Low high-density lipoprotein cholesterol and high triglycerides lipid profile in neuromyelitis optica spectrum disorder: Associations with disease activity and disability

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Low high-density lipoprotein cholesterol and high triglycerides lipid profile in neuromyelitis optica spectrum disorder: Associations with disease activity and disability Eun Bin Cho , Hye-Jin Cho , Misong Choi , Jin Myoung Seok , Hee Young Shin , Byoung Joon Kim , Ju-Hong Min PII: DOI: Reference:

S2211-0348(20)30057-2 https://doi.org/10.1016/j.msard.2020.101981 MSARD 101981

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Multiple Sclerosis and Related Disorders

Received date: Accepted date:

25 January 2020 3 February 2020

Please cite this article as: Eun Bin Cho , Hye-Jin Cho , Misong Choi , Jin Myoung Seok , Hee Young Shin , Byoung Joon Kim , Ju-Hong Min , Low high-density lipoprotein cholesterol and high triglycerides lipid profile in neuromyelitis optica spectrum disorder: Associations with disease activity and disability, Multiple Sclerosis and Related Disorders (2020), doi: https://doi.org/10.1016/j.msard.2020.101981

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Highlights 

The patients with AQP4-positive NMOSD had low high density lipoprotein (HDL) and high triglyceride (TG) regardless of disease activity compared to healthy controls



During attack, HDL-C levels were significantly lower compared to those in remission

 The TG level has positive correlation with EDSS scores at the time of sampling

Low high-density lipoprotein cholesterol and high triglycerides lipid profile in neuromyelitis optica spectrum disorder: Associations with disease activity and disability

Eun Bin Choa,b,c, Hye-Jin Choc, Misong Choic, Jin Myoung Seokd, Hee Young Shine, Byoung Joon Kimc,f and Ju-Hong Minc,f*

a

Department of Neurology, College of Medicine, Gyeongsang Institute of Health Science, Gyeongsang

National University, Jinju, Republic of Korea. b

Department of Neurology, Gyeongsang National University Changwon Hospital, Changwon, Republic

of Korea. c

Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine,

Seoul, Republic of Korea d

Department of Neurology, Soonchunhyang University Cheonan Hospital, Soonchunhyang University

College of Medicine, Cheonan, Republic of Korea e

Center for Health Promotion, Samsung Medical Center, Sungkyunkwan University School of Medicine,

Seoul, Republic of Korea f

Neuroscience Center, Samsung Medical Center, Seoul, Republic of Korea

Correspondence: Ju-Hong Min Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Ilwon-ro Gangnam-Gu, Seoul, 135-710, Korea Tel: +82-2-3410-3599 Cell phone: +82-10-9427-0592 E-mail: [email protected] †-

Equally contributed to the paper

Total word count:

Running title:

low high-density lipoprotein cholesterol and high triglycerides in neuromyelitis optica

spectrum disorder

Keywords: neuromyelitis optica, lipid profile, triglyceride, high-density lipoprotein, activity, disability

Abstract

Background: Altered lipid metabolism is a feature of systemic autoimmune diseases. Dyslipidemia is associated with the disease activity and progression in patients with multiple sclerosis (MS). However, in neuromyelitis optica spectrum disorder (NMOSD), changes in the lipid profile and the associations between specific lipid levels and disease activity/disability are unknown. Methods: Serum samples (N = 148) were collected from 53 patients with aquaporin-4 (AQP4)-positive NMOSD when they were not treated with lipid lowering agents. Fasting lipid (total cholesterol, triglyceride [TG], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C]) levels were compared between 39 patients with NMOSD, not taking steroids, and 142 age-, sex-, and body mass index-matched healthy controls. In addition, we analyzed the differences in the lipid profile between attack and remission samples and the associations between lipid profiles and clinical outcome in all 148 samples from 53 patients. The generalized estimating equation was used. Results: Patients with NMOSD showed lower HDL-C and higher TG levels compared to healthy controls (p = 0.017 and p < 0.001, respectively). HDL-C level was significantly lower during attack than remission (β = -7.851; p = 0.035), and TG level had positive correlation with EDSS scores (β = 0.014; p = 0.002) regardless of disease activity status. However, enhanced lesions on magnetic resonance imaging were not associated with lipid profiles. Conclusion: Dyslipidemia with low HDL-C and high TG correlated disease activity and disability in AQP4-positive NMOSD. It remains to be elucidated whether altered lipid metabolism contributes to deleterious immune response, possibly through inflammation, or is secondary to neurological disability in NMOSD.

