B cell, mantle cell, follicular or marginal zone lymphoma, and from healthy controls. This multimodal resource revealed quantitative and spatial aberrations of the T cell microenvironment across and within B-NHL entities. Quantitative differences in PD1+ TCF7- cytotoxic T cells, T follicular helper cells or IKZF3+ regulatory T cells were linked to their clonal expansion. The abundance of PD1+ TCF7- cytotoxic T cells was associated with poor survival. Our study portrays lymphoma-infiltrating T cells with unprecedented comprehensiveness and provides a unique resource for the investigation of lymphoma biology and prognosis.
Subject(s)
Lymphoma, B-Cell, Marginal Zone , T-Lymphocytes , Humans , T-Lymphocytes/pathology , B-Lymphocytes/pathology , Lymphoma, B-Cell, Marginal Zone/pathology , Transforming Growth Factor beta , Tumor MicroenvironmentABSTRACT
BACKGROUND: Macrophages play an important role in maintaining liver homeostasis and regeneration. However, it is not clear to what extent the different macrophage populations of the liver differ in terms of their activation state and which other liver cell populations may play a role in regulating the same. METHODS: Reverse transcription PCR, flow cytometry, transcriptome, proteome, secretome, single cell analysis, and immunohistochemical methods were used to study changes in gene expression as well as the activation state of macrophages in vitro and in vivo under homeostatic conditions and after partial hepatectomy. RESULTS: We show that F4/80+/CD11bhi/CD14hi macrophages of the liver are recruited in a C-C motif chemokine receptor (CCR2)-dependent manner and exhibit an activation state that differs substantially from that of the other liver macrophage populations, which can be distinguished on the basis of CD11b and CD14 expressions. Thereby, primary hepatocytes are capable of creating an environment in vitro that elicits the same specific activation state in bone marrow-derived macrophages as observed in F4/80+/CD11bhi/CD14hi liver macrophages in vivo. Subsequent analyses, including studies in mice with a myeloid cell-specific deletion of the TGF-ß type II receptor, suggest that the availability of activated TGF-ß and its downregulation by a hepatocyte-conditioned milieu are critical. Reduction of TGF-ßRII-mediated signal transduction in myeloid cells leads to upregulation of IL-6, IL-10, and SIGLEC1 expression, a hallmark of the activation state of F4/80+/CD11bhi/CD14hi macrophages, and enhances liver regeneration. CONCLUSIONS: The availability of activated TGF-ß determines the activation state of specific macrophage populations in the liver, and the observed rapid transient activation of TGF-ß may represent an important regulatory mechanism in the early phase of liver regeneration in this context.
Subject(s)
Liver Regeneration , Transforming Growth Factor beta , Animals , Mice , Gene Expression , Hepatocytes/metabolism , Macrophages/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
BACKGROUND AND OBJECTIVES: Immune responses in the central nervous system (CNS) are highly compartmentalized and cerebrospinal fluid (CSF) in particular often reflects CNS pathology better than peripheral blood. While CSF leukocytes are known to be distinct from blood, the immediate effects of peripheral leukocyte depletion on CSF leukocytes have not been studied in humans. METHODS: We here analyzed CSF and blood from two relapsing-remitting multiple sclerosis (RRMS) patients early after peripheral leukocyte depletion with the anti-CD52 antibody alemtuzumab compared to untreated RRMS and control patients using single cell RNA-sequencing. RESULTS: As expected for alemtuzumab, most leukocyte lineages including T cells were synchronously depleted from CSF and blood, while - surprisingly - pDCs were maintained in CSF but depleted from blood by alemtuzumab. Transcriptionally, genes associated with migration were elevated only in the CSF after alemtuzumab. Predicted cellular interactions indicated a central role of pDCs and enhanced migration signaling in the CSF after alemtuzumab. DISCUSSION: The CSF and blood compartments are thus partially uncoupled, emphasizing that the CNS is only partially accessible even for treatments profoundly affecting the blood.
