ABSTRACT
BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.
Subject(s)
Arthritis, Rheumatoid , Tumor Suppressor Protein p53 , Humans , Arthritis, Rheumatoid/metabolism , Cells, Cultured , Cytokines/metabolism , Fibroblasts/metabolism , Inflammation/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Synovial Membrane/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Thy-1 Antigens/immunologyABSTRACT
Huge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts in T cell biology. As such, flow cytometry gating strategies and markers, such as CD90, have been applied to indentify ILC. Here, we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly a sub-population of cells exhibit low or even no expression of this marker. CD90-negative and CD90-low CD127+ ILC were present amongst all ILC subsets in the gut. The frequency of CD90-negative and CD90-low CD127+ ILC was dependent on stimulatory cues in vitro and enhanced by dysbiosis in vivo. CD90-negative and CD90-low CD127+ ILC were a potential source of IL-13, IFNγ and IL-17A at steady state and upon dysbiosis- and dextran sulphate sodium-elicited colitis. Hence, this study reveals that, contrary to expectations, CD90 is not constitutively expressed by functional ILC in the gut.
Subject(s)
Colitis , Immunity, Innate , Humans , Colitis/metabolism , Cytokines/metabolism , Dysbiosis/metabolism , Lymphocytes/metabolism , Thy-1 Antigens/immunologyABSTRACT
Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.
Subject(s)
Dysbiosis/immunology , Gastrointestinal Microbiome/immunology , Immunity, Mucosal/immunology , Interleukins/immunology , Intestinal Mucosa/immunology , Stress, Psychological/immunology , Animals , Bacterial Adhesion/immunology , Crohn Disease/immunology , Crohn Disease/microbiology , Dysbiosis/microbiology , Enterobacteriaceae/classification , Enterobacteriaceae/genetics , Enterobacteriaceae/immunology , Escherichia coli/immunology , Escherichia coli/physiology , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Gastrointestinal Microbiome/genetics , Humans , Ileum/immunology , Ileum/microbiology , Ileum/pathology , Interleukins/metabolism , Male , Mice, Inbred C57BL , Receptors, Glucocorticoid/immunology , Receptors, Glucocorticoid/metabolism , Thy-1 Antigens/immunology , Thy-1 Antigens/metabolism , Interleukin-22ABSTRACT
BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease mediated by T helper type 2 (Th2) cells in acute phase. Group 2 innate lymphoid cells (ILCs) play a role in the initiation of the Th2 response. Although mold exposure is associated with the development of AD, studies on the underlying mechanisms are lacking. This study investigated whether group 2 ILCs are involved in inflammation in AD-like skin induced by Aspergillus fumigatus (Af). METHODS: We investigated changes of group 2 ILCs population in Af-induced AD-like skin lesions. To induce AD-like skin lesions, Af extracts were applied to the dorsal skin of BALB/c and Rag1-/- mice five times per week, with repeat exposures at 2-week intervals. RESULTS: The clinical parameters were higher in the Af-treated group than in the control group. Histologic findings revealed epiderrmal and dermal thickening as well as eosinophil and mast cell infiltration into the skin of Af-treated mice. Populations of group 2 ILCs in the skin were also significantly higher in the Af-treated group. In addition, interleukin-33 mRNA expression was significantly higher in the skin lesions of the Af-treated mice. In the Rag1-/- mice lacking mature lymphocytes, AD-like skin lesions were still induced by Af and ILCs depletion using an anti-CD90.2 mAb lowered the Af-induced inflammatory response. CONCLUSIONS: Group 2 ILCs may play a role in a murine model of Af-induced AD-like skin lesions.
