Identification of the flotillin-1/2 heterocomplex as a target of autoantibodies in bona fide multiple sclerosis

Abstract

BackgroundAutoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS).MethodsThe target antigen was identified by histo-immunoprecipitation using the patients’ sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells.ResultsSerum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors.ConclusionsAutoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1–2% of patients with bona fide MS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0900-z) contains supplementary material, which is available to authorized users.

A Spectrum of Neural Autoantigens, Newly Identified by Histo-Immunoprecipitation, Mass Spectrometry, and Recombinant Cell-Based Indirect Immunofluorescence

“…For some antigens like ATP1A3, ITPR1, or ROCK2 an expression in tumor samples of the respective index patient was observed, pointing to a paraneoplastic autoimmune background ( 20 , 31 , 36 ). Antigens like Flotillin1/2, GRIPAP1, or CLIP1 are also expressed in non-neural tissues without a tumor association ( 21 , 24 , 26 ). However, other autoantibodies, like AQP4 or DPPX which are established markers for neurological autoimmune diseases ( 37 , 38 ) show also expression in non-neuronal tissues.…”

“…For autoantibodies against ATP1A3, ITPR1, NCDN, and ROCK2 an association to autoimmune cerebellar syndromes has already been demonstrated by the authors ( 20 , 31 , 33 , 36 ). Moreover, anti-Flotillin1/2 autoantibodies were found to be present in multiple sclerosis patients ( 24 ). Thus, the antigen identification strategy that we present offers a potent instrument for identifying unknown autoantigens and contributes to better diagnosis of autoimmune diseases.…”

Myelinoclastic diffuse sclerosis (Schilder’s disease) is immunologically distinct from multiple sclerosis: results from retrospective analysis of 92 lumbar punctures

“…AQP4-IgG-positive NMO has been convincingly shown to be distinct from MS with regard to pathogenesis, prognosis and optimum treatment and—after a short period of disbelief and debates between “lumpers and splitters” [28]—is now considered a disease entity in its own right by virtually all experts in the field.MOG-IgG-positive encephalomyelitis (EM), which shares substantial clinical overlap with both MS and NMOSD, has also recently been shown to be an immunologically distinct entity in its own right [29]. The recent identification of novel autoantibodies against, for example, glial fibrillary acidic protein (GFAP) [30] or the flotillin-1/2 heterocomplex [31] in patients with CNS demyelination and reports on demyelination in patients with anti- N -methyl- d -aspartate receptor (NMDAR) encephalitis [32] have the potential to further challenge the idea of MS as an immunologically homogeneous disease in the future.Finally, recent studies [33, 34] suggesting substantial differences between MS and BCS in regard to both radiological and histopathological presentation and immunopathology have meanwhile also cast doubt on the concept of BCS being a “variant of MS”.…”

Flotillin‐1/2 autoimmunity in a patient with marginal zone lymphoma

“…The flotillin‐1/2 heterocomplex seems to be a potential autoantigen in a subset of approximately 1–2% of multiple sclerosis patients . Rarely, humoral central nervous system (CNS) autoimmunity against neural surface‐associated proteins has been linked to underlying latent hematological malignancies .…”

“…Anti‐flotillin‐1/2 antibodies, as identified by indirect immunofluorescence assay, were recently found in a subgroup of patients with the diagnosis of multiple sclerosis . Flotillin‐1/reggie‐2 and flotillin‐2/reggie‐1 are evolutionary, well‐conserved and ubiquitously expressed proteins, thus showing the highest abundance in striated muscle, brain, adipose and lung tissue.…”