- Email: [email protected]
Antibody response against HERV-W in patients with MOG-IgG associated disorders, multiple sclerosis and NMOSD
Journal of Neuroimmunology 338 (2020) 577110
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Antibody response against HERV-W in patients with MOG-IgG associated disorders, multiple sclerosis and NMOSD
T
Giannina Arrua, Elia Sechia, Sara Mariottob, Ignazio R. Zarboa, Sergio Ferrarib, Alberto Gajofattob, ⁎ Salvatore Monacob, Giovanni A. Deianaa, Marco Boc, Leonardo A. Sechic, Gian Pietro Sechia, a
Section of Neurology, Department of Clinical, Surgery and Experimental Sciences, University of Sassari, Italy Department of Neuroscience, Biomedicine and Movement Sciences, University of Verona, Italy c Department of Biomedical Sciences, University of Sassari, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: MS Myelin oligodendrocyte glycoprotein Neuromyelitis optica spectrum disorder Autoimmunity Human endogenous retroviruses
Increased expression of the retroviruses of HERV-W family has been linked to multiple sclerosis (MS) pathophysiology; nothing is known at the moment about MOG-IgG associated disorders. We compared antibody response against HERV-W peptides among patients with MOG-IgG associated disorders, multiple sclerosis (MS) and aquaporin-4 (AQP4)-IgG positive neuromyelitis optica spectrum disorder (NMOSD). A total of 102 serum samples were retrospectively analyzed. Antibody reactivity against HERV-W env peptides was similar in MOG-IgG associated disorders and MS, but different from AQP4-IgG positive NMOSD. Our findings expand the diagnostic role of HERV-W antibodies to the spectrum of demyelinating disorders associated with MOG-IgG.
1. Introduction Human endogenous retroviruses (HERVs) account for approximately 8% of human genome, being the result of ancestral infections with exogenous retroviruses and subsequent integration into germline DNA (Perron et al., 1997). Increased expression of the retroviruses of HERV-W family has been linked to multiple sclerosis (MS) pathophysiology (Perron et al., 1997) and has been proposed as a prognostic factor (Sotgiu et al., 2010) and a potential predictor of treatment response (Arru et al., 2014). Endogenous retroviruses likely exert their pathogenic effect by acting as activators/modulators of innate immunity, mainly through interaction with Toll-Like Receptors with a superantigen-like mechanism (Rolland et al., 2006). Through this mechanism HERV-W envelope proteins contribute to increased nitric oxide production in the brain, therefore influencing microglial migratory ability and reducing oligodendroglial differentiation capacity (Kremer et al., 2013). Myelin oligodendrocyte glycoprotein (MOG) is a central nervous system (CNS)-restricted protein expressed on the outermost surface of the myelin sheath and the plasma membrane of oligodendrocyte (Reindl et al., 2013). Over the last decade, a spectrum of CNS demyelinating diseases associated with autoantibodies directed against this protein (MOG-IgG) has been characterized (Lopez-Chiriboga et al.,
⁎
2018). MOG-IgG associated disorders show distinctive clinical and radiological features compared to MS, but frequently overlap those of aquaporin-4 (AQP4)-IgG positive neuromyelitis optica spectrum disorder (NMOSD) (Polman et al., 2011; Wingerchuk et al., 2015; Mariotto et al., 2017). We recently reported an increased humoral immune response towards some antigenic peptides of the HERV-W envelope proteins in MS patients but not in NMOSD patients (either AQP4-IgG positive or double-negative for AQP4-IgG and MOG-IgG), suggesting a possible role for HERV-W antibodies as discriminating biomarker between MS and NMOSD (Arru et al., 2017). Immunological response to HERV-W in patients with MOG-IgG associated disorders has yet to be investigated. We studied the humoral immune response against HERV-W peptides in patients with MOG-IgG associated disorders and made a comparison with that observed in MS and NMOSD. 2. Material and methods 2.1. Patients The study protocol was approved by the local ethics committee (Prot. 2457/2017, Azienda Sanitaria Locale 1, Sassari, Italy). All the included subjects provided written consent to sample storing and
Corresponding author at: Neurology Unit, AOU Sassari, Department of Clinical, Surgical and Experimental Sciences, Viale “S. Pietro” 10, 07100 Sassari, Italy. E-mail address: [email protected] (G.P. Sechi).
https://doi.org/10.1016/j.jneuroim.2019.577110 Received 17 July 2019; Received in revised form 18 October 2019; Accepted 4 November 2019 0165-5728/ © 2019 Elsevier B.V. All rights reserved.
