Anti-inflammatory effects of haptoglobin on LPS-stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb-Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

Macrophage modulation by polymerized hemoglobins: Potential as a wound-healing therapy.

Chronic skin wounds are hypoxic and are stalled in a pro-inflammatory state. Hemoglobin (Hb)-based oxygen carriers have shown potential in increasing oxygen delivery to aid wound healing. Macrophages also take up Hb, thus altering their phenotype and the regulation of inflammation. Herein, we compared the effect of Hb and polymerized Hbs (PolyHbs) on the phenotype of human macrophages. Macrophages were incubated with Hb or different forms of PolyHbs, and the inflammatory secretion profile was analyzed. PolyHbs were produced by polymerizing Hb in the relaxed (R) or tense (T) quaternary state and by varying the molar ratio of the glutaraldehyde crosslinking agent to Hb. Hb decreased the secretion of most measured factors. PolyHb treatment led to generally similar secretion profiles; however, Hb had more similar trends to R-state PolyHb. Ingenuity pathway analysis predicted positive outcomes in wound healing and angiogenesis for T-state PolyHb prepared with a 30:1 (glutaraldehyde:Hb) polymerization ratio. When tested in diabetic mouse wounds, T-state PolyHb resulted in the greatest epidermal thickness and vascular endothelial CD31 staining. Thus, the effects of PolyHb on macrophages are affected by the polymerization ratio and the quaternary state, and T-state PolyHb yields secretion profiles that are most beneficial in wound healing.