Deletion of TLR4 in Townes SS Mice Prevents Microvascular Stasis in Response to Hemin, LPS and Hypoxia/Reoxygenation: Role of Inflammation

We have previously shown that heme, a damage-associated molecular pattern (DAMP), released from sickle RBCs interacts with the innate immune toll-like receptor 4 (TLR4) on endothelium and blood cells to activate cell signaling. This activates the pro-inflammatory transcription factor NF-κB leading to the production of cytokines and adhesion molecules that promote inflammation, coagulation, and vaso-occlusion (VO). Previous studies in our lab have demonstrated that inhibition of TLR4 in Townes-SS mice with TAK242 reduces microvascular stasis in response to hemin, hypoxia/reoxygenation and LPS. To further delineate the role of TLR4 in sickle cell disease (SCD), we bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. These Tlr-/- Townes mice were backcrossed 10 generations to homogenize their genetic background with our Tlr4+/+ Townes colony. Townes-SS Tlr4-/- mice developed less microvascular stasis than Townes-SS Tlr4+/+ mice in response to challenges with heme infusion (4% vs 30% at 1 hr, p<0.05, Fig 1A); LPS infusion (13% vs 36% at 1 hr, p<0.05, Fig 1B); and hypoxia/reoxygenation (3% vs 20% at 1 hr, p<0.05, Fig 1C).

We next measured the complete blood counts, serum chemistries, and urine analytes in the Tlr4+/+ and -/- mice. We also quantified mRNA levels via qPCR for liver and kidney cytokines, adhesion molecule, heme oxygenase 1 (HO-1) and coagulation factors in all four genotypes (AA Tlr4+/+; AA Tlr4-/-; SS Tlr4+/+; SS Tlr4-/-). Hemoglobin levels in SS mice were lower than AA mice, but there were no differences between Tlr4+/+ and Tlr4-/- mice. WBC counts were greater in SS than AA mice, but there were no differences between Tlr4+/+ and Tlr4-/- mice. Bilirubin and liver function tests (LFTs) were higher in SS than AA, but there were no differences between Tlr4+/+ and Tlr4-/- mice. In the liver, adhesion molecules and HO-1 were higher in SS than AA, but there were no differences between Tlr4+/+ and Tlr4-/- mice. To define a potential mechanism for decreased microvascular stasis in response to hemin infusion in SS Tlr4-/-, we measured cytokines and adhesion molecule expression after heme challenge. When SS Tlr4+/+ mice were infused with hemin, we found increased expression of pro-inflammatory cytokines and cell adhesion molecules. SS Tlr4-/- mice infused with hemin had reduced expression of cytokines and cell adhesion molecules compared to SS Tlr4+/+ mice; most notably in IL-6 in the liver, adhesion molecules VCAM-1 and ICAM-1 in the liver and kidneys (p<0.05), and P-selectin and von Willebrand factor in the lungs (Fig 2). Our data thus far indicate that TLR4 of the innate immune system plays a critical role in VO and inflammation in challenged SS mice. We speculate that modulation of TLR4 with targeted inhibitors would be beneficial for SCD patients.

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