Journal of Neuroimmunology 338 (2020) 577110
G. Arru, et al.
Table 1 Demographical and clinical data of all investigated groups. Groups
Age, median ± SD
Sex, females / males, n
Disease duration / months 0–12 (%) 13–60 (%) > 60 (%)
Treatment
MOG-IgG+(# 22)
42 ± 15,98
10/12
13 (59) 4 (18) 5 (23)
MS (# 22)
44 ± 11,59
10/12
22 (100)
AQP4-IgG+(# 22) HCs (#36)
50 ± 13,67 49 ± 14,20
20/2 17/19
N.A. _
17 yes 5 no 0 yes 22 no N.A. _
N.A. = Not Available.
absorbance at 405 nm read on a VERSATunable Max microplate reader (Molecular Devices). Negative control wells were obtained by incubation of immobilized peptides with secondary Ab alone, and their mean values subtracted from all samples. Positive control sera were also included in all experiments. Results are expressed as means of triplicate 405 nm optical density (OD) values.
analysis for research purposes. Among serum samples referred for AQP4/MOG-IgG testing to the Laboratory of Neuropathology, University Hospital of Verona, Italy, we identified those of patients eventually diagnosed with MOG-IgG associated disorders between March 2014 and May 2017. Detailed clinical characteristics of these patients were previously reported (Mariotto et al., 2017). Identified samples were sex-matched with serum samples from: 1) a cohort of newly diagnosed relapsing-remitting MS patients at the Neurology Unit of the University Hospital of Sassari with at least two cerebrospinal fluid restricted oligoclonal bands and no prior treatment with disease modifying agents; and 2) control donors without known inflammatory disease of the CNS from the Blood Transfusion Centre of Sassari. We also included an unmatched cohort of 22 AQP4-IgG positive NMOSD patients from the Neuropathology Laboratory of Verona for which stored serum samples were available (these samples were included in our previous study and re-analyzed for the present study) (Arru et al., 2017). Table 1 shows the available basic demographical and clinical data of investigated groups. The diagnosis of MS, NMOSD and MOG-IgG associated disorders was based on the established criteria (Lopez-Chiriboga et al., 2018; Polman et al., 2011; Wingerchuk et al., 2015).
2.4. Statistical analysis The analysis was carried out using Graphpad Prism 6.0 software (San Diego, CA, USA). Continuous variables were presented as median (range), and categorical variables as numbers and percentages. Non parametric Kruskal-Wallis multiple comparison test was performed as post hoc for multiple comparisons, while the Mann-Whitney U test for unpaired data was used to compare two groups. A p-value < .008 was considered statistically significant after Bonferroni adjustment for multiple comparison between 4 groups. The diagnostic value of the indirect ELISA assays was evaluated by the receiver operating characteristic (ROC) curve. The optimal cut off values was chosen according to ROC analysis, setting specificity and sensitivity at 90% for all serum samples measured. 3. Results
2.2. Autoantibody testing A total of 102 samples were analyzed: 22 obtained from patients with MOG-IgG-associated disorders (10 females [45%], median age of 42 [range, 20–76]) years; 22 from MS patients (10 females [45%], median age of 44 [range, 20–63] years); 36 from unaffected controls (17 females [47%], median age of 49 [range, 25–81] years); and 22 from cases with AQP4-IgG positive NMOSD (20 females [91%] with a median age of 50 [range, 31–81] years). Patients in the AQP4-IgG positive NMOSD group were older compared to the other groups (p = .03). Sera of patients with MS and MOG-IgG associated disorders showed similarly increased antibody reactivity against all the four HERV-W peptides (Fig. 1). This reactivity was significantly higher for HERV-W env-su 93–108 and HERV-W env-su 129–143 compared to that observed in the other groups (Fig. 1A–B). In particular, antibodies against HERV-W env-su 93–108 were found in the sera of 20 out of 22 (91%) patients with MOG-IgG -associated disorders and in 19 out of 22 (86%) sera of MS patients, while only 7 out of 22 (32%) AQP4-IgG positive patients and 19 out of 36 (53%) controls were positive (AUC = 0.87, controls vs MOG-IgG -associated disorders vs MS vs AQP4-IgG positive NMOSD: p < .0001; MOG antibody-associated disorders vs AQP4-IgG positive NMOSD: p = .0001; MOG antibody-associated disease vs MS: p = ns) (Fig. 1A). Regarding the HERV-W env-su 129–143, specific antibodies were found in 18 out of 22 (82%) MOG antibody-associated disorders patients, 17 out of 22 (77%) MS patients, 11 out of 36 (31%) controls and 1 out of 22 (5%) AQP4-IgG positive patients (AUC = 0.97, controls vs MOG-IgG-associated disorders vs MS vs AQP4-IgG positive NMOSD, p < .0001; MOG-IgG-associated disorders vs AQP4-IgG positive NMOSD, p < .0001; MOG-IgG-associated disorders vs MS: p = ns) (Fig. 1B) Concerning HERV-W env-su 161–180, no statistical differences were observed regarding positivity against this peptide that was
