Non-Hematopoietic Stromal Cells Sense Toll-Like Receptor 4 Agonists and Consequently Enhance Myelopoiesis.

Abstract 2583

Hematopoiesis is tightly regulated by growth factors acting on stem and progenitor cells (HSPCs) in the bone marrow. During systemic infections cytokines are elevated in serum, myelopoiesis is enhanced, and myeloid CFUs and granulocytes increase in circulation. However, the underlying mechanisms of this “emergency” myelopoietic response have not been defined. Sensing of conserved pathogen-associated products by specialized pattern-recognition receptors such as Toll-like receptors (TLRs) is crucial for rapid responses to infection. Based on the well-known regulatory function of the BM microenvironment, we hypothesized that bone marrow stromal cells (BMSCs) express TLRs and possess all functional properties required to sense microbes and drive emergency myelopoiesis. Human BMSCs expressed Tlr1, Tlr5, and Tlr6 at similar levels and Tlr3 and Tlr4 mRNA at about 2-log higher compared to dendrititc cells (DCs). Stimulation of BMSCs with the TLR4 agonist LPS led to de novo expression of G-csf and Gm-csf, and increased M-csf, Il-6, and Il-11 expression. In line with this, LPS induced production of G-CSF and GM-CSF protein and significantly enhanced the secretion of M-CSF, IL-6, and IL-11. Using LPS-stimulated BMSC culture supernatant in myeloid CFU assays led to a 2.5-fold higher myeloid CFU activity compared to un-stimulated BMSC supernatant. This effect was partly mimicked by adding G-, M-, and GM-CSF to the methylcellulose cultures. Importantly, direct LPS stimulation of CB CD34⁺ cells had no effect. Furthermore, co-culture of BMSCs and CB CD34⁺ cells together with LPS for 12 days led to approximate 2-fold higher recovery of immuno-phenotypically primitive CD34⁺ cells, and retained up to 8-fold more CD34⁺ cells in divisions 0–3 as compared to LPS-free co-cultures as measured by CFSE dilution. When subjected to cytokine-supplemented myeloid CFU assays or transplanted into newborn RAG2^-/- γ_c^-/- mice to evaluate lymphoid differentiation, recovered CD34⁺ cells from LPS-stimulated BMSC cultures gave rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus proving maintenance of primitive hematopoietic progenitors. To elucidate the in vivo relevance of the findings and to clarify the contribution of stromal vs. hematopoietic cell expressed TLR4, we generated chimeras with TLR4^-/- hematopoiesis in a wild-type (WT) background (hematopoietic-TLR4^-/-) and WT hematopoiesis in a TLR4^-/- background (non-hematopoietic-TLR4-/-). Chimeric, WT, and TLR4^-/- mice were injected with LPS and hallmarks of myelopoietic responses such as G-CSF expression, myeloid cell mobilization from the BM, and increased myeloid cell production in the BM was evaluated. Significant G-csf mRNA induction could be observed in the BM of WT and hematopoietic-TLR4^-/- mice. To a much lesser, non-significant extent, this effect could be observed also in non-hematopoietic-TLR4^-/- mice, while no transcripts were detectable in TLR4^-/- mice. Accordingly, serum G-CSF levels significantly increased 10-fold in WT and hematopoietic-TLR4^-/- mice after LPS injection, but no increase was detectable in non-hematopoietic-TLR4^-/- and TLR4^-/- mice. LPS injection also resulted in a significant decrease in BM cellularity accompanied by an increase of spleen cell numbers only in WT and hematopoietic-TLR4^-/- mice. Furthermore, Gr-1^highCD11b^low/+ mature myeloid cells were significantly reduced whereas Gr-1^lowCD11b^low/+ immature promyelocytes and myelocytes significantly increased (2.5-fold) in the BM of WT and hematopoietic-TLR4^-/- mice. In contrast, similar changes in cellular composition could not be observed in TLR4^-/- and non-hematopoietic-TLR4^-/- mice, while a small, but still significant 1.25-fold increase in immature Gr-1^lowCD11b^low/+ cells was detectable in non-hematopoietic-TLR4^-/- mice. Finally, inflammation-induced Sca-1 upregulation on HSPCs and increasing frequencies of GMPs were only observed in WT and hematopoietic-TLR4^-/- mice. Collectively, our in vitro data demonstrate that human BMSCs are able to sense pathogens and stimulate emergency myelopoiesis but also prevent loss of HSPCs by enhancing their maintenance. Importantly, in vivo signaling via non-hematopoietic cell-expressed TLR4 is sufficient and is the main mechanism regulating both the release of mature myeloid cells from and the enhanced myeloid cell production in the bone marrow during systemic challenges.