CXCL12-Abundant Reticular (CAR) Cells Are Key Regulators for a Sustained Hematopoietic Response during Chronic Inflammation

The blood system is established and maintained by hematopoietic stem and progenitor cells (HSPCs) that are located in the bone marrow (BM) where they are surrounded by different types of hematopoietic and non-hematopoietic cells that together form a complex supportive microenvironment. It is well established that the microenvironment delivers a plethora of extrinsic signals that help to maintain stemness of HSCs and guide steady-state HSPC differentiation. The BM microenvironment also likely is a key player during infection and inflammation-induced demand-adapted hematopoiesis (e.g. Boettcher et al. Blood 2014; Manz and Boettcher Nat Immunol 2014).

We set out to study how the BM microenvironment supports the hematopoietic system in adaptation to severe systemic inflammation using lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly(I:C)) to mimic gram-negative bacterial or viral infection, respectively. Based on expression of CD45 and Ter119 in conjunction with Sca1, CD31, Tie2 and CD140b, we were able to flow-cytometrically define and isolate several cell populations enriched for distinct non-hematopoietic cell types in mouse BM, i.e. mesenchymal stromal cells (MSCs), CXCL12-abundant reticular cells (CAR cells), arteriolar endothelial cells (AECs) and sinusoidal endothelial cells (SECs).

Gene expression analyses for a number of prototypic inflammatory cytokines and chemokines revealed that interleukin-6 (IL-6) is highly significantly and specifically up-regulated in CAR cells during LPS stimulation. Extensive experimentation involving Il6 expression analyses from unseparated total tissue extracts (bone marrow, spleen, lung, heart, liver, kidney), and IL-6 levels in plasma and BM lysates from reciprocal BM chimeric Il6^-/- mice (Il6^-/-→WT, WT→ Il6^-/-) as well as conditional Il6^-/- mice (Lepr-Cre;Il6 ^flox/flox) demonstrated that CAR cells act as a primary source of IL-6 during LPS stimulation.

To test the putative functional consequences of IL-6 secretion from CAR cells during LPS-induced inflammation, we studied the hematopoietic response in LPS-treated WT and Il6^-/- mice. While there was neither a relative nor absolute difference in immunophenotypically defined HSPCs, myeloid progenitors or mature myeloid lineages following short-term LPS treatment (2 injections over 3 days), we observed a significant decrease in absolute numbers of cells along the myeloid lineage from the earliest HSPCs to the mature myeloid cells upon chronic-repetitive LPS stimulation (9 injections over 21 days) in Il6^-/- mice compared to WT mice. Moreover, the total number of colony-forming units granulocyte (CFU-G) was significantly reduced in the BM of LPS-treated Il6^-/- mice compared to WT mice. We observed the identical impaired hematopoietic responses in reciprocal BM chimeric Il6^-/- mice and conditional Il6^-/- mice (Lepr-Cre;Il6 ^flox/flox) corroborating that CAR cell-derived IL-6 is crucial for mounting an appropriate hematopoietic response upon LPS stimulation.

To address the question whether CAR cells may directly be able to sense Toll-like receptor (TLR) ligands (e.g. LPS is recognized by TLR4) we compared expression of Tlr1 - Tlr9 in MSCs, SECs, AECs, and CAR cells isolated from BM of WT mice to that found in pooled conventional and plasmacytoid dendritic cells (cDCs / pDCs) known to express different TLRs. Interestingly, the different BM niche cells expressed several TLRs at surprisingly high levels. To test whether IL-6 secretion following LPS stimulation is dependent on Tlr4-expressing CAR cells we generated reciprocal chimeric BM mice with a Tlr4-deficiency in either hematopoietic or non-hematopoietic cells. Our data demonstrated that Tlr4 expression on both hematopoietic and non-hematopoietic cells is equally contributing to IL-6 secretion during short-term LPS stimulation. However, following chronic-repetitive LPS stimulation, IL-6 secretion is completely dependent on Tlr4 expression in non-hematopoietic cells strongly suggesting that CAR cells sense and secrete IL-6 in that setting.

In summary, our data reveal that CAR cells play an essential role for a sustained hematopoietic response during chronic LPS treatment. IL-6 released from CAR cells acts as a critical growth stimulus for the early HSPC compartment to provide a reservoir of highly proliferative myeloid progenitors in order to counterbalance inflammation-driven cell consumption.