ABSTRACT
BACKGROUND: Vestibular schwannomas (VSs) remain a challenge due to their anatomical location and propensity to growth. Macrophages are present in VS but their roles in VS pathogenesis remains unknown. OBJECTIVES: The objective was to assess phenotypic and functional profile of macrophages in VS with single-cell RNA sequencing (scRNAseq). METHODS: scRNAseq was carried out in three VS samples to examine characteristics of macrophages in the tumour. RT-qPCR was carried out on 10 VS samples for CD14, CD68 and CD163 and a panel of macrophage-associated molecules. RESULTS: scRNAseq revealed macrophages to be a major constituent of VS microenvironment with three distinct subclusters based on gene expression. The subclusters were also defined by expression of CD163, CD68 and IL-1ß. AREG and PLAUR were expressed in the CD68+CD163+IL-1ß+ subcluster, PLCG2 and NCKAP5 were expressed in CD68+CD163+IL-1ß- subcluster and AUTS2 and SPP1 were expressed in the CD68+CD163-IL-1ß+ subcluster. RT-qPCR showed expression of several macrophage markers in VS of which CD14, ALOX15, Interleukin-1ß, INHBA and Colony Stimulating Factor-1R were found to have a high correlation with tumour volume. CONCLUSIONS: Macrophages form an important component of VS stroma. scRNAseq reveals three distinct subsets of macrophages in the VS tissue which may have differing roles in the pathogenesis of VS.
Subject(s)
Macrophages , Neuroma, Acoustic , Sequence Analysis, RNA , Single-Cell Analysis , Humans , Neuroma, Acoustic/genetics , Neuroma, Acoustic/pathology , Neuroma, Acoustic/metabolism , Single-Cell Analysis/methods , Macrophages/metabolism , Macrophages/pathology , Tumor Microenvironment/genetics , Female , Male , Middle Aged , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolismABSTRACT
BACKGROUND: Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren's syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. METHODS: We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. FINDINGS: Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. CONCLUSIONS: This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. FUNDING: Grant from F. Hoffmann-La Roche (Roche) AG.
Subject(s)
Arthritis, Rheumatoid , Synovial Membrane , Animals , Arthritis, Rheumatoid/genetics , Fibroblasts/metabolism , Phenotype , Stromal Cells/metabolismABSTRACT
Fibroblast-like synoviocytes (FLS) play an important role in maintaining joint homeostasis and orchestrating local inflammatory processes. When activated during injury or inflammation, FLS undergo transiently increased bioenergetic and biosynthetic demand. We aimed to identify metabolic changes which occur early in inflammatory disease pathogenesis which might support sustained cellular activation in persistent inflammation. We took primary human FLS from synovial biopsies of patients with very early rheumatoid arthritis (veRA) or resolving synovitis, and compared them with uninflamed control samples from the synovium of people without arthritis. Metabotypes were compared using NMR spectroscopy-based metabolomics and correlated with serum C-reactive protein levels. We measured glycolysis and oxidative phosphorylation by Seahorse analysis and assessed mitochondrial morphology by immunofluorescence. We demonstrate differences in FLS metabolism measurable after ex vivo culture, suggesting that disease-associated metabolic changes are long-lasting. We term this phenomenon 'metabolic memory'. We identify changes in cell metabolism after acute TNFα stimulation across disease groups. When compared to FLS from patients with early rheumatoid arthritis, FLS from patients with resolving synovitis have significantly elevated mitochondrial respiratory capacity in the resting state, and less fragmented mitochondrial morphology after TNFα treatment. Our findings indicate the potential to restore cell metabotypes by modulating mitochondrial function at sites of inflammation, with implications for treatment of RA and related inflammatory conditions in which fibroblasts play a role.
Subject(s)
Arthritis, Rheumatoid/immunology , Fibroblasts/immunology , Inflammation/immunology , Synoviocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Adult , Aged , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cells, Cultured , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Inflammation/metabolism , Inflammation/pathology , Male , Middle Aged , Oxidative Phosphorylation , Regression Analysis , Synoviocytes/metabolism , Synoviocytes/pathology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.
Subject(s)
Arthritis, Rheumatoid/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Receptor, Notch3/metabolism , Signal Transduction , Synovial Membrane/pathology , Animals , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/pathology , Endothelial Cells/pathology , Humans , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Receptor, Notch3/antagonists & inhibitors , Receptor, Notch3/deficiency , Receptor, Notch3/genetics , Thy-1 Antigens/metabolismABSTRACT
Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.