Subject(s)
Aspergillus fumigatus/immunology , Dermatitis, Atopic/immunology , Immunity, Innate , Animals , Antibodies, Monoclonal/therapeutic use , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/pathology , Disease Models, Animal , Female , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunoglobulin E/blood , Interleukin-33/genetics , Interleukin-33/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Skin/pathology , Th2 Cells/cytology , Th2 Cells/immunology , Th2 Cells/metabolism , Thy-1 Antigens/immunologyABSTRACT
CONTEXT: Modified BuShenYiQi formula (M-BYF) is derived from BuShenYiQi formula, used for the treatment of allergic asthma. The exact effect and mechanism of M-BYF on the improvement of asthma remain unclear. OBJECTIVE: We investigated the mechanism underlying the therapeutic effect of M-BYF on allergic asthma. MATERIALS AND METHODS: The asthma model was established in female BALB/c mice that were sensitized and challenged with ovalbumin (OVA). Mice in the treated groups were orally treated once a day with M-BYF (7, 14 and 28 g/kg/d) or dexamethasone before OVA challenge. Control and Model group received saline. Pathophysiological abnormalities and percentages of lung type 2 innate lymphoid cells (ILC2s) and Th9 cells were measured. Expression levels of type 2 cytokines and transcription factors required for these cells function and differentiation were analysed. Expression of vasoactive intestinal polypeptide (VIP)-VPAC2 signalling pathway-related proteins, and percentages of VIP expressing (VIP+) cells and VPAC2, CD90 co-expressing (VPAC2+CD90+) cells were detected. RESULTS: M-BYF alleviated airway hyperresponsiveness, inflammation, mucus hypersecretion and collagen deposition in asthmatic mice. M-BYF down-regulated percentages of ILC2s and Th9 cells with lower expression of GATA3, PU.1 and IRF4, reduced IL-5, IL-13, IL-9 and VIP production. The decrease in the expression of VIP-VPAC2 signalling pathway and percentages of VIP+ cells, VPAC2+CD90+ cells were observed after M-BYF treatment. The LD50 value of M-BYF was higher than 90 g/kg. DISCUSSION AND CONCLUSIONS: M-BYF alleviated experimental asthma by negatively regulating ILC2s and Th9 cells and the VIP-VPAC2 signalling pathway. These findings provide the theoretical basis for future research of M-BYF in asthma patient population.
Subject(s)
Anti-Asthmatic Agents/pharmacology , Asthma/drug therapy , Drugs, Chinese Herbal/pharmacology , Respiratory Hypersensitivity/drug therapy , Animals , Asthma/immunology , Dexamethasone/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/administration & dosage , Female , Immunity, Innate/drug effects , Lymphocytes/drug effects , Lymphocytes/immunology , Mice , Mice, Inbred BALB C , Receptors, Vasoactive Intestinal Peptide, Type II/metabolism , Respiratory Hypersensitivity/immunology , Signal Transduction/drug effects , Thy-1 Antigens/immunology , Vasoactive Intestinal Peptide/metabolismABSTRACT
We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN+ aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.
Subject(s)
Cholestasis/drug therapy , Fibroblasts/immunology , Immunotherapy , Liver Cirrhosis/drug therapy , Animals , Cholestasis/genetics , Cholestasis/immunology , Collagen/immunology , Fibroblasts/drug effects , Humans , Immunotoxins/administration & dosage , Liver Cirrhosis/genetics , Liver Cirrhosis/immunology , Mesothelin/genetics , Mesothelin/immunology , Mice , Thy-1 Antigens/genetics , Thy-1 Antigens/immunologyABSTRACT
Available therapies aimed at treating age-related osteoporosis are still insufficient. Therefore, designing reliable in vitro model for the analysis of molecular mechanisms underlying senile osteoporosis is highly required. We have isolated and characterized progenitor cells isolated from bone marrow (BMSCs) of osteoporotic mice strain SAM/P6 (BMSCSAM/P6 ). The cytophysiology of BMSCSAM/P6 was for the first time compared with BMSCs isolated from healthy BALB/c mice (BMSCBALB/c ). Characterization of the cells included evaluation of their multipotency, morphology and determination of specific phenotype. Viability of BMSCs cultures was determined in reference to apoptosis profile, metabolic activity, oxidative stress, mitochondrial membrane potential and caspase activation. Additionally, expression of relevant biomarkers was determined with RT-qPCR. Obtained results indicated that BMSCSAM/P6 and BMSCBALB/c show the typical phenotype of mesenchymal stromal cells (CD44+, CD73+, CD90+) and do not express CD45. Further, BMSCSAM/P6 were characterized by deteriorated multipotency, decreased metabolic activity and increased apoptosis occurrence, accompanied by elevated oxidative stress and mitochondria depolarisation. The transcriptome analyses showed that BMSCSAM/P6 are distinguished by lowered expression of molecules crucial for proper osteogenesis, including Coll-1, Opg and Opn. However, the expression of Trap, DANCR1 and miR-124-3p was significantly up-regulated. Obtained results show that BMSCSAM/P6 present features of progenitor cells with disturbed metabolism and could serve as appropriate model for in vitro investigation of age-dependent osteoporosis.
