Edited by: Janice C. Telfer, University of Massachusetts Amherst, United States
Reviewed by: Hao-Ching Wang, Taipei Medical University, Taiwan; Jesus Hernandez, Centro de Investigación en Alimentación y Desarrollo (CIAD), Mexico
This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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
Over recent years, there has been an increase in the use of oral passive immunotherapy, in humans as well as in livestock, partly in both cases to reduce antibiotic therapy or prophylaxis. The crucial role and specificity of the innate immune response in driving and controlling adaptive immune responses to particular pathogens is now beginning to be understood and manipulated. It has been demonstrated that the Th1/Th2 paradigm applies in chicken (
Since some PRR ligands display polysaccharidic motifs (
Therefore, the aim of the present study was to determine whether an ulvan extract of
Green tide algae
Sterile peripheral blood was obtained during routine follow-up of 28 days of age animals using heparin (20 U/ml) as anti-coagulant. The poultry veterinarians of the research team assessed the sanitary status before each experiment. Animals were raised without the use of antibiotics or any immune system stimulating chemicals from birth until blood sampling; the last vaccine was performed no later than day 12 of life for return to baseline immune parameters before the experiment. Blood from three chickens was pooled for each
Production of an oxidative burst by heterophils was quantified by oxidation of the non-fluorescent DCFH-DA (Dichlorofluorescein-diacetate, Sigma) to fluorescent DCF (Dichlorofluorescein) as described previously (
Heterophils degranulation was measured in five independent experiments in triplicate by quantifying the amount of β-D-glucuronidase activity in the culture medium following stimulation of the heterophils as previously described (
Monocytes were incubated with the ulvan extract in five independent experiments in duplicate. The cells were cultured in a RPMI-1640 glucose medium (2 g/l glucose) at 41°C with 5% of CO2, as previously described (
All experiments on heterophils as well as on monocytes were carried out with glycogen as a negative control (10 μg/ml), LPS (Sigma) as TLR4 agonist (10 μg/ml), Pam3CSK4 as TLR2 agonist (10 μg/ml, Invivogen, further designed as PAM) as previously described (
Inhibitors and their relative targets.
Chloroquinin | Cytoplasmic TLR (7/9/21) | 100 μM | InvivoGen |
2-aminopurine | TLR4/9/21 and PKR | 5 mM | InvivoGen |
OxPAPC | TLR2/4 | 50 μM | InvivoGen |
Polymyxin B | TLR4 | 100 μM | InvivoGen |
Anti-mTLR2-IgG | TLR2 | 0.66 nM | InvivoGen |
YM201636 | TLR9 | 5 μM | InvivoGen |
Gefitinib | NOD (RIP2) | 20 μM | InvivoGen |
Piceatannol | Dectin | 10 μM | InvivoGen |
Glybenclamide | NLRP3 | 50 μM | InvivoGen |
Parthenolide | NLRP1/3 | 40 μM | InvivoGen |
Wortmannin | PI3K | 40 nM | Sigma |
Gö 6983 | PKC | 100 nM | Sigma |
D609 | PLC | 100 μM | Sigma |
SB203580 | p38MAPK | 40 μM | InvivoGen |
SP600125 | JNK | 50 μM | InvivoGen |
PD98059 | ERK | 200 μM | InvivoGen |
Celastrol | NF-KB | 10 μM | InvivoGen |
Total RNA devoid of genomic DNA contamination was extracted with RNeasy Plus Mini Kit (Qiagen) and TRIzol® (Life Technologies) according to the manufacturers' instructions. Total RNA (200 ng) was used for first-strand cDNA synthesis with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). RT-qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) for all transcripts. All determinations were performed in duplicate and normalized against actin as the internal control gene. The results are expressed as the relative gene expression with the DeltaDeltaCt method. Fold change = 2−[(Ct target gene in sample−Ct actin in sample)−(Ct target gene in untreated cells−Ct actin in untreated cells)] (
Three hundred male broiler chickens (28 days old at day 0) with a Ross 308 genetic background, obtained from a local commercial hatchery, were used in three independent experiments. This research was approved by the Brest University ethics committee in compliance with French laws and regulations. The experiments were conducted on adult animals to allow sufficient volume for blood sampling and in order to have fully functional heterophils (
Individual quantification of plasma concentrations of glucuronidase activity and NO were performed as described above, while C-reactive protein (CRP), haptoglobin, interleukin1-β (IL1β), interferons α and γ (IFNα, IFNγ) concentrations were determined using ELISA kits as recommended by the manufacturer (Elabsciences). Heterophils and monocytes were purified as previously described to allow RT-qPCR experiments.
Comparisons between groups were performed on at least three independent experiments for
The protein content in the dry matter was 8.9 ± 0.3%. No fatty acids and no endotoxins could be detected. The average contents were 40.2 ± 0.7% for neutral sugars, 32.2 ± 0.8% for uronic acids, 8.3 ± 0.3% for sugar-bound sulfates. The monosaccharide composition evidenced the characteristic ulvan composition with, rhamnose, xylose, iduronic acid, and glucuronic acid (Figure
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.
Heterophils are considered as the poultry equivalents of mammalian neutrophils, and as such an integral part of the avian innate defenses against pathogens. Incubation with the ulvan extract leads to glucuronidase release by heterophils in a dose-dependent manner, with a peak at 3 h of incubation, as also observed for the positive controls LPS and PAM, but not the negative one, glycogen (Figure
Ulvan activates heterophils
Using selective inhibitors (Table
Heterophils are activated through TLR2 and TLR4 through PKC and PLC dependent mechanisms.