Introduction Neuromyelitis optica (NMO) is an autoimmune demyelinating disorder of the central nervous system (CNS), characterized by anti-aquaporin-4 (AQP4) antibody-mediated astrocytopathy. Pathologically, NMO lesions show active demyelination, inflammation and necrosis as well as AQP4 loss and astrocyte damage (Kawachi and Lassmann, 2017), which is distinct from demyelinating disorders as represented by multiple sclerosis (MS). Cholesterol is an essential component of an intact myelin sheath, mainly formed by oligodendrocytes and partly by plasma membranes of astrocytes and neurons (Dietschy and Turley, 2004). CNS and plasma cholesterol/lipoprotein compartments are strictly segregated by the blood–brain barrier (BBB); however, during oxidative stress, hydroxycholesterols (OHCs), metabolites of CNS cholesterol, can have damaging effects on BBB function and consequently on neuronal cells (Dias et al., 2014). In addition, cholesterol-enriched membrane microdomains such as lipid rafts and caveolae play a pivotal role in inflammatory and immune signaling (Fessler and Parks, 2011). Hypercholesterolemia enhances the inflammatory response in the vascular endothelium to induce monocyte adhesion, endothelial cell apoptosis, and vasodilation (Hansson and Libby, 2006). Conversely, inflammation can also lead to changes in lipid metabolism such as a decrease in highdensity lipoprotein cholesterol (HDL-C) level with impairment in reverse cholesterol transport (Esteve et al., 2005). Autoimmune disorders such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjogren syndrome have shown the association between dyslipidemia and systemic inflammation (de Carvalho et al., 2008; Lodde et al., 2006; McMahon et al., 2006). RA patients showed significantly reduced HDL-C and elevated triglyceride (TG) level; specifically, dysfunctional HDL-C exacerbated the inflammatory and atherogenic processes (Kim et al., 2016). High occurrence of dyslipidemia has been reported in MS patients, and adverse lipid profiles were also associated with increased disability and disease progression (Murali et al., 2019; Tettey et al., 2014; WeinstockGuttman, Bianca et al., 2011). However, there were few studies regarding lipid profile and disease activity in NMO. Because both peripheral and CNS inflammation play important roles in the immunopathogenesis of NMO (Uzawa et al., 2014), we expected that dyslipidemia might exist and was

associated with disease activity. In the present study, we compared the lipid profiles between patients with AQP4-positive NMOSD, during attack and remission, and healthy controls and the clinical relevance of dyslipidemia in NMOSD was elucidated.

Methods We reviewed consecutive patients with NMOSD, treated from January 1, 2012 to December 31, 2014 in Samsung Medical Center (Seoul, South Korea). A total of 54 patients (mean age 42.19 ± 16.16 years, female: male = 48:6) met the revised criteria for NMOSD with anti-AQP4 antibodies (Wingerchuk et al., 2015) measured by cell-based indirect immunofluorescence assay (Kang et al., 2012). We analyzed demographic, clinical, laboratory, and MRI data such as age, sex, body mass index (BMI), disease duration, disease activity states (attack or remission), number of relapses, Expanded Disability Status Scale (EDSS) score, medications including steroids and statins, lipid profiles (total cholesterol, TG, HDL-C, and low-density lipoprotein cholesterol; LDL-C), and the presence of enhancing lesions on magnetic resonance images (MRI) of brain/orbit/spinal cord, which were performed at attack. Attack (relapse) was defined as acute onset of neurologic symptoms lasting for 24 hours or more, and remission was stabilized neurologic symptoms at least 90 days after an attack. Venous blood samples were collected from the antecubital vein into vacuum tubes after a 12hour overnight fast. If the patients admitted during attack period, fasting blood samples were drawn within 24 hours of admission. We measured total cholesterol (normal range, 110−240 mg/dL), TG (normal range, 50−200 mg/dL), HDL-C (normal range, 30−50 mg/dL), and LDL-C (normal range, 40 − 130 mg/dL) levels by an enzymatic colorimetric method using a Modular D2400 (Roche Diagnostics, Basel, Switzerland). Due to repetitive blood sampling during the disease course, a total of 165 samples were available from 54 patients. Among them, we finally included 53 patients who had blood sample(s) (N = 148) not under the influence of statins. MRIs (brain, orbit, or spinal cord) obtained within one month from an attack were available for 40 patients. In addition, we collected the data of healthy controls who visited the Center for Health Promotion at our hospital for regular check-ups between January 2012 and December 2014. Healthy

controls did not have any history of neurological and medical disorders such as diabetes mellitus, dyslipidemia, and autoimmune diseases. A total of 15,232 subjects were found. This study was approved by the Institutional Review Board of Samsung Medical Center, and written informed consent was obtained from all patients. The requirement for informed consent in healthy controls was waived because we only used de-identified data collected for clinical purposes during health screening exams.

