TLR Agonist Induced Cytokine Production in Human Multipotent Mesenchymal Stromal Cells: A Potential Mechanism How Hematopoiesis Is Enhanced during Generalized Inflammatory Conditions.
Authors: Boettcher Steffen , Ziegler Patrick , Manz Markus G. 
Published: October 2019

Abstract
Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells. Their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like tissue damage and cancer. Human multipotent mesenchymal stromal cells (MSCs) have been shown to differentiate into various mesenchymal tissues such as bone, cartilage, and fat, as well as marrow and lymphoid organ stroma cells. Human MSCs are able to maintain CD34+ cells to some extent in vitro. Furthermore, it has been demonstrated that upon intra bone marrow transplantation into adult immunodeficient mice MSC derived cells support human hematopoiesis in vivo. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs in inflammatory conditions. MSCs from human bone marrow, cord blood, and umbilical cord whartons jelly were cultured by plastic adherence in IMDM 20% FCS, 1–8M dexamethasone (only during first 3 weeks), expanded for 2 passages, and subsequently analyzed. MSCs expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1, while G-CSF was rarely detectable. Consistently, respective cytokines were measured in supernatants at the following, declining levels (pg/ml): IL-6 (10000–10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF, whereas GM-CSF was rarely detectable, G-CSF, FLT3-L, and TPO were not detectable by ELISA. MSCs were further analyzed for expression of TLRs by semiquantitative RT-PCR. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional (BDCA-1+, CD14−, CD19−) and plasmacytoid (BDCA-4+,CD14−,CD19−) blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with LPS (TLR-4 agonist), MSCs produced previously undetectable G-CSF and GM-CSF, and M-CSF levels increased about 4 fold compared to untreated MSCs, whereas stimulation with CpG motifs (TLR-9 agonists) did not lead to changes in cytokine release detected in supernatants. In co-culture experiments using MSCs as a feeder layer for cord blood CD34+ cells, MSCs supported hematopoiesis and the development of myeloid cells. Importantly, MSC preconditioning with LPS led to an 1,7 fold increase in total hematopoietic cell number, while preconditioning with CpG had no measurable effect. These results demonstrate that MSCs express certain TLRs, and are capable to respond to pathogen associated molecules with an increase in secretion of hematopoiesis-relevant cytokines, and thus reveal a potential mechanism how hematopoiesis is enhanced during generalized inflammatory conditions, supporting e.g. in case of gram-negative sepsis efficient innate immune responses.