ABSTRACT
Tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with variable demand for insulin. Here, we asked how insulin-degrading enzyme (IDE) affects beta cell adaptation to metabolic and immune stress. C57BL/6 and autoimmune non-obese diabetic (NOD) mice lacking IDE were exposed to proteotoxic, metabolic, and immune stress. IDE deficiency induced a low-level UPR with islet hypertrophy at the steady state, rapamycin-sensitive beta cell proliferation enhanced by proteotoxic stress, and beta cell decompensation upon high-fat feeding. IDE deficiency also enhanced the UPR triggered by proteotoxic stress in human EndoC-ßH1 cells. In Ide-/- NOD mice, islet inflammation specifically induced regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. These findings establish a role of IDE in islet cell protein homeostasis, demonstrate how its absence induces metabolic decompensation despite beta cell proliferation, and UPR-independent islet regeneration in the presence of inflammation.
ABSTRACT
The chimeric antibodies anti-CD20 rituximab (Rtx) and anti-TNFα infliximab (Ifx) induce antidrug antibodies (ADAs) in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA)-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs) loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.
ABSTRACT
Autophagy is critical for maintaining cellular homeostasis. Organs such as the eye and brain are immunologically privileged. Here, we demonstrate that autophagy is essential for maintaining ocular immune privilege. Deletion of multiple autophagy genes in macrophages leads to an inflammation-mediated eye disease called uveitis that can cause blindness. Loss of autophagy activates inflammasome-mediated IL1B secretion that increases disease severity. Inhibition of caspase activity by gene deletion or pharmacological means completely reverses the disease phenotype. Of interest, experimental uveitis was also increased in a model of Crohn disease, a systemic autoimmune disease in which patients often develop uveitis, offering a potential mechanistic link between macrophage autophagy and systemic disease. These findings directly implicate the homeostatic process of autophagy in blinding eye disease and identify novel pathways for therapeutic intervention in uveitis.
Subject(s)
Autophagy , Eye Diseases/pathology , Inflammation/pathology , Macrophages/pathology , Animals , Autophagy/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Proteins/genetics , Cytokines/genetics , Cytokines/metabolism , Gene Deletion , Gene Expression Regulation , Humans , Inflammasomes/metabolism , Inflammation/genetics , Interleukin-1beta/metabolism , Macrophages/ultrastructure , Mice, Knockout , Polymorphism, Single Nucleotide/genetics , Uveitis/complications , Uveitis/genetics , Uveitis/pathologyABSTRACT
Macrophage dysfunction plays a pivotal role during neovascular proliferation in diseases of ageing including cancers, atherosclerosis and blinding eye disease. In the eye, choroidal neovascularization (CNV) causes blindness in patients with age-related macular degeneration (AMD). Here we report that increased IL10, not IL4 or IL13, in senescent eyes activates STAT3 signalling that induces the alternative activation of macrophages and vascular proliferation. Targeted inhibition of both IL10 receptor-mediated signalling and STAT3 activation in macrophages reverses the ageing phenotype. In addition, adoptive transfer of STAT3-deficient macrophages into eyes of old mice significantly reduces the amount of CNV. Systemic and CD163(+) eye macrophages obtained from AMD patients also demonstrate STAT3 activation. Our studies demonstrate that impaired SOCS3 feedback leads to permissive IL10/STAT3 signalling that promotes alternative macrophage activation and pathological neovascularization. These findings have significant implications for our understanding of the pathobiology of age-associated diseases and may guide targeted immunotherapy.
Subject(s)
Interleukin-10/metabolism , Macrophages/physiology , Macular Degeneration/immunology , Neovascularization, Pathologic/metabolism , STAT3 Transcription Factor/metabolism , Aged , Aged, 80 and over , Aging/metabolism , Animals , Eye/immunology , Eye/metabolism , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Porphyrins , RAW 264.7 Cells , Receptors, Interleukin-10/metabolism , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/metabolismSubject(s)
Adalimumab/chemistry , Adalimumab/therapeutic use , CD4-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/chemistry , Immunoglobulin Variable Region/chemistry , Natalizumab/chemistry , Natalizumab/therapeutic use , Amino Acid Sequence , HLA-DR Antigens/immunology , Humans , Immunoglobulin Variable Region/immunology , Protein DomainsABSTRACT
Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+) promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.
