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1.
Gut ; 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38857990

ABSTRACT

OBJECTIVE: Epigenetic mechanisms, including DNA methylation (DNAm), have been proposed to play a key role in Crohn's disease (CD) pathogenesis. However, the specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown. We set out to investigate the role of intestinal epithelial DNAm in CD pathogenesis. DESIGN: We generated 312 intestinal epithelial organoids (IEOs) from mucosal biopsies of 168 patients with CD (n=72), UC (n=23) and healthy controls (n=73). We performed genome-wide molecular profiling including DNAm, bulk as well as single-cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes evaluated using an organoid-lymphocyte coculture and a nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 (NLRC5) dextran sulphate sodium (DSS) colitis knock-out mouse model. RESULTS: We identified highly stable, CD-associated loss of DNAm at major histocompatibility complex (MHC) class 1 loci including NLRC5 and cognate gene upregulation. Single-cell RNA sequencing of primary mucosal tissue and IEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. Increased mucosal MHC-I and NLRC5 expression in adult and paediatric patients with CD was validated in additional cohorts and the functional role of MHC-I highlighted by demonstrating a relative protection from DSS-mediated mucosal inflammation in NLRC5-deficient mice. MHC-I DNAm in IEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature. CONCLUSIONS: Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.

2.
Clin Sci (Lond) ; 135(22): 2559-2573, 2021 11 26.
Article in English | MEDLINE | ID: mdl-34778899

ABSTRACT

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.


Subject(s)
Arthritis, Rheumatoid/metabolism , COVID-19/therapy , Pulmonary Alveolar Proteinosis/immunology , Pulmonary Surfactants/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Arthritis, Rheumatoid/therapy , Autoantibodies/chemistry , Bronchoalveolar Lavage Fluid , COVID-19/immunology , Choline/analogs & derivatives , Female , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Inflammation , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Pulmonary Alveolar Proteinosis/genetics , SARS-CoV-2/immunology , Surface-Active Agents
3.
Elife ; 92020 05 18.
Article in English | MEDLINE | ID: mdl-32420871

ABSTRACT

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.


Subject(s)
Alternaria/immunology , Antigens, Helminth/metabolism , Asthma/pathology , Interleukin-1 Receptor-Like 1 Protein/antagonists & inhibitors , Interleukin-33/antagonists & inhibitors , Nematospiroides dubius/metabolism , Animals , Cell Line , Humans , Immunologic Factors/metabolism , Interleukin-1 Receptor-Like 1 Protein/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nematospiroides dubius/immunology , Ovalbumin/immunology
4.
Eur J Immunol ; 47(6): 1075-1085, 2017 06.
Article in English | MEDLINE | ID: mdl-28383107

ABSTRACT

The TNF family cytokines B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) support plasma cell survival. It is known that inhibitors of BAFF only (BAFFR-Fc) or BAFF and APRIL (TACI-Fc) administered early enough in an NZB/NZW F1 mouse model of systemic lupus erythematosus (SLE) ameliorate clinical outcomes, pointing to a pathogenic role of BAFF. In the present study, TACI-Fc administrated at a later stage of disease, after onset of autoimmunity, decreased the number of bone marrow plasma cells and slowed down further formation of autoantibodies. TACI-Fc prevented renal damage during a 12-week treatment period regardless of autoantibody levels, while BAFFR-Fc did not despite a similar BAFF-blocking activity in vivo. TACI-Fc also decreased established plasma cells in a T-dependent hapten/carrier immunization system better than single inhibitors of BAFF or APRIL, and sometimes better than combined single inhibitors with at least equivalent BAFF and APRIL inhibitory activities. These results indicate that TACI-Fc can prevent symptoms of renal damage in a mouse model of SLE when BAFFR-Fc cannot, and point to a plasticity of plasma cells for survival factors. Targeting plasma cells with TACI-Fc might be beneficial to prevent autoantibody-mediated damages in SLE.