Serum AQP4-IgG were tested at the Laboratory of Neuropathology, University of Verona, Italy using a commercially available cell-based assay (Anti-Aquaporin-4 IIFT, Euroimmun, Lu ̈beck, Germany). MOGIgG were analyzed using recombinant live cell-based immunofluorescence assay with HEK293A cells transfected with full-length MOG, as previously described (Mariotto et al., 2017). None of the samples of the pathological cohorts studied tested positive for both antibodies (i.e., AQP4-IgG and MOG-IgG). 2.3. Antibody reactivity against HERV-W immunogenic peptides Antibody response against antigenic peptides derived from HERV-W envelope proteins including HERV-W env93–108 (NPSCPGGLGVTVCWTY), HERV-W env129–143 (VKEVISQLTRVHGTS), HERV-W env161–180 (HTRLVSLFNTTLTGLHEVSA), and HERV-W env248–262 (NSQCIRWVTPPTQIV) belonging to the surface region of the protein was carried out using Indirect Enzyme-Linked Immunosorbent Assays (ELISA) method. Ninety-six-well Nunc immunoplates were coated overnight at 4 °C with 10 μg/ml of peptides diluted in 0.05 M carbonate–bicarbonate buffer, pH 9.5 (Sigma). Plates were then blocked for 1 h at room temperature with 5% non-fat dried milk (Sigma) and washed twice with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-T). Sera samples were subsequently added at 1:100 dilution in PBS-T for 2 h at room temperature. After 5 washes in PBS-T, 100 μl of alkaline phosphatase-conjugated goat anti-human immunoglobulin G polyclonal Ab (1:1000; Sigma) was added for 1 h at room temperature. Plates were washed again 5 times in PBS-T and para nitrophenylphosphate (Sigma) added as substrate for alkaline phosphatase. Plates were incubated at 37 °C in the dark for 3–6 min and the 2
Journal of Neuroimmunology 338 (2020) 577110
G. Arru, et al.
Fig. 1. Detected values of antibodies against peptides derived from HERV-W envelope protein in serum. Twenty-two patients with MOG-IgG-associated disorders patients, 22 patients with multiple sclerosis (MS) patients, 22 patients with AQP4-IgG positive NMOSD, and 36 unaffected controls were screened for immunoglobulin G antibodies against HERV-Wenv93–108 (A), HERV-Wenv129–143 (B), HERV-Wenv161–180 (C) and HERV-Wenv248–262 (D) peptides. Scatter plots present median values with interquartile range. Cut-off values for positivity, indicated by dashed lines, were calculated by ROC analysis comparing values for disease group vs unaffected controls and considering a sensitivity and specificity of 90%. The p values for the multiple comparisons are also displayed.
different embryonic (perinatal) thymus exposure to the endogenous HERV-W envelope, which would be decreased or defective in such individuals. Moreover, it should be relevant to note that the antibody response we documented in the study does not have the magnitude of antibody response to infectious agents, when the adaptive immune system is involved in an antibody response to exogenous antigens. However, considered the accuracy of the antibody variations we observed, which is statistically significant and restricted to few peptides only, this should be explicated in the light of basal (auto-) immune response to self-antigens, which is naturally existing in healthy individuals and that, due to the endogenous nature of HERV-W envelope protein, will not generate a classical anti-infectious antibody response. Our observations provide important insight to better understand the known association between HERV-W and MS (Perron et al., 1997; Rolland et al., 2006). Although the pathogenic role of MOG-IgG has yet to be demonstrated, oligodendrocytes most likely represent the main cellular target in both MS and MOG-IgG-associated disorders and the antibody response against HERV-W that we observed might represent a consequence of the primary oligodendrocyte insult that occurs in both diseases. In particular, antibodies directed against HERV-W surface antigens would be produced via molecular mimicry with oligodendrocytes antigens, as suggested by recent findings showing the existence of cross-reactivity between MOG and some antigenic HERV-W envelope peptides (De Luca et al., 2019). A similar cell-specific rather than disease-specific association of anti-HERV-W antibodies would also be consistent with the scarce reactivity for HERV-W antigens that we have observed in patients with AQP4-IgG positive NMOSD, where the astrocytes are the primary disease target (Polman et al., 2011; Mariotto
recognized by 12 out of 36 (33%) controls, by 10 out of 22 (46%) patients with MOG-IgG-associated disorders, 13 out of 22 (59%) MS patients, and 13 out of 22 (59%) AQP4-IgG positive patients (AUC = 0.51, p = ns) (Fig. 1C). Finally, reactivity against HERV-W env-su 248–262 peptide was found in 14 out of 22 (64%) patients with MOG-IgG-associated disorders, in 17 out of 22 (82%) MS patients, and 12 out of 22 (55%) AQP4-IgG positive patients (AUC = 0.58, controls vs MOG-IgGassociated disorders vs MS vs AQP4-IgG positive NMOSD, p < .0001; MOG-IgG-associated disorders vs MS, p = ns). On the contrary, unaffected controls showed minimal to absent reactivity against this specific peptide with only 2 out of 36 (5%) samples being positive (Fig. 1D). Receiver operating characteristic (ROC) curves for the four HERV-W peptides studied are shown in Fig. 2. 4. Discussion Our results show similarly increased reactivity to particular peptides from HERV-W envelope in patients with MOG-IgG-associated disorders and relapsing-remitting MS, thus expanding the diagnostic role of HERV-W antibodies to the spectrum of demyelinating disorders associated with MOG-IgG. In addition, we confirm the lack of association between these antibodies and AQP4-IgG positive NMOSD (Arru et al., 2017). It is interesting to note that AQP4-IgG positive patients showed lower serum antibody levels when compared to healthy controls. Why this particular response occurs is unclear. Based on existing studies (Avrameas and Selmi, 2014; Palma et al., 2018), it may involve different levels of natural autoantibodies that may directly be linked to a 3