Subject(s)
Antibodies, Bacterial/immunology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Immunization , O Antigens/immunology , Salmonella typhimurium/immunology , Animals , Antibodies, Bacterial/blood , Antibody Formation , Antibody Specificity , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/genetics , Cross Protection , Disease Models, Animal , Epitopes/chemistry , Epitopes/immunology , Immunoglobulin G/blood , Mice , Models, Molecular , O Antigens/chemistry , O Antigens/genetics , Porins/chemistry , Porins/genetics , Porins/immunology , Protein Conformation , Salmonella Infections, Animal/immunology , Salmonella Infections, Animal/prevention & control , Sequence Analysis, ProteinABSTRACT
B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma.
Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Lymphoma, Large B-Cell, Diffuse/immunology , Proto-Oncogene Proteins c-bcl-6/metabolism , Adult , Aged , Aged, 80 and over , Animals , F-Box Proteins/metabolism , Female , Germinal Center/cytology , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Middle Aged , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein-Arginine N-Methyltransferases/metabolism , Proteolysis , Ubiquitin Thiolesterase/metabolism , UbiquitinationABSTRACT
Mesenchymal stromal cells (MSCs) upregulate podoplanin at sites of infection, chronic inflammation and cancer. Here, we investigated the functional consequences of podoplanin expression on the migratory potential of MSCs and their interactions with circulating platelets. Expression of podoplanin significantly enhanced the migration of MSCs compared to MSCs lacking podoplanin. Rac-1 inhibition altered the membrane localisation of podoplanin and in turn significantly reduced MSC migration. Blocking Rac-1 activity had no effect on the migration of MSCs lacking podoplanin, indicating that it was responsible for regulation of migration through podoplanin. When podoplanin-expressing MSCs were seeded on the basal surface of a porous filter, they were able to capture platelets perfused over the uncoated apical surface and induce platelet aggregation. Similar microthrombi were observed when endothelial cells (ECs) were co-cultured on the apical surface. Confocal imaging shows podoplanin-expressing MSCs extending processes into the EC layer, and these processes could interact with circulating platelets. In both models, platelet aggregation induced by podoplanin-expressing MSCs was inhibited by treatment with recombinant soluble C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b). Thus, podoplanin may enhance the migratory capacity of tissue-resident MSCs and enable novel interactions with cells expressing CLEC-2.
Subject(s)
Blood Platelets/physiology , Endothelium, Vascular/physiology , Membrane Glycoproteins/metabolism , Mesenchymal Stem Cells/metabolism , Thrombosis/metabolism , Cell Movement , Cells, Cultured , Endothelium, Vascular/pathology , Humans , Lectins, C-Type/metabolism , Membrane Glycoproteins/genetics , Microscopy, Confocal , Paracrine Communication , Platelet Aggregation , RNA, Small Interfering/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
OBJECTIVES: Controlled immune responses rely on integrated crosstalk between cells and their microenvironment. We investigated whether targeting proinflammatory signals from the extracellular matrix that persist during pathological inflammation provides a viable strategy to treat rheumatoid arthritis (RA). METHODS: Monoclonal antibodies recognising the fibrinogen-like globe (FBG) of tenascin-C were generated by phage display. Clones that neutralised FBG activation of toll-like receptor 4 (TLR4), without impacting pathogenic TLR4 activation, were epitope mapped by crystallography. Antibodies stained synovial biopsies of patients at different stages of RA development. Antibody efficacy in preventing RA synovial cell cytokine release, and in modulating collagen-induced arthritis in rats, was assessed. RESULTS: Tenascin-C is expressed early in the development of RA, even before disease diagnosis, with higher levels in the joints of people with synovitis who eventually developed RA than in people whose synovitis spontaneously resolved. Anti-FBG antibodies inhibited cytokine release by RA synovial cells and prevented disease progression and tissue destruction during collagen-induced arthritis. CONCLUSIONS: Early changes in the synovial microenvironment contribute to RA progression; blocking proinflammatory signals from the matrix can ameliorate experimental arthritis. These data highlight a new drug class that could offer early, disease-specific immune modulation in RA, without engendering global immune suppression.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Arthritis, Rheumatoid/immunology , Cellular Microenvironment/immunology , Immunotherapy/methods , Synovial Membrane/immunology , Animals , Antibodies, Monoclonal/immunology , Arthritis, Experimental , Collagen , Cytokines/metabolism , Disease Progression , Fibrinogen/immunology , Humans , Rats , Tenascin/metabolism , Toll-Like Receptor 4/immunologyABSTRACT
Systemic immunization with soluble flagellin (sFliC) from Salmonella Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103+CD11b+ cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4+ T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and CD11c-cre.Irf4fl/fl mice (which have reduced numbers of cDC2, particularly intestinal CD103+CD11b+ cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103+CD11b+ cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103+CD11b+ cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103+CD11b+ cDC2 in the induction of Th2-associated responses. CD11c-cre.Irf4fl/fl mice showed a reduced primary FliC-specific Th2-associated IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103+CD11b+ cDC2.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Dendritic Cells/metabolism , Flagellin/immunology , Animals , Antigens, CD/metabolism , CD11c Antigen/metabolism , Fluorescent Antibody Technique , Immunization , Immunohistochemistry , Integrin alpha Chains/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Mice , Mice, Knockout , Toll-Like Receptor 5/metabolismABSTRACT
Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases.
