Direct anti-inflammatory effects of angiotensin-(1\xe2\x80\x937) on microglia
Much evidence indicates that pro-inflammatory effects of the renin-angiotensin system (RAS) within the hypothalamus, including microglial activation and production of pro-inflammatory cytokines, play a role in chronic neurogenic hypertension. Our objective here was to examine whether angiotensin-(1\xe2\x80\x937) [Ang-(1\xe2\x80\x937)], a protective component of the RAS, exerts direct actions at microglia to counteract these pro-inflammatory effects. Mas, the Ang-(1\xe2\x80\x937) receptor, was shown to be present on cultured hypothalamic microglia. Treatment of these cells with Ang-(1\xe2\x80\x937) (100\xe2\x80\x931000 nM, 3\xe2\x80\x9312h) elicited significant decreases in basal levels of mRNAs for the pro-inflammatory cytokines interleukin-1\xce\xb2 (IL-1\xce\xb2) and tumor-necrosis factor \xce\xb1 (TNF\xce\xb1) and of the microglia-macrophage marker CD11b, and increases in basal levels of the anti-inflammatory cytokine interleukin-10. Incubation of microglial cultures with PRO (10\xe2\x80\x9350 nM; 6h) elicited significant increases in mRNAs for IL-1\xce\xb2, TNF\xce\xb1 and CD11b. The effects of PRO (10nM) on IL-1\xce\xb2 and TNF\xce\xb1 mRNAs, and TNF\xce\xb1 protein, were significantly attenuated by co-treatment with Ang-(1\xe2\x80\x937) (100 nM). Lastly, these actions of Ang-(1\xe2\x80\x937) were abolished by the Mas antagonist A-779, and were associated with reductions in NF-\xce\xbaB subunit expression. Collectively, these data provide the first evidence that Ang-(1\xe2\x80\x937) can exert direct effects at microglia to lower baseline and counteract PRO-induced increases in pro-inflammatory cytokines.
INTRODUCTION<!>Animals<!>Cell cultures and Treatments<!>Peptides<!>Immunocytochemistry<!>Real-time RT-PCR<!>TNF\xce\xb1 ELISA assay<!>Western Blot for NF-\xce\xbaB p65 in cultured microglia<!>Data analysis<!>Presence of Mas in microglial cells in culture<!>Angiotensin-(1\xe2\x80\x937) modulates baseline levels of cytokine mRNAs and CD11b in microglial cultures<!>Angiotensin-(1\xe2\x80\x937) decreases prorenin induced pro-inflammatory cytokine production in microglia<!>Mechanisms of Ang-(1\xe2\x80\x937)-mediated anti-inflammatory action<!>DISCUSSION<!>
<p>The renin-angiotensin system (RAS) exerts profound physiological effects in the brain to modulate blood pressure and fluid homeostasis. Angiotensin-II (Ang II), the principal active component of the RAS, acts via its type 1 receptors (AT1R) within forebrain circumventricular organs and the hypothalamus to increase sympathetic outflow and blood pressure, water intake and vasopressin secretion (Guyenet 2006, Krause et al. 2008, Lind et al. 1985). However, over activity of this Ang II/AT1R axis within the brain is a major contributor to certain diseases. For example, persistent AT1R activation by Ang II in cardiovascular control centers contributes to the sympathoexcitation and resulting high blood pressure that is characteristic of hypertension of neurogenic origin (de Kloet et al. 2013). The cardiovascular actions of RAS components within the brain are, however, not restricted to Ang II/AT1R. It is now well-recognized that (pro)renin (PRO) acting at its receptors (PRR) in the brain elicits both Ang II - dependent and independent effects on cardiovascular and fluid homeostasis, and contributes to the RAS involvement in neurogenic hypertension (Cuadra et al. 2010).