ALBERT

Description: Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function. To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2-/- + Tlr2+/+ marrow transplanted into Tlr2+/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r-/-) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs. Disclosures No relevant conflicts of interest to declare.

Description: Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

Description: Increased expression of Kruppel-like factor 7 (KLF7) is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia. The contribution of KLF7 to hematopoiesis has not been previously described. Herein, we characterized the effect on murine hematopoiesis of the loss of KLF7 and enforced expression of KLF7. Long-term multilineage engraftment of Klf7−/− cells was comparable with control cells, and self-renewal, as assessed by serial transplantation, was not affected. Enforced expression of KLF7 results in a marked suppression of myeloid progenitor cell growth and a loss of short- and long-term repopulating activity. Interestingly, enforced expression of KLF7, although resulting in multilineage growth suppression that extended to hematopoietic stem cells and common lymphoid progenitors, spared T cells and enhanced the survival of early thymocytes. RNA expression profiling of KLF7-overexpressing hematopoietic progenitors identified several potential target genes mediating these effects. Notably, the known KLF7 target Cdkn1a (p21Cip1/Waf1) was not induced by KLF7, and loss of CDKN1A does not rescue the repopulating defect. These results suggest that KLF7 is not required for normal hematopoietic stem and progenitor function, but increased expression, as seen in a subset of lymphoid leukemia, inhibits myeloid cell proliferation and promotes early thymocyte survival.

Description: 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. Disclosures No relevant conflicts of interest to declare.