A20 deficiency sensitizes pancreatic beta cells to cytokine-induced apoptosis in vitro but does not influence type 1 diabetes development in vivo

Dear Editor,

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the infiltration of inflammatory cells into the pancreatic islets of Langerhans, followed by the selective destruction of insulin-producing β-cells, resulting in hyperglycemia. One of the mechanisms causing β-cell death is the intra-islet release of inflammatory mediators such as interleukin-1β (IL-1β), tumor necrosis factor (TNF) and interferon-γ (IFN-γ) by activated immune cells.¹ Hence, the transcription factor NF-κB promotes pro-inflammatory and pro-apoptotic responses in β-cells on cytokine exposure. A transgenic mouse line in which NF-κB activation is attenuated specifically in β-cells conferred nearly complete protection against multiple low dose streptozotocin (MLDSTZ)-induced T1D.² Contrary, mice with constitutively active NF-κB signaling in β-cells spontaneously develop full-blown immune-mediated diabetes.³

The ubiquitin-editing enzyme A20 is a critical negative regulator of NF-κB signaling in response to multiple stimuli, including TNF and IL-1. Moreover, A20 can also act as a strong anti-apoptotic protein in specific cell types.⁴ A20 has been identified as the most highly upregulated anti-apoptotic protein in cytokine-stimulated primary islets and insulinoma cell lines.⁵ Consistent with this, overexpression of A20 in islets confers resistance to cytokine-mediated activation of NF-κB, protecting them from apoptosis in the early post-transplantation period.⁶ Interestingly, not only have NF-κB polymorphisms been identified in patients with T1D,⁷ also A20/TNFAIP3 has been identified as a T1D susceptibility locus in humans.⁸ Together, these data suggest an important role for A20 in β-cell function and T1D. Therefore, we generated and characterized A20-deficient mice which lack expression of A20 specifically in β-cells (Supplementary Figure 1A).

We first confirmed the anti-apoptotic function of A20 in β-cells, as primary islets isolated from β-cell-specific A20 knockout (A20^β−KO) mice were more susceptible to cytokine-induced cell death compared with wild-type islets (Supplementary Figure 1A). As A20 has a crucial role in β-cell survival in vitro, we next investigated whether A20^β-KO mice would be more susceptible to diabetes development when compared with wild-type littermates. A20^β-KO mice aged normally without any evidence of metabolic defects. Phenotypic analysis of A20^β-KO mice up to the age of 12 months revealed no pathological signs in the pancreas. A20^β-KO mice and control littermates were subjected to a model of T1D induced by MLDSTZ, however, both control and A20^β-KO mice developed a similar hyperglycemia, which was confirmed in a glucose tolerance test (ipGTT) performed 5 weeks after the first STZ injection (Supplementary Figure 1B). Next, we crossed A20^β-KO mice with C57BL6-Ins2^Akita/J mice, which carry a mutation in the insulin Ins2 gene that prevents normal folding and secretion and induces endoplasmic reticulum stress leading to β-cell death. Mice carrying the Ins2^Akita mutation become hyperglycemic very early in life, however, no differences could be observed in conditions of A20 deficiency in β cells. In agreement, ipGTT shows severe and similar defects in insulin secretion in both Ins2^Akita and A20^{β−KO/Akita} mice (Supplementary Figure 1C). Finally, A20^β-KO mice were backcrossed into a non-obese diabetic genetic background, and glucose levels were measured every week in order to follow diabetes development. Although only 40% of all mice developed diabetes, no differences could be detected between control and A20^β-KO mice (Supplementary Figure 1D). In conclusion, A20 deficiency in β cells does not affect β-cell apoptosis nor disease development in vivo.

The authors declare no conflict of interest.