Subject(s)
Multiple Sclerosis, Relapsing-Remitting , Multiple Sclerosis , Humans , Alemtuzumab/adverse effects , Multiple Sclerosis/drug therapy , Multiple Sclerosis/chemically induced , Central Nervous SystemABSTRACT
Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. We found that IL-6, measured in various cell types in post-MI hearts at the protein level and by quantitative PCR and RNAscope, was preferentially formed by cardiac fibroblasts (CFs). Single-cell RNA-Seq (scRNA-Seq) in infarcted mouse and human hearts confirmed this finding. We found that adenosine stimulated fibroblast IL-6 formation via the adenosine receptor A2bR in a Gq-dependent manner. CFs highly expressed Adora2b and rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans, scRNA-Seq revealed that Adora2B was also mainly expressed by fibroblasts. We assessed global IL-6 production in isolated hearts from mice lacking CD73 on T cells (CD4-CD73-/-), a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4-CD73-/- mice, suggesting adenosine-mediated modulation. Together, these findings demonstrate that post-MI IL-6 was mainly derived from activated CFs and was controlled by T cell-derived adenosine. We show that purinergic metabolic cooperation between CFs and T cells is a mechanism that modulates IL-6 formation by the heart and has therapeutic potential.
Subject(s)
Fibroblasts , Interleukin-6 , Myocardial Infarction , T-Lymphocytes , Animals , Humans , Mice , Adenosine/metabolism , Fibroblasts/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Mice, Inbred C57BL , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , T-Lymphocytes/metabolismABSTRACT
Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina, ultra-long Nanopore, and PacBio HiFi sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (GRCh38/hg38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.06%-0.49% in a 1000 Genomes Project read remapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.
Subject(s)
Histocompatibility Antigens Class II , Major Histocompatibility Complex , Humans , Haplotypes , Alleles , Histocompatibility Antigens Class II/genetics , Major Histocompatibility Complex/genetics , HLA Antigens/genetics , HLA-C Antigens/geneticsABSTRACT
Acute myocardial infarction (MI) induces an extensive sterile inflammation, which is dominated in the early phase by invading neutrophils and monocytes/macrophages. The inflammatory response after MI critically affects infarct healing and cardiac remodeling. Therefore, modulation of cardiac inflammation may improve outcome post MI. Insulin-like growth factor 1 (IGF1) treatment reduces infarct size and improves cardiac function after MI via IGF1 receptor mediated signaling in myeloid cells. Our study aimed to investigate the effect of IGF1 on neutrophil phenotype both in vitro and in vivo after MI. We show that IGF1 induces an anti-inflammatory phenotype in bone marrow derived neutrophils. On the molecular and functional level IGF1 treated neutrophils were indistinguishable from those induced by IL4. Surprisingly, insulin, even though it is highly similar to IGF1 did not create anti-inflammatory neutrophils. Notably, the IGF1 effect was independent of the canonical Ras/Raf/ERK or PI3K/AKT pathway, but depended on activation of the JAK2/STAT6 pathway, which was not activated by insulin treatment. Single cell sequencing analysis 3 days after MI also showed that 3 day IGF1 treatment caused a downregulation of pro-inflammatory genes and upstream regulators in most neutrophil and many macrophage cell clusters whereas anti-inflammatory genes and upstream regulators were upregulated. Thus, IGF1 acts like an anti-inflammatory cytokine on myeloid cells in vitro and attenuates the pro-inflammatory phenotype of neutrophils and macrophages in vivo after MI. IGF1 treatment might therefore represent an effective immune modulatory therapy to improve the outcome after MI.
Subject(s)
Insulin-Like Growth Factor I , Myocardial Infarction , Neutrophils , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Humans , Inflammation/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/pharmacology , Insulins/therapeutic use , Myocardial Infarction/metabolism , Neutrophils/metabolism , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
Uveitis describes a heterogeneous group of inflammatory eye diseases characterized by infiltration of leukocytes into the uveal tissues. Uveitis associated with the HLA haplotype B27 (HLA-B27) is a common subtype of uveitis and a prototypical ocular immune-mediated disease. Local immune mechanisms driving human uveitis are poorly characterized mainly due to the limited available biomaterial and subsequent technical limitations. Here, we provide the first high-resolution characterization of intraocular leukocytes in HLA-B27-positive (n = 4) and -negative (n = 2) anterior uveitis and an infectious endophthalmitis control (n = 1) by combining single-cell RNA-sequencing with flow cytometry and protein analysis. Ocular cell infiltrates consisted primarily of lymphocytes in both subtypes of uveitis and of myeloid cells in infectious endophthalmitis. HLA-B27-positive uveitis exclusively featured a plasmacytoid and classical dendritic cell (cDC) infiltrate. Moreover, cDCs were central in predicted local cell-cell communication. This suggests a unique pattern of ocular leukocyte infiltration in HLA-B27-positive uveitis with relevance to DCs.