</p><p>In contrast to Ang II and PRO, there is a growing body of evidence that angiotensin-(1–7) [Ang-(1–7)], derived primarily from the action of angiotensin converting enzyme 2 (ACE2) on Ang II, exerts effects via its receptor Mas that are opposite to those of Ang II and PRO (Gironacci et al. 2014), and as such has been suggested as a protective component of the RAS in cardiovascular control (Ferreira et al. 2010). Indeed, Mas immunofluorescence has been demonstrated in rat brain, particularly within forebrain areas that are involved in cardiovascular control and electrolyte balance, such as the hypothalamic paraventricular nucleus (PVN) (Becker et al. 2007), an area important in controlling sympathetic outflow (Coote 2005). Furthermore, Ang-(1–7) applied centrally has been shown to exert depressor effects (Campagnole-Santos et al. 1989), and viral-mediated increased expression of ACE2 in the PVN attenuates the hypertension induced by Ang II infusion (Sriramula et al. 2011).</p><p>While the above described actions of Ang II, PRO and Ang-(1–7) on cardiovascular homeostasis, and hypertension certainly involve effects via AT1R, PRR and Mas [the receptor for Ang-(1–7)] located on neurons, there is evidence from a number of brain areas that these receptors also exist upon microglia and astrocytes (Füchtbauer et al. 2011, Garrido-Gil et al. 2013, Guo et al. 2010, Kandalam et al. 2010, Miyoshi et al. 2008, Regenhardt et al. 2013, Valenzuela et al. 2010). This is important because there is accumulating evidence that RAS-induced neuroinflammation, in particular the activation of microglia and induction of pro-inflammatory cytokine expression and secretion, plays a key role in the chronic phase of RAS-induced neurogenic hypertension (Cardinale et al. 2012, Shi et al. 2010, Sriramula et al. 2013, Zubcevic et al. 2011). Indeed, recent in vitro studies from our group have demonstrated direct enhancement of microglial activation and pro-inflammatory cytokine production by PRO acting via PRR on these cells (Shi et al. 2014). When considering that the anti-hypertensive effects of ACE2/Ang-(1–7) are associated with potent anti-inflammatory actions (decreases in microglial activation and pro-inflammatory cytokine expression) in the hypothalamus, (Sriramula et al. 2011), it is pertinent to ask the question as to whether Ang-(1–7) can exert direct effects at microglia to counteract PRO-induced increases in pro-inflammatory cytokines.</p><p>In the present study we have utilized microglia cultured from rat hypothalamus to evaluate the direct effects of Ang-(1–7) on baseline and PRO-induced pro-inflammatory cytokine production, and the mechanisms of any observed Ang-(1–7) effects.</p><!><p>In this study we used newborn pups from timed pregnant (E16–18) SD rats. These animals were obtained from Charles River Farms (Wilmington, MA), and were housed individually in shoebox style forced-air cages, with access to tap water and food ad libitum and with a 12:12 hour light/dark cycle. All animal protocols were approved by the Institutional Animal Care and Use Committees of the University of Florida. In addition, the principles governing the care and treatment of animals, as stated in the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences (eighth ed., 2011), were followed at all times during this study.</p><!><p>Newborn SD rat pups were euthanized by exposure to 5% isoflurane followed immediately by decapitation. Brains were dissected and primary microglial cells were prepared from a hypothalamic block containing the PVN. Meninges and choroid plexus membranes were removed from brains, and the hypothalamus was dissected and minced with small scissors. The minced tissues were enzymatically digested by 0.25% Trypsin (Worthington Biochemical Corp., Lakewood, NJ) and 0.016% DNase I (Sigma, St. Louis, MO), and followed by centrifuging one time (300× g; 5 min) at room temperature. The pellet was re-suspended in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic cocktail (10,000 IU/ml Penicillin and 10,000 µg/ml Streptomycin; Cellgro, Manassas, VA), and filtered through a gauze sponge. The filtered cells were seeded onto 0.001% Poly-L-lysine (Sigma, St. Louis, MO) coated 75cm2 culture flasks at a density of 3–4 hypothalamic blocks/flask, and incubated with 5% CO2 at 37°C for 7 days without changing the medium. After this time period, cells floating in the culture medium were collected and transferred to 6-well plates for 3 hours, and the original flasks were fed with fresh medium to keep the cell growing. Microglia were collected in this way every 2–3 days for 2 weeks. Collected cells were >99% microglia based on the co-localization of Iba1 (ionized calcium-binding adaptor molecule 1) and DAPI (4',6-diamidino-2-phenylindole) immunostaining. After attachment to the new plates, microglia were fed with fresh DMEM (no FBS) and were ready for treatment with angiotensin peptides or PRO.