Reprogramming cholesterol metabolism in macrophages and its role in host defense against cholesterol-dependent cytolysins.

Cholesterol is a critical lipid for all mammalian cells, ensuring proper membrane integrity, fluidity, and biochemical function. Accumulating evidence indicates that macrophages rapidly and profoundly reprogram their cholesterol metabolism in response to activation signals to support host defense processes. However, our understanding of the molecular details underlying how and why cholesterol homeostasis is specifically reshaped during immune responses remains less well understood. This review discusses our current knowledge of cellular cholesterol homeostatic machinery and introduces emerging concepts regarding how plasma membrane cholesterol is partitioned into distinct pools. We then discuss how proinflammatory signals can markedly reshape the cholesterol metabolism of macrophages, with a focus on the differences between MyD88-dependent pattern recognition receptors and the interferon signaling pathway. We also discuss recent work investigating the capacity of these proinflammatory signals to selectively reshape plasma membrane cholesterol homeostasis. We examine how these changes in plasma membrane cholesterol metabolism influence sensitivity to a set of microbial pore-forming toxins known as cholesterol-dependent cytolysins that specifically target cholesterol for their effector functions. We also discuss whether lipid metabolic reprogramming can be leveraged for therapy to mitigate tissue damage mediated by cholesterol-dependent cytolysins in necrotizing fasciitis and other related infections. We expect that advancing our understanding of the crosstalk between metabolism and innate immunity will help explain how inflammation underlies metabolic diseases and highlight pathways that could be targeted to normalize metabolic homeostasis in disease states.

Subretinal Deposits in Pre-eclampsia and Malignant Hypertension: Implications for Age-Related Macular Degeneration.

PURPOSE: To describe the incidence of subretinal deposits that are similar in structure and stage on OCT imaging to subretinal drusenoid deposits (SDDs) in age-related macular degeneration (AMD) in patients with hypertensive choroidopathy secondary to severe pre-eclampsia and malignant hypertension (MHT) and the implications of this ischemic choroidopathy for the pathophysiologic characteristics of SDDs in AMD. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: Thirty-three pre-eclampsia patients and 25 MHT patients with serous retinal detachment (SRD) in at least 1 eye were included. METHODS: Serial multimodal images, including enhanced depth imaging spectral-domain OCT of eyes with hypertensive choroidopathy secondary to pre-eclampsia and MHT, were reviewed at 2 time points, the acute phase (within 4 weeks of initial hypertensive insult) and the recovery phase (beyond 4 weeks). MAIN OUTCOME MEASURES: Incidence of SDD-like lesions in patients with hypertensive choroidopathy secondary to pre-eclampsia and MHT. RESULTS: Subretinal drusenoid deposit-like lesions were observed exclusively in eyes with SRD. Serous retinal detachment occurred in 87.87% of eyes of pre-eclampsia patients and in 94% of eyes of MHT patients. Subretinal drusenoid deposit-like lesions occurred in 28.57% of all eyes with SRD, in 32.76% of eyes with SRD from the pre-eclampsia group, and in 23.40% of eyes with SRD from the MHT group. Vascular imaging suggested underlying choroidal ischemia in all patients (12 eyes) in which it was performed. CONCLUSIONS: Choroidal ischemia may be the underlying mechanism of SDD-like lesions in patients with pre-eclampsia and MHT choroidopathy. These findings potentially are of utmost importance in understanding the mechanism of the reticular macular disease subtype of AMD. Reticular macular disease is characterized by the known association of choroidal insufficiency and SDD, with choroidal insufficiency postulated, but not proven, to be causative. Pre-eclampsia and MHT choroidopathy seems to be a model for lesions similar to SDD in AMD developing based on choroidal insufficiency and, as such, may offer further insights into the pathoetiologic features of SDD in AMD.

