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[Retinal transcriptome profile in mice following dexamethasone treatment for endotoxin-induced uveitis].

ABSTRACT: OBJECTIVE:To investigate the changes in retinal transcriptome profile of mice with endotoxin-induced uveitis (EIU)following dexamethasone (DEX) treatment and explore the mechanisms underlying the therapeutic effect of DEX. METHODS:EIU was induced in BALB/c mice by intravitreal injection of 125 ng lipopolysaccharide (LPS), followed by topical applicationof DEX (0.1%) eye drops every 4 h for 24 h. The anterior chamber inflammation was examined with a slit lamp and the clinicalscores were assessed. The morphological changes in the eyes were assessed at 24 h after LPS injection. The retinas wereharvested for analysis of transcriptome profile using the next-generation sequencing (NGS)-based RNA sequencing (RNA-seq), and the expressions of the inflammatory cytokines and the differentially expressed genes (DEGs) were verified using real-timePCR. RESULTS:DEX alleviated the inflammatory response and reduced the mRNA expressions of IL-6, TNF- a, MCP-1 andICAM-1 at 24 h after LPS injection. A total of 52 DEGs were identified by RNA-seq. Within these DEGs, 37 genes were upregulated and 15 genes were down-regulated in LPS group as compared with DEX+LPS group. No significantly enriched GeneOntology (GO) terms was noted. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed 6up-regulated and 2 down-regulated KEGG pathways. RIG-I-like receptor signaling pathway and several immune- andinflammation-related genes including Ifit1, H2-T24, Mx2 and Eif2ak2 were significantly down regulated by DEX. Verificationwith RT-PCR yielded results consistent with these findings. CONCLUSIONS:DEX alleviates LPS-induced inflammatory response inthe retina of mice, and such protective effect is probably mediated by RIG-I like receptor signal pathway and the immune-andinflammation-related genes.

SUBMITTER: Peng YU

PROVIDER: S-EPMC6744048 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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[Retinal transcriptome profile in mice following dexamethasone treatment for endotoxin-induced uveitis].

Peng Y U YU Yiguo Qiu Q Ru Lin L Xinyu F U FU Bingtao Hao H Bo Lei L

Nan fang yi ke da xue xue bao = Journal of Southern Medical University 20180701 8

<h4>Objective</h4>To investigate the changes in retinal transcriptome profile of mice with endotoxin-induced uveitis (EIU)following dexamethasone (DEX) treatment and explore the mechanisms underlying the therapeutic effect of DEX.<h4>Methods</h4>EIU was induced in BALB/c mice by intravitreal injection of 125 ng lipopolysaccharide (LPS), followed by topical applicationof DEX (0.1%) eye drops every 4 h for 24 h. The anterior chamber inflammation was examined with a slit lamp and the clinicalscores ...[more]

PMID: 30187873

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Project description:PurposeEndotoxin-induced uveitis (EIU) is a well-established mouse model for studying human acute inflammatory uveitis. The purpose of this study is to investigate the genome-wide retinal transcriptome profile of EIU.MethodsThe anterior segment of the mice was examined with a slit-lamp, and clinical scores were evaluated simultaneously. The histological changes in the posterior segment of the eyes were evaluated with hematoxylin and eosin (H&E) staining. A high throughput RNA sequencing (RNA-seq) strategy using the Illumina Hiseq 2500 platform was applied to characterize the retinal transcriptome profile from lipopolysaccharide (LPS)-treated and untreated mice. The validation of the differentially expressed genes (DEGs) was analyzed with real-time PCR.ResultsAt the 24th hour after challenge, the clinical score of the LPS group was significantly higher (3.83±0.75, mean ± standard deviation [SD]) than that of the control group (0.08±0.20, mean ± SD; p<0.001). The histological evaluation showed a large number of inflammatory cells infiltrated into the vitreous cavity in the LPS group compared with the control group. A total of 478 DEGs were identified with RNA-seq. Among these genes, 406 were upregulated and 72 were downregulated in the LPS group. Gene Ontology (GO) enrichment showed three significantly enriched upregulated terms. Twenty-one upregulated and seven downregulated pathways were remarkably enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Eleven inflammatory response-, complement system-, fibrinolytic system-, and cell stress-related genes were validated to show similar results as the RNA-seq.ConclusionsWe first reported the retinal transcriptome profile of the EIU mouse with RNA-seq. The results indicate that the abnormal changes in the inflammatory response-, complement system-, fibrinolytic system-, and cell stress-related genes occurred concurrently in EIU. These genes may play an important role in the pathogenesis of EIU. This study will lead to a better understanding of the underlying mechanisms and shed light on discovering novel therapeutic targets for ocular inflammation.