</p><!><p>Ang II, Ang-(1–7) and A-779 were purchased from Bachem Bioscience (Torrance, CA). Human PRO was purchased from Innovative Research (Novi, MI).</p><!><p>Cultured microglial cells were washed with phosphate buffered saline (PBS; pH 7.2) and then fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were first incubated with PBS containing 0.1% Triton X-100 (PBST) for 10 min at room temperature, and were blocked in 10% goat serum in PBST for 1 hour at room temperature. Immunocytochemistry was first performed using a rabbit polyclonal antibody against Iba1 (1:200, overnight incubation at 4°C; Cat # 019-19741, Wako Chemicals USA, Richmond, VA). This was followed by incubation with a secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (1:500, 1 hour incubation at room temperature; Cat # A11008, Life Technologies, Grand Island, NY). After that, the cell sample was blocked again in 10% goat serum in PBST for 30 min at room temperature and incubated in a rabbit polyclonal antibody against Mas (1:50, 1 hour at room temperature; Cat # AAR-013, Alomone labs, Jerusalem, Israel) which was directly labeled with Alexa Fluor 594 using a Zenon Rabbit IgG labeling kit (Cat # Z-25307, Life Technologies, Grand Island, NY). Iba1and Mas immunoreactivities were viewed using an Olympus BX41 fluorescence microscope.</p><!><p>For mRNA analyses, cultured microglial cells were collected in lysis buffer. RNA was extracted from the samples using RNeasy Plus Mini kits (Qiagen, Valencia, CA). Purified RNA (200ng) was reverse transcribed with a high-capacity iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The mRNA levels of CD11b (αMβ2 integrin) (Rn00709342_m1), GFAP (Glial fibrillary acidic protein) (Rn00566603_m1), CD31 (Rn01467262_m1), NeuN (Rn01464214_m1), IL-1β (interleukin 1β) (Rn00580432_m1), TNFα (tumor necrosis factor alpha) (Rn99999017_m1), IL-10 (interleukin-10) (Rn00563409_m1), AT1R (angiotensin II type 1 receptor) (Rn00578456_m1), Glut-5 (glucose transporter type 5) (Rn00582000_m1), PRR (prorenin receptor) (Rn01430718_m1), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) subunits Nfkb1 (Rn01399583_m1) and Rela (Rn01502266_m1) were analyzed via quantitative real-time PCR using an ABI OneStep Plus machine (Applied Biosystems, Foster City, CA). Oligonucleotide primers and TaqMan probes were obtained from Applied Biosystems. Data were normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Rn99999916_s1).</p><!><p>TNFα protein levels in the culture medium and microglial cells were determined by using an ELISA kit (Cat # KRC3011, Invitrogen, Frederick, MD). Procedures were based on the manufacturer's protocol. Microglial cells were collected in cell extraction buffer (Invitrogen, Frederick, MD) supplemented with 1mM PMSF (phenylmethanesulfonyl fluoride) and protease inhibitor cocktail (Sigma, St. Louis, MO). Protein concentrations in cell lysates were measured using a Bradford assay. Duplicate samples were loaded onto standard 12 × 8 ELISA well strips coated with purified anti-rat TNFα antibody from the ELISA kit for a 2 hour incubation at room temperature. Bound TNFα was detected by a biotin-conjugated anti-rat TNFα detection antibody at room temperature, followed by incubation with Streptavidin-HRP (horseradish peroxidase) reaction enzyme for 30 min at room temperature. The optical density was measured at a wavelength of 450 nm with a Biotek (BioTek, Winooski, VT) plate reader. A standard curve was generated using serial dilutions of recombinant rat TNFα provided in the kit.