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. 2017 Sep 7;2(17):e94387.
doi: 10.1172/jci.insight.94387.

An ancestral retroviral protein identified as a therapeutic target in type-1 diabetes

Sandrine Levet  1 , Julie Medina  1 , Julie Joanou  1 , Amandine Demolder  1 , Nelly Queruel  1 , Kevin Réant  1 , Matthieu Normand  2 , Marine Seffals  2 , Julie Dimier  3 , Raphaële Germi  3   4 , Thomas Piofczyk  5 , Jacques Portoukalian  6 , Jean-Louis Touraine  6 , Hervé Perron  1   6   7
Affiliations

An ancestral retroviral protein identified as a therapeutic target in type-1 diabetes

Sandrine Levet et al. JCI Insight. .

Abstract

Human endogenous retroviruses (HERVs), remnants of ancestral viral genomic insertions, are known to represent 8% of the human genome and are associated with several pathologies. In particular, the envelope protein of HERV-W family (HERV-W-Env) has been involved in multiple sclerosis pathogenesis. Investigations to detect HERV-W-Env in a few other autoimmune diseases were negative, except in type-1 diabetes (T1D). In patients suffering from T1D, HERV-W-Env protein was detected in 70% of sera, and its corresponding RNA was detected in 57% of peripheral blood mononuclear cells. While studies on human Langerhans islets evidenced the inhibition of insulin secretion by HERV-W-Env, this endogenous protein was found to be expressed by acinar cells in 75% of human T1D pancreata. An extensive immunohistological analysis further revealed a significant correlation between HERV-W-Env expression and macrophage infiltrates in the exocrine part of human pancreata. Such findings were corroborated by in vivo studies on transgenic mice expressing HERV-W-env gene, which displayed hyperglycemia and decreased levels of insulin, along with immune cell infiltrates in their pancreas. Altogether, these results strongly suggest an involvement of HERV-W-Env in T1D pathogenesis. They also provide potentially novel therapeutic perspectives, since unveiling a pathogenic target in T1D.

Keywords: Autoimmunity; Beta cells; Diabetes; Endocrinology; Innate immunity.

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Conflict of interest statement

Conflict of interest: SL, JM, JJ, AD, NQ, and KR receive compensation for their work at GeNeuro Innovation. MN and MS receive compensation for their work at Excilone. TP receives compensation for his work at Biogazelle. HP receives compensation for his work at GeNeuro Innovation and is an inventor on patents owned by bioMérieux, INSERM, and GeNeuro but has transferred all his rights to bioMérieux or to GeNeuro under applicable laws for employed inventors.

