Abstract
BackgroundRetinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration.MethodsBV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT).ResultsThe in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration.ConclusionsThe AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.
Highlights
Retinal degenerative diseases significantly contribute to visual impairment and blindness
I3C reduces pro-inflammatory and enhances anti-oxidant gene expression in BV-2 cells We first examined whether treatment with the aryl hydrocarbon receptor (AhR) ligand I3C has an effect on a selected set of proinflammatory markers in microglia-like BV-2 cells
We aimed to confirm these data using Western blots. These experiments revealed that the LPS-induced expression of inducible Nitric oxide (NO)-synthase (i-NOS), IL 1ß, and cyclooxygenase 2 (COX2) was reduced by I3C on the protein level, whereas Heme oxygenase 1 (HMOX1) was increased and NADPH dehydrogenase quinone 1 (NQO1) was unchanged (Fig. 1i-l)
Summary
Retinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Phagocytes of the retina, microglia and macrophages, have been recognized as important factors in retinal degenerative diseases. These cells become amoeboid and migrate to subretinal (SR) space with upregulation of different pro-inflammatory molecules [2, 3]. Microglia enhance their phagocytosis capacity to resolve tissue damage. Persistent disease leads to chronically activate microglia, which may ingest cellular debris and healthy photoreceptors [4]. Microglia-targeted pharmacotherapy to mitigate chronic neuro-inflammation may be a promising treatment approach in retinal degenerative diseases
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