Abstract
Background:
Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein not previously described in the human central nervous system (CNS).
Objectives:
We determined MFAP4 CNS expression and measured cerebrospinal fluid (CSF) and serum levels.
Methods:
Tissue was sampled at autopsy from patients with acute multiple sclerosis (MS) (n = 3), progressive MS (n = 3), neuromyelitis optica spectrum disorder (NMOSD) (n = 2), and controls (n = 9), including 6 healthy controls (HC). MFAP4 levels were measured in 152 patients: 49 MS, 62 NMOSD, 22 myelin oligodendrocyte glycoprotein-associated disease (MOGAD), and 19 isolated optic neuritis (ION).
Results:
MFAP4 localized to meninges and vascular/perivascular spaces, intense in the optic nerve. At sites of active inflammation, MFAP4 reactivity was reduced in NMOSD and acute MS and less in progressive MS. CSF MFAP4 levels were reduced during relapse and at the onset of diseases (mean U/mL: MS 14.3, MOGAD 9.7, and ION 14.6 relative to HC 17.9. (p = 0.013, p = 0.000, and p = 0.019, respectively). Patients with acute ON (n = 68) had reduced CSF MFAP4 (mean U/mL: 14.5, p = 0.006). CSF MFAP4 levels correlated negatively with relapse severity (rho = −0.41, p = 0.017).
Conclusion:
MFAP4 immunoreactivity was reduced at sites of active inflammation. CSF levels of MFAP4 were reduced following relapse and may reflect disease activity.
Keywords: Inflammatory demyelinating diseases, central nervous system, relapse, microfibrillar-associated protein 4
Introduction
Inflammation during disease activity in inflammatory demyelinating diseases (IDDs) of the central nervous system (CNS) frequently associates with disruption of the blood–brain barrier (BBB) and clinical exacerbation.1 The three best-defined IDDs are multiple sclerosis (MS), aquaporin-4-IgG positive neuromyelitis optica spectrum disorder (NMOSD), and myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD). Relapse frequency and severity, together with the requirement for early institution of acute relapse immunotherapies,2,3 are critical to prevent or minimize disability and long-term outcome in IDDs.
Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein and a member of the fibrinogen-related domain family.4 MFAP4 is expressed in the perivascular ECM of microvessels, for example, in alveolar septa.5,6 MFAP4 binds to arginylglycyl-aspartic acid (RGD)-dependent cellular integrin receptors, including integrin αVβ3 and αVβ5.6,7 The ECM is a dynamic component of the neurovascular unit of BBB and ECM and its receptors and plays an important regulatory role in maintaining homeostasis and BBB integrity.4,8,9 Soluble MFAP4 is recognized as a marker of the matrix remodeling processes in peripheral vascular diseases.4,5,10 In a pilot study, we provided preliminary data that MFAP4 levels in cerebrospinal fluid (CSF) are reduced in patients with acute optic neuritis (ON) in population-based samples, and that MFAP4 is expressed in the optic nerve.11 Therefore, in two parallel and independent studies, we aimed to (1) determine the localization of MFAP4 in the human CNS (pathology part); and (2) measure CSF and serum MFAP4 levels in patients with IDDs (clinical part).
Materials and methods
Pathology study
Tissue was sampled at brain and spinal cord autopsy from patients with different stages of MS and NMOSD and from controls (Table 1). The tissues originated from Austria. For validation purposes, tissues sampled from NMOSD (1 optic nerve) from Japan, and HC (1 brain and 3 optic nerves) from Denmark, were investigated.
Table 1.
Pathology study: clinical information of patients with inflammatory demyelinating diseases of CNS and controls.
Characteristics | Control subjects | Acute MS | Progressive MS | NMOSD | Stroke |
---|---|---|---|---|---|
N | 7 | 3 | 3 | 2 | 3 |
Female/male ratio | 4:3 | 2:1 | 1:2 | 2:0 | 1:2 |
Disease duration (months) (mean) | – | 0.77 (1.3) | 222.67 (357) | 45.5 (192) | 5.57 (8.3) |
Course | – | Acute MS | PPMS (1) SPMS (2) |
Relapsing | Acute (1) Subacute (1) Chronic recurrent (1) |
Median age at death, years (range) | 60 (57) | 45 (10) | 46 (26) | 31.5 (23) | 74 (22) |
NMOSD: neuromyelitis optica spectrum disorder; PPMS: Primary progressive MS; SPMS: Secondary progressive course.
A total of seven control subjects without infection, neurological disease, brain, or optic nerve lesions were included (Table 1).