Subject(s)
Arthritis, Rheumatoid/metabolism , Fibroblasts/metabolism , Arthritis, Rheumatoid/genetics , Cadherins/genetics , Cadherins/metabolism , Cells, Cultured , Humans , Synovial Membrane/cytology , Synovial Membrane/metabolism , Thy-1 Antigens/genetics , Thy-1 Antigens/metabolism , TranscriptomeABSTRACT
INTRODUCTION: Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. METHODS: ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. RESULTS: We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. CONCLUSIONS: Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.
Subject(s)
Arthritis/metabolism , Stromal Cells/metabolism , Synovial Membrane/metabolism , Adult , Aged , Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Arthritis/diagnosis , Biomarkers/metabolism , CD55 Antigens/metabolism , Disease Progression , Endopeptidases , Female , Fibroblasts/metabolism , Gelatinases/metabolism , Humans , Male , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Middle Aged , Prognosis , Serine Endopeptidases/metabolism , Synovitis/diagnosis , Synovitis/metabolismABSTRACT
CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.
Subject(s)
Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , B-Lymphocytes/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/pathology , Arthritis, Rheumatoid/blood , B-Lymphocytes/pathology , Cell Differentiation , Cell Movement , Chemokine CXCL13/metabolism , Gene Expression Profiling , Humans , Inducible T-Cell Co-Stimulator Protein/metabolism , Interleukins/metabolism , Macrophage-Activating Factors , Positive Regulatory Domain I-Binding Factor 1 , Programmed Cell Death 1 Receptor/metabolism , Proto-Oncogene Proteins c-bcl-6/metabolism , Receptors, CXCR5/deficiency , Receptors, CXCR5/metabolism , Receptors, Chemokine/metabolism , Repressor Proteins/metabolism , Signaling Lymphocytic Activation Molecule Family/metabolism , Synovial Fluid/immunology , T-Lymphocytes, Helper-Inducer/metabolismABSTRACT
BACKGROUND: We investigated two distinct synovial fibroblast populations that were located preferentially in the lining or sub-lining layers and defined by their expression of either podoplanin (PDPN) or CD248, and explored their ability to undergo self-assembly and transmigration in vivo. METHODS: Synovial fibroblasts (SF) were cultured in vitro and phenotypic changes following stimulation with interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß1 were examined. To examine the phenotype of SF in vivo, a severe combined immunodeficiency (SCID) human-mouse model of cartilage destruction was utilised. RESULTS: SF in the lining layer in rheumatoid arthritis (RA) expressed high levels of PDPN compared to the normal synovium, whereas CD248 expression was restricted to sub-lining layer cells. TNF-α or IL1 stimulation in vitro resulted in an increased expression of PDPN. In contrast, stimulation with TGF-ß1 induced CD248 expression. In the SCID human-mouse model, rheumatoid SF recapitulated the expression of PDPN and CD248. Fibroblasts adjacent to cartilage expressed PDPN, and attached to, invaded, and degraded cartilage. PDPN+ CD248- SF preceded the appearance of PDPN- CD248+ cells in contralateral implants. CONCLUSIONS: We have identified two distinct SF populations identified by expression of either PDPN or CD248 which are located within different anatomical compartments of the inflamed synovial membrane. These markers discriminate between SF subsets with distinct biological properties. As PDPN-expressing cells are associated with early fibroblast migration and cartilage erosion in vivo, we propose that PDPN-expressing cells may be an attractive therapeutic target in RA.