No inhibition | 100.00 | 100.00 | ||
TLR2 | 48.54 ± 3.98 | 44.75 ± 2.49 | ||
TLR4 | 52.08 ± 5.15 | 57.43 ± 3.38 | ||
TLR2+4 | 10.08 ± 1.34 | 9.45 ± 4.52 | ||
TLR4+21 | 50.08 ± 6.11 | 48.58 ± 3.44 | ||
TLR21 | 98.47 ± 4.35 | None | 94.04 ± 2.16 | None |
Cytoplasmic TLR | 96.45 ± 3.17 | None | 96.58 ± 2.02 | None |
NOD | 95.61 ± 3.85 | None | 98.52 ± 2.35 | None |
Dectin | 98.72 ± 3.26 | None | 96.43 ± 3.54 | None |
NLRP3 | 94.24 ± 3.49 | None | 95.31 ± 2.81 | None |
NLRP1+3 | 98.80 ± 3.75 | None | 98.84 ± 2.64 | None |
PKC | 45.783 ± 3.26 | 59.73 ± 4.91 | ||
TLR2 + PKC | 18.19 ± 4.11 | 28.59 ± 3.16 | ||
TLR4 + PKC | 24.01 ± 5.22 | 30.97 ± 2.89 | ||
PLC | 58.15 ± 3.51 | 56.06 ± 4.99 | ||
TLR2 + PLC | 52.99 ± 4.88 | 25.63 ± 3.95 | ||
TLR4 + PLC | 23.15 ± 5.44 | 25.88 ± 3.34 | ||
PI3K | 64.85 ± 2.79 | 79.07 ± 5.48 | None | |
p38MAPK | 90.82 ± 2.92 | None | 86.96 ± 4.74 | None |
JNK | 90.62 ± 4.67 | None | 84.70 ± 5.84 | None |
ERK | 88.41 ± 2.49 | None | 85.49 ± 3.59 | None |
NF-KB | 86.68 ± 2.98 | None | 77.69 ± 9.04 | None |
We then investigated the intracellular proteins required for the degranulation and the oxidative burst. As shown in Table
The transcription pattern of IL1β, IFNα, IFNγ, IL8, and IL18 varied in a dose- and time-dependent manner in response to the ulvan extract (Table
Ulvan triggers cytokine transcription in heterophils.
IL1β | 2 h | 1.01 ± 0.11 | 1.00 ± 0.09a | 5.10 ± 0.46b | 17.10 ± 1.76c | 41.02 ± 3.60d | |
4 h | 0.99 ± 0.08 | 0.94 ± 0.09a | 6.35 ± 0.70b | 31.27 ± 3.83c | 92.71 ± 9.53d | ||
IFNα | 2 h | 1.06 ± 0.12 | 1.00 ± 0.14a | 2.24 ± 0.03b | 4.32 ± 0.31c | 17.61 ± 1.29d | |
4 h | 0.94 ± 0.09 | 0.98 ± 0.10a | 2.70 ± 0.26b | 16.43 ± 1.52c | 63.29 ± 5.67d | ||
IFNγ | 2 h | 1.09 ± 0.09 | 1.00 ± 0.10a | 6.51 ± 0.54b | 9.57 ± 1.02c | 37.60 ± 2.81d | |
4 h | 0.96 ± 0.10 | 0.97 ± 0.09a | 6.02 ± 0.69b* | 67.28 ± 6.50# | 294.94 ± 31.22& | ||
IL8 | 2 h | 0.97 ± 0.05 | 1.02 ± 0.02a | 2.43 ± 0.18b | 4.28 ± 0.26c | 5.54 ± 0.44d | None |
4 h | 1.05 ± 0.11 | 1.07 ± 0.10a | 2.06 ± 0.22b | 3.81 ± 0.36c | 6.38 ± 0.63d | ||
IL18 | 2 h | 1.02 ± 0.11 | 0.98 ± 0.10 | 1.20 ± 0.15 | 1.26 ± 0.12a | 2.27 ± 0.19b | |
4 h | 0.98 ± 0.09 | 0.97 ± 0.10a | 1.35 ± 0.12b | 2.57 ± 0.26c | 5.41 ± 0.55d | ||
TLR2 | 2 h | 1.05 ± 0.09 | 1.01 ± 0.10 | 1.13 ± 0.94a | 2.01 ± 0.13b | 4.61 ± 0.32c | |
4 h | 0.99 ± 0.09 | 0.97 ± 0.10a | 1.68 ± 0.12b | 4.91 ± 0.54c | 13.24 ± 1.30d | ||
TLR4 | 2 h | 0.99 ± 0.08 | 1.00 ± 0.10 | 1.25 ± 0.09 | 0.96 ± 0.09a | 2.62 ± 0.20b | |
4 h | 1.00 ± 0.09 | 0.97 ± 0.08 | 1.15 ± 0.09a | 2.77 ± 0.21b | 7.58 ± 0.68c |
Heterophil activation also resulted in raised transcription of TLR2 and TLR4 receptors, with TLR2 being the most affected (Table
Transcription in heterophils is TLR2 and TLR4 dependent and involves intracellular mediators.
