In the current study, the therapeutic effects of the cytostatic and immunomodulatory drug teriflunomide were evaluated in a mouse model of MG induced by AChR immunizations. Our results revealed that teriflunomide treatment after disease onset decreased the incidence and clinical disease scores of murine EAMG. Teriflunomide improved EAMG symptoms by reducing the absolute CD4+ T cell numbers and cytokine production, the number of certain plasma cell subsets and by decreasing long lived mature plasma cell expression (CD138 expression) and causing a subtle decrease in IgG1 type anti-AChR in comparison to other types of immunoglobulins. To our knowledge, this is the first study showing a clinical and immunological benefit of teriflunomide use after disease onset in an anti-AChR-mediated model of EAMG in mice. Yilmaz et al. have recently tested therapeutic administration of teriflunomide in MuSK mediated-EAMG and showed clinical benefit similar to the results presented herein (Yilmaz et al., 2021). AChR-specific autoantibody- mediated pathology account for 85% of MG cases in humans; while MuSK autoantibody-mediated MG accounts for 6% of the MG patient population (Fichtner et al., 2020; Wang and Yan, 2017). Therefore, the results of the current report have key implications for MG patients, and supports the use of teriflunomide as a therapeutic treatment.
In our hands, the clinical EAMG disease was first observed in some mice about the fourth week (ten days after the second immunization). Disease incidence and mean disease score reached a statistically significant level compared to the healthy control group at the sixth week after the first immunization (three weeks after the second AChR injection), which is consistent with the literature (Mantegazza et al., 2016; Shigemoto et al., 2015; Yilmaz et al., 2021). In some studies, the standards of mimicking MG treatment after disease onset is defined as the time when ≥ 60 % of mice develop clinical symptoms of the disease (Tuzun et al.,2015). In our experiment, this criterion was fulfilled three days after the third immunization and the treatment was started at this time. Teriflunomide treatment after disease onset reduced the anti-AChR antibody mediated EAMG incidence at 12th week, and the mean clinical MG scores starting at 10th week till the end of the experiment compared with untreated EAMG mice. The data indicate that teriflunomide’s clinical effects are detectable after three weeks of use. In Yilmaz et al.’s MuSK mediated EAMG model, the beneficial effect of teriflunomide was also detectable starting by the 11th week of disease induction, thus these independent studies corroborate each other’s findings. Although EAMG dependent weight loss was ameliorated by teriflunomide treatment in the Yilmaz et al. study (Yilmaz et al., 2021). Our EAMG+TF group showed no weight loss, but reduced weight gain compared with the EAMG group. The difference between the two studies could be explained by differences in the doses of teriflunomide, which was higher in the current study (Committee for Medicinal Products for Human Use-CHMP- 2013).
The primary mechanism of action of teriflunomide is through inhibition of mitochondrial enzyme DHO-DH. This enzyme is highly expressed in activated lymphocytes, both T and B cells which rely on de novo pyrimidine synthesis to meet their increased nucleotide demands. Because it is not a nucleotide analogue, and the resting lymphocytes can use salvage pathways to meet their pyrimidine needs, teriflunomide appears to affect mostly activated lymphocytes (Bar-Or et al., 2014). In vitro proliferation assays revealed that teriflunomide can inhibit proliferation of CD4+, CD8+ T cells as well as B and NK cells without reducing their survival (Li et al., 2013). Others have shown that, in vivo, teriflunomide was particularly effective in inhibiting the proliferation of T cells with high-affinity T cell receptors for the antigens (Posevitz et al., 2012).
In our experiments regarding the T cell compartment, the most notable changes were observed in CD4+ helper T cells with teriflunomide treatment. In the Yilmaz et al study, flow cytometric analyses of T cells (CD4+ or CD8+), B lymphocytes as well as natural killer (NK) cells revealed no significant difference between teriflunomide treated and untreated MuSK EAMG groups with respect to percentages of cells. However the changes in the absolute numbers of those cells have not been documented in that study (Yilmaz et al., 2021). Our experiments also did not reveal any reduction in the percentages/frequency of CD4+ T cells or their IL-2+, IL-17+, IL-22+, GM-CSF+ subsets between teriflunomide treated and untreated AChR EAMG groups. However, the absolute number of IFN-γ+, IL-17+, IL-2+, IL-22+ CD4+ T subsets as well as total CD4+ T cells were reduced significantly in the secondary lymphoid organs after teriflunomide treatment. The anti-proliferative effect of teriflunomide on CD4+ T cells was also evident in the thymus. Thus, helper T cells responses, particularly Th1, Th17 which were shown to play critical roles in the pathogenesis of EAMG, appeared to be inhibited by teriflunomide. This finding has been supported by the existing literature (Wang and Yan, 2017). When IFN-γ MFI values were examined, IFN-γ expression per cell was unaffected. These findings argue that teriflunomide, rather than inhibiting transcription/or translation events of these cytokine genes, acts by inhibiting proliferation of those T helper cell subsets. These data are in line with the human studies showing leukopenia/lymphopenia in MS patients who were treated with teriflunomide and the well-established negative impact of teriflunomide on highly proliferating T and B cells (Bar-Or et al., 2014; Confavreux et al., 2014; O'Connor et al., 2011). It is important to note that T and B cells’ frequency/percentage measurements do not always reflect the changes in the absolute number of cells, and discrepancies between some reports most likely result from the lack of absolute number calculations.
