NKT Cells Are Critical to Initiate an Inflammatory Response after Pseudomonas aeruginosa Ocular Infection in Susceptible Mice

Abstract

CD4+ T cells produce IFN-γ contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-γ and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (−/−; no T/NKT cells), B6.TCR Jα281−/− (NKT cell deficient), α-galactosylceramide (αGalCer) (anergized NKT cells) injected and IL-12p40−/− vs B6 controls were tested. IFN-γ mRNA was undetectable in RAG-1−/−- and αGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jα281−/−, αGalCer-treated, and IL-12p40−/− (activated CD4+ T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mφ; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-κB, and CD1d, and addition of rIFN-γ potentiated Mφ CD1d levels. Together, these data suggest that Langerhans cell/Mφ recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-γ production by NKT cells, that IFN-γ is required to optimally activate NK cells to produce IFN-γ, and that depletion of both NKT/NK cells results in earlier corneal perforation.

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

“…These observations are consistent with an earlier study showing that depletion of NK and NK-T cells in P. aeruginosa results in decreased IFN-␥ RNA expression, impaired bacterial killing, and accelerated corneal perforation (34).…”

Pattern recognition receptors in microbial keratitis

“…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. 84,85 In a murine model of corneal inflammation, Chinnery et al 83 demonstrated that TLR4 activation on resident corneal macrophages and DCs stimulated macrophage and neutrophil recruitment and induced corneal haze.…”

NKT Cells Are Required for Complete Freund’s Adjuvant-Mediated Protection from Autoimmune Diabetes

“…However, the cellular and molecular mechanisms by which CFA activates NK cells and prevents disease remains unknown. NK cell activation has been shown to be dependent on NKT cells and, at least in part, their release of IFN-g after aGC treatment or microbial infection (20,32,55). However, NKT cells are not required for NK cells to function, as steady-state levels of NK cell cytotoxicity are similar between NKT cell-deficient (CD1d 2/2 or Ja281 2/2 ) and wild-type mice (32).…”