Ulvan Activates Chicken Heterophils and Monocytes Through Toll-Like Receptor 2 and Toll-Like Receptor 4

Abstract

Responsiveness to invasive pathogens, clearance via the inflammatory response, and activation of appropriate acquired responses are all coordinated by innate host defenses. Toll-like receptor (TLR) ligands are potent immune-modulators with profound effects on the generation of adaptive immune responses. This property is being exploited in TLR-based vaccines and therapeutic agents in chickens. However, for administering the TLR agonist, all previous studies used in ovo, intra-muscular or intra-venous routes that cannot be performed in usual farming conditions, thus highlighting the need for TLR ligands that display systemic immune effects when given orally (per os). Here we have demonstrated that an ulvan extract of Ulva armoricana is able to activate avian heterophils and monocytes in vitro. Using specific inhibitors, we have evidenced that ulvan may be a new ligand for TLR2 and TLR4; and that they regulate heterophil activation in slightly different manner. Moreover, activation of heterophils as well as of monocytes leads to release pro-inflammatory cytokines, including interleukin1-β, interferon α and interferon γ, through pathways that we partly identified. Finally, when given per os to animals ulvan induces heterophils and monocytes to be activated in vivo thus leading to a transient release of pro-inflammatory cytokines with plasma concentrations returning toward baseline levels at day 3.

Keywords: TLR; avian; heterophil; inflammation; monocyte; ulvan.

Figure 1

Structure of the main disaccharide motifs present in ulvan. From the top to the bottom: β(1,4)-D-GlcA-α (1,4)-L-Rha 3 sulfate, β(1,4)-L-idoA-α (1,4)-L-Rha 3 sulfate, β(1,4)-D-xyl-α (1,4)-L-Rha 3 sulfate, β(1,4)-D-xyl 2-sulfate-α(1,4)-L-Rha 3 sulfate, where X represents the continuation of the polysaccharide chain. GlcA, glucuronic acid; Rha, rhamnose; IdoA, iduronic acid; Xyl, xylose.

Figure 2

Ulvan activates heterophils in vitro in a time- and dose-dependent manner. Heterophils (8 × 10⁶/ml) were incubated in vitro with different concentrations of ulvan, PAM as TLR2 agonist, LPS as TLR4 agonist, and glycogen as a control of polysaccharide specificity. Activation was evidenced by the quantification of degranulation as evidenced by β-D-glucuronidase ability to generate 4-methylum-belliferone (A); and the measurement of the oxidative burst as evidenced by fluorescein fluorescence (B). Data represents the mean ± SEM of five independent experiments in triplicate. *p < 0.05, **p < 0.01 for statistically different values for the same dose at different times. Different letters with the same color indicate statistically different values for the different doses at the same time (p < 0.05).

Figure 3

(A) Ulvan causes NO release by monocytes. Ulvan triggers NO release in a dose- and time dependent manner, measured using Griess' reagent. Data represents the mean ± SEM of four independent experiments in duplicate. Letters indicate statistically different values for the different doses at the same time with p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.005 when values are statistically different for the same dose at different times. (B) Ulvan acts through TLR2- and TLR4-dependent mechanisms. Monocytes were incubated in vitro with different concentrations of ulvan, PAM as TLR2 agonist, LPS as TLR4 agonist, and glycogen as a control of polysaccharide specificity. The monocytes were first incubated for 30 min with the inhibitors before addition of the ulvan extract at 25 μg/ml for 4 h. Data represents the mean ± SEM of four independent experiments in duplicate. Different symbols with the same color indicate statistically different values with p < 0.01, different numbers mean statistically different values with p < 0.005.

Figure 4

Ulvan acts in vivo when given per os. Ulvan was given per os at four different concentrations (0, 10, 25, 50 mg/l) to the animals at day 0. Blood samples (1ml per animal) were taken every day from day 0 to day 3. The NO concentration and β-D-glucuronidase activity were quantified individually in the plasma, the latter via the 4-methylum-belliferone formation. A dose response release is observed for both NO (A) and β-D-glucuronidase (B) with a peak at day 1. Data represents the mean ± SEM of three independent experiments with 25 chickens per group. Different letters with the same color indicate statistically different values for the different doses at the same time with p < 0.05, different symbols with the same color indicate statistically different values for the different doses at the same time with p < 0.01. *p < 0.05, **p < 0.01, and ***p < 0.005 when values are statistically different for the same dose at different times.

Figure 5

Heterophils respond to ulvan by modifying the transcription pattern of cytokine genes. Ulvan was given per os at four different concentrations (0, 10, 25, 50 mg/l) to the animals at day 0. Blood samples (1 ml per animal) were taken every day from day 0 to day 3. Cellular pellets of three animals were pooled to obtain sufficient number of cells for purification and RT-qPCR. Data represents the mean ± SEM of three independent experiments with 25 chickens per group. Different letters with the same color indicate statistically different values for the different doses at the same time with p < 0.05.

Figure 6

Cytokines mRNA are transcribed in monocytes in response to ulvan. Ulvan was given per os at four different concentrations (0, 10, 25, 50 mg/l) to the animals at day 0. Blood samples (1 ml per animal) were taken from day 0 to day 3. Cellular pellets of three animals were pooled to obtain sufficient number of cells for purification and RT-qPCR. Data represents the mean ± SEM of three independent experiments with 25 chickens per group. Different letters with the same color indicate statistically different values for the different doses at the same time with p < 0.05.

Figure 7

IL1β, IFNα, and IFNγ are released in a context of transient and moderate inflammation context. Ulvan was given per os at four different concentrations (0, 10, 25, 50 mg/l) to the animals at day 0. Blood samples (1 ml per animal) were taken every day from day 0 to day 3. ELISA were performed on each plasma to quantify the concentrations of IL1β and IFNα (A), IFNγ (B), C-Reactive Protein (C), and haptoglobin (D). Data represents the mean ± SEM of three independent experiments with 25 chickens per group. Different letters with the same color indicate statistically different values for the different doses at the same time with p < 0.05. *p < 0.05, when values are statistically different for the same dose at different times.