No inhibitor | 46.53 ± 1.64 | 22.12 ± 1.57 | 88.75 ± 3.49 | 4.08 ± 0.11 | 3.74 ± 0.16 | 6.56 ± 0.32 | 5.13 ± 0.33 |
TLR2 | 18.44 ± 2.57 |
11.41 ± 0.99 |
48.46 ± 1.19 |
1.70 ± 0.15 |
1.67 ± 0.19 |
2.37 ± 0.15 |
2.54 ± 0.14 |
TLR4 | 13.81 ± 1.45 |
10.27 ± 0.26 |
48.29 ± 0.75 |
1.93 ± 0.18 |
1.64 ± 0.08 |
2.22 ± 0.21 |
3.26 ± 0.28 |
TLR2+4 | 1.46 ± 0.13 |
1.15 ± 0.12 |
1.14 ± 0.15 |
0.96 ± 1.10 |
0.92 ± 0.10 |
2.14 ± 0.27 |
2.61 ± 0.19 |
TLR4+21 | 11.59 ± 1.08 |
10.67 ± 0.24 |
49.97 ± 0.76 |
1.95 ± 0.25 |
1.73 ± 0.17 |
2.50 ± 0.03 |
3.08 ± 0.04 |
TLR9 | 47.60 ± 1.46 | 21.61 ± 0.93 | 83.65 ± 6.98 | 4.015 ± 0.23 | 3.48 ± 0.26 | 6.51 ± 0.23 | 5.07 ± 0.38 |
Cytoplas mic TLR | 47.43 ± 2.11 | 20.56 ± 2.25 | 91.73 ± 5.58 | 3.87 ± 0.2 | 3.51 ± 0.20 | 6.12 ± 0.28 | 5.27 ± 0.15 |
NOD | 46.75 ± 2.00 | 19.86 ± 1.13 | 88.51 ± 4.14 | 4.096 ± 0.28 | 3.59 ± 0.12 | 6.24 ± 0.42 | 5.16 ± 0.06 |
Dectin | 47.21 ± 2.58 | 21.79 ± 1.61 | 88.03 ± 3.48 | 4.029 ± 0.05 | 3.88 ± 0.41 | 6.33 ± 0.33 | 5.28 ± 0.40 |
NLRP1+3 | 47.18 ± 2.54 | 22.97 ± 1.82 | 86.16 ± 4.20 | 4.10 ± 0.21 | 3.83 ± 0.29 | 6.59 ± 0.21 | 5.18 ± 0.28 |
NLRP3 | 47.42 ± 2.22 | 21.70 ± 2.42 | 85.59 ± 3.85 | 3.86 ± 0.26 | 3.79 ± 0.29 | 6.32 ± 0.22 | 4.43 ± 0.75 |
NF-KB | 6.2 ± 0.64 |
15.52 ± 1.19 |
64.23 ± 7.91 |
1.84 ± 0.22 |
1.81 ± 0.13 |
2.43 ± 0.19 |
1.72 ± 0.13 |
ERK | 7.51 ± 0.55 |
15.59 ± 1.57 |
66.51 ± 6.45 |
1.95 ± 0.10 |
2.06 ± 0.21 |
6.10 ± 0.59 | 4.36 ± 0.40 |
JNK | 2.80 ± 0.34 |
18.46 ± 1.79 | 89.81 ± 7.49 | 1.29 ± 0.14 |
1.24 ± 0.11 |
6.63 ± 0.66 | 4.17 ± 0.38 |
p38 MAPK | 2.92 ± 0.04 |
22.18 ± 1.96 | 85.79 ± 9.62 | 1.29 ± 0.14 |
1.42 ± 0.17 |
4.2 ± 0.31 | 3.77 ± 0.32 |
PLC | 48.44 ± 4.83 | 22.1 ± 1.65 | 83.52 ± 8.20 | 4.32 ± 0.41 | 4.35 ± 0.37 | 4.01 ± 0.42 | 3.36 ± 0.29 |
PKC | 48.83 ± 3.39 | 23.54 ± 1.78 | 79.41 ± 7.06 | 1.07 ± 0.08 | 4.29 ± 0.46 | 3.86 ± 0.37 | 3.39 ± 0.34 |
PI3K | 32.23 ± 2.34 | 23.69 ± 1.96 | 89.94 ± 8.40 | 4.06 ± 0.35 | 3.60 ± 0.35 | 16.87 ± 1.48 |
13.56 ± 1.66 |
TLR2 and TLR4 pathways regulate transcription in heterophils through common intracytoplasmic mediators.
