2029 – INTRAVITAL IMAGING OF CALCIUM NETWORK TOPOLOGY REVEALED THE IMMUNE-MODULATORY LOCI IN THE BONE MARROW

Abstract

Intercellular communications through calcium signaling orchestrates coordinated cell functions across diverse tissues. Bone marrow stromal cells (BMSCs) form an extended 3D network in the marrow and secrete cytokines essential for regulating blood and immune cell formation. Yet, the degree of coordination and heterogeneity among the BMSCs in the interconnected network remain poorly understood. Here, we developed an intravital calcium imaging platform using LepR-cre; Salsa6f mice, in which tdTomato and a calcium flux indicator (GCaMP6f) are expressed in BMSC lineages. We identified a subset of BMSCs that communicate via intercellular calcium waves (ICWs). The ICWs are provoked by acute inflammatory challenges (lipopolysaccharide [LPS], 1.4 mg/kg), and follow circadian rhythms. Further analyses from the single-cell RNA sequencing data suggest that the BMSC subset express high level of leukocyte chemoattractant Ccl2. Validations using intravital tracking confirmed that ICW initiation led to aggregation of CCR2+ leukocytes within 2 hours after LPS administration, forming “hot spots” within the systemic inflammatory landscape. As gap junctions have been reported to regulate timed cytokine secretion, we next generated triple transgenic LepR-cre; Cx43 floxed; Salsa6f mice. Cx43 deficiency reduced ICWs and led to a more diffused CCR2 cell distribution in contrast to the designated hot spots found in the WT mice. Functionally, Cx43 deficiency blunted the responses of IL-1β and IL-10 in the bone marrow after LPS challenge, whereas CXCL12 was not affected. Together, our intravital imaging toolbox uncovered the presence of immune modulatory loci in the bone marrow, where BMSCs coordinate through ICWs to work in spatially and temporally controlled manner.

Intercellular communications through calcium signaling orchestrates coordinated cell functions across diverse tissues. Bone marrow stromal cells (BMSCs) form an extended 3D network in the marrow and secrete cytokines essential for regulating blood and immune cell formation. Yet, the degree of coordination and heterogeneity among the BMSCs in the interconnected network remain poorly understood. Here, we developed an intravital calcium imaging platform using LepR-cre; Salsa6f mice, in which tdTomato and a calcium flux indicator (GCaMP6f) are expressed in BMSC lineages. We identified a subset of BMSCs that communicate via intercellular calcium waves (ICWs). The ICWs are provoked by acute inflammatory challenges (lipopolysaccharide [LPS], 1.4 mg/kg), and follow circadian rhythms. Further analyses from the single-cell RNA sequencing data suggest that the BMSC subset express high level of leukocyte chemoattractant Ccl2. Validations using intravital tracking confirmed that ICW initiation led to aggregation of CCR2+ leukocytes within 2 hours after LPS administration, forming “hot spots” within the systemic inflammatory landscape. As gap junctions have been reported to regulate timed cytokine secretion, we next generated triple transgenic LepR-cre; Cx43 floxed; Salsa6f mice. Cx43 deficiency reduced ICWs and led to a more diffused CCR2 cell distribution in contrast to the designated hot spots found in the WT mice. Functionally, Cx43 deficiency blunted the responses of IL-1β and IL-10 in the bone marrow after LPS challenge, whereas CXCL12 was not affected. Together, our intravital imaging toolbox uncovered the presence of immune modulatory loci in the bone marrow, where BMSCs coordinate through ICWs to work in spatially and temporally controlled manner.