Aims/hypothesis TNF-alpha plays a role in pancreatic beta cell loss in type 1 diabetes mellitus. In clinical interventions, TNF-alpha inhibition preserves C-peptide levels in early type 1 diabetes. In this study we evaluated the crosstalk of TNF-alpha, as compared with type I IFNs, with the type 1 diabetes candidate gene PTPN2 (encoding protein tyrosine phosphatase non-receptor type 2 [PTPN2]) in human beta cells. Methods EndoC-beta H1 cells, dispersed human pancreatic islets or induced pluripotent stem cell (iPSC)-derived islet-like cells were transfected with siRNAs targeting various genes (siCTRL, siPTPN2, siJNK1, siJNK3 or siBIM). Cells were treated for 48 h with IFN-alpha (2000 U/ml) or TNF-alpha (1000 U/ml). Cell death was evaluated using Hoechst 33342 and propidium iodide staining. mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) and protein expression by immunoblot. Results PTPN2 silencing sensitised beta cells to cytotoxicity induced by IFN-alpha and/or TNF-alpha by 20-50%, depending on the human cell model utilised; there was no potentiation between the cytokines. We silenced c-Jun N-terminal kinase (JNK)1 or Bcl-2-like protein 2 (BIM), and this abolished the proapoptotic effects of IFN-alpha, TNF-alpha or the combination of both after PTPN2 inhibition. We further observed that PTPN2 silencing increased TNF-alpha-induced JNK1 and BIM phosphorylation and that JNK3 is necessary for beta cell resistance to IFN-alpha cytotoxicity. Conclusions/interpretation We show that the type 1 diabetes candidate gene PTPN2 is a key regulator of the deleterious effects of TNF-alpha in human beta cells. It is conceivable that people with type 1 diabetes carrying risk-associated PTPN2 polymorphisms may particularly benefit from therapies inhibiting TNF-alpha.

Inhibition of the type 1 diabetes candidate gene PTPN2 aggravates TNF-α-induced human beta cell dysfunction and death

Marselli, Lorella;Marchetti, Piero;
2023-01-01

Abstract

Aims/hypothesis TNF-alpha plays a role in pancreatic beta cell loss in type 1 diabetes mellitus. In clinical interventions, TNF-alpha inhibition preserves C-peptide levels in early type 1 diabetes. In this study we evaluated the crosstalk of TNF-alpha, as compared with type I IFNs, with the type 1 diabetes candidate gene PTPN2 (encoding protein tyrosine phosphatase non-receptor type 2 [PTPN2]) in human beta cells. Methods EndoC-beta H1 cells, dispersed human pancreatic islets or induced pluripotent stem cell (iPSC)-derived islet-like cells were transfected with siRNAs targeting various genes (siCTRL, siPTPN2, siJNK1, siJNK3 or siBIM). Cells were treated for 48 h with IFN-alpha (2000 U/ml) or TNF-alpha (1000 U/ml). Cell death was evaluated using Hoechst 33342 and propidium iodide staining. mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) and protein expression by immunoblot. Results PTPN2 silencing sensitised beta cells to cytotoxicity induced by IFN-alpha and/or TNF-alpha by 20-50%, depending on the human cell model utilised; there was no potentiation between the cytokines. We silenced c-Jun N-terminal kinase (JNK)1 or Bcl-2-like protein 2 (BIM), and this abolished the proapoptotic effects of IFN-alpha, TNF-alpha or the combination of both after PTPN2 inhibition. We further observed that PTPN2 silencing increased TNF-alpha-induced JNK1 and BIM phosphorylation and that JNK3 is necessary for beta cell resistance to IFN-alpha cytotoxicity. Conclusions/interpretation We show that the type 1 diabetes candidate gene PTPN2 is a key regulator of the deleterious effects of TNF-alpha in human beta cells. It is conceivable that people with type 1 diabetes carrying risk-associated PTPN2 polymorphisms may particularly benefit from therapies inhibiting TNF-alpha.
2023
Roca-Rivada, Arturo; Marín-Cañas, Sandra; Colli, Maikel L; Vinci, Chiara; Sawatani, Toshiaki; Marselli, Lorella; Cnop, Miriam; Marchetti, Piero; Eizir...espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/1177605
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