Multiple sclerosis (MS) is a degenerative illness of the central nervous system (CNS) that results in demyelination and axonal injury resulting from the migration of peripherally activated immune cells into the CNS.¹ Both T- and B-cell reactivity to shared infectious and self-antigens (molecular mimicry) and nonspecific activation during infections (bystander activation) are key mechanisms driving MS-related immune activation, blood–brain barrier migration, CNS infiltration, and tissue damage.² Clinically isolated syndrome often indicates MS, usually with optic neuritis, brainstem, or spinal cord syndromes. Primary progressive MS, in 5% to 15% of cases, presents as gradual spastic paraparesis with single-system impairment.³ In early relapsing remitting MS (RRMS), relapse recovery often appears complete, despite underlying damage. As reserve declines, recovery lessens, leading to disability, and within 10 to 15 years, many progress to secondary progressive MS.⁴

The Middle East North Africa region falls in the low-to-moderate MS prevalence zone, but there is a trend toward increased MS prevalence over the last few decades, with a rate in the range of 30/100 000 – 84/100 000.⁵ According to the Middle East and North Africa Committee for Treatment and Research in Multiple Sclerosis (Middle East and North Africa Committee for Treatment and Research in Multiple Sclerosis) registry, MS onset was relapsing in 96% and progressive in 3.2% of cases.⁶ The exact MS prevalence in Egypt is unknown because of a lack of national surveys, but a retrospective meta-analysis across 10 referral centres estimated it at 1.41% (14.1 per 1000 neurological cases).⁷

In RRMS, symptoms develop gradually over days and last at least 24 h without fever or infection. A common first sign is unilateral optic neuritis with vision loss, eye pain, and altered colour vision. Vision loss stabilises within two weeks, but recovery may take longer and may be incomplete. Partial spinal cord myelitis causes gradual limb sensory and motor symptoms, with upper motor neuron signs such as spasticity, pyramidal weakness, hyperreflexia, and extensor plantar responses.⁸

Diagnosis of MS is clinical, based on history and examination, supported by investigations. The McDonald Criteria guide diagnosis requires dissemination in time and dissemination in space. Oligoclonal bands in cerebrospinal fluid (CSF) can indicate dissemination in time, supporting RRMS diagnosis in clinically isolated syndrome patients with magnetic resonance imaging (MRI) evidence of dissemination in space.⁹

The initiation of high-efficacy disease-modifying drugs (DMDs) may diminish disease activity, but there are concerns about their varying effects; therefore, fluid biomarkers that can forecast early disease activity following drug initiation may be beneficial.¹⁰

Loss of axons in MS impacts all types of fibres, resulting in as much as a 60% reduction in the spinal cord. This loss contributes to disability, cognitive deterioration, fatigue, shrinkage of the brain and cervical region, and inadequate response to treatment. Neurofilament light chain (NFL), which is released following axonal damage, can be found in CSF and blood, acting as a marker for disease progression and treatment effectiveness.¹¹

Chitinase 3-like 1 (CHI3L1) drives CNS inflammation by activating microglia, macrophages, and astrocytes, promoting cytokine release, blood–brain barrier disruption, and demyelination. It also induces neuronal apoptosis and inhibits neurite growth. As a key mediator of neuroinflammation and neurodegeneration, CHI3L1 is a promising therapeutic target in MS.¹²

The initiation, direction, and inhibition of CNS damage in MS are all roles of dendritic cells (DCs) that control T-cells, provoke an immune response, and ensure that the CNS immune system remains under surveillance. Classical DCs contribute to disease progression, but plasmacytoid dendritic cells (pDCs), which synthesise interferons, play a role in T-regulatory cell maturation and the resolution of illness.¹³ Plasmacytoid DCs represent a small subset of cells characterised by the expression of CD123, CD11c–, HLA-DR+, and express the blood DC antigen BDCA-2 (CD303) and BDCA-4 (CD304) antigens.¹⁴ Dendritic cells upregulate the expression of CCR7 and CXCR4, which enables them to migrate to lymph nodes, where they can present antigens to naïve T-cells, driving their polarisation toward pro-inflammatory T-cells.¹⁵ Dendritic cells with a stable, semi-mature phenotype and tolerogenic attributes are designated as tolerogenic DCs. Many tolerogenic DCs express programmed death-ligand (PD-L1 = CD274 and PD-L2), which binds to programmed cell death protein 1 (PD-1), expressed by T-cells, subsequently promoting tolerance via induction of clonal anergy and regulatory T-cells differentiation.¹⁶ Plasmacytoid DCs may support the expansion of myelin-specific regulatory T-cells through HLA-DR–mediated antigen presentation, as shown in MS models. CD274 on DCs may further suppress auto-reactive T-cell responses. Additionally, pDCs upregulate CCR7, supporting their migratory capacity.¹⁷

