Weddell seal (<i>Leptonychotes weddellii</i>) macrophages secrete protective molecules in response to hypoxia

Weddell seals are impressive divers and routinely dive between 20-400 m deep for ~30 minutes. To sustain bouts of diving, seals need to extend aerobic metabolism and as a result they have developed physiological mechanisms that efficiently cache and utilize oxygen stores. Chief among these mechanisms is the near-complete depletion of venous blood oxygen stores eliciting a hypoxemic state. In human clinical settings, hypoxemia induces the innate immune system and prompts signaling via inflammatory cytokines. One hypothesis for how seals sustain repeated diving without development of chronic injuries is the seal’s ability to dampen their inflammation response to diving but preserve these responses for other injuries. Macrophages are among the first immune cells recruited to the inflamed site and respond by upregulating both pro-and anti-inflammatory cytokines. To compare Weddell seal with human macrophage in vitro responses to inflammation we exposed primary cells to 4-h of 10 ng/mL lipopolysaccharide (LPS), a bacterial inflammatory stimulant. We found that both species produce a key pro-inflammatory cytokine, Il6, in response to LPS stimulation. However, human macrophages have notably higher induction of Il6 mRNA (35-fold increase from baseline, F 1,4 =80.31, P &lt;0.001, n=5), compared to seals (5-fold increase, F 1,4 =4.47, P =0.10, n=6). We next assessed macrophage cytokine responses following a multi-factor inflammatory treatment; LPS, hypoxia (1% O 2 ) or their combination. Seal macrophages increased expression of pro-inflammatory Tnfa only in response to LPS (LME, F 1,6 =6.35, P =0.45) but not hypoxia (F 1,6 = 2.04, P =0.20) or multi-factor LPS+hypoxia exposures (F 1,6 =0.46, P =0.52). On the other hand, human macrophages were responsive to hypoxia (F 1,3 = 1176.83, P =0.0001) with a significant interaction effect in the dual exposure (F 1,3 = 1992.87, P &lt;0.0001). Interestingly, Tnfa expression decreased in human cells when both LPS and hypoxia were combined (Tukey posthoc LPS versus LPS+hypoxia p&lt;0.0001), indicating potential dysfunction in inflammatory signaling in the face of multiple stressors. Seal macrophages show a blunted inflammation response to hypoxia, a potential adaptation to diving, but no evidence of transcriptional dysfunction even with multi-factor exposures. Similarly, Weddell seal macrophages maintained transcriptional response for Il1ra, an anti-inflammatory cytokine, in the multi-factor LPS+hypoxia exposures (F 1,6 = 0.859, P = 0.39) while the human macrophages were unable to mount any response (F 1,3 = 19.53, P = 0.02). Finally, conditioned media from both human and seal macrophages was collected after exposure to either normoxia or hypoxia and applied to a human macrophage cell line (THP-1) to determine if secreted molecules from either species could affect responses of these human cells to a subsequent 4-h of hypoxia. There was no significant difference in Il6 expression in THP-1 cells with either Weddell seal normoxic- or hypoxic-conditioned media (T 3.44 =1.87, P =0.15). This is unlike the human macrophage secretome, which increased Il6 expression with hypoxic conditioned media by 1.65-fold more (T 3.83 =4.27, P =0.014). Modifications of inflammatory signaling in Weddell seals emphasizes a new biomedical model to investigate innate immunity in a mammal that is repeatedly exposed to hypoxemia. NSF #1921491 NSF #1853326 NSF #2337400 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.