No inhibitor | 43.53 ± 3.51 | 21.33 ± 1.405 | 88.03 ± 1.89 | 4.31 ± 2.93 | 3.76 ± 0.36 | 6.40 ± 0.59 | 5.14 ± 0.56 |
TLR2 | 20.04 ± 2.41 | 11.76 ± 0.70 | 45.93 ± 4.42 | 1.84 ± 0.371 | 2.15 ± 0.22 | 2.71 ± 0.26 | 2.56 ± 0.11 |
TLR2 + NFKB | 7.21 ± 0.54 |
6.18 ± 0.56 |
25.81 ± 1.71 |
1.08 ± 0.11 |
1.03 ± 0.11 |
1.23 ± 0.18 |
1.45 ± 0.15 |
TLR2 + ERK | 7.50 ± 0.22 |
7.81 ± 0.65 |
26.49 ± 1.97 |
1.00 ± 0.03 |
0.97 ± 0.10 |
1.97 ± 0.18 | 3.44 ± 0.27 |
TLR2 + JNK | 3.45 ± 0.27 |
11.42 ± 0.98 | 41.81 ± 2.63 | 0.99 ± 0.07 |
0.45 ± 0.03 |
2.39 ± 0.24 | 3.65 ± 0.21 |
TLR2 + p38 | 3.11 ± 0.14 |
10.69 ± 0.82 | 45.14 ± 5.36 | 0.91 ± 0.05 |
0.53 ± 0.05 |
2.60 ± 0.30 | 3.83 ± 0.28 |
TLR2 + PLC | 19.41 ± 1.60 | 10.43 ± 1.29 | 42.46 ± 3.42 | 1.50 ± 0.08 | 1.87 ± 0.15 | 2.60 ± 0.23 | 3.23 ± 0.31 |
TLR2 + PKC | 19.72 ± 0.71 | 12.19 ± 1.16 | 43.52 ± 3.91 | 1.45 ± 0.12 | 1.78 ± 0.68 | 2.59 ± 0.27 | 3.08 ± 0.30 |
TLR2 + PI3K | 16.44 ± 0.44 | 11.33 ± 0.77 | 45.16 ± 4.13 | 1.50 ± 0.16 | 1.71 ± 0.13 | 4.04 ± 0.41 |
5.57 ± 0.49 |
TLR4 | 14.21 ± 0.80 | 10.46 ± 1.12 | 48.62 ± 4.17 | 1.86 ± 0.20 | 1.74 ± 0.18 | 2.41 ± 0.19 | 2.70 ± 0.21 |
TLR4 + NFKB | 5.78 ± 0.47 |
5.34 ± 0.48 |
27.13 ± 2.32 |
0.51 ± 0.03 |
0.71 ± 0.06 |
1.32 ± 0.14 |
1.01 ± 0.10 |
TLR4 + ERK | 7.62 ± 0.44 |
6.55 ± 0.72 |
22.36 ± 1.28 |
0.53 ± 0.04 |
0.73 ± 0.06 |
1.85 ± 0.18 | 2.49 ± 0.22 |
TLR4 + JNK | 3.25 ± 0.31 |
9.86 ± 0.86 | 41.62 ± 3.89 | 0.53 ± 0.04 |
0.81 ± 0.08 |
2.29 ± 1.17 | 2.07 ± 0.21 |
TLR4 + p38 | 2.01 ± 0.13 |
9.68 ± 0.86 | 43.38 ± 4.11 | 0.61 ± 0.06 |
0.49 ± 0.04 |
2.14 ± 0.22 | 2.43 ± 0.21 |
TLR4 + PLC | 13.44 ± 0.88 | 10.24 ± 1.01 | 44.05 ± 4.29 | 1.61 ± 0.17 | 1.75 ± 0.18 | 1.97 ± 0.20 | 2.22 ± 0.18 |
TLR4 + PKC | 13.21 ± 0.65 | 10.43 ± 0.96 | 46.20 ± 3.96 | 1.63 ± 0.21 | 1.55 ± 0.17 | 2.27 ± 0.20 | 2.17 ± 0.18 |
TLR4 + PI3K | 13.03 ± 0.76 | 11.76 ± 1.31 | 46.71 ± 4.55 | 1.48 ± 0.12 | 1.67 ± 0.14 | 4.43 ± 0.43 |
6.98 ± 0.61 |
Another leader cell in innate immunity is the monocyte, due to its ability to synthesize NO and cytokines, and to link innate and adaptive immunity (
The transcription of IFNβ, IL10, IL17, and IL18 genes was not affected by ulvan (Supplemental Table
Ulvan triggers cytokine transcription in monocytes.
IL1β | 2h | 1.00 ± 0.07 | 1.03 ± 0.08 | 1.20 ± 0.09 | 1.15 ± 0.09 | 1.26 ± 0.08 | p < 0.05 for ulvan 10μg/ml and 25μg/ml, p < 0.01 for 25μg/ml and 50 μg/ml |
4h | 0.92 ± 0.09 | 0.97 ± 0.07a | 1.63 ± 0.21b | 5.61 ± 0.48c* | 50.95 ± 3.72# | ||
IFNα | 2h | 0.99 ± 0.08 | 1.01 ± 0.08 | 1.13 ± 0.09 | 1.16 ± 0.11 | 1.51 ± 0.09 | p < 0.05 for ulvan 25μg/ml and p < 0.005 for 50 μg/ml |
4h | 1.00 ± 0.09 | 1.00 ± 0.09a | 2.58 ± 0.16b* | 21.41 ± 0.96# | 145.08 ± 12.69& | ||
IFNγ | 2h | 1.02 ± 0.09 | 0.98 ± 0.08a | 2.70 ± 0.012b | 4.07 ± 0.19c | 7.69 ± 0.25d | p < 0.05 for ulvan 25μg/ml and p < 0.005 for 50 μg/ml |
4h | 1.02 ± 0.08 | 0.97 ± 0.10a | 2.97 ± 0.21b* | 17.61 ± 1.53# | 147.51 ± 12.69& | ||
IL8 | 2h | 1.00 ± 0.10 | 0.98 ± 0.09 | 1.37 ± 0.12a | 2.74 ± 0.16b | 4.05 ± 0.33c | none |