Immunohistochemistry
The monoclonal anti-MFAP4 antibody (HG-HYB 7-14) stained for MFAP4, and the isotype control anti-OVA (HYB099-1) antibody was used as a negative control (State Serum Institute, Copenhagen, Denmark). C57BL/6/N Mfap4-deficient mice were immunized to produce monoclonal antibodies (HG HYB 7-14) against rMFAP4 as previously described.12 The specificity of the anti-MFAP4 antibody has previously been demonstrated.10 In brief, we have produced Mfap4-deficient mice and performed staining of MFAP4. Positive staining of MFAP4 was evident in wildtype and complete absence of staining was seen in Mfap4-deficient mice.13 4-μm-thick sections were cut from formalin-fixed, paraffin-embedded tissue blocks. The sections were mounted on FLEX IHC microscope slides (Dako/Agilent, Glostrup, Denmark), and they were dried at room temperature and baked at 60°C for 60 minutes before immunostaining. Staining was automated at the Discovery Ultra immunostainer (Ventana Medical Systems, Tucson, AZ) using the OmniMap anti-goat-HRP detection system (Ventana) and Discovery Purple chromogen kit (Ventana). Incubation with goat antihuman IgG Fc (Abcam, Cambridge, UK), diluted at 1:2000, was done for 32 minutes at 36°C. Epitope retrieval was performed in Cell Conditioning Solution 1 (CC1) for 32 minutes at 100°C. Nuclear counter-staining was performed using hematoxylin II (Ventana). Finally, the slides were washed, dehydrated, and coverslipped using an automated Dako coverslipper (Dako/Agilent).
In parallel sections, staining was performed as described for AQP4,14,15 glial fibrillary acidic protein (GFAP),14,15 and C9neo (a marker of membrane attack complex),14,15 hematoxylin and eosin. (HE),15 Iba-113,16 and Klüver-Barrera (KLb).13,16
In situ hybridization.
The RNA chromogenic in situ hybridization was performed using a well-established enhanced RNAscope 2.5 procedure.17 Paraffin sections of optic nerve were deparaffinized, rehydrated, and pretreated with H2O2 (blocking endogenous peroxidases), heat-treatment in Tris-EDTA (pH9) and Protease III treatment. Sections were hybridized with 20 probe pairs targeting nucleotide 109–1150 of human MFAP4 (NM_002404.2, 571221, ACD) at 40 C overnight. Negative controls were hybridized with probe diluent (300041, ACD).
Clinical study
Study population.
We performed a retrospective, multicenter study of 161 IDD patients recruited from eight different institutions (Region of Southern Denmark, n = 64; Greater Copenhagen Region, n = 25); Germany (n = 3); South Korea (n = 20); Italy (n = 10); France (n = 6); and USA (Mayo Clinic, n = 13; University of Colorado, n = 20). Inclusion criteria for the present investigation comprised age > 18 years, a diagnosis of MS according to the 201018 or 2017 MS criteria,19 a diagnosis of AQP4-IgG positive NMOSD according to the 2015 IPND consensus criteria,20 or a diagnosis of MOGAD.21
A relapse was defined as a new or worsening acute neurologic symptom lasting ⩾24 hours and not explained by fever, infection, or metabolic condition. Severity of the most recent relapse was rated on a three-step scale (mild, moderate, severe) and was based on change in baseline Expanded Disability Status Scale (EDSS)22 (EDSS < 4, 4–6.0, > 6.0, respectively) by the attending neurologist. Severity of visual loss was categorized as change from baseline, as mild (visual acuity (6/12 to 6/18), moderate (6/18 to 6/60), and severe (worse than 6/60) in line with World Health Organization ICD-11 definitions.23 Patients with a relapse within ⩽60 days prior to the CSF and serum collection were considered to be in the acute stage.
HC CSF and serum originated from 14 subjects without trauma or infection with median age 48.0 (22–63) years. In addition, sera from 50 healthy blood donors with median age 43.9 (18–65) years were analyzed. The data were reviewed in a blinded fashion. None of these persons had any morbidities.
Laboratory methods.
Blood and CSF were collected according to international research standards.24
Quantification of MFAP4 was done with an AlphaLISA assay (Perkin Elmer, MA, USA) at the University of Southern Denmark. Details of the procedure are described elsewhere.5 Samples were run in duplicates, and occasional samples with CV > 10% (12/161 samples 7.45%) were reanalyzed to obtain a valid measurement for every sample. Patients were tested for AQP4-IgG and MOG-IgG using live cell-based assays, as previously described.25,26
Statistics
Statistical analyses were performed using Stata 16 and 17 SE (StataCorp, USA). Multiple linear regression was used to compare MFAP4 across groups. Model validation was performed by graphical inspection of quantile plots of residuals and plots of residuals against fitted values. When model assumptions appeared violated, concentrations were analyzed following logarithmic transformation. Consequently, MFAP4 in serum and CSF were analyzed on a log10-scale. If necessary, the comparison was supplemented by an overall nonparametric Kruskal–Wallis test. Test for trend across ordered groups was performed equidistantly coding the groups and entering the grouping as continuous variable into the regression. Correlations were measured using Spearman’s rank correlation. This was a hypothesis-generating exploratory study so no adjustment for multiple comparisons was made. p-values below 0.05 were considered statistically significant.
Standard protocol approvals, registrations, and patient consents
The Research Ethical Committees for the Region of Southern Denmark approved the study protocol (ref. no. S-20130137 and ref. no. S-20080142) as did the Danish Data Protection Agency (ref. no. 14/26345). Approval by local ethical Institutional Review Boards from all centers were obtained following informed consent from the patients.
Results
Pathology
MFAP4 is expressed within the healthy CNS.
