Disulfide HMGB1 Reprograms Macrophages to Induce Alloimmunity via TLR4 and TLR9 Pathways Following Ischemia-Reperfusion Injury in Human Orthotopic Liver Transplantation
1Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, 2University of California Los Angeles (Multi-organ), Los Angeles, CA, 3Surgery, University of California, Los Angeles, Los Angeles, CA
Meeting: 2022 American Transplant Congress
Abstract number: 277
Keywords: Antigen presentation, Inflammation, Post-operative complications, Reactive oxygen species
Topic: Basic Science » Basic Science » 14 - Ischemia Reperfusion
Session Information
Session Name: Ischemia Reperfusion
Session Type: Rapid Fire Oral Abstract
Date: Monday, June 6, 2022
Session Time: 3:30pm-5:00pm
Presentation Time: 4:40pm-4:50pm
Location: Hynes Room 302
*Purpose: Human hepatocellular damage caused by ischemia reperfusion injury (IRI) generates the disulfide form of HMGB1 (diS HMGB1), eliciting myeloid cell activation and proinflammatory cytokine production. IRI is also linked to increased alloimmunity following human liver transplantation (LT). The role of the HMGB1-TLR4 and HMGB1-RAGE/TLR9 axes in mediating macrophage reprogramming and enhanced proinflammatory function is not well understood. We hypothesize that endogenous diS HMGB1 released from IRI-stressed liver allografts binds to TLR4 and RAGE/TLR9 to elicit signaling, macrophage reprogramming and enhanced macrophage proinflammatory function.
*Methods: Human peripheral blood monocytes were cultured with diS HMGB1 alone or with a TLR4 inhibitor (TLR4i), TLR9 inhibitor (TLR9i), TLR4i+TLR9i, or HMGB1 neutralizing antibody (αHMGB1) for 5 days to induce macrophage reprogramming. Non-immunogenic HMGB1 (aS HMGB1), LPS, and IL-4 served as controls. Macrophage phenotypes (T cell interaction: CD86, HLA-DR, PD-L1, TIM-3; Fc Engagement: CD16, CD64) and ROS production were assayed by flow cytometry on Day 5.
*Results: diS HMGB1 increased macrophage expression of CD86, HLA-DR, and CD64 and decreased PD-L1 compared to aS HMGB1. αHMGB1 treatment decreased CD86, HLA-DR, and CD64 and increased PD-L1 compared to diS HMGB1 alone. TIM-3 and CD16 remained unaffected by either condition. Blockade of diS HMGB1 with TLR4i alone significantly decreased CD64 and lowered CD86 and HLA-DR. TLR9i alone significantly decreased all three markers. Combined TLR4i+TLR9i synergistically decreased these markers, approaching levels observed with αHMGB1 treatment. PD-L1 remained unaffected by TLR inhibitors. diS HMGB1 induced the highest ROS production compared to LPS and IL-4 controls. Both αHMGB1 and TLR9i significantly decreased diS HMGB1-induced ROS production to similar levels. TLR4i had no impact on ROS production.
*Conclusions: This work reveals that diS HMGB1 has pleotropic effects on macrophage reprogramming depending upon interaction with specific PRRs. diS HMGB1 signaling via TLR4 reprograms macrophages with enhanced antigen presentation and Fc engagement functions contributing to both cellular and humoral alloimmune responses. Alternatively, diS HMGB1 reprograms macrophages for enhanced ROS production via TLR9 alone. These results imply neutralization of HMGB1, rather than TLR4/9 inhibition, during LT is a potential treatment for decreasing diS HMGB1-dependent post-LT alloimmunity in IRI+ patients.
To cite this abstract in AMA style:
Terry AQ, Sosa RA, Kaldas F, Busuttil R, Gjertson D, Kupiec-Weglinski J, Reed E. Disulfide HMGB1 Reprograms Macrophages to Induce Alloimmunity via TLR4 and TLR9 Pathways Following Ischemia-Reperfusion Injury in Human Orthotopic Liver Transplantation [abstract]. Am J Transplant. 2022; 22 (suppl 3). https://atcmeetingabstracts.com/abstract/disulfide-hmgb1-reprograms-macrophages-to-induce-alloimmunity-via-tlr4-and-tlr9-pathways-following-ischemia-reperfusion-injury-in-human-orthotopic-liver-transplantation/. Accessed May 16, 2025.« Back to 2022 American Transplant Congress