Dataset Information

Calreticulin and other components of endoplasmic reticulum stress in rat and human inflammatory demyelination.

ABSTRACT:

Background

Calreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This 'ecto-CRT' may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha) in biopsy and post-mortem human multiple sclerosis (MS) samples. We extend this work by analysing changes in expression of CRT, BiP, CHOP, XBP1 and p-eIF2 alpha in a rat model of inflammatory demyelination. Demyelination was induced in the spinal cord by intradermal injection of recombinant mouse MOG mixed with incomplete Freund's adjuvant (IFA) at the base of the tail. Tissue samples were analysed by semi-quantitative scoring of immunohistochemically stained frozen tissue sections. Data generated following sampling of tissue from animals with spinal cord lesions, was compared to that obtained using tissue derived from IFA- or saline-injected controls. CRT present in rat serum and in a cohort of human serum derived from 14 multiple sclerosis patients and 11 healthy controls was measured by ELISA.

Results

Stained tissue scores revealed significantly (p<0.05) increased amounts of CRT, CHOP and p-eIF2 alpha in the lesion, lesion edge and normal-appearing white matter when compared to controls. CHOP and p-eIF2 alpha were also significantly raised in regions of grey matter and the central canal (p<0.05). Immunofluorescent dual-label staining confirmed expression of these markers in astrocytes, microglia or neurons. Dual staining of rat and human spinal cord lesions with Oil Red O and CRT antibody showed co-localisation of CRT with the rim of myelin fragments. ELISA testing of sera from control and EAE rats demonstrated significant down-regulation (p<0.05) of CRT in the serum of EAE animals, compared to saline and IFA controls. This contrasted with significantly increased amounts of CRT detected in the sera of MS patients (p<0.05), compared to controls.

Conclusion

This data highlights the potential importance of CRT and other ER stress proteins in inflammatory demyelination.

SUBMITTER: Ni Fhlathartaigh M

PROVIDER: S-EPMC3893522 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Publications

Calreticulin and other components of endoplasmic reticulum stress in rat and human inflammatory demyelination.

Ní Fhlathartaigh Mary M McMahon Jill J Reynolds Richard R Connolly David D Higgins Eibhlín E Counihan Timothy T Fitzgerald Una U

Acta neuropathologica communications 20130715

<h4>Background</h4>Calreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This 'ecto-CRT' may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha ...[more]

PMID: 24252779

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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 (IFN?, IL-1?, or IFN?) 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 IFN?, IL-1?, or IFN?. Cells were exposed to either IFN? (1000?U/mL)?+?IL-1? (50?U/mL) or IFN? 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.ResultsIFN? + IL-1? and IFN? induced apoptosis of the cells after 48?h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFN? + IL-1? 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 IFN? + IL-1? (but not IFN?) 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 IFN? + IL-1? and IFN?, while phosphorylation of STAT2 was only activated by IFN? at 1-4 h. PDL1 expression was increased by both IFN? + IL-1? and IFN?.ConclusionsOur data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1? + IFN? and IFN?, 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.

Dataset Information

Calreticulin and other components of endoplasmic reticulum stress in rat and human inflammatory demyelination.

Background

Results

Conclusion

Publications

Calreticulin and other components of endoplasmic reticulum stress in rat and human inflammatory demyelination.

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