primed-mycobacterial-uveitis-pmu-as-model-for-post-infectious

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology:  ...

University of Bristol

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm) compared with untreated eyes (25 ± 5/mm; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.

Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9.

 Whether retinal microglia can maintain or restore immune homeostasis during and after inflammation is unclear. We performed single-eye mRNA-sequencing on microglia at different timepoints following a single inflammatory stimulus to characterise their transcriptome during and after resolution of endotoxin-induced uveitis (EIU).     (C57BL/6) male heterozygotes were administered tamoxifen via different regimes at 4-5 weeks of age. Four weeks post-tamoxifen, mice were injected intravitreally with 10 ng lipopolysaccharide (endotoxin induced uveitis, EIU). Six-hundred retinal microglia were obtained by FACS from individual naïve retinas and at 4 h, 18 h, and 2 weeks following EIU induction. Samples were sequenced to a depth of up to 16.7 million reads using the SMART-Seq v4 Ultra Low Input RNA kit. The data was analysed using Partek software and Ingenuity Pathway Analysis. Genes were considered differentially-expressed (DEG) if the FDR step-up -value was ≤0.05 and the fold-change was ≥±2.  Flow cytometric analysis indicates that the    strain is both sensitive (>95% tagging) and specific (>95% specificity) for microglia when tamoxifen is administered topically to the eye for 3 days. During "early" activation, 613 DEGs were identified. In contrast, 537 DEGs were observed during peak cellular infiltrate and none at 2 weeks, compared to baseline controls (1,069 total unique DEGs). Key marker changes were validated by qPCR, flow cytometry, and fluorescence microscopy. C5AR1 was identified and validated as a robust marker of differentiating microglial subsets during an LPS response.  Using EIU to provide a single defined inflammatory stimulus, mRNA-Seq identified acute transcriptional changes in retinal microglia which returned to their original transcriptome after 2 weeks. Yolk-sac derived microglia are capable of restoring their homeostatic state after acute inflammation.

Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation.

It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject demonstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized signals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentration, suggesting cell labelling is possible. ICG can label RPE and may be used as an additional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required.

Article	Year
Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 31981492	2020
Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation. Frontiers in immunology\| PubMed ID: 31993055	2019
Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina. PloS one\| PubMed ID: 32053618	2020

Oliver H. Bell

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