Multimodal analysis of ocular inflammation using endotoxin-induced uveitis

Abstract

Endotoxin-induced uveitis (EIU) in rodents is a model of acute Toll-like receptor 4 (TLR4)-mediated organ inflammation, and has been used to model human anterior uveitis, examine leukocyte trafficking and test novel anti-inflammatory therapeutics. Wider adoption has been limited by the requirement for manual, non-specific, cell-count scoring of histological sections from each eye as a measure of disease severity. Here, we describe a comprehensive and efficient technique that uses ocular dissection and multimodal tissue analysis. This allows matched disease scoring by multicolour flow cytometric analysis of the inflammatory infiltrate, protein analysis on ocular supernatants and qPCR on remnant tissues of the same eye. Dynamic changes in cell populations could be identified and mapped to chemokine and cytokine changes over the course of the model. To validate the technique, dose-responsive suppression of leukocyte infiltration by recombinant interleukin-10 was demonstrated, as well as selective suppression of the monocyte (CD11b+Ly6C+) infiltrate, in mice deficient for either Ccl2 or Ccr2. Optical coherence tomography (OCT) was used for the first time in this model to allow in vivo imaging of infiltrating vitreous cells, and correlated with CD11b+Ly6G+ counts to provide another unique measure of cell populations in the ocular tissue. Multimodal tissue analysis of EIU is proposed as a new standard to improve and broaden the application of this model.

Hypoxia inducible factors are dispensable for myeloid cell migration into the inflamed mouse eye

“…1b), consistent with reports that resident microglia express lower levels of Lysm compared to mature circulating myeloid cells2830. NK cells can be differentiated from both lymphoid and myeloid progenitors31 and whilst not a major population infiltrating the eye during EIU25, we included them in the analysis due to their expression of CD11b.…”

“…The myeloid infiltrate in EIU has been well characterized and is composed of predominantly neutrophils and inflammatory monocytes25. To confirm appropriate targeting of these infiltrating cells by Lysm -driven Cre recombinase (Cre) activity, we used confocal microscopy to analyse the anterior segment and retinal tissue from naïve and EIU-induced Lysm + /cre Rosa26 eYFP reporter mice.…”

“…The EIU stimulus, LPS, within the immune privileged ocular environment results in local production in the eye of various cytokines and chemokines including those relevant to the HIF pathway: MCP-1, MIP1α and MIP1β25, although production of M-CSF has not been reported. The subsequent myeloid infiltration from the circulating blood can be accurately quantified using multi-colour flow cytometry29 and biological effects observed25. The absence of a quantifiable phenotype in mutants where HIF alpha-subunits were either deleted or stabilized suggests no involvement of HIF pathways in myeloid migration in response to LPS.…”

“…In the mouse, it is characterized by an intraocular migration of myeloid cells from the blood, composed predominantly of neutrophils and inflammatory monocyte/macrophages. This myeloid infiltration can be enumerated by flow cytometry, peaking at 18 hours post induction and resolving with minimal tissue damage25. As LPS is a potent inducer of HIF stabilization26, we employed this model to investigate the importance of HIF pathways downstream of LPS induction on myeloid trafficking into inflamed ocular tissue in conditional knockout mice where HIF1a and HIF2a are either absent or stabilized in myeloid cells.…”

MMP-3 Deficiency Alleviates Endotoxin-Induced Acute Inflammation in the Posterior Eye Segment

“…To further confirm the inhibitory effect of MMP-3 on acute retinal inflammation, SD-OCT was applied as a non-invasive, in vivo imaging technique to quantify the number of infiltrating leukocytes in the vitreous cavity (adjacent to the optic nerve head) at 24 hpi, in both WT and MMP-3 −/− mice (Figure 4C,D) [41,42,43]. The number of leukocytes significantly increased, respectively, by 3- and 2-fold in the vitreous of WT and MMP-3 −/− eyes after induction of endotoxemia (Figure 4D).…”