The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment.

The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

A simplified protocol to induce hypoxia in a standard incubator: A focus on retinal cells.

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl2), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl2 concentrations used typically range from 100 to 600 µM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl2. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.

Endoplasmic Reticulum Stress, Oxidative Stress, and Rheumatic Diseases.

BACKGROUND: The endoplasmic reticulum (ER) is a multi-functional organelle responsible for cellular homeostasis, protein synthesis, folding and secretion. It has been increasingly recognized that the loss of ER homeostasis plays a central role in the development of autoimmune inflammatory disorders, such as rheumatic diseases. Purpose/Main contents: Here, we review current knowledge of the contribution of ER stress to the pathogenesis of rheumatic diseases, with a focus on rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We also review the interplay between protein folding and formation of reactive oxygen species (ROS), where ER stress induces oxidative stress (OS), which further aggravates the accumulation of misfolded proteins and oxidation, in a vicious cycle. Intervention studies targeting ER stress and oxidative stress in the context of rheumatic diseases are also reviewed. CONCLUSIONS: Loss of ER homeostasis is a significant factor in the pathogeneses of RA and SLE. Targeting ER stress, unfolded protein response (UPR) pathways and oxidative stress in these diseases both in vitro and in animal models have shown promising results and deserve further investigation.

The lupus susceptibility allele DRB1*03:01 encodes a disease-driving epitope.

The HLA-DRB1*03:01 allele is a major genetic risk factor in systemic lupus erythematosus (SLE), but the mechanistic basis of the association is unclear. Here we show that in the presence of interferon gamma (IFN-γ), a short DRB1*03:01-encoded allelic epitope activates a characteristic lupus transcriptome in mouse and human macrophages. It also triggers a cascade of SLE-associated cellular aberrations, including endoplasmic reticulum stress, unfolded protein response, mitochondrial dysfunction, necroptotic cell death, and production of pro-inflammatory cytokines. Parenteral administration of IFN-γ to naïve DRB1*03:01 transgenic mice causes increased serum levels of anti-double stranded DNA antibodies, glomerular immune complex deposition and histopathological renal changes that resemble human lupus nephritis. This study provides evidence for a noncanonical, antigen presentation-independent mechanism of HLA-disease association in SLE and could lay new foundations for our understanding of key molecular mechanisms that trigger and propagate this devastating autoimmune disease.

The role of probiotics on each component of the metabolic syndrome and other cardiovascular risks.

INTRODUCTION: Probiotics are defined as live microorganisms that when administered in adequate amounts confer health benefits to the host. The consumption of probiotics has gained increasing recognition from the scientific community due to the promising effects on metabolic health through gut microbiota modulation. AREAS COVERED: This article presents a review of scientific studies investigating probiotic species and their effects on different risk factors of the metabolic syndrome (MetS). This article also presents a summary of the major mechanisms involved with gut microbiota and the components of the MetS and raises the key issues to be considered by scientists in search of probiotics species for treatment of patients suffering from this metabolic disorder. EXPERT OPINION: Probiotics may confer numerous health benefits to the host through positive gut microbiota modulation. The strain selection is the most important factor for determining health effects. Further studies may consider gut microbiota as a novel target for prevention and management of MetS components and other cardiovascular risks.