Complement Activation Is Associated With Disease Severity in Multiple Sclerosis

Johanna Oechtering; Kerstin Stein; Sabine A Schaedelin; Aleksandra M Maceski; Annette Orleth; Stephanie Meier; Eline Willemse; Ferhan Qureshi; Ingmar Heijnen; Axel Regeniter; Tobias Derfuss; Pascal Benkert; Marcus D'Souza; Marguerite Limberg; Bettina Fischer-Barnicol; Lutz Achtnichts; Stefanie Mueller; Anke Salmen; Patrice H Lalive; Claire Bridel; Caroline Pot; Renaud A Du Pasquier; Claudio Gobbi; Heinz Wiendl; Cristina Granziera; Ludwig Kappos; Marten Trendelenburg; David Leppert; Jan D Luenemann; Jens Kuhle; for the Swiss MS Cohort Study

doi:10.1212/NXI.0000000000200212

. 2024 Feb 14;11(2):e200212. doi: 10.1212/NXI.0000000000200212

Complement Activation Is Associated With Disease Severity in Multiple Sclerosis

Johanna Oechtering

Johanna Oechtering, MD

¹From the Department of Neurology (J.O., A.M.M., A.O., S. Meier, E.W., T.D., M.D.S., M.L., B.F.-B., C. Granziera, L.K., D.L., J.K.); Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB) (J.O., S.A.S., A.M.M., A.O., S. Meier, E.W., T.D., P.B., M.D.S., M.L., B.F.-B., C. Granziera, L.K., D.L., J.K.), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland; Department of Neurology with Institute of Translational Neurology (K.S., H.W., J.D.L.), University Hospital 4 Münster, Germany; Clinical Trial Unit (S.A.S., P.B.), Department of Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland; Octavebio Bioscience (F.Q.), Menlo Park, CA; Division of Medical Immunology (I.H.), Laboratory Medicine, University Hospital Basel, Switzerland; Medica Laboratory (A.R.), Zürich; Department of Neurology (L.A.), Cantonal Hospital, Aarau; Department of Neurology (S. Mueller), Cantonal Hospital St. Gallen; Department of Neurology (A.S.), Inselspital, Bern University Hospital and University of Bern; Department of Clinical Neurosciences (P.H.L., C.B.), Division of Neurology; Diagnostic Department (P.H.L.), Division of Laboratory Medicine; Department of Pathology and Immunology (P.H.L.), Faculty of Medicine, University of Geneva; Division of Neurology (C.P., R.A.D.P.), Department of Clinical Neurosciences, Lausanne University Hospital (CHUV) and University of Lausanne; Neurocentre of Southern Switzerland (C. Gobbi), Multiple Sclerosis Centre, Ospedale Civico; Faculty of Biomedical Sciences (C. Gobbi), Università della Svizzera Italiana (USI), Lugano, Switzerland; Translational Imaging in Neurology (ThINk) Basel (C. Granziera), Department of Biomedical Engineering, Faculty of Medicine, University Hospital Basel and University of Basel; and Division of Internal Medicine (M.T.), University Hospital Basel and Clinical Immunology, Department of Biomedicine, University of Basel, Basel, Switzerland.

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¹, Kerstin Stein

Kerstin Stein, MSc

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¹, Sabine A Schaedelin

Sabine A Schaedelin, MSc

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¹, Aleksandra M Maceski

Aleksandra M Maceski, MSc

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¹, Annette Orleth

Annette Orleth, PhD

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¹, Stephanie Meier

Stephanie Meier, PhD

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¹, Eline Willemse

Eline Willemse, PhD

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¹, Ferhan Qureshi

Ferhan Qureshi, BS

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¹, Ingmar Heijnen

Ingmar Heijnen, MD

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¹, Axel Regeniter

Axel Regeniter, MD

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¹, Tobias Derfuss

Tobias Derfuss, MD

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¹, Pascal Benkert

Pascal Benkert, PhD

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¹, Marcus D'Souza

Marcus D'Souza, MD

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¹, Marguerite Limberg

Marguerite Limberg, MSc

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¹, Bettina Fischer-Barnicol

Bettina Fischer-Barnicol, MD

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¹, Lutz Achtnichts

Lutz Achtnichts, MD

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¹, Stefanie Mueller

Stefanie Mueller, MD

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¹, Anke Salmen

Anke Salmen, MD

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¹, Patrice H Lalive

Patrice H Lalive, MD

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¹, Claire Bridel

Claire Bridel, MD, PhD

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¹, Caroline Pot

Caroline Pot, MD

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¹, Renaud A Du Pasquier

Renaud A Du Pasquier, MD

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¹, Claudio Gobbi

Claudio Gobbi, MD

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¹, Heinz Wiendl

Heinz Wiendl, MD, PhD

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¹, Cristina Granziera

Cristina Granziera, MD, PhD

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¹, Ludwig Kappos

Ludwig Kappos, MD

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¹, Marten Trendelenburg

Marten Trendelenburg, MD

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¹, David Leppert

David Leppert, MD

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¹, Jan D Luenemann

Jan D Luenemann, MD, PhD

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^1,^✉, Jens Kuhle

Jens Kuhle, MD, PhD

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^1,^✉; for the Swiss MS Cohort Study¹

^✉

Correspondence Prof. Kuhle jens.kuhle@usb.ch or Prof. Luenemann jan.luenemann@ukmuenster.de

Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article.

J. Kuhle and J.D. Luenemann are co-senior authors.

The Article Processing Charge was funded by the authors.

Submitted and externally peer reviewed. The handling editor was Friedemann Paul, MD.

^✉

Corresponding author.

PMCID: PMC10913171 PMID: 38354323

Abstract

Background and Objectives

Histopathologic studies have identified immunoglobulin (Ig) deposition and complement activation as contributors of CNS tissue damage in multiple sclerosis (MS). Intrathecal IgM synthesis is associated with higher MS disease activity and severity, and IgM is the strongest complement-activating immunoglobulin. In this study, we investigated whether complement components (CCs) and complement activation products (CAPs) are increased in persons with MS, especially in those with an intrathecal IgM synthesis, and whether they are associated with disease severity and progression.

Methods

CC and CAP levels were quantified in plasma and CSF of 112 patients with clinically isolated syndrome (CIS), 127 patients with MS (90 relapsing-remitting, 14 primary progressive, and 23 secondary progressive), 31 inflammatory neurologic disease, and 44 symptomatic controls from the Basel CSF databank study. Patients with CIS/MS were followed in the Swiss MS cohort study (median 6.3 years). Levels of CC/CAP between diagnosis groups were compared; in CIS/MS, associations of CC/CAP levels with intrathecal Ig synthesis, baseline Expanded Disability Status Scale (EDSS) scores, MS Severity Score (MSSS), and neurofilament light chain (NfL) levels were investigated by linear regression, adjusted for age, sex, and albumin quotient.

Results

CSF (but not plasma) levels of C3a, C4a, Ba, and Bb were increased in patients with CIS/MS, being most pronounced in those with an additional intrathecal IgM production. In CIS, doubling of C3a and C4a in CSF was associated with 0.31 (CI 0.06–0.56; p = 0.016) and 0.32 (0.02–0.62; p = 0.041) increased EDSS scores at lumbar puncture. Similarly, doubling of C3a and Ba in CIS/MS was associated with 0.61 (0.19–1.03; p < 0.01) and 0.74 (0.18–1.31; p = 0.016) increased future MSSS. In CIS/MS, CSF levels of C3a, C4a, Ba, and Bb were associated with increased CSF NfL levels, e.g., doubling of C3a was associated with an increase of 58% (Est. 1.58; CI 1.37–1.81; p < 0.0001).

Discussion

CNS-compartmentalized activation of the classical and alternative pathways of complement is increased in CIS/MS and associated with the presence of an intrathecal IgM production. Increased complement activation within the CSF correlates with EDSS, future MSSS, and NfL levels, supporting the concept that complement activation contributes to MS pathology and disease progression. Complement inhibition should be explored as therapeutic target to attenuate disease severity and progression in MS.

