The central nervous system (CNS) is strongly connected to the peripheral immune system. A robust inflammatory response in the peripheral system can result in the infiltration of immune cells into the CNS [1]. Immune cells can migrate in and out of the CNS as a consequence of the increased permeability of the blood brain barrier (BBB), amplifying inflammatory processes in CNS and in the periphery. Therefore, peripheral immune cells may participate in the pathogenesis and progression of CNS diseases. Multiple sclerosis (MS) is a heterogeneous and complex autoimmune disease that is characterized by inflammation, demyelination, and axon degeneration in the CNS. The relevance of pro-inflammatory signaling in the pathogenesis of MS has been highlighted [2].

IL-6 is a key pro-inflammatory cytokine which is involved in autoimmunity. An alteration in IL-6 responsiveness is associated with the activation of T regulatory (Treg) populations [3]. IL-6 action is regulated by the expression of its receptor, both on the cell surface and in its soluble form, which, unlike the soluble receptors for other cytokines, tends to stabilize and strengthen the effect of IL-6.

Classical IL-6R signaling is the binding of IL-6 to the membrane-bound a-receptor (IL-6R, CD126). Binding is followed by dimerization of the signal-transducing b-receptor glycoprotein 130 (gp130) (CD130), which is ubiquitously expressed. Cells that do not express IL-6R obtain their IL-6 signals via trans-signaling. Enzymatic cleavage of the membrane receptor creates the soluble form of IL-6R (sIL-6R). Alternatively, the soluble form is produced by alternative splicing of the IL-6 receptor alpha transcript. IL-6/sIL-6R forms a signaling complex with gp130 on the cell surface. Further signaling through gp130 is mainly carried out by the Janus-kinase/signal transducer and activator of transcription (JAK/STAT). In contrast to classic signaling which is anti-inflammatory, trans-signaling is responsible for the pro-inflammatory effects of IL-6 [4] and the inhibited activity of Treg subpopulations. Activation of trans-signaling is tightly controlled at multiple levels. One of the natural inhibitors in human blood is the soluble form of gp130 (sgp-130). Sgp-130 inhibits IL-6 trans-signaling, whereas IL-6 classic signaling is largely unaffected [5].

The association between IL-6 and Relapsing-remitting multiple sclerosis (RRMS) pathogenesis is clear but there is some uncertainty about the role of IL-6 and IL-6 pathway components in blood. Specifically, whether the concentration of IL-6 in serum differs in patients with MS and whether this difference influences disease pathogenesis [6].

Recent studies have demonstrated phenotypic and functional changes in subpopulations of CD8⁺ Tregs in several CNS diseases including MS [7]. CD8⁺ T cells are age dependent and there is an exponential increase in the number of transcriptionally senescent CD8⁺ T cells. However, the role of aging in inducing changes in CD8⁺ Treg cells in MS is not well understood. In particular, how CD8⁺ T lymphocytes influence MS pathogenesis is unclear.

Here, we defined markers for IL-6 pathways in serum and regulatory CD8⁺ and CD4⁺ T cells in the blood of newly diagnosed RRMS females and analysed the associations among them and with age.

This study has described associations among IL-6 pathways in serum and regulatory CD4⁺ and CD8⁺ T cells in the blood of newly diagnosed female patients with RRMS and the influence of age. These associations confirm, extend and clarify the existing literature. There was no statistically significant difference between the RRMS and the healthy control group in the serum concentration of IL-6. However, there were significant differences between patients and controls in both TGF-$\beta$1 and sgp-130. In RRMS, an increased serum concentration of IL-6 has been observed, particularly in those patients with a longer history of disease [12]. Moreover, monocytes from MS patients have been shown to release more IL-6 [13].

The cells responsible for serum IL-6 and TGF-$\beta$1 have not been identified. Peripheral leukocytes that increased in number after relapse could be the source of these cytokines but these serum cytokines could come from the central nervous system. IL-6 and TGF-$\beta$1are produced by brain cells, including astrocytes, microglia, and neurons under pathological conditions [14] and individually variable cellular composition may affect the IL-6 level. Depression affects serum IL-6 concentration and depression is common in MS patients especially around diagnosis. The concentration of IL-6 in the plasma is elevated during acute depression in MS patients [15].

Activated TGF-$\beta$1 is required for both Treg and Th17 lineage maturation and it acts in combination with IL-6 [16]. However, in our study, TGF-$\beta$1 concentration was lower in the blood of RRMS patients. IL-6 and TGF-$\beta$1 levels were not correlated with each other. Interestingly, sgp-130 was significantly elevated in the serum of RRMS patients despite the lack of statistically significant changes in free IL-6 levels, free IL-6R$\alpha$ and IL-6/IL-6R$\alpha$ complexes. However, the concentration of sgp-130 did show a significant positive correlation with free IL-6R$\alpha$ in RRMS patients. The endogenous serum levels of IL-6R$\alpha$ and sgp-130 can either increase IL-6 trans-signaling via formation of IL-6/IL-6R$\alpha$ complexes or decrease trans-signaling via IL-6/IL-6R$\alpha$ /sgp-130 complexes.

