Multiple Sclerosis Is A Devastating Autoimmune Disease

Published Date: 02 Nov 2017

Administration of dehydroepiandrosterone (DHEA), the most abundant adrenal steroid in humans , has been shown to exert beneficial effects in EAE . However its mechanism of action, especially on the immune cells, is not understood. DHEA is additionally produced by neurons and glia (neurosteroid), affecting multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging . In peripheral tissues, DHEA is converted into androgens and/or estrogens, which also have variable immunomodulatory functions accompanied by several side effects . We have recently synthesized a DHEA derivative, BNN27 [(20R)-3β, 21-dihydroxy-17α, 20-epoxy-5-pregnene] (Fig. 1A) , deprived of estrogenic or androgenic actions, which possesses strong neuroprotective properties with an EC50 at nanomolar levels . Here, we investigated BNN27 as a potential immunomodulatory agent for established CNS autoimmunity.

We administered BNN27 into symptomatic (1-2 clinical score) mice with MOG35-55-induced EAE and they became almost free of paralysis within 2-3 days of treatment (mean maximum scores; 0.142857 ± 0.154303 for BNN27 group, versus 3 ± 0.408248 for PBS group, Fig. 1B and fig. S1A). We have also tested DHEA, the natural homologue to BNN27. In addition, as DHEA was reported to attenuate EAE when administered at disease induction, and well before clinical onset , it would be important to determine whether it would be beneficial to symptomatic mice. DHEA administration suppressed EAE, while control-treated mice presented severe symptoms (Fig. 1B and fig. S1A). CNS mononuclear cells from BNN27- and DHEA-treated mice were significantly lower in comparison to PBS-treated mice (fig. S1B). Spinal cord sections from BNN27- and DHEA-treated mice showed decreased degree of inflammation (Fig. 1C), a significant decrease in T cell infiltration (Fig. 1, D and E), as well as decreased microglial activation (fig. S1C). Whereas control-treated mice displayed substantial demyelination, BNN27- and DHEA-treated mice had intact spinal cords (Fig. 1E). MOG35-55 –specific responses of BNN27- or DHEA-treated mice were significantly suppressed (fig. S2A), characterized by decreased levels of IL-17, IFN-γ, osteopontin (Opn) and TNF-α (Fig.1F and fig. S2B). This was also accompanied by a significant reduction in the numbers of both TH17 (Fig. 1G) and TH1 cells (fig. S2C) in the draining lymph nodes (DLNs). The gene expression of the orphan nuclear receptor Rorc, master transcription factor of TH17 cells, as well as of the Il17 gene, were down-regulated in DLN cells from BNN27-treated EAE mice (fig. S2D).These findings suggest that BNN27, and DHEA, suppress pathogenic effector TH responses, effectively controlling ongoing CNS autoimmunity.

MOG35-55–specific responses of BNN27- or DHEA-treated mice were characterized by significantly increased production of the immunoregulatory cytokine IL-10 (Fig.1F and fig. S2B) along with increased frequency of IL-10-producing CD4+ T cells, as well as of IL-10/IL-17-producing CD4+ T cells in the DLNs and the CNS (Fig. 1G and fig. S2E). Both IL-10+ and IL-10/IL-17+ CD4+ T cell subsets have been demonstrated to be suppressive in EAE . We confirmed the up-regulation in the percentage and numbers of IL-10-producing CD4+ T cells using Il10gfp (Il10tm1.1Karp) mice that we treated with BNN27 after EAE clinical onset (fig. S3A). Isolated CNS mononuclear cells from BNN27-treated mice had increased expression of the Il10 gene (fig. S3A), as compared to PBS control-treated mice. In addition, CD4+ T cells from BNN27-treated mice had increased expression of the Il10ra gene (fig. S3A), indicating responsiveness to immune suppression by IL-10 . The secreted levels of TGF-β1 were not altered (fig. S2B). Notwithstanding its immune-regulatory functions, TGF-β1 can lead to TH17- or TH9-mediated pathology, depending on the cytokine milieu .

Furthermore, the percentage of CD4+ CD25+ Foxp3+ T regulatory cells was elevated in DLNs of BNN27-treated EAE mice, as compared to PBS-treated mice (fig. S3B). Similar immunomodulatory effects of BNN27 and DHEA in the course of EAE were also observed in mice euthanized at an earlier time point during disease peak (approximately day 17) (fig. S4, A and B). BNN27 and DHEA were equally suppressive even when administered at lower daily doses (≥ 1mg/day, data not shown). Overall, BNN27 and DHEA appear to suppress pathogenic TH1 and TH17 responses while increasing numbers of T cells with regulatory phenotype.