Introduction

Complex immune mechanisms involving T and B cells, antibodies, activated macrophages, and microglia along with the vulnerability of the target tissue account for the cascade of events that leads to the development and progression of multiple sclerosis (MS).¹ Histologic analysis of white matter lesions in MS patients with a relatively short disease course identified 4 different patterns in early phase MS pathogenesis; complement and immunoglobulin deposition are the typical features of the predominant pattern II.² In later phases of disease, this heterogeneity of lesion morphology seems to converge into a homogenous picture with one common pattern of antibody-mediated and complement-mediated myelin phagocytosis as the dominant mechanism of demyelination.^3-6 These findings were corroborated by further studies, reporting similar findings of complement components (CCs) and complement activation products (CAPs) next to immunoglobulins in MS plaques and in normal-appearing white matter.^6-12

A persistent intrathecal humoral immune response is a pathognomonic feature of MS: CSF-specific oligoclonal IgG bands (OCGBs) are present in up to 98%, and a quantitated intrathecal IgG and IgM synthesis according to the Reiber formula in 65%–72% and 20%–26% of patients, respectively.^13,14 The presence of intrathecal IgM synthesis is associated with higher disease activity and severity,^13-16 and other than IgG, in a dose-dependent manner.¹³ It is further associated with disproportionally higher spinal cord involvement in early MS and increased levels of neurofilament light chain (NfL) as a measure of neuroaxonal damage, highlighting its pathogenic relevance.¹⁷ As IgM is the most efficient immunoglobulin isotype for complement activation, these differences might be partially explained by an increased activation through the antibody-mediated classical pathway. Accordingly, CSF levels of C3 activation products correlate with those of IgM, but not of IgG or IgA synthesis.¹⁸

The classical complement pathway is initiated by antigen binding of IgM or IgG, and C1q then binds to the immune complex and triggers the sequential activation of subunits C1r and C1s. Activated C1s then further cascades the cleavage of components C2 and C4 into their parts, forming C4b2a, the classical pathway C3 convertase, cleaving C3 into C3a and C3b. This subsequently leads to the formation of C4b2a3b (C5 convertase). The alternative pathway serves as a powerful amplification loop. Hydrolyzed C3 binds to factor B, which is cleaved into Ba and Bb, forming the alternative pathway C3 convertase (C3bBb). All 3 complement pathways (classical, lectin, and alternative) eventually converge by assembling a C5 convertase, and C5b then triggers the formation of the terminal complement complex.⁶

We aimed to investigate complement activation in MS, especially in the context of an intrathecal IgM production and of those factors involved in the classical antibody-mediated pathway in patients with different disease subtypes and stages. We further investigated if there is an association with outcome parameters reflecting concurrent (Expanded Disability Status Scale score; EDSS) and future (MS Severity Score; MSSS) disease severity and NfL levels, specifically reflecting neuroaxonal damage.

Methods

Patients, Inclusion Criteria, and Data Collection

CSF and paired plasma samples were obtained from the CSF biobank of the University Hospital Basel from patients prospectively recruited into the Swiss MS cohort between 2012 and 2021.

We included all eligible patients with a CSF sample without erythrocyte contamination available with a first demyelinating event suggestive of MS (CIS), relapsing remitting MS (RRMS),¹⁹ secondary progressive MS (SPMS),²⁰ or primary progressive MS (PPMS)²¹ as well as symptomatic (SC) and inflammatory neurologic disease controls (INDC).²² First symptoms were defined as neurologic symptoms that lasted for ≥24 hours without fever, infection, or adverse reaction to a prescribed medication.²¹ Patients included were not treated with corticosteroids 30 days before lumbar puncture (LP).

RRMS had to have experienced at least 2 relapses before the LP.¹⁹ The inflammatory neurologic disease controls (INDC) consisted of patients with inflammatory neurologic CNS diseases different from MS.²² Symptomatic controls had neurologic symptoms without pathologic objective clinical or paraclinical findings, e.g., patients with migraine.²²

Demographic and clinical variables collected included sex, date of birth and in patients with CIS/MS onset of first symptoms, disease-modifying treatments, EDSS score at LP (in CIS, median 18 ([interquartile range (IQR) 9–54] days after onset), and at last follow-up visit (after median 6.3 [IQR 4.0–9.3] years). The standardized clinical assessments based on EDSS scores were performed by certified raters.^23,24 The MSSS was based on the EDSS at last follow-up visit.²⁵

Standard Protocol Approvals, Registrations, and Patient Consents

We included patients from the CSF biobank study of the Department of Neurology, University Hospital Basel, which were prospectively recruited into the Swiss MS cohort. These studies were approved by the local ethical committee, and patients were included after written informed consent.

Samples and Routine Cerebrospinal Fluid Analysis

CSF was centrifuged (400g for 10 minutes) within 30 minutes of collection, EDTA plasma samples were collected at the same time, centrifuged (2,000g for 10 minutes), and the supernatant frozen at −80°C. OCGBs were assessed by isoelectric focusing followed by immunofixation or immunoblotting. Testing of OCGBs was considered positive if pattern 2 or 3 (local synthesis of IgG within the CNS) was present.²⁶ CSF and serum concentrations of IgA, IgG, IgM, and albumin were measured nephelometrically. We used the derived CSF-to-serum quotients for Igs and albumin (Q_IgG, Q_IgM, Q_IgA, and Q_alb) for the calculations of the intrathecal Ig synthesis according to the formulas proposed by Reiber.¹⁴ The amount of intrathecal IgG, IgM, or IgA synthesis was expressed as intrathecal fraction in percentage of the total measured isotype concentration in CSF (IgG_{(IF = IntrathecalFraction)}, IgM_IF, and IgA_IF). The integrity of the blood-CSF barrier was determined by calculating the CSF/serum ratio for albumin (Q_alb).²⁶

Measurement of Complement Components and Their Activation Products

Samples were sent to the University Hospital of Münster (Münster, Germany) on dry ice. For the quantification of complement activation products (C4a, C3a, C5a, s-C5b9, Ba, and Bb) and components (C1q, C4, C3, C5, and Factor H and I) levels, samples were thawed at 37°C (water bath) and immediately transferred to ice to prevent complement activation and processed. Multiplex enzyme-linked immunosorbent assays (ELISAs) based on chemiluminescence were used according to the manufacturer's recommendations (Quidel, San Diego, CA; cat. number: A900, A917) to systematically profile components and activation products (in plasma and CSF samples) in duplicate determinations. Each plate contained samples from all different diagnosis groups to minimize consequences of potential interplate variation. Control samples provided by the manufacturer were included on each plate to ensure plate-to-plate consistency. Data points below lower limit of quantification (LLOQ) or above upper limit of quantification (ULOQ) were imputed (CSF: below LLOQ: 6.1%; above ULOQ: 0.6%; plasma: below LLOQ: 11.3%; above ULOQ: 0.02%; eTable 1, links.lww.com/NXI/A996). For data points above ULOQ, the respective threshold was used as a value, and for those below LLOQ, a random value between zero and LLOQ was imputed according to a uniform distribution.

Measurement of CSF Neurofilament Light Chain Levels

CSF NfL was measured in duplicate with the NF‐light^® assay (Quanterix, Billerica, MA). The mean intra-assay coefficient of variation (CV) of duplicate determinations for CSF NfL was 2.5%. Few samples with intra‐assay CVs >20% were repeatedly measured. The mean interassay CVs of internal quality controls for NfL were 6.8% (8.7 pg/mL), 4.4% (17.9 pg/mL), and 6.1% (106.5 pg/mL).

Statistics

Data are presented as median and IQR and as absolute and relative frequencies in case of categorical data. Differences in complement components and activation products' levels and the ratios C4a/C4, C3a/C3, and C5a/C5 in CSF or plasma as separate dependent variables between CIS, RRMS, SPMS, INDC vs SC (independent variables) were analyzed using linear regression models adjusted for age, sex, Q_alb, and treatment category at LP (vs untreated, respectively): platform (glatiramer acetate and interferons), orals (siponimod, fingolimod, dimethyl fumarate), and monoclonal antibodies (natalizumab, ocrelizumab, and rituximab) (eTable 2, links.lww.com/NXI/A996).

We analyzed associations of CSF immunoglobulin profiles with CC and CAP levels in CIS and MS combined. As intrathecal synthesis of Ig subtypes is not evenly and independently distributed and to analyze in relation to the same reference, patients were categorized in ascending order for the presence or absence of OCGB, IgG_IF, and IgM_IF (>0% vs 0%, respectively)^13,17:

OCGB⁻/IgG_IF⁻/IgM_IF⁻; n = 44,
OCGB⁺/IgG_IF⁻/IgM_IF⁻; n = 50,
OCGB⁺/IgG_IF⁺/IgM_IF⁻; n = 81, and
OCGB⁺/IgG_IF⁺/IgM_IF⁺; n = 55

Four (1.7%) patients were IgG_IF⁺ or IgM_IF⁺ in combination with OCGB⁻, and 5 (2.1%) patients had an OCGB⁺/IgG_IF⁻/IgM_IF⁺ profile and were excluded from this analysis. Using category 1 as reference, associations of the CSF Ig categories 2 to 4 (independent variables) with the 12 CC and CAP levels as well as the ratios C4a/C4, C3a/C3, and C5a/C5 in CSF or plasma as dependent variables, respectively, were analyzed by linear regression models adjusted for age, sex, Q_alb, and the 3 treatment categories (vs untreated, respectively) at LP.