Subject(s)
Disease Models, Animal , Lupus Erythematosus, Systemic/immunology , Plasma Cells/immunology , Transmembrane Activator and CAML Interactor Protein/administration & dosage , Animals , Autoantibodies/biosynthesis , Autoimmunity , B-Cell Activating Factor/antagonists & inhibitors , B-Cell Activating Factor/immunology , B-Cell Activation Factor Receptor/administration & dosage , B-Cell Activation Factor Receptor/immunology , B-Lymphocytes/immunology , Flow Cytometry , Kidney/immunology , Kidney/pathology , Lupus Erythematosus, Systemic/physiopathology , Lupus Erythematosus, Systemic/therapy , Mice , Plasma Cells/pathology , Transmembrane Activator and CAML Interactor Protein/immunology , Tumor Necrosis Factor Ligand Superfamily Member 13/antagonists & inhibitors , Tumor Necrosis Factor Ligand Superfamily Member 13/immunology
5.
J Exp Med ; 214(5): 1269-1280, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28356391

ABSTRACT

Toll-like receptors (TLRs) play an important role in immune responses to pathogens by transducing signals in innate immune cells in response to microbial products. TLRs are also expressed on B cells, and TLR signaling in B cells contributes to antibody-mediated immunity and autoimmunity. The SYK tyrosine kinase is essential for signaling from the B cell antigen receptor (BCR), and thus for antibody responses. Surprisingly, we find that it is also required for B cell survival, proliferation, and cytokine secretion in response to signaling through several TLRs. We show that treatment of B cells with lipopolysaccharide, the ligand for TLR4, results in SYK activation and that this is dependent on the BCR. Furthermore, we show that B cells lacking the BCR are also defective in TLR-induced B cell activation. Our results demonstrate that TLR4 signals through two distinct pathways, one via the BCR leading to activation of SYK, ERK, and AKT and the other through MYD88 leading to activation of NF-κB.


Subject(s)
B-Lymphocytes/physiology , Receptors, Antigen, B-Cell/physiology , Syk Kinase/physiology , Toll-Like Receptor 4/physiology , Animals , Female , Lipopolysaccharides/metabolism , Lymphocyte Activation/physiology , MAP Kinase Signaling System/physiology , Mice , NF-kappa B/physiology , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction/physiology
6.
MAbs ; 8(7): 1398-1406, 2016 10.
Article in English | MEDLINE | ID: mdl-27560702

ABSTRACT

Pulmonary alveolar proteinosis is associated with impaired alveolar macrophage differentiation due to genetic defects in the granulocyte macrophage colony-stimulating factor (GM-CSF) axis or autoantibody blockade of GM-CSF. The anti-GM-CSFRα antibody mavrilimumab has shown clinical benefit in patients with rheumatoid arthritis, but with no accompanying pulmonary pathology observed to date. We aimed to model systemic versus pulmonary pharmacodynamics of an anti-GM-CSFRα antibody to understand the pharmacology that contributes to this therapeutic margin. Mice were dosed intraperitoneal with anti-GM-CSFRα antibody, and pharmacodynamics bioassays for GM-CSFRα inhibition performed on blood and bronchoalveolar lavage (BAL) cells to quantify coverage in the circulation and lung, respectively. A single dose of 3 mg/kg of the anti-GM-CSFRα antibody saturated the systemic cellular pool, but dosing up to 10 times higher had no effect on the responsiveness of BAL cells to GM-CSF. Continued administration of this dose of anti-GM-CSFRα antibody for 7 consecutive days also had no inhibitory effect on these cells. Partial inhibition of GM-CSFRα function on cells from the BAL was only observed after dosing for 5 or 7 consecutive days at 30 mg/kg, 10-fold higher than the proposed therapeutic dose. In conclusion, dosing with anti-GM-CSFRα antibody using regimes that saturate circulating cells, and have been shown to be efficacious in inflammatory arthritis models, did not lead to complete blockade of the alveolar macrophages response to GM-CSF. This suggests a significant therapeutic window is possible with GM-CSF axis inhibition.


Subject(s)
Antibodies, Monoclonal/pharmacology , Macrophages, Alveolar/drug effects , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Animals , Arthritis, Rheumatoid/drug therapy , Bronchoalveolar Lavage Fluid , Female , Mice , Mice, Inbred BALB C
7.
J Biol Chem ; 290(26): 16330-42, 2015 Jun 26.
Article in English | MEDLINE | ID: mdl-25953898

ABSTRACT

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.