4h | 1.03 ± 0.09 | 0.09 ± 0.10 | 1.02 ± 0.11a | 1.92 ± 0.15b | 3.51 ± 0.28c | ||
IL13 | 2h | 1.02 ± 0.08 | 0.97 ± 0.09 | 1.07 ± 0.09 | 1.30 ± 0.11a | 2.72 ± 0.18b | p < 0.05 for ulvan 10μg/ml and 25μg/ml and p < 0.01 for 50 μg/ml |
4h | 1.00 ± 0.09 | 1.17 ± 0.16a | 2.48 ± 0.17b | 5.46 ± 0.51c* | 19.01 ± 1.41# | ||
TLR2 | 2h | 0.99 ± 0.09 | 0.96 ± 0.10a | 1.56 ± 0.13b | 1.81 ± 0.17c | 3.55 ± 0.25d | p < 0.05 for ulvan 25μg/ml and p < 0.01 for 50 μg/ml |
4h | 1.00 ± 0.09 | 0.98 ± 0.09a | 1.37 ± 0.10b | 6.60 ± 0.52c | 26.72 ± 1.94d | ||
TLR4 | 2h | 0.97 ± 0.08 | 1.00 ± 0.09 | 1.44 ± 0.12a | 2.26 ± 0.21b | 4.59 ± 0.33c | p < 0.05 for ulvan 10μg/ml and 25μg/ml and p < 0.01 for 50 μg/ml |
4h | 0.99 ± 0.09 | 1.10 ± 0.17a | 1.93 ± 1.14b | 6.60 ± 0.52c* | 26.72 ± 1.96# | ||
iNOS | 2h | 1.00 ± 0.10 | 0.09 ± 0.09 | 1.34 ± 0.10a | 3.52 ± 0.35b* | 19.89 ± 2.05# | p < 0.05 for ulvan 10μg/ml, p < 0.01 for 25μg/ml and p < 0.005 for 50 μg/ml |
4h | 0.97 ± 0.10 | 0.98 ± 0.09a | 4.47 ± 0.27b | 30.64 ± 2.16c# | 259.62 ± 11.59& |
Transcription in monocytes relies on TLR2 and TLR4 activation and requires intra-cellular mediators that differ according to the genes.
No inhibitor | 24.93 ± 2.43 | 42.96 ± 3.21 | 64.54 ± 5.01 | 12.09 ± 1.43 | 4.25 ± 0.50 | 12.39 ± 0.93 | 29.84 ± 2.18 | 53.59 ± 4.69 |
TLR2 | 12.24 ± 1.12a | 15.14 ± 1.53b | 28.89 ± 0.84a | 4.01 ± 0.38a | 2.14 ± 0.12a | 5.41 ± 0.27a | 15.62 ± 1.15a | 27.34 ± 2.23a |
TLR4 | 10.43 ± 0.97a | 11.79 ± 1.54b | 26.32 ± 0.62a | 5.04 ± 0.55a | 1.78 ± 0.12a | 8.69 ± 0.17a | 11.40 ± 1.01a | 25.58 ± 2.40a |
TLR2+4 | 1.16 ± 1.28c | 2.08 ± 0.19c | 1.09 ± 0.09c | 0.99 ± 0.08c | 1.21 ± 0.11a | 1.08 ± 0.10b | 1.16 ± 0.11c | 2.89 ± 0.30c |
TLR4+21 | 4.74 ± 0.35b | 11.79 ± 0.10b | 24.30 ± 0.71a | 5.74 ± 0.32a | 1.88 ± 0.19a | 7.78 ± 0.21a | 12.51 ± 0.14a | 25.87 ± 2.41a |
TLR9 | 20.13 ± 2.08 | 39.58 ± 2.85 | 58.44 ± 4.45 | 11.95 ± 1.53 | 4.23 ± 0.07 | 11.62 ± 0.84 | 25.09 ± 2.31 | 55.74 ± 2.30 |
Cytoplasmic TLR | 25.05 ± 3.69 | 41.88 ± 3.56 | 68.33 ± 6.04 | 11.78 ± 1.43 | 4.33 ± 0.19 | 12.36 ± 0.86 | 27.92 ± 2.47 | 54.58 ± 3.08 |
NOD | 26.57 ± 0.70 | 43.27 ± 2.27 | 67.48 ± 6.12 | 12.45 ± 1.47 | 4.11 ± 0.08 | 12.08 ± 0.99 | 29.67 ± 3.23 | 50.56 ± 3.95 |
Dectin | 24.49 ± 1.41 | 44.31 ± 2.36 | 66.87 ± 5.97 | 12.35 ± 1.74 | 4.13 ± 0.32 | 11.93 ± 1.02 | 30.55 ± 3.61 | 54.77 ± 4.38 |
NLRP1+3 | 24.73 ± 1.81 | 42.24 ± 2.27 | 67.30 ± 6.66 | 1.81 ± 0.96 | 4.24 ± 0.15 | 11.59 ± 0.99 | 29.67 ± 3.01 | 54.39 ± 4.70 |
NLRP3 | 25.31 ± 1.82 | 41.91 ± 1.36 | 64.07 ± 5.85 | 12.52 ± 1.60 | 4.48 ± 0.24 | 12.16 ± 1.05 | 27.78 ± 2.67 | 52.99 ± 3.36 |
NF-KB | 3.66 ± 0.32b | 5.96 ± 0.55c | 5.70 ± 0.52c | 2.88 ± 0.23a | 2.49 ± 0.19a | 5.34 ± 0.30a | 4.43 ± 0.36a | 8.92 ± 0.82c |
ERK | 6.34 ± 0.59b | 44.86 ± 4.81 | 44.61 ± 4.22a | 4.26 ± 0.38a | 4.12 ± 0.42 | 12.38 ± 1.12 | 32.51 ± 2.85 | 51.75 ± 3.85 |
JNK | 11.14 ± 1.12a | 44.12 ± 4.34 | 66.85 ± 5.86 | 4.17 ± 0.47a | 4.05 ± 0.37 | 11.72 ± 1.06 | 30.70 ± 2.72 | 55.49 ± 3.95 |
