Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cells Via Cell Autonomous and Cell Non-Autonomous Mechanisms

Toll like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that shape the innate immune system by identifying foreign pathogen-associated molecular patterns (PAMPS) and host-derived damage associated patterns (DAMPS). TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells (HSPCs). Of clinical significance, both lymphoproliferative and myelodysplastic syndromes have been linked to aberrant TLR signaling (Schuettpelz, et al., Front Immunol 2013; Varney, et al., Exp Hematol 2015). Despite extensive studies focused on the influence of TLRs through committed effector cell populations, more recent evidence suggests that these PRRs may elicit immune regulation from the more primitive level of hematopoietic stem cells (HSCs). As TLR2 is expressed on HSCs, in the present study, we sought to elucidate the effect of TLR2 signaling on HSCs, and determine the cell-autonomous versus non-autonomous effects of this signaling. To this end, we utilized the synthetic TLR2 agonist, PAM₃CSK₄, to assess the effects of augmented TLR2 signaling on HSC mobilization, function, cycling, and differentiation. In previous studies, we found that TLR2 is not required for HSC function (Schuettpelz et al., Leukemia 2014); however, in the present study, treatment of wild-type mice with PAM₃CSK₄ led to HSC expansion in both the bone marrow and spleen, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). Further, we observed increased HSC cycling and loss of function in competitive bone marrow transplantation assays in response to TLR2 agonist exposure. Treatment of chimeric animals (Tlr2^-/- + Tlr2^+/+ bone marrow transplanted into Tlr2^+/+ or Tlr2^-/- recipients) showed that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling. Analysis of serum, bone marrow, and spleen samples by cytokine expression arrays revealed an increase in G-CSF (serum) and TNFα (bone marrow) following TLR2 agonist treatment in wild-type mice. To further characterize the influence of these cytokines, respective receptor knockout models were employed. Inhibition of G-CSF enhanced HSC bone marrow expansion in response to PAM₃CSK₄, but partially rescued the expansion of spleen HSPCs. Likewise, loss of TNFa partially mitigated the expansion of spleen HSPCs in response to PAM₃CSK₄, and abrogated the PAM₃CSK₄-induced spleen HSC cycling. Further, we observed that loss of TNFa rescued the PAM₃CSK₄-mediated loss of bone marrow MEPs. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous cues, with G-CSF and TNFa contributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the particular cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway on HSCs in myelodysplastic syndrome (MDS) pathogenesis and other hematologic malignancies.