Suppression of neuroinflammation by an allosteric agonist and positive allosteric modulator of the α7 nicotinic acetylcholine receptor GAT107

Abstract

Background: The α7 nicotinic acetylcholine receptor (α7 nAChR) negatively regulates the synthesis and release of pro-inflammatory cytokines by immune cells. Our previous studies showed that in encephalitogenic T cells, α7 nAChR expression is upregulated and that activation of the cholinergic system can attenuate experimental autoimmune encephalomyelitis (EAE). GAT107 is an allosteric agonist and positive allosteric modulator (ago-PAM) of α7 nAChR that can produce persistent activation of this receptor. Therefore, in the present study, we investigated the effect of GAT107 on neuroinflammation in EAE, the animal model used for the study of multiple sclerosis (MS) via α7 nAChR, and the inflammatory pathways involved.

Methods: EAE was induced by administration of myelin oligodendrocyte glycoprotein (MOG_35-55) in C57BL/6 mice. EAE mice were treated with the ago-PAM GAT107 or a placebo for 9 days, starting from the day of EAE induction. Clinical assessment and immunological evaluation of immune cells and cytokine production was performed.

Results: Following activation of the α7 nAChR by GAT107 during EAE, disease severity was significantly reduced by 70% and was correlated with a reduction in the extent of neuroinflammation in the CNS. The treatment reduced encephalitogenic T cell proliferation and the production of pro-inflammatory cytokines, as well as increased the production of the anti-inflammatory cytokine IL-10. Furthermore, the expression of immune cell markers was altered by GAT107 treatment, which induced a significant reduction in macrophages, dendritic cells, and B cells, as well as a reduction in anti-MOG_35-55 antibodies. Additionally, GAT107 was found to directly activate α7 nAChR in murine macrophage RAW264.7 cells and in human PBMCs derived from MS patients and healthy donors.

Conclusions: Our results show that GAT107 can be a useful molecule for harnessing the cholinergic anti-inflammatory pathway for long-lasting and wide-ranging modulation and downregulation of neuroinflammation in EAE.

Keywords: B cells; Central nervous system inflammation; Immune cholinergic system; Multiple sclerosis; Selective allosteric agonist for α7 nAChR; α7 Nicotinic acetylcholine receptor.

Fig. 1

Amelioration of the clinical course of EAE on treatment with the ago-PAM GAT107. EAE was induced in wild-type (WT) mice by immunization with myelin oligodendrocyte glycoprotein (MOG)_35–55. The animals were given either placebo or GAT107 (10 mg/kg) twice daily by intraperitoneal injection. The results are expressed as the mean clinical score ± standard error (SE) of data from four separate experiments (n = 29 in the placebo group and n = 24 in GAT107-treated group) (**p < 0.001)

Fig. 2

Neuropathological features of spinal cords in EAE mice treated with GAT107. Spinal cords of mice from the placebo- and GAT107-treated groups were removed at day 30 after EAE induction. a Treatment with GAT107 (10 mg/day) for 8 days improved CNS inflammation and reduced the number of EAE lesions. Representative images (with hematoxylin and eosin staining) at × 20 magnification are shown for the b placebo- and c GAT107-treated cells. The arrows indicate the foci (n = 6 in the placebo group and n = 7 in GAT107-treated group) (**p < 0.001)

Fig. 3

Reduction in T cell ex vivo reactivity following GAT107 treatment. Proliferation of lymphocytes from placebo- and GAT107-treated mice was assessed based on [3H] thymidine incorporation in the presence of MOG_35–55 (a) or in the presence of ConA, anti-CD3 antibody, or LPS (b). IL-17, IFN-γ, IL-6, and IL-10 levels following MOG_35–55 stimulation were measured by ELISA, and the results are expressed as percentage of the mean secretion ± SE value of the placebo-treated group based on data from three separate experiments (c) (n = 6 for each group). The expression of immune cell surface markers was assessed by FACS, as described in the “Methods” (d). Following GAT107 treatment, a significant decrease in the expression of CD11b (macrophages), CD11c (dendritic cells), and CCR5-positive cells was observed, but there was no significant change in the number of CD4-, CD8-, and MHC class II-expressing cells (n = 3) (*p < 0.05, **p < 0.001)

Fig. 4

mRNA expression levels of foxp3, TGFβ, GATA3, T-bet, and RORγt following GAT107 treatment. Mice were treated with GAT107 (10 mg/day, i.p.) for 8 days from the day of EAE induction. One day later, pooled lymphocytes were obtained and activated with MOG_35–55 as described in the “Methods”. mRNA levels of foxp3, TGFβ, GATA3, T-bet, and RORγt were assessed by RT-PCR. The results are expressed as mean relative quantification value ± SE of three separate experiments (n = 6–7 in each group) (*p < 0.05)

Fig. 5

Level of B cell markers following GAT107 treatment. a Reduction in B200-positive cells following GAT107 treatment (n = 3) (*p < 0.05). b Reduction in anti-MOG_35–55 antibodies following GAT107 treatment. The serum antibody levels were detected with ELISA, as described in the “Methods”. Results are expressed as the mean of two separate experiments ± SE (n = 7) (*p < 0.05). c mRNA levels of APRIL, BAFF, BAFF-R, BCMA, CXCL13, OPN, and TACI were detected by RT-PCR at day 9 after EAE induction and following ex vivo reactivation with the MOG peptide. The results expressed as mean relative quantification value ± SE of two separate experiments (n = 5, *p < 0.05)

Fig. 6

Suppressive effect of GAT107 on inflammatory cytokine release from RAW267.4 cells. IL-6 production was measured in RAW267.4 cells stimulated with LPS (25 μg/ml) with and without α7 nAChR-targeting drugs. a Effects of ACh (100 μM) and GAT107 (10 μM) on LPS-dependent IL-6 release. b MLA (10 nM) antagonizes the effects of GAT107 (10 μM) on LPS-dependent IL-6 release (n = 4 biological replicates, two technical replicates each) (*p < 0.05, **p < 0.01)

Fig. 7

Suppressive effect of GAT107 on inflammatory cytokine release from PBMCs from HDs and MS patients. PBMCs were purified from samples from human donors, including healthy donors (a, b) and MS patients (c, d), and stimulated with ConA (5 μg/ml) with and without ACh (100 μM) or GAT107 (10 μM) for 24 h. (a + c) IL-17 and (b + d) IL-6 secretion were detected by ELISA (n = 17–19) (*p < 0.05)