Statistical analysis The statistical data were summarized as continuous and categorical variables. Continuous variables were reported as mean ± standard deviation or median (interquartile range), and categorical variables were presented as absolute and relative frequencies. For the comparison of lipid profiles between patients and healthy controls, we only included one blood sample per patient which was drawn when the patient had not been treated with steroid that can affect lipid profiles (Ettinger et al., 1987). And then, age-, sex-, and BMI-matched healthy controls were selected by a propensity score matching algorithm in a 1:4 (patient to control) ratio by using the R package MatchIt (R Foundation for Statistical Computing, Vienna, Austria). The propensity score matched groups were compared using the generalized estimating equations (GEE) approach. In addition, we analyzed the association between lipid profiles and clinical outcomes in all 148 samples from 53 patients. Age at sampling, sex, BMI, disease duration, time interval from first sampling within a patient, disease activity states (attack or remission), number of relapses, the use of oral steroid or immunosuppressant were used as covariates. The associations between each lipid level and disease activity states were also evaluated. We used GEE to take into accounts the correlations between observations on the same individual. The associations of lipid profiles with MRI findings at attack (N = 40) were also analyzed using GEE. Statistical analysis was performed using SPSS version 24 (SPSS, Inc., Chicago, IL, USA ) and graphed using the R package ggplot (R Foundation for Statistical Computing, Vienna, Austria).

Results

Altered lipid profiles in NMOSD patients The samples not under the influence of steroid were available in 39 patients with NMOSD. Among blood samples from 39 patients, 20 samples were collected at attack and 19 samples during remission. After performing propensity score matching for the entire healthy control candidates, a total of 142 age-, sex-, and BMI-matched healthy controls were created. The lipid profiles were compared between patients with NMOSD and matched healthy controls (Table 1). Serum TG levels were higher and HDL-C levels were lower in patients with NMOSD than in healthy controls (TG: 120.1±66.2 mg/dL vs. 93.9±62.3 mg/dL, p = 0.017; HDL-C: 50.8±18.1 mg/dL vs. 65.0±15.8 mg/dL, p < 0.001). Theses associations were maintained in both attack and remission samples (Figure 1). However, no differences were found in total cholesterol and LDL-c levels between patients and healthy controls.

Associations of lipid profiles with disease activity and disability in NMOSD patients On average, the study patients had available 3 (interquartile range [IQR], 1−4) serum samples. The median time between the first and the last blood draw was 3.9 (IQR, 2.3−8.0) years. Among 148 samples, 45 samples from 33 patients were collected at attack and 103 samples from 36 patients were during remission. During attack, HDL-C levels were significantly lower compared to those in remission (ßcoefficient = -7.851, p = 0.035), even after adjusting for age at sampling, sex, BMI, time interval from initial blood sampling, disease duration, number of relapses and the use of oral steroid or immunosuppressant, whereas total cholesterol, TG, and LDL-C levels were not significantly different between during attack and remission (Table 2). The level of HDL was also higher in patients taking oral prednisolone compared to those without (ß-coefficient = 14.442, p = 0.011; Table 2). The TG levels and BMI were correlated with EDSS scores at the time of sampling by multivariable analyses (ß-coefficient = 0.014, p = 0.002; ß-coefficient = -0.240, p = 0.004). However, other factors such as age at sampling, sex, disease duration, number of relapses until sampling, disease activity (attack or remission) at sampling, total cholesterol, HDL-C, and LDL-C levels, the use of oral steroid or immunosuppressant

were not associated (Table 3).

Association of lipid profile with MRI observations at relapse in NMOSD patients Forty samples were analyzed for the relationship between lipid profile and lesions on brain/spinal cord/optic MRI within 6.3 ± 9.8 days from blood sampling. Serum total cholesterol, TG, HDL-C, and LDL-C levels were not associated with the presence of enhanced lesions on MRI, after adjusting for the use of oral steroid or immunosuppressant.

