Zinc transporter 8 and MAP3865c homologous epitopes are recognized at T1D onset in Sardinian children

Abstract

Our group has recently demonstrated that Mycobacterium avium subspecies paratuberculosis (MAP) infection significantly associates with T1D in Sardinian adult patients. Due to the potential role played by MAP in T1D pathogenesis, it is relevant to better characterize the prevalence of anti-MAP antibodies (Abs) in the Sardinian population, studying newly diagnosed T1D children. Therefore, we investigated the seroreactivity against epitopes derived from the ZnT8 autoantigen involved in children at T1D onset and their homologous sequences of the MAP3865c protein. Moreover, sera from all individuals were tested for the presence of Abs against: the corresponding ZnT8 C-terminal region, the MAP specific protein MptD, the T1D autoantigen GAD65 and the T1D unrelated Acetylcholine Receptor. The novel MAP3865c281-287 epitope emerges here as the major C-terminal epitope recognized. Intriguingly ZnT8186-194 immunodominant peptide was cross-reactive with the homologous sequences MAP3865c133-141, strengthening the hypothesis that MAP could be an environmental trigger of T1D through a molecular mimicry mechanism. All eight epitopes were recognized by circulating Abs in T1D children in comparison to healthy controls, suggesting that these Abs could be biomarkers of T1D. It would be relevant to investigate larger cohorts of children, followed over time, to elucidate whether Ab titers against these MAP/Znt8 epitopes wane after diagnosis.

Figure 1. Prevalence of Abs against homologous ZnT8 and MAP3865c transmembrane epitopes in Type 1 Diabetes (T1D), and healthy controls (HCs) Sardinian children.

Sera were tested for their reactivity against plate-coated with MAP3865c_125–133(A) and its homologous ZnT8_178–186 (B); and with MAP3865c_133–141 (C) and its homologous ZnT8_186–194 (D). The dotted line lines indicate the cut-off for positivity used in each assay, as calculated by ROC analysis. The percent fraction of Ab+ sera is indicated on top of each distribution, while bars indicate the corresponding median±interquartile range. AUC and p values are given in the top right corner. Figure shows representative experiments out of three performed.

Figure 2. Prevalence of Abs against MAP3865c epitopes falling into the region of homology comprising the polymorphic Znt8 325^th residue in 29 Type 1 Diabetes (T1D) and 30 healthy controls (HCs) Sardinian children.

Sera were tested for their reactivity against plate-coated with MAP3865c_246–252 (A) MAP3865c_256–262 (B) MAP3865c_261–267 (C) and MAP3865c_281–287 (D) peptides. Data representation is the same as in Fig. 1.

Figure 3. Correlation between titers of RSR ZnT8 Ab and MAP reactive Abs.

Correlation is shown between titers of Abs recognizing (A) ZnT8RSR Abs and MAP3865c_281–287 C-terminal epitope; (B) ZnT8RSR Abs and MptD MAP specific protein. Each circle represents the titer of one T1D child. The dotted line lines indicate the cut-off for positivity used in each assay, as calculated by ROC analysis.