Subject(s)
Cell Differentiation/genetics , Mesenchymal Stem Cells/immunology , Osteogenesis/genetics , Osteoporosis/genetics , 5'-Nucleotidase/genetics , 5'-Nucleotidase/immunology , Animals , Cell Differentiation/immunology , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/immunology , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/immunology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred BALB C , Osteoblasts/immunology , Osteoblasts/metabolism , Osteogenesis/immunology , Osteoporosis/immunology , Osteoporosis/pathology , Stem Cells/immunology , Stem Cells/metabolism , Thy-1 Antigens/genetics , Thy-1 Antigens/immunologyABSTRACT
Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques (p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.
Subject(s)
Antigens, Surface/metabolism , Hepatocyte Growth Factor/analysis , Microspheres , Antibodies/immunology , Antigens, Surface/analysis , Antigens, Surface/immunology , Cell Line , Culture Media/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Fluoresceins/chemistry , Hepatocyte Growth Factor/immunology , Hepatocyte Growth Factor/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Immunoassay/methods , Thy-1 Antigens/analysis , Thy-1 Antigens/immunology , Thy-1 Antigens/metabolismABSTRACT
Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM+) fraction, which diminished with age. The mesenchymal fraction composed of CD90+ and CD90+CD73+ cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM+ cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM+ cells supported by CD90+ subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90+ and CD90+PDGFRα+ cells. In postnatal lung, a subset of CD90+ cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90+CD146+ cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.
Subject(s)
Immunomodulation/immunology , Mesenchymal Stem Cells/metabolism , Thy-1 Antigens/immunology , Thy-1 Antigens/metabolism , Adolescent , Biomarkers/metabolism , CD146 Antigen/immunology , CD146 Antigen/metabolism , Cell Separation/methods , Child , Child, Preschool , Epithelial Cell Adhesion Molecule/immunology , Epithelial Cell Adhesion Molecule/metabolism , Female , Humans , Immunologic Factors/immunology , Immunologic Factors/metabolism , Infant , Infant, Newborn , Male , Mesenchymal Stem Cells/immunology , Microvessels/immunology , Microvessels/metabolism , Pericytes/immunology , Pericytes/metabolism , Prospective StudiesABSTRACT
Increased T helper (Th)1/Th17 immune responses are a hallmark of Crohn's disease (CD) immunopathogenesis. CD90+ (myo-)fibroblasts (MFs) are abundant cells in the normal (N) intestinal mucosa contributing to mucosal tolerance via suppression of Th1 cell activity through cell surface membrane-bound PD-L1 (mPD-L1). CD-MFs have a decreased level of mPD-L1. Consequently, mPD-L1-mediated suppression of Th1 cells by CD-MFs is decreased, yet the mechanism responsible for the reduction in mPDL-1 is unknown. Increased expression of matrix metalloproteinases (MMPs) has been reported in CD. Herein we observed that when compared to N- and ulcerative colitis (UC)-MFs, CD-MFs increase in LPS-inducible levels of MMP-7 and -9 with a significant increase in both basal and inducible MMP-10. A similar pattern of MMP expression was observed in the CD-inflamed mucosa. Treatment of N-MFs with a combination of recombinant human MMP-7, -9 and -10 significantly decreased mPD-L1. In contrast, inhibition of MMP activity with MMP inhibitors or anti-MMP-10 neutralizing antibodies restores mPD-L1 on CD-MFs. CD-MFs demonstrated reduced capacity to suppress Th1 and Th17 responses from activated CD4+ T cells. By contrast, supplementation of the CD-MF:T-cell co-cultures with MMP inhibitors or anti-MMP neutralizing antibodies restored the CD-MF-mediated suppression. Our data suggest that (i) increased MMP-10 expression by CD-MFs and concomitant cleavage of PD-L1 from the surface of CD-MFs are likely to be one of the factors contributing to the decrease of mPD-L1-mediated suppression of Th1/Th17 cells in CD; and (ii) MMPs are likely to have a significant role in the intestinal mucosal immune responses.
