Docosahexaenoic Acid Decreases Pro-Inflammatory Mediators in an <i>In Vitro</i> Murine Adipocyte Macrophage Co-Culture Model

<div><p>Paracrine interactions between adipocytes and macrophages contribute to chronic inflammation in obese adipose tissue. Dietary strategies to mitigate such inflammation include long-chain polyunsaturated fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, which act through PPARγ-dependent and independent pathways. We utilized an <i>in vitro</i> co-culture model designed to mimic the ratio of macrophages:adipocytes in obese adipose tissue, whereby murine 3T3-L1 adipocytes were cultured with RAW 264.7 macrophages in direct contact, or separated by a trans-well membrane (contact-independent mechanism), with 125 µM of albumin-complexed DHA, EPA, palmitic acid (PA), or albumin alone (control). Thus, we studied the effect of physical cell contact versus the presence of soluble factors, with or without a PPARγ antagonist (T0070907) in order to elucidate putative mechanisms. After 12 hr, DHA was the most anti-inflammatory, decreasing MCP1 and IL-6 secretion in the contact system (−57%, −63%, respectively, p≤0.05) with similar effects in the trans-well system. The trans-well system allowed for isolation of cell types for inflammatory mediator analysis. DHA decreased mRNA expression (p<0.05) of <i>Mcp1</i> (−7.1 fold) and increased expression of the negative regulator, <i>Mcp1-IP</i> (+1.5 fold). In macrophages, DHA decreased mRNA expression of pro-inflammatory M1 polarization markers (p≤0.05), <i>Nos2</i> (iNOS; −7 fold), <i>Tnfα</i> (−4.2 fold) and <i>Nfκb</i> (−2.3 fold), while increasing anti-inflammatory <i>Tgfβ1</i> (+1.7 fold). Interestingly, the PPARγ antagonist co-administered with DHA or EPA in co-culture reduced (p≤0.05) adiponectin cellular protein, without modulating other cytokines (protein or mRNA). Overall, our findings suggest that DHA may lessen the degree of MCP1 and IL-6 secreted from adipocytes, and may reduce the degree of M1 polarization of macrophages recruited to adipose tissue, thereby decreasing the intensity of pro-inflammatory cross-talk between adipocytes and macrophages in obese adipose tissue.</p></div

M1 and M2 macrophage polarization marker mRNA expression in trans-well co-cultured macrophages.

<p>mRNA expression of key (A) M1 (<i>Nos2</i>, <i>Nfκb</i> and <i>Tnfα</i>), (B) M2 (<i>Arg1</i>, <i>Mrc2</i>) polarization genes, and (C) regulatory cytokines (<i>Tgfβ1</i> and <i>Il-10</i>) from macrophages harvested from the trans-well system at 12 hr. 0 hr  =  serum starved adipocytes alone prior to co-culture and fatty acid treatment, (+)  =  positive control; co-cultured adipocytes and macrophages plus 25 µM BSA, DHA  =  co-cultured adipocytes and macrophages in the presence of 125 µM DHA, EPA =  co-cultured adipocytes and macrophages in the presence of 125 µM EPA, and PA =  co-cultured adipocytes and macrophages in the presence of 125 µM PA. Values are mean fold change ± SEM. The experiment was independently conducted 2 times (in triplicate) for a final sample size of n = 6. A different letter indicates treatments are significantly different from each other, p≤0.05.</p

Adipokine secretion in the trans-well versus contact co-culture system.

<p>Adipokine secretion after the 12-culture incubation in the contact (A) and trans-well (B) treated co-culture conditions, and (C) a comparison of secreted MCP1, IL-6 and TNFα between the contact and trans-well co-culture systems. Note: no IL-10 was detected in the trans-well system (B). 0 hr  =  serum starved adipocytes alone prior to co-culture and fatty acid treatment, (−)  =  negative control; adipocytes alone treated with 25 µM BSA, (+)  =  positive control; co-cultured adipocytes and macrophages plus 25 µM BSA, DHA =  co-cultured adipocytes and macrophages in the presence of 125 µM DHA, EPA =  co-cultured adipocytes and macrophages in the presence of 125 µM EPA, and PA =  co-cultured adipocytes and macrophages in the presence of 125 µM PA. Values are means ± SEM. The experiment was independently conducted 3 times for a final sample size of n =  6–9. A different letter or an asterisk (*) indicates treatments are significantly different from each other, p≤0.05.</p

mRNA expression of inflammatory mediators in trans-well co-cultured adipocytes.