Associations of the 12 CC and CAP levels (separate independent variables) with EDSS at LP in CIS and with MSSS at last follow-up and CSF NfL levels in CIS/MS (dependent variables, respectively) were analyzed by using linear regression, adjusted for age, sex, and Q_alb. The NfL analyses were additionally adjusted for the 3 treatment categories (vs untreated, respectively) at LP.

NfL as well as CC and CAP levels were log transformed before analysis to better comply with normal assumption. To facilitate the estimates interpretation, a logarithm with base 2 was used in those analyses including CC and CAP as separate independent variables. Estimates were further back transformed to indicate the effect in percentages. Statistical analyses were performed using R (version 3.6.3).

Data Availability

Data are available on reasonable request.

Results

Patients and Clinical Data

CSF and plasma samples were available from 112 patients with CIS (2 without plasma), 90 RRMS (2 without plasma), 23 SPMS, 14 PPMS, 44 SC, and 31 INDC. Patients with progressive MS were older and showed higher EDSS scores than CIS/RRMS (Table 1; diagnoses of SC and INDC are listed in eTable 3, links.lww.com/NXI/A996).

Table 1.

Demographic, Clinical, and CSF Characteristics at Lumbar Puncture

	CIS	RRMS	SPMS	PPMS	SC	INDC
n	112	90	23	14	44	31
Demographic and clinical data
Female (n, %)	80 (71.4)	64 (71.1)	13 (56.5)	8 (57.1)	29 (65.9)	14 (45.2)
Age at LP (y)	34.4 (26.7–43.8)	38.0 (31.4–48.1)	53.7 (48.9–59.6)	48.5 (46.7–57.8)	42.7 (25.2–52.2)	57.4 (36.7–71.3)
Disease duration at LP (mo)^a	0.6 (0.3–1.8)	56.1 (15.6–91.7)	253.1 (147.0–332.1)	50.0 (15.2–81.3)	—	—
EDSS at LP	2.0 (1.5–2.5)	2.5 (2.0–3.5)	6.0 (4.0–7.0)	3.5 (3.0–4.0)	—	—
Untreated (n, %)	111 (99.1)	70 (77.8)	15 (65.2)	13 (92.9)	—	—
Follow-up available (n, %)	88 (78.6)	64 (71.1)	19 (82.6)	9 (64.3)	—	—
Follow-up time (y)	6.4 (4.0–9.1)	6.6 (3.8–9.7)	6.0 (4.3–7.1)	8.2 (5.8–10.7)	—	—
CSF data
Cell count	4.3 (2.0–9.0)	3.0 (1.0–6.0)	1.0 (0.3–2.2)	1.7 (1.1–2.9)	1.0 (0.3–1.7)	24.6 (7.0–76.9)
Q_alb	4.5 (3.6–6.4)	5.4 (3.9–7.3)	5.5 (3.8–7.7)	5.0 (4.4–7.2)	4.3 (3.6–5.1)	9.5 (7.8–12.4)
OCGB⁺ (%)	90 (80.4)	73 (82.0)	17 (85.0)	11 (78.6)	0 (0)	13 (41.9)
IgG_IF⁺ (%)	66 (58.9)	53 (59.6)	9 (45.0)	8 (57.1)	0 (0)	7 (22.6)
IgM_IF⁺ (%)	31 (27.7)	24 (27.0)	2 (10.0)	3 (21.4)	0 (0)	8 (25.6)
IgA_IF⁺ (%)	5 (4.5)	7 (7.9)	1 (5.0)	1 (7.1)	0 (0)	4 (12.9)
OCGB⁻/IgG_IF⁻/IgM_IF⁻ (%)^b	22 (19.6)	16 (17.8)	3 (13.0)	3 (21.4)	44 (100.0)	17 (54.8)
OCGB⁺/IgG_IF⁻/IgM_IF⁻ (%)^b	21 (18.7)	18 (20.0)	8 (34.8)	3 (21.4)	0 (0)	5 (16.1)
OCGB⁺/IgG_IF⁺/IgM_IF⁻ (%)^b	38 (33.9)	31 (34.4)	7 (30.4)	5 (35.7)	0 (0)	1 (3.2)
OCGB⁺/IgG_IF⁺/IgM_IF⁺ (%)^b	28 (25.0)	22 (24.4)	2 (8.7)	3 (21.4)	0 (0)	6 (19.4)

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Abbreviations: ⁺ = presence of OCGB or IgG_IF/IgM_IF/IgA_IF; ⁻ = absence of OCGB or IgG_IF/IgM_IF/IgA_IF; CIS = clinically isolated syndrome; Ig G/M/A_IF = immunoglobulin G/M/A intrathecal fraction; INDC = inflammatory neurologic disease controls; IQR = interquartile range; OCGB = oligoclonal IgG bands; PPMS = primary progressive MS; Q_alb = albumin quotient; RRMS = relapsing-remitting MS; SC = symptomatic controls; SPMS = secondary progressive MS.

Median and IQR are displayed if not mentioned otherwise.

Since first MS symptoms.

Five (1.6%) OCGB⁻/IgG_IF⁺ or OCGB⁻/IgM_IF⁺ patients (1 RRMS, 3 SPMS, 1 INDC) and 6 (1.9%) OCGB⁺/IgG_IF⁻/IgM_IF⁺ patients (3 CIS, 2 RRMS, 1 INDC) excluded from this classification.¹³

Complement Components, Activation Products, and Q_alb, Age, and Sex

CSF

Q_alb was positively associated with all complement components and activation products (all p < 0.001) except C4, i.e., concentrations were higher with increasing Q_alb (e.g., doubling of Q_alb was associated with 18% higher C4a). Age increased C4a by 1% per year (p = 0.0065); furthermore, effects of age were seen for C3, Ba, Bb, and Factor I (eTable 4, links.lww.com/NXI/A996). CC and CAP were not different between sexes.

Plasma

Q_alb showed no associations with plasma CC and CAP, while age was associated with Ba and s-C5b9. Male patients had 17%/13% lower C4a and C3a (both p < 0.01) levels than female patients (eTable 5, links.lww.com/NXI/A996).

Complement Components and Activation Products' Levels in CIS/Multiple Sclerosis and Control Groups

CSF

C4a was increased in all stages of CIS/MS vs SC, which was most pronounced in patients with progressive MS (PPMS: Est 1.53, 95% CI 1.16–2.01, p = 0.0026, i.e., levels were increased by 53% vs SC; SPMS by 58%, p = 0.0005; RRMS by 41%; p < 0.0001; CIS by 19%, p = 0.0277; Table 2, Figure 1, eFigure 1 for raw values, links.lww.com/NXI/A996). Instead, levels of C4 were decreased, and subsequently, differences of the C4a/C4 ratio vs SC became more pronounced, especially in SPMS and PPMS (Table 2).

Table 2.

CSF Complement Components or Activation Products' Levels in CIS/MS and Inflammatory Neurologic Disease Controls (vs Symptomatic Controls)

