IL-1ß expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function.

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1ß (IL-1ß) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1ß signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1ß and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1ß expression.

TGF-ß signaling in myeloproliferative neoplasms contributes to myelofibrosis without disrupting the hematopoietic niche.

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-ß to these alterations by abrogating TGF-ß signaling in bone marrow mesenchymal stromal cells. Loss of TGF-ß signaling in Osx-Cre-targeted MSCs prevented the development of myelofibrosis in both MPLW515L and Jak2V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-ß signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPLW515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPLW515L was intact in Osx-Cre Smad4fl/fl recipients, demonstrating that SMAD4-independent TGF-ß signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPLW515L. Together, these data show that JNK-dependent TGF-ß signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.

Microbiota Signals Suppress B Lymphopoiesis With Aging in Mice.

Aging is associated with significant changes in hematopoiesis that include a shift from lymphopoiesis to myelopoiesis and an expansion of phenotypic hematopoietic stem cells (HSCs) with impaired self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SPF mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. The expansion of phenotypic and functional HSCs that occurs with aging is similar in SPF and GF mice. However, HSCs from young GF mice have increased lymphoid lineage output, and the aging-associated expansion of myeloid-biased HSCs is significantly attenuated in GF mice. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress B lymphopoiesis at multiple stages of development and contribute to the expansion of myeloid-biased HSCs that occurs with aging.

Radiation induces iatrogenic immunosuppression by indirectly affecting hematopoiesis in bone marrow.

The immune system plays a vital role in cancer therapy, especially with the advent of immunotherapy. Radiation therapy induces iatrogenic immunosuppression referred to as radiation-induced lymphopenia (RIL). RIL correlates with significant decreases in the overall survival of cancer patients. Although the etiology and severity of lymphopenia are known, the mechanism(s) of RIL are largely unknown. We found that irradiation not only had direct effects on circulating lymphocytes but also had indirect effects on the spleen, thymus, and bone marrow. We found that irradiated cells traffic to the bone marrow and bring about the reduction of hematopoietic stem cells (HSC) and progenitor cells. Using mass cytometry analysis (CyTOF) of the bone marrow, we found reduced expression of CD11a, which is required for T cell proliferation and maturation. RNA Sequencing and gene set enrichment analysis of the bone marrow cells following irradiation showed down-regulation of genes involved in hematopoiesis. Identification of CD11a and hematopoietic genes involved in iatrogenic immune suppression can help identify mechanisms of RIL.

Canonical signaling by TGF family members in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.

Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche. Prior studies showed that signaling from members of the transforming growth factor (TGF) superfamily in mesenchymal stromal cells is required for normal niche development. Here, we assessed the impact of TGF family signaling on niche maintenance and stress responses by deleting Smad4 in mesenchymal stromal cells at birth, thereby abrogating canonical TGF signaling. No alteration in the number or spatial organization of CXCL12-abundant reticular (CAR) cells, osteoblasts, or adipocytes was observed in Osx-Cre, Smad4fl/fl mice, and expression of key niche factors was normal. Basal hematopoiesis and stress erythropoiesis responses to acute hemolytic anemia were normal. TGF-ß potently inhibits stromal CXCL12 expression in vitro; however, G-CSF induced decreases in bone marrow CXCL12 expression and subsequent hematopoietic stem/progenitor cell mobilization were normal in Osx-Cre, Tgfbr2fl/fl mice, in which all TGF-ß signaling in mesenchymal stromal is lost. Finally, although a prior study showed that TGF-ß enhances recovery from myeloablative therapy, hematopoietic recovery following single or multiple doses of 5-flurauracil were normal in Osx-Cre, Tgfbr2fl/fl mice. Collectively, these data suggest that TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.

TGF-ß Signaling Plays an Essential Role in the Lineage Specification of Mesenchymal Stem/Progenitor Cells in Fetal Bone Marrow.

Mesenchymal stromal cells are key components of hematopoietic niches in the bone marrow. Here we abrogated transforming growth factor ß (TGF-ß) signaling in mesenchymal stem/progenitor cells (MSPCs) by deleting Tgfbr2 in mesenchymal cells using a doxycycline-repressible Sp7 (osterix)-Cre transgene. We show that loss of TGF-ß signaling during fetal development results in a marked expansion of CXCL12-abundant reticular (CAR) cells and adipocytes in the bone marrow, while osteoblasts are significantly reduced. These stromal alterations are associated with significant defects in hematopoiesis, including a shift from lymphopoiesis to myelopoiesis. However, hematopoietic stem cell function is preserved. Interestingly, TGF-ß signaling is dispensable for the maintenance of mesenchymal cells in the bone marrow after birth under steady-state conditions. Collectively, these data show that TGF-ß plays an essential role in the lineage specification of fetal but not definitive MSPCs and is required for the establishment of normal hematopoietic niches in fetal and perinatal bone marrow.

Bone marrow dendritic cells regulate hematopoietic stem/progenitor cell trafficking.

A resident population of dendritic cells (DCs) has been identified in murine bone marrow, but its contribution to the regulation of hematopoiesis and establishment of the stem cell niche is largely unknown. Here, we show that murine bone marrow DCs are perivascular and have a type 2 conventional DC (cDC2) immunophenotype. RNA expression analysis of sorted bone marrow DCs shows that expression of many chemokines and chemokine receptors is distinct from that observed in splenic cDC2s, suggesting that bone marrow DCs may represent a unique DC population. A similar population of DCs is present in human bone marrow. Ablation of conventional DCs (cDCs) results in hematopoietic stem/progenitor cell (HSPC) mobilization that is greater than that seen with ablation of bone marrow macrophages, and cDC ablation also synergizes with G-CSF to mobilize HSPCs. Ablation of cDCs is associated with an expansion of bone marrow endothelial cells and increased vascular permeability. CXCR2 expression in sinusoidal endothelial cells and the expression of two CXCR2 ligands, CXCL1 and CXCL2, in the bone marrow are markedly increased following cDC ablation. Treatment of endothelial cells in vitro with CXCL1 induces increased vascular permeability and HSPC transmigration. Finally, we show that HSPC mobilization after cDC ablation is attenuated in mice lacking CXCR2 expression. Collectively, these data suggest that bone marrow DCs play an important role in regulating HSPC trafficking, in part, through regulation of sinusoidal CXCR2 signaling and vascular permeability.