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Abstract Cellular adaptation to low oxygen tension triggers primitive pathways that ensure proper cell function. Conditions of hypoxia and low glucose are characteristic of injured tissues and hence successive waves of inflammatory cells must be suited to function under low oxygen tension and metabolic stress. While Hypoxia-Inducible Factor (HIF)-1α has been shown to be essential for the inflammatory response of myeloid cells by regulating the metabolic switch to glycolysis, less is known about how HIF1α is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1α (IRE1α/XBP1) axis in order to initiate HIF1α-dependent production of cytokines such as IL1β, IL6 and VEGF-A. Knockout of either HIF1α or IRE1α in myeloid cells ameliorates vascular phenotypes in a model of retinal pathological angiogenesis driven by sterile inflammation. Thus, pathways associated with ER stress, in partnership with HIF1α, may co-regulate immune adaptation to low oxygen.

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia and dyslipidemia, leads to nonproliferative diabetic retinopathy (NPDR). NPDR is associated with blood-retina barrier disruption, plasma exudates, microvascular degeneration, elevated inflammatory cytokine levels, and monocyte (Mo) infiltration. Whether and how the diabetes-associated changes in plasma lipid and carbohydrate levels modify Mo differentiation remains unknown. Here, we show that mononuclear phagocytes (MPs) in areas of vascular leakage in DR donor retinas expressed perilipin 2 (PLIN2), a marker of intracellular lipid load. Strong upregulation of PLIN2 was also observed when healthy donor Mos were treated with plasma from patients with T2DM or with palmitate concentrations typical of those found in T2DM plasma, but not under high-glucose conditions. PLIN2 expression correlated with the expression of other key genes involved in lipid metabolism (ACADVL, PDK4) and the DR biomarkers ANGPTL4 and CXCL8. Mechanistically, we show that lipid-exposed MPs induced capillary degeneration in ex vivo explants that was inhibited by pharmaceutical inhibition of PPARγ signaling. Our study reveals a mechanism linking dyslipidemia-induced MP polarization to the increased inflammatory cytokine levels and microvascular degeneration that characterize NPDR. This study provides comprehensive insights into the glycemia-independent activation of Mos in T2DM and identifies MP PPARγ as a target for inhibition of lipid-activated MPs in DR.

Summary: When researchers submit a protocol for peer review and publication, they receive feedback from reviewers to help improve the usability of the protocol. These authors can be the perfect peer reviewers helping propel research forward. They can use their technical expertise and sharpened writing skills to help improve the main aspects of published protocols, namely their clarity and reproducibility. This backstory chronicles the journey of Dr. Guillaume Blot, from a junior researcher and author to a protocol reviewer. For complete details, please refer to Blot et al. (2021).

Abstract Background Rhegmatogenous retinal detachment (RD) involving the macula is a major cause of visual impairment despite high surgical success rate, mainly because of cone death. RD causes the infiltration of activated immune cells, but it is not clear whether and how infiltrating inflammatory cells contribute to cone cell loss. Methods Vitreous samples from patients with RD and from control patients with macular hole were analyzed to characterize the inflammatory response to RD. A mouse model of RD and retinal explants culture were then used to explore the mechanisms leading to cone death. Results Analysis of vitreous samples confirms that RD induces a marked inflammatory response with increased cytokine and chemokine expression in humans, which is closely mimicked by experimental murine RD. In this model, we corroborate that myeloid cells and T-lymphocytes contribute to cone loss, as the inhibition of their accumulation by Thrombospondin 1 (TSP1) increased cone survival. Using monocyte/retinal co-cultures and TSP1 treatment in RD, we demonstrate that immune cell infiltration downregulates rod-derived cone viability factor (RdCVF), which physiologically regulates glucose uptake in cones. Insulin and the insulin sensitizers rosiglitazone and metformin prevent in part the RD-induced cone loss in vivo, despite the persistence of inflammation Conclusion Our results describe a new mechanism by which inflammation induces cone death in RD, likely through cone starvation due to the downregulation of RdCVF that could be reversed by insulin. Therapeutic inhibition of inflammation and stimulation of glucose availability in cones by insulin signaling might prevent RD-associated cone death until the RD can be surgically repaired and improve visual outcome after RD. Trial registration ClinicalTrials.gov NCT03318588

Abstract Background Age-related macular degeneration is characterized by the accumulation of subretinal macrophages and the degeneration of cones, but mainly of rods. We have previously shown that Mononuclear Phagocytes-derived IL-1β induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1β but the mechanism is unknown. Methods Retinal explants were culture in the presence of human monocytes or IL-1β and photoreceptor cell survival was analyzed by TUNEL labeling. Glutamate concentration and transcription levels of gene involved in the homeostasis of glutamate were analyzed in cell fractions of explant cultured or not in the presence of IL-1β. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-β or monocytes. Results We here show that IL-1β does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1β leads to glutamate-induced rod photoreceptor cell death as it increases the extracellular glutamate concentrations in the retina through the inhibition of its conversion to glutamine in Müller cells, increased release from Müller cells, and diminished reuptake. The inhibition of non-NMDA receptors completely and efficiently prevented rod apoptosis in retinal explants cultured in the presence of IL-1β or, more importantly, in vivo, in a model of subretinal inflammation. Conclusions Our study emphasizes the importance of inflammation in the deregulation of glutamate homeostasis and provides a comprehensive mechanism of action for IL-1β-induced rod degeneration.

Summary: The classical aortic ring model is well suited for deciphering pro-angiogenic processes. Here, we propose simple modifications of the standard protocol to study various anti-angiogenic processes from growth arrest to capillary degeneration. Aortic rings are cultured under basal conditions for 6 days to allow physiological vessel sprouting and then split into treatment groups to follow capillary growth or degeneration for an additional 2 days.

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon beta (IFN-beta) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-beta gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-beta promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-beta promoter chromatin structure.