Elevated C‐X‐C motif ligand 13 and B‐cell‐activating factor levels in neuromyelitis optica during remission

Abstract Background Discovery of specific antibodies against astrocytic water channel aquaporin‐4 (AQP4), which is produced by plasma cells, in the serum of neuromyelitis optica (NMO) confirmed the pathogenic role of B cells in NMO. C‐X‐C motif ligand 13 (CXCL13) and B‐cell‐activating factor (BAFF) are two crucial factors for antibody production. Relevant studies have focused on the acute phase of NMO. However, CXCL13 and BAFF levels during remission, remain to be elucidated. Objective To evaluate serum levels of CXCL13 and BAFF in NMO and multiple sclerosis (MS) patients during remission and explore their correlation with immunosuppressive agents and clinical features in NMO. Methods Serum CXCL13 and BAFF were measured by enzyme‐linked immunosorbent assay (ELISA) in NMO patients, MS patients, and controls. Results Serum CXCL13 levels of NMO patients (n = 24) were significantly higher than those of controls (n = 22) (p = .001), but CXCL13 levels of MS patients (n = 20) and controls (n = 22) did not differ significantly (p = .279). Although the three groups showed no differences in serum BAFF levels, serum BAFF levels of NMO patients without immunosuppressive treatment (n = 8) were significantly elevated compared with those of NMO patients with immunosuppressive therapy (n = 16) (p = .003) and controls (n = 22) (p = .024). In NMO patients, CXCL13 levels were correlated with onset age (p = .026) and duration to the last relapse (p = .003). Conclusion During remission, serum CXCL13 and BAFF levels have not decreased to normal in NMO patients, and B‐cell‐related autoimmune response persists. Immunosuppressive therapy decreased serum BAFF levels, but did not affect CXCL13 expression.

C-X-C motif ligand 13 is currently considered a potential biomarker of NMO. Serum and cerebrospinal fluid (CSF) concentrations of CXCL13 are reportedly elevated in NMO patients during relapse; furthermore, CXCL13 levels have a higher trend in NMO than in MS (Alvarez et al., 2013;Festa et al., 2009;Quan et al., 2013;Zhong et al., 2011). Meanwhile, studies have demonstrated that CXCL13 levels have a strong correlation with some clinical measures and other biomarkers. CSF CXCL13 levels are correlated with Expanded Disability Status Scale scores, annualized relapse rates (ARR), CSF white blood cells counts, and other measures in NMO and MS (Alvarez et al., 2013;Zhong et al., 2011), and serum CXCL13 levels are correlated with remission extent and magnetic resonance imaging activity during the first and second years, but not with interferon-β1b or glatiramer acetate levels in MS patients (Festa et al., 2009). To date, most studies regarding CXCL13 have focused on the acute phases of NMO and MS. CXCL13 concentrations during remission remains unclear, and the correlation between serum CXCL13 levels and the use of immunosuppressive therapy (azathioprine, steroids), the first-line treatment of NMO during remission, has not been studied.
B-cell-activating factor, also known as B lymphocyte stimulator or the tumor necrosis factor (TNF) superfamily member 13B (TNFSF13B), is another key cytokine in B-cell development (Schneider et al., 1999).
BAFF levels are increased in systemic lupus erythematosus, rheumatoid arthritis, and Sjӧgren syndrome, among other conditions (Morais, Vilas-Boas, & Isenberg, 2015). Elevated CSF BAFF levels are also observed in the acute relapse phase of NMO, which differs from that of MS (Quan et al., 2013;Vaknin-Dembinsky, Brill, Orpaz, Abramsky, & Karussis, 2010;Wang et al., 2012). Although no differences were seen in serum BAFF levels among NMO patients, MS patients, and healthy donors, increased serum BAFF levels were found in MS patients treated with interferonβ (Krumbholz et al., 2008;Vaknin-Dembinsky et al., 2010) and NMO patients treated with rituximab (Gredler et al., 2013). In other words, common therapeutic modalities used to treat NMO and MS therapies affect serum BAFF levels. During remission, most patients with NMO receive treatment consisting of azathioprine and steroids, two nonspecific immunosuppressive agents. We suspected that immunosuppressive therapy could inhibit BAFF expression and that higher serum BAFF levels might exist in NMO patients without treatment during remission, which would indicate persistent humoral immune dysfunction.
Here, we aimed to confirm whether serum CXCL13 and BAFF levels were abnormal in NMO and MS patients during remission and whether we could discriminate between the two diseases accordingly.
We also investigated the effect of immunosuppressive therapy on CXCL13 and BAFF expression in NMO.

| Patients and controls
Twenty-four NMO patients who met the 2006 Wingerchuk diagnostic criteria (Wingerchuk, Lennon, Pittock, Lucchinetti, & Weinshenker, 2006) and 20 MS patients who met the 2010 McDonald's diagnostic criteria (Polman et al., 2011) from Beijing Tiantan Hospital were enrolled. Serum samples were collected during remission of MS and NMO (>1 month from the last relapse and without new symptoms).
Three of the relapsing-remitting MS (RRMS) patients were using Betaseron and the other 17 RRMS patients had no treatments. A total of 16 NMO patients were using immunosuppressive therapy (oral glucocorticoids, n = 7; azathioprine, n = 2; oral glucocorticoids combined with azathioprine, n = 7); Twenty-two volunteers without CNS inflammatory demyelinating diseases were recruited as controls. The study was approved by the local ethics committee (IRB of Beijing Tiantan Hospital Affiliated to Capital Medical University, No. KY2015-003-02) and informed consent was obtained from all participants.

