Synergy between viral and bacterial toll-like receptors leads to amplification of inflammatory responses and preterm labor in the mouse

Abstract

Toll-like receptors (TLRs) recognize molecular constituents of pathogens and activate host innate immune responses. TLR2 responds to Gram-positive organisms and components of their cell walls. TLR3 responds to double-stranded RNA (an intermediate in viral replication). A mouse macrophage cell line (RAW 264.7) and freshly obtained mouse peritoneal macrophages were treated in tissue culture for 5 or 10 h with either peptidoglycan (PGN; a TLR2 ligand, 1 μg/ml), polyinosinic:cytidylic acid (poly(I:C); a TLR3 ligand, 10 μg/ml), both PGN and poly(I:C), or neither. Total RNA was extracted, and RT-PCR was performed. A mouse model of preterm birth induced by intrauterine injection of TLR ligands was used to test in vivo effects. Compared to stimulation with either PGN or poly(I:C) alone, stimulation of macrophages with both ligands (whether simultaneously or sequentially) resulted in synergistic expression of inflammatory mediators, including inducible nitric oxide synthase, interleukin 1 beta, tumor necrosis factor alpha, and the chemokine CCL5 (RANTES). Using peritoneal macrophages obtained from mutant and control mice, this synergy was determined to be dependent upon TLR2 and the TLR-related intracellular adaptor proteins MYD88 and TICAM1 (TRIF). Simultaneous administration of both PGN and poly(I:C) to pregnant mice also produced dramatic synergy in the occurrence of preterm delivery. These results support a possible role for viral infection in preterm labor. Synergy in the mechanisms of parturition suggests the existence of a "two-hit" trigger mechanism that minimizes responses to stimuli of limited biological significance while providing an efficient amplification strategy for rapid activation of labor in response to multiple or more severe insults.

FIG. 1.

Synergy between TLR2 and TLR3 activation. RAW 264.7 cells were stimulated with either PBS, PGN (1 μg/ml), poly(I:C) (10 μg/ml), or both PGN and poly(I:C) for 5 h. Real-time PCR was performed for IL1B, NOS2, CCL5 (RANTES), TNF, TLR2, and TLR3, normalized to the housekeeping gene GAPDH and with average expression for the combined (PGN plus poly(I:C)) group in each case set to 100. Error bars represent mean ± SD. P values were calculated for all four treatments simultaneously by ANOVA (n = 3 replicates/condition). Depicted is a representative example from among three repeat experiments.

FIG. 2.

Priming of cells for synergistic expression of cytokines and chemokines occurs with either PGN or poly(I:C). RAW 264.7 cells were treated in tissue culture with either PGN (1 μg/ml), poly(I:C) (10 μg/ml), or both PGN and poly(I:C) either simultaneously or sequentially for 5–10 h. For sequential incubations, cultures were incubated with the first reagent for 5 h, washed with PBS, and then incubated for an additional 5 h with the second reagent. Real-time PCR was performed using GAPDH as a control gene. The average expression of combined treatment with PGN plus poly(I:C) for 5 h was set to 100. Error bars represent mean ± SD. P values for the four treatment groups calculated by ANOVA are indicated separately for each set of culture conditions (n = 3 replicates/condition). Depicted is a representative example from among three repeat experiments.

FIG. 3.

Synergy between PGN and poly(I:C) requires functional TLR2, MYD88, and TICAM1. Primary mouse peritoneal macrophages extracted from TLR2-deficient (Tlr2KO) and wild-type (C57BL/6J) control mice and from MYD88-deficient (Myd88KO), TICAM1-deficient (Ticam1KO), doubly deficient (DBL KO), and wild-type (B6129F2/J) controls were stimulated with either PGN (1 μg/ml), poly(I:C) (10 μg/ml), or both for 5 h. Duplex RT-PCR was performed for IL1B, TNF, and CCL5 (RANTES), normalized to the housekeeping gene GAPDH. Means ± SD are shown for triplicate wells. Average expression for wild-type macrophages treated with combined PGN plus poly(I:C) was set to 100. P values for the four treatment groups calculated by ANOVA are indicated separately for each genotype. Absent P values indicate unmeasurable values. Depicted is a representative example from among three repeat experiments.

FIG. 4.

Synergistic effect of intrauterine administration of PGN and poly(I:C) on the occurrence (A) and timing (B) of preterm delivery in Day 14.5 pregnant mice. Preterm delivery was defined as delivery of at least one pup within 48 h (all deliveries occurred in less than 36 h). Delivery data for PGN and poly(I:C) alone were presented previously as single points of dose-response curves [8]. P values are by Fisher exact test (A) and a 3 × 4 contingency table (B).