CCR6 is a chemokine receptor essential for the function of effector TH17 cells, as well as for their entry into the CNS and the establishment of EAE . Analysis of CCR6 expression in DLN TH17 cells from BNN27-, DHEA- or PBS-treated EAE mice at early disease stages showed that BNN27- and DHEA-treated mice had significantly lower CCR6 expression on TH17 cells, compared to PBS-treated EAE mice (Fig. 1H and fig. S3C), suggesting that BNN27 and DHEA affect the infiltration of inflammatory TH17 cells into the CNS.

EAE can be transferred to recipients by autoreactive CD4+ T cells . Rag1-/- mice that received CD4+ T cells from BNN27-treated EAE donors had significantly milder paralysis and delayed disease onset (Fig. 2A), as compared to Rag1-/- mice that received equal numbers of CD4+ T cells from PBS-treated donors. Suppressed disease score was accompanied by significantly increased production of IL-10 and decreased production of IL-17 and IFN-γ (Fig. 2B). CD4+ T cells from DHEA-treated mice had reduced pathogenic potential upon transfer as well (fig. S5). These findings suggest that effector CD4+ T cells from BNN27- or DHEA-treated EAE mice remain significantly less pathogenic even after transfer into lymphopenic, untreated recipients.

We also tested whether BNN27 had therapeutic effects on symptomatic SJL mice that suffered from relapsing-remitting EAE induced by immunization with proteolipid protein peptide PLP139-151, a different to MOG autoantigen. Therapeutic administration of BNN27 significantly suppressed disease (Fig. 2C and fig. S6A), decreasing levels of IFN-γ and IL-17 in response to PLP139-151 peptide (Fig. 2D) and reducing numbers of isolated CNS mononuclear cells (fig. S6B), as well as numbers of DLN TH17 and TH1 cells (fig. S6C) and CNS TH17 cells (Fig.2E). Numbers of CCR6+ TH17 cells were also decreased (fig. S6C) following BNN27 treatment. The gene expression of Rorc, was down-regulated in DLN cells from BNN27-treated EAE mice (fig. S6D). In parallel, IL-10 levels, as well as spinal cord IL-10- and IL-10/IL-17-producing CD4+ T cells were significantly increased in BNN27-treated EAE mice (Fig. 2, D and E). Similar to BNN27, DHEA administration had beneficial effects on EAE of SJL mice (fig. S7, A to E). Thus, BNN27 and DHEA effectively suppress ongoing autoreactive responses independently of the inducing autoantigen and the type of EAE.

Of note, disease remained suppressed, even when we re-challenged DHEA- (fig. S8A) and BNN27 -treated (data not shown) mice with MOG35-55/IFA to induce relapse. MOG35-55-specific secretion of IFN-γ and IL-17 remained significantly lowered, while IL-10 secretion remained significantly elevated (fig. S8B). When we re-injected DHEA- (fig. S8C) and BNN27-treated (data not shown) mice with pertussis toxin to facilitate blood-brain barrier breakdown, their clinical phenotype, as well as MOG35-55–specific IFN-γ and IL-17 secretion remained significantly suppressed (fig. S8D). Again, IL-10 secretion in DHEA-treated EAE mice remained significantly up-regulated (fig. S8D). Thus, certain pathogenic triggers cannot cancel disease suppression conferred by BNN27 or DHEA. The suppressive effects of BNN27 or DHEA were long-lasting; symptomatic EAE mice treated with BNN27 or DHEA for 15 days remained free of paralysis for 50 more days with no treatment (Fig. 2F).

To test whether BNN27-mediated suppression was targeted mainly to ongoing autoreactive responses, we immunized BNN27- and DHEA-treated EAE mice to an irrelevant antigen (ovalbumin). This resulted in a successful TH1 immune response to this new antigen (fig. S8E, F). We observed a similar enhanced TH1 immune response when we challenged BNN27- and DHEA-treated mice with inactivated lentivirus (data not shown). These findings suggest that BNN27 and DHEA act effectively against the myelin-specific ongoing autoreactive responses without dampening the protective responses induced by pathogen-mimicking agents, consistent also with previous studies showing that DHEA enhances anti-microbial immunity . Similarly, administration of BNN27 or DHEA, starting at the time of EAE induction with MOG35-55 in CFA (containing inactivated M. tuberculosis) resulted in significantly enhanced TH1 responses (fig. S9B). However, disease severity was milder (fig. S9A), accompanied by decreased CNS inflammation (fig. S9D), as well as decreased IL-17/IFN-γ ratio (fig. S9C). Overall, it appears that BNN27 and DHEA act more effectively when the autoimmune response is established, which is of clinical importance. This could be attributed to differential regulation of the entry of inflammatory TH1 and TH17 cells into the CNS .

