Objective: To analyze the relationship of aqueous interleukin-6 (IL-6) with diabetic retinopathy (DR) severity. Design: Prospective, controlled trial at a tertiary academic medical center. Subjects: Three hundred twenty-eight eyes of 164 adult type II diabetic patients with varying levels of DR. Methods: A total of 328 eyes of 164 diabetic patients were enrolled based on DR severity: 46 eyes of 23 patients in the no DR group, 236 eyes of 118 patients in the moderate nonproliferative DR (NPDR) group, and 46 eyes of 23 patients in the proliferative DR (PDR) group. ETDRS visual acuity, spectral-domain OCT, and color fundus photographs were taken at baseline. Blood draw and aqueous sampling of each eye was performed. Blood glucose and hemoglobin A1c (HbA1c) were measured. Main Outcomes Measures: Aqueous IL-6 was measured using a microparticle bead-based multiplex assay. Results: Median HbA1c differed between the 3 DR groups (P = 0.03), but there was no correlation between IL-6 and HbA1c (rho = 0.08, P = 0.179). Baseline aqueous IL-6 levels were significantly different between DR groups. Median IL-6 and interquartile range was 5.40 pg/mL (2.99-8.77) for eyes in the no DR group, 9.25 pg/mL (5.35-22.35) for eyes in the moderate NPDR group, and 15.71 pg/mL (9.24-48.58) for eyes in the PDR group (P < 0.001). Median central subfield thickness (CST) did not differ significantly between the 3 groups (P = 0.351), but there was a significant positive correlation between IL-6 and CST (rho = 0.18, P = 0.001). There was also a significant positive correlation between IL-6 and macular volume (rho = 0.12, P = 0.031). Increased IL-6 was significantly associated with increased odds of having DME (odds ratio = 1.00, P = 0.015). Conclusions: We report the largest analysis of aqueous IL-6 in diabetic eyes. We observed that IL-6 is significantly associated with DR severity and DME. Our results considerably strengthen the prevailing evidence that IL-6 is a key contributor to the pathogenesis of DR and may represent both a biomarker of disease activity and a novel therapeutic target.

PURPOSE:Inflammatory cytokines are key mediators of diabetic macular edema (DME). Prior assessments are limited by study design and small sample size. We rigorously analyzed the association of 24 inflammatory cytokines with DME in a prospective trial of 328 eyes of 164 adult type II diabetic patients with different stages of diabetic retinopathy severity. METHODS:Early Treatment Diabetic Retinopathy Study visual acuity, fundus photography, and optical coherence tomography (OCT) of each eye were taken at baseline. Bilateral aqueous sampling was performed. A microparticle bead-based multiplex assay was used to measure aqueous levels of 24 inflammatory cytokines. Diabetic macular edema was defined as central subfield thickness >320 µ m on OCT with the presence of intraretinal fluid. RESULTS:Median and interquartile range of vascular endothelial growth factor A, IL-6, and IL-8 were 148.70 pg/mL (103.93-232.96), 7.67 pg/mL (4.64-18.90), and 11.14 pg/mL (6.48-17.46) in eyes without DME, respectively, and 181.32 pg/mL (141.82-307.81), 16.53 pg/mL (8.15-48.68), and 15.95 (10.67-26.53) in eyes with DME, respectively. Positive odds ratios with 95% confidence intervals were observed for IL-8 (2.73 [1.41-5.29]; P = 0.003), FMS-like tyrosine kinase 3 ligand (FLT-3L) (2.27 [1.22-4.23]; P = 0.01), FGF-2 (1.74 [1.06-2.86]; P = 0.029), IL-6 (2.93 [1.62-5.31]; P < 0.001), and IL-10 (2.79 [1.50-5.21]; P = 0.001). CONCLUSION:We report that IL-8, FLT-3L, FGF-2, IL-6, and IL-10 were significantly elevated in eyes with DME.

