Lipopolysaccharide-driven Th2 cytokine production in macrophages is regulated by both MyD88 and TRAM

Abstract

Gram-negative bacterial lipopolysaccharide (LPS) activates macrophages by interacting with Toll-like receptor 4 (TLR4) and triggers the production of various pro-inflammatory Th1 type (type 1) cytokines such as IFNgamma, TNFalpha, and IL8. Though some recent studies cited macrophages as potential sources for Th2 type (type 2) cytokines, little however is known about the intracellular events that lead to LPS-induced type 2 cytokines in macrophages. To understand the mechanisms by which LPS induces type 2 cytokine gene expression, macrophages were stimulated with LPS, and the expression of IL-4 and IL-5 genes were examined. LPS, acting through TLR4, activates both type 1 and type 2 cytokine production both in vitro and in vivo by using macrophages from C3H/HeJ or C3H/HeOuJ mice. Although the baseline level of both TNFalpha and IL-4 protein was very low, TNFalpha was released rapidly after stimulation (within 4 h); however, IL-4 was released after 48 h LPS stimulation in secreted form. Silencing of myeloid differentiation protein (MyD88) and TRIF-related adaptor molecule (TRAM), using small interfering RNA abolished IL-4 induction induced by LPS whereas silencing of TRAM has no effect on TNFalpha induction, thereby indicating that LPS-induced TNFalpha is MyD88-dependent but IL-4 is required both MyD88 and TRAM. These findings suggest a novel function of LPS and the signaling pathways in the induction of IL-4 gene expression.

FIGURE 1.

LPS induces type 1 and type 2 cytokine production in the murine model of lung inflammation. C3H/HeOuJ mice were treated intranasally with LPS (0.3 mg/kg) in 50 μl of sterile saline. After 1, 7, or 14 days, mice were sacrificed, and the BAL fluid was assessed for the total cell counts (A) as well as levels of cytokines, including: TNFα (B), IL-4 (C), and IL-5 (D). Cytokine levels were measured by ELISA. Data are expressed as mean ± S.E. of three different experiments, and each had five mice per group. Significance (p < 0.05), indicated by an asterisk, is LPS-challenged animals versus saline-treated animals.

FIGURE 2.

LPS induces IL-4 and TNFα gene expression in macrophages. A, RAW264.7, MH-S, and BMDMs cells were plated at a density of 1 × 10⁶ cells per well in 6-well plates. A day later the cells were stimulated with or without LPS (10 ng/ml) for varying times (1, 2, 4, 8, or 24 h for TNF-α) and (8, 24, 36, or 48 h for IL-4). The cells were then lysed with TRIzol, total RNA was extracted, and cDNA was reverse-transcribed. The cDNA was then analyzed for IL-4 (A) and TNF-α (B) mRNA by real time gene amplification using relevant primers and Sybr green 1. Error bars represent S.D. from the average of three readings. The figures are representatives of five independent experiments done in triplicates. C, LPS induces IL-4 protein release in macrophages. RAW264.7 cells plated at a density of 3 × 10⁵ cells per well in 24-well plate tissue culture plates were stimulated with LPS for varying times. The supernatants were analyzed for murine IL-4 (C) or TNF-α (D) by ELISA according to the manufacturer's instructions. All the experiments were repeated three times and yielded similar results. Significance (p < 0.05), indicated by *, is LPS-treated cells versus LPS-untreated cells.

FIGURE 3.

LPS-induced IL-4 release is TLR4-dependent in macrophages and in the murine model of lung inflammation. RAW264.7 cells were pretreated with functional antibody of TLR4 (TLR4fAb) for 1 h and then stimulated with LPS for 72 h. The supernatant was collected, and IL-4 ELISA was done using the manufacturer's protocol (A). BMDMs from C3H/HeOuJ (B) and C3H/HeJ (C) mice were stimulated with or without LPS for 12, 24, or 48 h. The cells were lysed with TRIzol and frozen at −80 °C. On the day of the experiment, the samples were thawed, total RNA was extracted, and cDNA was reverse-transcribed. The cDNA was then amplified by real time PCR using IL-4 primers and Sybr Green 1. The experiment is a representative of three independent experiments using two mice each time. Results are expressed as mean ± S.D. C3H/HeOuJ and C3H/HeJ mice were treated intranasally with LPS (0.3 mg/kg) in 50 μl of sterile phosphate-buffered saline. After 2 weeks, mice were sacrificed, and the BAL fluid was assessed for total cell counts (D and E), and IL-4 was measured by ELISA (F and G). Data are expressed as mean ± S.E. of three different experiments, and each had five mice per group. *, p < 0.05 compared with CON; **, p < 0.05 compared with LPS treatment without TLR4 fAb.

FIGURE 4.

LPS-induced Th2 response is MyD88/TRAM-dependent in murine macrophages. RAW264.7 cells (A) were co-transfected with pcDNA3-MyD88DN and pIL4-luc then stimulated with LPS for 24 h following which the luciferase activity was measured. *, p value < 0.05. RAW264.7 (B), MH-S (C), and BMDM (D), cells were co-transfected with siRNA MyD88 or siRNA TRAM or both together with pIL4-luc. One day post-transfection, the whole cell lysate was separated by SDS-PAGE and blotted with anti-MyD88 or anti-TRAM antibodies. Another set of cells were stimulated with media or LPS for 48 h, and then luciferase gene activity was determined with 3010 luminometer. The experiment is a representative of three independent experiments. Results are expressed as mean ± S.D. Statistical analysis was done using Student's t test, and p value < 0.05 was considered significant.

FIGURE 5.

LPS-induced IL-4 protein release requires both MyD88 and TRAM pathways, while LPS-induced TNF-α protein release is MyD88-dependent and TRAM-independent. RAW264.7 cells were transfected with siRNA against MyD88 or siRNA against TRAM or both together. A day later, the cells were stimulated with LPS for the indicated times. The supernatants were collected and were utilized to measure IL-4 (A) and TNF-α (B) levels by ELISA, following the manufacturer's protocol. The experiment was repeated three times with similar results. Results are expressed as mean ± S.D. Statistical analysis was done using the Student's t test, and a p value < 0.05 was considered significant.

FIGURE 6.

Schematic representation depicting LPS, acting through TLR4, activates both Th1 and Th2 cytokine production in macrophages. As indicated, LPS is an early Th1 cytokine inducer, which is MyD88-dependent, and LPS is also a delayed Th2 cytokine inducer that is both MyD88- and TRAM-dependent.