Myeloablative hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for both malignant and benign diseases of the bone marrow. However, HSCT is frequently complicated by acute graft-versus-host disease (aGVHD) wherein donor T lymphocytes target recipient tissue antigens. Animal models and human correlative data suggest that lipopolysaccharides (LPS, or “endotoxin”) derived from Gram-negative bacteria play a key role in HSCT-provoked aGVHD. Conditioning regimen-induced damage to the intestinal tract allows increased translocation of LPS from intestinal bacterial flora, triggering production of pro-inflammatory cytokines (e.g., TNF-α) that activate allo-immune reactions culminating in aGVHD. Our preliminary data indicate humoral and cellular changes consistent with LPS-induced systemic inflammation, including up-regulation of Toll-like receptor 4 (TLR4), a component of the LPS receptor. We also find that myeloablation-related neutropenia results in severely reduced plasma levels of bactericidal/permeability-increasing protein (BPI), a potent neutrophil-derived endotoxin-neutralizing host protein, and that individuals with lower baseline levels of plasma BPI have a significantly increased risk for aGVHD. Thus, both published and preliminary data suggest that replenishing BPI might shield patients from endotoxin and thereby reduce incidence of aGVHD.
In this context, a pilot phase I/II study is planned to examine the safety, tolerability, and pharmacokinetics of rBPI21, a 21 kDa recombinant N-terminal fragment of human BPI with an excellent safety profile and in vivo LPS-neutralizing activity, administered to individuals undergoing myeloablative allogeneic HSCT. We propose to take advantage of this pilot study as a unique opportunity to probe the relationship between endotoxin-directed innate immunity and aGVHD in patients undergoing HSCT. As rBPI21 is a selective inhibitor of endotoxin, our approach will assess the role of endotoxemia in triggering innate immune responses in vivo during HSCT.
We propose to examine banked samples from a cohort of HSCT donor:patient pairs (N = 150) to determine if common BPI genotypes are associated with aGVHD, a screen that may, for the first time, identify BPI genotypes that modify risk of aGVHD. A potential mechanism for such association will be explored by correlating aGVHD-associated BPI genotypes with BPI expression.
Comparison of blood plasma derived from control (observational study, N = 30) and treated (rBPI21 pilot study, N = 30) cohorts with respect to total and bioactive endotoxin, as well as plasma-mediated modulation of exogenous endotoxin bioactivity will characterize endotoxin-directed humoral innate immunity during myeloablative HSCT. In addition, measurement of in vivo production (plasma samples) of LPS-modulating acute phase reactants (LPS-binding protein and soluble CD14) and cytokines (IL-6, MCP-1, and sTNFRs) with distinct temporal patterns of up-regulation after HSCT will reveal the impact of endotoxin neutralization on the post-HSCT inflammatory response in vivo.
Finally, we will determine the effects of rBPI21 infusion on functional expression of the tri-partite endotoxin receptor comprised of membrane CD14/TLR4/MD-2. Surface expression of CD14, MD-2 and TLR4, as well as spontaneous and endotoxin-induced production of intracellular TNF-α will be characterized by flow cytometry, defining relationships between bioactivity of endotoxin and endotoxin receptor expression.
Overall these studies will provide fresh insights into the role of endotoxin-directed innate immunity in triggering aGVHD.