Zinc Transporter 8 and MAP3865c Homologous Epitopes are Recognized at T1D Onset in Sardinian Children

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.


Introduction
Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease resulting from the destruction of insulin-secreting pancreatic b cells. Although it is established that this autoimmune disease stems from a combination of genetic predisposition and environmental factors, the latter remain elusive [1]. During the period preceding T1D clinical onset, autoantibodies (aAbs) directed to islets antigens such as insulin, glutamic acid decarboxylase (GAD65), insulinoma associated protein-2 and zinc transporter 8 (Znt8) may be detectable for months up to years before disease onset [2], and progressively wane after diagnosis [3]. Wenzlau et al. reported that 60-80% of recent-onset T1D harbor antibodies against Znt8 C terminal domain [4].
Our recent studies suggested that Mycobacterium avium subspecies paratuberculosis (MAP) infection significantly associates with T1D in Sardinian population [5]. We reported that anti-MAP and anti-ZnT8 antibodies (Abs) targeting homologous membrane-spanning sequences are cross-reactive and capable of eliciting strong immune responses in T1D adult patients [6], opening the possibility of a molecular mimicry mechanism precipitating disease.
Sardinia is one of the regions with the highest incidence of T1D and multiple sclerosis worldwide, displaying a much higher T1D prevalence compared to other Mediterranean populations [7]. Several factors, such as genetic isolation, have likely contributed to the fixing of predisposing alleles. MAP infection is estimated to affect approximately 60% of Sardinian herds [6], and this mycobacterium can be found in pasteurized milk products and may be asymptomatically transmitted to humans [8]. Due to the potential role played by MAP in T1D pathogenesis, it is relevant to better characterize the prevalence of anti-MAP Abs in the Sardinian population. To this end, we investigated the seroreactivity against the previously identified ZnT8/MAP epitopes, in children at T1D-onset and compared to healthy controls (HCs) [6]. Moreover, sera from all individuals were tested for the presence of Abs against 4 newly identified putatively relevant Cterminal MAP3865c epitopes (MAP3865c 246-252 , MAP3865c 256-262 , MAP3865c 261-267 and MAP3865c 281-287 ), the corresponding ZnT8 C-terminal region, the MAP specific protein MptD, the T1D autoantigen GAD65 and the T1D unrelated Acetilcoline Receptor (ACHR).

Ethical Statement
Blood samples were collected after obtaining informed written consents from the guardians of all subjects. The study protocols were approved by the ethics committee of the University of Sassari and Cagliari, Sardinia, Italy.

ELISA
Indirect enzyme-linked immunosorbent assay (ELISA) to detect Abs specific for MAP3865c peptides and MptD protein were performed as described elsewhere [6]. The cut-off for positivity was calculated by ROC analysis, setting specificity at 93.3% (i.e., Ab+ HCs #6.7%). The percent fraction of Ab+ sera, Area Under ROC Curve(AUC), and p values after Fisher exact test are indicated. Results were normalized to a strongly positive control serum included in all experiments, the reactivity of which was set at 10.000 arbitrary units (AU)/ml. The statistical significance was determined using Graphpad Prism 6.0 software.
ElisaRSR TM ZnT8 Ab TM (RSR Limited, United Kingdom) kit for the quantitative determination of autoantibodies (aAbs) to the ZnT8 C-terminal region (Znt8 275-369 ) in serum was carried out according to the manufacturer's instructions.
The concentration of both human GAD65 and human Acetylcholine Receptor (ACHR) Abs in serum samples from T1D patients and matched HCs was determined using two different ELISA kit (MBS, The Netherlands) following the manufacturer's instructions.