Uveitis is a form of inflammation in the eye. It can occur in response to infection, or when the immune system mistakenly attacks the eye, in what is known as autoimmune uveitis. In approximately 80 percent of cases, the front part of the eye is affected. During an inflammatory episode, the liquid inside the front part of the eye fills with immune cells, but the nature of these cells remains unknown. This is because uveitis is rare, and doctors cannot routinely take samples from inside the eyes of affected individuals to diagnose the disease. This lack of samples makes research into this disease challenging. There are two main groups of immune cells that could be responsible for uveitis: myeloid cells and lymphoid cells. Myeloid cells form the first line of immune defense against infection by non-specifically attacking and removing pathogens . Lymphoid cells form the second line of immune defense, attacking specific pathogens. Lymphoid cells also have long-term memory, meaning they can 'remember' previous infections and fight them more effectively. Lymphoid cells receive instructions from a type of myeloid cell called a dendritic cell about what to attack. Dendritic cells relay their instructions to lymphoid cells using molecules called human leukocyte antigens (HLA). Autoimmune uveitis affecting the front part of the eye is common in individuals with an HLA type called HLA-B27, suggesting that communication between dendritic and lymphoid cells plays an important role in this type of inflammation. To make the most of limited patient samples, Kasper et al. used single cell techniques to examine the immune cells from the fluid inside the eye. Six samples came from people with autoimmune uveitis, and one from a person with an eye infection. The infection sample contained mainly myeloid cells that might attack bacteria responsible for the infection. In contrast, the autoimmune uveitis samples contained mainly lymphoid cells. Of these samples, four were from individuals with the gene that codes for the HLA-B27 molecule. These samples had a unique pattern of immune cells, with more dendritic cells than the samples from individuals that did not have this gene. This study included only a small number of individuals, but it shows that analysing single immune cells from the eye is possible in uveitis. This snapshot could help researchers understand the local immune response in the eye, and find an optimal treatment.
Subject(s)
Dendritic Cells/classification , HLA-B27 Antigen/immunology , Uveitis, Anterior/pathology , Endophthalmitis/pathology , Female , Humans , Lymphocytes , Male , Myeloid Cells , Sequence Analysis, RNA , Uveitis, Anterior/immunologyABSTRACT
Nine strains of a Rodentibacter-related bacterium were isolated over a period of 38 years from a laboratory mouse (Mus musculus), seven laboratory rats (Rattus norvegicus) and a Syrian hamster (Mesocricetus auratus) in Düsseldorf and Heidelberg, Germany. The isolates are genotypically and phenotypically distinct from all previously described Rodentibacter species. Sequence analysis of 16S rRNA and rpoB gene sequences placed the isolates as a novel lineage within the genus Rodentibacter. In addition to the single-gene analysis, the whole genome sequence of the strain 1625/19T revealed distinct genome-to-genome distance values to the other Rodentibacter species. The genomic DNA G+C content of strain 1625/19T was 40.8âmol% within the range of Rodentibacter. At least six phenotypic characteristics separate the new isolates from the other Rodentibacter species, with Rodentibacter heylii being the most closely related. In contrast to the latter, the new strains display ß-haemolysis and are ß-glucuronidase, d-mannitol and sorbitol positive, but fail to produce lysine decarboxylase and trehalose. The genotypic and phenotypic differences between the novel strains and the other closely related strains of the genus Rodentibacter indicate that they represent a novel species within the genus Rodentibacter, family Pasteurellaceae, for which the name Rodentibacter haemolyticus sp. nov. is proposed. The type strain 1625/19T, (=DSM 111151T=CCM 9081T), was isolated in 2019 from the nose of a laboratory mouse (Mus musculus) in Düsseldorf, Germany.
Subject(s)
Mesocricetus/microbiology , Mice/microbiology , Pasteurellaceae , Phylogeny , Rats/microbiology , Animals , Animals, Laboratory/microbiology , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Germany , Pasteurellaceae/classification , Pasteurellaceae/isolation & purification , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.
Subject(s)
Fibroblasts/metabolism , Myocardium/metabolism , Pericardium/physiology , Stromal Cells/metabolism , Transcriptome , Animals , Gene Expression Profiling , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , RNA , Sequence Analysis, RNA , Single-Cell Analysis , WT1 Proteins/metabolismABSTRACT
Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C3 barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C4 maize (Zea mays L.) plants. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which abBS cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of abBS cells and support a modification to the canonical phloem loading pathway in maize.