<p>The mRNA expression of key inflammatory (A) cytokines (<i>Il-6</i> and <i>Mcp1</i>), (B) signalling intermediates (<i>Nfκb</i>, <i>Tlr4</i> and <i>Tlr2</i>) and (C) negative feedback factors (<i>Mcp1-IP</i> and <i>Socs3</i>) from adipocytes harvested from the trans-well system after 12 hr of co-culture. 0 hr  =  serum starved adipocytes alone prior to co-culture and fatty acid treatment, (−)  =  negative control; adipocytes alone treated with 25 µM BSA, (+)  =  positive control; co-cultured adipocytes and macrophages plus 25 µM BSA, DHA  =  co-cultured adipocytes and macrophages in the presence of 125 µM DHA, EPA =  co-cultured adipocytes and macrophages in the presence of 125 µM EPA, and PA =  co-cultured adipocytes and macrophages in the presence of 125 µM PA. Values are mean fold change ± SEM. The experiment was independently conducted 3 times (in triplicate) for a final sample size of n = 6–9. A different letter indicates treatments are significantly different from each other, p≤0.05.</p

Effect of diet on splenic CD4⁺ T cell polarization.

<p>Splenic CD4⁺ cells were purified by positive selection and cultured for 3 d under A) Th17 or B) Treg polarizing conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049739#s2" target="_blank">Materials and Methods</a>, n = 3−4 TNBS treated mice/dietary group). Bars represent mean values ± SEM. Bars not sharing a common letter are significantly different (P≤0.05).</p

Visceral adipose tissue macrophage infiltration.

<p>Stromal vascular cells (SVC) were isolated and quantified from total visceral adipose tissue (HF and HF-FO groups, n = 3 vehicle controls and 6–8 TNBS-treated mice, LF n = 4 pooled samples comprised of 3–4 mice/treatment). A) percentage of F4/80⁺ CD11b⁺ cells (total macrophages), B) percentage of F4/80⁺ CD11c⁺ cells (M1 macrophages), C) percentage of F4/80⁺ CD206⁺ cells (M2 macrophages). Data were analyzed by two-way ANOVA (main effects: diet and treatment) and bars represent mean values ± SEM. Bars not sharing a common letter are significantly different (<i>P</i>≤0.05).</p

Colon histological disease scores for TNBS-treated mice.

<p>Colonic mucosal injury (0–3) and inflammation (0–3) scores were assessed in a blinded manner by a board-certified pathologist (B. Weeks) and combined for a total score (0–6). Representative images (100 × magnification) are shown for the HF, HF-FO and LF TNBS-treated groups, respectively (panels A-C) and a representative image of a HF vehicle control (panel D) is shown. E) Combined injury/inflammation histological score within the distal colon (n = 10−14 TNBS treated mice/diet). Data were analyzed using the Kruskal-Wallis test followed by Wilcoxon two-sample testing, and bars represent median values. Bars not sharing a common letter are significantly different (P≤0.05).</p

Visceral adipose tissue mRNA expression¹.

1<p>Values are means ± SEM (n = 5−10/dietary group). Data were analyzed by two-way ANOVA (main effects: diet and treatment). For all genes, there was no effect of treatment (i.e., TNBS versus vehicle, <i>P</i>>0.05), therefore, only the main effect of diet is shown. Within individual genes, values not sharing a lower case letter denote significant differences (<i>P</i>≤0.05). Data were normalized to ribosomal 18S.</p

Effect of diet and colitis on splenic T cell subsets.

<p>A) Tregs (CD4⁺ FOXP3⁺), B) Th17 (CD4⁺ IL17A⁺), and C) Th1 (CD4⁺ IFNγ⁺) cell populations (n = 3–6 vehicle controls and n = 6−12 TNBS treated mice/dietary group). Bars represent mean values ± SEM. Bars not sharing a common letter are significantly different (P≤0.05).</p

Characterization of the diet-induced obese phenotype.

<p>C57BL/6 mice were fed a high diet (HF), high fat diet supplemented with FO (HF-FO) or a low fat (LF) control diet for 12 weeks (n = 12−17 TNBS-treated and 4–6 vehicle controls/diet). Mice were presensitized with 1% TNBS or vehicle control (week 11), followed by a 2.5% TNBS enema or vehicle control (week 12) and sacrificed 3 d post-TNBS. A) Changes in body weight over time. B) Visceral adipose tissue weight from individual visceral depots (perinephric, mesenteric and epididymal) or combined (total visceral adipose). Serum concentrations of C) insulin, D) leptin, E) resistin and F) adiponectin. All data were analyzed by two-way ANOVA (main effects: diet and treatment) and <i>P</i>-values are shown. Bars represent means ± SEM and statistical significance was (<i>P</i>≤0.05). Panel A) asterisk (*) indicates statistically significant time points where the HF and HF-FO groups differed from LF (<i>P</i>≤0.05). Panels B-F) bars not sharing a common letter differ (<i>P</i>≤0.05).</p