We have also tested the ability of BNN27 to control established EAE in 2D2 TCR Tg mice that are greatly susceptible, as they bear high numbers of MOG35-55–specific CD4+ T cells . Administration of BNN27 in symptomatic EAE 2D2 TCR Tg mice resulted in significantly milder symptoms, as compared to PBS-treated mice (mean maximum scores; 2.25± 0.25 for BNN27 group, versus 3.75±0.25 for PBS group, Fig. 3A and Suppl. Video). MOG35-55–specific responses of BNN27- treated 2D2 TCR Tg mice were significantly suppressed (fig. S10A), characterized by decreased IL-17, IFN-γ and IL-6 secretion (Fig.3B and fig. S10B), while IL-10 was up-regulated (Fig.3B and fig. S10B). Expression levels of Rorc and Ccr6 were also down-regulated in DLN 2D2 CD4+ T cells from BNN27-treated mice, (Fig. 3C and fig. S10B), compared to control. Therefore, BNN27 was also effective in suppressing EAE and TH17 responses in highly predisposed 2D2 TCR Tg mice.

BNN27 was also effective in controlling the activation of 2D2 TCR Tg T cells in vitro. Culture of DLN cells from 2D2 TCR Tg mice with MOG35-55 in the presence of 100 nM BNN27 or DHEA also resulted in a remarkable decrease of the percentages of IL-17+CD4+ T cells, while the percentage of IL-10+IL-17+ T cells among TH17 cells was significantly increased (Fig. 3D). Additionally, expression levels of Il17, Il6, Ccr6 and Rorc were down-regulated in 2D2 CD4+ T cells cultured in the presence of BNN27 and DHEA (fig. S10C). Both agents exerted similar effects in ex vivo re-stimulated DLN CD4+ T cells from mice with EAE (data not shown). We also observed direct effects of BNN27 on purified CD4+ T cells, as BNN27 suppressed the anti-CD3/anti-CD28 –driven CD4+ T cell proliferation (fig. S10D). To test the effects of BNN27 and DHEA on ongoing TH17 responses, we re-stimulated DLN cells from mice with EAE (10 days after immunization) with MOG35-55 in the presence of IL-6, TGF-β and IL-23, known to maintain TH17 responses, in the presence of BNN27 or DHEA or RPMI, for 7 days . Both steroids decreased the percentages of IL-17+ CD4+ T cells (Fig. 3E) and IL-17 secretion (Fig.3G), concomitant with increased percentages of IL-10+ IL-17+ CD4+ T cells among TH17 cells (fig. S10E) and elevated IL-10 secretion (Fig. 3F). Both steroids did not affect the numbers of 7AAD-positive CD4+ T cells (data not shown). BNN27 and DHEA were ineffective in suppressing IL-17 secretion and IL17+ CD4+ T cell percentages when CD4+ T cells were Il10-deficient (Fig. 3, E and G). Furthermore, sorted IL-10- CD4+ T cells from Il10gfp EAE mice that were stimulated in TH17 maintenance conditions (as in Fig. 3E) in the presence of BNN27 were unable to revert to an IL-10-producing population (fig. S11A). In this setting, IL-17+ CD4+ T cells were not suppressed by BNN27 (fig. S11A). However, in parallel cultures of sorted IL-10+ CD4+ T cells, we observed expansion of IL-10+ CD4+ T cells in the presence of BNN27 (fig. S11B). It appears that the suppressive function of BNN27 relies on the presence of IL-10-producing cells. In addition, these findings indicate that the beneficial effects of BNN27 in EAE involve direct suppressive effects on immune cells.