Subject(s)
Endoplasmic Reticulum Stress/drug effects , Gene Deletion , Insulysin/chemistry , Multiprotein Complexes/metabolism , Schizosaccharomyces/enzymology , TOR Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Apoptosis/drug effects , Cytoprotection/drug effects , Dithiothreitol/pharmacology , Endopeptidases/chemistry , Endopeptidases/metabolism , Genetic Complementation Test , Genome, Fungal/genetics , Humans , Insulin/chemistry , Insulin/metabolism , Insulysin/metabolism , Mechanistic Target of Rapamycin Complex 1 , Models, Molecular , Molecular Sequence Data , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/metabolism , Sequence Alignment , Sirolimus/pharmacology , Tunicamycin/pharmacologyABSTRACT
Cyclin-dependent kinase (CDK) inhibitors have been considered as excellent drug candidates for cancer therapy owing to their potential capacity to restore cell cycle control. The first generation of CDK inhibitors showed modest clinical advantages that could be attributed to off-target effects preventing them from reaching therapeutic concentrations. A phase I dose-escalation study using the second generation multi-CDK inhibitor PHA-793887 was conducted on a total of 19 patients with advanced refractory malignancies in two sites in Europe: the University of Leeds and St. James's Institute of Oncology, Leeds, UK, and the Institut Gustave Roussy, Villeujf, France (IGR). Fifteen patients were treated at IGR. Six among these patients manifested the reactivation of herpes virus replication. In vitro experiments revealed that PHA-793887 severely impaired signaling by toll-like receptors (such as TLR3, TLR4 and TLR9) in dendritic cells (DC), thus suppressing the production of multiple cytokines (type 1 interferon, interleukin-6,-10, -12, and tumor necrosis factorα) by mature DC, as well as the DC-stimulated production of interferon-γ by natural killer cells. Pharmacological inhibition of glycogen synthase-3ß (GSK-3ß), one of the off-targets of PHA-793887, did not cause such immunological defects. Altogether, these data underscore a hitherto unsuspected immunosuppressive effect of PHA-793887.
Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Herpesviridae Infections/chemically induced , Neoplasms/drug therapy , Pyrazoles/adverse effects , Pyrazoles/pharmacology , Pyrroles/adverse effects , Pyrroles/pharmacology , Toll-Like Receptors/antagonists & inhibitors , Adult , Aged , Cells, Cultured , Cyclin-Dependent Kinases/metabolism , Disease Susceptibility/chemically induced , Disease Susceptibility/pathology , Female , Herpesviridae/drug effects , Herpesviridae/physiology , Herpesviridae Infections/pathology , Humans , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Male , Middle Aged , Neoplasms/enzymology , Neoplasms/pathology , Pyrazoles/therapeutic use , Pyrroles/therapeutic use , Signal Transduction/drug effects , Signal Transduction/immunology , Toll-Like Receptors/metabolismABSTRACT
In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopic localization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as an engulfment signal for antigen-presenting cells.(1) Here, we report that yet another ER protein, the lysyl-tRNA synthetase (KARS), was exposed on the surface of stressed cells, on which KARS co-localized with CRT in lipid rafts. Depletion of KARS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KARS was also found in the supernatant of stressed cells. Recombinant KARS protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, recombinant KARS protein was unable to stimulate macrophages in vitro. These results underscore the contribution of KARS to the emission of (one of) the principal signal(s) of immunogenic cell death, CRT exposure.
Subject(s)
Calreticulin/metabolism , Cell Membrane/metabolism , Cytotoxicity, Immunologic , Lysine-tRNA Ligase/metabolism , Amino Acid Sequence , Anthracyclines/pharmacology , Cell Death/drug effects , Cell Membrane/drug effects , Cytotoxicity, Immunologic/drug effects , HeLa Cells , Humans , Lysine-tRNA Ligase/chemistry , Molecular Sequence Data , Protein Transport/drug effects , Signal Transduction/drug effectsABSTRACT
IgA nephropathy (IgAN), the most common primary glomerulonephritis in the world, is characterized by IgA immune complex-mediated mesangial cell proliferation. The transferrin receptor (TfR) was identified previously as an IgA1 receptor, and it was found that, in biopsies of patients with IgAN, TfR is overexpressed and co-localizes with IgA1 mesangial deposits. Here, it is shown that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (three- to four-fold increase) in quiescent human mesangial cells (HMC). IgA-induced but not cytokine-induced HMC proliferation is dependent on TfR engagement as it is inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. It is dependent on the continued presence of IgA1 rather than on soluble factors released during IgA1-mediated activation. In addition, pIgA1-induced IL-6 and TGF-beta production from HMC was specifically inhibited by mAb A24, confirming that pIgA1 triggers a TfR-dependent HMC activation. Finally, upregulation of TfR expression induced by sera from patients with IgAN but not from healthy individuals was dependent on IgA. It is proposed that deposited pIgA1 or IgA1 immune complexes could initiate a process of auto-amplification involving hyperexpression of TfR, allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation and activation, features that are commonly implicated in the chronicity of mesangial injuries observed in IgAN and that could explain the recurrence of IgA1 deposits in the mesangium after renal transplantation.