Loss of bone marrow adrenergic beta 1 and 2 receptors modifies transcriptional networks, reduces circulating inflammatory factors, and regulates blood pressure

Abstract

Hypertension (HTN) is a prevalent condition with complex etiology and pathophysiology. Evidence exists of significant communication between the nervous system and the immune system (IS), and there appears to be a direct role for inflammatory bone marrow (BM) cells in the pathophysiology of hypertension. However, the molecular and neural mechanisms underlying this interaction have not been characterized. Here, we transplanted whole BM cells from the beta 1 and 2 adrenergic receptor (AdrB1(tm1Bkk)AdrB2(tm1Bkk)/J) knockout (KO) mice into near lethally irradiated C57BL/6J mice to generate a BM AdrB1.B2 KO chimera. This allowed us to evaluate the role of the BM beta 1 and beta 2 adrenergic receptors in mediating BM IS homeostasis and regulating blood pressure (BP) in an otherwise intact physiological setting. Fluorescence-activated cell sorting demonstrated that a decrease in systolic and mean BP in the AdrB1.B2 KO chimera is associated with a decrease in circulating inflammatory T cells, macrophage/monocytes, and neutrophils. Transcriptomics in the BM identified 7,419 differentially expressed transcripts between the C57 and AdrB1.B2 KO chimera. Pathway analysis revealed differentially expressed transcripts related to several cell processes in the BM of C57 compared with AdrB1.B2 KO chimera, including processes related to immunity (e.g., T-cell activation, T-cell recruitment, cytokine production, leukocyte migration and function), the cardiovascular system (e.g., blood vessel development, peripheral nerve blood flow), and the brain (e.g., central nervous system development, neurite development) among others. This study generates new insight into the molecular events that underlie the interaction between the sympathetic drive and IS in modulation of BP.

Keywords: adrenergic receptors; bone marrow; chimera; immune system; sympathetic drive.

Fig. 1.

Whole bone marrow (BM) ablation and reconstitution with AdrB1.B2 knockout (KO) BM significantly reduced systolic blood pressure (SBP, in A) and mean blood pressure (MBP, in B) in AdrB1.B2 KO chimera at night (dark red bar, P < 0.05). This was accompanied by a significant reduction in the CD4⁺ T cells, CD11b⁺ macrophage/monocytes, and CD184⁺.CD62L⁺ neutrophil levels in the circulation of the AdrB1.B2 KO chimera mice as measured at night (dark bars, P < 0.05) (C, D). C: examples of raw flow cytometry data for circulating T cells (top), macrophage/monocytes (middle), and neutrophils (bottom) in the C57 and AdrB1.B2 KO chimera mice. *P < 0.05 vs. C57 chimera.

Fig. 2.

Fluorescence-activated cell sorting for Sca⁺/C-ckit⁺/Lin⁻ (SKL) markers of hematopoietic stem cells (HSCs) presented as % of circulating mononuclear cells (MNCs) following the irradiation/reconstitution recovery confirms no pathological changes in levels of total HSCs in the C57 and AdrB1.B2 KO chimera mice. Top: raw data with global gating of HSCs in circulation (far left), gating for Lin⁻ cells (second from left), and representative raw data panels for circulating Sca⁺Ckit⁺ cells in C57 (third from left), and AdrB1.B2 KO chimera mice (far right). The graph shows means (± SE; n = 8 per group).

Fig. 3.

Histology in the BM of C57 and AdrB1.B2 KO chimera. No significant histologic differences were detected between the BM of C57 control (A, ×100 magnification, bar = 200 μm; B, ×400 magnification, bar = 50 μm) and AdrB1.B2 KO chimera mice (C, ×100 magnification, bar = 200 μm; D, ×400 magnification, bar = 50 μm). Each section is markedly cellular (normal for mice), and there are sufficient numbers of megakaryocytes, erythroid precursors, and myeloid precursors. Black arrows, megakaryocytes; red arrows, erythrocyte precursors; yellow arrows, myeloid precursors.

Fig. 4.

Cluster analysis of BM expression profiles in C57 chimera and AdrB1.B2 KO chimera. Expression profiles using the whole transcriptome were distinct in the BM between the 2 groups. The numbers represent each individual. Q. Norm, quantile normalization.

Fig. 5.

A transcriptional network for T-cell interaction in the BM of AdrB1.B2 KO chimera compared with C57 chimera mice was downregulated. Red indicates that relative mRNA levels are increased for the transcript, and blue indicates that relative mRNA levels are decreased for the transcript. Different shades of color are reflective of the change in relative abundance. Gene identifiers are provided in the abbreviation list. The square entities refer to cell processes, the oval entities refer to proteins in general, triangles represent ligands, and receptors are those molecules embedded in the membrane with the mushroom cap (e.g., CD4). The arrows indicate direction of entity regulation, while the red hue around the transcript points out CD4, a key gene in the network.

Fig. 6.

Transcriptional networks for cell processes related to lymphocytes in the BM of AdrB1.B2 KO chimera compared with C57 chimera mice were differentially expressed. Red indicates that relative mRNA levels are increased for the transcript and blue indicates that relative mRNA levels are decreased for the transcript. Different shades of color are reflective of the change in relative abundance. Gene identifiers are provided in the abbreviation list and supplemental legend to Fig. 5.

Fig. 7.

Quantitative real-time PCR for Cxcl1 (A), Itga4 (B), and Ms4a4b (C) in the BM of c57 and AdrB1.B2 chimera mice (n = 8 per group). Quantitative real-time PCR for beta adrenergic receptor in circulating mononuclear cells (MNCs) of C57 and AdrB1.B2 KO chimera mice following the irradiation/reconstitution recovery confirms the KO phenotype of the AdrB1.B2 KO chimera (D). Fc, fold change. Shown are the biological replicates as well as the means (± SE).