The authors have no proprietary or commercial interest in any materials discussed in this article.

Objective: A biomarker to classify diabetic retinopathy (DR) severity and guide preventative treatment is a major unmet need. We analyzed the relationship of aqueous VEGF-A with DR severity to determine if VEGF-A is a candidate biomarker. Design: A prospective, controlled trial at a tertiary academic medical center. Subjects: One hundred seven eyes of 107 patients without diabetes and 328 eyes of 164 adult type II diabetic patients with varying levels of DR. Methods: Aqueous was sampled in 107 consecutive eyes without diabetes undergoing elective vitrectomy surgery. All diabetic eyes had aqueous sampling of both eyes, ETDRS visual acuity, spectral-domain OCT, and color fundus photograph testing. Blood glucose and hemoglobin A1c (HbA1c) were measured. Aqueous VEGF-A was measured using a microparticle bead-based multiplex assay. Main Outcome Measures: Aqueous VEGF-A, DR severity, and diabetic macular edema (DME). Results: Median and interquartile range (IQR) aqueous VEGF-A levels for the 107 eyes without diabetes was 55.74 pg/ml (23.56–76.72). Median (IQR) VEGF-A levels increased with DR severity: 100.34 pg/ml (72.31–126.07) in the no DR group, 162.69 pg/ml (113.76–233.89) in the moderate nonproliferative DR (NPDR) group, and 295.48 pg/ml (193.80–409.99) in the proliferative DR (PDR) group (P < 0.001). The moderate NPDR group was further subdivided based on ETDRS severity into mild-moderate NPDR (N = 154 eyes) and moderate-severe NPDR (N = 82 eyes). Median (IQR) VEGF-A levels were 147.24 pg/ml (108.15–187.50) and 206.75 pg/ml (136.18–303.25) in the mild-moderate and moderate-severe NPDR groups, respectively. Using the no DR group as reference, odds ratios with 95% confidence intervals for VEGF-A were 2.72 (1.3–5.7) in the mild-moderate NPDR (P = 0.008), 6.79 (2.75–16.73) in the moderate-severe NPDR (P < 0.001), and 15.02 (4.86–46.44) in the PDR (P < 0.001) groups, respectively. After controlling for gender, age, and HbA1c, increasing VEGF-A was significantly correlated with increasing DR severity (P < 0.001), but not with the presence of DME (P = 0.39). Conclusions: Aqueous VEGF-A levels tightly correlate with DR severity and support VEGF-A as a novel biomarker to classify DR and guide early intervention. Use of VEGF-A levels may guide preventative treatment, reduce the risk of progression and vision loss, and allow optimal allocation of health care resources. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Objective:We analyzed the cross-sectional associations of 24 inflammatory cytokines with diabetic retinopathy (DR) severity. Design:Prospective, clinical trial at a tertiary academic medical center. Subjects:Three hundred twenty-eight eyes of 164 patients with diabetes with varying severity of DR, including none. Methods:All diabetic eyes had aqueous sampling of both eyes, ETDRS visual acuity, and color fundus photographs. Three groups were enrolled according to grading of baseline color fundus photographs: 23 (46 eyes) patients with diabetes with no DR, 118 (236 eyes) patients with diabetes with moderate nonproliferative DR (NPDR), and 23 (46 eyes) patients with diabetes with proliferative DR. The moderate NPDR group was further subdivided into mild-moderate and moderate-severe groups based on the ETDRS severity scale. Blood was drawn to measure hemoglobin A1c. A microparticle bead-based multiplex assay was used to measure: fibroblast growth factor-2, eotaxin, granulocyte colony-stimulating factor, FMS-like tyrosine kinase 3, GRO, interleukin (IL)-10, monocyte chemotactic protein (MCP)-3, macrophage-derived chemokine, soluble CD40L, IL-17A, IL-1 receptor antagonist, IL-1β, IL-2, IL-4, IL-6, IL-8, induced protein 10, MCP-1, macrophage inflammatory protein-1β, tumor necrosis factor-α, VEGF-A, regulated on activation normal T expressed and secreted, and platelet-derived growth factor-AA and -AB/BB. Triplicate testing of all cytokines was performed. Main Outcome Measures:Aqueous cytokines, DR severity, hemoglobin A1c. Results:Median and interquartile ranges of VEGF-A by grade of eye were 100.57 (80.93-145.67), 153.40 (112.86-206.24), 223.45 (135.27-319.21), and 295.60 (177.46-388.89) pg/mL among no DR, mild-moderate NPDR, moderate-severe NPDR, and proliferative DR groups, respectively. Median and interquartile ranges of IL-6 were 5.45 (3.16-7.86), 8.28 (4.78-20.68), 12.80 (8.24-27.49), and 17.14 (10.31-52.61) pg/mL and of IL-8 were 7.06 (4.10-13.08), 10.53 (6.45-15.85), 16.25 (11.61-24.10), and 20.61 (14.00-27.65) pg/mL among no DR, mild-moderate NPDR, moderate-severe NPDR, and proliferative DR groups, respectively. Similar positive linear relationships were seen with IL-4 and MCP-1. Compared with the no DR group, significant progressive odds ratios were observed among all DR severity groups for VEGF-A, IL-6, IL-8, and IL-4. Significant progressive odds ratios from mild-moderate NPDR to moderate-severe NPDR were observed for FMS-like tyrosine kinase 3, IL-10, induced protein 10, MCP-1, macrophage-derived chemokine, and platelet-derived growth factor-AA. The majority of cytokines demonstrated no relationship with hemoglobin A1c. Conclusions:We report that several key inflammatory cytokines demonstrate cross-sectional associations with DR severity. Our results lend support to the presumption that inflammatory cytokines are important biomarkers that associate with DR severity and may represent novel targets for inhibition. Financial Disclosures:The authors have no proprietary or commercial interest in any materials discussed in this article.