Journal of Neuroimmunology 338 (2020) 577110
G. Arru, et al.
Fig. 2. ROC curves relative to immunoglobulin G antibodies against HERV-Wenv93–108 (A), HERV-Wenv129–143 (B), HERV-Wenv161–180 (C) and HERV-Wenv248–262 (D) peptides in serum of studied cohorts. ROC analysis was calculated through Graphpad Prism 6.0 software.
Declaration of Competing Interest
et al., 2017). Of note, although the primary target in NMOSD are astrocytes, over time a severe oligodendrocyte loss may also occur (Wingerchuk et al., 2015). So, this could also contribute to molecular mimicry. In conclusion, HERV-W might represent a common initial insult resulting in different inflammatory and clinical manifestations (MS vs MOG-IgG associated disorders), according to different biological and environmental substrates (Rolland et al., 2006; Jakel et al., 2019). Our findings are limited by the retrospective design of the study and the small sample size, which need replication of the results. The MS patients that we included were not routinely screened for MOG-IgG and AQP4-IgG as part of their diagnostic work-up but they all had typical clinical, cerebrospinal fluid (i.e. presence of oligoclonal bands), and MRI characteristics at onset, so that indiscriminate antibody testing in these patients was not recommended (Jarius et al., 2018). Lastly, the patients in the AQP4-IgG positive group were different from patients in the other cohorts (i.e., older and almost exclusively female), in accordance with the general demographic characteristics of this condition. However, we previously showed that antibody response against HERV-W is significantly different between AQP4-IgG positive cases and MS patients, even after accounting for age and sex (Arru et al., 2017).
The authors declare no competing interest. References Arru, G., Leoni, S., Pugliatti, M., Mei, A., Serra, C., Delogu, L.G., et al., 2014. Natalizumab inhibits the expression of human endogenous retroviruses of the W family in multiple sclerosis patients: a longitudinal cohort study. Mult. Scler. 20 (2), 174–182. https:// doi.org/10.1177/1352458513494957. Arru, G., Sechi, E., Mariotto, S., Farinazzo, A., Mancinelli, C., Alberti, D., et al., 2017. Antibody response against HERV-W env surface peptides differentiates multiple sclerosis and neuromyelitis optica spectrum disorder. Mult. Scler. J. Exp. Transl. Clin. 3 (4). https://doi.org/10.1177/2055217317742425. 2055217317742425. Avrameas, S., Selmi, C., 2014. Natural autoantibodies in the physiology and pathophysiology of the immune system. J. Autoimmun. 41, 46–49. https://doi.org/10.1016/j. jaut.2013.01.006. De Luca, V., Higa, A.M., Romano, C.M., Mambrini, G.P., Peroni, L.A., Trivinho-Strixino, F., et al., 2019. Cross-reactivity between myelin oligodendrocyte glycoprotein and humanendogenous retrovirus W protein: nanotechnological evidence for the potential trigger of multiple sclerosis. Micron. 120, 66–73. https://doi.org/10.1016/j. micron.2019.02.005. Jakel, S., Agirre, E., Falcao, A.M., van Bruggen, D., Lee, K.W., Knuesel, I., et al., 2019. Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature. 566 (7745), 543–547. https://doi.org/10.1038/s41586-019-0903-2. Jarius, S., Paul, F., Aktas, O., Asgari, N., Dale, R.C., de Seze, J., et al., 2018. MOG encephalomyelitis : international recommendations on diagnosis and antibody testing. J. Neuroinflammation 15 (1), 134. Kremer, D., Schichel, T., Forster, M., Tzekova, N., Bernard, C., van der Valk, P., et al., 2013. Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation. Ann. Neurol. 74 (5), 721–732. https://doi.org/ 10.1002/ana.23970. Lopez-Chiriboga, A.S., Maied, M., Fryer, J., Dubey, D., McKeon, A., Flanagan, E.P., et al., 2018. Association of MOG-IgG serostatus with relapse after acute disseminated encephalomyelitis and proposed diagnostic criteria for MOG-IgG-associated disorders. JAMA Neurol. 75 (11), 1355–1363. https://doi.org/10.1001/jamaneurol.2018.1814. Mariotto, S., Ferrari, S., Monaco, S., Benedetti, M.D., Schanda, K., Alberti, D., et al., 2017. Clinical spectrum and IgG subclass analysis of anti-myelin oligodendrocyte glycoprotein antibody-associated syndromes: a multicenter study. J. Neurol. 264 (12), 2420–2430.