Subject(s)
Arthritis, Rheumatoid/pathology , Fibroblasts/cytology , Synovial Membrane/cytology , Aged , Animals , Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Cartilage, Articular/metabolism , Cell Differentiation/physiology , Cytokines/metabolism , Female , Flow Cytometry , Heterografts , Humans , Male , Membrane Glycoproteins/metabolism , Mice , Mice, SCID , Microscopy, Fluorescence , Middle Aged , Polymerase Chain Reaction , Transendothelial and Transepithelial Migration/physiologyABSTRACT
Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T cells (T-bet(+) IFN-γ(+) CD4 T cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B-cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL-4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL-12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection-induced, Th1-mediated inflammation.
Subject(s)
Cytokines/immunology , Dendritic Cells/immunology , Flagellin/immunology , Salmonella Infections/immunology , Salmonella typhimurium/immunology , Th1 Cells/immunology , Animals , Cytokines/genetics , Dendritic Cells/pathology , Flagellin/genetics , Immunization , Mice , Mice, Knockout , Salmonella Infections/genetics , Salmonella Infections/prevention & control , Salmonella typhimurium/genetics , Th1 Cells/pathology , Toll-Like Receptor 5/genetics , Toll-Like Receptor 5/immunologyABSTRACT
Although a history of cleft palate is the most common cause of velopharyngeal dysfunction (VPD), there are other disorders that can also cause hypernasality and/or nasal emission. These include other structural anomalies of the velopharyngeal valve (velopharyngeal insufficiency), neurophysiological disorders that result in inadequate velopharyngeal movement (velopharyngeal incompetence), and even faulty articulation placement in the pharynx (velopharyngeal mislearning). Unfortunately, individuals with non-cleft causes of hypernasality and/or nasal emission do not typically present at a cleft palate/craniofacial center where there are professionals who specialize in the evaluation and treatment of these disorders. As a result, they are often misdiagnosed and do not receive appropriate treatment. In this review, we present various conditions that can cause hypernasality and/or nasal emission during speech. We discuss appropriate treatment based on the underlying cause of the condition. It is important that pediatric otolaryngologists are able to recognize these disorders so that affected patients are referred to specialists in velopharyngeal dysfunction for treatment.
Subject(s)
Velopharyngeal Insufficiency/diagnosis , Velopharyngeal Insufficiency/etiology , Voice Disorders/etiology , Child , Cleft Palate/complications , Humans , Pharynx/physiopathology , Velopharyngeal Insufficiency/therapy , Voice Disorders/physiopathology , Voice Disorders/therapy , Voice QualityABSTRACT
The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4(+) T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ⼠30-fold increase in Sca-1(hi) progenitors and a corresponding loss of Sca-1(lo/int) subsets. Most strikingly, the capacity of donor Sca-1(hi) cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1(hi) c-kit(int) cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.
Subject(s)
Bone Marrow Cells/immunology , Salmonella Infections, Animal/immunology , Stem Cells/immunology , Animals , Antigens, Ly/immunology , Bone Marrow Cells/microbiology , Bone Marrow Cells/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/microbiology , Homeostasis/immunology , Interferon-gamma/immunology , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Salmonella/immunology , Salmonella Infections, Animal/pathology , Stem Cells/microbiology , Stem Cells/pathologyABSTRACT
Mucosal immunity is poorly activated after systemic immunization with protein Ags. Nevertheless, induction of mucosal immunity in such a manner would be an attractive and simple way to overcome the intrinsic difficulties in delivering Ag to such sites. Flagellin from Salmonella enterica serovar Typhimurium (FliC) can impact markedly on host immunity, in part via its recognition by TLR5. In this study, we show that systemic immunization with soluble FliC (sFliC) drives distinct immune responses concurrently in the mesenteric lymph nodes (MLN) and the spleen after i.p. and s.c. immunization. In the MLN, but not the spleen, sFliC drives a TLR5-dependent recruitment of CD103(+) dendritic cells (DCs), which correlates with a diminution in CD103(+) DC numbers in the lamina propria. In the MLN, CD103(+) DCs carry Ag and are the major primers of endogenous and transgenic T cell priming. A key consequence of these interactions with CD103(+) DCs in the MLN is an increase in local regulatory T cell differentiation. In parallel, systemic sFliC immunization results in a pronounced switching of FliC-specific B cells to IgA in the MLN but not elsewhere. Loss of TLR5 has more impact on MLN than splenic Ab responses, reflected in an ablation of IgA, but not IgG, serum Ab titers. Therefore, systemic sFliC immunization targets CD103(+) DCs and drives distinct mucosal T and B cell responses. This offers a potential "Trojan horse" approach to modulate mucosal immunity by systemically immunizing with sFliC.
