Cell-free hemoglobin triggers macrophage cytokine production via TLR4 and MyD88.

Cell-free hemoglobin (CFH) is elevated in the airspace of patients with acute respiratory distress syndrome (ARDS) and is sufficient to cause acute lung injury in a murine model. However, the pathways through which CFH causes lung injury are not well understood. Toll-like receptor 4 (TLR4) is a mediator of inflammation after detection of damage- and pathogen-associated molecular patterns. We hypothesized that TLR4 signaling mediates the pro-inflammatory effects of CFH in the airspace. After intra-tracheal CFH, BALBc mice deficient in TLR4 had reduced inflammatory cell influx into the airspace (bronchoalveolar lavage [BAL] cell counts, median TLR4KO:0.8x104 /mL [IQR 0.4-1.2x104 /mL], wild-type (WT): 3.0x104 /mL [2.2-4.0x104 /mL], p<0.001) and attenuated lung permeability (BAL protein, TLR4KO: 289mg/mL [236-320], WT: 488mg/mL [422-536], p<0.001). These mice also had attenuated production of IL-1β, IL-6, and TNF-α in the airspace. C57Bl/6 mice lacking TLR4 on myeloid cells only (LysM.Cre+/- TLR4fl/fl ) had reduced cytokine production in the airspace after CFH, without attenuation of lung permeability. In vitro studies confirm that WT primary murine alveolar macrophages exposed to CFH (0.01-1mg/mL) had dose-dependent increases in IL-6, IL-1 b, CXCL-1, TNF-a, and IL-10 (p<0.001). Murine MH-S alveolar-like macrophages show TLR4-dependent expression of IL-1b, IL-6, and CXCL-1 in response to CFH. Primary alveolar macrophages from mice lacking TLR4 adaptor proteins MyD88 or TRIF revealed that MyD88KO macrophages had 71-96% reduction in CFH-dependent pro-inflammatory cytokine production (p<0.001), whereas macrophages from TRIFKO mice had variable changes in cytokine responses. These data demonstrate that myeloid TLR4 signaling through MyD88 is a key regulator of airspace inflammation in response to CFH.