Subject(s)
B7-H1 Antigen/metabolism , Cell Membrane/metabolism , Crohn Disease/metabolism , Fibroblasts/metabolism , Matrix Metalloproteinases/metabolism , Thy-1 Antigens/metabolism , B7-H1 Antigen/immunology , Cell Membrane/immunology , Crohn Disease/immunology , Crohn Disease/pathology , Female , Fibroblasts/immunology , Fibroblasts/pathology , Humans , Matrix Metalloproteinases/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Thy-1 Antigens/immunologyABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with no effective treatment. Cancer cells, especially cancer stem cells (CSCs), redirect immune cells to evade immune surveillance and even coopt these immune cells to support their growth and metastasis. However, the identification of CSCs and how CSCs interact with immune cells in PDAC remain uncharacterized. Here, we report that CD90 is expressed on both stromal and tumor cells and that high expression of CD90 is related to a poor prognosis in patients with PDAC. The CD90 highly expressed (CD90hi) population in PDAC cells harbors high stemness features and tumorigenicity. Notably, CD90 acts as an anchor for monocyte/macrophage adhesion, providing a physical interaction between CD90hi cells and monocytes/macrophages. In response, the crosstalk between CD90hi cells and monocytes/macrophages promotes immunosuppressive features of immune cells, which enhance the stemness and epithelial-mesenchymal transition (EMT) of PDAC cells. Moreover, PD-L1 is dominantly expressed in the CD90hi population, providing another strategy for these cells to evade immune surveillance. These findings provide an understanding of the biological significance of CD90 expression in PDAC cells and uncover a novel mechanism for how "stem-like" PDAC cells evade immune surveillance.
Subject(s)
Carcinoma, Pancreatic Ductal/immunology , Neoplastic Stem Cells/immunology , Pancreatic Neoplasms/immunology , Thy-1 Antigens/immunology , Animals , B7-H1 Antigen/biosynthesis , B7-H1 Antigen/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Heterografts , Humans , Immune Tolerance , Macrophages/immunology , Mice , Mice, Nude , Monitoring, Immunologic , Monocytes/immunology , Pancreatic Neoplasms/pathology , Stromal Cells/immunology , Stromal Cells/pathology , Thy-1 Antigens/biosynthesisABSTRACT
OBJECTIVE: The aim of this study was to investigate whether bone marrow stem cells (MSCs) derived exosomes in rats could promote osteoblast proliferation and improve osteoporosis via inhibiting cell apoptosis. MATERIALS AND METHODS: MSCs in rats were isolated and cultured, followed by the identification of surface antigens via flow cytometry. The differentiation of MSCs was detected by alizarin red staining and oil red staining. After extraction from MSCs by ultracentrifugation, the size distribution of exosomes was detected by tunable resistive pulse sensing (TRPS). Specific antigens in MSCs-derived exosomes were determined by flow cytometry. Furthermore, the proliferation and viability of hFOB1.19 cells treated with MSCs-derived exosomes were detected by cell count kit-8 (CCK-8) assay. The effect of MSCs-derived exosomes on cell apoptosis was evaluated by flow cytometry. Protein expression levels of apoptosis-related genes in hFOB1.19 cells were detected by Western blot. RESULTS: MSCs differentiated into osteoblasts and lipoblasts under different treatments. Meanwhile, MSCs-derived exosomes exhibited typical elongated morphology after isolation and culture for 1 and 3 days, respectively. Functionally, MSCs-derived exosomes could promote the viability of hFOB1.19 cells, and significantly increase the expression level of GLUT3. In addition, MSCs-derived exosomes remarkably downregulated apoptosis-related genes and decreased apoptosis in hFOB1.19 cells. CONCLUSIONS: MSCs-derived exosomes could promote osteoblast proliferation via inhibiting cell apoptosis, eventually improving osteoporosis.