Project description:PurposeUveitis is a common cause of vision loss. The renin angiotensin system (RAS), which plays a vital role in cardiovascular system, is a potent mediator of inflammation and has been implicated in the pathogenesis of uveitis. A newly identified axis of RAS, ACE2/Ang-(1-7)/Mas, has emerged as a novel target because it counteracts the deleterious effect of angiotensin II. The purpose of this study was to investigate the effect of endogenous ACE2 activation in preventing endotoxin-induced uveitis (EIU) in mice.MethodsACE2 activator diminazene aceturate (DIZE) was administered both systemically and locally. For systemic administration, female BALB/c mice received intraperitoneal injection of DIZE (60 mg/kg body weight [BW]) for 2 days prior to lipopolysaccharide (LPS) intravitreal injection (125 ng) to induce uveitis. For local study, DIZE was given at 0.5, 0.1, and 0 mg/mL as eyedrops six times per day for 2 days before LPS injection. The anterior segment of the mice was examined at 12, 24, 48, and 72 hours after LPS injection, and clinical scores were determined at the same time. Morphology and infiltrating inflammatory cells were evaluated after 24 hours. The mRNA levels of inflammatory cytokines were analyzed by real-time RT-PCR. ACE2 activity was determined using a self-quenching fluorescent substrate.ResultsAt 24 hours, the clinical score of mice treated with DIZE systemically was significantly lower (mean, ∼1.75) than the saline vehicle group (mean, ∼4) (P < 0.001). Histological examination showed 63.4% reduction of infiltrating inflammatory cells in the anterior segment and 57.4% reduction in the posterior segment of DIZE-treated eyes. The number of CD45(+) inflammatory cells in the vitreous of the DIZE-treated group was decreased (43.3%) compared to the vehicle group (P < 0.01). The mRNA levels of inflammatory cytokines were significantly reduced in the DIZE-treated group (P < 0.01, P < 0.001). The number of infiltrating inflammatory cells was also significantly reduced in eyes that received topical administration of DIZE: 73.8% reduction in the 0.5 mg/mL group and 51.7% reduction in the 0.1mg/mL group compared to the control group. DIZE treatment resulted in significantly increased ACE2 activity in the retina (P < 0.001).ConclusionsEndogenous ACE2 activation by DIZE has a preventive effect on LPS-induced ocular inflammation in the EIU mouse model. These results support the notions that RAS plays a role in modulating ocular immune response and that enhancing ACE2 provides a novel therapeutic strategy for uveitis.

Project description:Essentials Glucocorticoids are associated with an increased risk of thrombosis. Healthy volunteers received dexamethasone or placebo in an endotoxin lung instillation model. Dexamethasone suppressed thrombin generation in bronchoalveolar lavage. Glucocorticoids inhibit endotoxin induced pulmonary coagulopathy.SummaryBackground Activation of local and systemic coagulation is a common finding in patients with pneumonia. There is evidence that glucocorticoids have procoagulant activity in the circulation, particularly in the context of inflammation. The effects of glucocorticoids on local pulmonary coagulation have not yet been investigated. Objective To use a human model of lung inflammation based on the local instillation of endotoxin in order to investigate whether glucocorticoids alter pulmonary coagulation. Methods Twenty-four healthy volunteers were randomized to receive either dexamethasone or placebo in a double-blind trial. Endotoxin was instilled via bronchoscope into right or left lung segments, followed by saline into the contralateral site. Six hours later, a bilateral bronchoalveolar lavage (BAL) was performed and coagulation parameters were measured. Results Endotoxin induced activation of coagulation in the bronchoalveolar compartment: the level of prothrombin fragment 1 + 2 (F1 + 2 ) was increased three-fold (248 pmol L-1 , 95% confidence interval [CI] 43-454 versus 743 pmol L-1 , 95% CI 437-1050) and the level of thrombin-antithrombin complex (TATc) was increased by ~ 50% (31 μg L-1 , 95% CI 18-45 versus 49 μg L-1 , 95% CI 36-61) as compared with saline-challenged segments. Dexamethasone reduced F1 + 2 (284 pmol L-1 , 95% CI 34-534) and TATc (9 μg L-1 , 95% CI 0.7-17) levels almost to those measured in BAL fluid from the saline-instilled segments in the placebo group. Dexamethasone even profoundly reduced F1 + 2 levels (80%) in saline-instilled lung segments (50 pmol L-1 , 95% CI 12-87). In contrast, dexamethasone had no effect on systemic F1 + 2 levels. Conclusions Dexamethasone inhibits endotoxin-induced coagulopathy in lungs. This trial is the first to provide insights into the effects of glucocorticoids on pulmonary coagulation in response to endotoxin.

Project description:(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.

Dataset Information

[Retinal transcriptome profile in mice following dexamethasone treatment for endotoxin-induced uveitis].

Publications

[Retinal transcriptome profile in mice following dexamethasone treatment for endotoxin-induced uveitis].

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