Bioluminescence for in vivo detection of cell-type-specific inflammation in a mouse model of uveitis

Abstract

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells, T cells, and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75% of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7, the ocular T cell population increases to 50% of CD45 + cells, leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible (< 1% of CD45 + cells), the ocular B cell population increases to > 4% by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models.

Systemic prime exacerbates the ocular immune response to heat-killed Mycobacterium tuberculosis

“…Mice were maintained with standard chow and medicated water (acetaminophen 300 mg/kg) ad libitum, with 12 h light: 12 h dark cage lighting under specific pathogen-free conditions. PMU induction in mice is a modification of a protocol previously published in rabbits ( Mruthyunjaya et al, 2006 ; Cousins, 1992 ; Jaffe et al, 1998 ) and rats( K. L. Pepple et al, 2015 ; Kathryn L. Pepple, Wilson, and Van Gelder, 2018 ) and has been reported previously ( Gutowski et al, 2017 ; John et al, 2020 , 2021 ). Briefly, on day -7 animals receive a subcutaneous injection of 100 μg killed mycobacterium tuberculosis H37Ra antigen (#231141, Difco Laboratories, Detroit, MI) in a 0.1 cc emulsion of incomplete Freund’s adjuvant (#263910, Difco Laboratories, Detroit, MI) divided with half the dose to each hip.…”

“…OCT imaging was performed as described previously ( John et al, 2020 ). Briefly, OCT images were acquired using the Bioptigen Envisu R2300.…”

Characterization of Gene Therapy Associated Uveitis Following Intravitreal Adeno-Associated Virus Injection in Mice

“…After OCT imaging, the animal was euthanized and both right (injected) and left (fellow, uninjected) eyes were enucleated and intraocular contents collected separately as described by John et al 14 Briefly, each eye was rinsed in a flow buffer, placed in 50 μL of flow buffer, the cornea and lens removed, and the aqueous humor, iris, vitreous, retina, and retinal pigmented epithelium were collected. The intraocular contents were incubated with 10 mg/mL of DNAse (Roche, Germany) and 0.5 mg/mL of collagenase (Roche, Germany) at 37°C for 25 [30-F11, 565967]) for 30 minutes at 4°C.…”

A Review of the Various Roles and Participation Levels of B-Cells in Non-Infectious Uveitis

“…IL-10-producing plasmablasts (140) lack CD20. Bregs expressing high levels of PD-L1 also resist anti-20 therapy primarily due to their high levels of BAFF receptor (BAFF-R) expression (141). The efficacy of rituximab against uveitis may partly come from the expansion of these resistant regulatory B cells.…”