MFAP4 was expressed in the connective tissue spaces of CNS from all controls, including meninges and the vascular/perivascular spaces (Figure 1). MFAP4 immunoreactivity was present in the ECM in the intima and adventitia of larger vessels (Figure 1(a)–(c), (e) and (f)), pial vessels (Figure 1(e) and (f)) and choroid plexus (Figure 1(d)), and to a variable extent in the ECM of meninges and perivascular connective tissue (Figure 1(a)–(f)).
Figure 1.
MFAP4 immunoreactivity in the control brain.
Micrographs show tissue sampled from control brain. (a–f) Sections were stained for MFAP4 using monoclonal antibodies against MFAP4. (a–c, e, and f) MFAP4 was expressed to a variable extent in the connective tissue spaces of meninges and around vessels, (e and f) including pial vessels and (d) choroid plexus, denoted by black arrows. Scale bars = 200 μm (a), 100 μm (c), 50 μm, (b, d, f), and 20 μm (e).
Widespread MFAP4 immunoreactivity throughout the optic nerve.
There was much more connective tissue around the vessels of the optic nerve than in the brain parenchyma, occasionally forming septae between nerve fiber bundles. MFAP4 reactivity predominated in these connective tissue bundles using immunostaining (purple) for MFAP4 (Figure 2(a)). In situ hybridization analysis demonstrated that MFAP4 was expressed in small vessels and the capillaries in healthy optic nerve (Figure 2(b)). Negative controls showed lack of staining (Figure 2(c) and (d)).
Figure 2.
MFAP4 immunoreactivity in a control human optic nerve.
Micrographs show longitudinal sections of the optic nerve. (a) Immunostaining for MFAP4 indicated in purple. Insert in (a) shows the whole tissue cross section of the eye and the red box indicates the magnified area. (b) Punctuate in situ hybridization (indicated in red) of MFAP4 mRNA. (c) Corresponding negative controls, respectively, with isotype antibody control staining or (d) in situ hybridization without probe pair. Widespread MFAP4 immunoreactivity was observed throughout in the optic nerve, including the perivascular areas and in small vessels and capillaries (a and b). Areas of MFAP4 immunoreactivity are marked by black arrows. Lack of MFAP4 detection in negative controls (c and d). Scale bars = 250 μm (a and c); 100 μm (b and d).
Reduction of MFAP4 reactivity at sites of active inflammation.
At sites of inflammation, documented by the presence of inflammatory infiltrates in parallel sections, MFAP4 expression was diminished in NMOSD (Figure 3(a)) in areas that co-localized with loss of AQP4 (Figure 3(b)), GFAP staining (Figure 3(f)), and increased microglia activation with Iba-1 reactivity (Figure 3(e)). MFAP4 positive staining was preserved and co-localized (Figure 3(c)) with AQP4 (Figure 3(d)) and GFAP (top of Figure 3(f)) immunoreactivities in the perilesional area. This observation was confirmed in optic nerve tissue from an NMOSD patient showing a reduction of MFAP4 reactivity co-incident with loss of AQP4 expression and deposition of C9neo and infiltration of neutrophils and macrophages (Supplemental Figure 1).
Figure 3.
MFAP4-negative staining co-localizes with loss of AQP4 in NMOSD.
Micrographs show sections of the mesencephalon from an NMOSD patient. Sections were stained for MFAP4 (a and c), AQP4 (b and d), Iba1 (e), and GFAP (f) (indicated by brown staining).
(b) MFAP4-negative staining co-localizes with loss of AQP4; (c) MFAP4 positive staining area adjacent (left) to a lesion; (d) AQP4 positive area adjacent (right) to a lesion; (e) microglia activation, Iba-1 reactivity in corresponding areas to (a) and (b); and (f) GFAP staining: reduction of GFAP in areas corresponding to areas in (a), (b), and (e). Areas of histopathological changes are marked with arrows. Original magnification 4× (a–f).
In acute MS, demyelination, and cellular infiltration (Figure 4(a)) coincided with diminished MFAP4 immunoreactivity (Figure 4(b) and (c)). Weak MFAP4 reactivity was seen at the sites of preserved myelin (Figure 4(b) (insert), and in the perilesional demyelination area.
Figure 4.
MFAP4 immunoreactivity is diminished in acute MS.
Micrographs show sections of an active parenchymal brain lesion in an MS patient. Sections were stained for hematoxylin and eosin. (HE), Klüver–Barrera (KLb) (HE-KLb) (a) and MFAP4 (b and c, brown staining). (a) The image shows demyelination and massive inflammation, with myelin staining in bottom-left corner. (b) MFAP4 staining reveals a similar pattern to the myelin staining (a) (insert b), with additional staining at vessel in the center. (c) The bottom of the same areas as shown in (a) and (b) is illustrated by MFAP4-stained sections with MFAP4-positive staining area adjacent (below) to a lesion. In (c) the bottom of the same areas as shown in (a) and (b) has MFAP4 positive staining in the area adjacent to a lesion. Original magnification 25× (a–c).
