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Valosin-containing protein-regulated endoplasmic reticulum stress causes NOD2-dependent inflammatory responses.

ABSTRACT: NOD2 polymorphisms may affect sensing of the bacterial muramyl dipeptide (MDP) and trigger perturbed inflammatory responses. Genetic screening of a patient with immunodeficiency and enteropathy revealed a rare homozygous missense mutation in the first CARD domain of NOD2 (ENST00000300589; c.160G > A, p.E54K). Biochemical assays confirmed impaired NOD2-dependent signaling and proinflammatory cytokine production in patient's cells and heterologous cellular models with overexpression of the NOD2 mutant. Immunoprecipitation-coupled mass spectrometry unveiled the ATPase valosin-containing protein (VCP) as novel interaction partner of wildtype NOD2, while the binding to the NOD2 variant p.E54K was abrogated. Knockdown of VCP in coloncarcinoma cells led to impaired NF-κB activity and IL8 expression upon MDP stimulation. In contrast, tunicamycin-induced ER stress resulted in increased IL8, CXCL1, and CXCL2 production in cells with knockdown of VCP, while enhanced expression of these proinflammatory molecules was abolished upon knockout of NOD2. Taken together, these data suggest that VCP-mediated inflammatory responses upon ER stress are NOD2-dependent.

SUBMITTER: Ghalandary M

PROVIDER: S-EPMC8913691 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Valosin-containing protein-regulated endoplasmic reticulum stress causes NOD2-dependent inflammatory responses.

Ghalandary Maryam M Li Yue Y Fröhlich Thomas T Magg Thomas T Liu Yanshan Y Rohlfs Meino M Hollizeck Sebastian S Conca Raffaele R Schwerd Tobias T Uhlig Holm H HH Bufler Philip P Koletzko Sibylle S Muise Aleixo M AM Snapper Scott B SB Hauck Fabian F Klein Christoph C Kotlarz Daniel D

Scientific reports 20220310 1

NOD2 polymorphisms may affect sensing of the bacterial muramyl dipeptide (MDP) and trigger perturbed inflammatory responses. Genetic screening of a patient with immunodeficiency and enteropathy revealed a rare homozygous missense mutation in the first CARD domain of NOD2 (ENST00000300589; c.160G > A, p.E54K). Biochemical assays confirmed impaired NOD2-dependent signaling and proinflammatory cytokine production in patient's cells and heterologous cellular models with overexpression of the NOD2 mu ...[more]

PMID: 35273242

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Project description:BackgroundAdult human pancreatic beta cells are the "gold standard" for studies on diabetes pathogenesis, but their use is limited by insufficient availability and variable quality. An important effort has recently taken place to differentiate beta cells from human induced pluripotent stem cells (iPSCs) and validate their use for diabetes research. We presently used a 7-stage protocol to generate beta cells from human iPSC and evaluated whether these cells are responsive to the pro-inflammatory cytokines (IFNγ, IL-1β, or IFNα) that play a role in type 1 diabetes.MethodsThe iPSC-derived islet-like cell clusters contained 40-50% beta and 10-15% alpha cells and expressed the receptors for IFNγ, IL-1β, or IFNα. Cells were exposed to either IFNγ (1000 U/mL) + IL-1β (50 U/mL) or IFNα alone (2000 U/mL) for 24/48 h. Apoptosis was quantified using Hoechst/propidium iodide staining or the RealTime Glo Apoptosis Kit (Promega). After treatment, CXCL10 secretion was quantified by ELISA. The expression of multiples genes (Ins, Gcg, Nkx2.2, Nkx6.1, Pdx1, Mafa, BiP, Chop, Atf3, CXCL10, CXCL9, CCL5, and HLA-ABC) was quantified by RT-qPCR. Phosphorylation state and total expression of STAT1/STAT2, as well as expression of PDL1 and of the ER chaperone BiP, were quantified by Western blotting. The co-localization of HLA-ABC or cleaved caspase-3 and Ins/Gcg expression was assessed by immunohistochemistry. The presence of HLA-ABC at the plasma membrane was measured by flow cytometry.ResultsIFNγ + IL-1β and IFNα induced apoptosis of the cells after 48 h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFNγ + IL-1β induced a pro-inflammatory phenotype, including increased CXCL10, CXCL9, and CCL5 expression; CXCL10 secretion; and HLA-ABC expression. HLA overexpression was confirmed at the protein level by Western blotting and flow cytometry. Exposure to IFNγ + IL-1β (but not IFNα) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1 h by IFNγ + IL-1β and IFNα, while phosphorylation of STAT2 was only activated by IFNα at 1-4 h. PDL1 expression was increased by both IFNγ + IL-1β and IFNα.ConclusionsOur data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1β + IFNγ and IFNα, by activating the same pathogenic processes as adult human primary beta cells. These cells thus represent a valuable tool for future research on the pathogenesis of type 1 diabetes.

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Valosin-containing protein-regulated endoplasmic reticulum stress causes NOD2-dependent inflammatory responses.

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Valosin-containing protein-regulated endoplasmic reticulum stress causes NOD2-dependent inflammatory responses.

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