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Intrinsic anti‐inflammatory properties of serum in deep‐diving seals

Diving seals are repeatedly exposed to hypoxia‐reoxygenation and other pro‐inflammatory stimuli such as shear stresses in alveoli from lung collapse and re‐expansion. Moreover, they have a known hyperlipidemic status, which is associated with vascular inflammatory stress in humans and mice. To investigate potential protective strategies in free‐living populations of deep‐diving seals, we examined baseline levels of cytokine biomarkers, as well as the inflammatory response of IL6 gene expression in seal blood cells exposed to an endotoxin (LPS, lipopolysaccharide, 1–1000ng/mL). Despite aspects of their basic physiology that predisposes other mammals to inflammatory stress, cytokine production in healthy Weddell seals (adults and pups) and Northern elephant seals (adults only) following LPS incubation was blunted (10–100X increase in plasma IL6), compared to human samples assayed under the same conditions (>1000X increase). These plasma samples, further assayed using a cytokine panel validated for pinnipeds, revealed that many cytokines were below detectable levels in Weddell seals, with only IL6, IL10 and TNFα exhibiting a dose‐response to LPS. Under isolated, in vitro conditions, Weddell seal monocytes maintained the expected, robust response to stimulation with LPS endotoxin. However, when this response was compared under typical cell culture conditions (using media supplemented with FBS) versus media supplemented with an equivalent amount of Weddell seal serum (WSS), IL6 expression from stimulated monocytes exposed to WSS declined significantly (LPS dose*serum‐type 2way repeated‐measures ANOVA across all doses: F1,6= 8.5, p=0.027 for FBS vs WSS), as did cytokines detected in the cell culture media. To further elucidate the potential anti‐inflammatory properties of seal serum, we repeated the experiment comparing FBS to de‐complemented WSS in RAW cells, a mouse macrophage line. These results validated our findings from seal monocytes, demonstrating that mouse cytokine expression (IL6, TNFα, and Mcp1), was consistently reduced in cells cultured with WSS compared to FBS. Finally, we confirmed that these results held in RAW cells exposed to other TLR ligands, including Pam‐3‐Cys, Poly I:C and CpG DNA (TNFα expression in WSS