Toll-Like Receptor Signaling Inhibits Eosinophilopoiesis.

Abstract 1558

Eosinophils and their progenitors are robustly elevated in the bone marrow in response to diverse stimuli including helminth infection and allergic triggers. In contrast, bacterial infection results in diminution in the number of circulating eosinophils. Markedly reduced peripheral blood eosinophils of sudden onset but prolonged duration is generally a consequence of acute bacterial infection. Further, eosinopenia has been shown to be a sensitive marker of sepsis and correlates with severity of disease in critically ill patients. While the initial eosinopenic response is believed to be secondary to rapid migration of circulating eosinophils to the site of infection, the mechanism for prolonged eosinophil depletion with bacterial infection remains undefined. As Toll-like receptors (TLRs) have been shown to be expressed by early hematopoietic progenitors and by mature eosinophils, we initially investigated TLR expression during eosinophil differentiation. In a culture system we developed to differentiate mature eosinophils from low density bone marrow progenitors, IL-5 stimulation of progenitors resulted in induced expression of seven TLRs, including TLR1 (4-fold), TLR4 (5-fold), TLR6 (2-fold), TLR7 (6-fold), TLR8 (4-fold), TLR9 (3-fold), and TLR13 (8-fold), when compared to expression in the progenitors prior to IL-5-induced differentiation. Notably, a majority of the TLRs (TLR1, TLR4, TLR7, TLR8, TLR9, and TLR13) were induced specifically early in eosinophil differentiation after 4 days of IL-5 stimulation and prior to expression of surface markers, including Siglec-F and CCR3, associated with mature eosinophils. To study the effects of TLR signal transduction on eosinophil development, we exposed bone marrow progenitors to TLR agonists for 18 hours after 4 days of IL-5 stimulation and then subsequently induced further eosinophil differentiation with IL-5 stimulation for another 8 days. We measured effects of TLR signaling on cell number, eosinophil differentiation, cytokine production and protease activity. LPS stimulation resulted in a reduction in total cell numbers by more than 65% with immature eosinophils a predominant cell type. In addition, LPS stimulation of progenitors resulted in significantly increased cytokine and chemokine production, including IL-6 (80-fold) and CCL22 (2-fold), and protease activity (2-fold) after 4–6 days of subsequent IL-5 stimulation compared to IL-5 stimulation alone. Similarly, stimulation of eosinophil progenitors with the TLR1/TLR2 agonist PAM3CSK4 or TLR6/TLR2 agonist PAM2CSK4 for 18 hours resulted in markedly decreased total cell numbers in a dose-dependent manner after 3 days of subsequent IL-5 stimulation. Together, these data suggest that TLR signaling in progenitors results in inhibition of IL-5-stimulated eosinophil development with aberrant cytokine, chemokine and protease production. As eosinophil granules have been shown to have anti-bacterial properties and eosinophilia improves survival rate in an experimental bacterial peritonitis model, delineation of the mechanism of infection-induced eosinopenia may lead to novel adjuvant therapeutics for bacteremic critically ill patients.