Effects by anthrax toxins on hematopoiesis: a key role for cytokines as mediators.

An understanding of anthrax toxins on the emerging immune system and blood production are significant to medicine. This study examined the effects of anthrax toxin on hematopoiesis and determined roles for cytokines. Anthrax holotoxin toxin is three components: protective antigen (PA) binds to the target cell and mediates the entry of lethal factor (LF) and edema factor (EF). Anthrax toxin dramatically inhibits signaling in immune cells. We first identified the cell subsets that interacted with the protective antigen (PA) and then studied the effects on hematopoietic progenitors in clonogenic assays: granulocytic-monocytic (CFU-GM) and late erythroid (CFU-E). Multi-color immunofluorescence with FITC-PA indicated its interaction with early and late myeloid cells. Clonogenic assays, in the presence or absence of holotoxin and individual toxin proteins resulted in significant suppression by hologenic toxic alone, despite the presence of growth-promoting cytokines. Antibodies to anthrax receptor (ATR1) reversed the suppressive effects, indicating specificity. Monomeric proteins showed different effects on myeloid and erythroid progenitors. Suppression was not due to cell death, based on undetectable active caspase 3. Cytokine array analyses with supernatants from toxin-stimulated stroma showed an increase in the hematopoietic suppressor, MIP-1α. This finding, in addition to our previous studies, showing an increase in IL-10, suggested indirect roles for cytokines in toxin-mediated hematopoietic suppression. The chemokine, SDF-1α was increased. Since SDF-1 is involved in the mobilization of hematopoietic cells, it is likely that anthrax holotoxin could induce cell exit from BM. In summary, anthrax holotoxin, but not individual toxins, exerted hematopoietic effects on myeloid and erythroid progenitors via specific receptor, partly through the induction of cytokines.

Non-canonical effects of anthrax toxins on haematopoiesis: implications for vaccine development.

Anthrax receptor (ATR) shares similarities with molecules relevant to haematopoiesis. This suggests that anthrax proteins might bind to these mimicking molecules and exert non-specific haematopoietic effects. The haematopoietic system is the site of immune cell development in the adult. As such, ATR ligand, protective antigen (PA) and the other anthrax proteins, lethal factor, edema factor, could be significant to haematopoietic responses against Bacillus anthracis infection. Because haematopoiesis is the process of immune cell development, effects by anthrax proteins could be relevant to vaccine development. Here, we report on effects of anthrax proteins and toxins on early and late haematopoiesis. Flow cytometry shows binding of PA to haematopoietic cells. This binding might be partly specific because flow cytometry and Western blots demonstrate the presence of ATR1 on haematopoietic cell subsets and the supporting stromal cells. Functional studies with long-term initiating cell and clonogenic assays determined haematopoietic suppression by anthrax toxins and stimulation by monomeric proteins. The suppressive effects were not attributed to cell death, but partly through the induction of haematopoietic suppressors, interleukin (IL)-10 and CCL3 (MIP-1alpha). In summary, anthrax proteins affect immune cell development by effects on haematopoiesis. The type of effect, stimulation or suppression, depend on whether the stimulator is a toxin or monomeric protein. The studies show effects of anthrax proteins beginning at the early stage of haematopoiesis, and also show secondary mediators such as IL-10 and CCL3. The roles of other cytokines and additional ATR are yet to be investigated.