ASSOCIATION OF TREATMENT RESPONSE WITH QUANTITATIVE CHANGES IN CHOROIDAL NEOVASCULARIZATION AND CHOROIDAL VESSEL IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To evaluate the association between treatment response and quantitative morphological changes in choroidal neovascularization and outer choroidal vessels using optical coherence tomography angiography (OCTA) and en face OCT in neovascular age-related macular degeneration (nAMD). METHODS: We retrospectively analyzed 75 eyes of typical nAMD patients and 53 polypoidal choroidal vasculopathy eyes of 124 patients with OCTA performed at least 6 months after initial antivascular endothelial growth factor treatment. Quantitative parameters, including vessel area, vessel diameter, branch vessel length, fractal dimension, and lacunarity were analyzed based on en face images of the choroidal neovascularization and choroidal vessel in Haller's layer. Parameters associated with loss of logarithm of the minimum angle of resolution visual acuity with the basis of 0.3 and the treatment interval (good vs. poor responder based on 12 weeks) were analyzed. Analyses were conducted for "before OCTA" (initial visit to OCTA) and "after OCTA" (OCTA to 6 months post-OCTA). RESULTS: In typical nAMD, visual acuity loss before OCTA was associated with a higher SD of choroidal neovascularization diameter and lower choroidal fractal dimension. Visual acuity loss after OCTA in typical nAMD was associated with higher lacunarity of the choroid. Poor responders before OCTA were not associated with any factor. Poor responders after OCTA were associated with a lower SD of outer choroidal vessel diameter in typical nAMD. In polypoidal choroidal vasculopathy, no factor was associated with clinical outcomes in either period. CONCLUSION: Quantitative analyses of choroidal neovascularization on OCTA and choroidal vessels on en face OCT provide information about treatment response, including changes in visual acuity and treatment interval, in nAMD.

Serous retinal detachment in preeclampsia and malignant hypertension.

OBJECTIVES: To compare and evaluate the characteristics of hypertensive choroidopathy with serous retinal detachment in preeclampsia and malignant hypertension (HTN) and explore choroidal ischemia as a pathogenesis using multimodal imaging. METHODS: A retrospective multicenter case series. Medical charts were reviewed. Clinical characteristics and multimodal imaging, including optical coherence tomography (OCT) and OCT angiography (OCTA), were evaluated. RESULTS: Fifty-three eyes of 29 preeclampsia patients and 45 eyes of 24 HTN patients were included. There were no differences in age, follow-up duration, baseline visual acuity, central macular thickness (CMT), or subfoveal choroidal thickness (CT) between the two groups. Blood pressure parameters, including systolic blood pressure, diastolic blood pressure, and pulse rate, were significantly higher in the HTN group. After serous retinal detachment resolved, both CMT (p < 0.001) and CT (p = 0.003) decreased more in the preeclampsia group. Hypertensive retinopathy features, including hemorrhage, exudates, cotton-wool spots, and optic disc edema, were predominantly found in the HTN group (p = 0.001). Final visual acuity was better in the preeclampsia group than in the HTN group (p = 0.048). Poor visual prognostic factors included the presence of retinopathy features (p = 0.005) and retinal detachment in the macula (p = 0.017). CONCLUSION: Choroidal circulation may be affected earlier than retinal circulation by elevated blood pressure, presumably because of anatomical differences and autoregulatory mechanisms in the retinal vasculature. Serous retinal detachment with hypertensive choroidopathy presented with choroidal thickening that decreased after resolution, but the residual flow defects observed in the choriocapillaris on OCTA confirmed the long-hypothesized notion that ischemia is a mechanism underlying hypertensive choroidopathy.

Changes in Stromal and Luminal Areas of the Choroid in Pachychoroid Diseases: Insights Into the Pathophysiology of Pachychoroid Diseases.

Purpose: To evaluate and compare changes in choroidal vascular and stromal areas in patients with three major pachychoroid diseases for better insight into the pathophysiology of these diseases. Methods: Eighty-six eyes of 86 patients (50 men and 36 women; mean age, 49.1 years) were evaluated, including 21 patients with chronic central serous chorioretinopathy (CSC), 14 with pachychoroid pigment epitheliopathy (PPE), 19 with pachychoroid neovasculopathy (PNV), 14 with myopic choroidal neovascularization (mCNV), and 18 controls. Multimodal retinal imaging, including enhanced-depth imaging optical coherence tomography (EDI-OCT), was performed. Each EDI-OCT image was binarized with ImageJ software, and luminal (dark pixels) and stromal (light pixels) areas were calculated (3000 µm wide in the subfoveal choroid centered on the fovea). Results: The subfoveal choroidal thickness (SFCT) was greater in the three pachychoroid groups than in the control group (430.01 vs. 282.61 µm, P < 0.001). There was no significant difference in SFCT among the three pachychoroid groups. The luminal-to-total choroidal ratio (L/C) was highest (ANOVA, P = 0.001) and the stromal-to-total choroidal ratio (S/C) lowest in the CSC group (ANOVA, P = 0.001). Interestingly, stromal area changes were not correlated with SFCT in the CSC and PNV groups, in contrast to the good correlation between luminal area changes and SFCT in these groups. Conclusions: The eyes of CSC patients had significantly smaller choroidal stromal areas than those of controls or of PPE, PNV, or mCNV patients. The differences in choroidal stromal area, L/C, and S/C in different pachychoroid diseases may reflect different predominant pathogenic processes.