Discussion The links between the altered blood lipid profile and disease activity in NMOSD have been poorly understood. Our findings showed that the patients with NMOSD had higher TG and lower HDLC levels, regardless of disease activity, compared to healthy subjects. In addition, low HDL-C and elevated TG levels were associated with disease activity and disability, respectively, in NMOSD. Dyslipidemia has been reported in various autoimmune disorders. Patients with RA had elevated TG and reduced HDL-C levels, especially during high disease activity status, and this profile was associated with systemic inflammation (Kim et al., 2016; Rodriguez-Carrio et al., 2017). In SLE, the ‘lupus pattern’ lipid profile (high TG and low HDL-C level) is well-known and it was reported that increased TNF-ɑ, a proinflammatory cytokine, could induce dyslipoproteinemia, insulin resistance, and endothelial cell activation (Borba et al., 2006; Svenungsson et al., 2003). Lower HDL-C levels and/or higher TG levels were also observed in patients with SS, early polymyositis, and dermatomyositis (Lodde et al., 2006; Wang et al., 2014; Wang et al., 2013). Regarding MS, heterogeneous results have been reported for dyslipidemia, but most studies showed higher total cholesterol and/or higher LDL-C levels compared with healthy controls (Zhornitsky et al., 2016). The levels of apolipoprotein A-I (apoAI), the most abundant component of HDL, and/or HDL-C seemed to be decreased in MS compared to healthy controls, but there were also inconsistent results (Gafson et al., 2018; Murali et al., 2019). Recently, it was also reported that serum apoA-I level was significantly lower in patients with NMO than in subjects with acute transverse myelitis (ATM) (Zhong, Y.-H. et al., 2013). Another study

showed that NMO patients had higher TG, total cholesterol, and LDL-C levels than healthy controls, although HDL-C levels were not different between the two groups (Li et al., 2010). However, this study included patients with NMO at attack who met the diagnostic criteria for NMO in 1999 (Wingerchuk et al., 1999), which did not require the presence of AQP4-ab; therefore, seronegative patients with optic neuritis and myelitis may affect their results (Li et al., 2010). In addition, acute steroid therapy could elevate HDL-C level probably offsetting decreased HDL-C (Zhong, Y.-H. et al., 2013). Therefore, we newly elucidated altered lipid profiles in patients with seropositive NMOSD, high TG and low HDL-C levels, which resembled to those in patients with autoimmune disorders such as RA and SLE. In the present study, serum TG levels have positive association with EDSS scores and HDLC level was significantly lower at attack than during remission. There are lines of evidence that lipids influence inflammation and vice versa. Triglycerides and triglyceride-rich lipoprotein remnants are associated with upregulated pro-inflammatory cytokine production and enhanced inflammatory response and monocyte activation (Alipour et al., 2008). During acute phase reaction, HDL-C and apoA-I levels are reduced and the proteins associated with HDL is altered influencing anti-oxidant and anti-inflammatory properties of HDL (Norata et al., 2012). HDL has an ability to modulate cholesterol content in cell lipid rafts affecting a series of responses in immune cells including macrophages, B and T lymphocytes. In addition, ApoA-I reduces inflammation in the CNS by preventing contact between T cells and macrophages (Norata et al., 2012). Therefore, high TG and low HDL lipid profiles in our patients may indicate high disease activity of seropositive NMOSD by inflammatory mediators such as proinflammatory cytokines. There were very few studies regarding lipid profile and disease activity in NMO, but they were mostly in line with our results. A recent study showed that TG level was positively associated with poor recovery in first-attacked NMOSD (Wu et al., 2019). The association between serum apoA-I and EDSS scores in patients with NMO was not found, although TG and HDL-C levels were not evaluated (Zhong, Y.H. et al., 2013). Although one study for NMO revealed that HDL-C level during attack was positively correlated with number of relapses, they indicated that the predictive value of this result must be limited considering that HDL-C plays as an anti-inflammatory factor (Li et al., 2010). Most MS studies indicated that total cholesterol and LDL-C levels were associated with EDSS

score, however, a few studies have analyzed TG or HDL-C (Zhornitsky et al., 2016). Higher baseline TG level was associated with a worse EDSS score in MS (Weinstock-Guttman, B. et al., 2011) and a relapse in the first demyelinating event (Tettey et al., 2017). The concentration of TG-rich very low density lipoprotein also showed high correlation with EDSS score after controlling for age, sex, disease duration and BMI (Gafson et al., 2018). In addition, higher HDL-C level in MS patients was associated with lower contrast-enhancing lesion volumes on brain MRI (Palavra et al., 2013; Tettey et al., 2014; Weinstock-Guttman, Bianca et al., 2011; Weinstock-Guttman, B. et al., 2011). In these studies, the working hypothesis was that the pro-inflammatory and thrombogenic processes associated with dyslipidemia could plausibly contribute to disease progression in MS. In NMOSD, the mechanisms by which serum lipid may influence disease activity and disability and the usefulness of lipid lowering therapy on disease prognosis should be established in the near future. This study had several limitations. First, the number of patients was relatively small, however, repeated measures during disease course could partly overcome this weakness. Second, we did not assess the level of other inflammatory parameters, such as CRP or cytokines (e.g. interleukin-6) nor assessed their association with the level of lipids, which could be more informative about lipid profile changes in NMOSD. Third, this was a cross-sectional study, which cannot conclude the cause and effect relationship between lipid profile and clinical outcomes. Finally, although we tried to use samples from patients not under the influence of drugs to compare with healthy controls, we analyzed all NMOSD samples to elucidate the effect of TG and HDL-C on disease disability or activity; the adjustment of the confounding factors could be limited. In our knowledge, this is the first study showing high TG and low HDL lipid profile in AQP4positive NMOSD during remission as well as attack. In addition, this lipid profile was associated with disease activity and disability in NMOSD. Further prospective large-cohort research is needed to investigate the role of TG and HDL-C as biomarkers and to elucidate whether altered lipid profiles are caused by or result in inflammation in NMOSD patients.

