Agnes Schröder

Immune cells control skin lymphatic electrolyte homeostasis and blood pressure

The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

Cutaneous Na+ Storage Strengthens the Antimicrobial Barrier Function of the Skin and Boosts Macrophage-Driven Host Defense

Immune cells regulate a hypertonic microenvironment in the skin; however, the biological advantage of increased skin Na(+) concentrations is unknown. We found that Na(+) accumulated at the site of bacterial skin infections in humans and in mice. We used the protozoan parasite Leishmania major as a model of skin-prone macrophage infection to test the hypothesis that skin-Na(+) storage facilitates antimicrobial host defense. Activation of macrophages in the presence of high NaCl concentrations modified epigenetic markers and enhanced p38 mitogen-activated protein kinase (p38/MAPK)-dependent nuclear factor of activated T cells 5 (NFAT5) activation. This high-salt response resulted in elevated type-2 nitric oxide synthase (Nos2)-dependent NO production and improved Leishmania major control. Finally, we found that increasing Na(+) content in the skin by a high-salt diet boosted activation of macrophages in a Nfat5-dependent manner and promoted cutaneous antimicrobial defense. We suggest that the hypertonic microenvironment could serve as a barrier to infection.

Spooky sodium balance

Current teaching states that when sodium intake is increased from low to high levels, total-body sodium (TBNa) and water increase until daily sodium excretion again equals intake. When sodium intake is reduced, sodium excretion briefly exceeds intake until the excess TBNa and water are eliminated, at which point sodium excretion again equals intake. However, careful balance studies oftentimes conflict with this view and long-term studies suggest that TBNa fluctuates independent of intake or body weight. We recently performed the opposite experiment in that we fixed sodium intake for several weeks at three levels of sodium intake and collected all urine made. We found weekly (circaseptan) patterns in sodium excretion that were inversely related to aldosterone and directly to cortisol. TBNa was not dependent on sodium intake but instead exhibited far longer (≥ monthly) infradian rhythms independent of extracellular water, body weight, or blood pressure. The findings are consistent with our ideas on tissue sodium storage and its regulation that we developed on the basis of animal research. We are implementing (23)Na-magnetic resonance imaging (MRI) to pursue open questions on sodium balance in patients. Our findings could be relevant to therapeutic strategies for hypertension and target-organ damage.

Salt-dependent chemotaxis of macrophages

Besides their role in immune system host defense, there is growing evidence that macrophages may also be important regulators of salt homeostasis and blood pressure by a TonEBP-VEGF-C dependent buffering mechanism. As macrophages are known to accumulate in the skin of rats fed under high salt diet conditions and are pivotal for removal of high salt storage, the question arose how macrophages sense sites of high sodium storage. Interestingly, we observed that macrophage-like RAW264.7 cells, murine bone marrow-derived macrophages and peritoneal macrophages recognize NaCl hypertonicity as a chemotactic stimulus and migrate in the direction of excess salt concentration by using an in vitro transwell migration assay. While RAW264.7 cells migrated toward NaCl in a dose-dependent fashion, no migratory response toward isotonic or hypotonic media controls, or other osmo-active agents, e.g. urea or mannitol, could be detected. Interestingly, we could not establish a specific role of the osmoprotective transcription factor TonEBP in regulating salt-dependent chemotaxis, since the specific migration of bone marrow-derived macrophages following RNAi of TonEBP toward NaCl was not altered. Although the underlying mechanism remains unidentified, these data point to a thus far unappreciated role for NaCl-dependent chemotaxis of macrophages in the clearance of excess salt, and suggest the existence of novel NaCl sensor/effector circuits, which are independent of the TonEBP system.

NF-κB inhibitor targeted to activated endothelium demonstrates a critical role of endothelial NF-κB in immune-mediated diseases

Significance The transcription factor NF-κB is crucially involved in the pathogenesis of inflammatory diseases and represents a target for treatment. However, a general blockade of NF-κB by small-molecule inhibitors is associated with serious side effects due to the importance of NF-κB in cellular survival and function of various organs. This paper demonstrates that cell type-specific NF-κB inhibition can be achieved using multi-modular fusion proteins, which exclusively target activated endothelial cells. Inhibition of NF-κB within activated endothelium potently ameliorated peritonitis and arthritis in mice, indicating that endothelial NF-κB might be a valid target in inflammatory diseases. Importantly, this strategy enables the targeting of other cell types and intracellular signaling pathways. Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inflammatory genes crucially involved in the pathogenesis of inflammatory diseases. NF-κB governs the expression of adhesion molecules that play a pivotal role in leukocyte–endothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a “sneaking ligand construct” (SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i) an E-selectin targeting domain, (ii) a Pseudomonas exotoxin A translocation domain, and (iii) a NF-κB Essential Modifier-binding effector domain interfering with NF-κB activation. The E-selectin–specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inflammatory cells. Furthermore, SLC1 treatment significantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the pathogenesis of arthritis and can be selectively inhibited in a cell type- and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specific manner and enables selective targeting of distinct cell populations to improve effectiveness and risk–benefit ratios of therapeutic interventions.

High salt intake reprioritizes osmolyte and energy metabolism for body fluid conservation

Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter–driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions.

Elementary immunology: Na(+) as a regulator of immunity.

