Therapeutic targeting of cellular senescence in diabetic macular edema: preclinical and phase 1 trial results.

Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .

Perilipin 2-positive mononuclear phagocytes accumulate in the diabetic retina and promote PPARγ-dependent vasodegeneration.

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.

HIF1α-dependent hypoxia response in myeloid cells requires IRE1α.

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 IL1ß, 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.

Past history of obesity triggers persistent epigenetic changes in innate immunity and exacerbates neuroinflammation.

Age-related macular degeneration is a prevalent neuroinflammatory condition and a major cause of blindness driven by genetic and environmental factors such as obesity. In diseases of aging, modifiable factors can be compounded over the life span. We report that diet-induced obesity earlier in life triggers persistent reprogramming of the innate immune system, lasting long after normalization of metabolic abnormalities. Stearic acid, acting through Toll-like receptor 4 (TLR4), is sufficient to remodel chromatin landscapes and selectively enhance accessibility at binding sites for activator protein-1 (AP-1). Myeloid cells show less oxidative phosphorylation and shift to glycolysis, ultimately leading to proinflammatory cytokine transcription, aggravation of pathological retinal angiogenesis, and neuronal degeneration associated with loss of visual function. Thus, a past history of obesity reprograms mononuclear phagocytes and predisposes to neuroinflammation.

Why STAR Protocols authors make excellent protocol reviewers.

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).

Reproducing diabetic retinopathy features using newly developed human induced-pluripotent stem cell-derived retinal Müller glial cells.

Muller glial cells (MGCs) are responsible for the homeostatic and metabolic support of the retina. Despite the importance of MGCs in retinal disorders, reliable and accessible human cell sources to be used to model MGC-associated diseases are lacking. Although primary human MGCs (pMGCs) can be purified from post-mortem retinal tissues, the donor scarcity limits their use. To overcome this problem, we developed a protocol to generate and bank human induced pluripotent stem cell-derived MGCs (hiMGCs). Using a transcriptome analysis, we showed that the three genetically independent hiMGCs generated were homogeneous and showed phenotypic characteristics and transcriptomic profile of pMGCs. These cells expressed key MGC markers, including Vimentin, CLU, DKK3, SOX9, SOX2, S100A16, ITGB1, and CD44 and could be cultured up to passage 8. Under our culture conditions, hiMGCs and pMGCs expressed low transcript levels of RLPB1, AQP4, KCNJ1, KCJN10, and SLC1A3. Using a disease modeling approach, we showed that hiMGCs could be used to model the features of diabetic retinopathy (DR)-associated dyslipidemia. Indeed, palmitate, a major free fatty acid with elevated plasma levels in diabetic patients, induced the expression of inflammatory cytokines found in the ocular fluid of DR patients such as CXCL8 (IL-8) and ANGPTL4. Moreover, the analysis of palmitate-treated hiMGC secretome showed an upregulation of proangiogenic factors strongly related to DR, including ANG2, Endoglin, IL-1ß, CXCL8, MMP-9, PDGF-AA, and VEGF. Thus, hiMGCs could be an alternative to pMGCs and an extremely valuable tool to help to understand and model glial cell involvement in retinal disorders, including DR.

Modifications to the classical rat aortic ring model to allow vascular degeneration studies.

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.

Insulin inhibits inflammation-induced cone death in retinal detachment.

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.

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.

IL-1ß induces rod degeneration through the disruption of retinal glutamate homeostasis.

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-1ß induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1ß but the mechanism is unknown. METHODS: Retinal explants were culture in the presence of human monocytes or IL-1ß 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-1ß. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-ß or monocytes. RESULTS: We here show that IL-1ß does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1ß 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-1ß 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-1ß-induced rod degeneration.

In vitro, in cellulo and structural characterizations of the interaction between the integrase of Porcine Endogenous Retrovirus A/C and proteins of the BET family.

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.

Quantitative transmission characteristics of different H5 low pathogenic avian influenza viruses in Muscovy ducks.

EU annual serosurveillance programs show that domestic duck flocks have the highest seroprevalence of H5 antibodies, demonstrating the circulation of notifiable avian influenza virus (AIV) according to OIE, likely low pathogenic (LP). Therefore, transmission characteristics of LPAIV within these flocks can help to understand virus circulation and possible risk of propagation. This study aimed at estimating transmission parameters of four H5 LPAIV (three field strains from French poultry and decoy ducks, and one clonal reverse-genetics strain derived from one of the former), using a SIR model to analyze data from experimental infections in SPF Muscovy ducks. The design was set up to accommodate rearing on wood shavings with a low density of 1.6 ducks/m(2): 10 inoculated ducks were housed together with 15 contact-exposed ducks. Infection was monitored by RNA detection on oropharyngeal and cloacal swabs using real-time RT-PCR with a cutoff corresponding to 2-7 EID50. Depending on the strain, the basic reproduction number (R0) varied from 5.5 to 42.7, confirming LPAIV could easily be transmitted to susceptible Muscovy ducks. The lowest R0 estimate was obtained for a H5N3 field strain, due to lower values of transmission rate and duration of infectious period, whereas reverse-genetics derived H5N1 strain had the highest R0. Frequency and intensity of clinical signs were also variable between strains, but apparently not associated with longer infectious periods. Further comparisons of quantitative transmission parameters may help to identify relevant viral genetic markers for early detection of potentially more virulent strains during surveillance of LPAIV.

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

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.

A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells.

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.

