Signaling Mediated by Toll-Like Receptor 5 Sensing of Pseudomonas aeruginosa Flagellin Influences IL-1β and IL-18 Production by Primary Fibroblasts Derived from the Human Cornea

Abstract

Pseudomonas aeruginosa is the principal cause of bacterial keratitis worldwide and overstimulation of the innate immune system by this organism is believed to contribute significantly to sight loss. In the current study, we have used primary human corneal fibroblast (hCF) cells as an ex vivo model of corneal infection to examine the role of P. aeruginosa flagellum and type three secretion system (TTSS) in inducing inflammasome-associated molecules that trigger IL-1β and IL-18 production during the early stages of the infection. Our results show that P. aeruginosa infection stimulated the non-canonical pathway for IL-1β and IL-18 expression and pathway stimulation was influenced predominantly by the flagellum. Both IL-1β and IL-18 cytokines were expressed intracellularly during bacterial infection, but only the former was released and detected in the extracellular environment. We also investigated the signaling pathways in hCFs mediated by Toll-Like Receptor (TLR)4 and TLR5 sensing of P. aeruginosa, and our data show that the signal triggered by TLR5-flagellin sensing significantly contributed to IL-1β and IL-18 cytokine production in our model. Our study suggests that IL-18 expression is wholly dependent on extracellular flagellin sensing by TLR5, whereas IL-1β expression is also influenced by P. aeruginosa lipopolysacharide. Additionally, we demonstrate that IL-1β and IL-18 production by hCFs can be triggered by both MyD88-dependent and -independent pathways. Overall, our study provides a rationale for the development of targeted therapies, by proposing an inhibition of flagellin-PRR-signaling interactions, in order to ameliorate the inflammatory response characteristic of P. aeruginosa keratitis.

Effect of Different Antibiotic Chemotherapies on Pseudomonas aeruginosa Infection In Vitro of Primary Human Corneal Fibroblast Cells

“…Recently, we have shown with adherence and gentamicin exclusion assays that P. aeruginosa can adhere to primary hCFs and subsequently invade ( Cendra et al, 2017 ). In the current study, MOI-dependent levels of PAO1 adherence and subsequent invasion were observed after 3 h of infection ( Figure 3 ).…”

“…Infection of hCFs with P. aeruginosa PAO1 induces significant extracellular release of the cytokine IL-1β ( Wong et al, 2011 ; Cendra et al, 2017 ). Since exacerbation of the inflammatory response is a critical event during eye infection, we tested the hypothesis that antibiotic treatments not only reduced bacterial infection but were also anti-inflammatory, by influencing IL-1β cytokine production.…”

“…A Cochrane review of studies of steroid use in microbial keratitis proposed the earlier use of anti-inflammatories to reduce damaging effects from the infective process ( Herretes et al, 2014 ). Recently, we described the mechanisms whereby P. aeruginosa infection induces inflammasome-associated molecules in primary hCFs in vitro , which leads principally to the extracellular release of the cytokine IL-1β ( Cendra et al, 2017 ). In our current study, the most effective anti-inflammatory antibiotics were CIP and GEN, which reduced IL-1β production from hCFs infected with high bacterial MOI (=100).…”

“…Recently, we reported that P. aeruginosa can adhere to human primary corneal fibroblasts (hCFs) in vitro and subsequently invade. We also reported that hCFs were a source of IL-1β secretion in response to challenge with P. aeruginosa in vitro ( Cendra et al, 2017 ). IL-1β is a pleiotropic cytokine that is involved in several inflammatory processes involving a variety of different eukaryotic cells ( Ren and Torres, 2009 ) and over-stimulated IL-1β expression can contribute to impaired healing ( Okamoto et al, 2004 ; Stapleton et al, 2008 ).…”

Amino acids 89–96 of Salmonella flagellin: a key site for its adjuvant effect independent of the TLR5 signaling pathway

“…However, the production of pro-IL-1β and pro-IL-18 can be influenced by TLR5 signaling. 12 These observations may explain why reduced TLR5 activity was accompanied by decreased NLRC4 signaling in aa 89-96 fliC → flaA mutant flagellin.…”