</p><!><p>Microglial cells were collected in PBS and total protein was extracted by RIPA lysis buffer (Cell Signaling Technology Inc., Beverly, MA) containing protease inhibitor cocktail (Sigma, St. Louis, MO) and phosphatase inhibitor cocktails (Sigma, St. Louis, MO). Protein concentrations were quantified using a Bradford assay. Protein samples were loaded (20µg/lane) and separated on 4–15% SDS polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), which were blocked in 5% w/v nonfat dry milk in 1 X tris-buffered saline (TBS) with 0.1% Tween 20 (1 X TBST) at room temperature for 1 h. Membranes were rinsed with 1X TBST and incubated overnight at 4°C with a rabbit monoclonal antibody against serine 536 phosphorylated NF-κB p65 (1:1000; Cat # 3033, Cell Signaling Technology Inc., Beverly, MA) in 1X TBST with 5% BSA, then washed with 1 X TBST, and incubated with horseradish peroxidase-coupled secondary antibody (1:5000, Cell Signaling Technology Inc., Beverly, MA) for 1 h at room temperature. After washing, protein bands were detected with enhanced chemiluminescence substrate (PerkinElmer Inc., Waltham, MA) for 1 min at room temperature and exposed to X-ray film (Carestream Health Inc., Rochester, NY). The signal intensity of the primary antibody binding was quantified by Image J software 1.45s (National Institutes of Health, Bethesda, MD) and normalized to a loading control Cofilin (rabbit polyclonal antibody, 1:100,000, Abcam, Cambridge, MA).</p><!><p>Data are expressed as means ± SEM. Statistical significance was evaluated as specified in the figure legends, either with the use of Student's t-test when comparing only two data groups, or with a one-way ANOVA followed by Bonferroni analysis for multiple comparisons. Statistical comparisons were performed using Prism5 (GraphPad Prism Software Version 5.04, La Jolla, CA). Differences were considered significant at p<0.05.</p><!><p>Real time PCR analyses revealed that microglial cultures prepared from the hypothalamus of SD rat pups express high levels of mRNA for the microglial marker CD11b, and that the presence of mRNAs for astrocyte marker GFAP, for the endothelial cell marker CD31, and for the neuronal marker NeuN in these cultures was almost undetectable (Figure 1A). Immunostaining revealed that the vast majority of cells present in hypothalamic microglial cultures from SD rats contained immunoreactivity for the microglial marker Iba1 (Figure 1B). Mas immunoreactivity was also present and completely co-localized with Iba1 in these cultures (Figures 1C–E). Omission of the Iba1 antibody or Mas antibody resulted in no-specific staining (Figures 1F,G). The specificity of the Mas antibody used here was demonstrated by us previously, using brain tissues from a Mas-deficient mouse (Regenhardt et al. 2013). Collectively, the data indicated that these cultures were appropriate for investigating the effects of Ang-(1–7) on microglia.</p><!><p>Considering the data presented in Figure 1, we tested whether Ang-(1–7) was able to affect the baseline levels of mRNAs for pro- and anti-inflammatory cytokines in microglia in culture. Treatment of hypothalamic microglial cultures from SD rats with Ang-(1–7) (100nM, 3 –12h) elicited significant (P<0.05) decreases in the basal levels of mRNAs for the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor-necrosis factor α (TNFα), (Figures 2A, B). A higher concentration of Ang-(1–7) (1000 nM) did not elicit greater decreases in IL-1β and TNFα mRNAs (Figures 2D, E). Ang-(1–7) (100nM, 12h) also produced a significant increase in the basal mRNA levels for anti-inflammatory cytokine IL-10 in hypothalamic microglia (Figures 2C, F). Longer term (24h) incubations following a single application of Ang-(1–7) produced no significant changes in pro- or anti-inflammatory cytokine mRNAs (data not shown). We also tested whether Ang-(1–7) treatment affected the baseline levels of mRNA for the microglial marker CD11b, increased expression of which corresponds to the degree of microglial activation (González-Scarano et al. 1999). The data in Figure 2G demonstrate that Ang-(1–7) (100nM, 3 –12h) significantly decreases CD11b mRNA levels at each time point.