Redundant and cooperative interactions between TLR5 and NLRC4 in protective lung mucosal immunity against Pseudomonas aeruginosa

Abstract

Flagellin is the major structural component of flagella expressed by Pseudomonas aeruginosa (PA) and other bacteria. This protein has been shown to activate the Toll-like receptor TLR5 and the Nod-like receptor Nlrc4/Ipaf, culminating in the expression of innate cytokines and antimicrobial molecules. In this study, we tested the hypothesis that TLR5 and Nlrc4 in combination are required for maximal protective lung innate mucosal immunity against PA. To test this hypothesis, we compared innate immune responses in wild-type (WT) C57B6 mice challenged with PA intratracheally to those observed in mice genetically deficient in TLR5 (TLR5(-/-)) or Nlrc4 (Nlrc4(-/-)) alone or in combination (TLR5/Nlrc4(-/-)). As compared to WT, TLR5(-/-) and Nlrc4(-/-) mice, we observed a significant increase in mortality in TLR5/Nlrc4(-/-) mice, which was associated with a >5,000-fold increase in lung PA colony-forming units and systemic bacterial dissemination. The increased mortality observed in double-deficient mice was not attributable to differences in lung leukocyte influx or lung injury responses. Levels of biologically active IL-1β and IL-18 were reduced in the bronchoalveolar lavage fluid from PA-infected Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, indicating the requirement for Nlrc4-dependent caspase-1 activation. Similarly, decreased production of biologically active IL-1β and activation of caspase-1 was observed in PA-stimulated pulmonary macrophages isolated from Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, whereas the expression of iNOS and the production of NO were significantly reduced in cells from double-mutant but not single-mutant mice. Collectively, our findings indicate that TLR5 and Nlrc4 have both unique and redundant roles in lung antibacterial mucosal immunity, and the absence of both pathogen recognition receptors results in an increase in susceptibility to invasive lung infection.

Fig. 1

Effect of TLR5, Nlrc4 or TLR5/Nlrc4 gene deletion on survival after i.t. PA challenge. Mice were administered 7 × 10⁵ CFU i.t., and then survival was assessed up to 6 days. Results shown are combined from two separate experiments; n = 8–10 mice per group. * p < 0.05 as compared to the other groups.

Fig. 2

Effect of TLR5, Nlrc4 or TLR5/Nlrc4 gene deletion on lung and spleen PA CFU after i.t. PA challenge. Mice were administered 5 × 10⁵ CFU i.t., and then PA CFU were quantitated in lung (a) and spleen (b) 24 h later. Data are expressed as log₁₀, combined from two separate experiments; n = 6–8 per group. * p < 0.0001 as compared to the other groups.

Fig. 3

Effect of TLR5/Nlrc4 gene deletion on total BALF cells (a) and BALF PMN (b) after PA challenge. Mice were administered 5 × 10⁵ CFU i.t., and then total cells and PMN were quantitated at 6 and 18 h postchallenge; n = 4–6 per group.

Fig. 4

Effect of TLR5, Nlrc4 or TLR5/Nlrc4 gene deletion on BALF albumin levels. Mice were administered 5 × 10⁵ CFU i.t., and then BALF albumin levels were quantitated at 6 and 18 h postchallenge; n = 4–6 per group. * p < 0.01 as compared to other groups.

Fig. 5

Effect of TLR5, Nlrc4 or TLR5/Nlrc4 gene deletion on BALF cytokine/chemokine levels. Mice were administered 5 × 10⁵ CFU i.t., and then BALF TNF-α (a), KC/CXCL1 (b), IL-1β (c) and IL-18 (d) levels were quantitated by ELISA at 18 h postchallenge; n = 4–6 per group. * p < 0.05 as compared to infected WT control mice.

Fig. 6

Effect of TLR5, Nlrc4 or TLR5/Nlrc4 gene deletion on the expression of IL-1β, NO and caspase-1 activation in PA-stimulated PM in vitro. Expression of IL-1β and iNOS mRNA (a), levels of IL-β and NO in PM-conditioned media (b), and amount of total and active (p10) forms of caspase-1 by Western blotting (c). n = 4–6 per group for mRNA and protein studies, whereas Western blotting is representative of 2 separate experiments. * p < 0.05 as compared to PM from WT mice.