BACKGROUND/AIMS:To assess 24 pro-inflammatory cytokines in the aqueous and serum of normal patients without diabetes. METHODS:94 consecutive eyes of 94 patients undergoing elective vitrectomy surgery were prospectively enrolled. Blood and aqueous sampling were done in the operating room. A microparticle bead-based multiplex assay was used to measure aqueous and serum levels of: fibroblast growth factor-2, eotaxin, granulocyte colony stimulating factor (G-CSF), FMS-like tyrosine kinase 3 ligand (FLT-3L), growth-regulated alpha protein (GROa), interleukin (IL)-10, monocyte chemotactic protein (MCP)-3, macrophage-derived chemokine (MDC), soluble CD40L (sCD40L), IL-17A, interleukin-1 receptor antagonist (IL-1RA), IL-1β, IL-2, IL-4, IL-6, IL-8, interferon gamma-induced protein 10 (IP-10), MCP-1, macrophage inflammatory protein-1β, tumour necrosis factor-α, vascular endothelial growth factor (VEGF), regulated on activation normal T expressed and secreted (RANTES), platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB. RESULTS:A significant positive correlation between aqueous and serum levels was observed for FLT-3L (r=0.45; p<0.001) and positive trends were observed for eotaxin (r=0.27; p=0.04), MDC (r=0.34; p<0.01) and RANTES (r=0.33; p=0.007). There were no significant differences observed based on age or gender. In contrast, aqueous cytokine levels for G-CSF, GROa and MCP-1 were significantly higher in pseudophakic eyes compared with phakic eyes: G-CSF (16.52 pg/mL±22.39 vs 2.77 pg/mL±6.80, p<0.0001), GROa (12.45 pg/mL±6.54 vs 8.26 pg/mL±5.48, p=0.001) and MCP-1 (1340.51 pg/mL±565.21 vs 830.44 pg/mL±454.36, p<0.0001), respectively. CONCLUSIONS:We report the first characterisation of 24 different pro-inflammatory cytokines in 94 patients without diabetes. The absence of the crystalline lens was associated with significantly higher aqueous levels of G-CSF, GROa and MCP-1. TRIAL REGISTRATION NUMBER:NCT04505566.