QUANTIFICATION OF RETINAL VESSEL TORTUOSITY IN DIABETIC RETINOPATHY USING OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To investigate the association of vessel tortuosity with severity of diabetic retinopathy (DR) using optical coherence tomography angiography. METHODS: We retrospectively analyzed 30 healthy eyes and 121 eyes of diabetic subjects with no DR, mild nonproliferative DR (NPDR), moderate to severe NPDR and proliferative DR (PDR). Binarized images were used to quantify the vessel tortuosity, vessel density, foveal avascular zone (FAZ) area, and FAZ acircularity. The vessels were divided vertically as superficial retinal layer and deep retinal layer, and horizontally as circular areas with 3 mm and 1.5 mm diameters. Analysis of variance was performed for multiple comparisons. Correlation analysis evaluated the association between the quantified parameters. RESULTS: Compared with healthy eyes, vessel tortuosity increased as DR severity was more in NPDR, but decreased in PDR (P = 0.033). The decrease in vessel density and the increase in both FAZ area and FAZ acircularity were consistent, while DR approached PDR. Among all parameters, statistically significant difference between no DR and mild NPDR was observed only in vessel tortuosity, especially within the 1.5 mm area of superficial retinal layer (P = 0.011). Correlations of vessel tortuosity with FAZ area and acircularity were confined to the 3 mm and 1.5 mm areas of superficial retinal layer (r = -0.185, P = 0.023 for FAZ area; r = 0.268, P = 0.001 for FAZ acircularity), while vessel density strongly correlated with FAZ parameters in the superficial retinal layer and deep retinal layer. CONCLUSION: Vessel tortuosity increased as the stage of NPDR was more severe, but decreased in PDR. The vessel tortuosity determined using optical coherence tomography angiography might be a useful parameter indicating the progression to PDR, circumventing the risk from invasive conventional angiography.

Structure of rhamnose-binding lectin CSL3: unique pseudo-tetrameric architecture of a pattern recognition protein.

The crystal structure of the L-rhamnose-binding lectin CSL3 was determined to 1.8 A resolution. This protein is a component of the germline-encoded pattern recognition proteins in innate immunity. CSL3 is a homodimer of two 20 kDa subunits with a dumbbell-like shape overall, in which the N- and C-terminal domains of different subunits form lobe structures connected with flexible linker peptides. The complex structures of the protein with specific carbohydrates demonstrated the importance of the most variable loop region among homologues for the specificity toward oligosaccharides. CSL3 and Shiga-like toxin both use Gb(3) as a cellular receptor to evoke apoptosis. They have very different overall architecture but share the separation distance between carbohydrate-binding sites. An inspection of the structure database suggested that the pseudo-tetrameric structure of CSL3 was unique among the known lectins. This architecture implies this protein might provide a unique tool for further investigations into the relationships between architecture and function of pattern recognition proteins.

Chemotactic migration of human mesenchymal stem cells and MC3T3-E1 osteoblast-like cells induced by COS-7 cell line expressing rhBMP-7.

During bone development, remodeling, and repair, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells (MPCs) that enter into the osteoblastic lineage, and enhance the recruitment of MPCs and osteogenic cells. The process of migration is believed to be regulated, in part, by growth factors stored within the bone matrix, which are released by bone resorption. In this study, primary human mesenchymal stem cells (hMSCs) and MC3T3-E1 osteoblasts were examined for chemotaxis in response to recombinant human BMP-7 (rhBMP-7) produced in COS-7 cells (co-culture system). In order to produce BMP-7 transfected cells (BTCs), which serve as suppliers of rhBMP-7 under in vitro culture conditions, the encoding DNA was transferred into the pTARGET expression vector and introduced into COS-7 cells by conventional genetic engineering techniques. In cell culture studies, the rhBMP-7 produced in BTCs stimulated the specific activity of ALP, the production of cAMP in response to PTH, and the synthesis of osteocalcin. Migration assays were conducted with a computer-aided time-lapse video-microscopy system, to allow the rapid and precise analysis of cell migration and for the dynamic measurement of cell position and morphology. The migration distance and speed of the MC3T3-E1 cells, or hMSCs, co-cultured with BTCs, using a band-type seeding method, were significantly increased (p < 0.001), compared to those of the MC3T3-E1 cells (or hMSCs) only. In conclusion, these studies revealed that rhBMP-7 plays a role in the migration of bone-forming cells, and that the co-culture model (co-culture of bone-forming cells with BMP-7-producing cells) using a computer-aided, time-lapse video-microscopy system, is useful for the chemotactic migration assay of other chemotactic growth factors.

In vitro bioassay of endotoxin using fluorescein as a pH indicator in a macrophage cell culture system.

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH-indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS-detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.