Multiple sclerosis displays a distinct gender bias towards increased susceptibility and disease severity in females with relapsing-remitting disease. However, women with MS experience remarkable recoveries during pregnancy, indicating a crucial protective role of sex hormones with both estrogen and estriol treatments suggested to have therapeutic potential in treatment of MS. Since MS is often considered to be a T cell-dependent autoimmune disease, the initial focus of immunomodulatory role of estrogen was on T cells. However, our prior studies demonstrated that the protective effects of E2-treatment are not mediated through direct action on the encephalitogenic T cells, and that both immune and CNS cells are required in propagating immunomodulatory effects of E2 [12
]. Exact molecular mechanisms in mediating the regulatory effects by E2 in EAE have not yet been identified, thus necessitating an ongoing search to determine the crucial immunomodulatory players. Tregs are prime immunoregulatory candidates since their numbers increased by E2 in EAE-protected mice [13
]. Surprisingly, our contemporary study demonstrated that E2 can fully protect mice against clinical and histological signs of EAE in the absence of Treg cells (Offner, manuscript submitted), suggesting an alternative pathway for E2-mediated protection against EAE. Since the E2-implanted Foxp3-depleted mice demonstrated an increase in PD-L1, CD80 and CD86 expression by B cells, we further investigated the role of B cells in E2-mediated protection against EAE.
Our studies demonstrate for the first time that protective effects of E2 vs. EAE require B cells, and further implicate immunosuppressive IL-10-producing CD1dhigh
B cell subpopulation as a likely contributor. The role of B cells in MS and EAE include both pathogenic and regulatory mechanisms. Multiple studies have demonstrated that B cells are crucial for EAE pathogenesis [14
]. However, studies have also consistently demonstrated that depletion of CD20+
B cells before EAE induction leads to increased levels of CNS-infiltrating encephalitogenic T cells and enhanced disease severity [6
In a similar vein, E2 treatment of the µMT−/−
mice delayed but could not inhibit the trafficking of immune cells into the CNS, thus permitting severe inflammatory reactions, demyelination and severe EAE. Besides an increase of the pro-inflammatory chemokines like MCP-1 and RANTES in the CNS, µMT−/−
mice demonstrated a significant increase specifically in CXCL13. CXCL13 gains importance in MS since its expression in the intrameningeal follicles rich in B cells [27
] and also within inflammatory cells bearing a macrophage-like morphology from autopsied MS lesions is detected [28
]. However, since our study demonstrates higher levels of CXCL13 in absence of B cells, it could be due to subsets of CD4+
T cells which are able to infiltrate the CNS.
B cells contribute to immune responses by functioning as antigen-presenting cells and by secreting cytokines [23
]. Thus, in addition to their possible pathogenic role in producing demyelinating antibodies, presenting myelin autoantigens to T cells [29
] or secreting effector cytokines, B cells from MS patients may also secrete anti-inflammatory cytokines [30
],implicating a potential for modulating disease progression. Particular stages of B cell differentiation are sensitive to effects of E2 [31
], with ERα being implicated in the development of mature B-lymphocytes [33
]. In the present study E2 could modulate T cell proliferative responses by acting on B cells in an ERα-specific manner, consistent with our previous studies that failed to show a direct effect of E2 on T cells.
Our recent studies have demonstrated a critical role for PD-1 in E2-mediated protection of EAE [24
]. Also, the crucial role of PD-L1 rather than PD-L2 in inhibition of EAE has been demonstrated [35
]. Here, we demonstrate a significant increase in the expression of PD-L1 on B cells in E2-treated WT mice after induction of EAE, although no differences were observed after treatment with E2 prior to immunization. Moreover, our study for the first time demonstrates a critical role of PD-L1 in E2-mediated protection, with E2-implanted PD-L1−/−
mice showing no protection from clinical and histological EAE. Thus, B cells mediate immunomodulatory effects of E2 on encephalitogenic T cells through ERα and the negative co-stimulatory PD1/PD-L1 pathway.
E2 can best mediate its protective effects when administered prior to EAE initiation rather than during the induction phase post-immunization [8
]. Anti-CD20-depletion of B cells prior to EAE induction leads to exacerbation of the disease with increased encephalitogenic T cell infiltration into the CNS while B cell depletion during disease progression suppressed EAE symptoms [6
], thus demonstrating reciprocal roles of B cells in different phases of EAE. Increase in EAE severity upon B cell depletion before disease initiation resulted from depletion of a rare IL-10-producing CD1dhigh
subpopulation of B cells characterized as B regulatory (Breg) cells [6
In our study, percentages of this CD1dhigh
Breg cell subpopulation increased significantly in WT E2-implanted mice. Studies in a contact hypersensitivity model showed that transfer of IL-10-producing B cells into B-deficient or B-depleted mice inhibited CD4+
T cell-dependent inflammatory responses [7
]. Moreover, suppressive function of CD1dhigh
B cells increased post-immunization [7
] and was dependent on their ability to produce IL-10 [7
], thus indicating that this antigen-induced IL-10-producing Breg cell subset can downregulate T cell activation. Recently, it has been proposed that Breg cells (i.e. IL-10-producing B cells) have a protective role during EAE initiation (congruent with the data in this manuscript), whereas Treg cells have a therapeutic role in the later stages after onset of clinical disease [36
]. Further studies would be needed to characterize IL-10 production by the CD1dhigh
B cells. However, an abrogation of IL-10 production after E2 treatment in µMT−/−
mice strongly implicates B cells as the source of IL-10 in the WT E2-treatedmice.
In summary, B cells play an indispensible role in E2-mediated protection against EAE. Absence of B cells abrogates the protective effects of E2 and leads to a significantly increased infiltration of inflammatory cells into the CNS of mice with EAE. E2 mediates its immunomodulatory effects on B cells via ERα and PD-L1 and B cells primed with E2 are able to significantly suppress T cell proliferative responses. Finally, this is the first study to implicate CD1dhigh
B cells (IL-10-producing Breg-cells) as possible key contributors induced by E2 treatment. These results suggest an important protective role for E2-primed B-regulatory cells in MS and have strong implications for patients receiving current MS therapies such as Rituximab™ that cause B cell depletion [37
]. Our study implicates that caution be used during such B cell-depleting therapies since the non-selective elimination of B cells may interrupt the beneficial E2 interactions that could promote anti-inflammatory influence on other immune cells leading to remissions and would preclude potential therapeutic application of E2 given alone or in combination with other regulatory agents [39