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Table 1 Lipid profiles in 39 NMOSD patients (at attack or during remission) and healthy controls NMOSD

Healthy control*

(N = 39)

(N = 142)

Age at sampling

43.6±16.8

44.6±12.4

0.820

Female (%)

19 (95.0)

72 (94.7)

1.000

BMI, kg/m2

22.0±2.8

22.0±4.0

0.451

15.3 (1.3−43.3)

N/A

8 (21)

N/A

Total cholesterol, mg/dL

181.8±49.6

191.9±35.9

0.252

TG, mg/dL

120.1±66.2

93.9±62.3

0.017

HDL-C, mg/dL

50.8±18.1

65.0±15.8

<0.001

LDL-C, mg/dL

113.4±37.7

118.2±33.7

0.535

Disease duration, month‡ Use of immunosuppressant†† (%)

p-value†

NMOSD, neuromyelitis optica spectrum disorder; N, number; BMI, body mass index; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; N/A, not applicable *

age-, sex-, and BMI-matched healthy controls by a propensity score matching (1:4)

†

Generalized estimating equations (GEE) method was used for the matched groups

‡

median (interquartile range)

††

azathioprine, mycophenolate, cyclophosphamide, cyclosporin, and methotrexate

Table 2 Factors associated with high-density lipoprotein cholesterol levels in patients with neuromyelitis optica spectrum disorder Multivariable analysis (p<0.1 in Uni)

Univariable analysis Beta coefficient -0.072

Standard Error 0.172

pvalue* 0.674

Beta coefficient

Standard Error

pvalue*

Sex

-13.652

4.188

0.001

-9.133

3.291

0.006

BMI

-0.480

0.889

0.589

Disease duration

-0.465

0.385

0.227

Time from first sampling

0.001

0.002

0.483

Samples at attack

-11.789

4.317

0.006

-7.581

3.594

0.035

Total attacks until sampling

-0.341

0.628

0.587

Use of steroids

16.154

5.700

0.005

14.442

5.696

0.011

Use of immunosuppressant

3.502

5.803

0.546

Age at sampling

Uni., univariable analysis; BMI, body mass index *

Generalized estimating equations (GEE) method was used for the repeated samples.

Table 3 Association between demographic factors, lipid profiles and neurological disability, measured by the Expanded Disability Status Scale (EDSS) score in patients with neuromyelitis optica spectrum disorder Multivariable analysis (p<0.1 in Uni)

Univariable analysis Beta coefficient 0.011

Standard Error 0.031

pvalue* 0.710

Sex

0.594

1.001

0.555

BMI

-0.178

0.083

0.032

Disease duration

0.055

0.030

0.373

Time from first sampling

0.000

0.002

0.552

Samples at attack

0.318

0.740

0.667

Total attacks until sampling

0.062

0.082

0.451

Total cholesterol

-0.002

0.007

0.717

Triglyceride

0.011

0.005

0.020

HDL-C

-0.016

0.024

0.517

LDL-C

0.001

0.008

0.923

Use of steroids

0.330

0.999

0.739

Use of immunosuppressant

0.053

0.764

0.945

Age at sampling

Beta coefficient

Standard Error

pvalue*

-0.240

0.083

0.004

0.014

0.004

0.002

Uni., univariable analysis; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol *

Generalized estimating equations (GEE) method was used for the repeated samples.

Figure 1 Comparison of serum triglyceride (A) and high-density lipoprotein cholesterol (B) levels between neuromyelitis optica spectrum disorder (NMOSD) patients and healthy controls (HCs). Attack and remission samples of NMOSD and corresponding HC samples were separately represented. TG, triglyceride; HDL-C, high-density lipoprotein cholesterol

Conflicts of interests The author declares that there is no conflict of interest.

Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03934476).