Endotoxin-induced uveitis is primarily dependent on radiation-resistant cells and on MyD88 but not TRIF

Abstract

TLR4 activation by LPS (endotoxin) is mediated by the MyD88 and TRIF intracellular signaling pathways. We determined the relative activation of these pathways in murine ocular tissue after LPS exposure. Additionally, we explored whether BM-derived or non-BM-derived cells were the major contributors to EIU. Mice deficient in TRIF or MyD88 and their congenic (WT) controls received 250 ng ultrapure LPS ivt at 0 h. Ocular inflammation was assessed by histological analysis at 4, 6, and 24 h, and additionally, in MyD88(-/-) mice, intravital microscopy was performed at 4 h and 6 h to assess adherent, rolling, and infiltrating cells in the iris vasculature and tissue. Cytokines associated with the MyD88 and TRIF intracellular signaling pathways were analyzed in ocular tissue at 4 h. BM chimeric mice (WT→WT, TLR4(-/-)→WT, WT→TLR4(-/-)) received 250 ng LPS by ivt injection, and ocular tissues were examined by histology at 6 h. Lack of MyD88 resulted in a markedly diminished cellular response and reduced production of MyD88-related cytokines 4 h post-LPS treatment. In contrast, lack of TRIF led to reduced production of TRIF-related cytokines and no change in the cellular response to LPS. Therefore, the MyD88 pathway appears to be the dominant TLR4 pathway in EIU. Only WT → TLR4(-/-) chimeric mice were resistant to EIU, and this suggests, surprisingly, that non-BM-derived (radiation-resistant) cells in the eye play a greater role than BM-derived cells.

Impact of IL-1 signalling on experimental uveitis and arthritis

“…At the indicated times, mice were killed, and the eyes, ankles and knees were dissected, fixed in 10% neutral-buffered formalin, and prepared for paraffin embedding and sectioning as previously reported 25 26. Tissue sections (7 µm thick) were stained with H&E, and the severity of inflammatory changes was quantified by a masked observer.…”

“…Dissected ankles, knees and eyes were homogenised in lysis buffer containing protease inhibitors 25 27. Protein concentrations were quantified by BCA assay (BioRad, Hercules, California, USA).…”

Unexpected Roles for Toll-Like Receptor 4 and TRIF in Intraocular Infection with Gram-Positive Bacteria

“…In an endotoxin-induced uveitis (EIU) mouse model, the TLR4/MyD88-dependent pathway contributed to inflammation (43)(44)(45). However, in the EIU model, some of TRIFdependent cytokines were found to be proinflammatory (46), while other TRIF-dependent cytokines were anti-inflammatory (43,47).…”

“…In an endotoxin-induced uveitis (EIU) mouse model, the TLR4/MyD88-dependent pathway contributed to inflammation (43)(44)(45). However, in the EIU model, some of TRIFdependent cytokines were found to be proinflammatory (46), while other TRIF-dependent cytokines were anti-inflammatory (43,47). In Pseudomonas aeruginosa keratitis models, MyD88-and TRIF-mediated signaling following LPS recognition by TLR4 has been reported (48)(49)(50).…”

Role of toll-like receptors in human iris pigment epithelial cells and their response to pathogen-associated molecular patterns

“…In addition, expression of TLR2 in human conjunctival epithelial cells was shown to play a significant role in the chronic ocular inflammatory response to Staphylococcus aureus [3]. Kezic et al [4] suggested that both epithelial cells and immune cells play a role in ocular inflammation. Specifically, radiation-resistant, non-bone marrow derived ocular cells, such as iris endothelial cells or nonpigmented ciliary body epithelial cells, play a greater role in the development of endotoxin-induced uveitis (EIU) than bone marrow-derived macrophages and dendritic cells residing in the eye [4].…”