In progressive MS, MFAP4 immunoreactivity was seen in vascular/perivascular spaces and meninges and cortical sulcus (Supplemental Figure 2(a)). In addition, MFAP4 immunoreactivity was diminished in lesions with demyelination in progressive MS to a lesser degree than in acute MS (Supplemental Figure 2(b)). In patients with stroke, MFAP4 immunoreactivity was detected to a similar degree as in the other controls in the connective tissue spaces, including meninges and ECM in the intima and adventitia of larger vessels ( Supplemental Figure 2(c) and (d)).
Clinical study
Demographics and disease characteristics.
Serum and CSF were collected from 161 patients with IDDs. Of the 161 patients, 9 were excluded because either AQP4-IgG or MOG-IgG were not tested. Of the resulting 152 patients, 62 were AQP4-IgG positive NMOSD, 22 MOG-IgG positive (MOGAD), and 68 were double-seronegative. In all, 19 patients out of the 68 had isolated optic neuritis (ION) and 49 patients had MS. Samples from MS patients were obtained before treatment with corticosteroids or disease-modifying drugs (Table 2 and Supplemental Figure 3).
Table 2.
Clinical and demographic data of the study population.
No of seronegative/% | NMOSD+ AQP4-IgG | MOGAD+ MOG-IgG | p Value | ||
---|---|---|---|---|---|
Number | 68 | 62 | 22 | ||
Gender | Female | 46 (68%) | 57 (92%) | 8 (36%) | <0.001 |
Male | 22 (32%) | 5 (8%) | 14 (64%) | ||
Ethnicity | African American | 2 (3%) | 0 | 0 | <0.001 |
Asian | 3 (4%) | 20 (32%) | 1 (5%) | ||
Caucasian | 61 (90%) | 38 (61%) | 19 (86%) | ||
Latin American | 1 (1%) | 0 | 0 | ||
not specified | 1 (%) | 4 (6%) | 2 (9%) | ||
Clinical presentation at the most recent relapse prior to LP | ON | 68 (100%) | 7 (11%) | 7 (32%) | |
SC | 0 | 29 (45%) | 3 (14%) | ||
ON + SC | 0 | 3 (5%) | 0 | ||
Brain | 0 | 0 | 4 (18%) | ||
Brainstem | 0 | 2 (3%) | 0 | ||
SC + brainstem | 0 | 1 (2%) | 0 | ||
Missing data | 0 | 24 (39%) | 8 (36%) | <0.001 | |
Age at lumbar puncture, median (IQR) | 39.6 (28.4, 49.4) | 45.0 (32.5, 53.2) | 48.6 (33.3, 56.5) | 0.069 | |
Disease duration (months), median (IQR) | 27.2 (21.5, 31.5) | 104.9 (39.9, 162.2) | 37.85 (16.6, 65.2) | <0.001 | |
Therapy at time of LP | None | 68 (100%) | 32 (52%) | 16 (73%) | <0.001 |
Yes | 0 | 30 (48%) | 6 (27%) | ||
AZT | 10 (16%) | 1 (5%) | |||
RIT | 3 (5%) | ||||
MMF | 7 (11%) | ||||
MTO | 1 (2%) | ||||
Steroids | 6 (10%) | 3 (14%) | |||
PLEX | 1 (2%) | ||||
Combination | 2 (3%) | 2 (10%) | |||
Was sample taken within 60 days of any attack? | No | 8 (12%) | 29 (48%) | 3 (18%) | <0.001 |
Yes | 58 (88%) | 31 (52%) | 14 (82%) | 0.40 | |
Days from most recent attack to LP, median (IOR) | 17 (10, 34) | 8.5 (3, 22) | 11 (5, 24) |
No correlation between MFAP4 serum and CSF levels
Serum MFAP4 (sMFAP4) levels in IDD patients did not differ (mean U/mL: 27.4) compared with HC (19.9; p = 0.149). When stratified by IDD type, the mean sMFAP4 level was elevated only in MS patients (mean U/mL: 32.0 MS vs. 19.9 HC; p = 0.025). No correlation was observed between sMFAP4 and CSF MFAP4 levels in IDD patients (rho = 0.14 p = 0.25), and in the fraction of patients with MS (rho 0.15 = p = 0.265).
Association of serum and CSF MFAP4 levels with age and gender
Serum and CSF MFAP4 levels were not associated with sex. CSF MFAP4 values had a positive correlation with age in IDD patients (n = 133), p = 0.000, rho = 0.34 (95% CI = 0.18; 0.49), not statistically significant in HC, p = 0.184, rho = 0.46 (95% CI = −0.22; 1.15). When stratified by diagnosis, CSF MFAP4 levels were associated with age only in patients with MS (n = 48), p = 0.000, rho = 0.42. (95% CI = 0.20; 0.64).