5. Conclusions Our findings do not support an exclusive association between HERV-W and MS. The different antibody response against HERV-W in patients with AQP4-IgG positive NMOSD compared to patients with MS and MOG-IgG associated disorders might be explained by the different disease-targets in these disorders (i.e., astrocytes vs oligodendrocytes). Funding The authors declare that no funding has been received. 4
Journal of Neuroimmunology 338 (2020) 577110
G. Arru, et al. Palma, J., Tokarz-Deptuła, B., Deptuła, J., Deptuła, W., 2018. Natural antibodies – facts known and unknown. Cent. Eur. J. Immunol. 43 (4), 466–475. https://doi.org/10. 5114/ceji.2018.81354. Perron, H., Garson, J.A., Bedin, F., Beseme, F., Paranhos-Baccala, G., Komurian-Pradel, F., et al., 1997. Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis. Proc. Natl. Acad. Sci. U. S. A. 94 (14), 7583–7588. Polman, C.H., Reingold, S.C., Banwell, B., Clanet, M., Cohen, J.A., Filippi, M., et al., 2011. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann. Neurol. 69 (2), 292–302. Reindl, M., Di Pauli, F., Rostásy, K., Berger, T., 2013. The spectrum of MOG autoantibodyassociated demyelinating diseases. Nat. Rev. Neurol. 9 (8), 455–461. https://doi.org/
10.1038/nrneurol.2013.118. Rolland, A., Jouvin-Marche, E., Viret, C., Faure, M., Perron, H., Marche, P.N., 2006. The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses. J. Immunol. 176 (12), 7636–7644. https://doi.org/10.4049/jimmunol.176.12.7636. Sotgiu, S., Mameli, G., Serra, C., Zarbo, I.R., Arru, G., Dolei, A., 2010. Multiple sclerosisassociated retrovirus and progressive disability of multiple sclerosis. Mult. Scler. 16 (10), 1248–1251. https://doi.org/10.1177/1352458510376956. Wingerchuk, D.M., Banwell, B., Bennett, J.L., Cabre, P., Carroll, W., Chitnis, T., et al., 2015. International consensus diagnostic criteria for neuromyelitis optica spectrum disorders. Neurology. 85 (2), 177–189.
5
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Antibody response against HERV-W in patients with MOG-IgG associated disorders, multiple sclerosis and NMOSD
T
Giannina Arrua, Elia Sechia, Sara Mariottob, Ignazio R. Zarboa, Sergio Ferrarib, Alberto Gajofattob, ⁎ Salvatore Monacob, Giovanni A. Deianaa, Marco Boc, Leonardo A. Sechic, Gian Pietro Sechia, a
Section of Neurology, Department of Clinical, Surgery and Experimental Sciences, University of Sassari, Italy Department of Neuroscience, Biomedicine and Movement Sciences, University of Verona, Italy c Department of Biomedical Sciences, University of Sassari, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: MS Myelin oligodendrocyte glycoprotein Neuromyelitis optica spectrum disorder Autoimmunity Human endogenous retroviruses
Increased expression of the retroviruses of HERV-W family has been linked to multiple sclerosis (MS) pathophysiology; nothing is known at the moment about MOG-IgG associated disorders. We compared antibody response against HERV-W peptides among patients with MOG-IgG associated disorders, multiple sclerosis (MS) and aquaporin-4 (AQP4)-IgG positive neuromyelitis optica spectrum disorder (NMOSD). A total of 102 serum samples were retrospectively analyzed. Antibody reactivity against HERV-W env peptides was similar in MOG-IgG associated disorders and MS, but different from AQP4-IgG positive NMOSD. Our findings expand the diagnostic role of HERV-W antibodies to the spectrum of demyelinating disorders associated with MOG-IgG.
