HERV-W and Mycobacterium avium subspecies paratuberculosis Are at Play in Pediatric Patients at Onset of Type 1 Diabetes

The etiology of T1D remains unknown, although a variety of etiological agents have been proposed as potential candidates to trigger autoimmunity in susceptible individuals. Emerging evidence has indicated that endogenous human retrovirus (HERV) may play a role in the disease etiopathogenesis; although several epigenetic mechanisms keep most HERVs silenced, environmental stimuli such as infections may contribute to the transcriptional reactivation of HERV-Wand thus promote pathological conditions. Previous studies have indicated that also Mycobacterium avium subspecies paratuberculosis (MAP) could be a potential risk factor for T1D, particularly in the Sardinian population. In the present study, the humoral response against HERV-W envelope and MAP-derived peptides was analyzed to investigate their potential role in T1D etiopathogenesis, in a Sardinian population at T1D onset (n = 26), T1D (45) and an age-matched healthy population (n = 45). For the first time, a high serum-prevalence of anti-Map and anti-HERV-W Abs was observed in pediatric patients at onset of T1D compared to T1D patients and healthy controls. Our results support the hypothesis that external infections and internal reactivations are involved in the etiology of T1D, and that HERV-W activation may be induced by infectious agents such as MAP.


Introduction
Type 1 diabetes (T1D) is an autoimmune disorder afflicting millions of people around the world, characterized by T cell infiltration within pancreatic islets and marked by the production of autoantibodies to the beta-cell [1]. The destruction of 90% of beta cells is related to the onset of the clinical symptoms [2]. The major genetic determinant of T1D are polymorphisms within the HLA region [3], although over 60 non-HLA loci T1D susceptibility loci have been identified [4]. However, the genetic drift alone cannot explain the increase in the incidence of T1D, and environmental factors are thought to play a critical role in triggering islet autoimmunity [5,6]. A variety of studies suggested that the environmental agents trigger disease development in genetically susceptible subjects [7,8]. The loss of self-tolerance and the development of the immune response can be a consequence of the mechanism of cross-reactivity associated with the molecular mimicry provoked by infectious factor [9]. A number of factors, including diet, intestinal dysbiosis, viral infections such as enteroviruses (coxsackieviruses, rotaviruses, cytomegalovirus, parvovirus), have been explored as potential risk factor for the disease progression, but no causal relationship has been established [8].
Previous studies have indicated that Mycobacterium avium subspecies paratuberculosis (MAP) could be a potential risk factor for T1D [10,11]. MAP is the etiological agent of Johne's disease, a chronic and inflammatory bowel disease that affects ruminants [12]. The primary route of MAP transmission is oral-fecal through ingestion of contaminated milk, water or the environment caused by infected animals [13][14][15].
MAP is able to invade the intestinal mucosa by interacting with enterocytes [16]. MAP is endemic in the ruminant population of Sardinian Island, an area with one of the highest incidences of T1D in the world [17] and numerous clinical studies have linked MAP to T1D in the Sardinian population [10]. We previously identified different MAP peptides based on the sequence homologies present with human proteins including zinc transporter protein 8 (ZnT8) and with proinsulin (PI) and use them in ELISA [10].
Emerging evidence has also indicated that human endogenous retrovirus (HERV) may play a role in T1D etiopathogenesis [18,19], and it has been reported to function also as a superantigen due to the ability to induce a strong T cell responses [20].
Part of the human genome, about 8%, is composed of HERV, which until recently were considered junk DNA. The integrations of the provirus genome into the DNA of germinal cells are the remnants of infections that occurred over several million years; however, numerous nonsense mutations have rendered them defective [21]. The envelope proteins of HERV-W (HERV-Wenv) has been detected in serum, in peripheral blood mononuclear cells and within the pancreas of patients with T1D [18][19][20], also, in an in vitro study in transgenic mice, HERV-Wenv repressed internal insulin secretion [22,23]. The monoclonal antibody GNbAC1, has been developed to neutralize the effects of HERV-Wenv, and is currently in phase IIa clinical trials as a possible HERV-based therapeutic approach in T1D [24]. Interestingly, not all HERVs remain silenced, and they can be reactivated under certain pathological condition such as infection [24]. The aim of this study was to investigate the role of MAP and HERV in a Sardinian pediatric population with T1D, analyzing the humoral response against homologues peptides derived from both pathogens.
Collectively, data obtained from this study support the involvement of MAP and to HERV-Wenv in the loss of immune tolerance that leads to autoimmunity in T1D. Furthermore, a positive correlation between anti-MAP and anti-HERV-Wenv antibodies observed, suggested a potential relationship between MAP seropositivity and reactivation of HERV.

Patients
A total of 45 children with T1D pediatric and young patients (median age 12.6, n = 18 females and n = 27 males mean years with T1D = 6) and 26 patients with onset of T1D (median age 7.8, n = 7 females and n = 19 males) recruited at the time of diagnosis and blood sampling was performed before starting with insulin therapy, were enrolled in this retrospective study. The inclusion criteria were diagnosis of T1D upon manifestation of the clinical symptoms such as severe hyperglycemia e/o ketoacidosis, testing for the presence of classical islet autoantibodies, as well as levels of glycated hemoglobin, according to the American Diabetes Association criteria [25]. Clinical data of T1D patients are available in Supplementary Table S1. Forty-five healthy and age-matched (median age 11.8 years, 29 females, 16 males) volunteers without autoimmune diseases and inflammatory episodes in the last 2 months served as controls. Patients were recruited at the Pediatrics department of the AOU of Sassari and Department of Experimental and Clinical Medicine of the University of Sassari, while controls samples were obtained from the Department of Endocrinology of the University Hospital (AOU) of Sassari and written informed consent from a parent or legal tutor was obtained for all study participants. All methods were performed in accordance with regional and national regulations.

