Crosstalk between notch and toll-like receptor signaling pathways in macrophages and its roles in autoimmune diseases

Abstract

Macrophages are innate immune cells which their plasticity depends on the microenvironment. Various activators act on macrophages by induction of cascade signaling pathways including Notch signaling, resulting in secretion of diverse cytokines and inflammatory mediators. Consequently, inflammatory macrophages are associated with autoimmune diseases including multiple sclerosis. Stimulation of macrophages by IFNγ and lipopolysaccharide (LPS) leads to activation and secretion of various inflammatory related cytokines, including IL-12 and IL-10. In this study, we investigated the impacts of Notch1 deletion in LPS-activated macrophages in an Experimental autoimmune encephalomyelitis (EAE) model and the role of Notch signaling in regulating the expression of IL-12p40. Moreover, the role of Notch signaling in mediating IL-10 production in LPS/immune complex-activated macrophages is investigated. To examine the impact of Notch1 deficiency in activated macrophages, an adoptive transfer of activated macrophages derived from Notch1fl/fl X Mx1cre+/- (N1KO) mice or wild type mice was performed before the induction of EAE. Mice receiving activated N1KO macrophages showed a delay in the onset and decreased severity of EAE, compared to mice receiving wild type activated macrophages. In vitro re-stimulation of splenocytes by MOG35-55 peptide from these mice revealed that cells from mice receiving N1KO macrophages produced significantly less IL-17, compared to the control mice whereas IFNγ production was similar in both conditions. Furthermore, activated N1KO macrophages in vitro produced less IL-6 and exhibited lower CD80 expression. Activated N1KO macrophages did not exhibit any defect in IL-12p40/70 production whereas activated macrophages derived from CSL/Rbp-jκfl/fl X Mx1cre+/- (CSL/RBP-Jκ KO) mice phenocopied that of the gamma secretase (GSI) treatment which inhibits activation of Notch receptors. Furthermore, the nuclear translocation of NF-κB subunit c-Rel, one of the key regulatory factors in controlling il12p40 transcription, was compromised in GSI-treated and CSL/RBP-Jκ KO, but not in the N1KO macrophages. These results suggest that Notch1 expression in macrophages may affect the onset and severity of EAE through decreased IL-6 and CD80 which is involved in Th17 but not in Th1 response. Moreover, our findings suggest that Notch1, but not CSL/RBP-Jκ or gamma secretase activity is dispensable for IL-12p40/70 production in macrophages. Unlike the phenotype of LPS-activated macrophages, activation of macrophages with LPS in the presence of immune complex results in high level of IL-10 and low level of IL-12 production. Moreover, we investigated and shown that the production of IL-10 in macrophages was LPS-dependent and the activation of macrophages by IFNγ/LPS together with immune complex impacted the production of IL-10, IL-12, IL-6 but not TNFα. IFNγ/LPS+immune complex activated macrophages expressed Notch signaling molecules and the activation of Notch signaling was initiated after LPS-stimulation. The activation of Notch signaling depended upon NF-κB and MAPK pathways similar to that of LPS stimulation alone. Using pharmacological inhibitors to inhibit key signaling pathways, we confirmed that IL-10 production in LPS/immune complex activated macrophages were regulated by NF-κB, MAPK and PI3K. Furthermore, inhibition of Notch signaling using GSI resulted in the reduction of IL-10 production in the all LPS-stimulated condition. Finally, we found that Notch signaling affected the activation of NF-κB and MAPK signaling but not PI3K in this setting.