Subject(s)
Apoptosis/physiology , Bone Marrow Cells/metabolism , Cell Proliferation/physiology , Exosomes/transplantation , Osteoblasts/cytology , Osteoporosis/therapy , Animals , Antigens, CD34/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line , Exosomes/genetics , Exosomes/immunology , Humans , Osteoblasts/immunology , Osteogenesis/immunology , Osteogenesis/physiology , Rats, Sprague-Dawley , Thy-1 Antigens/immunologyABSTRACT
Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the nature of disease progression and its regulation remain unclear. In the present study, we monitored the course of anti-Thy1 nephritis from days 1 to 5 and established gene expression profiles at each time point using microarrays to explore the development of inflammation. According to the gene expression profiles, macrophage infiltration (triggered by CCL2 activation) was evident on day 1 and enhanced inflammation over the next few days. We screened for genes with expression levels similar to CCL2 and found that the upregulation of the circadian gene albumin D-site-binding protein (DBP) was involved in CCL2 activation in mesangial cells. More importantly, CCL2 expression showed oscillatory changes similar to DBP, and DBP induced peak CCL2 expression at 16:00 a clock on day 1 in the anti-Thy1 nephritis model. We knocked down DBP through transfection with a small interfering RNA (siRNA) and used RNA sequencing to identify the DBP-regulated TNF-α-CCL2 pathway. We performed chromatin immunoprecipitation sequencing (ChIP-Seq) and the dual luciferase assay to show that DBP bound to the TRIM55 promoter, regulating gene expression and in turn controlling the TNF-α-CCL2 pathway. In conclusion, DBP-regulated circadian CCL2 expression by the TRIM55-TNF pathway in injured mesangial cells at an early stage, which promoted macrophage recruitment and in turn triggered infiltration and inflammation in a model of anti-Thy1 nephritis.
Subject(s)
Chemokine CCL2/metabolism , Circadian Rhythm , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Isoantibodies/immunology , Nephritis/immunology , Thy-1 Antigens/immunology , Transcription Factors/metabolism , Animals , Chemokine CCL2/genetics , DNA-Binding Proteins/genetics , Glomerular Mesangium/immunology , Glomerular Mesangium/metabolism , Macrophages/immunology , Macrophages/metabolism , Nephritis/metabolism , Nephritis/pathology , Rats , Rats, Sprague-Dawley , Transcription Factors/geneticsABSTRACT
The opportunistic fungal pathogen Candida albicans frequently causes diseases such as oropharyngeal candidiasis (OPC) in immunocompromised individuals. Although it is well appreciated that the cytokine IL-17 is crucial for protective immunity against OPC, the cellular source and the regulation of this cytokine during infection are still a matter of debate. Here, we directly visualized IL-17 production in the tongue of experimentally infected mice, thereby demonstrating that this key cytokine is expressed by three complementary subsets of CD90+ leukocytes: RAG-dependent αß and γδ T cells, as well as RAG-independent ILCs. To determine the regulation of IL-17 production at the onset of OPC, we investigated in detail the myeloid compartment of the tongue and found a heterogeneous and dynamic mononuclear phagocyte (MNP) network in the infected tongue that consists of Zbtb46-Langerin- macrophages, Zbtb46+Langerin+ dendritic cells (DCs) and Ly6C+ inflammatory monocytes. Of those, the Langerin+ DC population stands out by its unique capacity to co-produce the cytokines IL-1ß, IL-6 and IL-23, all of which promote IL-17 induction in response to C. albicans in the oral mucosa. The critical role of Langerin+ DCs for the innate IL-17 response was confirmed by depletion of this cellular subset in vivo, which compromised IL-17 induction during OPC. In conclusion, our work revealed key regulatory factors and their cellular sources of innate IL-17-dependent antifungal immunity in the oral mucosa.