Reduction of CSF MFAP4 levels in the acute stage
On average, CSF MFAP4 levels in relapsing IDD patients were marginally lower compared with HC (mean U/mL: 15.2 vs. 17.9; p = 0.050), Figure 5(a). When stratified by diagnosis, CSF MFAP4 levels were decreased significantly in MOGAD (mean U/mL: 13.1 vs. 17.9; p = 0.020), ION (mean U/mL: 14.6 vs. 17.9; p = 0.019), and MS (mean U/mL: 15.0 vs. 17.9; p = 0.020) relative to HC (Figure 5(b)). CSF MFAP4 levels in MS and NMOSD were not significantly different in the acute stage compared with the chronic stage. In MOGAD CSF MFAP4 levels were decreased significantly in the acute stage relative to chronic stage (mean U/mL: 13.1 vs. 21.1; p = 0.001), independent of age.
Figure 5.
Reduction of CSF MFAP4 in the acute stage.
Comparing IDD patients in the acute stage and HC showed a trend toward (a) decreased CSF MFAP4 levels (p = 0.05), (b) significantly in MOGAD, ION, and MS.
(c) Comparison between CSF MFAP4 levels in Caucasians and non-Caucasians showed lower CSF MFAP4 levels in Caucasians. (d) Comparing Caucasian IDD patients in the acute stage and HC showed significantly decreased CSF MFAP4 levels.
CSF MFAP4 levels are affected by number of relapses
Non-Caucasian patients (n = 26, predominantly with NMOSD (20), consisting of Afro-Americans,2 Asians,24 and Latin Americans1) experienced more attacks (median 2) before sample collection than did Caucasians (n = 87), (median 1) (p = 0.000). Comparison between age, sex, and severity of attacks in non-Caucasian patients versus Caucasians did not reveal any differences.
Caucasian patients (n = 68) had lower CSF MFAP4 levels than non-Caucasian patients (n = 23) (mean U/mL: 14.9 vs. 17.9; p = 0.0097) in all samples as well as in samples obtained during the acute stage (mean U/mL: 14.5 vs. 17.8; p = 0.005) (Figure 5(c)). Comparison of Caucasian IDD patients in the acute stage and HC showed decreased CSF MFAP4 levels (mean 14.5 U/mL) relative to HC (17.9; p = 0.007), (Figure 5(d)). The levels of CSF MFAP4 were not influenced by medical treatment (data not shown). Notably, number of relapses prior to lumbar puncture and CSF MFAP4 levels were correlated (rho = 0.33, p = 0.004) (95% CI = 0.10: 0.55) independent of age, (Figure 6(a)). Furthermore, a positive correlation was observed between disease duration prior to lumbar puncture and CSF MFAP4 levels (rho = 0.22, p = 0.032) (95% CI = 0.02–0.44), independent of age. We therefore examined 53 patients with samples obtained at onset of disease, that is, presenting attack, of whom 6 had NMOSD, 7 MOGAD, 19 ION, and 21 MS. For the total group CSF MFAP4 levels were lower compared with HC (mean U/mL: 14.3 vs. 17.9; p = 0.013), (Figure 6(b)). CSF MFAP4 levels from MOGAD patients (mean U/mL: 9.7) and ION patients (mean U/mL: 14.6) were decreased relative to HC (mean U/mL: 17.9; p = 0.000 and p = 0.019), respectively (Figure 6(c)).
Figure 6.
A positive correlation between number of relapses and CSF MFAP4 levels.
(a) Number of relapses was associated with higher CSF MFAP4 levels. (b) CSF MFAP4 decreased relative to HC in patients at presenting attack, (c) specifically for MOGAD patients and ION patients. (d) In MOGAD the CSF MFAP4 levels decreased more significantly relative to HC in patients with less relapses: >2 relapses (mean U/mL: 13.1; p = 0.02), 2 relapses (mean U/mL: 12.3; p = 0.006), one relapse (mean U/mL: 9.7; p = 0.000).
(e) CSF MFAP4 levels in the acute stage within ⩽60 days was positively associated with time from recent relapse to sample collection (rho = 0.61), and (f) associated strongly with time to sample collection within ⩽15 days (rho = 1) in MOGAD.
In MOGAD, the CSF MFAP4 levels were lower than in HC. The decrease was less prominent in the MOGAD group with increasing number of relapses (D): >2 relapses (mean U/mL: 13.1; p = 0.02), 2 relapses (mean U/mL: 12.3; p = 0.006) (Figure 6(d)).
CSF MFAP4 levels and time from onset of recent relapse to sample collection
Univariate analysis indicated in patients with MOGAD in the acute stage a correlation between CSF MAP4 levels and time from onset of recent relapse to sample collection (rho = 0.61, p = 0.007) (95% CI = 0.08: 0.36), (Figure 6(e)), and strongly associated with time to sample collection within ≤ 15 days (rho = 1), (Figure 6(f)). Based on a linear regression model, average MFAP4 levels increased to 0.22 U/mL per day following attack onset. From these data we have estimated a time limit on sampling CSF as 6.2 weeks.
CSF MFAP4 levels as a biomarker for acute optic neuritis
Patients with acute ON relapse (n = 68) had reduced CSF MFAP4 levels (mean U/mL: 14.5 vs. 17.9; p = 0.006) (Figure 7(a)), compared with levels in IDD patients with cerebral, spinal, or brainstem relapse (other CNS attacks, (n = 28)) (16.6; p = 0.048), and in ON patients with samples taken in the chronic stage (n = 10) (17.8; p = 0.031), independent of age, disease duration, and number of relapses.
