Direct pro-inflammatory effects of prorenin on microglia

Abstract

Neuroinflammation has been implicated in hypertension, and microglia have been proposed to play an important role in the progression of this disease. Here, we have studied whether microglia are activated within cardiovascular regulatory area(s) of the brain during hypertension, especially in high blood pressure that is associated with chronic activation of the renin-angiotensin-system. In addition, we determined whether prorenin, an essential component of the renin-angiotensin-system, exerts direct pro-inflammatory effects on these microglia. Our data indicate that two rodent models which display neurogenic hypertension and over activation of the renin-angiotensin-system in the brain (sRA mice and spontaneously hypertensive rats) exhibit microglial activation, and increased levels of pro-inflammatory cytokines, in the paraventricular nucleus of the hypothalamus, an area crucial for regulation of sympathetic outflow. Further, the renin-angiotensin-system component prorenin elicits direct activation of hypothalamic microglia in culture and induction of pro-inflammatory mechanisms in these cells, effects that involve prorenin receptor-induced NFκB activation. In addition, the prorenin-elicited increases in cytokine expression were fully abolished by microglial inhibitor minocycline, and were potentiated by pre-treatment of cells with angiotensin II. Taken together with our previous data which indicate that pro-inflammatory processes in the paraventricular nucleus are involved in the hypertensive action of renin-angiotensin-system, the novel discovery that prorenin exerts direct stimulatory effects on microglial activation and pro-inflammatory cytokine production provides support for the idea that renin-angiotensin-system -induced neurogenic hypertension is not restricted to actions of angiotensin II alone.

Figure 1. Levels of pro-inflammatory cytokines within central cardiovascular control centers of control and sRA mice.

Levels of IL-1β (A) and TNFα (B) mRNAs and TNFα protein (C) were measured in the AV3V, PVN, NTS and RVLM of control and sRA mice as described in the Methods. Data are mean ± SEM., from 6 control and 6 sRA mice for the mRNA and TNFα protein measurements. * P<0.05 vs. respective control.

Figure 2. Microglial activation in the PVN of sRA mice and SHR.

(A) Control vs. sRA mice: Panels (i) and (ii) are representative bright field micrographs showing Iba1 immunoreactivity in the PVN of control and sRA mice. Panels (iii-v) are bar graphs comparing the microglial fractional area (iii), and the levels of PRR (iv) and IL-10 (v) mRNAs in the PVN of control and sRA mice. Data are means ± SEM from the numbers of mice indicated within the bars. *P<0.05 vs. respective control. (B) SHR vs. WKY rats: Panels (i) and (ii) are representative bright field micrographs showing Iba1 immunoreactivity in the PVN of WKY rats and SHR. Panels (iii-vii) are bar graphs comparing the microglial fractional area (iii), and the levels of PRR (iv), IL-1β v), TNFα (vi) and IL-10 (vii) mRNAs in the PVN from both strains. Data are means ± SEM from the numbers of mice indicated within the bars. *P<0.05 vs. respective control.

Figure 3. PRR expression and function in microglia.

(A) (i) Representative fluorescence micrographs showing immunoreactive PRR (P) and Iba1 co-localized (P+I) in mouse N-9 microglial cells and SD rat primary microglia. (B) CD11b protein expression was analyzed in N-9 cells and SD rat primary microglia by western blotting following 24 hr treatment with either control media (DMEM) or prorenin (20 nmol/L). Top: Representative immunoblots showing CD11b and β-actin (loading control) protein bands under each treatment condition; Bottom: Bar graphs are band density ratios of CD11b protein normalized against β-actin. Data are means ± SEM, n = 6 experiments.

Figure 4. Prorenin increases pro-inflammatory cytokine levels in microglia.

(A) Mouse N-9 cells (i–ii) and SD rat primary microglial cells (iii–iv) were treated with prorenin (20 nmol/L) in the absence or presence of losartan (1 µmol/L) for 24 hr, followed by analysis of IL-1β and TNFα mRNA levels as detailed in the Methods. Data are means ± SEM, n = 3 experiments. * P<0.05 vs. respective control. (B) Levels of IL-1β and TNFα mRNAs were measured after 6 hr treatment with prorenin (5–50 nmol/L) in cultured microglial cells prepared from WKY rats and SHR. Data are means ± SEM, n = 4–5 experiments. All prorenin-treated groups were significantly different vs. their respective controls (P<0.05). * P<0.05 vs. respective WKY rat group. (C) Levels of IL-1β and TNFα mRNAs were analyzed after 24 hr treatment of SD rat primary microglial cells with control solution (DMEM) or prorenin (20 nmol/L) in the absence or presence of the indicated concentrations of HRP. Data are means ± SEM from n = 3 experiments. * P<0.05 vs. respective control; † P<0.05 vs. prorenin-alone group.

Figure 5. Prorenin increases TNFα levels in N-9 microglial cells.

N-9 cells were treated with control medium or prorenin (20 nmol/L) for 6 hr, followed by analysis of TNFα-positive cells using flow cytometry (A–C) and released TNFα by ELISA assay (D). FSC: forward scatter. Data are means ± SEM from n = 3 experiments. * P<0.05 vs. control.

Figure 6. NFκB activation mediated prorenin-induced cytokine production.

(A) N-9 (i–ii) and primary microglial cells (iii–iv) were treated with prorenin (10–50 nmol/L), and cell lysates were analyzed by real-time RT-PCR for NFκB1 and NFκBia. Data are mean ± SEM, * P<0.05 vs. control, n = 5 each group. (B) Representative images of NFκB immunoreactivity and Dapi nuclear stain in N-9 (i) and primary microglial cell (ii) 24 hr post control (upper panels) and prorenin (20 nmol/L, lower panels) treatments. (C) N-9 (i–ii) and primary microglia (iii–iv) were treated with control solution (DMEM) or prorenin (20 nmol/L) in the presence or absence of PDTC (50 µmol/L), and IL-1β and TNFα mRNA levels were quantified by real-time RT-PCR. Experiments were performed triplicate. Data are mean ± SEM, * P<0.05 vs. control, n = 5 each group.

Figure 7. Ang II potentiates prorenin-induced TNFα production in microglia.

Mouse N-9 cells were pretreated with control media (DMEM) or Ang II (100 nmol/L) for 12 h, followed by treatment with DMEM or prorenin (20 nmol/L) for 6 hr. Next, TNFα positive cells were analyzed by flow cytometry. Data are means ± SEM from n = 6 experiments. * P<0.05 vs. control; † P<0.05 vs. prorenin without Ang II pretreatment.

Figure 8. Minocycline inhibits prorenin induced pro-inflammatory cytokine production in microglial cells.

Mouse N-9 (A) and SD rat primary microglial cells (B) were treated with control solution (DMEM) or prorenin (20 nmol/L) in the presence or absence of minocycline (1 µmol/L) for 24 hr; cell lysates were analyzed for IL-1β and TNFα mRNAs. Data are means ± SEM from 6 experiments. * P<0.05 vs. control.