Subject(s)
Antigens, Surface/immunology , Candida albicans/immunology , Candidiasis, Oral/immunology , Dendritic Cells/immunology , Interleukin-17/biosynthesis , Lectins, C-Type/immunology , Mannose-Binding Lectins/immunology , Mouth Mucosa/immunology , Animals , Candidiasis, Oral/microbiology , Cytokines/immunology , Female , Flow Cytometry , Interleukin-1beta/biosynthesis , Interleukin-23/biosynthesis , Interleukin-23/immunology , Interleukin-6/biosynthesis , Leukocytes/immunology , Male , Mice , Mice, Inbred C57BL , Mononuclear Phagocyte System/immunology , Mouth Mucosa/cytology , Mouth Mucosa/microbiology , Neutrophils/immunology , Specific Pathogen-Free Organisms , Spleen/cytology , Spleen/immunology , Thy-1 Antigens/immunology , Tongue/cytology , Tongue/immunology , Tongue/microbiologyABSTRACT
PURPOSE OF REVIEW: Stroma is a broad term referring to the connective tissue matrix in which other cells reside. It is composed of diverse cell types with functions such as extracellular matrix maintenance, blood and lymph vessel development, and effector cell recruitment. The tissue microenvironment is determined by the molecular characteristics and relative abundances of different stromal cells such as fibroblasts, endothelial cells, pericytes, and mesenchymal precursor cells. Stromal cell heterogeneity is explained by embryonic developmental lineage, stages of differentiation to other cell types, and activation states. Interaction between immune and stromal cell types is critical to wound healing, cancer, and a wide range of inflammatory diseases. Here, we review recent studies of inflammatory diseases that use functional genomics and single-cell technologies to identify and characterize stromal cell types associated with pathogenesis. RECENT FINDINGS: High dimensional strategies using mRNA sequencing, mass cytometry, and fluorescence activated cell-sorting with fresh primary tissue samples are producing detailed views of what is happening in diseased tissue in rheumatoid arthritis, inflammatory bowel disease, and cancer. Fibroblasts positive for CD90 (Thy-1) are enriched in the synovium of rheumatoid arthritis patients. Single-cell RNA-seq studies will lead to more discoveries about the stroma in the near future. SUMMARY: Stromal cells form the microenvironment of inflamed and diseased tissues. Functional genomics is producing an increasingly detailed view of subsets of stromal cells with pathogenic functions in rheumatic diseases and cancer. Future genomics studies will discover disease mechanisms by perturbing molecular pathways with chemokines and therapies known to affect patient outcomes. Functional genomics studies with large sample sizes of patient tissues will identify patient subsets with different disease phenotypes or treatment responses.
Subject(s)
Arthritis, Rheumatoid/genetics , Inflammatory Bowel Diseases/genetics , RNA, Messenger/metabolism , Stromal Cells/cytology , Arthritis, Rheumatoid/immunology , Cell Differentiation , Chemokines , Endothelial Cells/cytology , Endothelial Cells/immunology , Fibroblasts/cytology , Fibroblasts/immunology , Flow Cytometry , Genomics , Humans , Inflammatory Bowel Diseases/immunology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/immunology , Pericytes/cytology , Pericytes/immunology , Rheumatic Diseases/genetics , Rheumatic Diseases/immunology , Sequence Analysis, RNA , Single-Cell Analysis , Stromal Cells/immunology , Synovial Membrane/cytology , Thy-1 Antigens/immunologyABSTRACT
A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease.
Subject(s)
Anti-Glomerular Basement Membrane Disease/immunology , Epithelial Cells/immunology , Glomerulosclerosis, Focal Segmental/immunology , Hyaluronan Receptors/immunology , Kidney Glomerulus/immunology , Albuminuria/genetics , Albuminuria/immunology , Albuminuria/metabolism , Animals , Anti-Glomerular Basement Membrane Disease/genetics , Anti-Glomerular Basement Membrane Disease/metabolism , Anti-Glomerular Basement Membrane Disease/pathology , Autoantibodies/immunology , Cell Movement , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Epithelial Cells/metabolism , Epithelial Cells/pathology , Extracellular Matrix Proteins/metabolism , Genetic Predisposition to Disease , Glomerulosclerosis, Focal Segmental/genetics , Glomerulosclerosis, Focal Segmental/metabolism , Glomerulosclerosis, Focal Segmental/pathology , Granulocytes/immunology , Granulocytes/metabolism , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Macrophages/immunology , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Signal Transduction , Thy-1 Antigens/genetics , Thy-1 Antigens/immunology , Thy-1 Antigens/metabolismABSTRACT
There is a lack of effective targeted therapies for the treatment of complement dependent diseases. We developed two recombinant Fc multimers, G207 and G211, with limited ability to interact with low/moderate affinity FcγRs, but with high avidity for C1q. These drugs effectively inhibited complement dependent cytotoxicity (CDC) in vitro, and prevented the deposition of C1q, C3b and MAC, on the surface of Ab-opsonized cells. Importantly, these inhibitory effects were both C1q dependent and independent. In order to determine the biologic relevance of our findings, we evaluated the clinical efficacy of these drugs in three different animal models, acute RBC hemolysis, anti-Thy-1 nephritis and passive Heymann's nephropathy (PHN), in which disease pathophysiology relies preferentially on complement activation. While G207 was protective in the anti-Thy-1 nephritis and PHN models, G211 was protective in all of the models tested and could effectively treat PHN. In the anti-Thy-1 nephritis model, G211 prevented the characteristic histologic changes associated with the disease and limited glomerular deposition of C3. Collectively, these data suggest that "complement preferential" Fc multimers offer a novel approach to the treatment of complement mediated diseases.