1. Introduction Human endogenous retroviruses (HERVs) account for approximately 8% of human genome, being the result of ancestral infections with exogenous retroviruses and subsequent integration into germline DNA (Perron et al., 1997). Increased expression of the retroviruses of HERV-W family has been linked to multiple sclerosis (MS) pathophysiology (Perron et al., 1997) and has been proposed as a prognostic factor (Sotgiu et al., 2010) and a potential predictor of treatment response (Arru et al., 2014). Endogenous retroviruses likely exert their pathogenic effect by acting as activators/modulators of innate immunity, mainly through interaction with Toll-Like Receptors with a superantigen-like mechanism (Rolland et al., 2006). Through this mechanism HERV-W envelope proteins contribute to increased nitric oxide production in the brain, therefore influencing microglial migratory ability and reducing oligodendroglial differentiation capacity (Kremer et al., 2013). Myelin oligodendrocyte glycoprotein (MOG) is a central nervous system (CNS)-restricted protein expressed on the outermost surface of the myelin sheath and the plasma membrane of oligodendrocyte (Reindl et al., 2013). Over the last decade, a spectrum of CNS demyelinating diseases associated with autoantibodies directed against this protein (MOG-IgG) has been characterized (Lopez-Chiriboga et al.,
⁎
2018). MOG-IgG associated disorders show distinctive clinical and radiological features compared to MS, but frequently overlap those of aquaporin-4 (AQP4)-IgG positive neuromyelitis optica spectrum disorder (NMOSD) (Polman et al., 2011; Wingerchuk et al., 2015; Mariotto et al., 2017). We recently reported an increased humoral immune response towards some antigenic peptides of the HERV-W envelope proteins in MS patients but not in NMOSD patients (either AQP4-IgG positive or double-negative for AQP4-IgG and MOG-IgG), suggesting a possible role for HERV-W antibodies as discriminating biomarker between MS and NMOSD (Arru et al., 2017). Immunological response to HERV-W in patients with MOG-IgG associated disorders has yet to be investigated. We studied the humoral immune response against HERV-W peptides in patients with MOG-IgG associated disorders and made a comparison with that observed in MS and NMOSD. 2. Material and methods 2.1. Patients The study protocol was approved by the local ethics committee (Prot. 2457/2017, Azienda Sanitaria Locale 1, Sassari, Italy). All the included subjects provided written consent to sample storing and
Corresponding author at: Neurology Unit, AOU Sassari, Department of Clinical, Surgical and Experimental Sciences, Viale “S. Pietro” 10, 07100 Sassari, Italy. E-mail address: [email protected] (G.P. Sechi).
https://doi.org/10.1016/j.jneuroim.2019.577110 Received 17 July 2019; Received in revised form 18 October 2019; Accepted 4 November 2019 0165-5728/ © 2019 Elsevier B.V. All rights reserved.
Journal of Neuroimmunology 338 (2020) 577110
G. Arru, et al.
Table 1 Demographical and clinical data of all investigated groups. Groups
Age, median ± SD
Sex, females / males, n
Disease duration / months 0–12 (%) 13–60 (%) > 60 (%)
Treatment
MOG-IgG+(# 22)
42 ± 15,98
10/12
13 (59) 4 (18) 5 (23)
MS (# 22)
44 ± 11,59
10/12
22 (100)
AQP4-IgG+(# 22) HCs (#36)
50 ± 13,67 49 ± 14,20
20/2 17/19
N.A. _
17 yes 5 no 0 yes 22 no N.A. _
N.A. = Not Available.
absorbance at 405 nm read on a VERSATunable Max microplate reader (Molecular Devices). Negative control wells were obtained by incubation of immobilized peptides with secondary Ab alone, and their mean values subtracted from all samples. Positive control sera were also included in all experiments. Results are expressed as means of triplicate 405 nm optical density (OD) values.
analysis for research purposes. Among serum samples referred for AQP4/MOG-IgG testing to the Laboratory of Neuropathology, University Hospital of Verona, Italy, we identified those of patients eventually diagnosed with MOG-IgG associated disorders between March 2014 and May 2017. Detailed clinical characteristics of these patients were previously reported (Mariotto et al., 2017). Identified samples were sex-matched with serum samples from: 1) a cohort of newly diagnosed relapsing-remitting MS patients at the Neurology Unit of the University Hospital of Sassari with at least two cerebrospinal fluid restricted oligoclonal bands and no prior treatment with disease modifying agents; and 2) control donors without known inflammatory disease of the CNS from the Blood Transfusion Centre of Sassari. We also included an unmatched cohort of 22 AQP4-IgG positive NMOSD patients from the Neuropathology Laboratory of Verona for which stored serum samples were available (these samples were included in our previous study and re-analyzed for the present study) (Arru et al., 2017). Table 1 shows the available basic demographical and clinical data of investigated groups. The diagnosis of MS, NMOSD and MOG-IgG associated disorders was based on the established criteria (Lopez-Chiriboga et al., 2018; Polman et al., 2011; Wingerchuk et al., 2015).