OBJECTIVE:To analyze the relationship of aqueous interleukin-6 (IL-6) with diabetic retinopathy (DR) severity. DESIGN:Prospective, controlled trial at a tertiary academic medical center. SUBJECTS:Three hundred twenty-eight eyes of 164 adult type II diabetic patients with varying levels of DR. METHODS:A total of 328 eyes of 164 diabetic patients were enrolled based on DR severity: 46 eyes of 23 patients in the no DR group, 236 eyes of 118 patients in the moderate nonproliferative DR (NPDR) group, and 46 eyes of 23 patients in the proliferative DR (PDR) group. ETDRS visual acuity, spectral-domain OCT, and color fundus photographs were taken at baseline. Blood draw and aqueous sampling of each eye was performed. Blood glucose and hemoglobin A1c (HbA1c) were measured. Aqueous IL-6 was measured using a microparticle bead-based multiplex assay. MAIN OUTCOMES MEASURES:Aqueous IL-6 levels, HbA1c, DR severity, Diabetic macular edema (DME). RESULTS:Median HbA1c differed between the 3 DR groups (P = 0.03), but there was no correlation between IL-6 and HbA1c (ρ = 0.08, P = 0.179). Baseline aqueous IL-6 levels were significantly different between DR groups. Median IL-6 and interquartile range was 5.40 pg/mL (2.99-8.77) for eyes in the no DR group, 9.25 pg/mL (5.35-22.35) for eyes in the moderate NPDR group, and 15.71 pg/mL (9.24-48.58) for eyes in the PDR group (P < 0.001). Median central subfield thickness (CST) did not differ significantly between the 3 groups (P = 0.351), but there was a significant positive correlation between IL-6 and CST (ρ = 0.18, P = 0.001). There was also a significant positive correlation between IL-6 and macular volume (ρ = 0.12, P = 0.031). Increased IL-6 was significantly associated with increased odds of having DME (odds ratio = 1.00, P = 0.015). CONCLUSIONS:We report the largest analysis of aqueous IL-6 in diabetic eyes. We observed that IL-6 is significantly associated with DR severity and DME. Our results considerably strengthen the prevailing evidence that IL-6 is a key contributor to the pathogenesis of DR and may represent both a biomarker of disease activity and a novel therapeutic target. FINANCIAL DISCLOSURE(S):Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

The power of CRISPRi to decrease targeted gene expression for clinical applications has been inhibited by delivery challenges. Existing constructs are too large to fit within the ∼4.7 kb packaging size limitation of adeno-associated virus (AAV), the only FDA approved viral vector for clinical use. Therefore, we optimized CRISPRi components to generate a single AAV vector that contains all functional elements and effectively knocks down expression of an endogenous gene in vivo. First, we increased nuclear targeting of Staphylococcus aureus deactivated Cas9 (SadCas9) 4-fold by using a helical linker and the c-Myc nuclear localization signal. Second, we identified an amino-terminal Krüppel associated box (KRAB) construct as the most effective in decreasing expression of target genes in vitro. Third, we optimized promoters for guide RNA and evaluated mini-promoters for expression of KRAB-SadCas9 in liver cells. Our final construct decreased protein convertase subtilisin/kexin type 9 (Pcsk9) mRNA and secreted protein 5-fold in vitro. The corresponding AAV2/8 vector was localized in nuclei of liver cells and decreased Pcsk9 mRNA and serum protein levels by 30% in vivo. This single AAV approach provides a potential clinically translatable method for decreasing targeted gene transcription by CRISPRi in vivo.

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings.

Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.