		SC	CIS^a	RRMS^a	SPMS^a	PPMS^a	INDC^a
C4a	Est 95% CI		1.19 (1.02–1.40)	1.41 (1.19–1.67)	1.58 (1.22–2.04)	1.53 (1.16–2.01)	1.39 (1.10–1.75)
	p		0.0277	<0.0001	0.0005	0.0026	0.0054
	Con. (ng/mL)	16.5 (13.7–22.0)	20.1 (14.7–28.7)	24.4 (17.4–36.0)	30.4 (21.2–48.8)	29.9 (20.2–39.9)	31.3 (22.0–43.7)
C4	Est 95% CI		0.87 (0.78–0.97)	0.88 (0.78–1.00)	0.88 (0.73–1.05)	0.85 (0.70–1.04)	0.89 (0.75–1.05)
	p		0.0168	0.0491	0.1680	0.1076	0.1600
	Con. (µg/mL)	18.5 (15.5–20.7)	16.9 (13.6–19.7)	17.0 (13.8–19.7)	17.7 (14.4.–19.2)	17.4 (14.1–18.0)	18.8 (15.6–21.5)
C4a/C4	Est 95% CI		1.37 (1.12–1.67)	1.59 (1.29–1.97)	1.79 (1.27–2.53)	1.79 (1.27–2.53)	1.56 (1.17–2.09)
	p		0.0020	<0.0001	0.0004	0.0001	0.0027
	Quotient	0.9 (0.7–1,2)	1.1 (0.9–1.9)	1.4 (1.0–2.3)	1.6 (1.3–3.4)	2.0 (1.2–2.2)	1.6 (1.0–3.1)
C3a	Est 95% CI		1.55 (1.29–1.86)	1.84 (1.51–2.25)	1.64 (1.22–2.22)	1.80 (1.30–2.48)	2.79 (2.13–3.66)
	p		<0.0001	<0.0001	0.0012	0.0004	<0.0001
	Con. (ng/mL)	1.7 (1.5–2.5)	3.1 (2.1–3.8)	3.8 (2.6–5.6)	4.0 (2.9–5.2)	4.3 (3.0–6.5)	7.6 (4.3–17.1)
C3	Est 95% CI		0.87 (0.65–1.16)	0.76 (0.56–1.04)	1.08 (0.68–1.72)	0.66 (0.40–1.09)	0.75 (0.49–1.14)
	p		0.3479	0.0896	0.7535	0.1040	0.1792
	Con. (µg/mL)	10.9 (5.5–20.3)	9.2 (4.7–20.6)	8.7 (5.2–18.4)	16.4 (9.7–28.1)	8.7 (6.3–12.6)	14.2 (6.9–21.9)
C3a/C3	Est 95% CI		1.78 (1.27–2.49)	2.41 (1.68–3.48)	1.52 (0.88–2.63)	2.73 (1.52–4.90)	3.73 (2.28–6.11)
	p		0.0009	<0.0001	0.1016	0.0009	<0.0001
	Quotient	0.2 (0.1–0.3)	0.3 (0.2–0.6)	0.4 (0.2–0.8)	0.2 (0.1–0.4)	0.5 (0.4–0.7)	0.6 (0.2–1.4)
C5a	Est 95% CI		1.27 (0.86–1.88)	1.04 (0.68–1.59)	1.35 (0.72–2.52)	1.04 (0.53–2.03)	1.66 (0.94–2.92)
	p		0.2241	0.8492	0.3528	0.9172	0.0821
	Con. (ng/mL)	0.04 (0.02–0.07)	0.05 (0.02–0.09)	0.05 (0.02–0.10)	0.09 (0.03–0.16)	0.05 (0.04–0.06)	0.15 (0.09–0.21)
C5	Est 95% CI		0.95 (0.81–1.11)	0.88 (0.74–1.04)	0.96 (0.75–1.24)	1.10 (0.84–1.43)	1.10 (0.88–1.38)
	p		0.4913	0.1349	0.7602	0.5017	0.3960
	Con. (µg/mL)	0.16 (0.13–0.21)	0.17 (0.13–0.23)	0.19 (0.14–0.28)	0.23 (0.14–0.54)	0.24 (0.20–0.36)	0.47 (0.30–0.80)
C5a/C5	Est 95% CI		1.34 (0.88–2.05)	1.18 (0.75–1.87)	1.40 (0.71–2.77)	0.95 (0.45–1.96)	1.50 (0.81–2.78)
	p		0.1697	0.4681	0.3326	0.8799	0.1959
	Quotient	0.23 (0.13–0.39)	0.23 (0.13–0.54)	0.25 (0.12–0.49)	0.23 (0.14–0.47)	0.21 (0.12–0.28)	0.32 (0.19–0.72)
Ba	Est 95% CI		1.16 (1.01–1.33)	1.20 (1.03–1.39)	1.18 (0.95–1.47)	1.13 (0.89–1.43)	1.90 (1.56–2.33)
	p		0.0377	0.0018	0.0191	0.32755	<0.0001
	Con. (ng/mL)	6.7 (5.1–8.3)	8.3 (5.8–10.3)	9.5 (6.7–12.9)	10.1 (7.3–18.2)	11.6 (7.9–13.7)	31.4 (14.7–45.1)
Bb	Est 95% CI		1.21 (0.96–1.53)	1.26 (0.98–1.63)	1.22 (0.83–1.79)	1.26 (0.84–1.91)	2.064(1.44–2.)
	p		0.1149	0.0782	0.3140	0.2654	<0.0001
	Con. (ng/mL)	3.8 (2.7–6.3)	5.1 (3.3–7.5)	6.2 (4.0–8.7)	6.6 (4.8–9.4)	6.9 (4.9–8.7)	16.0 (10.5–27.5)
C1q	Est 95% CI		1.06 (0.95–1.19)	1.05 (0.93–1.18)	1.10 (0.92–1.31)	1.10 (0.91–1.33)	1.57 (1.34–1.84)
	p		0.2673	0.4303	0.3006	0.3169	<0.0001
	Con. (µg/mL)	0.19 (0.16–0.23)	0.22 (0.17–0.25)	0.21 (0.18–0.26)	0.25 (0.19–0.31)	0.22 (0.17–0.32)	0.35 (0.27–0.51)
Factor H	Est 95% CI		0.93 (0.86–0.99)	0.95 (0.88–1.02)	0.89 (0.80–1.00)	0.99 (0.88–1.11)	1.15 (1.04–1.27)
	p		0.0266	0.1474	0.0464	0.8228	0.0061
	Con. (µg/mL)	2.7 (2.6–3.0)	2.7 (2.5–2.9)	2.8 (2.6–3.2)	2.9 (2.7–3.3)	3.1 (2.6–3.5)	3.8 (3.3–5.3)
Factor I	Est 95% CI		0.92 (0.81–1.05)	1.00 (0.86–1.15)	0.87 (0.70–1.08)	1.04 (0.83–1.30)	1.22 (1.01–1.48)
	p		0.2166	0.9606	0.2000	0.7541	0.0441
	Con. (ng/mL)	106 (84–144)	103 (78–141)	129 (96–191)	138 (97–221)	163 (104–241)	277 (182–465)
sC5b-9	Est 95% CI		1.11 (0.75–1.66)	0.88 (0.57–1.35)	1.13 (0.60–2.16)	0.95 (0.48–1.90)	1.48 (0.83–2.65)
	p		0.6046	0.5606	0.7021	0.8888	0.1884
	Con. (ng/mL)	3.0 (1.0–9.2)	3.8 (1.6–10,5)	4.2 (1.9–10.7)	5.6 (3.5–14.5)	4.7 (2.7–8.4)	18.2 (11.2–34.5)

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Abbreviations: CI = 95% confidence interval; CIS = clinically isolated syndrome; Con. = concentration; Est = estimate; INDC = inflammatory neurologic disease controls; IQR = interquartile range; LP = lumbar puncture; mcg = microgram; ml = milliliter; ng = nanogram; p = p-value; PPMS = primary progressive MS; RRMS = relapsing-remitting MS; SPMS = secondary progressive MS; SC = symptomatic controls; Q_{Alb =} albumin quotient.

Median and IQR raw concentrations/quotients are displayed.

Vs symptomatic controls (SC). Estimates are adjusted for age, sex, Q_Alb, and treatment categories—platform, orals, and monoclonal antibodies (vs untreated, respectively). Associations of age and Q_Alb are displayed in eTable 4 (links.lww.com/NXI/A996).

Marginal effects are displayed as derived from the multivariable analyses (Table 2). (A) C4a was increased in CIS by 19% (Est. 1.19; p = 0.0277), 41% in RRMS (p < 0.0001), and 39% in INDC (p = 0.0054), and this increase was most pronounced in SPMS (58%; p = 0.0005) and PPMS (53%; p = 0.0026) vs SC. (B) C3a levels were increased in all CIS/MS groups and INDC (CIS 55%, RRMS 84%, SPMS 64%, PPMS 80%, and INDC 179%; all p ≤ 0.001). (C) Ba was increased in CIS (16%; p = 0.0377), RRMS (20%; p = 0.0178), and INDC (90%; p < 0.0001). (D) Bb levels were only higher in INDC (104%; p < 0.0001) vs SC. CAP = complement activation products; CC = complement components; CIS = clinically isolated syndrome; Est. = estimate; INDC = inflammatory neurologic disease controls; PPMS = primary progressive MS; RRMS = relapsing-remitting MS; SPMS = secondary progressive MS; SC = symptomatic controls; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Accordingly, levels of C3a were strongly increased vs SC in CIS (55%), RRMS (84%), SPMS (64%), and PPMS (80%, all p ≤ 0.001) with even stronger effects for the C3a/C3 ratio in CIS, RRMS, and PPMS. Different from C4a, where levels in progressive MS were numerically higher than in INDC, INDC showed higher C3a levels than in all stages of CIS/MS.

Ba was increased in CIS (16%) and RRMS (20%) vs SC. No differences between CIS/MS and SC were observed for later CC and CAP C5a, C5, s-C5b9, C1q, and Factor H and I. INDC showed highest levels for Ba, Bb, C1q, and Factor H and I (Table 2).