Subject(s)
Phloem/metabolism , Plant Leaves/metabolism , Plant Proteins/metabolism , Zea mays/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Phloem/genetics , Plant Leaves/genetics , Plant Proteins/genetics , Zea mays/geneticsABSTRACT
BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis. METHODS: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4-/-, Abcb4-/-/Tgr5Tg and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4-/- mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models. RESULTS: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4-/- livers, in Abcb4-/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4-/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfß2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4-/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4-/- livers. CONCLUSIONS: Reduced TGR5 levels in BECs from patients with PSC and Abcb4-/- mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells.
Subject(s)
Biliary Tract/drug effects , Cholangitis, Sclerosing/genetics , Down-Regulation/drug effects , Receptors, G-Protein-Coupled/drug effects , Animals , Biliary Tract/metabolism , Cholangitis, Sclerosing/drug therapy , Cholangitis, Sclerosing/physiopathology , Disease Models, Animal , Down-Regulation/genetics , Down-Regulation/physiology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/physiology , Liver/drug effects , Liver/pathology , Mice , Receptors, G-Protein-Coupled/metabolism , Virulence FactorsABSTRACT
BACKGROUND: Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts. METHODS: We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche. RESULTS: Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes. CONCLUSIONS: With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.
Subject(s)
Cerebellar Neoplasms , Extracellular Vesicles , Medulloblastoma , Cerebellar Neoplasms/genetics , Hedgehog Proteins/genetics , Humans , Male , Medulloblastoma/genetics , TranscriptomeABSTRACT
Human genomes contain about 100,000 LINE-1 (L1) retroelements, of which more than 100 are intact. L1s are normally tightly controlled by epigenetic mechanisms, which often fail in cancer. In bladder urothelial carcinoma (UC), particularly, L1s become DNA-hypomethylated, expressed and contribute to genomic instability and tumor growth. It is, however, unknown which individual L1s are activated. Following RNA-immunoprecipitation with a L1-specific antibody, third generation nanopore sequencing detected transcripts of 90 individual elements in the VM-Cub-1 UC line with high overall L1 expression. In total, 10 L1s accounted for >60% of the reads. Analysis of five specific L1s by RT-qPCR revealed generally increased expression in UC tissues and cell lines over normal controls, but variable expression among tumor cell lines from bladder, prostate and testicular cancer. Chromatin immunoprecipitation demonstrated active histone marks at L1 sequences with increased expression in VM-Cub-1, but not in a different UC cell line with low L1 expression. We conclude that many L1 elements are epigenetically activated in bladder cancer in a varied pattern. Our findings indicate that expression of individual L1s is highly heterogeneous between and among cancer types.
Subject(s)
Long Interspersed Nucleotide Elements/genetics , Retroelements/genetics , Testicular Neoplasms/genetics , Aged , Aged, 80 and over , Chromatin Immunoprecipitation , DNA Methylation/genetics , DNA Methylation/physiology , Female , Histones/metabolism , Humans , Immunoprecipitation , Male , Middle Aged , Nanopore Sequencing , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The histone demethylase UTX (gene: KDM6A) directs cell and tissue differentiation during development. Deleterious mutations in KDM6A occur in many human cancers, most frequently in urothelial carcinoma. The consequences of these mutations are poorly understood; plausibly, they may disturb urothelial differentiation. We therefore investigated the effects of UTX siRNA-mediated knockdown in two in vitro models of urothelial differentiation; namely, primary cultures of urothelial epithelial cells treated with troglitazone and PD153035 and the immortalized urothelial cell line HBLAK treated with high calcium and serum. In both models, efficient UTX knockdown did not block morphological and biochemical differentiation. An apparent delay was due to a cytotoxic effect on the cell cultures before the initiation of differentiation, which induced apoptosis partly in a p53-dependent manner. As a consequence, slowly cycling, smaller, KRT14high precursor cells in the HBLAK cell line were enriched at the expense of more differentiated, larger, proliferating KRT14low cells. UTX knockdown induced apoptosis and enriched KRT14high cells in the BFTC-905 papillary urothelial carcinoma cell line as well. Our findings suggest an explanation for the frequent occurrence of KDM6A mutations across all stages and molecular subtypes of urothelial carcinoma, whereby loss of UTX function does not primarily impede later stages of urothelial differentiation, but favors the expansion of precursor populations to provide a reservoir of potential tumor-initiating cells.