Subject(s)
Antigens, CD/biosynthesis , Dendritic Cells/immunology , Flagellin/administration & dosage , Flagellin/immunology , Forkhead Transcription Factors/biosynthesis , Immunoglobulin A/biosynthesis , Integrin alpha Chains/biosynthesis , Lymph Nodes/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cells, Cultured , Coculture Techniques , Dendritic Cells/metabolism , Forkhead Transcription Factors/physiology , Immunization , Lymph Nodes/cytology , Lymph Nodes/metabolism , Mesentery , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mucous Membrane/cytology , Mucous Membrane/immunology , Mucous Membrane/metabolism , Salmonella Infections/metabolism , Salmonella Infections/pathology , Salmonella Infections/prevention & control , Salmonella typhimurium , Spleen/cytology , Spleen/immunology , Spleen/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid Vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.
Subject(s)
Antigens, Bacterial/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/microbiology , Polysaccharides, Bacterial/biosynthesis , Salmonella typhi/immunology , Animals , Antibodies, Bacterial/biosynthesis , Antibodies, Bacterial/physiology , Antigens, Bacterial/biosynthesis , B-Lymphocyte Subsets/transplantation , Mice , Mice, Inbred C57BL , Mice, Knockout , Peritoneal Cavity/cytology , Peritoneal Cavity/microbiology , Peritoneum/cytology , Peritoneum/immunology , Peritoneum/metabolism , Polysaccharides, Bacterial/immunology , Porins , Salmonella Vaccines/administration & dosage , Salmonella Vaccines/biosynthesis , Salmonella Vaccines/immunology , Typhoid Fever/immunology , Typhoid Fever/metabolism , Typhoid Fever/prevention & controlABSTRACT
Thymic atrophy is a frequent consequence of infection with bacteria, viruses, and parasites and is considered a common virulence trait between pathogens. Multiple reasons have been proposed to explain this atrophy, including premature egress of immature thymocytes, increased apoptosis, or thymic shutdown to prevent tolerance to the pathogen from developing. The severe loss in thymic cell number can reflect an equally dramatic reduction in thymic output, potentially reducing peripheral T cell numbers. In this study, we examine the relationship between systemic Salmonella infection and thymic function. During infection, naive T cell numbers in peripheral lymphoid organs increase. Nevertheless, this occurs despite a pronounced thymic atrophy caused by viable bacteria, with a peak 50-fold reduction in thymocyte numbers. Thymic atrophy is not dependent upon homeostatic feedback from peripheral T cells or on regulation of endogenous glucocorticoids, as demonstrated by infection of genetically altered mice. Once bacterial numbers fall, thymocyte numbers recover, and this is associated with increases in the proportion and proliferation of early thymic progenitors. During atrophy, thymic T cell maturation is maintained, and single-joint TCR rearrangement excision circle analysis reveals there is only a modest fall in recent CD4(+) thymic emigrants in secondary lymphoid tissues. Thus, thymic atrophy does not necessarily result in a matching dysfunctional T cell output, and thymic homeostasis can constantly adjust to systemic infection to ensure that naive T cell output is maintained.
Subject(s)
Recovery of Function/immunology , Salmonella Infections/immunology , Thymus Gland/immunology , Thymus Gland/pathology , Animals , Atrophy , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/microbiology , Cell Differentiation/immunology , Cell Movement/immunology , Mice , Salmonella Infections/pathology , Salmonella Infections/physiopathology , Salmonella typhimurium/immunology , Thymus Gland/microbiologyABSTRACT
As employers look for ways to reduce rising health care costs, worksite health promotion interventions are increasingly being used to improve employee health behaviors. An alternative approach to traditional worksite health promotion programs is the implementation of environmental and/or policy changes to encourage employees to adopt healthier behaviors. This review examines the evidence for the effectiveness of worksite health promotion programs using environmental and/or policy changes either alone or in combination with individually focused health behavior change strategies. A review of the relevant literature, published between 1995 and 2010, identified 27 studies that met all inclusion criteria. Limited evidence was found for the effectiveness of environmental and/or policy changes alone (n = 11) to change employee behavior, but more promising results were identified with multicomponent interventions (n = 16). There is a strong need for improvement in the design and evaluation of future health promotion programs focusing solely on environmental and/or policy changes at the worksite.