Katrina J. Binger

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.

Interferon-γ Signaling Inhibition Ameliorates Angiotensin II–Induced Cardiac Damage

Angiotensin (Ang) II induces vascular injury in part by activating innate and adaptive immunity; however, the mechanisms are unclear. We investigated the role of interferon (IFN)-&ggr; and interleukin (IL)-23 signaling. We infused Ang II into IFN-&ggr; receptor (IFN-&ggr;R) knockout mice and wild-type controls, as well as into mice treated with neutralizing antibodies against IL-23 receptor and IL-17A. Ang II–treated IFN-&ggr;R knockout mice exhibited reduced cardiac hypertrophy, reduced cardiac macrophage and T-cell infiltration, less fibrosis, and less arrhythmogenic electric remodeling independent of blood pressure changes. In contrast, IL-23 receptor antibody treatment did not reduce cardiac hypertrophy, fibrosis, or electric remodeling despite mildly reduced inflammation. IL-17A antibody treatment behaved similarly. In the kidney, IFN-&ggr;R deficiency reduced inflammation and tubulointerstitial damage and improved glomerular filtration rate. Nonetheless, albuminuria was increased compared with Ang II–treated wild-type controls. The glomeruli of Ang II–treated IFN-&ggr;R knockout mice exhibited fewer podocytes, less nephrin and synaptopodin staining, and impaired podocyte autophagy. Thus, IFN-&ggr; blockade, but not IL-23 receptor antibody treatment, protects from Ang II–induced cardiac damage and electric remodeling. In the kidney, IFN-&ggr; signaling acts in a cell type–specific manner. Glomerular filtration rate is preserved in the absence of the IFN-&ggr;R, whereas podocytes may require the IFN-&ggr;R in the presence of Ang II for normal integrity and function.

High salt reduces the activation of IL-4- and IL-13-stimulated macrophages

A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt-induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis.

Candesartan attenuates diabetic retinal vascular pathology by restoring glyoxalase-I function.

OBJECTIVE Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy. RESEARCH DESIGN AND METHODS Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats. RESULTS In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO•), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO•. Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO•, and restored GLO-I. CONCLUSIONS We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.

Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (Tm) from 58.7 °C for native apoA-I to 48.2 °C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 Å and 9.92 Å, indicating a cross-β amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation.

Apolipoprotein C-II Amyloid Fibrils Assemble via a Reversible Pathway that Includes Fibril Breaking and Rejoining

Alzheimers and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.

RILLKKMPSV Influences the Vasculature, Neurons and Glia, and (Pro)Renin Receptor Expression in the Retina

The (pro)renin receptor [(P)RR] is implicated in organ pathology. We examined the cellular location of the (P)RR and whether a putative (P)RR antagonist, RILLKKMPSV, corresponding to the handle region of the prorenin prosegment (handle region peptide [HRP]) influences angiogenesis, inflammation, and neuronal and glial function in rat retina. The (P)RR was localized to retinal vessels, endothelial cells, and pericytes, but most immunolabeling was in ganglion cells and glia. HRP (1 mg/kg per day by IP injection) reduced physiological angiogenesis in developing retina. Moreover, HRP (0.1 mg/kg per day by subcutaneous minipump) reduced pathological retinal angiogenesis, inflammation, and vascular endothelial growth factor and intercellular adhesion molecule-1 mRNA in rats with oxygen-induced retinopathy (OIR) to an extent similar to valsartan (10 mg/kg per day, IP). In contrast to its effects on vasculature, HRP compromised the electroretinogram in shams and OIR and increased phosphorylated extracellular-signal–related protein kinase 1/2 immunolabeling in shams but not in OIR, whereas valsartan did not affect the electroretinogram and reduced extracellular-signal–related protein kinase 1/2 immunolabeling in OIR. Retinal (P)RR mRNA levels were increased in OIR; HRP, but not valsartan, increased (P)RR mRNA levels in shams, whereas both HRP and valsartan reduced (P)RR mRNA levels in OIR. A control peptide (VSPMKKLLIR, 0.1 mg/kg per day) did not influence retinal vasculopathy or function. Circulating HRP levels in rats administered 1 mg/kg per day HRP were undetectable (<3 pmol/L). We conclude that HRP had protective effects on the retinal vasculature similar to those of valsartan; however, unlike valsartan, HRP injured neuro-glia, which may involve the (P)RR, although the undetectable circulating HRP level makes a direct effect of HRP on retinal (P)RR function unlikely.