In our experiments, teriflunomide did not reduce total CD8+ T cell numbers in vivo in EAMG mice, be it in lymph nodes, spleen or thymus. However, we observed a reduction in CD40L+ CD8+ T cell numbers (activated CD8+ T cells) in the thymus. Both CD4+ and CD8+ T cells are shown to be involved in the pathogenesis of EAMG in rats and mice, unlike human MG, which occurs predominantly through CD4+ T cell and B-cell mediated pathology (Wang and Yan, 2017; Zhang et al., 1996). Accordingly, genetic or antibody-mediated depletion of CD8+ or CD4+ T cells suppressed the disease (Zhang et al., 1996). It is unclear why teriflunomide has a more robust impact on preferentially CD4+ T cell compartment in the murine EAMG model. This could be related to differential reliance of CD4+ and CD8+ T cells on DHO-DH and requires further study. Although a recent study suggested that 12-month use of teriflunomide in relapsing- remitting MS patients selectively reduced CD8+ memory T cells, absolute numbers of cells have not been examined in that study (Tilly et al., 2021). Another report performed on seven multiple sclerosis (MS) patients by Gandoglia et al. showed a trend towards reduction in helper T cells after teriflunomide use (Gandoglia et al., 2017).
Both leflunomide and teriflunomide have been shown to inhibit B cell proliferation through inhibition of DHO-DH and other targets such as cyclin D3 and cyclin A expression (Ringshausen et al., 2008). In the peripheral blood of teriflunomide receiving MS patients, absolute numbers of mature, regulatory or CD19+ total B cells were significantly reduced (Gandoglia et al., 2017). The only report investigating B cells in the EAMG mice model was Yilmaz et al.’s anti-MuSK mediated EAMG study. In that study, B cell percentages were found not to be significantly altered, while the absolute B cell counts were not examined (Yilmaz et al., 2021). Our data revealed that total CD19+ B cells as well as plasma cells (CD19+/low CD138) absolute numbers were not significantly altered by teriflunomide treatment. On the other hand, Lambda+ plasma cell absolute numbers were significantly reduced in the spleen of EAMG+TF mice compared with the untreated EAMG group. Additionally, CD138 expression (mean fluorescence intensity showing mean antigen expression), which is high in long lived mature plasma cells responsible for the production of IgG, were significantly reduced in the lymph nodes and the spleen suggesting that teriflunomide may result in significant changes in the plasma cell functions affecting antibody production in a selective way (Bortnick and Allman, 2013; Nutt et al., 2015).
In our experiments, teriflunomide treatment appears to increase IgM isotype, while it also causes a subtle decrease in IgG1 level. Although IgG1 levels were still comparable between EAMG and EAMG+TF mice groups in the 12-week post EAMG induction, IgG1 levels at 12th week of disease induction was more significant in the EAMG group compared to EAMG+TF group when compared to their corresponding basal levels. These data argue that the drop in IgG1 levels EAMG+TF mice group is continuing, yet undetectable at this time point. Indeed, in the anti- MuSK mediated EAMG model of Yilmaz et al. serum IgG1 levels significantly reduced after teriflunomide treatment at the 14th week of disease induction (Yilmaz et al., 2021). Besides reduced IgG deposition at the neuromuscular junctions was reported in that study, supporting our data. Yılmaz et al did not measure serum IgM levels in their study (Yilmaz et al., 2021).