By targeting DCs, DMDs may provide a viable strategy for the treatment of MS, and research on the effects of DMDs on DCs is rare. The induction of tolerogenic DCs, which possess significant therapeutic potential, has been demonstrated to counter autoimmune reactions.¹⁸

The National MS Society has identified more than 136 studies to evaluate the different therapeutic options for MS. The United States Food and Drug Administration has approved the following drugs for RRMS: injectables (interferon and glatiramer acetate), oral therapies (fingolimod, teriflunomide, dimethyl fumarate, siponimod, cladribine), infusion therapies (natalizumab, ocrelizumab, alemtuzumab, and rituximab), and other (azathioprine, laquinimod, cyclophosphamide, daclizumab, dalfampridine, mitoxantrone, glucocorticoids, intravenous immunoglobulin, dalfampridine).¹⁹

Interferon beta-1A (IFN-b-1A) decreases the frequency of relapses, progression of disability, MRI lesion count, and provides cognitive advantages.²⁰ Fingolimod, which modulates the S1P receptor, sequesters naïve and memory T-cells within lymph nodes, resulting in reduced lymphocyte levels in the blood and addressing all forms of MS.²¹ Rituximab, which targets CD20+ B-cells, lessens inflammation, decreases relapses, and results in fewer new brain lesions in RRMS.²²

The aim of our study was to evaluate the effect of DMDs such as IFN-b-1A, fingolimod and B-cell (rituximab) therapy on NFL and CHI3L1 serum levels, and some biomarkers of pDCs in patients diagnosed with RRMS.

The 44 MS patients were subdivided by treatment type as follows: 17 patients on IFN-b-1A, 20 patients on fingolimod, and 7 patients on B-cell therapy (rituximab).

Neuronal and axonal destruction are among the pathological characteristics of MS.²⁷ Research indicates that high-efficacy DMDs can be introduced to reduce disease activity, prevent disability, and help regulate disease progression.^28,29,30

The pathophysiology of RRMS changes over time, and as more treatment choices become available, there is a need for more methods to measure disease activity and response to therapy, such as biomarkers in body fluids.^31,32 Neurofilament light chain, an axonal protein, and CHI3L1, a glial glycoprotein in the CNS, have been shown to serve as biomarkers for inflammation and axonal injury in MS.³³ Enhanced comprehension of their reaction to DMDs may facilitate their incorporation into clinical uses and treatment monitoring.³⁴

To our view, investigations of the combined serum levels of NFL and CHI3L1 and the role of tolerogenic pDCs in response to DMDs for MS treatment are rare.

The age and gender profile of the patients in this study are consistent with previous reports in the literature, for example Zakaria et al., who reported in 2016 that the mean age of their patients was 31.87 ± 8.7, and 72% were female.³⁵ We reported that the fingolimod group had higher mean ages than the IFN-b-1A group. In contrast to fingolimod, which is reserved for more advanced instances that typically worsen as patients age, the majority of female RRMS patients in our study prefer to use IFN-b-1A more frequently at lower ages during the childbearing period. Macaron et al. in 2023 reported that as patients age, they may be exposed to more DMDs with varying mechanisms of action.³⁶

We found that the employment rates in the B-cell therapy group were 57.1%, 85% for fingolimod, and 94.1% for IFN-b-1A. The lower employment rate observed in the B-cell therapy group may be attributable to their significantly higher EDSS score, which ranged from 4 to 7, with a median of 5. The association between unemployment and physical disability in MS has been widely reported,³⁷ with 82% of individuals employed at an EDSS of 0, but only 25% employed at an EDSS of 6.5.³⁸

All patients in our study underwent MRI before treatment initiation. The B-cell therapy group demonstrated a higher percentage of brain atrophy, cord atrophy, and cord lesions compared to the other treatment groups. IFN-b-1A was linked to the lowest rates of MRI lesions and atrophy, which could be attributed to its classification as a first-line treatment for the early stages of RRMS. In contrast, the increased percentage of brain atrophy and spinal cord lesions associated with B-cell therapy may result from its application in more advanced disease stages. Multiple sclerosis patients on IFN-b-1A for a year, who exhibit at least two of three variables (relapses, increased disability, or MRI activity), are candidates for treatment modification because of disease progression risk.³⁹