Plasma

Few differences between groups were observed in plasma: PPMS showed 33% increased C3a (p = 0.0198) and C3 was 14% reduced in RRMS (p = 0.0290) in comparison with SC (eTable 5, eFigure 2 for raw values, links.lww.com/NXI/A996).

CSF Complement Components and Activation Products' Levels in Ig Categories in CIS/Multiple Sclerosis

In OCGB⁺/IgG_IF⁻/IgM_IF⁻, CSF C3a was increased by 25% (p = 0.0225) (Table 3, Figure 2), while OCGB⁺/IgG_IF⁺/IgM_IF⁻ had higher levels for several early complement activation components vs OCGB⁻/IgG_IF⁻/IgM_IF⁻ (C4a: 28%; C3a: 67%; Ba: 20%; Bb: 37%; all p < 0.01). These findings were consistently most pronounced in OCGB⁺/IgG_IF⁺/IgM_IF⁺ (C4a: 58%; C3a: 134%; both p < 0.0001; Ba: 35%; Bb: 53%; both p < 0.01). In addition, in patients with an intrathecal IgM synthesis, the C1q level was increased by 23% (p = 0.0005). OCGB⁺/IgG_IF⁺/IgM_IF⁺ showed 16% decreased C4 levels (p = 0.0151). C3a/C3 and C4a/C4 ratios supported these findings with most pronounced increases in OCGB⁺/IgG_IF⁺/IgM_IF⁺ (Table 3). In plasma, no such patterns were found (data not shown).

Table 3.

Associations of CSF Immunoglobulin Categories With Complement Components and Activation Products' Levels in CSF in CIS/MS

		OCGB⁺/IgG_IF⁻/IgM_IF^−a, n = 50	OCGB⁺/IgG_IF⁺/IgM_IF^−a, n = 81	OCGB⁺/IgG_IF⁺/IgM_IF^+a, n = 55
C4a	Est 95% CI	1.15 (0.96–1.38)	1.28 (1.09–1.52)	1.58 (1.31–1.90)
C4a	p	0.1228	0.0033	<0.0001
C4	Est 95% CI	1.09 (0.96–1.25)	1.04 (0.92–1.18)	0.84 (0.73–0.97)
C4	p	0.2000	0.5595	0.0151
C4a/C4	Est 95% CI	1.06 (0.85–1.32)	1.24 (1.01–1.52)	1.87 (1.49–2.35)
C4a/C4	p	0.6226	0.0393	<0.0001
C3a	Est 95% CI	1.25 (1.03–1.50)	1.67 (1.41–1.99)	2.34 (1.91–2.82)
C3a	p	0.0225	<0.0001	<0.0001
C3	Est 95% CI	1.06 (0.76–1.49)	1.30 (0.95–1.77)	1.11 (0.78–1.57)
C3	p	0.7249	0.1054	0.5671
C3a/C3	Est 95% CI	1.17 (0.80–1.72)	1.29 (0.91–1.84)	2.11 (1.42–3.14)
C3a/C3	p	0.4190	0.1555	0.0003
C5a	Est 95% CI	0.86 (0.55–1.32)	1.06 (0.71–1.58)	1.25 (0.80–1.97)
C5a	p	0.4867	0.7640	0.3246
C5	Est 95% CI	1.01 (0.85–1.20)	0.94 (0.80–1.10)	1.05 (0.87–1.25)
C5	p	0.9313	0.4275	0.6271
C5a/C5	Est 95% CI	0.85 (0.53–1.37)	1.13 (0.73–1.76)	1.20 (0.73–1.97)
C5a/C5	p	0.50623	0.5758	0.4698
Ba	Est 95% CI	1.08 (0.93–1.25)	1.20 (1.05–1.38)	1.35 (1.16–1.57)
Ba	p	0.3298	0.0092	0.0002
Bb	Est 95% CI	1.21 (0.93–1.56)	1.37 (1.08–1.74)	1.53 (1.17–2.00)
Bb	p	0.1577	0.0096	0.0021
C1q	Est 95% CI	0.99 (0.89–1.11)	1.05 (0.95–1.16)	1.23 (1.09–1.37)
C1q	p	0.9235	0.3799	0.0005
Factor H	Est 95% CI	1.02 (0.95–1.09)	1.02 (0.96–1.09)	1.02 (0.95–1.09)
Factor H	p	0.3449	0.4855	0.6465
Factor I	Est 95% CI	0.99 (0.85–1.15)	1.04 (0.91–1.19)	1.11 (0.95–1.30)
Factor I	p	0.8625	0.5853	0.1926
sC5b-9	Est 95% CI	0.94 (0.59–1.51)	1.45 (0.94–2.23)	1.32 (0.81–2.15)
sC5b-9	p	0.8012	0.0943	0.2599

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Abbreviations: 95% CI: 95% confidence interval; CSF IgG_IF/M_IF^+/−: presence/absence of immunoglobulin G/M intrathecal fraction; Est: estimate; OCGB^+/−: presence/absence of oligoclonal IgG bands, p: p-value; Q_alb: albumin quotient.

vs category OCGB⁻/IgG_IF⁻/IgM_IF⁻(n = 44); adjusted for age, sex, Q_alb, and treatment categories—platform, orals, and monoclonal antibodies (vs untreated, respectively).

Marginal effects are displayed as derived from the multivariable analyses (Table 3). (A) In comparison with OCGB⁻/IgG_IF⁻/IgM_IF⁻, C4a was increased in OCGB⁺/IgG_IF⁺/IgM_IF⁻ (28% increase; p = 0.0033) and was most pronounced in OCGB⁺/IgG_IF⁺/IgM_IF⁺ (58%; p < 0.0001). (B) C3a levels were increasing along the CSF categories (vs OCGB⁻/IgG_IF⁻/IgM_IF⁻), and this increase was most pronounced in OCGB⁺/IgG_IF⁺/IgM_IF⁺ (134% increase; p < 0.0001). (C) Ba levels were increased in category OCGB⁺/IgG_IF⁺/IgM_IF⁻ (vs pattern OCGB⁻/IgG_IF⁻/IgM_IF⁻) by 20% (p = 0.0092) but were most pronounced in OCGB⁺/IgG_IF⁺/IgM_IF⁺ patients (increase 35%; p = 0.0002). (D) Bb levels were increased in category OCGB⁺/IgG_IF⁺/IgM_IF⁻ (vs pattern OCGB⁻/IgG_IF⁻/IgM_IF⁻) by 37% (p = 0.0096) but were most pronounced in OCGB⁺/IgG_IF⁺/IgM_IF⁺ patients (53%; p = 0.0021). CAP = complement activation products; CC = complement components; CIS = clinically isolated syndrome; CSF IgG_IF/M_IF^+/− = presence/absence of immunoglobulin G/M intrathecal fraction; OCGB^+/− = presence/absence of oligoclonal IgG bands. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Associations of CSF Complement Components and Activation Products With Clinical Disability Scores in CIS/Multiple Sclerosis

In multivariable analyses, average EDSS in CIS was increased by 0.32 steps per doubling of C4a (95% CI 0.02–0.62, p = 0.041) and by 0.31 steps per each C3a concentration doubling (Est. 0.31, 95% CI 0.06–0.56, p = 0.016). MSSS in CIS/MS at last follow-up was increased by 0.61 (CI 0.19–1.03, p = 0.0053) steps per each doubling of C3a, and in addition, increased Ba levels were associated with a 0.74 (CI 0.18–1.31, p = 0.0106) steps higher MSSS (Table 4, Figure 3, A and B).

Table 4.