</p><!><p>Our objective in this series of experiments was to test whether Ang-(1–7) would diminish the stimulatory effects of PRO on pro-inflammatory cytokines in microglial cultures. The presence of PRR mRNA in microglial cultures prepared from the hypothalamus of newborn SD rats was demonstrated by quantitative real time PCR analyses (data not shown). Incubation of these microglial cultures with PRO (10, 20 or 50nM; 6h) elicited concentration-dependent and highly significant increases in the levels of IL-1β and TNFα mRNAs (Figures 3A, B) when compared with control treatments. PRO (10–50nM; 6h) also elicited significant, but more minor, increases in CD11b mRNA (Figure 3C). In previous studies we demonstrated that these effects of PRO on pro-inflammatory cytokine expression in hypothalamic microglial cultures are independent of Ang II generation (Shi et al. 2014). The reported Kd range for the PRR is 1–8 nM (Nabi et al. 2006, Batenburg et al. 2007), and in subsequent experiments we used PRO at a dose of 10 nM. The stimulatory action of PRO (10nM; 6h) on IL-1β and TNFα mRNAs was attenuated by co-treatment with Ang-(1–7) (100nM) (Figures 4A, B). Interestingly, PRO-induced increases in CD11b were not affected by Ang-(1–7) treatment (data not shown).</p><p>The inhibitory effects of Ang-(1–7) on pro-inflammatory cytokines in hypothalamic microglia were also apparent at the protein level. Treatment of microglial cultures from SD rats with PRO (10nM) for 6 or 12 h significantly increased TNFα protein levels in the growth medium when compared with controls (Figures 5A, B), suggesting that PRO increases TNFα secretion from microglia. However, the stimulatory effects of PRO (10nM; 6h or 12h) on TNFα protein in the growth medium were not altered by co-treatment with Ang-(1–7) (100nM) (Figures 5A, B). Treatment of hypothalamic microglial cultures with PRO (10nM; 12h) also induced significant increases in TNFα protein levels in the cell lysate compared with controls. This stimulatory action of PRO on TNFα protein within cells was significantly attenuated (by ~37%) by co-treatment with Ang-(1–7) (100nM) (Figure 5C).</p><!><p>To determine the mechanisms underlying the anti-inflammatory effects of Ang-(1–7), we first assessed whether the actions of Ang-(1–7) were Mas-dependent. Hypothalamic microglial cultures from SD rats were incubated with Ang-(1–7) (100nM) in the absence or presence of the Mas inhibitor A779 (1µM, 6h). The data in Figure 6A & 6B demonstrate that the respective reductions of basal TNFα mRNA and IL-1β mRNA levels induced by Ang-(1–7) were inhibited by co-treatment with A779.</p><p>Previous data from our group have demonstrated that PRO-induced increases in pro-inflammatory cytokine mRNA expression in hypothalamic microglia are dependent on NF-κB (Shi et al. 2011). Thus, we measured the effects of Ang-(1–7) on the expression of mRNAs for the NF-κB complex subunits, Nfkb1 (NF-κB p50) and Rela (NF-κB p65) in hypothalamic microglia. Treatment of microglial cultures with Ang-(1–7) [100nM, 6h] significantly decreased the basal mRNA levels for both Nfkb1 (NF-κB p50) and Rela (NF-κB p65), when compared with controls (Figure 6C, D). Western blot experiments indicated that Ang-(1–7) [100nM, 6h] treatment elicited a trend to decrease baseline protein levels of phospho-NF-κB p65 (P-NF-κB p65) (Figure 6E,F).</p><!