Effect of haptoglobin (purified from human serum) on gene expression in monocyte-derived macrophages.

Haptoglobin is a major serum protein that sequesters free hemoglobin. Moreover, haptoglobin regulates the inflammatory response in the absence of hemoglobin. Conflicting functional outcomes are reported, whereas involvement of NF?B is suggested consistently. Our data indicate that purified haptoglobin induces NF?B-dependent transcription through toll-like receptor 4. Notably, haptoglobin itself is dispensable for this effect. We show here that haptoglobin binds lipopolysaccharides from different bacterial species with micromolar affinities. This finding explains previous divergent observations. Pro-inflammatory responses elicited by purified haptoglobin are caused by heterogeneous lipopolysaccharides associated with the protein. Given its abundance in human serum, haptoglobin constitutes a buffer for serum-borne lipopolysaccharide. Restricted lipopolysaccharide availability dampens inflammatory responses. Concordantly, NF?B activation in primary macrophages is delayed relative to stimulation with pure lipopolysaccharide. Our findings warrant evaluation of therapeutic benefits of haptoglobin for non-hemolytic conditions as well as re-evaluation of purification strategies and will furthermore allow to disentangle mechanisms of haptoglobin-dependent immunoregulation. Overall design: Monocyte-derived macrophages from three healthy female donors were treated with vehicle, 25 µg/ml mixed-type haptoglobin, or haptoglobin and 50 µM arachidonic acid for 2, 4, 6, and 8 hours for comparative RNA-seq.