Our results suggest that most free IL-6 cannot bind IL-6R$\alpha$, suggesting that higher amounts of endogenous sgp-130 in the blood do not effectively inhibit IL-6 trans-signaling and that free IL-6 can allow simultaneous classic and trans-signaling in RRMS. Alternatively, sgp-130 could form short-lived complexes that quickly associate and dissociate and increase the half-life of IL-6 and enable both classic and trans-signaling. However, the mechanism regulating the sgp-130 level has not been determined. The expression of sgp-130 is influenced by various factors such as chemical substances as well as other proteins or hormones [17]. For paracrine and autocrine trans-signaling, even modest quantities of IL-6/IL-6R$\alpha$ complexes could stimulate gp-130 on nearby cells.

It is not known if the concentration of IL-6/IL-6R$\alpha$ complexes is greater at foci of inflammation. Proteolytic cleavage produces large amounts of IL-6R$\alpha$ in the CNS and shedding may greatly increase the concentration of sgp-130. In remission sIL-6R/IL-6 complexes can promote outgrowth of neurites and increase neuron survival; they may also promote remyelination and regeneration of nerves [18]. However, elevated values of IL-6R$\alpha$ were observed only in patients during the acute phase of disease and not in patients in remission [15].

A possible explanation is that endogenous sgp-130 might inhibit the recognition of IL-6/IL-6R$\alpha$ complexes in serum. Endogenous sgp-130 can bind IL-6/IL-6R$\alpha$ complexes, but the low concentration of endogenous serum sgp-130 may only capture a small proportion of IL-6/IL-6R$\alpha$ in IL-6/IL-6R$\alpha$/sgp-130 complexes. Consequently, it may not nullify the majority of the endogenous trans-signaling complexes. Experimentally, 400 ng/mL of endogenous sgp-130 is able to reduce IL-6/IL-6R$\alpha$ complex detection by about 26% [19]. Both IL-6R$\alpha$ and sgp-130 are increased in the serum of MS patients but sgp-130 was decreased in the CSF of MS patients [20, 21]. Another study supported elevated sIL-6R but not sgp-130 in RRMS patients [22].

Detection of cytokine receptors is often a more robust guide to immunological mechanisms, because cytokines have a short half-life in blood [23]. Here, we analysed the classic IL-6 pathway receptors and STAT activation in CD4⁺ and CD8⁺ Treg subsets. IL-6 signaling through gp130 triggers JAK1, and dimerization and phosphorylation of STAT3, followed by translocation to the nucleus and transcription of target genes. The mitogen activated protein kinase (MAPK) pathway is activated by IL-6, leading to additional gene transcription [24].

We found striking differences in CD126 and CD130 expression between healthy donors and RRMS in peripheral blood CD8⁺CD122⁺ Tregs. A higher frequency of the CD126 membrane receptor and increased CD130 expression were observed in the CD8⁺CD122⁺ T cell subset of RRMS patients, confirming a previous report [25]. This result indicates the increased involvement of the classical IL-6 activation pathway in CD8⁺CD122⁺ Tregs within RRMS. In RRMS patients, CD8⁺CD122⁺CD126⁺ Tregs showed a significant positive association with levels of free IL-6 in serum. RRMS patients have a 3-fold lower percentage of CD8⁺CD122⁺ T cells than healthy donors and the distribution of CD8⁺CD25⁺FoxP3 and CD4⁺CD25⁺FoxP3⁺ Tregs was unchanged [10]. Therefore, in RRMS fewer cells are available for classical IL-6 signaling. This is particularly important, as classical IL-6 signaling has homeostatic and anti-inflammatory effects [26]. The increased expression of CD126 and CD130 could be a mechanism that supports anti-inflammatory effects of the reduced population of CD8⁺CD122⁺ T cells.

Surprisingly, despite increased levels of IL-6R and gp-130 in CD8⁺CD122⁺ T cells, these cells did not exhibit sensitivity to IL-6 and IL-2, as indicated by the unchanged frequency of phosphorylated STAT3 and STAT5, and inhibited STAT3 and STAT5 MFI, respectively. Further the concentration of pSTAT3 was negatively correlated with the serum concentration of IL-6R$\alpha$. A Possible explanation is that phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) signaling can influence JAK/STAT signaling by influencing the expression in mouse primary T cells of suppressor of cytokine signaling (SOCS) [27].

The PI3K/Akt signaling pathway, is involved in multiple signaling pathways which control the development, differentiation, maturation, migration and activation of CD8⁺ T cells. Defective signaling by STAT5 in response to cytokines such as IL-2 and IL-15 is associated with constitutively active Akt in CD8 T cells [28]. Another explanation is that activation of STAT3 leads to the expression of SOCS3; this molecule negatively regulates the IL-6 signaling pathway. However, there was no difference in SOCS1 and SOCS3 expression in blood between RRMS patients and healthy controls [29].

