Involvement of bone marrow cells and neuroinflammation in hypertension

Abstract

Rationale: Microglial activation in autonomic brain regions is a hallmark of neuroinflammation in neurogenic hypertension. Despite evidence that an impaired sympathetic nerve activity supplying the bone marrow (BM) increases inflammatory cells and decreases angiogenic cells, little is known about the reciprocal impact of BM-derived inflammatory cells on neuroinflammation in hypertension.

Objective: To test the hypothesis that proinflammatory BM cells from hypertensive animals contribute to neuroinflammation and hypertension via a brain-BM interaction.

Methods and results: After BM ablation in spontaneously hypertensive rats, and reconstitution with normotensive Wistar Kyoto rat BM, the resultant chimeric spontaneously hypertensive rats displayed significant reduction in mean arterial pressure associated with attenuation of both central and peripheral inflammation. In contrast, an elevated mean arterial pressure along with increased central and peripheral inflammation was observed in chimeric Wistar-Kyoto rats reconstituted with spontaneously hypertensive rat BM. Oral treatment with minocycline, an inhibitor of microglial activation, attenuated hypertension in both the spontaneously hypertensive rats and the chronic angiotensin II-infused rats. This was accompanied by decreased sympathetic drive and inflammation. Furthermore, in chronic angiotensin II-infused rats, minocycline prevented extravasation of BM-derived cells to the hypothalamic paraventricular nucleus, presumably via a mechanism of decreased C-C chemokine ligand 2 levels in the cerebrospinal fluid.

Conclusions: The BM contributes to hypertension by increasing peripheral inflammatory cells and their extravasation into the brain. Minocycline is an effective therapy to modify neurogenic components of hypertension. These observations support the hypothesis that BM-derived cells are involved in neuroinflammation, and targeting them may be an innovative strategy for neurogenic resistant hypertension therapy.

Keywords: autonomic nervous system; bone marrow cells; hypertension; immune system; microglia.

Figure 1. Pro-inflammatory markers are elevated in the bone marrow (BM) of two rat models of hypertension

A. BM mononuclear cells (MNCs) from the SHR show increased mRNAs for CCL2, CSF2, IFNγ, and IL1β compared to WKY (n=4 per group). *p < 0.05 vs WKY. B. CCL2 levels are elevated in SHR BM supernatant, serum, and cerebrospinal fluid (CSF) (n=4 per group). *p < 0.05, **p < 0.01 vs WKY; & p < 0.05 vs serum. C. BM MNCs from chronic Ang II infusion in SD rats have increased mRNA of CCL2, CCR2, Hif1α, IL1β, Nos2, TLR4, and TNFα compared to saline-infused SD control rats (n=6 per group). *p < 0.05 vs control.

Figure 2. Bone marrow (BM) reconstitution modulates blood pressure and autonomic function in SHR and WKY rats

A. Mean arterial pressure (MAP) was measured directly by radiotelemetry (n=5 per group). Reconstitution of the WKY rat with SHR bone marrow increases MAP. Conversely, reconstitution of the SHR with WKY bone marrow lowers MAP. B. Spectral analysis of the systolic blood pressure (SBP) and pulse interval (PI) waveforms of telemetry (n=5 per group). sBRG: spontaneous baroreflex gain, LF: low frequency, VLF: very low frequency. C. Plasma norepinephrine (NE) is elevated in the WKY-SHR vs WKY-WKY. There is no change in the SHR rats with WKY bone marrow (n=5 per group). *p < 0.05, **p < 0.01 vs WKY-WKY; #p < 0.05, ##p < 0.01 vs SHR-SHR.

Figure 3. Bone marrow (BM) reconstitution with SHR cells alters blood perfusion of the hind limbs in the WKY

A. Hind paw perfusion in arbitrary units (AU) over five minutes of recording time. B. Rate of blood perfusion change over the first 1.5 minutes and stabilized blood perfusion of the hind paw are both decreased in WKY-SHR vs WKY-WKY. C. Photograph indicating region of interest on both hind paws. D. Representative blood flow intensity maps during the last minute of the sample period. The color scale is from blue to red, lower to higher flow. (n=3–4 per group) *p < 0.05 vs WKY-WKY.

