Differential effects of a saturated and a monounsaturated fatty acid on MHC class I antigen presentation - PubMed (original) (raw)

Differential effects of a saturated and a monounsaturated fatty acid on MHC class I antigen presentation

S R Shaikh et al. Scand J Immunol. 2008 Jul.

Abstract

Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids.

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Figures

Figure 1

Figure 1

PA-treated APC are resistant to lysis by activated CTL. (A) Representative experiment comparing specific lysis of APC as a function of effector to lipid-treated targets. Target T2-Kb cells were incubated with 250–500 _μ_M PA or OA complexed to BSA, or BSA alone, for 12 h at 37 °C, peptide pulsed, labelled with 51Cr and mixed with activated 2C CTL for 4 h. 51Cr release was measured in terms of radioactive counts in the supernatants normalized to maximal release and corrected for leakage, as described in the Materials and methods. (B) Sample histogram of CTL lysis measured by Annexin V-Cy5 staining using flow cytometry. Control or lipid-treated T2-Kb-YFPs were incubated with activated CTL for 2 h, at which time apoptotic cells were identified as YFP+ Annexin V-Cy5+7-AAD−. (C) Average change in percent specific lysis at E/T = 5/1. Data in C (average ± SE) are from five independent measurements (**P < 0.01).

Figure 2

Figure 2

PA-treatment of APC inhibits their ability to activate naïve T cells. (A) Sample flow cytometry fluorescence dot plots of naïve T-cell activation, assessed by staining with anti-mouse CD69-PECy7 and CD8-PE. Cells are gated on live CD8+ T cells. The numbers in the upper right quadrant are the percentage of CD69+ T cells activated with T2-Kb cells treated with 500 _μ_M BSA, PA, or OA. T cell only is the negative control. (B) Change in percentage of activated CD69+ T cells with and without lipid treatment. (C) Geometric mean of CD69 surface expression of CD8+ T cells stimulated with BSA-, PA-, or OA-treated T2-Kbs. T2-Kb cells were treated with 250–500 _μ_M PA or OA for 12 h at 37 °C, peptide pulsed, and then mixed with naïve T cells isolated from the spleens of 2C transgenic mice at a ratio of 1/1. Activation of naïve T cells was measured with flow cytometry 6 h later. Data in B and C (average ± SE) are from three independent measurements (*P < 0.01).

Figure 3

Figure 3

Fatty acid treatment of APC lowers MHC class I surface expression. T2-Kb cells were treated for 12 h with 250–500 _μ_M BSA, PA, or OA at 37 °C and pulsed with 1 nM SIY. MHC class I surface expression was measured with antibody binding (20.8.4s) using flow cytometry. (A) Sample flow cytometry histogram of 20.8.5-Cy5 staining for MHC class I. (B) Median fluorescence intensity (MFI) values (average ± SE) of 20.8.5-Cy5 staining for MHC class I on T2-Kb cells treated with BSA, PA or OA from three independent measurements (*P < 0.05).

Figure 4

Figure 4

PA-treatment of APC inhibits conjugation with activated CTL. (A) Sample flow cytometry dot plots of activated CTL, T2-Kb-YFPs, and conjugates. T2-Kb-YFP cells were treated with BSA, PA, or OA for 12 h, peptide pulsed, and mixed with DiD loaded CTL at a ratio of 1/1. Conjugates were identified as YFP+DiD+ after 15 min of incubation at 37 °C. The numbers in the upper right quadrant are the percentage of T cells conjugated. (B) Change in the rate of APC–T-cell conjugation (percentage of T cells conjugated to APC). Data are pooled from two separate experiments. (C) Change in APC–T-cell conjugation (average ± SE) relative to the BSA control from three to four independent measurements at 15 min (*P < 0.01).

Figure 5

Figure 5

OA-treatment of APC prevents the effects of PA on antigen presentation. (A) Treatment of T2-Kbs with 500 _μ_M PA + 500 _μ_M OA does not affect cell viability. Sample flow cytometry dot plots of Annexin V-Cy5, 7-AAD staining. Cells are greater than 90% viabile (lower left quadrant). (B) CTL lysis of T2-Kb-YFP cells treated with BSA or 500 _μ_M PA + 500 _μ_M OA. Cells were treated for 12 h at 37 °C, peptide pulsed, and mixed with activated CTL (E/T = 5) for 2 h at which time apoptotic cells were identified as YFP+ Annexin V-Cy5+ 7AAD−. (C) Change (average ± SE) in lysis is from two to three independent measurements (**P < 0.01).

Figure 6

Figure 6

Accumulation of fatty acids into triglyceride-rich lipid droplets protects against changes in antigen presentation. (A) Confocal fluorescence microscopy images of C1C12 BODIPY (green), Nile Red (red), or C16BODIPY (yellow) in T2-Kb cells treated for 12 h with BSA, 500 _μ_M PA, 500 _μ_M OA, or 500 _μ_M PA + 500 _μ_M OA. Images are false coloured and the nucleus is stained with DAPI (blue). C1C12BODIPY and Nile Red report on accumulation of monounsaturated fatty acids in neutral lipid stores and C16BODIPY reports on incorporation of palmitate. (B) Box plot statistics for the fluorscence microscopy data represented in A for n = 31–43 cells per treatment condition. The frequency is area (pixels2) occupied by C1C12BODIPY, Nile red, or C16BODIPY in lipid droplets of T2-Kb cells above a defined threshold. Note that non-overlapping notches (dashed line) represent statistical significance [27].

Figure 7

Figure 7

PA and OA exert differential effects on the structure of the plasma membrane. (A) The effects of PA- and OA-treatment on plasma membrane structure were assessed using time-resolved fluorescence anisotropy measurements. Data were analysed using an empirical sum-of-three exponentials model, which quantifies probe order in terms of (A) the order parameter, S, and probe rotational dynamics in terms of (B) the average rotational correlation time. (C) Changes in actin remodeling of the plasma membrane was assessed with phalloidin (red) binding on APC using confocal fluorescence microscopy. (D) Box plot statistics for the fluorescence microscopy data represented in C for n = 25–30 cells per condition. The frequency is pixel intensity of phalloidin after subtraction of background. Note that non-overlapping notches (dashed line) of the box plots represents statistical significance [27]. T2-Kb cells were treated for 12 h with BSA, 500 _μ_M PA, 500 _μ_M OA, or 500 _μ_M PA + 500 _μ_M OA. Values in A–B are average ± SE from two independent experiments (*P < 0.05, **P < 0.01, P < 0.001).

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