Macrophages polarization is mediated by the combination of PRR ligands and distinct inflammatory cytokines

Abstract

Macrophages recognize microbes through Pattern Recognition Receptors (PRRs), and then release pro-inflammatory and anti-inflammatory cytokines. Recent studies have highlighted that collaboration between different PRRs. However, these studies have neglected the crosstalk between various PRRs on macrophages. In the present study, we investigated the interplay of nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2) and TLRs (TLR1, 2, 3, 4, 5, 6, 7, 8) in terms of macrophage activation, the expression and production of cytokines. The macrophages were stimulated with a single PRR ligand or a combination of TLR and NOD ligands. After 8 h of incubation, the mRNA expression of interleukin-1β (IL-1β), IL-4, IL-6, IL-10, IL-12p35, IL-12p40, IL-13, and interferon-γ (IFN-γ) was evaluated. The production of these cytokines was also measured. NOD2 synergized with TLR3 agonists on enhancement of IL-10 release. However, the combination of NOD1 with TLR3 ligands showed little effect on IL-10 production. Moreover, NOD2 inhibited the percentages of CD11b + F4/80 + cells activated by TLR3 agonist.

Keywords: Macrophage; NLRs; interleukin; pattern recognition receptor; toll-like receptor.

Figure 1

The qRT-PCR and ELISA results of IL-6, IL-10, IL-13 and IL-1β when RAW264.7 cells were stimulated with iE-DAP, MDP, Pam3CSK4, FSL-1, Poly (I:C), LPS, Rec FLA-ST and R848 alone. Data showed the mean and standard deviation values of results, with statistically significant difference, *and **, P < 0.05, and P < 0.01, respectively, versus normal secretion level (medium only).

Figure 2

iE-DAP or MDP had synergistic or inhibitory interactions with each type of TLR ligand on IL-6 and IL-10 production. The supernatant was collected after RAW264.7 cells were stimulated 24 h with combination of iE-DAP or MDP with single TLR ligand. The amount of these cytokines was measured via ELISA. Data showed the mean and standard deviation values of ELISA results with statistically significant difference, * and **, P < 0.05, and P < 0.01, respectively, versus normal secretion level (medium only).

Figure 3

FACS plots showed percentages of CD11b + F4/80 + cells in upper right quadrant. A. iE-DAP promoted the double-positive cells activated by FSL-1 and LPS, while MDP enhanced the ratio of CD11b + F4/80 + cells induced by Pam3CSK4 and FSL-1. B. iE-DAP decreased the percentage of double-positive cells which were elicited by Pam3CSK4and Poly (I:C). By contrast, MDP inhibited the percentages of CD11b + F4/80 + cells induced by Poly (I:C) and recFLA-ST.