Recently published data support the concept that classical IL-6 signaling through STAT3 is anti-inflammatory while trans-signaling by IL-6 is pro-inflammatory. IL-6 trans-signaling could inhibit the differentiation of regulatory T-cells [4]. Our results show that classical IL-6 signaling in CD8⁺CD122⁺ T cells is disrupted in RRMS. This disruption could contribute to the dysregulation of Treg function and enhance the inflammatory process provoked by CD25-positive CD4 and CD8 Tregs.

No differences in the levels of CD126 and CD130 were observed in CD25-positive Tregs (CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD25⁺FoxP3⁺) between healthy donors and patients with RRMS but these cells exhibited sensitivity to IL-6 stimulation, resulting in an increase in the intracellular percentage of pSTAT3. This implicates trans-signaling of IL-6 in these cells. Interestingly, the percentage of pSTAT3-positive cells in the CD4⁺CD25⁺FoxP3⁺ population was positively correlated with the level of TGF-$\beta$1 and negatively correlated with IL-6R$\alpha$ in the serum of RRMS patients.

CD4⁺ T cells strongly express IL-6R. This expression is lost in activated T cells during inflammation, probably due to proteolytic cleavage of the extracellular membrane protein domain [30]. On the other hand, CD8⁺ T cells do not produce A disintegrin and metalloprotease 17 (ADAM17) and consequently do not express sIL-6R [25]. CD126 expression on CD8⁺ T cells is inhibited by STAT5 [31]. These results suggest that induction of the IL-6 pathway may depend on CD25 expression by Tregs.

Age-related changes in pSTAT5 expression across all Treg populations: CD8⁺CD122⁺, CD8⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺FoxP3⁺ in healthy controls were absent in RRMS patients, suggesting that RRMS may interfere with normal immune aging patterns, possibly by promoting a sustained inflammatory state that overrides senescence of T cells. STAT5 phosphorylation maintains the expression of CD25 and supports Treg stability during aging [32].

MS patients show early immunosenescence and have fewer naïve T cells, and fewer functional regulatory T cells [33]. Although, the adaptive immune system shows reduced efficacy earlier in men [34], here we demonstrated disrupted frequency of pSTAT5 expression in Treg populations in RRMS females.

The main limitation of our study was the small size of the study population. Statistical power is influenced by the number of individuals tested as well as the size of the effect and statistical test. Although all patients were female to avoid confounding between differences due to disease and potential differences due to gender, inclusion of only one gender in studies is the second limitation of the study. The results strongly indicate that further studies are needed to explain the role of key signaling pathways in the development of Tregs populations and RRMS pathogenesis.

The level of IL-6 pathway markers in serum reflects both classic and trans-signaling in RRMS patients. The CD8⁺CD122⁺ Treg subset in RRMS patients exhibited an increased level of markers associated with classical IL-6R signaling without STAT3 phosphorylation. The observed alterations in the expression of receptors and in signaling activity may contribute to the dysregulation of CD8⁺CD122⁺ Treg function. For CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD25⁺FoxP3⁺ Tregs, no changes in classical IL-6R surface markers were observed in RRMS, but these cells exhibited sensitivity to IL-6 stimulation, resulting in an increase in the percentage of pSTAT3 in cells, suggesting IL-6 trans-signaling in these cells. Our findings underscore the complex interplay between IL-6 signaling and age-related immune regulation. Age-related changes in pSTAT5 expression across all Treg populations in healthy controls were absent in RRMS patients. The study enhances our understanding of the peripheral immune system changes associated with CNS pathology and disease progression. It also opens a new avenue to identify biomarkers of disease progression and potentially offers a new approach to manipulate the immune system to protect the CNS.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

MK: conceptualization, investigation, methodology, data analysis, writing; MM: conceptualization, investigation, methodology, data analysis, review and editing draft; LS: investigation, review and editing draft; MMC: data analysis, visualization, editing draft; RR: resources, design of the work, review and editing draft; AK: resources, design of the work, review and editing draft; MS: data analysis, review and editing draft; KDŁ: conceptualization, funding acquisition, project administration, supervision, data analysis, writing, review and editing draft. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and was approved by the local ethics committee at the Military Institute of Hygiene and Epidemiology (No. 17/2019). All patients or their families/legal guardians gave written informed consent before enrollment.

We are grateful to Małgorzata Malinska (Department of Neurology, Military Institute of Aviation Medicine, Warsaw, Poland) for the excellent clinical support, Beata Kon for the statistical analysis and all patients who agreed to donate blood for research.

This work was supported by the TEAM TECH/2017-4/22 project carried out within the TEAM TECH programme of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development Fund.

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/JIN36207.