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) 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 mice would be more susceptible to diabetes development when compared with wild-type littermates. A20 mice aged normally without any evidence of metabolic defects. Phenotypic analysis of A20 mice up to the age of 12 months revealed no pathological signs in the pancreas. A20 mice and control littermates were subjected to a model of T1D induced by MLDSTZ, however, both control and A20 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 mice with C57BL6-Ins2Akita/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 Ins2Akita 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 Ins2Akita and A20 mice (Supplementary Figure 1C). Finally, A20 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 mice (Supplementary Figure 1D). In conclusion, A20 deficiency in β cells does not affect β-cell apoptosis nor disease development in vivo.