Figures

Figure 1
Figure 1. HERV-W-Env is expressed in human T1D patients.
(A) Serum from controls (n = 93) and T1D (n = 30) were analyzed by sandwich ELISA using monoclonal antibodies raised against HERV-W-Env. Each sample has been tested in triplicate. Results are presented as mean of the triplicate for each donor and as mean ± SEM for each group. Significance determined by Mann Whitney U test. (B) PBMC RNA was extracted from controls (n = 26) and T1D (n = 23) and analyzed by qRT-PCR assay. HERV-W-env expression was normalized to housekeeping genes B2M and YWHAZ using qbase+ and is expressed as calibrated normalized relative quantities (CNRQ). Each qPCR was done in duplicate, individual CNRQ values are plotted, and mean ± SEM for each group is shown. Significance determined by unpaired t test. (C) HERV-W-Env expression was assessed on pancreas slices from controls (n = 19) and T1D (n = 20) stained using GN_mAb_Env03 and automatically quantified. The HERV-W-Env positive area was reported to the total pancreatic tissue area. Two slides per individuals were quantified — one in the head and one in the tail. Results are presented as mean of the 2 slides for each donor and as mean ± SEM for each group. Significance determined by Mann Whitney U test. Individuals from cohorts presented in A–C were classified into either positive or negative groups for HERV-W-Env based on thresholds set at mean + 2 SD of control group. Cutoffs were set at 0.231 OD450nm for ELISA, at 1.356 CNRQ for qRT-PCR, and at 21.43% for pancreas IHC. Results are presented as contingency plots representing the number of individuals positive (in orange) or negative (in black). Significance determined by χ2 test. (D) Pancreas slices of three nPOD donors stained with GN_mAb_Env03 and adjacent slides coimmunostained with insulin (red) and glucagon (brown) are presented. Pancreatic Langerhans islets are highlighted with red dotted line. nPOD case ID is indicated in the upper right corner, with block number and pancreas zone (PH, pancreas head; PT, pancreas tail). Scale bars: 100 μm. ***P < 0.001, ****P < 0.0001.
Figure 2
Figure 2. Characteristics of HERV-W-Env expression in pancreas and during the course of the disease.
(A) Three nPOD T1D cases stained with GN_mAb_Env03 are presented at 0.5× original magnification, and corresponding higher magnification 3× and 10×. nPOD case ID, block number, and pancreas zone are indicated on the left, and disease duration is indicated on the right. Scale bars: 0.5 ×, 5mm; 3×, 500 μm; 10×, 250 μm. (B–D) For each of the 3 cohorts presented in Figure 1, A–C, HERV-W-Env expression is presented as a function of the disease duration, the dotted line representing the positivity cutoff. T1D duration is presented as function of HERV-W-Env protein expression in serum as determined by ELISA (B), as a function of HERV-W-env gene expression in PBMC (C), or as a function of HERV-W-Env expression in pancreas as determined by IHC (D).
Figure 3
Figure 3. Proinflammatory features associated with HERV-W-Env expression within the pancreas.
(A) Pancreas of nPOD T1D cases 6036 and 6054 were immunostained with GN_mAb_Env03 (upper panels), and adjacent slides were immunostained with anti-CD3 (orange) and anti-CD68 (red) (lower panels). Lower panels display the same areas as upper panels. Scale bars: 100 μm. (B–G) Percentages of CD68+ (B) and CD3+ (D) cells in the exocrine pancreas are presented for controls (n = 19) and T1D patients (n = 20) and as a function of HERV-W-Env+ area (C and E, respectively) (Figure 1C). The relationship between CD3+ and CD68+ cells for each T1D donor is presented in F, which allowed to determine 3 groups: (i) CD3low/CD68low, (ii) CD3high/CD68low, and (iii) CD68high/CD3low. Based on this plot, the cutoff for CD3+ cells is set at 0.065%, and for CD68+ cells, it is set at 0.1%. The HERV-W-Env+ area presented in Figure 1C was reanalyzed in these 3 groups and was presented as mean for each individual and mean ± SEM for each group (G). Significance determined by Mann Whitney U test (B and D), by Spearman test (C), and by Kruskal-Wallis test followed by Dunn multiple comparison test (G). *P < 0.05, **P < 0.01, ****P < 0.0001.
Figure 4
Figure 4. Transgenic mice expressing HERV-W-env display hyperglycemia and pancreatic abnormalities.