The skin can serve as an interstitial Na+ reservoir. Local tissue Na+ accumulation increases with age, inflammation and infection. This increased local Na+ availability favors pro-inflammatory immune cell function and dampens their anti-inflammatory capacity. In this review, we summarize available data on how NaCl affects various immune cells. We particularly focus on how salt promotes pro-inflammatory macrophage and T cell function and simultaneously curtails their regulatory and anti-inflammatory potential. Overall, these findings demonstrate that local Na+ availability is a promising novel regulator of immunity. Hence, the modulation of tissue Na+ levels bears broad therapeutic potential: increasing local Na+ availability may help in treating infections, while lowering tissue Na+ levels may be used to treat, for example, autoimmune and cardiovascular diseases.

Cardiovascular and Renal Effects of High Salt Diet in GDNF+/- Mice with Low Nephron Number

Aims: To test the suggested association of low nephron number and later development of renal and cardiovascular disease we investigated the effects of high sodium diet in heterozygous GDNF+/- mice. Methods: Aged wild type and GDNF+/- mice were grouped together according to high sodium (HS, 4%) or low sodium (LS, 0.03%) diet for 4 weeks. The heart, the aorta and the kidneys were processed for morphometric and stereological evaluations and TaqMan PCR. Results: On HS GDNF+/- mice showed significantly higher drinking volume and urine production than wt and mean arterial blood pressure tended to be higher. Heart weight was higher in GDNF+/- than in wt, but the difference was only significant for LS. HS significantly increased cardiac interstitial tissue in GDNF+/-, but not in wt. On LS GDNF+/- mice had significantly larger glomeruli than wt and HS led to an additional two fold increase of glomerular area compared to LS. On electron microscopy glomerular damage after HS was seen in GDNF+/-, but not in wt. Dietary salt intake modulated renal IL-10 gene expression in GDNF+/-. Conclusion: In the setting of 30% lower nephron number HS diet favoured maladaptive changes of the kidney as well as of the cardiovascular system.

Mild Salt-Sensitive Hypertension in Genetically Determined Low Nephron Number is Associated with Chloride but Not Sodium Retention

Background/Aims: One potential pathomechanism how low nephron number leads to hypertension in later life is altered salt handling. We therefore evaluated changes in electrolyte and water content in wildtype (wt) and GDNF+/- mice with a 30% reduction of nephron number. Methods: 32 GDNF+/- and 36 wt mice were fed with low salt (LSD, 0.03%, normal drinking water) or high salt (HSD, 4%, 0.9% drinking water) diet for 4 weeks. Blood pressure was continuously measured by telemetry in a subgroup. At the end of the experiment and after standardized ashing processes electrolyte- and water contents of the skin and the total body were determined. Results: We found higher blood pressure in high salt treated GDNF+/-compared to wt mice. Of interest, we could not confirm an increase in total-body sodium as predicted by prevailing explanations, but found increased total body and skin chloride that interestingly correlated with relative kidney weight. Conclusion: We hereby firstly report significant total body and skin chloride retention in salt sensitive hypertension of GDNF+/-mice with genetically determined lower nephron number. Thus, in contrast to the prevailing opinion our data argue for the involvement of non-volume related mechanisms.

OP0023 Low Salt Diet Ameliorates Collagen-Induced Arthritis

Background There is a complex relationship between nutrition and the immune system, which is only partially understood. Especially, the impact of nutrition on inflammatory autoimmune diseases like rheumatoid arthritis (RA) requires further investigations. It was recently shown that an increased salt intake aggravates the clinical symptoms in an animal model of multiple sclerosis by inducing pathogenic Th17 cells [1]. However the effect of dietary factors such as low salt consumption on the inflammatory status in arthritis is poorly investigated. Objectives In this study we examined the effect of low salt diet in a mouse model of arthritis and identified its underlying mechanisms. Methods Two weeks before immunization with bovine type II collagen (bCII) in complete Freunds adjuvant (CFA) the experimental diet was started in DBA/1 mice. The low salt group (n=9) received a diet containing a sodium content <0.03% and tap water for 62 days, whereas the high-salt group (n=10) was fed with a diet containing 4% NaCl and 0.9% saline solution. Arthritis severity was assessed by clinical scoring using a graded scale (0-4). Hind paws were prepared for histological analysis. Levels of anti-CII autoantibodies were measured by ELISA. Homogenates from hind paws were prepared and cytokines were assessed by ELISA. mRNA levels of inflammatory cytokines in the spleen were determined by qRT-PCR. Results In CIA the low salt diet decreased severity and incidence of arthritis compared to the high salt group. The histology showed reduced infiltrations with inflammatory cells after low salt diet. Also cartilage breakdown and bone destruction was less pronounced in the low salt compared to the high salt group. ELISA experiments showed that the level of pathogenic IgG2a antibodies against CII was markedly elevated in high salt mice, whereas low salt consumption reduced anti-CII IgG2a levels significantly. CII-specific IgG1 titers were increased in the low salt versus the high salt group. This resulted in decreased IgG2a:IgG1 ratio in the low salt group and indicates a shift toward a more Th2-dominated immune response. Already 20 days after immunization we observed decreased levels of MCP-1 and IL-1β and an increase in IL-10 in paw extracts of mice which had received a low salt diet. Further, in low salt mice mRNA levels of IFNγ, IL-6 and IL-1β were reduced. Conclusions Lowering the salt intake attenuates clinical and histopathological manifestations in collagen-induced arthritis. The low salt diet protects against antibody-mediated joint destruction through a Th2 shift and a decreased expression of potential mediators of inflammation. We conclude that a low salt diet could be a useful supplementary nutritional therapy of immune-mediated arthritides. References Kleinewietfeld, M., et al., Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature, 2013. 496(7446): p. 518-22. Disclosure of Interest None declared