Subject(s)
B-Cell Activating Factor/metabolism , B-Cell Maturation Antigen/metabolism , Transmembrane Activator and CAML Interactor Protein/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 13/metabolism , Animals , B-Cell Activating Factor/chemistry , B-Cell Activating Factor/genetics , B-Cell Activation Factor Receptor/genetics , B-Cell Activation Factor Receptor/metabolism , B-Cell Maturation Antigen/genetics , Dimerization , Humans , Ligands , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Binding , Signal Transduction , Transmembrane Activator and CAML Interactor Protein/genetics , Tumor Necrosis Factor Ligand Superfamily Member 13/chemistry , Tumor Necrosis Factor Ligand Superfamily Member 13/genetics
8.
J Immunol ; 194(10): 4650-6, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25862820

ABSTRACT

Signals from the BCR are required for Ag-specific B cell recruitment into the immune response. Binding of Ag to the BCR induces phosphorylation of immune receptor tyrosine-based activation motifs in the cytoplasmic domains of the CD79a and CD79b signaling subunits, which subsequently bind and activate the Syk protein tyrosine kinase. Earlier work with the DT40 chicken B cell leukemia cell line showed that Syk was required to transduce BCR signals to proximal activation events, suggesting that Syk also plays an important role in the activation and differentiation of primary B cells during an immune response. In this study, we show that Syk-deficient primary mouse B cells have a severe defect in BCR-induced activation, proliferation, and survival. Furthermore, we demonstrate that Syk is required for both T-dependent and T-independent Ab responses, and that this requirement is B cell intrinsic. In the absence of Syk, Ag fails to induce differentiation of naive B cells into germinal center B cells and plasma cells. Finally, we show that the survival of existing memory B cells is dependent on Syk. These experiments demonstrate that Syk plays a critical role in multiple aspects of B cell Ab responses.


Subject(s)
Antibody Formation/immunology , B-Lymphocytes/immunology , Immunologic Memory/immunology , Intracellular Signaling Peptides and Proteins/immunology , Lymphocyte Activation/immunology , Protein-Tyrosine Kinases/immunology , Animals , Cell Differentiation/immunology , Cell Survival/immunology , Flow Cytometry , Mice , Mice, Mutant Strains , Syk Kinase
9.
PLoS One ; 8(4): e61350, 2013.
Article in English | MEDLINE | ID: mdl-23620746

ABSTRACT

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called "reverse signalling". In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.


Subject(s)
B-Cell Activating Factor/metabolism , Cell Membrane/metabolism , Myeloid Cells/cytology , Myeloid Cells/metabolism , Signal Transduction , Transmembrane Activator and CAML Interactor Protein/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 13/metabolism , Animals , B-Cell Activation Factor Receptor/metabolism , Chromatography, Gel , HEK293 Cells , Humans , Macrophages/metabolism , Mice , Receptors, Fc/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Fusion Proteins/metabolism , Solubility , TWEAK Receptor
10.
Immunity ; 38(3): 475-88, 2013 Mar 21.
Article in English | MEDLINE | ID: mdl-23453634

ABSTRACT

Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This "tonic" BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors.


Subject(s)
B-Cell Activation Factor Receptor/immunology , B-Lymphocytes/immunology , Intracellular Signaling Peptides and Proteins/immunology , Protein-Tyrosine Kinases/immunology , Receptors, Antigen, B-Cell/immunology , Signal Transduction/immunology , 3-Phosphoinositide-Dependent Protein Kinases , Animals , B-Cell Activating Factor/immunology , B-Cell Activating Factor/pharmacology , B-Cell Activation Factor Receptor/genetics , B-Cell Activation Factor Receptor/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , CD79 Antigens/immunology , CD79 Antigens/metabolism , Cell Survival/drug effects , Cell Survival/genetics , Cell Survival/immunology , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Flow Cytometry , Gene Expression Profiling , Immunoblotting , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Models, Immunological , Oligonucleotide Array Sequence Analysis , Phosphatidylinositol 3-Kinases/immunology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proteins/genetics , Proteins/immunology , Proteins/metabolism , RNA, Untranslated , Receptor Cross-Talk/immunology , Receptors, Antigen, B-Cell/metabolism , Signal Transduction/drug effects , Syk Kinase , Tamoxifen/pharmacology
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