Myeloablative Hematopoietic Stem Cell Transplantation (HSCT) Is Accompanied by Endotoxemia, Activation of Endotoxin-Directed Innate Immunity, and Deficiency of Endogenous Proteins That Limit Endotoxin- Induced TNF Production

Abstract

Background: HSCT is an important treatment modality complicated by a number of immune-mediated toxicities, including acute graft vs host disease (aGVHD). In animal HSCT models, endotoxemia-induced production of TNF contributes to development of inflammatory regimen related toxicity and aGVHD. We tested the hypothesis that in human HSCT, myeloablative chemoradiotherapy regimens contribute to the early development of both endotoxemia and profound deficiency in host defense molecules that normally serve to inhibit endotoxin signaling, effects that may activate endotoxin–directed innate immunity and contribute to development of toxicities including aGVHD.Methods: Peripheral blood was obtained prospectively from HSCT recipients (N =35) at Baseline (B) prior to the start of conditioning, Days 0 (Day of HSCT), 7, 14, 21, and 28 and from their HSCT donors. Results on soluble biomarkers were also validated with frozen plasma samples from a retrospective HSCT cohort (N=21). Plasma was analyzed for endotoxin (Limulus amobocyte lysate and endotoxin activity assays), endotoxin-interactive proteins: bactericidal/permeability-increasing protein (BPI), LPS-binding protein (LBP), soluble CD14 (sCD14) and soluble MD-2 (sMD-2), as well as chemokine ligand 5 (CCL5)/Regulated upon Activation Normal T Cell Expressed and Secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1), chemokines that inhibit Toll-like receptor-4 (TLR4)-mediated TNF and IL-6 production. Monocyte expression of LPS-receptor components mCD14 and TLR4 was measured by flow cytometry. Whole blood RNA was isolated for analysis of TLR transcriptome activation using quantitative real-time PCR.Findings: During myeloablative HSCT, endotoxemia was detectable by two independent methods (LAL and EAA) and was accompanied by a profound deficiency in plasma concentrations of BPI and RANTES that fell >10-fold from baseline (B) to D7 (p < 0.001). At Day 0, monocyte surface expression of mCD14 fell and TLR4 increased (p < 0.05) and selective activation of a TLR pathway transcriptional response was detected in whole blood, suggesting early exposure to bioactive endotoxin. Febrile neutropenia was accompanied by a 10-fold decline by D7 in plasma concentrations of BPI and CCL5/RANTES and increased IL-6, consistent with an acute phase response. Subsequently, levels of endotoxin-modulating proteins LBP (D7–14) and sCD14 (D7, 21, and 28) increased (p < 0.05). Spontaneous TNF production was inversely correlated with plasma CCL2/MCP- 1, a chemokine known to inhibit TLR4-mediated TNF. Low plasma concentrations of RANTES/CCL5 (D0 and D7) significantly correlated with subsequent risk of aGVHD (p < 0.05).Interpretation: During myeloablative HSCT, endotoxemia occurs in the context of deficient BPI and RANTES, potentially contributing to TLR4-mediated production of TNF and consequent inflammatory sequelae, including aGVHD. Therefore, therapies aimed at enhancing endotoxin-neutralization, such as replenishment of deficient endotoxin antagonists, may be effective approaches to reduce toxicities associated with myeloablative HSCT. We are investigating this hypothesis in an ongoing phase I/II clinical trial of intravenous administration of the endotoxin-neutralizing anti-infective protein rBPI21 (Opebecan; XOMA (US) LLC) to reduce inflammatory, regimen-related toxicity in patients undergoing myeloablative HSCT.