Neovascularization Is Attenuated With Aldosterone Synthase Inhibition in Rats With Retinopathy

Neovascularization is a hallmark feature of retinopathy of prematurity and diabetic retinopathy. Type 1 angiotensin receptor blockade reduces neovascularization in experimental retinopathy of prematurity, known as oxygen-induced retinopathy (OIR). We investigated in OIR whether inhibiting aldosterone with the aldosterone synthase inhibitor FAD286 reduced neovascularization as effectively as angiotensin receptor blockade (valsartan). OIR was induced in neonatal Sprague-Dawley rats, and they were treated with FAD286 (30 mg/kg per day), valsartan (10 mg/kg per day), or FAD286+valsartan. The cellular sources of aldosterone synthase, the mineralocorticoid receptor, and 11&bgr;-hydroxysteroid dehydrogenase 2 were evaluated in retinal cells involved in neovascularization (primary endothelial cells, pericytes, microglia, ganglion cells, and glia). In OIR, FAD286 reduced neovascularization and neovascular tufts by 89% and 67%, respectively, and normalized the increase in vascular endothelial growth factor mRNA (1.74-fold) and protein (4.74-fold) and was as effective as valsartan and FAD286+valsartan. In retina, aldosterone synthase mRNA was reduced with FAD286 but not valsartan. Aldosterone synthase was detected in microglia, ganglion cells, and glia, whereas mineralocorticoid receptor and 11&bgr;-hydroxysteroid dehydrogenase 2 were present in all of the cell types studied. Given the location of aldosterone synthase in microglia and their contribution to retinal inflammation and neovascularization in OIR, the effects of FAD286 on microglial density were studied. The increase in microglial density (ionized calcium binding adaptor protein 1 immunolabeling) in OIR was reduced with all of the treatments. In OIR, FAD286 reduced the increase in mRNA for tumor necrosis factor-&agr;, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and monocyte chemoattractant molecule 1. These findings indicate that aldosterone inhibition may be a potential treatment for retinal neovascularization.

Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils

The in vivo aggregation of proteins into amyloid fibrils suggests that cellular mechanisms that normally prevent or reverse this aggregation have failed. The small heat‐shock molecular chaperone protein αB‐crystallin (αB‐c) inhibits amyloid formation and colocalizes with amyloid plaques; however, the physiological reason for this localization remains unexplored. Here, using apolipoprotein C‐II (apoC‐II) as a model fibril‐forming system, we show that αB‐c binds directly to mature amyloid fibrils (Kd 5.4 ± 0.5 μM). In doing so, αB‐c stabilized the fibrils from dilution‐induced fragmentation, halted elongation of partially formed fibrils, and promoted the dissociation of mature fibrils into soluble monomers. Moreover, in the absence of dilution, the association of αB‐c with apoC‐II fibrils induced a 14‐fold increase in average aggregate size, resulting in large fibrillar tangles reminiscent of protein inclusions. We propose that the binding of αB‐c to fibrils prevents fragmentation and mediates the lateral association of fibrils into large inclusions. We further postulate that transient interactions of apoC‐II with αB‐c induce a fibril‐incompetent monomeric apoC‐II form, preventing oligomerization and promoting fibril dissociation. This work reveals previously unrecognized mechanisms of αB‐c chaperone action in amyloid assembly and fibril dynamics, and provides a rationale for the in vivo colocalization of small heat‐shock proteins with amyloid deposits.—Binger, K. J., Ecroyd, H., Yang, S., Carver, J. A., Howlett, G. J., Griffin, M. D. W. Avoiding the oligomeric state: αB‐crystallin inhibits fragmentation and induces dissociation of apolipoprotein C‐II amyloid fibrils. FASEB J. 27, 1214–1222 (2013). www.fasebj.org

Macrophages in homeostatic immune function

Macrophages are not only involved in inflammatory and anti-infective processes, but also play an important role in maintaining tissue homeostasis. In this review, we summarize recent evidence investigating the role of macrophages in controlling angiogenesis, metabolism as well as salt and water balance. Particularly, we summarize the importance of macrophage tonicity enhancer binding protein (TonEBP, also termed nuclear factor of activated T-cells 5 [NFAT5]) expression in the regulation of salt and water homeostasis. Further understanding of homeostatic macrophage function may lead to new therapeutic approaches to treat ischemia, hypertension and metabolic disorders.