This study was designed to determine the contributions of the TTS system and LPS of P. aeruginosa in the pathogenesis of acute lung injury and bacteria-induced death. To understand the role of TLR4 signaling in acute lung injury and sepsis, we compared LPS-resistant C3H/HeJ mice, which are refractory to the biological effects of LPS due to the mutation of tlr4, to LPS-sensitive C3H/FeJ mice . The two strains of mice received tracheal instillation of either cytotoxic wild type P. aeruginosa PA103 or an isogenic strain PA103ΔUT which is missing two critical TTS toxins, ExoU and ExoT . Three different doses of bacteria were tested in these mice. ExoU is a cytotoxin inducing necrotic cell death by a mechanism associated with its phospholipase activity . ExoT is a 53-kDa exoenzyme S possessing ADP-ribosyltransferase and small GTPase-activating activities. ExoT blocks epithelial wound healing . The administration of large doses of PA103ΔUT did not cause any lung injury nor affect the mortality of any strain of mice. Large doses of wild type PA103 caused acute injury, bacteremia and death in the both strains of mice. Although there appeared to be minor differences in survival, there were no statistically significant differences in the mortality rates between the strains of mice infected with large doses of PA103. These results suggest that the administration of large doses of bacteria overwhelms host defense, even in mice with an intact LPS-signaling pathway. Furthermore, although there was abnormal clearance of the non-cytotoxic PA103ΔUT in the C3H/HeJ mice in comparison to the C3H/FeJ mice, PA103ΔUT did not cause lung injury. As no lung injury occurred, PA103ΔUT did not disseminate, and all the mice survived. These results demonstrate that organ injury and mortality were not directly affected by P. aeruginosa LPS or the TLR4 signaling of the infected mice.
In contrast, an intact TLR4 signaling pathway was clearly essential for host protection from virulent PA103. More C3H/HeJ mice died from a medium dose of PA103, and some of C3H/HeJ mice still died even from the lowest dose of PA103. When lung injury was measured after a short interval, there was no statistical difference between the acute lung injury produced by similar doses in either the C3H/HeJ or C3H/FeJ mice. Therefore, a given dose of cytotoxic bacteria produced a reproducible quantity of lung injury, in a short interval, in both mice. However, when the infections were allowed to persist for a longer interval, the number of cytotoxic bacteria in the C3H/HeJ multiplied to a greater extent and caused lung edema later. The lack of bacterial clearance in the C3H/HeJ mice ultimately led to relatively larger quantities of lung injury, even when smaller doses of cytotoxic bacteria had been administered. The bacteria were able to multiply to injurious levels, implying that, in the absence of normal TLR4 signaling, cytotoxic PA103 can avoid the host immune clearance, disseminate into the bloodstream, and cause the death of the infected animals. The low leukocyte number and a lack of TNFα production in the infected airspace in the C3H/HeJ mice could indicate the lack of a normal inflammatory response that clears the bacteria from the infected site in these mice missing normal TLR4 signaling. Therefore, immune responses modulated by TLR4 signaling are important in the protecting hosts from cytotoxic P. aeruginosa expressing TTS toxins.
LPS is a static component of gram-negative bacteria, while the virulence associated with the TTS system relies on active energy production by live bacteria; the TTS apparatus can be upregulated and TTS toxins need to be translocated using bacterial energy [10, 11, 13]. Gram-negative bacteria such as E. coli and P. aeruginosa possess LPS as a component of their cell membranes, although there are many variations in its pattern . However, only pathogenic strains of gram-negative bacteria possess the TTS system [33, 34]. In P. aeruginosa, only strains expressing the TTS system can cause significant acute necrotic cell death and tissue damage, while non-cytotoxic P. aeruginosa missing TTS but still possessing LPS do not lead to tissue damage [5, 7]. Recently, there have been reports of endogenous stimulators of TLRs [25, 26]. For TLR4, heparan sulfate, hyaluronan, HSP60 and HSP70, surfactant protein A, and β-defensin 2 have been all implicated as possible endogenous ligands or stimulators [25, 26, 35]. These facts suggest that TLRs play a role as surveillance receptors for tissue injury and tissue remodelling as well as for infection . If TLR4 recognizes not only LPS but also degradation products of endogenous macromolecules from necrotic cells and microorganisms, and if this recognition provokes responses to limit tissue injury and induce remodelling, a lack of normal TLR4 signaling must be disadvantageous during infections involving severe tissue damage. The activation of this surveillance system must be more important when cytotoxic P. aeruginosa infects and causes tissue injury. Without having this surveillance system, hosts may not be able to survive infection involving severe tissue damage. Members of the TLR and interleukin-1 receptor (IL-1R) superfamily share an intracytoplasmic Toll-IL-1 receptor (TIR) domain, which mediates recruitment of the interleukin-1 receptor-associated kinase (IRAK) complex via TIR-containing adapter molecules. Recently, 3 unrelated children with an inherited IRAK-4 deficiency were reported. They were prone to infections caused by pyogenic bacteria . Their blood and fibroblast cells did not activate NF-κB or mitogen-activated protein kinase (MAPK) and failed to induce downstream cytokines in response to any of the known ligands of TIR-bearing receptors. These findings suggest that in humans the normal TLR signaling pathway is crucial for protective immunity against specific virulent bacteria and is redundant against most other microorganisms. Our results suggest that, in P. aeruginosa infections, the identification of the phenotype or genotype of P. aeruginosa could be useful clinically.