Our study showed that serum NFL levels at a cut-off ≤ 6.600 pg/mL and CHI3L1 levels ≤ 10.25 ng/mL were elevated in MS patients compared to non-MS controls, which was consistent with previous findings.^40,41,42 As axonal injury increases, the progression of disability becomes more severe. Serum NFL was significantly correlated with EDSS (r = 0.560, p ≤ 0.001), aligning with larger studies involving 814 and 607 patients, which found a weak yet significant association between EDSS and serum NFL levels, showing a 12% and 8% NFL increase per EDSS step.^43,44 Furthermore, serum NFL is correlated with CHI3L1, suggesting that glial activation is associated with axonal injury and the progression of disability in MS.⁴⁵

Comparing the serum NFL levels across the three treatment groups, we observed the highest levels in the B-cell therapy group compared with fingolimod (p = 0.005) and IFN-b-1A (p < 0.001). This could be attributed to the association of NFL levels with MRI findings and higher EDSS in this group, as short-term prognostic studies^42,44,45,46 have demonstrated that NFL levels correlate with EDSS deterioration, relapse frequency, and brain atrophy measured by MRI.

Chitinase 3-like 1 is a potential therapeutic target for the treatment of MS. Strategies to suppress CHI3L1 may reduce neuroinflammation, aid remyelination, and protect axons.⁴⁷ Our results showed that serum CHI3L1 levels were higher in the B-cell therapy group compared with IFN-b-1A and fingolimod groups, with a significant positive correlation with EDSS (r = 0.422, p ≤ 0.004), contrasting with the findings of a study that reported that CHI3L1 levels were not associated with DMD type but rather with long-term DMD use.⁴⁷ Being a proinflammatory marker secreted by various immune cells such as macrophages, synoviocytes, neutrophils, and endothelial cells, it is anticipated that it plays a role in the pathogenesis of MS. However, we cannot conclude that IFN-b-1A and fingolimod directly inhibit its levels in comparison to B-cell therapies; their effects may be more associated with the stages of disability and inflammation in the B-cell therapy group. Larger studies on CHI3L1 serum levels are needed for it to be evaluated as a biomarker for DMDs response.

Dendritic cells are essential for eliciting either an inflammatory or tolerogenic response. Disease-modifying drugs can significantly affect DC function in MS. However, unlike T- and B-cells, the effects of DMDs on DCs are less well studied, although tolerogenic DC induction is recognised for its therapeutic potential in preventing autoimmune reactions.¹⁸

In this study, we investigated the role of pDCs in MS by examining the percentage of pDCs and their tolerogenic and migratory functions in response to three DMD groups. We found that, although there was an increase in the percentage of pDCs, which ranged from 0.0 to 71.0 in patients with MS compared to the healthy group (0.0–4.0), it was not statistically significant between the two groups or across the treatment groups. This may be due to the absence of CSF samples in our study and the fact that our patients were in the remitting stage. Previous reports have shown that pDC levels increase in the CSF and demyelinating lesions during disease exacerbations.⁴⁸ Since pDC function differs between tissues, their blood levels may not accurately reflect their activity in lymph nodes or the CNS. As a result, sampling from peripheral blood might overlook important migratory or immunoregulatory functions of pDCs that are only observable in their native locations which would be more informative by the CSF samples. In remission and under effect of DMDs, inflammation tends to be lower, which could lead to less activation or migration of pDCs compared to periods of active disease. This inactive immune environment might obscure changes in pDC function that occur during more active disease phases.

We assessed pDC tolerogenic behaviour by assessing PD-L1 expression on pDCs and their migration by identifying CCR7. No significant difference was observed between patients with MS and controls or across treatment groups, but we found a significant positive correlation between NFL, CHI3L1, and pDCs’ migratory function. Plasmacytoid DCs, constituting only 0.3% – 0.5% of peripheral blood, are challenging to study, and their role in MS is still unclear.⁴⁹

The limitation of this study was obtaining CSF samples which could reveal localised immune signatures not captured peripherally and provide more sensitive CNS data alongside peripheral samples to better analyse pDC, NFL and CHI3L1 correlation, but our patients declined this procedure. Further studies are necessary to examine the long-term clinical outcomes of various DMDs and to establish NFL and CHI3L1 levels as potential drug response biomarkers. The small B-cell therapy cohort likely reflects its use mainly in highly active RRMS, initial use in primary progressive MS, and limited use in stable RRMS, according to the protocol of the Committee of National Egyptian guidelines, Ministry of Health and Neurology Scientific Committee. Few studies have explored the role of pDCs in MS or the effects of DMDs on these cells. Modifying pDCs could emerge as a strategy to enhance immune control in MS, although their specific role in MS requires further clarification, particularly regarding the effects of DMDs.