Associations of CC and CAP in CSF With EDSS at LP in CIS, MSSS in CIS/MS, and CSF NfL Levels

	EDSS at LP (n = 105)^a			MSSS at last follow-up (n = 168)^b			CSF NfL levels (n = 235)
	Est.	95% CI	p Value	Est.	95% CI	p Value	Est.	95% CI	p Value
C4a	0.32	0.02 to 0.62	0.0415	0.38	0.14 to 0.91	0.1558	1.35	1.14 to 1.60	0.0005
C4	−0.25	−0.60 to 0.09	0.1553	0.20	−0.44 to 0.85	0.5420	0.89	0.70 to 1.13	0.3340
C3a	0.31	0.06 to 0.56	0.0157	0.61	0.19 to 1.03	0.0053	1.58	1.37 to 1.81	<0.0001
C3	−0.01	−0.17 to 0.16	0.9385	0.04	−0.24 to 0.31	0.7987	1.06	0.96 to 1.16	0.2500
C5a	−0.06	−0.19 to 0.08	0.4098	−0.04	−0.26 to 0.17	0.6879	1.04	0.97 to 1.12	0.2940
C5	−0.07	−0.45 to 0.31	0.7144	0.27	−0.25 to 0.79	0.3067	0.94	0.82 to 1.07	0.3369
Ba	0.27	−0.13 to 0.66	0.1870	0.74	0.18 to 1.31	0.0106	1.42	1.16 to 1.75	0.0010
Bb	0.03	−0.17 to 0.23	0.7419	0.24	−0.13 to 0.61	0.2005	1.17	1.04 to 1.32	0.0115
C1q	0.16	−0.31 to 0.63	0.4996	0.51	−0.32 to 1.33	0.2312	1.15	0.95 to 1.40	0.1613

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Abbreviations: 95% CI = 95% confidence interval; CAP = complement activation products; CC = complement components; CIS = clinically isolated syndrome; EDSS = Expanded Disability Status Scale score; Est = estimate; LP = lumbar puncture; MSSS = MS Severity score; NfL = neurofilament light chain; p = p-value; Q_alb = albumin quotient.

For EDSS at LP (dependent variable) and MSSS (dependent variable) at last follow-up, estimates represent additive effects of doubling of each CSF CC or CAP (independent variable, besides age, sex, Q_alb). For CSF NfL levels (dependent variable), estimates represent multiplicative effects of doubling of each CSF CC or CAP (independent variable, besides age, sex, Q_alb, and treatment categories—platform, orals, and monoclonal antibodies (vs untreated, respectively). Separate models per CC or CAP per line.

In 7 patients with CIS, the EDSS score at LP was not available.

Sixty-seven patients with CIS/MS had no follow-up/EDSS score at last visit, and in 4 patients, no Q_alb was available.

Estimates are displayed as derived from the multivariable analyses (A/B/C adjusted for age, sex, and Q_alb and C also adjusted for treatment categories at LP) (Table 4). Separate models per CC and CAP per line are shown. (A) EDSS at LP (n = 105) was on average increased by 0.32 points per doubling of CSF C4a levels (p = 0.0415) and for C3a by 0.31 points (p = 0.0157). (B) MSSS at last follow-up (n = 168) was 0.61 points (p = 0.0053) higher when doubling CSF C3a levels and 0.74 points higher (p = 0.0106) per doubling of Ba. Marginal effects are displayed as derived from the multivariable analyses adjusted for age, sex, and Q_alb (Table 4). (C) In CIS/MS (n = 235), doubling of several activation components was associated with higher CSF NfL levels (C4a: 35% higher CSF NfL levels (p = 0.0005); C3a: 58% (p < 0.0001); Ba: 42% (p = 0.0010); Bb 17% (p = 0.0115)) (Table 4). CAP = complement activation products; CC = complement components; CIS = clinically isolated syndrome; EDSS = Expanded Disability Status Scale score; LP = lumbar puncture; MSSS = MS Severity Score; NfL = neurofilament light chain; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Associations of CSF Complement Components and Activation Products With NfL Levels in CIS/Multiple Sclerosis

CSF levels of several CAP were associated with higher CSF NfL levels in multivariable analysis with most pronounced effects for C4a and C3a: doubling of C3a showed 58% higher CSF NfL levels (p < 0.0001). Similar results were seen for C4a (35%; p = 0.0005), Ba (42%; p = 0.0010), and Bb (17%, p = 0.0115) (Table 4, Figure 3C).

Discussion

Our study demonstrates that activation products of the classical and alternative pathways, particularly C3a, C4a, Ba, and Bb, are increased in the CSF of patients with CIS and MS in comparison with symptomatic controls. Increased levels of C4a, reflecting complement activation through the classical, antibody-mediated pathway, were most pronounced in progressive MS, even relative to INDC.

Our data corroborate earlier findings of increased levels of C3 activation products in the CSF of patients with CIS.²⁷ C4 levels were decreased, likely because of compound consumption. Later cascade complement activation products such as C5a or s-C5b9 were not increased in patients with CIS/MS, in line with previous findings.¹⁸

In accordance with others,^28,29 we did not find a consistent pattern of increased CC or CAP in plasma of patients with CIS/MS compared with symptomatic controls, suggesting that complement activation seems to be a part of a compartmentalized inflammatory process behind the blood-brain barrier.

We found associations between the albumin quotient, which was used as a proxy of the blood-brain barrier function and almost all CC and CAP levels in CSF. Their concentrations in plasma are in general substantially higher, so this can be explained by diffusion along the gradients. Subsequently, a proportion of CC and CAP in CSF should have their origin in the circulation.

However, it was shown that complement factors can also be produced by brain resident cells,³⁰ especially C1q and C3 seem to play a crucial pathophysiologic role and are upregulated in the more diseased MS brain.^8,12,31,32 The strongest increase of early complement cascade activation products (and upregulation of C1q) was observed in the OCGB⁺/IgG_IF⁺/IgM_IF⁺ pattern, while this was less pronounced in patients with only additional IgG_IF on top of OCGB, indicating main involvement of the classical pathway. The classical pathway of complement is triggered when antigen-bound IgM or IgG antibody molecules bind C1, which consists of the multimeric pattern recognition molecule C1q and a heterotetramer of the proteases C1r and C1s.³³ C1q binds a single IgG Fc segment with very low affinity. By contrast, antigen-driven antibody clustering and Fc-Fc interactions allow for multivalent C1q binding and optimal complement activation.³⁴ In pentameric star-shaped IgM molecules, Fc regions are already close to each other and cell surface binding of IgM readily exposes C1q binding sites.³⁵ These structural differences might account for the higher level of complement activation in patients with intrathecal IgM vs IgG production. Both antibody classes, however, require antigen binding for optimal complement activation, and the specificity of CSF-derived C1q binding of IgG and IgM remains to be elucidated.

The complement signature in patients with MS identified in this study is compatible with antibody-mediated activation of the classical pathway. In addition, we observed higher levels of activation products of the alternative pathway (AP) of complement. The AP of complement is continuously activated and can act as an amplification loop for all 3 pathways. It is initiated by the generation and binding of C3b. Initial antibody-mediated activation of complement can, therefore, trigger alternative pathway activation and thus lead to the amplification of complement activation.^33,36

The association of intrathecal activation products of the classical and alternative pathways (C4a, C3a, and Ba) with concurrent and future disease severity, as reflected by EDSS and MSSS, is a strong indicator for a critical role of the complement system in both inflammatory and neurodegenerative pathomechanisms of MS. It is of interest that the dose-dependent associations of NfL CSF levels with complement activation products are further indication that neuroaxonal damage may be driven by early complement cascade activation, i.e., in the absence of later cascade effector level complement activation beyond C3. These findings are paralleled by a large body of evidence from histopathologic studies suggesting complement activation as a driver of early plaque formation,² for both demyelination and axonal injury.^37,38 IgG and IgM colocalized with complement C3b on demyelinated axons and oligodendrocytes and antibody-antigen immunocomplexes were detected in foamy macrophages in active lesion areas.¹⁰ Recombinant monoclonal IgG1 antibodies from MS patients' CSF plasmablasts bound to conformational proteolipid protein 1 membrane complexes, when injected into the mouse brain with human complement recapitulated histologic features of MS pathology including complement deposition.³⁹

In the cortical and deep gray matter of patients with progressive MS, C1q depositions and activated complement component B and C3b were significantly increased, especially in areas with elevated numbers of complement receptor–positive microglia.¹² These findings are in line with a recent study demonstrating increased immunoreactivity for C1q, C4d, C3b, and Bb in thalamic lesions with an active inflammatory pathology in patients with progressive MS.⁴⁰

Complement activation is not restricted to lesional tissue but extends into normal-appearing white matter, the periplaque rims, perivascular inflammatory infiltrates, and astrocytic gliosis.

Diffuse axonal injury and microglial activation as well as clusters of microglia around damaged axons coated with C3b can be found, specifically after longer disease duration.^9,41,42 It is however not clear whether this is causatively linked to damage or may be a reactive mechanism to remove debris of damaged axons in a chronic stage.⁸

One limitation of our study is the lack of MRI data, and future scientific efforts will aim at providing further validation of our study results and an investigation of treatment effects on CC and CAP levels in longitudinal CSF samples.