Figure 4. Peripheral inflammatory cells and activated microglia in the hypothalamic paraventricular nucleus (PVN) are decreased in the SHR following reconstitution with WKY bone marrow

A. Specific inflammatory cell populations are increased in the circulation of WKY-SHR, including CD4⁺/CD8⁺/CD25⁺ and CD3⁺/CD45⁺ T-cells; these were decreased in the SHR-WKY. Additionally, CD4⁻/CD5⁻/CD8⁻/CD90⁺ were decreased in the WKY-SHR, and increased in the SHR-WKY (n=5–8 per group). B. Representative images at 10x and 40x magnification of Iba1+ microglia in the PVN. Scale bar is 100µm in 10x, and 30µm in 40x. C. Quantification of activated microglia in the PVN: number of microglia and % of activated microglia per 40,000µm², cell body area, are increased in WKY-SHR; these values are decreased in SHR-WKY (n=5 per group). CD11b mRNA was higher in the SHR-SHR compared to WKY-WKY control, and restored in the SHR-WKY (n=5 per group). However, no changes were detected in WKY-SHR vs WKY-WKY. *p < 0.05, **p < 0.01 vs WKY-WKY; #p < 0.05, ##p < 0.01 vs SHR-SHR.

Figure 5. Oral minocycline (mino) attenuates mean arterial pressure (MAP) and peripheral inflammation in SHRs

A. MAP measure by telemetry indicates that mino attenuates the development of HTN in the SHR (n=5-6 per group). B. Specific inflammatory cell populations were increased in the blood in SHRs, including CD4⁺/CD8⁺/CD25⁺, CD4⁺/CD8⁺, and CD68⁺. Mino treatment lowers these ratios back to control (n=5-6 per group). C. Following a similar trend, CD4⁺/CD8⁺/CD25⁺ and CD4⁺/CD8⁺ cells were increased in the bone marrow (BM) and decreased by mino treatment. CD4⁻/CD5⁻/CD8⁻/CD90⁺ cells (APCs) were lower in SHRs compared to WKY, and restored by oral mino (n=4-8 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs WKY control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs SHR control.

Figure 6. Oral minocycline (mino) attenuates mean arterial pressure (MAP) and peripheral inflammation in chronic Ang II infusion

A. Mino attenuates the development of HTN in the chronic Ang II infusion model (n=4 per group). This effect was consistent over 7 weeks of treatment. B. Specific inflammatory cell populations were increased in the blood in chronic Ang II infusion, including CD4⁺/CD8⁺/CD25⁺, CD4⁺/CD8⁺, and CD68⁺. Mino treatment lowers these ratios back to control (n=4-8 per group). C. Following a similar trend, CD4⁺/CD8⁺/CD25⁺ and CD4⁺/CD8⁺ cells were increased in the bone marrow (BM) and decreased by mino treatment. CD4⁻/CD5⁻/CD8⁻/CD90⁺ cells (APCs) were lower in chronic Ang II infusion, and restored by oral mino (n=4-8 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ## p < 0.01, ###p < 0.001 vs Ang II.

Figure 7. Chronic Ang II infusion increases bone marrow derived microglia/macrophages in the hypothalamic paraventricular nucleus (PVN)

A. Representative images at 10x magnification from the PVN of experimental groups. GFP+ cells are bone marrow derived, and Iba1+ cells indicate microglia/macrophages. Scale bar is 100µm; images taken at Bregma −1.80mm; PVN and third ventricle (3V) are labeled for orientation. B. Higher magnification (40x) images of GFP+/Iba1+ cells in the PVN. Scale bar is 30µm. C. Quantification of GFP+/Iba1+ cells in the PVN reveals an increase in the number of cells in chronic Ang II infusion group, which is decreased by minocycline (mino) treatment. CCL2 content in the CSF was also attenuated by mino (n=5-8 per group). D. Ang II treatment (1µM) of primary hypothalamic neurons induces an increase in CCL2 mRNA and CCL2 protein in the cell culture media. *p < 0.05, **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ###p < 0.001 vs Ang II.

Figure 8. Proposed hypothesis for the extravasation of bone marrow cells to the hypothalamic paraventricular nucleus (PVN) and the involvement of neuroinflammation in hypertension (HTN)

Pro-hypertensive signals such as angiotensin II (Ang II) activate PVN preautonomic neurons to increase in sympathetic nerve activity (SNA) and cause release of C-C chemokine ligand 2 (CCL2). The increased SNA impacts the bone marrow (BM) resulting in an increase in inflammatory cells (IC) and decrease in angiogenic progenitor cells (APCs). This imbalance is associated with vascular pathology and increase in blood pressure. Additionally, some of these inflammatory progenitors migrate to the PVN as a result of an increased neuronal release of CCL2 where they differentiate into BM-derived microglia/macrophages. Both resting microglia and BM-derived microglia/macrophages are activated to release an array of cytokines, chemokines, and reactive oxygen species (ROS) which will further increase preautonomic neuronal activity. This leads to a state of sustained sympatho-excitation which will result in a perpetuation of high blood pressure and ultimately established hypertension.