p38 MAPK | 4.02 ± 0.35b | 43.36 ± 3.98 | 64.97 ± 4.79 | 4.58 ± 0.34a | 4.55 ± 0.31 | 13.03 ± 1.23 | 29.95 ± 2.62 | 57.23 ± 4.21 |
PLC | 14.71 ± 1.13a | 43.87 ± 4.08 | 68.40 ± 5.95 | 11.50 ± 1.06 | 4.23 ± 0.41 | 12.85 ± 1.25 | 30.69 ± 2.96 | 54.75 ± 5.22 |
PKC | 13.1 ± 1.11a | 41.42 ± 4.12 | 65.92 ± 5.19 | 11.55 ± 1.14 | 4.12 ± 0.39 | 12.69 ± 1.30 | 31.33 ± 3.12 | 53.74 ± 5.29 |
PI3K | 13.41 ± 1.24a | 41.15 ± 3.75 | 65.73 ± 6.23 | 3.67 ± 0.19a | 4.23 ± 0.43 | 18.74 ± 1.82a | 57.72 ± 4.16b | 117.73 ± 9.86c |
TLR2 and TLR4 pathways regulate transcription in monocytes through common intracytoplasmic mediators.
No inhibitor | 25.39 ± 0.66 | 43.40 ± 2.18 | 66.39 ± 2.67 | 11.90 ± 1.09 | 4.41 ± 0.39 | 12.22 ± 1.13 | 29.45 ± 2.28 | 55.72 ± 4.63 |
TLR2 | 12.22 ± 1.58 | 15.06 ± 1.48 | 27.39 ± 1.41 | 4.48 ± 0.23 | 2.16 ± 0.16 | 5.98 ± 0.52 | 15.14 ± 1.48 | 26.14 ± 2.13 |
TLR2 + NFKB | 1.41 ± 0.24b | 0.88 ± 0.21b | 1.83 ± 0.38b | 1.19 ± 0.21a | 0.90 ± 0.10a | 0.87 ± 0.09a | 1.04 ± 0.11b | 1.03 ± 0.10b |
TLR2 + ERK | 3.25 ± 0.31b | 12.97 ± 1.97 | 9.36 ± 0.77a | 1.43 ± 0.11a | 2.04 ± 0.018 | 2.63 ± 0.24 | 13.21 ± 1.19 | 22.68 ± 2.53 |
TLR2 + JNK | 5.24 ± 0.41a | 13.69 ± 0.96 | 27.46 ± 2.59 | 1.78 ± 0.13a | 2.04 ± 0.17 | 3.14 ± 0.25 | 15.38 ± 1.49 | 26.15 ± 2.05 |
TLR2 + p38 | 1.87 ± 0.15b | 13.00 ± 1.41 | 24.93 ± 1.57 | 1.63 ± 0.14a | 2.00 ± 0.11 | 3.08 ± 0.29 | 14.73 ± 1.31 | 25.53 ± 2.24 |
TLR2 + PLC | 5.66 ± 0.34a | 14.63 ± 2.72 | 27.80 ± 2.29 | 4.39 ± 0.46 | 2.03 ± 0.20 | 3.24 ± 0.23 | 14.33 ± 1.07 | 24.09 ± 2.19 |
TLR2 + PKC | 4.74 ± 0.30a | 12.49 ± 1.18 | 26.58 ± 2.64 | 4.49 ± 0.42 | 2.11 ± 0.18 | 3.24 ± 0.31 | 14.91 ± 1.44 | 25.95 ± 1.99 |
TLR2 + PI3K | 3.19 ± 1.20b | 13.09 ± 1.54 | 28.13 ± 1.83 | 2.77 ± 0.10a | 2.14 ± 0.21 | 10.76 ± 1.03a | 30.82 ± 2.96a | 48.23 ± 4.29a |
TLR4 | 10.44 ± 1.50 | 11.123 ± 1.17 | 21.99 ± 2.04 | 5.55 ± 0.48 | 1.85 ± 0.13 | 8.09 ± 0.88 | 15.99 ± 1.48 | 5.561 ± 5.26 |
TLR4 + NFKB | 1.73 ± 0.14b | 0.94 ± 0.18b | 2.32 ± 0.52b | 1.91 ± 0.10a | 0.92 ± 0.08a | 1.68 ± 1.14b | 1.06 ± 0.10b | 0.98 ± 0.11b |
TLR4 + ERK | 5.85 ± 0.54a | 11.60 ± 1.08 | 14.45 ± 1.48a | 2.29 ± 0.25a | 1.73 ± 0.14 | 10.94 ± 1.05 | 15.64 ± 1.41 | 5.66 ± 0.52 |
TLR4 + JNK | 7.60 ± 0.72a | 11.99 ± 2.03 | 21.21 ± 2.19 | 2.11 ± 0.23a | 1.78 ± 0.15 | 10.88 ± 1.01 | 15.30 ± 1.49 | 5.30 ± 0.52 |
TLR4 + p38 | 1.68 ± 0.13b | 12.42 ± 1.57 | 7.12 ± 0.68 | 2.31 ± 0.21a | 1.78 ± 0.12 | 11.27 ± 1.12 | 14.06 ± 1.33 | 5.75 ± 0.51 |
TLR4 + PLC | 7.34 ± 0.72a | 10.98 ± 1.06 | 22.88 ± 2.34 | 5.51 ± 0.59 | 1.78 ± 0.16 | 10.68 ± 1.02 | 14.84 ± 1.41 | 5.84 ± 0.57 |
TLR4 + PKC | 7.47 ± 0.74a | 10.85 ± 1.09 | 20.78 ± 2.56 | 5.30 ± 0.48 | 1.79 ± 0.13 | 9.76 ± 0.95 | 15.71 ± 1.47 | 5.78 ± 5.57 |
TLR4 + PI3K | 6.99 ± 0.69a | 10.76 ± 1.07 | 20.06 ± 1.95a | 2.24 ± 0.21a | 1.76 ± 0.16 | 18.74 ± 1.32a | 28.815 ± 2.85a | 10.670 ± 1.57a |
To address the potential biological effects of ulvan