Preclinical studies using experimental allergic encephalomyelitis (EAE) models of MS showed that reducing complement activation can be protective from neural tissue damage. Different strategies were using a cobra venom factor as a tool to consume complement⁴³ and untargeted or targeted pharmacologic inhibition of complement activation.⁴⁴ Treatment with recombinant soluble human complement receptor-1 (CR1), inhibiting C3 and C5 convertase, significantly reduced clinical disease severity, inhibited CNS inflammation, almost blocked demyelination, and markedly reduced tissue deposition of C1, C3, and C9 in an antibody-mediated demyelinating EAE rat model.⁴⁵ In a recent study, a C1q-blocking antibody efficiently reduced C1q staining intensity in the brain, and at a later EAE timepoint, also cells reflecting reactive gliosis in the white matter were significantly reduced. This suggested that pharmacologic inhibition of early complement activation might be a potential therapeutic avenue to address chronic inflammation in the white matter.³¹ Hence, complement inhibition beyond the blood-brain barrier might be a novel therapeutic approach to attenuate disease severity in MS and might be one of the highly needed tools to combat ongoing smoldering progression.

Acknowledgment

The authors express their deep thankfulness to patients and relatives for their participation and support, study nurses for their motivated collaboration and recruitment efforts, and the administrative personnel of the CSF databank study and the Swiss Multiple Sclerosis Cohort study.

Glossary

CAP: complement activation products
CC: complement components
CI: confidence interval
CIS: clinically isolated syndrome
DMT: disease-modifying treatment
EDSS: Expanded Disability Status Scale score
Est: estimate
INDC: inflammatory neurologic disease controls
IgG_IF/IgM_IF: immunoglobulin G/M intrathecal fraction
IQR: interquartile range
LLOQ: lower limit of quantification
LP: lumbar puncture
MS: multiple sclerosis
MSSS: Multiple Sclerosis Severity Score
NfL: neurofilament light chain
OCGB: oligoclonal IgG bands
PPMS: primary progressive multiple sclerosis
RRMS: relapsing-remitting multiple sclerosis
SC: symptomatic controls
SPMS: secondary progressive multiple sclerosis
ULOQ: upper limit of quantification

Appendix. Authors

Name	Location	Contribution
Johanna Oechtering, MD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; analysis or interpretation of data
Kerstin Stein, MSc	Department of Neurology with Institute of Translational Neurology, University Hospital 4 Münster, Germany	Major role in the acquisition of data
Sabine A. Schaedelin, MSc	Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research; Clinical Trial Unit, Department of Clinical Research, University Hospital Basel, University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; analysis or interpretation of data
Aleksandra M. Maceski, MSc	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data
Annette Orleth, PhD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Major role in the acquisition of data
Stephanie Meier, PhD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Eline Willemse, PhD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Ferhan Qureshi, BS	Octavebio Bioscience, Menlo Park, CA	Drafting/revision of the manuscript for content, including medical writing for content
Ingmar Heijnen, MD	Division of Medical Immunology, Laboratory Medicine, University Hospital Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data
Axel Regeniter, MD	Medica Laboratory, Zürich, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Tobias Derfuss, MD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Pascal Benkert, PhD	Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research; Clinical Trial Unit, Department of Clinical Research, University Hospital Basel, University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Marcus D'Souza, MD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Marguerite Limberg, Msc	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Major role in the acquisition of data
Bettina Fischer-Barnicol, MD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data
Lutz Achtnichts, MD	Department of Neurology, Cantonal Hospital, Aarau, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Stefanie Mueller, MD	Department of Neurology, Cantonal Hospital St. Gallen, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Anke Salmen, MD	Department of Neurology, Inselspital, Bern University Hospital and University of Bern, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Patrice H. Lalive, MD	Department of Clinical Neurosciences, Division of Neurology; Diagnostic Department, Division of Laboratory Medicine; Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Claire Bridel, MD, PhD	Department of Clinical Neurosciences, Division of Neurology, Geneva University Hospital, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Caroline Pot, MD	Division of Neurology, Department of Clinical Neurosciences, Lausanne University Hospital (CHUV) and University of Lausanne, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Renaud A. Du Pasquier, MD	Division of Neurology, Department of Clinical Neurosciences, Lausanne University Hospital (CHUV) and University of Lausanne, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Claudio Gobbi, MD	Neurocentre of Southern Switzerland, Multiple sclerosis centre, Ospedale Civico; Faculty of biomedical Sciences, Università della Svizzera Italiana (USI), Lugano, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Heinz Wiendl, MD, PhD	Department of Neurology with Institute of Translational Neurology, University Hospital 4 Münster, Germany	Drafting/revision of the manuscript for content, including medical writing for content
Cristina Granziera, MD, PhD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research; Translational Imaging in Neurology (ThINk) Basel, Department of Biomedical Engineering, Faculty of Medicine, University Hospital Basel and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Ludwig Kappos, MD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Marten Trendelenburg, MD	Division of Internal Medicine, University Hospital Basel and Clinical Immunology, Department of Biomedicine, University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; study concept or design
David Leppert, MD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content
Jan D. Luenemann, MD, PhD	Department of Neurology with Institute of Translational Neurology, University Hospital 4 Münster, Germany	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; analysis or interpretation of data
Jens Kuhle, MD, PhD	Department of Neurology; Multiple Sclerosis Centre and Research Center for Clinical Neuroimmunology and Neuroscience (RC2NB), Departments of Biomedicine and Clinical Research, University Hospital and University of Basel, Switzerland	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; analysis or interpretation of data

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Study Funding

This investigation was supported by the Swiss Multiple Sclerosis Society (research grant 2021/10). The Swiss MS Cohort study received funding from the Swiss Multiple Sclerosis Society and grant funding from Biogen, Bristol Myers Squibb, Celgene, Merck, Novartis, Roche, and Sanofi. The authors acknowledge support from the German Research Foundation (LU 900/3-1 to J.D.L. and through the Collaborative Research Centre SFB-TRR128 ′Initiating/Effector vs Regulatory Mechanisms in Multiple Sclerosis—Progress Towards Tackling the Disease' SFB/TR128 to H.W. and J.D.L).