Figure 7.
CSF MFAP4 as a marker for acute optic neuritis relapse.
(a and b) Patients with acute ON relapse had significantly reduced CSF MFAP4 levels.
When we stratified patients by diagnosis (ON-MS, ON-NMOSD, ON-MOGAD, and ION), CSF MFAP4 levels were decreased in all groups: ON-MOGAD (mean 12.2, p = 0.005), ON-NMOSD (mean 12.8, p = 0.028), ION (mean 14.6, p = 0.025), and ON-MS (mean 15.0, p = 0.026) compared with HC (mean 17.9), (Figure 7(b)).
Correlation between CSF MFAP4 levels and relapse severity
MFAP4 levels were not associated with EDSS in IDD patients. In total, a relapse severity score at the most recent relapse prior to lumbar puncture was available for 44 IDD patients. Relapse severity score was associated with lower CSF MFAP4 levels independent of number of relapses in acute IDDs (rho = −0.41, p = 0.017) (95% CI = −0.13: −0.69), (Figure 8).
Figure 8.
CSF MFAP4 levels reflect relapse severity.
A negative correlation between CSF MFAP4 levels and relapse severity was observed.
Discussion
Our data demonstrate MFAP4 protein immunoreactivity in the vascular/perivascular spaces and meninges, particularly of the optic nerve. At sites of active inflammation in tissue from NMOSD and acute MS, MFAP4 immunoreactivity was reduced, suggesting that inflammation altered the composition of extracellular matrix. Clinically, CSF MFAP4 levels were reduced in the acute stage during relapses of IDDs and more pronounced in patients with MOGAD and acute ON relapse. In MOGAD we observed an association between CSF MFAP4 levels and time from onset of recent relapse. A positive correlation was observed between number of relapses and CSF MFAP4 levels, suggesting a dynamic reorganization of MFAP4 in CNS as a reaction to inflammatory relapses. Relapse severity was negatively associated with CSF MFAP4 levels, suggesting that MFAP4 may serve as a potential marker of disease activity and relapse severity. Overall, the clinical findings are consistent with the pathological data since a reduction of CSF MFAP4 occurred in acute disease stages.
The recognized cellular receptor for MFAP4 is integrin αVβ3.10 Two main receptors/adhesion proteins are involved in the cell–cell and cell–matrix interactions of the BBB: dystroglycan and integrins.9,10 In our study, MFAP4 was absent in CNS during relapse as documented by the presence of inflammatory infiltrates in parallel sections, suggesting that MFAP4 remodeling may occur as a consequence of BBB damage and vascular reorganization. Notably, in situ hybridization of the optic nerve demonstrated mRNA MFAP4 expression in the capillaries.
Reduction of MFAP4 reactivity at sites of active inflammation in NMOSD and active MS suggests that MFAP4 reflect CNS tissue damage and possibly relate to astrocytes and oligodendrocytes being the target as well as the source of MFAP4. In line with pathology data, substantial CSF MFAP4 levels reduction was seen in the acute stage, and the reduction was more pronounced in patients with MOGAD and acute ON. Notably, the reduction of CSF MFAP4 correlated with the severity of all types of relapses.
This study raises a potential role of MFAP4 in severity of relapses. The pathology data were collected without knowledge of clinical data, including CSF and serum levels and vice versa, diminishing bias in the study. Patients originated from a large cohort of samples from multiple centers, which increases the generalizability of the findings. The study conclusions are limited by the cross-sectional design of our study; longitudinal design with consecutive samples will be necessary for further validation, including treatment effects. A large prospective study designed to validate these observations in our study would be an obvious continuation.
MFAP4 immune reactivity is increased in liver fibrosis, cardiovascular disorders, and asthma.27,28 In these diseases the MFAP4 levels are increased in serum, whereas we in this study observed that CSF levels in acute IDDs were decreased.
In the present study, the mean serum MFAP4 level was elevated only in MS patients. However, no correlation was observed between sMFAP4 and CSF MFAP4 levels including the MS patients. CSF MFAP4 levels were decreased in patients with MS, in line with the pathology data. All controls in our study were without morbidity whereas limited information was obtained on comorbidity in the IDD patients. Future studies will illuminate the putative effects of comorbidity.
In conclusion, MFAP4 is expressed in the human CNS and diminished at sites of active inflammation. CSF MFAP4 levels were lower in the acute stage, especially in patients with MOGAD and acute ON, where it correlated negatively with the severity of the relapse. Our findings underscore the timepoint of MFAP4 determinations relative to an attack and suggest a role for MFAP4 of disease activity and relapse severity.
Supplementary Material
Acknowledgements
We thank Dr. Christine Nilsson for her assistance in sample logistics and Simon B. M. Kristensen, Ph.D., for his expert assistance in data management. We thank Tine Rasmussen, Malene H. Nielsen and Lone Christiansen for their expert technical assistance.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research was supported by the Danish Multiple Sclerosis Society (A40208, A38376, A35152), The University of Southern Denmark, and the Slagelse Hospital Research Fund.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: S.S., M.N.O., M.W., S.M., S.T.L., K.S., and N.A. have nothing to disclose.