Treatment of EAE mice with BNN27 results in the induction of CD4+ T cells with regulatory phenotype that could potentially suppress autoreactive responses. CD4+ T cells from BNN27-treated EAE mice were co-transferred into lymphopenic Rag1-/- recipients, in combination with CFSE-labeled effector CD4+ T cells from donors with EAE. Rag1-/- recipients adoptively co-transferred with CD4+ T cells from BNN27-treated EAE donors had significantly decreased numbers of proliferating CFSE-labeled effector CD4+ T cells in the DLNs, delayed disease onset and significantly reduced disease clinical score compared to control recipients (Fig. 4, A and B). We have also examined whether TH cells from BNN27- and DHEA–treated EAE mice could suppress ongoing EAE upon adoptive transfer into symptomatic wild type mice. Transfer of CD3+ or CD4+ T cells from BNN27- and DHEA-treated mice suppressed the ongoing disease of recipients (Fig.4, C and D and fig. S12A). Significantly reduced ratios of IL-17+/IFN-γ+ T cells in the CNS (fig. S12B), and of IL-17/IFN-γ levels secreted by DLN cells (fig. S12C) were found in recipients of CD3+ cells from BNN27 or DHEA- treated EAE donors, compared to control recipients. Significant suppression of the disease was also found in recipient mice with ongoing EAE adoptively transferred with DLN (fig. S13) and with CD11c+ cells from BNN27- or DHEA- treated EAE mice (Fig. 4E). In fact, adoptive transfer of CD11c+ cells from BNN27- or DHEA- treated mice into recipients with ongoing EAE significantly decreased the level of IL-17, IFN-γ (fig. S14A) and the numbers of CCR6+ TH17 cells in DLNs (fig. S14B). It is possible that Treg cells induced by BNN27 educate DCs to become tolerogenic . However, co-culture of BNN27-pre-treated naïve CD11c+ cells together with CD4+ T cells from day 10- EAE mice resulted in a significant up-regulation of IL-10 secretion by the CD4+ T cells (data not shown), indicating that BNN27 has also a direct effect on DCs. Taken together, these findings suggest that treatment of BNN27 or DHEA induces, besides functional regulatory T cells, additional immune cell populations with suppressing properties of ongoing EAE in vivo.

To elucidate the role of IL-10 in BNN27- and DHEA-mediated EAE suppression, we tested the ability of these neurosteroids to control disease in Il10-/- EAE mice. Both BNN27 and DHEA were ineffective to suppress the disease in the absence of IL-10 (Fig. 4F). In contrast to wild type EAE mice, BNN27 and DHEA did not suppress the secretion of IL-17 in Il10-/- EAE mice (Fig. 4G). We have also tested the ability of neutralizing antibodies against IL-10 to reverse the suppressive effects of BNN27 or DHEA in wild type EAE mice. Antibody-neutralization of IL-10 blunted the suppressive effects of BNN27 and DHEA, increasing the severity of EAE to levels observed in PBS-treated control EAE mice (fig. S15, A and B). CD4+ T cells from BNN27-treated Il10-/- EAE mice preserved their pathogenicity after transfer to Rag1-/- recipients (fig. S15C). In vitro, 2D2 TCR CD4+ T cells from BNN27-treated EAE mice were stronger suppressors of CFSE-labeled 2D2 TCR CD4+ T effector cells, than 2D2 TCR CD4+ T cells from PBS-treated EAE mice (Fig.4H). Blockade of IL-10 reversed this suppression (Fig.4H). We conclude that IL-10 induction mediates, to a great extent, the suppressive effects of the BNN27 treatment in EAE.

We have also tested whether BNN27 and DHEA affect the differentiation of naïve human CD4+ T cells towards TH17. BNN27 at 100 nM in combination with a mixture of TGF-β, IL-6, IL-21 and IL-1β significantly decreased the secretion of IL-17 in human CD4+ T cells in culture (Fig. 4I), reducing also the number of IL-17+ CD4+ T cells (Fig. 4J). It is of note that BNN27 decreased the numbers of both TH1 and TH17 cells, in the absence of polarizing factors (Fig. 4J). DHEA had similar immunomodulatory effects (fig. S16, A-C). Notably, in the same cell cultures, DHEA increased the number of human IL-10+ CD4+ T cells (fig. S16C), compared to CD4+ T cells, stimulated with the mixture of TGF-β, IL-6, IL-21 and rIL-1β alone. We have also assessed the effects of BNN27 and DHEA in T cells, isolated from eight MS patients with RR-MS (Table S1). BNN27 and DHEA significantly decreased the release of IL-17, IFN-γ and IL-4 cytokines, while IL-10 and TNF-α remained unaffected (Fig. 4K). In MS, IL-4 secretion inhibits Treg cell induction . Our data suggest that in addition to EAE mice, BNN27 and DHEA may limit human autoreactive TH17 responses in MS patients by interfering with both TH17 polarization and ongoing TH17 responses.