The NO concentration was quantified in plasma from individual chickens as described above, since it mirrors, at least in part, monocyte activation. A dose- and time-dependent release was observed with maximal concentrations at day 1 with values of 9.99 ± 0.85 μM for the negative control, 29.72 ± 1.34 μM with 10 mg/l ulvan, 48.22 ± 1.51 μM with 25 mg/l ulvan, and 81.8 ± 6.50 μM with 50 mg/l ulvan (Figure
Ulvan acts
Release of granules by heterophils, and potentially by monocytes, was measured by quantification of the amount of β-D-glucuronidase activity. As shown in Figure
In order to gain insight into the mechanisms of action of ulvan, RT-qPCR analyses were carried out on monocytes and heterophils purified from blood samples taken at 0, 24, 48, and 72 h after ulvan administration. Since blood samples were only 1 ml per animal, samples were first centrifuged to separate the plasma from the cells. The cellular pellets of three animals were then pooled to obtain sufficient amount of cells for purification.
Heterophils responded as early as day 1 by tuning on the transcription of the pro-inflammatory genes for IL1β, IFNα, IFNγ, and to a lower extent those of IL8, TLR2, and TLR4 (Figure
Heterophils respond to ulvan by modifying the transcription pattern of cytokine genes. Ulvan was given
The transcription patterns observed for the monocytes appeared to differ somewhat from the ones for heterophils. Common to both cellular types is the fact that the acute phase of the response was detected at day 1. However, despite increased fold changes, the transcription of IL1β gene was less affected in monocytes than in heterophils with values at day 1 ranging only from 0.89 ± 0.08 with 10 mg/l ulvan to 7.63 ± 0.82 with 50 mg/l ulvan (Figure
Cytokines mRNA are transcribed in monocytes in response to ulvan. Ulvan was given
To address whether the variations in mRNA amounts correlated with cytokines release, ELISA assays were performed for IL1β, IFNα, and IFNγ (Figure
IL1β, IFNα, and IFNγ are released in a context of transient and moderate inflammation context. Ulvan was given
As IFNα and IFNγ may bridge over other cellular populations for activation, and as autocrine and/or paracrine loops may occur, we assessed the extent of the inflammation initiated by the ulvan, at an early stage by quantifying C - Reactive Protein (CRP), and at a later stage by measuring haptoglobin. CRP was synthetized by the liver in response to ulvan in a dose-dependent manner. The plasmastic CRP concentrations reached 1.08 ± 0.06 ng/ml without ulvan supplementation and rose at day 1 to 9.74 ± 0.36 ng/ml with 10 mg/l ulvan and 37.49 ± 0.85 ng/ml with 50 mg/l ulvan (Figure
The immune system is constantly exposed to a large variety of threatening and potentially damaging agents and uses complex cellular and molecular mechanisms to determine the appropriate response to each situation: Whether to activate the adaptive immunity or if the innate immune response may be sufficient. The latter one involves different populations of mononuclear cells (monocytes, macrophages, NK, NKT, B, and γδ T lymphocytes) and polynuclear cells.
