Human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice

Abstract

Multiple sclerosis (MS) is a complex multifactorial disease of the central nervous system (CNS) for which animal models have mainly addressed downstream immunopathology but not potential inducers of autoimmunity. In the absence of a pathogen known to cause neuroinflammation in MS, Mycobacterial lysate is commonly used in the form of complete Freund's adjuvant to induce autoimmunity to myelin proteins in Experimental Allergic Encephalomyelitis (EAE), an animal model for MS. The present study demonstrates that a protein from the human endogenous retrovirus HERV-W family (MSRV-Env) can be used instead of mycobacterial lysate to induce autoimmunity and EAE in mice injected with MOG, with typical anti-myelin response and CNS lesions normally seen in this model. MSRV-Env was shown to induce proinflammatory response in human macrophage cells through TLR4 activation pathway. The present results demonstrate a similar activation of murine dendritic cells and show the ability of MSRV-Env to trigger EAE in mice. In previous studies, MSRV-Env protein was reproducibly detected in MS brain lesions within microglia and perivascular macrophages. The present results are therefore likely to provide a model for MS, in which the upstream adjuvant triggering neuroinflammation is the one detected in MS active lesions. This model now allows pre-clinical studies with therapeutic agents targeting this endogenous retroviral protein in MS.

Figure 1. Env-SU induces the maturation of murine DC.

DC from wild-type C57Bl/6 mice were stimulated with LPS (2 µg/mL) or Env-SU (2 µg/mL). After 24 h of incubation, cells were analysed by cytofluorometry for MHC class II and CD11c surface markers expression. DC were harvested, washed in PBS with 3% FCS and 0.16% sodium azide and immunostained for MHCII (monoclonal IA^b-PE antibody) and for CD11c (monoclonal CD11c-APC antibody) for 30 min at 4°C. The conjugated Abs used for staining were all obtained from BD Biosciences. A propidium iodide staining has been used to exclude dead cells. Double positive cells for CD11c and MHC-II (upper right quadrant) represents mature DC. Upper panel: Graphic distribution of cells according to immunostaining intensity for CD11c (abscissa) and MHCII (ordinate) without stimulation (left), after stimulation by LPS (center) or after stimulation by MSRV-Env (right). Lower panel: Corresponding percentage of cells in each quadrant, below each graphic. Results are representative for three experiments.

Figure 2. Env-SU induced the production of pro-inflammatory cytokine in DC culture.

Murine Bone marrow DC were stimulated with LPS (2 µg/mL), PSB (100 µg/mL) and different amounts of Env-SU for 24 h. Culture supernatants were then analysed by ELISA for IL-6 (A), TNFα (B) and IL-12p70 (C) production. In (D), the specific inhibition of MSRV-Env stimulated DC by anti-Env neutralizing monoclonal antibody (GN-mAb_03) is shown, compared to an irrelevant anti-MSRV Gag (GN-mAb_12) or to diluents without antibody. IL-6 levels obtained in non-stimulated DC (medium) as negative control, or stimulated with LPS as positive control are also shown. IL-6 production was measured by ELISA in supernatants after 24 h of incubation (ELISA standard deviations <5%). These results are representative of three experiments realised under identical conditions.

Figure 3. Comparison of IL6 production in Env-SU stimulated murine DC derived from TLR4 KO and CD14 KO mice compared with wild-type mice.

IL-6 production in bone marrow DC culture supernatants of TLR4 KO (A) and CD14 KO (B) mice compared to wild-type C57BL/6 mice after 24 h of stimulation with LPS, PSB and graded dose of Env-SU. Percentages shown in the histogram correspond to the percentage of residual IL6 secretions dosed in cells supernatants of TLR4 or CD14 KO mice relative to wild-type mice. Data presented are representative of three experiments under identical conditions.

Figure 4. MSRV induced EAE like symptoms in C57Bl/6 mice.

Experiments were performed on 6–8 weeks-old C57BL/6 female mice. Mice were immunized subcutaneously with 200 µg of MOG 35–55 per animal emulsified in CFA containing 400 µg of Mycobacterium tuberculosis H37RA, or an emulsion containing 10 µg or 50 µg of Env-SU in IFA, or an emulsion containing 20 µg or 50 µg of full-length Env protein (in C), or MOG with IFA alone. Immediately thereafter and again 2 days later, the mice received an intraperitoneal injection of 200 ng of Bordetella pertussis toxin in PBS. Mice of IFA, CFA or Env groups were examined daily and clinical score were then monitored on the clinical scale of 0 to 6. Mean scores and standard deviation (A–B) or SEM (C) for each group of mice are presented. The values are representative for three experiments with Env-SU and more than three with full-length MSRV-Env (with protocol variants) that repeatedly showed similar induction of EAE.

Figure 5. Histological analysis of cervical spinal cord (mouse #1-1).

A. Microscopic changes in transverse section of spinal cord are shown (100×). Multiple inflammatory foci and some multifocal inflammatory lesions are present in the leptomeninges, around blood vessels in the leptomeninges and white matter, and parenchyma of the white matter (arrows). There is also vacuolation in the white matter that is consistent with edema and demyelination. B. Luxol fast blue stained section of cervical spinal cord (100×). This section is from the same block of tissue as the H&E stained section shown in A. Areas of demyelination are visible within white matter (lighter blue stained areas, in the outer parts of the spinal cord; arrows). C. H&E stained transverse section of thoracic spinal cord (100×). Four multifocal inflammatory lesions are present in the leptomeninges, around blood vessels in the leptomeninges and white matter (box and arrows). There also is vacuolation in the white matter that is consistent with edema and demyelination (box). D. H&E stained section of thoracic spinal cord (400×). Detail of the above slide (boxed area). Two multifocal inflammatory lesions are shown (arrows).

Figure 6. EAE mice induced by Env-SU display anti-MOG autoimmunity in splenocytes cultures.

A. γIFN secretion of pooled splenocytes (2 mice for the IFA group and 3 mice for the Env-SU group) of EAE mice assessed by ELISA of culture supernatants 24 h after stimulation. B. Kinetics of γIFN production upon MOG recall on pooled splenocytes of IFA (n = 3) and Env-SU (n = 3) injected EAE mice. IFN-γ secretion is assessed in culture supernatants after 24 h, 48 h and 72 h of incubation. Results are representative of two experiments.