IL4 induces IL6-producing M2 macrophages associated to inhibition of neuroinflammation in vitro and in vivo

Abstract

Background: Myeloid cells, such as macrophages and microglia, play a crucial role in neuroinflammation and have been recently identified as a novel therapeutic target, especially for chronic forms. The general aim would be to change the phenotype of myeloid cells from pro- to anti-inflammatory, favoring their tissue-trophic and regenerative functions. Myeloid cells, however, display a number of functional phenotypes, not immediately identifiable as pro- or anti-inflammatory, and associated to ambiguous markers.

Methods: We employed in vitro assays to study macrophage polarization/differentiation in the presence of classical polarizing stimuli such as IFNγ (pro-inflammatory) and IL4 (anti-inflammatory). We induced neuroinflammation in mice by immunization with a myelin antigen and treated diseased mice with intracisternal delivery of an IL4-expressing lentiviral vector. We analyzed clinical, pathological, and immunological outcomes with a focus on myeloid cells.

Results: We found that IL6, usually considered a pro-inflammatory cytokine, was released in vitro by macrophages treated with the anti-inflammatory cytokine IL4. We show the existence of macrophages expressing IL6 along with classical anti-inflammatory markers such as CD206 and demonstrate that these cells are immunosuppressive in vitro. In neuroinflamed mice, we show that IL4 delivery in the central nervous system (CNS) is associated with clinical and pathological protection from disease, associated with increased IL6 expression in infiltrating macrophages.

Conclusions: IL6 is known to mediate both pro- and anti-inflammatory effects, having two distinct ways to induce cell-signaling: either through the membrane bound receptor (anti-inflammatory) or through trans-signaling (pro-inflammatory). We show here that IL6-expressing macrophages are associated to protection from neuroinflammation, suggesting that IL6 anti-inflammatory properties prevail in the CNS, and calling for a general reconsideration of IL6 in macrophage polarization.

Keywords: Autoimmunity; EAE; IL4; IL6; M2 macrophages.

Fig. 1

Gene expression of typical M1 and M2 markers in bone marrow-derived macrophages stimulated in vitro. a–f Real-time RT-PCR for Ym1, CCL17, IL6, iNos, IL-1β, and TNF-α, in macrophages non stimulated (NS) and stimulated with different doses of IFN-γ (M1) and IL4 (M2). Gapdh has been used as a housekeeping gene. Data are shown as fold induction (FI ± standard deviation) over NS

Fig. 2

rIL4 induces IL6 expression in CD206⁺ macrophages. a, b Immunofluorescent staining for CD206 (green) and IL6 (red) reveals co-localization of IL6 and CD206 expression in bone marrow-derived (BM) macrophages, exposed to rIL4 at 20 ng/mL, but not in peritoneal (PEC) macrophages. a ×100, scale bar = 10 μm. b ×40, scale bar = 50 μm

Fig. 3

IL4-stimulated macrophages releasing IL6 are immune-regulatory. IL4-stimulated bone marrow-derived macrophages (M2) reduce IL-2 release from MOG-TCR transgenic T lymphocytes (2D2) stimulated with MOG_35–55 (a), as compared to non stimulated macrophages (NS), while releasing high levels of IL6 (b) as measured by ELISA

Fig. 4

IL4 gene therapy induces M2 markers and IL6 expression in CNS-infiltrating CD11b⁺ cells in vivo. mRNA levels of IL6, IL-1β, and YM1 were measured by real-time RT-PCR in CD11b⁺ cells recovered from the CNS of EAE mice treated intracisternally with an IL4-expressing (IL4) or a GFP-expressing (GFP) lentivirus. IL4 gene therapy induces a decrease of IL-1β (a), and an increase of IL6 (b) and Ym1 (c), as compared to control-treated mice. n = 3 for each group. Gapdh has been used as a housekeeping gene. Data are shown as arbitrary units (AU ± standard deviation). *P < 0.05 (t test)

Fig. 5

IL4 gene therapy upregulates CD206 in CNS-infiltrating macrophages of EAE mice. Immunofluorescence spinal cord sections of C57Bl/6 mice affected by EAE and treated, at 12 d.p.i, with the GFP-expressing (GFP, a) or the IL4-expressing lentivirus (IL4, b). The staining (CD206 in green, Iba1 in red, nuclei in blue, as indicated) highlights upregulation of CD206 in Iba1⁺ macrophages in EAE mice treated with IL4 gene therapy (b) as compared to mice treated with the GFP-expressing control virus (a). a, b ×40, scale bar 10 μm. Scale quantification of CD206⁺ macrophages in spinal cords is shown (± standard deviation) in (c). *P < 0.05 (t test)

Fig. 6

CNS IL4 gene therapy inhibits clinical and pathological signs of EAE. Clinical course of EAE mice intracisternally injected with the IL4-expressing (IL4, open dots) or the GFP-expressing (GFP, closed dots) lentivirus is shown either as median (a) or mean (±standard deviation) (b) values (EAE score is non-parametric). Arrows indicate the day of virus injection (day 12 post immunization). IL4-treated EAE mice are protected both in terms of clinical severity (including cumulative and maximum score, a–d), and demyelination (g) and axonal loss (h). The protective effect of IL4 gene therapy is associated to an increased number of infiltrating CD3⁺ T cells (e), but no modulation of IB4⁺ myeloid cells (f). ***P < 0.0001 (Mann–Whitney in a, t test in g); *P < 0.05 (Mann–Whitney in c, d, t test in e)