Jason C McCartt, MD, Christopher W Mangieri, MD, Colin D Brown, MD, Prasad M Balakrishna, PhD. Dwight D. Eisenhower Army Medical Center
Damage-associated molecular patterns (DAMPs) have been linked to initiating sterile systemic inflammatory responses following traumatic injury. Specifically the high-mobility group box 1 (HMGB-1) protein has been isolated as a major mediator in the initiation of the pro-inflammatory cascade. HMGB1 and other damage associated molecules activate toll like receptor-4 (TLR4) to release inflammatory cytokines such tumor necrosis factor α (TNFα). Multiple approaches to inhibit this initial trigger have been hypothesized to be effective therapeutic interventions. Among these are buffering of HMGB1 actions by binding proteins and small molecule inhibitors of TLR4 receptor signaling. We hypothesized that inhibition of HMGB1-TLR4 signaling pathway will decrease the release of inflammatory cytokines by immune cells. In order to test this hypothesis and compare the potency of the HMGB1 binding proteins and TLR4 inhibitors, we used murine macrophage culture system (RAW cells). HMGB1 from three different sources caused significant increases in TNFα release.
Different HMGB1 binding proteins (antibodies, soluble TLR4-Fc chimera, soluble RAGE-Fc chimera) did not decrease TNFα release caused by HMGB1. The ability of these proteins to bind HMGB1 was confirmed by in vitro binding assays. We found that these proteins had a cytokine inducing effect of their own. A common feature of all these diverse proteins is the presence of Fc-immunoglobin motif in their structure. These results suggest that activation of a TLR4 independent pathway, most likely Fc-receptor activation, is responsible for their cytokine induction ability.
TAK-242 and Parthenolide are two commercially available small molecule inhibitors of TLR4 receptor signaling. In RAW cell cultures both compounds inhibited TNFα release caused by HMGB1. However, inhibition of TNFα release by Parthenolide was associated with cytotoxicity. TAK-242 caused a dose-dependent inhibition with an IC50 of 16.12 ± 1.9 nM. Furthermore, inhibition caused by TAK-242 was relatively long lasting. Pretreatment of cells with TAK-242 for 2 hours significantly inhibited the HMGB1-induced TNFα release over the next 24 hours. Future studies on the time-course and reversibility of TAK-242 actions will pave the way for in vivo experiments for preclinical development of this drug.
Presented at the SAGES 2017 Annual Meeting in Houston, TX.
Abstract ID: 80177
Program Number: MSS13
Presentation Session: Full-Day Military Surgical Symposium – Basic Science Presentations
Presentation Type: MSSPodium