2.4. Statistical analysis The analysis was carried out using Graphpad Prism 6.0 software (San Diego, CA, USA). Continuous variables were presented as median (range), and categorical variables as numbers and percentages. Non parametric Kruskal-Wallis multiple comparison test was performed as post hoc for multiple comparisons, while the Mann-Whitney U test for unpaired data was used to compare two groups. A p-value < .008 was considered statistically significant after Bonferroni adjustment for multiple comparison between 4 groups. The diagnostic value of the indirect ELISA assays was evaluated by the receiver operating characteristic (ROC) curve. The optimal cut off values was chosen according to ROC analysis, setting specificity and sensitivity at 90% for all serum samples measured. 3. Results
2.2. Autoantibody testing A total of 102 samples were analyzed: 22 obtained from patients with MOG-IgG-associated disorders (10 females [45%], median age of 42 [range, 20–76]) years; 22 from MS patients (10 females [45%], median age of 44 [range, 20–63] years); 36 from unaffected controls (17 females [47%], median age of 49 [range, 25–81] years); and 22 from cases with AQP4-IgG positive NMOSD (20 females [91%] with a median age of 50 [range, 31–81] years). Patients in the AQP4-IgG positive NMOSD group were older compared to the other groups (p = .03). Sera of patients with MS and MOG-IgG associated disorders showed similarly increased antibody reactivity against all the four HERV-W peptides (Fig. 1). This reactivity was significantly higher for HERV-W env-su 93–108 and HERV-W env-su 129–143 compared to that observed in the other groups (Fig. 1A–B). In particular, antibodies against HERV-W env-su 93–108 were found in the sera of 20 out of 22 (91%) patients with MOG-IgG -associated disorders and in 19 out of 22 (86%) sera of MS patients, while only 7 out of 22 (32%) AQP4-IgG positive patients and 19 out of 36 (53%) controls were positive (AUC = 0.87, controls vs MOG-IgG -associated disorders vs MS vs AQP4-IgG positive NMOSD: p < .0001; MOG antibody-associated disorders vs AQP4-IgG positive NMOSD: p = .0001; MOG antibody-associated disease vs MS: p = ns) (Fig. 1A). Regarding the HERV-W env-su 129–143, specific antibodies were found in 18 out of 22 (82%) MOG antibody-associated disorders patients, 17 out of 22 (77%) MS patients, 11 out of 36 (31%) controls and 1 out of 22 (5%) AQP4-IgG positive patients (AUC = 0.97, controls vs MOG-IgG-associated disorders vs MS vs AQP4-IgG positive NMOSD, p < .0001; MOG-IgG-associated disorders vs AQP4-IgG positive NMOSD, p < .0001; MOG-IgG-associated disorders vs MS: p = ns) (Fig. 1B) Concerning HERV-W env-su 161–180, no statistical differences were observed regarding positivity against this peptide that was
Serum AQP4-IgG were tested at the Laboratory of Neuropathology, University of Verona, Italy using a commercially available cell-based assay (Anti-Aquaporin-4 IIFT, Euroimmun, Lu ̈beck, Germany). MOGIgG were analyzed using recombinant live cell-based immunofluorescence assay with HEK293A cells transfected with full-length MOG, as previously described (Mariotto et al., 2017). None of the samples of the pathological cohorts studied tested positive for both antibodies (i.e., AQP4-IgG and MOG-IgG). 2.3. Antibody reactivity against HERV-W immunogenic peptides Antibody response against antigenic peptides derived from HERV-W envelope proteins including HERV-W env93–108 (NPSCPGGLGVTVCWTY), HERV-W env129–143 (VKEVISQLTRVHGTS), HERV-W env161–180 (HTRLVSLFNTTLTGLHEVSA), and HERV-W env248–262 (NSQCIRWVTPPTQIV) belonging to the surface region of the protein was carried out using Indirect Enzyme-Linked Immunosorbent Assays (ELISA) method. Ninety-six-well Nunc immunoplates were coated overnight at 4 °C with 10 μg/ml of peptides diluted in 0.05 M carbonate–bicarbonate buffer, pH 9.5 (Sigma). Plates were then blocked for 1 h at room temperature with 5% non-fat dried milk (Sigma) and washed twice with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-T). Sera samples were subsequently added at 1:100 dilution in PBS-T for 2 h at room temperature. After 5 washes in PBS-T, 100 μl of alkaline phosphatase-conjugated goat anti-human immunoglobulin G polyclonal Ab (1:1000; Sigma) was added for 1 h at room temperature. Plates were washed again 5 times in PBS-T and para nitrophenylphosphate (Sigma) added as substrate for alkaline phosphatase. Plates were incubated at 37 °C in the dark for 3–6 min and the 2
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Fig. 1. Detected values of antibodies against peptides derived from HERV-W envelope protein in serum. Twenty-two patients with MOG-IgG-associated disorders patients, 22 patients with multiple sclerosis (MS) patients, 22 patients with AQP4-IgG positive NMOSD, and 36 unaffected controls were screened for immunoglobulin G antibodies against HERV-Wenv93–108 (A), HERV-Wenv129–143 (B), HERV-Wenv161–180 (C) and HERV-Wenv248–262 (D) peptides. Scatter plots present median values with interquartile range. Cut-off values for positivity, indicated by dashed lines, were calculated by ROC analysis comparing values for disease group vs unaffected controls and considering a sensitivity and specificity of 90%. The p values for the multiple comparisons are also displayed.