T.M. has received speaker honoraria from Tanabe Mitsubishi Pharma, Novartis Pharma., Alexion Pharma., Teijin Pharma., Viela Bio, and Biogen Idec Japan; he has received research support from Cosmic Corporation, and Medical and Biological Laboratories Co.; and grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology.
J.F. has served on scientific advisory boards for and received funding for travel related to these activities as well as honoraria from Biogen Idec, Merck Serono, Sanofi-Aventis, Teva, Novartis, and Almirall.
S.H. is a scientific advisor for Alcon, Santen, Sanofi, and Thea.
S.MA. has received support for attending scientific meetings by Merck and Euroimmun and received speaker honoraria from Biogen.
K.F. has received grants from the Ministry of Education of Japan, Ministry of Health, Welfare and Labor of Japan, and received personal fees from Roche/Chugai, Alexion, Viela Bio/ Horizon Therapeutics, Biogen, Eisai, Mitsubishi Tanabe, Novartis, Astellas, Tejin, Takeda, UCB, Merck Biopharma, Abbvie, Japan Tobacco, and Asahi Kasei Medical.
K.R. has received research support from Novartis, Merck Serono, German Ministry of Education and Research, European Union (821283-2), Stiftung Charité (BIH Clinical Fellow Program), and Arthur Arnstein Foundation; and received travel grants from the Guthy-Jackson Charitable Foundation.
T.L.A. has received nonfinancial support from Amgen, and free reagents from 10X genomics and ABD bioscience for projects outside the submitted work.
R.M. reports personal fees from Alexion, Horizon Therapeutics, Merck, Biogen, Roche, and UCB, outside the submitted work.
E.F. has served on advisory boards for Alexion, Genentech, and Horizon Therapeutics. He has received speaker honoraria from Pharmacy Times, and royalties from UpToDate. He was a site primary investigator in a randomized clinical trial on Inebilizumab in neuromyelitis optica spectrum disorder run by Medimmune/Viela-Bio/Horizon Therapeutics. He has received funding from the NIH (R01NS113828), is a member of the medical advisory board of the MOG project, and an editorial board member of the Journal of the Neurological Sciences and Neuroimmunology Reports.
S.P. has received personal compensation for serving as a consultant for Genentech, Sage Therapeutics, and Astellas. He has received personal compensation for serving on scientific advisory boards or data safety monitoring boards for F. Hoffman-LaRoche AG, Genentech, and UCB. His institution has received compensation for serving as a consultant for Astellas, Alexion, and Viela Bio/MedImmune. All compensation is paid to Mayo Clinic. He has received research support from Alexion, Viela Bio/MedImmune, Roche/Genentech. He has a patent, Patent# 8,889,102 (Application#12-678350, Neuromyelitis Optica Autoantibodies as a Marker for Neoplasia)—issued; a patent, Patent# 9,891,219B2 (Application#12-573942, Methods for Treating Neuromyelitis Optica (NMO) by Administration of Eculizumab to an individual that is Aquaporin-4 (AQP4)-IgG Autoantibody positive)—issued.
H.J.K. received a grant from the National Research Foundation of Korea and research support from Aprilbio and Eisai; received consultancy/speaker fees from Alexion, Aprilbio, Altos Biologics, Biogen, Celltrion, Daewoong, Eisai, GC Pharma, HanAll BioPharma, Handok, Horizon Therapeutics (formerly Viela Bio), MDimune, Mitsubishi Tanabe Pharma, Merck Serono, Novartis, Roche, Sanofi, Teva-Handok, and UCB; is a co-editor for the Multiple Sclerosis Journal and an associate editor for the Journal of Clinical Neurology.
J.B. reports personal fees from Roche, Genentech, Horizon, Chugai Pharma, Clene Nanoscience, Reistone-Bio, Beigene, grants and personal fees from Alexion, grants from National Institutes of Health, and has a patent Aquaporumab issued.
F.P. has served on the scientific advisory boards of Novartis and MedImmune; received travel funding and/or speaker honoraria from Bayer, Novartis, Biogen, Teva, Sanofi-Aventis/Genzyme, Merck Serono, Alexion, Chugai, MedImmune, and Shire; is an associate editor of Neurology: Neuroimmunology & Neuroinflammation; is an academic editor of PLoS ONE; consulted for Sanofi Genzyme, Biogen, MedImmune, Shire, and Alexion; received research support from Bayer, Novartis, Biogen, Teva, Sanofi-Aventis/Geynzme, Alexion, and Merck Serono; and received research support from the German Research Council, Werth Stiftung of the City of Cologne, German Ministry of Education and Research, Arthur Arnstein Stiftung Berlin, EU FP7 Framework Program, Arthur Arnstein Foundation Berlin, Guthy-Jackson Charitable Foundation, and NMSS.
