Adrenergic signaling and hematopoietic stem cell function in the bone marrow niche rely on peri-arteriolar NADPH oxidase (NOX) and connexin gap junctions. Free

Hematopoietic stem cell (HSC) activity is tightly regulated by the bone marrow (BM) microenvironment, often referred to as “niche”. The HSC niche is abundantly innervated by adrenergic nerves and comprises different niche cell types, among which mesenchymal stem cells (MSCs) orchestrate the retention and maintenance of HSCs. We have previously identified adrenergic nerves as a key regulator of niche remodeling and HSC function (Nature Medicine, 2018); however, the downstream mechanisms remain unknown. An intriguing observation about the distribution of nerves in the BM parenchyma is that the bulk of innervation is concentrated on arteriolar blood vessels, while sinusoids are denuded of innervation. However, reticular MSCs (reticMSCs) that are primarily targeted by adrenergic signals to mediate circadian egress of hematopoietic stem and progenitor cells (HSPCs) into peripheral blood (by the production of CXCL12) are peri-sinusoidal and don't directly interact with nerve terminals. This raises important questions as to how neural signals are transmitted in the niche to mediate circadian oscillations and HSC function.

Here we report a novel neural signal transmission mechanism, mediated by perivascular MSC networks and reliant on connexin gap junctions (CxGJs) and NADPH oxidase (NOX), a major source of cellular superoxide. Our studies revealed circadian oscillations of superoxide in reticMSCs (P<0.0001), which were abrogated in sympathectomized mice (P<0.0001) or mice deficient in β-adrenergic receptors (βARs) (Adrb2^–/–; Adrb3^–/–; P=0.0043). Similar to chemical denervation, neutralization of superoxide in mice with N-Acetyl-L-Cysteine (NAC) abrogated circadian oscillation of HSPC egress to peripheral blood and elevated BM CXCL12 levels (P=0.014), suggesting a functional role for superoxide downstream of βAR signaling. Furthermore, inactivation of NOX (Cyba^fl/fl; Myh11-creER^T2) or deletion of Connexin 43 (Cx43^fl/fl; Myh11-creER^T2) in a subset of peri-arteriolar MSCs (periMSCs), which are directly innervated by adrenergic fibers, recapitulated these phenotypes, leading to a complete loss in oscillations of blood HSPCs. Strikingly, loss of either peri-arteriolar NOX or Cx43 led to 2-fold expansion of reticMSCs (P=0.0058 for Cyba andP=0.018 for Cx43 deletion) that exhibited enhanced adipogenic differentiation potential (2-fold; P=0.0123) and upregulation of pathways involved in proliferation, DNA repair, and inflammatory signaling, and downregulation of adrenoreceptor signaling and expression of extracellular matrix (ECM) components, as determined by RNA sequencing.

As the Myh11-creER^T2 transgene does not recombine in reticMSCs, our findings suggest that the loss of peri-arteriolar NOX and CxGJ compromises intracellular communication between peri-arteriolar and sinusoidal MSC networks, possibly facilitated by superoxide to transmit neural signals in the BM parenchyma. To model stromal communication, we developed a novel ex vivo stromal biomimetic microenvironment that enables the isolation, expansion, and co-culture of reticular and peri-arteriolar MSCs to model niche cellular communication. Using this system, we show that βAR signaling induces superoxide production in periMSCs, which depends on NOX and protein kinase C (PKC). Moreover, periMSCs transmitted superoxide to reticMSCs (P=0.003), which was mediated by NOX and Cx43, as deletion of either in periMSCs abolished superoxide transfer to reticMSCs. These experiments establish a proof-of-concept demonstrating that in periMSCs, βAR signaling drives NOX-derived superoxide production, which is relayed to reticMSCs via CxGJ.

Investigating the functional consequence of NOX perturbation in the peri-arteriolar niche, we detected 2-fold expansion of phenotypic HSCs in Cyba^fl/fl; Myh11-creER^T2 mice (P=0.0058), which was recapitulated in both Cx43^fl/fl; Myh11-creER^T2 and Adrb2^–/–; Adrb3^–/– mice but not upon loss of NOX in reticMSC (Cyba^fl/fl; Lepr-cre). Competitive HSC transplantation revealed reduced HSC engraftment (P=0.043) and poor contribution to the myeloid lineage (P=0.028), which was further deteriorated following secondary transplantation (P<0.0001) and likely mediated by remodeling of the BM stromal niche in Cyba^fl/fl; Myh11-creER^T2 donors. Together, our findings uncover a previously unrecognized neuro-stromal communication mechanism within the BM niche and establish peri-arteriolar NOX and CxGJs as critical mediators of BM stromal homeostasis and HSC function.