| Enzyme-linked immunosorbent assay
The serum samples were centrifuged with 1220g for 10 min and

| Statistical analysis
The data were analyzed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA. RRID: SCR_002865). The different groups were compared with nonparametric Mann-Whitney U test or Student's t test according to normality. Correlations between CXCL13/BAFF levels and clinical features (onset age, annualized relapse rate, disease course, and time from the last relapse) were analyzed using Spearman's rank test.

| Compliance with ethical standards
This study was approved by the local ethics committee (IRB of Beijing Tiantan Hospital Affiliated to Capital Medical University, No. KY2015-003-02) and informed consent was obtained from all participants.

| Patient demographics
The mean ages of the groups (NMO, MS, and control) were similar.
The demographics and clinical features of NMO and MS patients are shown in Table 1 and details in Table 2.

| Disease duration and duration to the last relapse in NMO
There were no significant differences in terms of disease duration and duration to the last relapse between NMO patients treated with immunosuppressive agents (group A, n = 16) and NMO patients who were not treated with immunosuppressive agents (group B, n = 8) (Figure 1b).

| CXCL13 correlation with clinical features in NMO
In NMO patients, CXCL13 was correlated with onset age

| DISCUSSION
Here, we analyzed serum CXCL13 and BAFF levels in NMO  were 500 times higher than those in the CSF, which demonstrates that NMO-IgG is produced in the peripheral blood but not in the intrathecal area (Takahashi et al., 2007). Research to date has indicated that B-cellmediated inflammation exists not only in the CNS but also in the peripheral blood of NMO patients. Therefore, peripheral B-cell-mediated inflammation is correlated with disease activity in NMO. In this study, we detected crucial peripheral factors involved in B-cell-mediated inflammatory reaction. CXCL13 interacts with CXCR5 on B cells and promotes B-cell development within the secondary lymphoid tissues by chemotactic recruitment (Legler et al., 1998). BAFF is a major factor for peripheral immature B lymphocyte survival and differentiation in response to signals via the B-cell receptor (Batten et al., 2000). Both play important roles in B-cell-mediated inflammation.
In the relapse phase, CD138 + HLA − DR + plasma blasts are increased in the peripheral blood of patients with NMO (Chihara et al., 2013) and cause severe inflammatory activity. The serum gradients of CXCL13 are higher in NMO patients within 2 months of symptom relapse than in noninflammatory neurological disease controls (Alvarez et al., 2013 (Frischer et al., 2009), but studies have seldom focused on the remission period of NMO.
Our research focused on NMO patients during remission and resulted in a few main findings. First, we found significantly increased CXCL13 levels in NMO, a finding that is consistent with previous studies of the relapse phase (Alvarez et al., 2013;Quan et al., 2013). discriminate between the two diseases by CSF and serum CXCL13 concentrations, even in the acute phase (Alvarez et al., 2013). Azathioprine is a purine synthesis inhibitor that interferes with cellular proliferation, particularly leukocytes. Azathioprine in combination with oral prednisolone has been shown to be an effective treatment for NMO and has been recommended as the first-line treatment during remission (Sellner et al., 2010). Our study revealed that BAFF was one of its targets and confirmed the necessity of use in NMO remission. There were no significant differences between serum CXCL13 levels in NMO patients with immunosuppressive therapy and other NMO patients. CXCL13 is secreted in the secondary lymphoid organs by follicular dendritic cell and macrophages (Carlsen et al., 2004;Gunn et al., 1998). We suspect that immunosuppressive treatment had little influence on these cells and could not affect CXCL13 expression. One murine model study showed that anti-CXCL13 antibody was effective in the treatment of autoimmune disorders (Klimatcheva et al., 2015). Despite other studies that have demonstrated that CXCL13 is elevated in many inflammatory CNS diseases and is not specific to NMO and MS, our findings show that therapy targeting CXCL13 may also be useful for NMO (Kothur, Wienholt, Brilot, & Dale, 2016).
Finally, we discovered that serum CXCL13 levels declined with prolonged time to the last relapse, which suggests that it is correlated with disease activity. High concentrations of CXCL13 were unfavorable for NMO recovery. However, immunosuppressive treatment was ineffective for CXCL13 expression. Accordingly, we suspect that therapy targeting CXCL13 in combination with immunosuppressive treatment may be more useful in reducing NMO recurrence. Furthermore, this study showed that serum CXCL13 levels were positively correlated with NMO onset age. This may mean that NMO patients with an older onset age may have a more severe extent of inflammation.

| CONCLUSION
In summary, serum levels of both CXCL13 and BAFF in NMO patients remained higher during remission, which shows that humoral immune dysfunction persisted during NMO remission.
Furthermore, immunosuppressive therapy can decrease serum BAFF levels but only slightly affects CXCL13 levels. As such, we suspect that immunosuppressive treatment is necessary and useful and that CXCL13 targeting may be another effective therapy for reducing NMO recurrence. This study was limited in patients who were not examined before and after treatment and during remission, and this may be a potential reason that significant differences in BAFF levels were not observed between NMO patients with and without treatment.

AUTHOR CONTRIBUTIONS
Su Wang, Tao Yang, and Jianglong Wan participated in the collection of samples and carried out CXCL13 and BAFF ELISA tests. Su Wang and Yongchao Zhang performed the statistical analysis. Yongping Fan was the designer of the study and took overall clinical and research responsibility. All authors provided their final approval of the content of this manuscript.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.