><p>The observations made in this study support the conclusion that Ang-(1–7) can exert direct effects at microglia to lower baseline and counteract PRO-induced increases in pro-inflammatory cytokines. The major findings of this study include: (i) Mas, the receptor for Ang-(1–7) is present in microglial cultures prepared from the hypothalamus; (ii) Ang-(1–7) was able to decrease the baseline mRNA expression for pro-inflammatory cytokines and CD11b, and increase the basal mRNA levels for the anti-inflammatory cytokine IL-10 in hypothalamic microglia in culture; (iii) Ang-(1–7) diminished the stimulatory actions of PRO on pro-inflammatory cytokines in microglial cultures; (iv) Ang-(1–7) decreased the PRO-induced increase in cellular TNFα protein levels in microglia; (v) The anti-inflammatory actions of Ang-(1–7) in hypothalamic microglia were Mas-dependent, and were associated with decreased expression of NF-κB subunits.</p><p>The importance of these observations is that they provide a mechanistic basis for the beneficial effects of this peptide in disease states that involve RAS-induced activation of the neuroimmune system (de Kloet et al. 2013). One such disease state is drug-resistant or neurogenic hypertension, which is characterized by sustained sympathoexcitation (Esler 2011, Takahashi 2012). Both Ang II/AT1R and PRO/PRR mechanisms contribute to this sustained hypertension (Cuadra et al. 2010, de Kloet et al. 2013), with a recent study indicating that Ang II-induced hypertension is dependent upon central PRR (Li et al. 2012). Further, a part of these effects of Ang II and PRO involves induction of and association with pro-inflammatory mechanisms including activation of microglia in the hypothalamus (Shi et al. 2011, Shi et al. 2014). Our current observations that Ang-(1–7) decreases pro-inflammatory cytokine production and secretion and also CD11b by direct Mas-mediated actions at microglia help to explain the findings that over expression of the Ang-(1–7) forming enzyme ACE2 in the hypothalamic PVN attenuates hypertension induced by Ang II infusion and also the associated increase in the expression of the IL-1β, TNFα and interleukin-6 at this site (Sriramula et al. 2011). Another such disease state is stroke, which results in a severe intracerebral pro-inflammatory effect that contributes to neuronal death (Iadecola & Anrather 2011). Data from our group and others have demonstrated that Ang-(1–7) applied centrally can reduce infarct size and improve behavioral outcomes in ischemic stroke (Mecca et al. 2011, Jiang et al. 2012), and we have also demonstrated that Ang-(1–7) can increase the lifespan of stroke prone spontaneously hypertensive rats (spSHR), a model of hemorrhagic stroke (Regenhardt et al. 2014). In the case of ischemic stroke, Ang-(1–7) elicited a significant decrease in the post-stroke induction of pro-inflammatory cytokines in the cerebral cortex, and in the spSHR Ang-(1–7) decreased microglial numbers within the striatum (Regenhardt et al. 2014). Our previous data indicate that Mas are present on microglia within the cerebral cortex (Regenhardt et al. 2013), and based on our findings in the current study and on preliminary findings that Ang-(1–7) decreases IL-1β and TNFα mRNA levels in microglia cultured from cerebral cortex (data not shown), we can speculate that the beneficial actions of Ang-(1–7) in both kinds of stroke at least include direct actions at cortical microglia to exert anti-inflammatory effects. A further pathological state that involves RAS-induced activation of the neuroimmune system is Parkinson's Disease, characterized by a loss of dopaminergic neurons in the substantia nigra. Studies have indicated the presence of PRR immunoreactivity on neurons and microglia within the substantia nigra, and also suggest that these receptors contribute to dopaminergic cell death (Valenzuela et al., 2010).