We have recently shown that DHEA binds with high affinity to NGF receptors . Indeed, DHEA exerts at least part of its neuroprotective actions by directly interacting with TrkA and p75NTR receptors (KD: 5-10 nM), efficiently inducing TrkA phosphorylation at tyrosine-490, and p75NTR interaction with its effector proteins RIP2 and TRAF6 . Based on these observations, in the present study we tested the ability of BNN27 to bind to NGF receptors. BNN27 effectively competed binding of [3H]-DHEA to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA or p75NTR receptors with Ki at nanomolar levels (Fig. 4L). Immune cells, such as T cells and macrophages strongly express NGF receptors ,. BNN27 at 100 nM effectively induced the phosphorylation of TrkA at tyrosine 490 in isolated CD4+ T cells from EAE mice (Fig. 4M). BNN27 at 100 nM facilitated the association of p75NTR receptor to its effector protein RIP2 in mouse CD4+ T cells in culture (Fig. 4M). Consistently, in cultures of DLN cells from EAE-induced mice, a synthetic inhibitor of TrkA phosphorylation and an antibody against p75 abrogated the induction of IL-10 by BNN27 or DHEA (Fig. 4N and fig. S17A). In addition, the suppressive effects of BNN27 and DHEA on Ccr6 gene expression (as in fig. S10C), were no longer observed when we cultured CD4+ T cells in the presence of a synthetic inhibitor of TrkA receptor (fig. S17B). In the same cultures, TrkA inhibition, as well as blocking of p75 receptor reversed the suppression of IL-17 secretion (data not shown). Our findings suggest that NGF receptors contribute to the suppressive effects of BNN27 and DHEA.

Several studies demonstrate the involvement of the NGF system in EAE and MS. NGF was shown to protect marmosets against EAE by down-regulating the production of IFN-γ by CNS-infiltrating T cells, and up-regulating the production of IL-10 by glial cells in inflammatory lesions . NGF administration suppressed EAE , while neutralization of NGF or deficiency in p75NTR receptor expression exacerbated EAE in mice . Interestingly, enhanced expression of NGF receptors was reported in EAE and MS lesions, most probably as a rescue response . Disease up-regulation of NGF receptors in the EAE and MS lesions may enhance the efficacy of BNN27 in suppressing neuroinflammation.

A DHEA metabolite towards its conversion to androgens and estrogens, 5-androsten-3β, 17β-diol (ADIOL), exerts anti-inflammatory effects on microglia and astrocytes in EAE mice, acting via estrogen receptor beta (ERβ) . Our experiments showed that both DHEA and BNN27 dampened IL-6 secretion, as well as iNOS expression, compared to control-treated primary microglia and BV2 cells (fig. S17, C and D). Also, Il10 expression was elevated in BV2 cultured in the presence of DHEA and BNN27, although it was not statistically significant (fig. S17D). Both DHEA and its metabolite ADIOL, were shown to bind, the former with low and the latter with high affinity, to estrogen receptor beta (ERβ Ki: 200 nM and 1 nM respectively) . In contrast to DHEA and ADIOL, BNN27 was unable to compete binding of [3H]-Estradiol to cytosolic preparations isolated from HEK293 cells transfected with the cDNA of ERbeta receptors (fig. S18A). ADIOL was unable to reverse binding of [3H]-DHEA to membranes isolated from HEK293TrkA transfectants while effectively competing its binding to membranes from HEK293p75NTR transfectants (Ki: 1.05+x nM) (fig. S18B). Moreover, ADIOL at 100 nM facilitated, within 20 min, the association of p75NTR receptor to its effector protein RIP2 in mouse CD4+ T cells in culture (Fig. 4N). Our findings suggest that, in addition to ERβ receptors, p75NTR receptors may also contribute to the previously observed suppressive effects of ADIOL in EAE mice. Glial cells strongly express p75NTR receptors, which hold a central role in the control of their differentiation, migration, proliferation and activation . For example, it is possible that ADIOL suppresses microglia activation via ERβ receptors, while affecting astrocytes through p75NTR receptors.

Our data also point to IL-10-mediated suppression of ongoing autoimmune responses by DHEA. DHEA and ADIOL activate ERβ receptors , are intermediates in estrogen and androgen biosynthesis , and their administration not only impairs the endocrine system long-term, but also poses a risk for estrogen- and androgen- dependent tumors, particularly in genetically predisposed patients (Miller et al. Steroids, 2013). Immunomodulatory synthetic derivatives, which lack estrogenic and androgenic properties, are far more suitable for treatment of chronic inflammatory conditions. Here we show that the derivative BNN27, a neuroprotective microneurotrophin , is a strong inducer of IL-10-mediated immune regulation and successfully suppresses pathogenic autoreactive responses and disease. BNN27 can serve as prime molecule for development of specific immunotherapies that induce IL-10-expressing T cells, pivotal in the regulation of immune responses and in the maintenance of immunological tolerance . Moreover, our findings add to insights on the crosstalk between the immune system and neurotrophic components .