Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice

Abstract

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice.This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated GRP78 and reduced spontaneous NETosis of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate, characterized by reduced frequencies of effector memory CD4 + T cells and a modest reduction in the frequency of IFNγ-producing CD4 + and CD8 + T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes.

Citrullination and PAD Enzyme Biology in Type 1 Diabetes – Regulators of Inflammation, Autoimmunity, and Pathology

“…Not surprisingly, based on the high levels of PAD4 in neutrophils and their important role in histone 3 citrullination and induction of NETosis, as outlined above, in vivo PAD inhibition was shown to reduce NET formation and associated NET-induced tissue damage (204,205). Other ameliorating disease effects were associated with a decrease in circulating pro-inflammatory cytokine levels, such as IL-6, TNFa and IL-1b (206,207), an increase in Treg populations (200) or a shift in T-lymphocyte populations from Th1/Th17 towards Th2 (201,208). The latter is thought to be mediated through direct inhibition of citrullination of the transcription factors RORgT and GATA3 (see also above) (72).…”

“…As such, amelioration or even reversal of disease in case of intervention therapies, and delayed initiation or complete protection in case of preventive therapies, was shown in mouse models of RA, MS, SLE, UC, inflammatory bowel disease, and recently also in T1D [reviewed by (198,199)]. In general, mechanistic insights from these studies have taught us that targeting PAD making use of pan-PAD inhibitors effectively decreases protein citrullination levels in the inflamed target tissues, as measured mainly by LC-MS/MS, PAD activity assays or Western blotting using anti-citrulline Ab (200)(201)(202)(203). Such decreased protein citrullination can evidently have an effect on the autoreactive responses against citrullinated autoantigens, both in terms of autoantibody and T-cell responses, as shown in some published studies (200,201).…”

“…In general, mechanistic insights from these studies have taught us that targeting PAD making use of pan-PAD inhibitors effectively decreases protein citrullination levels in the inflamed target tissues, as measured mainly by LC-MS/MS, PAD activity assays or Western blotting using anti-citrulline Ab (200)(201)(202)(203). Such decreased protein citrullination can evidently have an effect on the autoreactive responses against citrullinated autoantigens, both in terms of autoantibody and T-cell responses, as shown in some published studies (200,201). Next to this, it has become clear that other mechanisms are involved in the observed protection, pointing towards more general effects on innate and adaptive immunity.…”

“…Of interest in the field of T1D, a recent study from the Overbergh laboratory showed a complete protection against diabetes development in the NOD mouse, by daily subcutaneous injections of BB-Cl-amidine (1µg/g body weight) (200). BB-Cl-amidine is a pan-PAD inhibitor that, similar to its mother compound (Cl-amidine), irreversibly inactivates PAD enzymes through covalent modification of an important cysteine for the activity of the enzymes (209).…”

“…BB-Cl-amidine is a pan-PAD inhibitor that, similar to its mother compound (Cl-amidine), irreversibly inactivates PAD enzymes through covalent modification of an important cysteine for the activity of the enzymes (209). Remarkably, diabetes protection was observed when starting treatment at 8 weeks of age, a time point at which insulitis is already ongoing, but hyperglycemia has not developed (200). This observation tempts us to conclude that citrullination may play a role in amplification of the disease rather than being an initial trigger in breaking immune tolerance.…”