different embryonic (perinatal) thymus exposure to the endogenous HERV-W envelope, which would be decreased or defective in such individuals. Moreover, it should be relevant to note that the antibody response we documented in the study does not have the magnitude of antibody response to infectious agents, when the adaptive immune system is involved in an antibody response to exogenous antigens. However, considered the accuracy of the antibody variations we observed, which is statistically significant and restricted to few peptides only, this should be explicated in the light of basal (auto-) immune response to self-antigens, which is naturally existing in healthy individuals and that, due to the endogenous nature of HERV-W envelope protein, will not generate a classical anti-infectious antibody response. Our observations provide important insight to better understand the known association between HERV-W and MS (Perron et al., 1997; Rolland et al., 2006). Although the pathogenic role of MOG-IgG has yet to be demonstrated, oligodendrocytes most likely represent the main cellular target in both MS and MOG-IgG-associated disorders and the antibody response against HERV-W that we observed might represent a consequence of the primary oligodendrocyte insult that occurs in both diseases. In particular, antibodies directed against HERV-W surface antigens would be produced via molecular mimicry with oligodendrocytes antigens, as suggested by recent findings showing the existence of cross-reactivity between MOG and some antigenic HERV-W envelope peptides (De Luca et al., 2019). A similar cell-specific rather than disease-specific association of anti-HERV-W antibodies would also be consistent with the scarce reactivity for HERV-W antigens that we have observed in patients with AQP4-IgG positive NMOSD, where the astrocytes are the primary disease target (Polman et al., 2011; Mariotto
recognized by 12 out of 36 (33%) controls, by 10 out of 22 (46%) patients with MOG-IgG-associated disorders, 13 out of 22 (59%) MS patients, and 13 out of 22 (59%) AQP4-IgG positive patients (AUC = 0.51, p = ns) (Fig. 1C). Finally, reactivity against HERV-W env-su 248–262 peptide was found in 14 out of 22 (64%) patients with MOG-IgG-associated disorders, in 17 out of 22 (82%) MS patients, and 12 out of 22 (55%) AQP4-IgG positive patients (AUC = 0.58, controls vs MOG-IgGassociated disorders vs MS vs AQP4-IgG positive NMOSD, p < .0001; MOG-IgG-associated disorders vs MS, p = ns). On the contrary, unaffected controls showed minimal to absent reactivity against this specific peptide with only 2 out of 36 (5%) samples being positive (Fig. 1D). Receiver operating characteristic (ROC) curves for the four HERV-W peptides studied are shown in Fig. 2. 4. Discussion Our results show similarly increased reactivity to particular peptides from HERV-W envelope in patients with MOG-IgG-associated disorders and relapsing-remitting MS, thus expanding the diagnostic role of HERV-W antibodies to the spectrum of demyelinating disorders associated with MOG-IgG. In addition, we confirm the lack of association between these antibodies and AQP4-IgG positive NMOSD (Arru et al., 2017). It is interesting to note that AQP4-IgG positive patients showed lower serum antibody levels when compared to healthy controls. Why this particular response occurs is unclear. Based on existing studies (Avrameas and Selmi, 2014; Palma et al., 2018), it may involve different levels of natural autoantibodies that may directly be linked to a 3
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Fig. 2. ROC curves relative to immunoglobulin G antibodies against HERV-Wenv93–108 (A), HERV-Wenv129–143 (B), HERV-Wenv161–180 (C) and HERV-Wenv248–262 (D) peptides in serum of studied cohorts. ROC analysis was calculated through Graphpad Prism 6.0 software.
Declaration of Competing Interest
et al., 2017). Of note, although the primary target in NMOSD are astrocytes, over time a severe oligodendrocyte loss may also occur (Wingerchuk et al., 2015). So, this could also contribute to molecular mimicry. In conclusion, HERV-W might represent a common initial insult resulting in different inflammatory and clinical manifestations (MS vs MOG-IgG associated disorders), according to different biological and environmental substrates (Rolland et al., 2006; Jakel et al., 2019). Our findings are limited by the retrospective design of the study and the small sample size, which need replication of the results. The MS patients that we included were not routinely screened for MOG-IgG and AQP4-IgG as part of their diagnostic work-up but they all had typical clinical, cerebrospinal fluid (i.e. presence of oligoclonal bands), and MRI characteristics at onset, so that indiscriminate antibody testing in these patients was not recommended (Jarius et al., 2018). Lastly, the patients in the AQP4-IgG positive group were different from patients in the other cohorts (i.e., older and almost exclusively female), in accordance with the general demographic characteristics of this condition. However, we previously showed that antibody response against HERV-W is significantly different between AQP4-IgG positive cases and MS patients, even after accounting for age and sex (Arru et al., 2017).
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5. Conclusions Our findings do not support an exclusive association between HERV-W and MS. The different antibody response against HERV-W in patients with AQP4-IgG positive NMOSD compared to patients with MS and MOG-IgG associated disorders might be explained by the different disease-targets in these disorders (i.e., astrocytes vs oligodendrocytes). Funding The authors declare that no funding has been received. 4
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