When both studies of teriflunomide in EAMG are evaluated together, it can be reported that this drug may cause some selective changes in the antibody responses. Upon considering the basic mechanism of action of teriflunomide that is inhibiting the rapidly progressing T and B cells, to explain these selective changes in the antibody responses seems to be difficult. Through its basic mechanism of inhibitory action on DHO-DH, teriflunomide has been proposed to have direct suppressive effect on B cells, since it reduced proliferation and lipopolysaccharide stimulated Ig secretion of B cells (Siemasko et al., 1996). Siemasko et al. in their in vivo study found that leflunomide decreases both IgG and IgM secretion, in a manner that is mostly decreasing IgG levels (Siemasko et al., 1998). They also discovered that leflunomide has some DHO-DH independent functions including tyrosine kinase inhibitory activity causing an indirect effect on B cells, in such a way that it decreases IL-4 driven class switch recombination into IgG1, causing a reduction in IgG1 production (Siemasko et al., 1998; Claussen et al., 2012). Our results, with the increase in IgM and subtle decrease in IgG1 levels, are in accordance with the inhibition of this class switch. Similarly, in a clinical study comparing the serum immunoglobulin levels during teriflunomide and ofatumumab (human anti CD-20 monoclonal antibody) treatment, the proportion of patients with IgG levels below the lower limit of the normal and the proportion of patients with IgM levels below the lower limit of the normal were found to be 22.9% and 6.6% in teriflunomide using patients, and 14.2% and 17.7% in ofatumumab using patients respectively. It is interesting to see that the proportion of IgG decrease with teriflunomide use was higher than the proportion of IgM decrease below the normal lower limits. The proportion of decrease in Ig G level with teriflunomid is also higher than that of ofatumumab, which is a drug totally active on B lymphocytes (Wiendl et al)., 2020).
In our study, an absolute clinical improvement was observed with teriflunomide use but exact mechanisms underlying this improvement seem difficult to explain. The impact of teriflunomide on B cells, either directly on B cells, or indirectly through CD4+ T cells might lead to the clinical ameloriation of the disease. The decrease in the number of plasma cells, the reduction of the mean CD 138+ expression (CD138 mean fluorescent intensity) meaning reduced activity of long lived mature plasma cells, as well as lower numbers of the helper T cells which may have taken a toll on B cell affinity maturation and the subtle decrease in IgG1, possibly resulting from the drug’s inhibitory action on tyrosine kinase, might have caused the therapeutic changes in the immunological events at neuromuscular junctions. (Bortnick and Allman, 2013; Khodadadi et al., 2019; Nutt et al., 2015, A, A6). IgG1 is the major immunoglobulin in the pathogenesis of AChR autoantibody-mediated MG (Lefvert et al., 1981; Rodgaard et al., 1987) and may exert its effects via complement activation, by blocking AChR signaling, or by inducing internalization of the receptor from the cell membrane (Ey et al., 1979). IgM to IgG1 isotype switch is induced by IL-4 and IL-21 cytokines (Moens and Tangye, 2014), and whether their levels are altered after teriflunomide treatment in vivo requires further study.
In this study, it is exceptionally interesting to see that how a drug shows selectivity in its actions on cell types that is to say affecting helper T cells (even especially IFN-g secreting helper T cells) more than cytotoxic T cells, affecting mostly more mature plasma cells out of all B cells and affecting some types of immunoglobulins more. These findings are most probably due to some regulations and interactions of the immune mechanisms in relation to the effects and the dose of the drug. Clausen et al in their review explained that most of kinase inhibitory actions of teriflunomide were observed in vitro in higher concentrations (at least one order of magnitude higher) than those used to block DHO-DH, They also claimed that it is difficult to understand whether these effects would be reliable in vivo (Claussen et al., 2012). Indeed, our study may also show the higher concentration effects of the teriflunomide due to its use in a high dose. Likewise the differences of our findings from those of Yilmaz et al. may be explained by the differences in the doses of the drug, which was about 200-250 mg/day for each mouse in the current study and 30 mg/day in the Yilmaz et al’s study (Yilmaz et al., 2021).
This study has a wide range of investigations with T cells, B cells and cytokines in the spleen, lymph node and the thymus in a detailed way and with serum anti-AChR immunoglobulins in the murine AChR-induced EAMG model. Nevertheless, due to some practical limitations, the memory T and B cells, and more elaborate studies in the thymus could not be performed. Furthermore the prolonging of the treatment period would yield more pronounced results regarding the immunoglobulins. Finally, the stimulation of T cells was preferred to be performed with PMA/Ionomycin rather than more specific AChR peptide stimulation, due to its capability of giving stronger signals as a valuable method.
In conclusion, our study shows that teriflunomide has clinical benefits and prevented the progression of the disease in a murine model of MG through different possible mechanisms including suppression of immune responses by reducing the number of cytokine-producing T cells, by changing the functions of plasma cells and by leading selective changes in anti-AChR antibody quantities and types. These mechanisms need to be verified with some other studies. Additionally, extending the follow-up period in future studies and performing the experiments with different doses of teriflunomide could definitely be more informative. The data presented herein suggests that teriflunomide may be a suitable candidate for use in MG patients and even in chronic inflammatory neuromuscular diseases owing to its widespread effect on the immune system and its low side effects. Further studies including human trials in MG patients would be beneficial.