Inhibition of CD40-TRAF6 interactions by the small molecule inhibitor 6877002 reduces neuroinflammation

Abstract

Background: The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation.

Methods: Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice).

Results: We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models.

Conclusions: Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.

Keywords: Co-stimulation; EAE; Inflammation; Monocytes; Multiple sclerosis.

Fig. 1

SMI treatment of monocytes inhibits CD40-induced trans-endothelial migration by limiting ROS production. Human monocytes were treated with either SMI 6877002 (1–10 μM) or vehicle for 1 h and stimulated with G28.5 (agonistic CD40 antibody) for 16 h, or pretreated with G28.5 for 1 h and then stimulated with SMI 6877002 for 16 h. a Monocyte trans-endothelial migration was studied in vitro using hCMEC/D3 cells by Transwell migration [31]. b ROS production measured as mean fluorescence intensity (MFI) of rhodamine 123. c CD40-induced monocyte migration in the presence or absence of the ROS scavenger luteolin (50 μM), measured in time-lapse migration. Experiments were performed in triplicate using buffy coats from 3 (1A), 4 (1B), or 3 (1C) human donors, and results are presented as the mean ± SEM. */#P < 0.05, **/##P < 0.01,***/###P < 0.001 as determined by Student’s t test. #luteolin vs control

Fig. 2

CD40-TRAF6-inhibiting SMI 6877002 improved the inflammatory phenotype of monocytes. Human monocytes were treated with SMI 6877002 (2.5 μM) for 1 h and stimulated with G28.5 (agonistic CD40 antibody) for 16 h. a CD14⁺ (classical), CD14⁺/CD16⁺ (intermediate), and CD16⁺ (non-classical) subsets of monocytes were measured by flow cytometry. b HLA-DR, CD80, and CD86 mean fluorescent intensity (MFI) measured in the classical monocyte subset (CD14⁺) by flow cytometry. c TNF, IL-6, and IL-10 production were measured by ELISA. Experiments were performed in triplicate using buffy coats of 2 (flow cytometry) or 4 (ELISA) human donors, and results are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t test

Fig. 3

SMI 6877002 treatment ameliorates severe paralysis in rats subjected to EAE. a EAE was induced, and animals were treated with 10 μmol/kg SMI 6877002 or vehicle from days 6 to 11 after induction. Clinical scores were observed daily. Experiments were performed with 6 animals in the control group and 11 animals in the EAE and SMI groups. b Immunofluorescence analysis of rat EAE cerebellum to determine macrophage and T cell infiltration into the CNS parenchyma. Sections were stained for ED1 (in red for macrophages), R7.3 (in red for T cells), and laminin (in green for localization). Representative images from three animals per group sacrificed at the peak of the disease. Scale bar 25 μm. c Gene expression in rat spinal cord during peak of disease was measured by qPCR. mRNA expression levels of TNF, NOS2, MMP9, CD80, CD86, and CD40 presented as relative expression compared to GAPDH. Expression was measured in three animals per group. d Quantified numbers of CD68 (for macrophages)- and CD3 (for T cells)-positive immune cell infiltrates in spinal cord tissues collected at day 20 of EAE. For each animal, the amount of infiltrates was counted on four levels, 5 mm between sections. Results are presented as the mean ± SEM. *P < 0.05, ***P < 0.001 as determined by Student’s t test

Fig. 4

Prolonged SMI treatment does not affect EAE development in mice. Mice were treated with vehicle or 10 μmol/kg SMI 6877002 starting 3 days prior to EAE induction until 17 days after immunization. a Clinical scores of mice treated with vehicle or SMI 6877002. Experiments were performed with 14 animals in the vehicle- and SMI-treated groups and 6 control animals without EAE induction. b SMI 6877002 treatment reduces the percentage of Mac3⁺ cells in the cerebellum of EAE mice at the peak of disease (scale bar Mac3 staining 400 μm), but has no effect on T cell infiltration into the cerebellum (scale bar CD3 staining 200 μm). c Flow cytometric analysis demonstrates that SMI 6877002 treatment results in a shift in the CD4/CD8 T cell balance in lymph nodes. Analysis was performed in lymph nodes of six animals per group sacrificed at the peak of disease. Results are presented as the mean ± SEM. *P < 0.05, ***P < 0.001 as determined by Student’s t test