ABSTRACT
Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) - an autoimmune disease of the CNS - increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.
Subject(s)
Cerebrospinal Fluid/immunology , Leukocytes/immunology , Multiple Sclerosis/immunology , Animals , B-Lymphocytes/immunology , Blood Cells/metabolism , Central Nervous System/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Gene Expression Profiling , Humans , Leukocytes/metabolism , Mice , Multiple Sclerosis/blood , Multiple Sclerosis/cerebrospinal fluid , Phenotype , Single-Cell Analysis , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolismABSTRACT
Currently, the clinical diagnosis of schizophrenia relies solely on self-reporting and clinical interview, and likely comprises heterogeneous biological subsets. Such subsets may be defined by an underlying biology leading to solid biomarkers. A transgenic rat model modestly overexpressing the full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1) protein (tgDISC1 rat) was generated that defines such a subset, inspired by our previous identification of insoluble DISC1 protein in post mortem brains from patients with chronic mental illness. Besides specific phenotypes such as DISC1 protein pathology, abnormal dopamine homeostasis, and changes in neuroanatomy and behavior, this animal model also shows subtle disturbances in overarching signaling pathways relevant for schizophrenia. In a reverse-translational approach, assuming that both the animal model and a patient subset share common disturbed signaling pathways, we identified differentially expressed transcripts from peripheral blood mononuclear cells of tgDISC1 rats that revealed an interconnected set of dysregulated genes, led by decreased expression of regulator of G-protein signaling 1 (RGS1), chemokine (C-C) ligand 4 (CCL4), and other immune-related transcripts enriched in T-cell and macrophage signaling and converging in one module after weighted gene correlation network analysis. Testing expression of this gene network in two independent cohorts of patients with schizophrenia versus healthy controls (n = 16/50 and n = 54/45) demonstrated similar expression changes. The two top markers RGS1 and CCL4 defined a subset of 27% of patients with 97% specificity. Thus, analogous aberrant signaling pathways can be identified by a blood test in an animal model and a corresponding schizophrenia patient subset, suggesting that in this animal model tailored pharmacotherapies for this patient subset could be achieved.
Subject(s)
Biomarkers/blood , Gene Regulatory Networks , Schizophrenia , Signal Transduction/genetics , Animals , Chemokine CCL4/blood , Cohort Studies , Disease Models, Animal , Male , Nerve Tissue Proteins/genetics , RGS Proteins/blood , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Schizophrenia/blood , Schizophrenia/classification , Schizophrenia/genetics , Schizophrenia/immunology , Sensitivity and SpecificityABSTRACT
BACKGROUND: Serine/threonine kinase 33 (STK33) has been shown to be conserved across all major vertebrate classes including reptiles, mammals, amphibians and fish, suggesting its importance within vertebrates. It has been shown to phosphorylate vimentin and might play a role in spermatogenesis and organ ontogenesis. In this study we analyzed the genomic locus and expression of stk33 in the class Aves, using a combination of large scale next generation sequencing data analysis and traditional PCR. RESULTS: Within the subclass Palaeognathae we analyzed the white-throated tinamou (Tinamus guttatus), the African ostrich (Struthio camelus) and the emu (Dromaius novaehollandiae). For the African ostrich we were able to generate a 62,778 bp long genomic contig and an mRNA sequence that encodes a protein showing highly significant similarity to STK33 proteins from other vertebrates. The emu has been shown to encode and transcribe a functional STK33 as well. For the white-throated tinamou we were able to identify 13 exons by sequence comparison encoding a protein similar to STK33 as well. In contrast, in all 28 neognath birds analyzed, we could not find evidence for the existence of a functional copy of stk33 or its expression. In the genomes of these 28 bird species, we found only remnants of the stk33 locus carrying several large genomic deletions, leading to the loss of multiple exons. The remaining exons have acquired various indels and premature stop codons. CONCLUSIONS: We were able to elucidate and describe the genomic structure and the transcription of a functional stk33 gene within the subclass Palaeognathae, but we could only find degenerate remnants of stk33 in all neognath birds analyzed. This led us to the conclusion that stk33 became a unitary pseudogene in the evolutionary history of the class Aves at the paleognath-neognath branch point during the late cretaceous period about 100 million years ago. We hypothesize that the pseudogenization of stk33 might have become fixed in neognaths due to either genetic redundancy or a non-orthologous gene displacement and present potential candidate genes for such an incident.