(A and B) Seven-week-old CAG-Env transgenic mice expressing of HERV-W-env and control C57Bl/6J mice of the same age were fasted for 5 hours before measuring glycemia (A) and insulinemia (B). Results are presented as individual values and as mean ± SEM. n = 10 or 11 mice in each group. Significance determined by unpaired t test (glycemia) or by Mann-Whitney U test, one-tailed (insulinemia). (C) Pancreas sections of 12-month-old CAG-Env mice (n = 5 females and n = 6 males) and C57BL/6J mice (n = 5 females and n = 7 males) were stained with HES (Hematoxylin Eosin Saffran). Representative images of C57BL/6J (left panels) and CAG-Env (right panels) mouse pancreata were presented at 10× (upper panels) and 40× (lower panels) original magnification. Fatty infiltrates are highlighted with green arrows and immune cells infiltrates with orange arrows. Scale bars: upper panels, 200 μm; lower panels, 50 μm. (D and E) Pancreas sections from 12-month-old CAG-Env mice (n = 5 females and n = 6 males) and C57BL/6J mice (n = 5 females and n = 7 males) were analyzed for immune cell infiltration (D) and fat infiltration (E). A gradation from 0–3 was affected to each slide, 0 corresponding to no infiltration of fat or immune cells and 3 corresponding to high level of infiltrations. Two sections distant by 500 μm were analyzed for each mouse, blindly and independently by 2 people, and their evaluations were averaged for each slide. Results are presented as mean of the 2 slides for each mouse and as mean ± SEM for each group. Significance was determined by Mann Whitney U test. *P < 0.05; **P < 0.01, ****P < 0.0001.
Figure 5
Figure 5. Direct pathogenic effects of HERV-W-Env toward pancreatic β cells.
(A) Human Langerhans islets have been exposed for 48 hours to HERV-W-Env (0, 25, 50, 100 ng/ml), and insulin secretion has been measured in response to 20 mM glucose stimulation. Results are presented as individual values and as mean ± SEM. Each condition has been performed in triplicate and in 3 independent experiments. Significance determined by Bonferroni’s test. (B) INS1E cells have been exposed for 72 hours to increasing concentrations of HERV-W-Env (0, 10, 25, 50, 100, 400 ng/ml). Insulin secretion has been measured either in absence of glucose (baseline) or in response to 10 mM glucose stimulation. Results are presented as the difference in concentration between glucose stimulation and baseline. Each condition has been performed in triplicate and in 2 independent experiments, and data are presented as individual values and as mean ± SEM. Significance determined by Bonferroni’s test. (C) Human Langerhans islets have been exposed for 48 hours to HERV-W-Env (0 and 100 ng/ml) and to GNbAC1 (3 μg/ml) or placebo. Insulin secretion has been measured in response to 20 mM glucose stimulation. Results are presented as individual values and as mean ± SEM. Each condition has been performed in triplicate and in 3 independent experiments. Significance determined by unpaired t test. (D) Effect of HERV-W-Env on human Langerhans islet viability has been assessed using human islet microtissues exposed to 0, 100, and 200 ng/ml of HERV-W-Env for 48 hours before being lysed. Lysates were analyzed for ATP content using Promega CellTiter-Glo Luminescent Cell Viability Assay. Results are presented as individual values and as mean ± SEM. Each condition has been performed six times, and 1 experiment has been performed. Significance determined by Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 6
Figure 6. Current working model.
This diagram illustrates the current working model in which the present study is integrated. Data presented demonstrate that HERV-W-Env protein is expressed in T1D patients and particularly in their pancreas (Figure 1), where it may promote β cell dysfunction (Figure 5) and macrophages recruitment (Figure 3), possibly leading to hyperglycemia and decrease levels of insulin (Figures 4 and 5). As reported by other studies, HERV-W-Env displays other pathogenic properties that may be relevant in T1D pathogenesis. In particular, HERV-W-Env promotes autoimmunity (23) and impairs TLR4+ cells such as endothelial cells (49) and Schwann cells (50), both of which are associated with major T1D comorbidities. The underlying question is why the HERV-W-env gene has been transcriptionally activated and translated into a pathogenic protein. An explanation relies upon its transactivation and epigenetic dysregulation by infectious agents, as it has already been demonstrated (33, 60, 61). In T1D, the question of such dysregulation by enterovirus is raised (12), as well as the role of EBV (62). This diagram illustrates how, in a global pathogenic cascade leading to the disease, HERV-W-Env expression is placed at a crucial position, between multiple environmental factors operating in genetically susceptible individuals and multiple clinical outcomes. This positioning provides a rationale for the neutralization of HERV-W-Env as a therapeutic target in T1D. Within this framework, GNbAC1 — a humanized monoclonal IgG4 antibody neutralizing HERV-W-env — is currently tested in a phase IIa clinical trial in T1D patients (NCT03179423).
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