</p><p>Despite the findings made in the current study, a number of questions remain to be answered. First, since Ang-(1–7) does not completely decrease both basal and PRO-induced increases in pro-inflammatory cytokines in microglia, the efficacy of its effects as an anti-inflammatory mechanism may be questioned. We would perhaps not expect Ang-(1–7) to completely decrease cytokine expression within microglia while they are in a basal or non-stimulated state, but the failure of this peptide to abolish the actions of PRO might mean that it is not the only counteracting mechanism involved in tempering the pro-inflammatory mechanisms of hypothalamic microglia. In addition, it is also possible that a greater effect of Ang-(1–7) would have been revealed by utilizing a wider range of concentrations or treatment times with this peptide.</p><p>A further question concerns the signaling pathways that mediate the anti-inflammatory actions of Ang-(1–7) in microglia. Based on our data, they are Mas-dependent, and our data indicate involvement of the NF-κB pathway. However, the exact locus or upstream mechanism involved in this Ang-(1–7) action is not known. It has been shown that PRO induces Erk1/Erk2 mitogen-activated protein kinase (MAPK) activation in mesangial cells via its membrane receptor PRR, and this effect is independent of a possible generation or action of Ang II (Nguyen et al. 2002). In contrast, Ang-(1–7) inhibited Ang II stimulation of MAPK (Erk1/Erk2) activities in vascular smooth muscle cells (VSMCs) (Tallant et al. 2003). Another study indicates that Erk acts upstream of NF-κB, and it has been shown that Erk regulation of NF-κB activation involves increased IκB phosphorylation and increased NF-κB DNA binding activity (Dhawan et al. 2002). We have previously demonstrated that PRO-induced pro-inflammatory cytokine production in microglial cultures is dependent on NF-κB activation (Shi et al. 2011). Given that NF-κB activation is a key step in initiating pro-inflammatory cytokine production in microglia, Ang-(1–7) could function as a counter player to PRO elicited cytokine production. Taken together, these data support the hypothesis that Ang-(1–7) may exert its anti-inflammatory action at microglia by inhibition of Erk/NF-κB pathway. A further realistic possibility is that the intracellular signaling pathways responsible for the anti-inflammatory action of Ang-(1–7) are independent from the pathways that are involved in the pro-inflammatory actions of PRO, especially as Ang-(1–7) can decrease basal cytokine expression.</p><p>Additionally, while the present study is focused on the effects of Ang-(1–7) on microglial activation, it will also be very important to determine whether Ang-(1–7) and PRO might affect the migration of microglia. A further question is whether the current cell culture findings translate to the in vivo situation and whether Ang-(1–7) has similar direct anti-inflammatory effects at microglia in the PVN, and whether these effects contribute to the antihypertensive actions of this peptide?</p><p>In summary, this study demonstrates a direct action of Ang-(1–7) on microglia to lower basal- and PRO-stimulated increases in the levels of pro-inflammatory cytokines. While these effects of Ang-(1–7) are only partial, they are significant. Considering that pro-inflammatory effects of RAS components such as Ang II and PRO in the CNS play causative roles in chronic neurogenic hypertension, further exploration of the anti-inflammatory effects of Ang-(1–7) in vivo may lead to a novel therapeutic avenue for this disease.</p><!><p>ARRIVE guidelines have been followed:</p><p>Yes</p><p>=> if No, skip complete sentence</p><p>=> if Yes, insert "All experiments were conducted in compliance with the ARRIVE guidelines."</p><p>Conflicts of interest: None</p><p>=> if 'none', insert "The authors have no conflict of interest to declare."</p><p>=> otherwise insert info unless it is already included</p>