Subject(s)
Complement C1q/immunology , Complement System Proteins/metabolism , Erythrocytes/physiology , Immune System Diseases/therapy , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Animals , Cells, Cultured , Complement C3/metabolism , Complement System Proteins/immunology , Cytotoxicity, Immunologic , Disease Models, Animal , Glomerulonephritis, Membranous , Hemolysis , Humans , Immune System Diseases/immunology , Molecular Targeted Therapy , Protein Binding , Protein Multimerization , Receptors, Fc/metabolism , Thy-1 Antigens/immunology , Transgenes/geneticsABSTRACT
Antibody-mediated crosslinking of Thy-1 [also known as cluster of differentiation (CD)90], results in a T cell receptor (TcR)like signal; however, the impact of Thy1 stimulation in comparison to TcR stimulation on T cell activation and effector function has yet to be fully elucidated. In the present study, the outcome of Thy1 and TcRinduced stimulation of T cells was investigated in mice, using fragment crystalizable (Fc) receptorbound antibodies and costimulatory signals provided by syngeneic lipopolysaccharidematured bone marrowderived dendritic cells. Compared with TcR signaling, Thy1 signaling initiated a less robust proliferative response in T cells, as determined by tritiatedthymidine incorporation. In addition, enzymelinked immunosorbent assays revealed that interleukin2 production was reduced, and the expression of CD25 and cyclin D3 was weaker in Thy1stimulated cells, as determined by western blotting; however, the expression of cyclindependent kinase 6 was similar to that in TcRinduced T cells. Furthermore, western blotting demonstrated that the phosphorylation of ζ-chainassociated protein kinase 70 and extracellular signalregulated kinase 1/2 was delayed following Thy1 stimulation. DNA fragmentation assays revealed that cytotoxic effector function was also slower to develop in Thy1stimulated T cells, required more time to be effective and was largely Ca2+independent; these findings suggested that Fas ligand rather than granuleassociated perforin was involved in T cell effector function. In conclusion, the present results suggested that Thy1 signaling may contribute to the regulation of T cell homeostasis and the development of nonspecific T cellmediated cytotoxicity. However, further studies are required to elucidate the exact physiological roles of TcRlike signals that result from Thy1 crosslinking and to investigate the molecular mechanisms that are involved.
Subject(s)
Calcium/metabolism , Cytotoxicity, Immunologic , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thy-1 Antigens/immunology , Animals , Antibodies, Monoclonal/immunology , Cell Line , DNA Replication , Dendritic Cells/immunology , Dendritic Cells/metabolism , Fas Ligand Protein/metabolism , Interleukin-2/biosynthesis , Interleukin-2 Receptor alpha Subunit/metabolism , Lymphocyte Activation , Mice , Phosphorylation , ZAP-70 Protein-Tyrosine Kinase/metabolismABSTRACT
IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.
Subject(s)
Hematopoietic Stem Cells/immunology , Immunity, Innate , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin Receptor Common gamma Subunit/immunology , Animals , Antibodies, Monoclonal/administration & dosage , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Granuloma/immunology , Granuloma/microbiology , Immunocompromised Host/immunology , Interferon-gamma/immunology , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Killer Cells, Natural/immunology , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/immunology , Liver/cytology , Liver/immunology , Liver/microbiology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mycobacterium avium/growth & development , Mycobacterium avium/immunology , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/immunology , Spleen/cytology , Spleen/immunology , Thy-1 Antigens/genetics , Thy-1 Antigens/immunologyABSTRACT
Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.