Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation

Abstract

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Interferon-γ-induced MD-2 Protein Expression and Lipopolysaccharide (LPS) Responsiveness in Corneal Epithelial Cells Is Mediated by Janus Tyrosine Kinase-2 Activation and Direct Binding of STAT1 Protein to the MD-2 Promoter

“…Chimeric Mice-Bone marrow cells from C57BL/6TgN (ACTbEGFP) 10sb (eGFP) mice or MD-2 Ϫ/Ϫ were isolated as described (17). Recipient mice received 2 ϫ 600 gray doses of whole body irradiation 3 h apart.…”

Pattern recognition receptors in microbial keratitis

“…79 TLR4 must be associated with co-stimulatory molecule MD-2 to recognise LPS; 81 accessory proteins lipopolysaccharide-binding protein and CD14 transfer the LPS molecules from the bacteria to the TLR4/MD-2 complex. 82 Resident myeloid cells are capable of conferring LPS responsiveness, 83 but although TLR4 is also expressed on human corneal epithelial cells, MD-2 is not detectable. It is thought that MD-2 expression is enabled by the infiltration of immune cells such as natural killer cells, which activate the JAK2/STAT1 transcription pathway and produce IFN-γ.…”

“…In addition to the MyD88 pathway, a non-MyD88 pathway via TRIF exists which has also been found to be present in P. aeruginosa keratitis. 83 MyD88/TRIF pathways induce production of a chemokine known as CXCL1/KC which recruits neutrophils to the cornea from limbal blood vessels. MyD88/TRIF pathways also promote the production of IL-1α and IL-1β, which creates a positive feedback loop via the interleukin 1 receptor IL-1R1 interaction with MyD88.…”

Immunomodulatory Peptide IDR-1018 Decreases Implant Infection and Preserves Osseointegration

“…Rigorously cleaned implants were incubated with 100 lL of the bacterial suspensions for 20 minutes at room temperature with gentle shaking, rinsed three times in phosphate-buffered saline (PBS), and immediately implanted into pilot holes in the middiaphysis of the femur [4,5] of 6-to 8-week-old female C57BL/6 J or macrophage Fas-induced apoptosis (MAFIA) mice. Macrophage recruitment was measured in the MAFIA mice, in which the monocyte/macrophage-specific colony-stimulating factor receptor promoter drives both expression of enhanced green fluorescent protein and a Fas apoptosis system activated by the dimerization drug, AP20187 [8,10]. MAFIA mice are on the C57BL/6 J background and have a normal phenotype unless exposed to AP20187.…”