Salt Intake Activates Peripheral Th17 Immune Cells And Increases Expression of AT1a Receptors in the PVN Facilitated by an IL17/Microglia Dependent Mechanisms
Niousha Ahmari, Rebeca Arocha, Wendi L. Malphurs, Eliza Balazic, Christopher J. Martyniuk and Jasenka Zubcevic
Dept of Physiology, University of Florida, Gainesville, FL
Introduction: Arterial hypertension (HTN) is often salt sensitive and accompanied by renin-angiotensin (RAS) and immune system activation both centrally and in periphery. In a salt-sensitive model, rats fed a moderately high salt diet (2% NaCl) show neuroadaptations that increase excitability of PVN sympathetic control neurons without developing overt HTN. Here, we show that moderately elevated dietary salt increases peripheral IL17 expression in T lymphocytes and contributes to the salt-sensitivity of arterial HTN, possibly by microglial-facilitated priming of the pre-sympathetic circuitry. Methods: Adult Sprague-Dawley rats were placed on either a normal (0.4% NaCl) or moderately elevated salt (2% NaCl) diet for two weeks. Blood and bone marrow were collected to determine levels of CD4+IL17+ T lymphocytes by FACS, and brains were processed to determine levels of CD4+IL17+ T lymphocytes in the PVN by IHC. Western blotting was performed in whole PVN ex vivo to confirm protein levels of NFkB. Real Time PCR tested the levels of IL17a, IL17a receptor (IL17aR), and angiotensin II type 1a receptor (AT1aR), as well as SOD2 and nNOS in the PVN. Lastly, primary microglial cultures were incubated with either aCSF vehicle or IL17a (4 hours), following which the treated microglia were rinsed out and co-incubated with primary neuronal cultures (6 hours). Real time PCR was performed in neurons to determine relative expression levels of AT1aR, IL17aR, nNOS and SOD2. Results: Moderately high salt intake increased expression of IL17a in CD4+ T cells by ~50%, with no change in the total CD4+ T cell population. IHC revealed a ~3-fold increase in CD4+IL17+ T lymphocytes in the PVN of rats on salt diet. This was accompanied by a ~4-fold increase in Iba1+ cells in the PVN of rats on salt diet. Western blotting indicated a ~30% increase in NFkB protein levels in the PVN of rats on salt diet, and real time PCR revealed a ~70-fold increase in the relative expression levels of IL17a, a ~4-fold increase in IL17aR, and a ~2-fold increase in AT1aR in the PVN of rats on salt diet. Additionally, we observed a ~20% reduction in relative expression levels of nNOS, and a ~60% reduction in SOD2 gene expression in the PVN of rats on salt diet. Real time PCR in neuronal cultures co-incubated with IL17a-treated microglia in vitro revealed a ~30-fold increase in relative expression levels of AT1aR, and a ~180-fold increase in IL17aR expression, with no changes in either nNOS or SOD2. Conclusion: A moderately high salt diet is not sufficient to raise blood pressure in rats. Nevertheless, it does activate a population of IL17-expressing immune cells, which are capable of infiltrating the PVN and generating microglial inflammatory responses. We propose that these mechanisms contribute to molecular adaptations of PVN pre-sympathetic neurons that primes them to secondary pro-hypertensive and/or immune challenges, which ultimately cause overt HTN.