Author Of 1 Presentation
PS06.05 - B cells regulate chronic CNS inflammation in an IL-10-dependent manner
Abstract
Background
Several lines of evidence indicate essential roles for B cells in the pathogenesis of multiple sclerosis (MS). B cells act as potent antigen-presenting cells and throughout the chronic course of MS, B cell-follicle like structures can be found in the meninges of MS patients. However, whether and how B cells interact with CNS-resident cells, such as microglia and astrocytes to possibly modulate chronic progression of MS remains unclear.
Objectives
In the present study, we aimed at analyzing the interaction of B cells with CNS-resident cells in modulation of chronic CNS inflammation.
Methods
Primary microglia and astrocytes were generated from newborn C57BL/6 mice and were incubated with activated B cells or their supernatants. IL-6 and IL-10 production was abolished by genetic ablation or neutralization of IL-6 or IL-10 using specific antibodies. Thereafter, CNS resident cells were co-cultured with MOG-specific T cells. Further, C57BL/6 mice were depleted of B cells by 3 weekly subcutaneous injections of 0.2 mg murine anti-CD20 prior to immunization with MOG peptide p35-55, a setting in which B cells remain naïve. Microglial and astrocytic activation/modulation was assessed by ELISA, flow cytometry, immunohistochemistry and qRT-PCR.
Results
Incubation of primary microglia or astrocytes with IL-10-neutralized B cell supernatant or co-culture with IL-10-deficient B cells resulted in increased pro-inflammatory cytokine production, an upregulation of co-stimulatory molecules as well as an enhanced capacity to activate T cells as antigen-presenting cells. In vivo, depletion of naïve B cells worsened clinical severity of experimental encephalomyelitis (EAE) and increased the number of CNS infiltrating immune cells. Exacerbation was associated with an enhanced expression of molecules involved in antigen-presentation on microglia cells as well as an upregulation of pro-inflammatory gene products in astrocytes.
Conclusions
These findings highlight that B cells substantially alter the functional status of microglia and astrocytes in chronic CNS inflammation. Specifically, B cell-derived IL-10 is capable of diminishing the inflammatory responses of CNS-resident microglia and astrocytes. Our observation suggests that regulatory B cell function may be important in controlling CNS intrinsic inflammation associated with clinical progression.
Presenter Of 1 Presentation
PS06.05 - B cells regulate chronic CNS inflammation in an IL-10-dependent manner
Abstract
Background
Several lines of evidence indicate essential roles for B cells in the pathogenesis of multiple sclerosis (MS). B cells act as potent antigen-presenting cells and throughout the chronic course of MS, B cell-follicle like structures can be found in the meninges of MS patients. However, whether and how B cells interact with CNS-resident cells, such as microglia and astrocytes to possibly modulate chronic progression of MS remains unclear.
Objectives
In the present study, we aimed at analyzing the interaction of B cells with CNS-resident cells in modulation of chronic CNS inflammation.
Methods
Primary microglia and astrocytes were generated from newborn C57BL/6 mice and were incubated with activated B cells or their supernatants. IL-6 and IL-10 production was abolished by genetic ablation or neutralization of IL-6 or IL-10 using specific antibodies. Thereafter, CNS resident cells were co-cultured with MOG-specific T cells. Further, C57BL/6 mice were depleted of B cells by 3 weekly subcutaneous injections of 0.2 mg murine anti-CD20 prior to immunization with MOG peptide p35-55, a setting in which B cells remain naïve. Microglial and astrocytic activation/modulation was assessed by ELISA, flow cytometry, immunohistochemistry and qRT-PCR.
Results
Incubation of primary microglia or astrocytes with IL-10-neutralized B cell supernatant or co-culture with IL-10-deficient B cells resulted in increased pro-inflammatory cytokine production, an upregulation of co-stimulatory molecules as well as an enhanced capacity to activate T cells as antigen-presenting cells. In vivo, depletion of naïve B cells worsened clinical severity of experimental encephalomyelitis (EAE) and increased the number of CNS infiltrating immune cells. Exacerbation was associated with an enhanced expression of molecules involved in antigen-presentation on microglia cells as well as an upregulation of pro-inflammatory gene products in astrocytes.
Conclusions
These findings highlight that B cells substantially alter the functional status of microglia and astrocytes in chronic CNS inflammation. Specifically, B cell-derived IL-10 is capable of diminishing the inflammatory responses of CNS-resident microglia and astrocytes. Our observation suggests that regulatory B cell function may be important in controlling CNS intrinsic inflammation associated with clinical progression.
Author Of 1 Presentation
P0334 - Evobrutinib, a highly selective BTK inhibitor, prevents antigen-activation of B cells and ameliorates experimental autoimmune encephalomyelitis (ID 1125)
Abstract
Background
Background: B cells are key mediators of inflammatory processes in multiple sclerosis, a notion substantiated by the success of B-cell depletion therapies; however, overall depletion does not only target pathogenic B cells but can also affect regulatory B-cell properties. An alternative strategy may be the specific inhibition of Bruton’s tyrosine kinase (BTK), which is centrally involved in B-cell receptor (BCR) signaling and subsequently mediates B-cell activation and differentiation. BTK inhibitors therefore hold the promise to control pathogenic functions such as antigen presentation and cytokine release.
Objectives
Objectives: To evaluate the BTK inhibitor evobrutinib in a mouse model of experimental autoimmune encephalomyelitis (EAE).
Methods
Methods: C57Bl/6 mice received oral evobrutinib or vehicle starting 7 days before immunization with conformational MOG1-117 protein (a B cell–mediated model of EAE). EAE severity was assessed for 60 days using a standard scale. B-cell maturation and activation markers on B and T cells were analyzed by flow cytometry on day 12 post immunization. T cell proliferation and differentiation were assessed after a 3-day co-culture with BTKi-treated B cells. Intracellular calcium flux was analyzed using calcium-sensitive dyes and BCR or T cell receptor (TCR) stimulation. BTK expression and phosphorylation as well as cytokine production were assessed on healthy human B cells via PhosFlow protocols or ELISA, respectively.
Results
Results: Evobrutinib showed a dose-dependent amelioration of EAE severity throughout the 60-day observation period. Evobrutinib led to an accumulation of follicular type (FO) II B cells and a corresponding reduction in FO I B cells, a BTK-dependent transition. Expression of CD86, CD69, and major histocompatibility complex class II on B cells, and CD25 and CD69 on T cells, was reduced. Evobrutinib inhibited the B cell-mediated proliferation and proinflammatory differentiation of T cells. BCR-mediated mobilization of excitatory calcium was reduced by evobrutinib, while TCR signaling remained unaffected. In human B cells, BTK expression and phosphorylation were depending on the maturation of B cells, while the overall cytokine release was inhibited by evobrutinib.
Conclusions
Conclusion: Evobrutinib efficiently reduces BTK-dependent signaling after BCR stimulation, preventing B-cell activation, proinflammatory differentiation, and function. This translates into reduced CNS inflammation and clinical amelioration in a B cell–mediated EAE model.