Thinning of the RPE and choroid associated with T lymphocyte recruitment in aged and light-challenged mice

International audienceThe choroidal vasculature is essential when it comes to bringing oxygen and nutrients to the functioning retina and evacuating debris resulting from the normal visual cycle. Choroidal thinning is a common feature in many human eye diseases, including high myopia [1,2] and retinitis pigmentosa [3,4], and has been reproducibly observed with age [5-7]. However, the association between choroidal thinning and age-related macular degeneration (AMD) remains controversial. Some authors have reported the loss of choriocapillaries in eyes with exudative AMD [8], and choroidal thinning has been detected in some studies [9-11]. Choroidal thinning has also been associated with geographic atrophy (GA), the dry form of late AMD [12-15]. A morphometric analysis by Ramrattan et al. more than two decades ago showed a decrease in choriocapillary density and diameter with age and in GA, but choroidal thinning was only significant with age [6]. Moreover, it has been reported that the choriocapillaries and choroid are thinner in areas where the RPE has degenerated [8]. However, all studies agree that aging is associated with significant choroidal thinning [16-18]. The exact mechanisms behind choroidal thinning with age or disease are not clear. The RPE is a monolayer of pigmented cells situated between photoreceptors and Bruch's membrane; its plays an essential role in the visual cycle. RPE65, which is also called 11-cis retinol isomerase and is strongly expressed in the RPE, participates in the production of 11-cis retinal [19], which is essential for photoreceptor function [20]. Mutations in the RPE65 gene cause progressive photoreceptor degeneration [21,22] and adult RPE65 −/

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.: DLL4's opposing effects in choroidal neovascularization

International audienceInflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV

Understanding the dynamics of Toll-like Receptor 5 response to flagellin and its regulation by estradiol

© 2017 The Author(s). Toll-like receptors (TLRs) are major players of the innate immune system. Once activated, they trigger a signalling cascade that leads to NF-ΰ B translocation from the cytoplasm to the nucleus. Single cell analysis shows that NF-ΰ B signalling dynamics are a critical determinant of transcriptional regulation. Moreover, the outcome of innate immune response is also affected by the cross-talk between TLRs and estrogen signalling. Here, we characterized the dynamics of TLR5 signalling, responsible for the recognition of flagellated bacteria, and those changes induced by estradiol in its signalling at the single cell level. TLR5 activation in MCF7 cells induced a single and sustained NF-k B translocation into the nucleus that resulted in high NF-k B transcription activity. The overall magnitude of NF-k B transcription activity was not influenced by the duration of the stimulus. No significant changes are observed in the dynamics of NF-k B translocation to the nucleus when MCF7 cells are incubated with estradiol. However, estradiol significantly decreased NF-k B transcriptional activity while increasing TLR5-mediated AP-1 transcription. The effect of estradiol on transcriptional activity was dependent on the estrogen receptor activated. This fine tuning seems to occur mainly in the nucleus at the transcription level rather than affecting the translocation of the NF-k B transcription factor

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. Methods: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8+/− mice expressing ß-galactosidase. Aged Mfge8+/− and Mfge8−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. Results: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8−/− mice as compared to controls. Conclusions: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD

Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration

International audiencePhysiologically, the retinal pigment epithelium (RPE) expresses immunosuppressive signals such as FAS ligand (FASL), which prevents the accumulation of leukocytes in the subretinal space. Age-related macular degeneration (AMD) is associated with a breakdown of the subretinal immunosuppressive environment and chronic accumulation of mononuclear phagocytes (MPs). We show that subretinal MPs in AMD patients accumulate on the RPE and express high levels of APOE. MPs of Cx3cr1 À/À mice that develop MP accumulation on the RPE, photoreceptor degeneration, and increased choroidal neovascularization similarly express high levels of APOE. ApoE deletion in Cx3cr1 À/À mice prevents patho-genic age-and stress-induced subretinal MP accumulation. We demonstrate that increased APOE levels induce IL-6 in MPs via the activation of the TLR2-CD14-dependent innate immunity receptor cluster. IL-6 in turn represses RPE FasL expression and prolongs subretinal MP survival. This mechanism may account, in part, for the MP accumulation observed in Cx3cr1 À/À mice. Our results underline the inflammatory role of APOE in sterile inflammation in the immunosuppressive subretinal space. They provide rationale for the implication of IL-6 in AMD and open avenues toward therapies inhibiting pathogenic chronic inflammation in late AMD