Disclosure

K. Stein, S.A. Schaedelin, A.M. Maceski, A. Orleth, S. Meier, E. Willemse, I. Heijnen, A. Regeniter, P. Benkert, M. Limberg, and B. Fischer-Barnicol report no conflicts of interest. J. Oechtering received research support from the Swiss MS Society and served on advisory boards for Roche and Merck. F. Qureshi is an employee of Octave Bioscience, Inc. T. Derfuss received speaker fees, research support, travel support, and/or served on Advisory Boards, data safety monitoring boards, or Steering Committees of Actelion, Alexion, Celgene, Polyneuron, Novartis Pharma, Merck Serono, Biogen, Teva, Bayer-Schering, GeNeuro, Mitsubishi Pharma, MedDay, Roche, and Genzyme. M. D'Souza has received travel support from Bayer AG, Biogen, Teva Pharmaceuticals, and Sanofi Genzyme and research support from the University Hospital Basel. L. Achtnichts served on scientific advisory boards for Celgene, Novartis Pharmaceuticals, Merck, Biogen, Sanofi Genzyme, Roche, and Bayer; received funding for travel and/or speaker honoraria from Celgene, Biogen, Sanofi Genzyme, Novartis, Merck Serono, Roche, Teva, and the Swiss MS Society; and research support from Biogen, Sanofi, Genzyme, and Novartis. S. Mueller received speaker fees, research support, travel support, and/or served on advisory boards by Almirall, Alexion, Bayer, Biogen, Bristol Myers Squibb, Celgene, Genzyme, Merck-Serono, Teva, Novartis, and Roche. A. Salmen received speaker honoraria for activities with Bristol Myers Squibb, CSL Behring, Novartis, and Roche and research support from the Baasch Medicus Foundation, the Medical Faculty of the University of Bern, and the Swiss MS Society. P.H. Lalive received honoraria for speaking and/or travel expenses from Biogen, Merck, Novartis, and Roche; consulting fees from Biogen, GeNeuro, Merck, Novartis, Roche; research support from Biogen, Merck, and Novartis. None were related to this work. C. Bridel served on scientific advisory boards for Biogen, Novartis, and BMS. C. Pot received consulting fees and/or travel compensation, used exclusively for research support, for activities with Biogen, Merck, Novartis, Roche, and Sanofi Genzyme. R.A. Du Pasquier has served on scientific advisory boards for Biogen, Celgene, Merck, Novartis, Roche, and Sanofi. He has received funding for travel or speaker honoraria from Roche. H. Wiendl receives honoraria for acting as a member of Scientific Advisory Boards for Janssen, Merck, and Novartis as well as speaker honoraria and travel support from Alexion, Amicus Therapeuticus, Biogen, Biologix, Bristol Myers Squibb, Cognomed, F. Hoffmann-La Roche Ltd., Gemeinnützige Hertie-Stiftung, Medison, Merck, Novartis, Roche Pharma AG, Genzyme, TEVA, and WebMD Global. Prof. Wiendl is acting as a paid consultant for Biogen, Bristol Myers Squibb, EMD Serono, Idorsia, Immunic, Novartis, Roche, Sanofi, the Swiss MS Society, and UCB. His research is funded by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgesellschaft (DFG), Deutsche Myasthenie Gesellschaft e.V., Alexion, Amicus Therapeutics Inc., Argenx, Biogen, CSL Behring, F. Hoffmann-La Roche, Genzyme, Merck KgaA, Novartis Pharma, Roche Pharma, and UCB Biopharma. C. Gobbi: The University Hospital Basel (USB), as an employer of Cristina Granziera, has received the following fees which were used exclusively for research support: (1) advisory board and consultancy fees from Actelion, Novartis, Genzyme, and F. Hoffmann-La Roche; (2) speaker fees from Biogen and Genzyme-Sanofi; and (3) research support from F. Hoffmann-La Roche Ltd. Before her employment at USB, she has also received speaker honoraria and travel funding from Novartis. L. Kappos 's: employer (University Hospital Basel), has received and dedicated to research support steering committee, advisory board, and consultancy fees (Abbvie, Actelion, Almirall, Auriga Vison AG, Bayer HealthCare, Biogen, Eisai, EMD Derono Inc, Genzyme, Genentech Inc, F. Hoffmann-La Roche, Japan Tobacco, Janssen Pharmaceuticals Inc, Merck, Minoryx Therapeutics SL, Novartis, Sanofi, Santhera, Senda Biosciences, Shionogi BV, and TG Therapeutics); speaker fees (Bayer HealthCare, Biogen, Celgene, Genzyme, Janssen Pharmaceuticals Inc, Merck, Novartis, Roche, and Sanofi); support of educational activities (Allergan, Bayer HealthCare, Biogen, CSL Behring, Genzyme, Merck, Novartis, Roche, Pfizer, Sanofi, Shire, and Teva); license fees for Neurostatus products; and grants (Bayer HealthCare, Biogen, European Union, Innosuisse, Merck, Novartis, Roche Research Foundation, Swiss MS Society, and Swiss National Research Foundation). M. Trendelenburg has research collaborations with Roche, Novartis, and Idorsia (all Switzerland). D. Leppert is a Chief Medical Officer of GeNeuro. J.D. Luenemann received speaker fees, research support, travel support, and/or served on advisory boards by Abbvie, Alexion, Argenx, Biogen, Merck, Novartis, Roche, Sanofi, and Takeda. J. Kuhle received speaker fees, research support, travel support, and/or served on advisory boards by Swiss MS Society, Swiss National Research Foundation (320030_189140/1), University of Basel, Progressive MS Alliance, Bayer, Biogen, Bristol Myers Squibb, Celgene, Merck, Novartis, Octave Bioscience, Roche, and Sanofi. Go to Neurology.org/NN for full disclosures.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data are available on reasonable request.

[R1] 1.Attfield KE, Jensen LT, Kaufmann M, Friese MA, Fugger L. The immunology of multiple sclerosis. Nat Rev Immunol. 2022;22(12):734-750. doi: 10.1038/s41577-022-00718-z [DOI] [PubMed] [Google Scholar]

[R2] 2.Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol. 2000;47(6):707-717. doi: [DOI] [PubMed] [Google Scholar]

[R3] 3.Breij EC, Brink BP, Veerhuis R, et al. Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol. 2008;63(1):16-25. doi: 10.1002/ana.21311 [DOI] [PubMed] [Google Scholar]

[R4] 4.Ingram G, Loveless S, Howell OW, et al. Complement activation in multiple sclerosis plaques: an immunohistochemical analysis. Acta Neuropathol Commun. 2014;2:53. doi: 10.1186/2051-5960-2-53 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Loveless S, Neal JW, Howell OW, et al. Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis. Brain Pathol. 2018;28(4):507-520. doi: 10.1111/bpa.12546 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Morgan BP, Gommerman JL, Ramaglia V. An “Outside-In” and “Inside-Out” consideration of complement in the multiple sclerosis brain: lessons from development and neurodegenerative diseases. Front Cell Neurosci. 2020;14:600656. doi: 10.3389/fncel.2020.600656 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Compston DA, Morgan BP, Campbell AK, et al. Immunocytochemical localization of the terminal complement complex in multiple sclerosis. Neuropathol Appl Neurobiol. 1989;15(4):307-316. doi: 10.1111/j.1365-2990.1989.tb01231.x [DOI] [PubMed] [Google Scholar]

[R8] 8.Michailidou I, Naessens DM, Hametner S, et al. Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: implication for disease pathogenesis. Glia. 2017;65(2):264-277. doi: 10.1002/glia.23090 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Prineas JW, Kwon EE, Cho ES, et al. Immunopathology of secondary-progressive multiple sclerosis. Ann Neurol. 2001;50(5):646-657. doi: 10.1002/ana.1255 [DOI] [PubMed] [Google Scholar]

[R10] 10.Sadaba MC, Tzartos J, Paino C, et al. Axonal and oligodendrocyte-localized IgM and IgG deposits in MS lesions. J Neuroimmunol. 2012;247(1-2):86-94. doi: 10.1016/j.jneuroim.2012.03.020 [DOI] [PubMed] [Google Scholar]

[R11] 11.Storch MK, Piddlesden S, Haltia M, Iivanainen M, Morgan P, Lassmann H. Multiple sclerosis: in situ evidence for antibody- and complement-mediated demyelination. Ann Neurol. 1998;43(4):465-471. doi: 10.1002/ana.410430409 [DOI] [PubMed] [Google Scholar]

[R12] 12.Watkins LM, Neal JW, Loveless S, et al. Complement is activated in progressive multiple sclerosis cortical grey matter lesions. J Neuroinflammation. 2016;13(1):161. doi: 10.1186/s12974-016-0611-x [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Complement Activation Is Associated With Disease Severity in Multiple Sclerosis

Johanna Oechtering, MD

Kerstin Stein, MSc

Sabine A Schaedelin, MSc

Aleksandra M Maceski, MSc

Annette Orleth, PhD

Stephanie Meier, PhD

Eline Willemse, PhD

Ferhan Qureshi, BS

Ingmar Heijnen, MD

Axel Regeniter, MD

Tobias Derfuss, MD

Pascal Benkert, PhD

Marcus D'Souza, MD

Marguerite Limberg, MSc

Bettina Fischer-Barnicol, MD

Lutz Achtnichts, MD

Stefanie Mueller, MD

Anke Salmen, MD

Patrice H Lalive, MD

Claire Bridel, MD, PhD

Caroline Pot, MD

Renaud A Du Pasquier, MD

Claudio Gobbi, MD

Heinz Wiendl, MD, PhD

Cristina Granziera, MD, PhD

Ludwig Kappos, MD

Marten Trendelenburg, MD

David Leppert, MD

Jan D Luenemann, MD, PhD

Jens Kuhle, MD, PhD

Abstract

Background and Objectives

Methods

Results

Discussion

Introduction

Methods

Patients, Inclusion Criteria, and Data Collection

Standard Protocol Approvals, Registrations, and Patient Consents

Samples and Routine Cerebrospinal Fluid Analysis

Measurement of Complement Components and Their Activation Products

Measurement of CSF Neurofilament Light Chain Levels

Statistics

Data Availability

Results

Patients and Clinical Data

Table 1.

Complement Components, Activation Products, and Qalb, Age, and Sex

CSF

Plasma

Complement Components and Activation Products' Levels in CIS/Multiple Sclerosis and Control Groups

CSF

Table 2.

Figure 1. Adjusted Group Differences for CSF CC and CAP in Patients With CIS/MS and Inflammatory Neurologic Disease Controls vs Symptomatic Controls.

Plasma

CSF Complement Components and Activation Products' Levels in Ig Categories in CIS/Multiple Sclerosis

Table 3.

Figure 2. Adjusted Group Differences for CSF CC and CAP Stratified by Ig CSF Categories in Patients With CIS/MS.

Associations of CSF Complement Components and Activation Products With Clinical Disability Scores in CIS/Multiple Sclerosis

Table 4.

Figure 3. Adjusted Associations Between CC and CAP in CSF With (A) EDSS at LP in CIS and B) MSSS at Last Follow-up in Patients With CIS/MS and (C) NfL Levels in CIS/MS.

Associations of CSF Complement Components and Activation Products With NfL Levels in CIS/Multiple Sclerosis

Discussion

Acknowledgment

Glossary

Appendix. Authors

Study Funding

Disclosure

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Complement Components, Activation Products, and Q_alb, Age, and Sex