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Dietary Arachidonate in Milk Replacer Triggers Dual Benefits of PGE 2 Signaling in LPS-challenged Piglet Alveolar Macrophages

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Dietary Arachidonate in Milk Replacer Triggers Dual Benefits of PGE 2 Signaling in LPS-challenged Piglet Alveolar Macrophages

Kathleen R Walter et al. J Anim Sci Biotechnol.

Abstract

Background: Respiratory infections challenge the swine industry, despite common medicinal practices. The dual signaling nature of PGE2 (supporting both inflammation and resolution) makes it a potent regulator of immune cell function. Therefore, the use of dietary long chain n-6 PUFA to enhance PGE2 effects merits investigation.

Methods: Day-old pigs (n = 60) were allotted to one of three dietary groups for 21 d (n = 20/diet), and received either a control diet (CON, arachidonate = 0.5% of total fatty acids), an arachidonate (ARA)-enriched diet (LC n-6, ARA = 2.2%), or an eicosapentaenoic (EPA)-enriched diet (LC n-3, EPA = 3.0%). Alveolar macrophages and lung parenchymal tissue were collected for fatty acid analysis. Isolated alveolar macrophages were stimulated with LPS in situ for 24 h, and mRNA was isolated to assess markers associated with inflammation and eicosanoid production. Culture media were collected to assess PGE2 secretion. Oxidative burst in macrophages was measured by: 1) oxygen consumption and extracellular acidification (via Seahorse), 2) cytoplasmic oxidation and 3) nitric oxide production following 4, 18, and 24 h of LPS stimulation.

Results: Concentration of ARA (% of fatty acids, w/w) in macrophages from pigs fed LC n-6 was 86% higher than CON and 18% lower in pigs fed LC n-3 (P < 0.01). Following LPS stimulation, abundance of COX-2 and TNF-α mRNA (P < 0.0001), and PGE2 secretion (P < 0. 01) were higher in LC n-6 PAM vs. CON. However, ALOX5 abundance was 1.6-fold lower than CON. Macrophages from CON and LC n-6 groups were 4-fold higher in ALOX12/15 abundance (P < 0.0001) compared to LC n-3. Oxygen consumption and extracellular acidification rates increased over 4 h following LPS stimulation (P < 0.05) regardless of treatment. Similarly, increases in cytoplasmic oxidation (P < 0.001) and nitric oxide production (P < 0.002) were observed after 18 h of LPS stimulation but were unaffected by diet.

Conclusions: We infer that enriching diets with arachidonic acid may be an effective means to enhance a stronger innate immunologic response to respiratory challenges in neonatal pigs. However, further work is needed to examine long-term safety, clinical efficacy and economic viability.

Keywords: Arachidonic acid; Cyclooxygenase; Eicosanoid; Eicosapentaenoic acid; Inflammation; LPS; Lipid mediator class switch; Lipoxin; Porcine alveolar macrophage.

Conflict of interest statement

All animal protocols for this study were approved and carried out in accordance to the Institutional Animal Care and Use Committee of North Carolina State University.Not applicable.The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Dietary LC n-6 PUFA increases COX-2 mRNA abundance and eicosanoid production in porcine alveolar macrophages. Relative expression level of (a) COX-2 mRNA and (b) PGE2 production in alveolar macrophages isolated from piglets fed milk replacer with varying fatty acid composition. Measurements were made after 24 h of culture in absence (Basal) or presence of LPS. RNA values are mean fold changes relative to basal CON alveolar macrophages. Fatty acid effect, LPS effect and fatty acid-LPS interaction were evaluated. Values are represented as least square means ± SEM, (a) n = 12; (b) n = 20. Bars lacking a common letter differ
Fig. 2
Fig. 2
Dietary LC n-6 modifies lipoxygenase and cytokine mRNA abundance associated with a pro-inflammatory response in porcine alveolar macrophages. Relative mRNA abundance of (a) ALOX-5, (b) TNF-α, and (c) IL-6 in alveolar macrophages from piglets fed milk replacer with varying fatty acid composition. Measurements were made after 24 h of culture in absence (Basal) or presence of LPS. Values are mean fold changes relative to basal CON alveolar macrophages. Fatty acid effect, LPS effect and fatty acid-LPS interaction were assessed. Values are represented as least square means ± SEM, n = 12. Bars lacking a common letter differ
Fig. 3
Fig. 3
Long chain n-6 supplementation initiates initial stages of lipid-mediator class switching in porcine alveolar macrophages. Relative mRNA abundance of (a) ALOX-12/15 and (b) IL-10 in alveolar macrophages from piglets fed milk replacer with varying fatty acid composition. Measurements were made after 24 h of culture in absence (Basal) or presence of LPS. Values are mean fold changes relative to basal CON alveolar macrophages. Fatty acid effect, LPS effect and fatty acid-LPS interaction were assessed. Values represented as least square means ± SEM, n = 12. Bars lacking a common letter differ
Fig. 4
Fig. 4
LPS stimulation in porcine alveolar macrophages alters oxidative burst and cellular stress regardless of long chain PUFA enrichment. Representative trace of oxidative burst by measurements of (a) OCR, (b) ECAR and cellular stress by measurement of (c) NO and (d) cytoplasmic oxidation in PAM isolated from piglets fed milk replacer with varying fatty acid composition. Measurements were made during culture in absence (Basal) or presence of LPS at varying time points over the course of 24 h. Fatty acid effect, LPS effect and fatty acid-LPS interaction were assessed. Values are least square means ± SEM, n = 5. ECAR, extracellular acidification rate; NO, nitric oxide; OCR, oxygen consumption rate

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