Neutrophils Regulate Bone Formation Via IL-1b Secretion

The bone marrow (BM) is surrounded by a shell of cortical bone that protects the hematopoietic stem cells (HSC) from external aggressions. Within the BM, progenitors for bone and blood coexist and, intriguingly, the same mesenchymal progenitors that give rise to bone also form niches that regulate hematopoiesis. Whether, and if so how, the hematopoietic system regulates the formation of its protective osteal shell is poorly understood. Among HSC descendants, innate immune cells have been involved in maintaining homeostasis in multiple organs, and neutrophils in particular have been shown to regulate the BM stroma both at steady-state and during stress, and to contribute to vascular repair. Hence, we examined MRP8cre iDTR mice (iDTR) which allow for inducible neutropenia upon diphtheria toxin injection. We found that acute neutropenia in these mice caused bone mass reduction (48% reduction) as determined by μCT, while no changes in osteoclast numbers. We confirmed this reduction using the calcein double labeling assay, altogether indicating that neutrophils regulate bone formation. We found a similar osteoporotic phenotype in MRP8cre CXCR4fl mice (CXCR4ΔN), in which only the return of circulating neutrophils to the BM is compromised, which suggested that it is peripheral-experienced neutrophils returning to the BM that exert the regulatory effect on the bone-producing mesenchymal cells. Concomitant with bone loss, we found that the number of hematopoietic progenitors were elevated in both in the iDTR and CXCR4ΔN models as assessed by flow cytometry, CFU-C assays and limited dilution reconstitution assays, suggesting that neutrophils counteract natural, age-related alterations in the BM. To identify the mechanisms by which neutrophils regulate bone formation, we searched for factors differentially produced by neutrophils returning to the BM compared with marrow-residing neutrophils. Transcriptomic analyses of neutrophils that migrated to the BM in a parabiotic model identified 8 soluble factors that were not produced by immature neutrophils. Among these factors, we found that interleukin 1 beta (Il1b) was highly expressed in returning neutrophils by qRT-PCR and protein levels, and single-cell RNAseq demonstrated that the production of Il1b was a hallmark of neutrophil maturation and egress into blood. To explore whether this cytokine mediated the osteogenic program of neutrophils in the BM, we generated mice with neutrophil-specific deletion of Il1b ; MRP8cre Il1bfl mice (Il1bΔN). Single-cell RNA-seq analysis of CD45-Ter119-CD31-CXCL12+ stroma cells from control CXCL12GFP mice, iDTR CXCL12GFP and Il1bΔN CXCL12GFP demonstrated marked anomalies in the differentiation of mesenchymal stem cell (MSC) into osteoblast lineage cells (OLC) and, to a lesser extent, adipocytic-lineage cells in the absence of neutrophils or neutrophil-derived Il1b. In vitro cultures confirmed a modulatory effect of Il1b on MSC differentiation. These findings correlated with the close proximity of neutrophils to CXCL12GFP+ MSC over random distribution, and with high expression of interleukin 1 receptor type1 (Il1r1) in OLC, altogether suggesting that neutrophils modulate the osteogenic program of MSC through Il1b secretion. To explore physiological contexts in which neutrophils modulate bone formation, we first examined the circadian transcriptome of total BM cells and found that Il1b was one of the few cytokines whose transcript levels in the BM followed a tight circadian pattern. We found that depletion of neutrophils fully ablated Il1b expression at all time points, raising the possibility that neutrophils enable diurnal differentiation of mesenchymal cells to produce bone. Indeed, the calcein double labeling assay assays revealed marked circadian patterns of bone synthesis that were significantly blunted in neutropenic and Il1bΔN mice. We propose that bone and blood lineages have co-evolved as an interlocked system that ensures both physical protection and cellular output of this organ.