Complement Factor H and Apolipoprotein E Participate in Regulation of Inflammation in THP-1 Macrophages

The alternative pathway (AP) of complement is constantly active in plasma and can easily be activated on self surfaces and trigger local inflammation. Host cells are protected from AP attack by Factor H (FH), the main AP regulator in plasma. Although complement is known to play a role in atherosclerosis, the mechanisms of its contribution are not fully understood. Since FH via its domains 5-7 binds apoliporotein E (apoE) and macrophages produce apoE we examined how FH could be involved in the antiatherogenic effects of apoE. We used blood peripheral monocytes and THP-1 monocyte/macrophage cells which were also loaded with acetylated low-density lipoprotein (LDL) to form foam cells. Binding of FH and apoE on these cells was analyzed by flow cytometry. High-density lipoprotein (HDL)-mediated cholesterol efflux of activated THP-1 cells was measured and transcriptomes of THP-1 cells using mRNA sequencing were determined. We found that binding of FH to human blood monocytes and cholesterol-loaded THP-1 macrophages increased apoE binding to these cells. Preincubation of fluorescent cholesterol labeled THP-1 macrophages in the presence of FH increased cholesterol efflux and cholesterol-loaded macrophages displayed reduced transcription of proinflammatory/proatherogenic factors and increased transcription of anti-inflammatory/anti-atherogenic factors. Further incubation of THP-1 cells with serum reduced C3b/iC3b deposition. Overall, our data indicate that apoE and FH interact with monocytic cells in a concerted action and this interaction reduces complement activation and inflammation in the atherosclerotic lesions. By this way FH may participate in mediating the beneficial effects of apoE in suppressing atherosclerotic lesion progression.Peer reviewe

Sexual Inequality in Tuberculosis

Olivier Neyrolles and Lluis Quintana-Murci review the evidence on why tuberulosis notification is twice as high in men as in women in most countries

Estradiol Regulates Expression of Estrogen Receptor ERα46 in Human Macrophages

BACKGROUND:Monocytes and macrophages are key innate immune effector cells that produce cytokines and chemokines upon activation. We and others have shown that 17beta-estradiol (E2) has a direct role in the modulation of monocyte and macrophage immune function. However, relatively little is known about the ability of E2 to regulate isoform expression of estrogen receptors (ERs) in these cells. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we quantify expression of ERalpha and ERbeta in human monocytes and macrophages. We also show for the first time that the N-terminal truncated ERalpha variant, ERalpha46, is expressed in both cell types. Promoter utilization studies reveal that transcription of ERalpha in both cell types occurs from upstream promoters E and F. Treatment with E2 induces ERalpha expression in macrophages but has no effect on ERbeta levels in either cell type. During monocyte-to-macrophage differentiation, ERalpha is upregulated in a time-dependent manner. Previous studies by our group demonstrated that E2 treatment attenuates production of the chemokine CXCL8 in an ER-dependent manner. We now show that ERalpha expression levels parallel the ability of E2 to suppress CXCL8 production. CONCLUSIONS/SIGNIFICANCE:This work demonstrates for the first time that human macrophages predominantly express the truncated ER variant ERalphap46, which is estradiol-inducible. This is mediated through usage of the ERalpha F promoter. Alternative promoter usage may account for tissue and cell type-specific differences in estradiol-induced effects on gene expression. These studies signify the importance of ERalpha expression and regulation in the ability of E2 to modulate innate immune responses