Docosahexaenoic acid prevents dendritic cell maturation, inhibits antigen-specific Th1/Th17 differentiation and suppresses experimental autoimmune encephalomyelitis

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Abstract

Docosahexaenoic acid (DHA), the most abundant essential n  3 polyunsaturated fatty acid in the CNS, emerged recently together with eicosapentaenoic acid (EPA) and DHA/EPA metabolic derivatives as a major player in the resolution of inflammation. Protective anti-inflammatory effects of DHA were reported in clinical studies and animal models of colitis, sepsis, and stroke. Here we report for the first time a beneficial effect of dietary n  3 fatty acids in experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. In the present study we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) in CD4+ T cell stimulation and differentiation. Pretreatment of DC with DHA prevented LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of costimulatory molecules and lack of proinflammatory cytokine production (IL-12p70, IL-6, and IL-23). DHA-treated DC were poor stimulators of antigen-specific T cells in terms of proliferation and Th1/Th17 differentiation. This was associated with an increase in p27(kip1), a cell cycle arresting agent, and with decreases in Tbet, GATA-3, and RORγt, master transcription factors for Th1, Th2, and Th17. In contrast, T cells co-cultured with DC-DHA express higher levels of TGFβ and Foxp3, without exhibiting a functional Treg phenotype. Similar to the in vitro results, the beneficial effect of DHA in EAE was associated with reduced numbers of IFNγ- and IL-17-producing CD4+ T cells in both spleen and CNS.

Introduction

In contrast to n  6 polyunsaturated fatty acids (PUFA) such as arachidonic acid (AA) which mediate predominantly proinflammatory effects, the n  3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are mostly anti-inflammatory. The n  3 PUFAs were reported to be protective in animal models of Alzheimer’s and Parkinson’s disease, in the ischemia–reperfusion model of stroke, and more recently in spinal cord and traumatic brain injury models (Bailes and Mills, 2010, Bousquet et al., 2008, Calon et al., 2004, Lopez-Vales et al., 2010, Marcheselli et al., 2003). In human subjects, high intake of dietary DHA and EPA affected the expression of more than 1000 genes, reducing proinflammatory and atherogenic related gene expression (Bouwens et al., 2009). In patients with multiple sclerosis (MS), several studies reported lower levels of inflammatory cytokines and MMP9 and significant improvement in quality of life following 3–6 months of n  3 fatty acids supplementation (Gallai et al., 1995, Shinto et al., 2009, Weinstock-Guttman et al., 2005).

Although MS etiology remains elusive, the pathology relies mainly on autoimmune mechanisms that involve activated antigen-specific T cells as major players in addition to macrophages, dendritic cells (DC) and B cells (Frohman et al., 2006, Slavin et al., 2010). Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for MS. Multiple T cell subsets including CD4+ Th1 and Th17, γδT cells, CD8+ and Treg have been shown to be involved in EAE, with Th1 and Th17 as major pathogenic cells (Slavin et al., 2010). DC play an essential role in antigen presentation and T cell activation in EAE, with CNS perivascular conventional/myeloid DC (cDC/mDC) activating myelin-specific T cells and inducing differentiation into encephalitogenic Th1/Th17 (Bailey et al., 2007, Slavin et al., 2010).

DC represent an essential cellular link between innate and adaptive immunity. DC are professional antigen presenting cells whose major role is the uptake, processing and presentation of antigens to naïve CD4+ and CD8+ T cells. Following antigen uptake, cDC mature, upregulating MHCII and co-stimulatory molecules, secreting various cytokines and chemokines, and acquiring a new chemokine receptor pattern which enables them to migrate to neighboring lymph nodes. CD4+ T cell activation and differentiation in vivo is mediated through cognate interactions of naïve T cells with antigen-presenting cells, primarily cDC, which provide signaling through MHCII/antigen complexes and costimulatory molecules. Cytokines secreted by mature cDC play an important role in CD4+ T cell differentiation, with IL-12 supporting Th1, and IL-6, TGFβ and IL-23 supporting Th17 differentiation. We and others reported that exposure to DHA maintains cDC stimulated with TLR ligands in an immature state characterized by low MHCII, CD40, CD80, CD86, and CCR7 expression, and lack of inflammatory cytokine production (Kong et al., 2010, Wang et al., 2007, Zapata-Gonzalez et al., 2008, Zeyda et al., 2005). In our previous study (Kong et al., 2010) we did not address the effects of DHA-treated cDC on T cell activation and differentiation. Here we report for the first time that exposure to DHA prevents cDC to induce antigen-specific naïve CD4+ T cells to differentiate into Th17 cells, and that DHA-treated DC arrest T cells in G0/G1 phase through increased expression of p27(kip1). We also report for the first time that dietary DHA reduces EAE disease severity and that this correlates with a significant reduction in both Th1 and Th17 cells in spleen and the CNS.

Section snippets

Mice

B10.A mice (I-Ek), C57BL/6 mice (H-2b), TCR-Cyt-5CC7-I/Rag1−/− transgenic (PCCF-specific TCR Tg; I-Ek) and C57BL/6-Tg (Tcra2D2, Tcrb2D2)1Kuch/J (MOG35–55 specific TCR) were purchased from Jackson Laboratory (Bar Harbor, ME) and Taconic (Hudson, NY). Transgenic mice were bred and maintained in the Temple University School of Medicine animal facility (Philadelphia, PA) under pathogen-free conditions. All mice used were between 6 and 10 weeks of age. Mice were handled and housed in accordance with

DHA prevents LPS-induced DC maturation and cytokine production

Purified CD11c+ bone marrow-derived DC were exposed to DHA for 24 h followed by LPS stimulation. In previous experiments we established the optimal dose of DHA as 50 μM (Kong et al., 2010). Twenty-four hours later we analyzed the levels of CD40, CD80, CD86, PD-L1, and PD-L2 by flow cytometry. In immature DC, DHA treatment inhibited the expression of MHCII and increased slightly the expression of CD40 and CD86 (Table 1). As reported previously, we found that DHA prevented the upregulation of

Discussion

Initially the anti-inflammatory effects of DHA and EPA have been attributed to the inhibition of cycloxygenase-mediated AA metabolism. However, recent studies established that DHA and EPA also alter the composition of lipid rafts in immune cells, act as ligands for nuclear receptors such as peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXR), and generate endogenous metabolites involved in the resolution of inflammation such as resolvins, docosatrienes and

Conflict of Interest

All authors declare that there are no conflicts of interest.

Acknowledgments

This study was supported by the following grants: NIH/NIAID 2RO1AI052306 and Pennsylvania CURE Tobacco Settlement Formula (DG).

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