Results
Abs responses against eight MAP/Znt8 peptides, four falling into the transmembrane region previously described [6], and four homologues to the human C-terminal Znt8 immunogenic region, were analyzed in 29 new-onset T1D children, and 30 age matched HCs using indirect ELISA. All eight peptides were highly recognized showing detectable reactivity. Results are summarized in Figure 1 and Figure 2.
The specificity of the MAP/Znt8 peptide based ELISA was further validated performing the RSR ZnT8 Ab ELISA test. Commercially available RSR ZnT8 Ab ELISA kit searches and identifies Abs against C-terminal residues 275-369 inclusive of the human ZnT8. After carrying out the RSR ZnT8 Ab ELISA 13 positives among 29 T1D (44.8%) were promptly identified, whereas none of the HCs was positive. The transmembrane MAP3865c/ZnT8 pairs of homologues peptides and the four Cterminal MAP3865c peptides were even more sensitive than the RSR ZnT8 Ab ELISA kit, in fact a range spanning from 8 to 16 (27.6-55.2%) out of 29 T1D patients, were correctly identified. Noteworthy, the best correlation between the RSR ZnT8 Ab ELISA kit and the C-terminal MAP3865c peptide is shown in Figure 3A.
Abs against the MAP-specific protein MptD were searched by indirect ELISA, highlighting that 14 out of 29 T1D children (48.3%) vs 1 out of 30 (3.3%) HCs were MptD Abs+ (p,0.0001). A correlation between Abs recognizing ZnT8RSR and Abs recognizing MptD MAP specific protein is shown in Fig. 3B.
Abs against GAD65, another major T1D autoantigen, and a totally T1D-unrelated protein ACHR (negative control) were also searched.
A similar percentage of Ab+ was found against GAD65 and MAP3865c peptides, whereas no blood sample of diabetic patients was reported to be positive for ACHR protein. Indeed, 12 out of 29 (41.4%) of T1D children and 2 out of 30 HCs (6.7%) were found to be positive after carrying out GAD65 ELISA testing.
If we look at T1D subjects, the high frequencies of Abs reacting against a MAP-specific protein MptD, together with the lack of Ab+ towards the T1D-unrelated protein ACHR, highlight the specificity of the MAP3865c/Znt8 based ELISA.

Discussion
The objective of this study was to investigate whether ZnT8/ MAP peptides were recognized in children at T1D-onset. We asked whether Abs against these epitopes may be markers of T1D in this pediatric population of Sardinia. This hypothesis is sustained by two major pieces of evidence.
Firstly, we have recently reported that MAP3865c and ZnT8 homologous sequences were cross-recognized by Abs in Sardinian T1D adults [6]. Secondly, it has been demonstrated that MAP infection triggers a specific humoral response in Sardinian T1D patients, which display high frequencies of Abs reacting against several mycobacterial proteins [10].
It is known that the number of newly diagnosed cases positive for ZnT8 Abs is correlated with an older age at diagnosis of T1D in children and decline following diagnosis [3]. A trend towards an older age at T1D diagnosis was observed in children positive for Abs against Znt8 178-186 (mean age at diagnosis 11.663.8 vs 7.663.9). Of note, titers against MAP3865c 133-141 and ZnT8 186-194 epitopes were similar to the one displayed by MAP3865c 125-133 , Znt8 178-186 epitopes in T1D and healthy children. Indeed, Abs against MAP3865c 125-133 , Znt8 178-186 had been previously found in 65.4% and 68% respectively, T1D adult from Sardinia. It is to establish if this is due to a loss of tolerance toward these epitopes in central lymphoid organs as well as in mature lymphocytes in the periphery. We here provide evidence for an association between Abs positivity for ZnT8 and MAP homologue epitopes in Sardinian T1D children. We were also able to demonstrate the specificity of the recognition, on account of the high frequencies of Abs reacting against MAP specific MptD protein, and the total lack of Ab+ towards ACHR (negative control) protein. The 2 T1D MptD negative patients (but positive for MAP3865c) may be explained by the lower sensitivity of the MptD ELISA (due to the poorly conserved conformation of the protein purified). Likewise, the C-terminal specificity of the MAP peptide based ELISA was confirmed by the RSR ZnT8 Ab ELISA results. Of note all children Abs+ for MAP3865c 133-141 were also positive for its homolog ZnT8 186-194 whereas only 7 out of 10 (70%) of children Abs+ for MAP3865c 125-133 were as well positive for ZnT8 178-186. This is a first step in understanding the possible role played by these epitopes in initiating autoimmunity. It is still  early to conclude that these peptides are potential biomarker for disease prediction in T1D at risk children. Further work needs to analyze larger cohorts of children followed over time to elucidate whether Ab titers against these MAP/Znt8 epitopes also decrease after diagnosis. Moreover, it will be relevant to clarify whether MAP infection and seroreactivity appears before or after T1D onset. It would be also interesting to look for the presence of MAP in intestinal tissues of T1D patients [15]. This would be the first step to sort out among different interpretations: is MAP an innocent bystander that merely colonizes the host? Is its presence due to a secondary infection not involved in T1D etiology? Or is MAP a primary infectious agent triggering b-cell autoimmunity?

Author Contributions
Conceived and designed the experiments: LAS. Performed the experiments: SM DC. Analyzed the data: LAS SM MZ. Contributed reagents/ materials/analysis tools: CR MP. Wrote the paper: SM LAS.