Endothelial Cells Are Essential to Sense Lipopolysaccharide in a MYD88-Dependent Manner and to Subsequently Induce Emergency Myelopoiesis

Abstract 641

Severe systemic infections evoke a number of characteristic clinical signs such as fever, neutrophilia and the appearance of immature myeloid precursors in the circulation (left-shift). This reflects a well-regulated hematopoietic response program to enhance myeloid cell output during times of increased hematopoietic demand, a condition which is referred to as 'emergency myelopoiesis'. Important molecular components of the emergency myelopoiesis cascade, such as cytokines and transcription factors involved, have been elucidated. However, the initial steps of emergency myelopoiesis involving pathogen recognition and translation into accelerated bone marrow (BM) myelopoiesis have only been inferred from findings on Toll-like receptor (TLR) expression on immature hematopoietic stem and progenitor cells (HSPCs) as well as on mature hematopoietic cells (e.g. macrophages). Accordingly, it has been assumed that both immature as well as mature hematopoietic cells are involved in sensing infection and inducing emergency myelopoiesis directly and indirectly, respectively. Surprisingly, by generating reciprocal BM chimeric animals mice with TLR4^−/− hematopoiesis on a wild-type (WT) nonhematopoietic background (TLR4^−/−→WT mice) and WT hematopoiesis on a TLR4^−/− nonhematopoietic background (WT→TLR4^−/−mice), we demonstrated that LPS-Induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells (Boettcher et al., J Immunol. 2012 Jun 15;188(12):5824–8.). However, the precise identity and localization of the nonhematopoietic cell type crucial for sensing gramnegative infection-derived lipopolysaccharide (LPS) has remained elusive to date.

We now have addressed this fundamental question using BM transplantation experiments and Cre-loxP recombination technology. BM chimeric mice with a myeloid differentiation primary response gene 88 (Myd88)-deficiency in the hematopoietic lineage (MYD88^−/−→WT mice) showed a normal LPS response indistinguishable to control (WT→WT) mice, while knocked out Myd88 within the nonhematopoietic compartment (WT→MYD88^−/− mice) led to a non-responsiveness towards LPS similar to controls (Myd88^−/−→Myd88^−/− mice). These results are in line with our earlier data, thus confirming the critical role of the TLR4/MYD88 pathway in nonhematopoietic cells for the induction of emergency myelopoiesis. In order to specifically delete TLR-MyYD88-downstream signaling in various nonhematopoietic cells including BM Nestin+ mesenchymal stem cells (MSCs) and their progeny, perivascular cells, endothelial cells, and hepatocytes, we generated Nes-Cre;Myd88^fl/fl, Pdgfrb-Cre;Myd88^fl/fl, Tek-Cre;Myd88^fl/fl, and Alb-Cre;Myd88^fl/fl mice, respectively. We observed a normal increase in the frequency of BM CD11b⁺Gr-1^low immature myeloid precursors accompanied by a decrease of BM CD11b⁺Gr-1^high mature myeloid cells upon LPS stimulation characteristic for efficient emergency myelopoiesis in Nes-Cre;Myd88^fl/fl, Pdgfrb-Cre;Myd88^fl/fl, and Alb-Cre;Myd88^fl/fl mice as compared to control mice. Furthermore, we measured highly-elevated plasma G-CSF levels in these mouse strains upon LPS injection. Hence, intact TLR signaling in mesenchymal stromal cells incl. Nestin+ MSCs, perivascular cells as well as hepatocytes is dispensable for induction of emergency myelopoiesis. Strikingly, Tek-Cre;Myd88^fl/fl mice were completely non-responsive towards LPS stimulation as assessed by the above-mentioned parameters. Our results thus demonstrate a fundamental and unanticipated role of the endothelium for sensing of systemically spread pathogens and subsequent stimulation of BM emergency myelopoiesis.