Blood Samples
Five to eight mL of peripheral blood was taken from each individual in EDTA tubes which was processed within 12 h of collection. Plasma was obtained using Ficoll-Paque ® (Cynthia Europe Milano, Italy) according to the protocol. Plasma rates was immediately transferred into a clean polypropylene tube and apportioned into 0.5 mL aliquots and transported at -20 • C for short-term storage (<6 months) and −80 • C for long-term storage (>6 months).

Peptides
Peptides showed in Table 1
Each sample was run in duplicated, and normalization was performed with a positive control (absorbance reactivity set at 1.0 arbitrary units) included in each assay. Background activity was calculated as the mean signal of an immobilized peptide with secondary Ab alone.

Statistical Analysis
GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA) was used for calculating the sensitivity and specificity, false negative and false positive values. Mann-Whitney test were used to analyze non-parametric data. For evaluation of diagnostic values of Ab, receiver operating characteristic curve (ROC) and area under the curve (AUC) was established. Fisher's exact test and Spearman's were used for comparison and correlation between MAP and HERV-Wenv peptides, respectively; p < 0.05 was considered statistically significant.

Seroreactivity against HERV-W Antigen
Concerning HERV-W peptides, significant differences in Ab distribution were also detected between the patient groups and the control group. Anti-HERV-Wenv 93-108 Abs were detected in T1D patients ( Figure 2D).

Discussion
The incidence of type 1 diabetes in children has increased worldwide, and it is therefore important to find markers to enable early diagnosis and new treatments. Despite intensive research, the etiology of T1D remains unknown, and a multiplicity of etiological agents has been proposed as potential candidates to triggers autoimmunity in susceptible individuals with T1D [27].
The present study analyzed the potential involvement of MAP and HER-W as contributor factors in T1D etiopathogenesis, based on previous reports. Indeed, in recent studies a direct correlation between anti-MAP Abs and anti-HERV-Wenv has been observed in a cohort of patient with T1D [28].
This study demonstrates for the first time a high seroprevalence of anti-Map and anti-HERV-W Abs in pediatric patients with T1D from Sardinia. Our results support the hypothesis that both pathogens are linked to T1D etiology and also that HERV-W activation can be induced by certain infectious agent such as MAP. To note, a possible connection between MAP and HERV-W was also showed in multiple sclerosis (MS) where it was detected a partial decrease of anti-MAP Abs and anti-HERV-Wenv in the serum of patients following treatment with natalizumab [29].
These differences may be the first step in suggesting MAP infection as a plausible circumstance capable of inducing the expression of HERV-W antigens which in turn lead to immune imbalance.
The significant results we have obtained are in line with this hypothesis, given the high reactivity expressed in patients at the onset for all peptide fragments evaluated of MAP and HERV-Wenv compared to a healthy population of corresponding age and geographical background but also compared to the population with diabetes developed for years. Interestingly, in young people receiving insulin therapy, we observed a lower antibody titer for HERV-Wenv than young people of the same age at the start of the disease. It was seen that in MAP infection Th17-derived cytokine genes were down-regulated, these play an important role in the early phase of mycobacterial infection [30], in particular IL-26, which induces immune cell initiation and direct pathogen killing [31] and IL-17F, which provides protective immunity against intracellular pathogens through modulation of the Th1 response and neutrophil recruitment [32,33].
MAP also influences the expression of interferon regulatory genes, with reduced expression of IRF4 inducing an under-regulation of the Th1 immune response, thus enhancing the persistent survival of MAP [34], and an over-regulation of IRF5 and IRF7 that may result in inhibition of T-cell proliferation through tryptophan depletion mediated by indoleamine 2,3-dioxygenase that subsequently leads to an immunosuppressive state [35].
Several factors may contribute to the transcriptional reactivation of HERV-W and may promote pathological conditions [36], some environmental stimuli such as infection immune factors and oxidative stress seems to contribute by hindering the binding of methyl groups transferred by DNA methyltransferases and enhancing gene expression following inhibition of histone deacetylases through interaction with specific binding sites in HERV promoter regions [37].
Adhesion of MAP to the mucosa lining the small intestine and subsequent uptake by M-cells and enterocytes may play a role in triggering antibody production [16] another contribution of MAP in inducing latent infection in humans could result from molecular mimicry with homologous epitopes such as ZnT8 and PI, leading to autoimmune responses [38,39].
In addition, oxidative processes have been demonstrated in patients with Crohn's disease and in MAP-infected cattle, where an increase in selenium-dependent glutathione peroxidase (GPx) activity has been seen [40]. This probably contribute to HERV transcriptional reactivation by hindering the binding of methyl groups transferred by DNA methyltransferases and enhancing gene expression following inhibition of histone deacetylases and consequently promote disease conditions [37].
The hypothesis that MAP may be involved in HERV-W transactivation is also supported by the presence of highly expressed CD68+ in granulomatous lesions of infected goats, indicating high lysosome counts and acid phosphatase activity [41], similarly, increased expression of CD68+ infiltrating macrophages has been reported in the pancreas of T1D patients, in this case linked to HERV-W expression [22].
As humans are not the main target of MAP, it may act indirectly on immune homeostasis as a consequence of survival mechanisms that elude host defense against pathogens and conditions that favor the expression of HERV-W antigens. In conclusion, we have demonstrated high seropositivity against MAP and HERV-W peptides with a significant prevalence at disease onset and at the same time the moderate and high correlations present between the two agents; further investigation would help to elucidate the role of MAP as a plausible trigger for the expression of HERV-W antigens which in turn lead to immune imbalance and subsequent disease onset. Additional follow up studies of Ab-titers over longer time frames are necessary to determine the role of both pathogens in T1D.