RASSF1C, an isoform of the tumor suppressor RASSF1A, promotes the accumulation of beta-catenin by interacting with betaTrCP.

The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with betaTrCP. Binding of RASSF1C to betaTrCP involves serine 18 and serine 19 of the SS(18)GYXS(19) motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by betaTrCP; however, surprisingly, the association between RASSF1C and betaTrCP does not occur via the betaTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the beta-catenin oncogene, due to inhibition of its betaTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for beta-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on beta-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis.

Luman, a new partner of HIV-1 TMgp41, interferes with Tat-mediated transcription of the HIV-1 LTR.

In our search for new partners of the HIV-1 envelope glycoprotein (Env), we found that the cytoplasmic domain of the TMgp41 (TMgp41 CD) subunit of HIV-1 Env interacted with Luman, a transcription factor of the CREB/ATF family. Luman is anchored in the endoplasmic reticulum membrane and subjected to activation by regulated intramembrane proteolysis (RIP). The RIP process permits the release of the activated amino-terminal fragment of Luman into the cytoplasm, and its import into the nucleus. Here, we demonstrate that interaction between the TMgp41 CD and Luman requires a region encompassing the b-Zip and TM domains of Luman and decreases the stability of this factor. Moreover, we found that overexpression of a constitutively active form of Luman in cells transfected with HXB2R HIV-1 provirus decreased the intracellular expression of Gag and Env and led to a decrease in virion release. This negative effect of activated Luman on HIV-1 production was correlated to the inhibition of Tat transactivation of the HIV-1 LTR, which might be related to an interaction of activated Luman with Tat. Altogether, these results show that Luman acts as a partner of two major HIV-1 proteins: the TMgp41 Env subunit and Tat. The interaction between the TMgp41 subunit of Env and Luman affects the stability of the full-length Luman protein, the precursor of the activated, nuclear form of Luman, which acts negatively on Tat-mediated HIV-1 transactivation.

Tail-interacting protein TIP47 is a connector between Gag and Env and is required for Env incorporation into HIV-1 virions.

The presence of the envelope glycoprotein Env in HIV-1 virions is essential for infectivity. To date, the molecular mechanism by which Env is packaged into virions has been largely unknown. Here, we show that TIP47 (tail-interacting protein of 47 kDa), which has been shown to interact with Env, also binds the MA (matrix) domain of HIV-1 Gag protein and that these three proteins form a ternary complex. Mutations in Gag that abrogate interaction with TIP47 inhibit Env incorporation and virion infectivity as well as colocalization between Gag and Env. We also show that TIP47 silencing impairs Env incorporation and infectivity and abolishes coimmunoprecipitation of Gag with Env. In contrast, overexpression of TIP47 increases Env packaging. Last, we demonstrate that TIP47 can interact simultaneously with Env and Gag. Taken together, our results show that TIP47 is a cellular cofactor that plays an essential role in Env incorporation, allowing the encounter and the physical association between HIV-1 Gag and Env proteins during the viral assembly process.

HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse.

Dendritic cells (DCs) are essential components of the early events of HIV infection. Here, we characterized the trafficking pathways that HIV-1 follows during its capture by DCs and its subsequent presentation to CD4(+) T cells via an infectious synapse. Immunofluorescence microscopy indicates that the virus-containing compartment in mature DCs (mDCs) co-labels for the tetraspanins CD81, CD82, and CD9 but contains little CD63 or LAMP-1. Using ratio imaging of pH-reporting fluorescent virions in live DCs, we show that HIV-1 is internalized in an intracellular endocytic compartment with a pH of 6.2. Significantly, we demonstrate that the infectivity of cell-free virus is more stable at mildly acidic pH than at neutral pH. Using electron microscopy, we confirm that HIV-1 accumulates in intracellular vacuoles that contain CD81 positive internal membranes but overlaps only partially with CD63. When allowed to contact T cells, HIV-1-loaded DCs redistribute CD81, and CD9, as well as internalized HIV-1, but not the immunological synapse markers MHC-II and T-cell receptor to the infectious synapse. Together, our results indicate that HIV-1 is internalized into a non-conventional, non-lysosomal, endocytic compartment in mDCs and further suggest that HIV-1 is able to selectively subvert components of the intracellular trafficking machinery required for formation of the DC-T-cell immunological synapse to facilitate its own cell-to-cell transfer and propagation.

Targeting of the human immunodeficiency virus type 1 envelope to the trans-Golgi network through binding to TIP47 is required for env incorporation into virions and infectivity.

Here, we report that human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is located mainly in the trans-Golgi network (TGN) due to determinants present in the cytoplasmic domain of the transmembrane gp41 glycoprotein (TMgp41). Internalization assays demonstrated that Env present at the cell surface returns to the TGN. We found that the cytoplasmic domain of TMgp41 binds to TIP47, a protein required for the transport of mannose-6-phosphate receptors from endosomes to the TGN. Overexpression of a mutant of TIP47 affected the transport of Env from endosomes to the TGN. Retrograde transport of Env to the TGN requires a Y(802)W(803) diaromatic motif present in the TMgp41 cytoplasmic domain. Mutation of this motif abolished both targeting to the TGN as well as interaction with TIP47. These data support the view that binding of TIP47 to HIV-1 Env facilitates its delivery to the TGN. Lastly, we show that virus mutated in the Y(802)W(803) motif is poorly infectious and presents a defect in Env incorporation, supporting a model in which retrograde transport of Env is implicated in the optimization of fully infectious HIV-1 production.