G.L.S. is an inventor of patents owned by the University of Southern Denmark
WO2014114298, WO2019086580, WO2014114298. B.G.W. receives royalties from RSR Ltd, Oxford University, Hospices Civil de Lyon, and MVZ Labor PD Dr. Volkmann und Kollegen GbR for a patent of NMO-IgG as a diagnostic test for neuromyelitis optica spectrum disorders, served on the adjudication committee for clinical trials conducted by MedImmune/VielaBio, Alexion, and UCB Biosciences and consulted for Chugai/Roche/Genentech, Horizon Therapeutics and Mitsubishi Tanabe regarding neuromyelitis optica spectrum disorders. He has received honoraria for speaking at internal meetings of Genentech, Novartis, and Horizon and at external meetings for Roche.
H.L. has received fees for lectures from Merck, Novartis, Bristol Myers Squibb, and Sanofi-Aventis; and served as a consultant for Biogen Idec, and Roche.
Footnotes
Consent of Publication
Informed consent was obtained from all subjects involved in the study.
Ethical Approval and Consent to Participate
The Research Ethical Committees for the Region of Southern Denmark approved the study protocol (ref. nos S-20130137 and S-20080142) as did the Danish Data Protection Agency (ref. no. 14/26345). Approval by local ethical Institutional Review Boards from all centers for this multicenter study were obtained following informed consent from the patients. All patients gave written consent to participate after verbal and written descriptions of the study. Data were processed in accordance with the European Union General Data Protection Regulations.
Supplemental Material
Supplemental material for this article is available online.
Contributor Information
Sara Samadzadeh, Department of Regional Health Research, University of Southern Denmark, Odense, Denmark/Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark/Department of Neurology, Slagelse Hospital, Slagelse, Denmark/Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité—Universitätsmedizin Berlin, Berlin, Germany.
Mads Nikolaj Olesen, Department of Regional Health Research, University of Southern Denmark, Odense, Denmark/Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark/Department of Neurology, Slagelse Hospital, Slagelse, Denmark/Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
Martin Wirenfeldt, Department of Regional Health Research, University of Southern Denmark, Odense, Denmark/Department of Pathological Anatomy and Molecular Biology, Hospital South West Jutland, Esbjerg, Denmark.
Sören Möller, Open Patient Data Explorative Network, Odense University Hospital, University of Southern Denmark, Odense, Denmark.
Tatsuro Misu, Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Kerstin Soelberg, Department of Regional Health Research, University of Southern Denmark, Odense, Denmark.
Jette Lautrup Frederiksen, Danish Multiple Sclerosis Center, Department of Neurology, Copenhagen University Hospital—Rigshospitalet, Glostrup, Denmark.
Steffen Heegaard, Departments of Ophthalmology and Pathology, Rigshospitalet, Glostrup, Denmark.
Sara Mariotto, Neurology Unit, Department of Neurosciences, Biomedicine, and Movement Sciences, University of Verona, Verona, Italy.
Kazuo Fujihara, Department of Multiple Sclerosis Therapeutics, Fukushima Medical University School of Medicine, Fukushima, Japan/Multiple Sclerosis and Neuromyelitis Optica Center, Southern Tohoku Research Institute for Neuroscience, Koriyama, Japan.
Klemens Ruprecht, Department of Neurology, Charité—Universitätsmedizin Berlin, Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
Thomas Levin Andersen, Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark/Department of Pathology, Odense University Hospital, Odense, Denmark.
Romain Marignier, Service de Neurologie, Sclérose en Plaques, Pathologies de la Myéline et Neuro-inflammation, Hôpital Neurologique Pierre Wertheimer, Hospices Civils de Lyon, Lyon, France.
Søren Thue Lillevang, Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
Eoin P Flanagan, Department Neurology and Center for MS and Autoimmune Neurology, Mayo Clinic, Rochester, MN, USA.
Sean J Pittock, Department Neurology and Center for MS and Autoimmune Neurology, Mayo Clinic, Rochester, MN, USA.
Ho Jin Kim, Department of Neurology, Research Institute and Hospital of National Cancer Center, Goyang, Republic of Korea.
Jeffrey L Bennett, Department of Neurology & Ophthalmology, Programs in Neuroscience & Immunology University of Colorado, Anschutz, CO, USA.
Friedemann Paul, Experimental and Clinical Research Center, Max Delbrueck Center for Molecular Medicine and Charité—Universitätsmedizin Berlin, Berlin, Germany.
Grith Lykke Sorensen, Cancer and Inflammation, Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
Brian G Weinshenker, Department of Neurology, University of Virginia, Charlottesville, VA, USA.
Hans Lassmann, Center for Brain Research, Medical University of Vienna, Vienna, Austria.
Nasrin Asgari, Department of Regional Health Research, University of Southern Denmark, Odense, Denmark/Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark Department of Neurology, Slagelse Hospital, Slagelse, Denmark/Open Patient Data Explorative Network, Odense University Hospital, University of Southern Denmark, Odense, Denmark.
Availability of Data and Materials
The Principal Author has full access to the data used in the analyses in the manuscript. Anonymized data of this study will be available from the corresponding author on reasonable request from any qualified investigator, following the EU General Data Protection Regulation.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The Principal Author has full access to the data used in the analyses in the manuscript. Anonymized data of this study will be available from the corresponding author on reasonable request from any qualified investigator, following the EU General Data Protection Regulation.