The primary polymorphonuclear leukocyte in chicken is the heterophil. It provides a rapid deployment of the effector arm of the innate immune system in birds, displaying a variety of pathogen recognition receptors, including toll-like receptors (TLRs), which account for the recognition of a multitude of pathogens. The TLR family in chickens consists of ten genes, where TLR2 and TLR4 are orthologs to mammal TLRs (
In our experiments, heterophils constitutively express TLR2 and TLR4, as previously reported (
As human TLR4 has been described to bind palmitic acid (
Results for the degranulation are consistent with the ones for the burst, thus confirming that ulvan is a less potent activator of chTLR2 and chTLR4 than PAM and LPS
When analyzing the transduction pathways, neither the burst nor the degranulation were statistically significantly affected by the specific inhibitors against p38MAPK, JNK, ERK, NF-κB, as previously described (
Two proteins appear as the main regulators of both degranulation and oxidative burst, PKC and PLC. Interestingly, for the burst, they both seem to act simultaneously on chTLR4 and chTLR2 transduction pathways and with the same efficiency (Table
Due to the lack of myeloperoxidase, avian heterophils produce only weak amounts of NO and no Neutrophil Extracellular Trap as myeloperoxidase is required for this release (
In a second step, we examined whether chTLR2 and chTLR4 activation with ulvan may result in modifications of the transcription pattern for heterophils and monocytes. We focused on cytokines involved in the innate immune response. As previously described, we have observed that TLR stimulation on heterophils
This mechanism may be common to heterophils and monocytes, as the same chTLR2 and chTLR4 regulation pattern arises in monocytes. In addition, we observed that the iNOS gene is regulated in a similar manner to the TLR2 and TLR4 genes in monocytes. Our previous hypothesis may thus also apply to this gene, in order to allow a sufficient amount of NO to be released for pathogen killing, but not too excessive or too long lasting in order to avoid cellular damage to healthy cells. Our results are consistent with those of Peroval et al for the avian HD11 macrophage cell line with the same inhibitors for PI3K, (wortmaninn), NF-κB, p38MAPK and ERK (PD98059), with PAM and LPS as agonists for TLR2 and TLR4, respectively (
However, the transcriptional regulation appears to differ somewhat between heterophils and monocytes for IL1β, IL8, IL18, IFNα, and IFNγ, thus suggesting that other cell-type specific mechanisms could be involved. This would for instance explain the discrepancy of IL13 gene regulation observed between the two cellular types.
Nevertheless, in both cellular types TRL4 and TLR2 share common intracellular mediators. Within a cellular type each mediator acts in a similar extent on TLR2 and TRR4 pathways (Tables
Given the
Plasma concentration of CRP rose in a dose dependent-manner to values that are above the normal (1.56–8.6 ng/ml, according to the manufacturer) except for the control group and the 10 mg/l ulvan group. All groups were back to normal as soon as day 2. Furthermore, haptoglobin concentrations did not vary and remained within normal range for all the groups (93–186 ng/ml, according to the manufacturer). Ulvan thus appears to induce a transient and moderate inflammation. In addition, as no change in the animals' behavior was observed for the three independent experiments (from 1 week before the experiment until slaughtering), and as Ct value and Delta Ct values of all the studied genes are extremely close between the
We have evidenced, for the first time to our knowledge, that β-D-glucuronidase, a lysosomal enzyme, was released in a dose dependent manner at day 1 after ulvan oral intake. This reflects heterophil activation and potentially monocyte activation (
In line with this result, NO was also present in plasma. Nevertheless, we cannot exclude that cellular types other than monocytes may contribute to NO synthesis, as demonstrated for instance in mammalian endothelial cells (
We did not detect any variation for IFNβ mRNA,
ChIFNα is strongly induced in response to a number of viral infections, such as influenza A virus and Newcastle disease virus (
In addition, IFNα has been described to promote murine NK cells expansion by protecting them from fratricide (
As in mammals, chIFNγ is essential for host defense against intracellular pathogens and a hallmark of Th1 immunity (
Moreover, St Paul et al reported IL1β, IFNα, and IFNγ genes to be more transcribed in the spleen of birds that received a mixture of TLR4 and TLR21 agonists (
Finally, we cannot rule out that ulvan also act through the modulation of GALT functions. TLR2 and TLR4 expression has been described throughout the avian digestive tract despite the fact that the subpopulations were not purified (
We report for the first time that ulvan activates TLR4 and TLR2 on avian heterophils and monocytes.
Further translational and fundamental studies are necessary to fully understand its mode of action.
NG, FB, ML, and PC contributed to the conception and design of the study, including the protocol for the ethical committee. CG and PC prepared the ulvan extract. FB, CG, OM, BQ, and ML prepared and performed the
FB, CG, ML, and PC were employed by company Amadeite. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was financed by a grant from the BPI-France/ISI Ulvans project (n°: I1110001W) to Amadéite company, Bréhan (France), a project certified by Pôle Mer Bretagne.
The authors would like to acknowledge Michael Theron, head of the biology department UBO, the ORPHY team (Christine Moisan, Christelle Goanvec) for their support and the lipidocean plateform (Fabienne Guerard, Philippe Soudant, Fabienne Legrand) for its help in analyzing lipids.
The Supplementary Material for this article can be found online at:
RMN proton analysis. The samples were dissolved on 99.97% atome D2O and subjected to RMN proton analysis. The RMN proton spectrum was registered at 298 K on a Bruker Avance 500 spectrometer with a inversed cryogenic probe 5 mm 1H/13C/15N TCI. The isotopic shifts were referenced with respect to an external standard (trimethylsilypropionic acid). No suppression of the HOD signal was performed.
Lipids were extracted with CHCl3/MeOH (2/1; v/v) and the amount of palmitic acid quantified using GC-FID (gas chromatography with flame ionization detector) according to Le Croizier et al. (
Primers sequences and relative references.
Fold change that do not vary statistically in heterophils
Fold change that do not vary statistically in monocytes
Animals weight during the three
Fold change that do not vary statistically in heterophils
Δ-Ct values for all the studied genes in heterophils during the three
Fold change that do not vary statistically in monocytes
Δ-Ct values for all the studied genes in monocytes during the three