Effects of dietary lysine restriction on inflammatory responses in piglets

The aim of this study was to investigate the effects of lysine restriction on inflammatory responses in piglets. 38 male piglets with similar body weight of 9.62 kg were randomly divided into control group (basal diet) and lysine-restricted group (diet containing 70% lysine of the control diet). The results showed that lysine restriction increased the serum concentration of IgG an IgM. Piglets fed the lysine-restricted diet exhibited overexpression of interleukin-8 (IL-8) in the kidney (P < 0.05) and IL-6 and IL-4 in the spleen (P < 0.05). The mRNA abundances of IL-4 in the kidney (P < 0.05) and IL-10 in the liver (P < 0.05) were significantly lower in the lysine-restricted group compared with the control group. Meanwhile, lysine restriction increased the mRNA level of Tlr8 in the kidney (P < 0.05) but decreased the mRNA level of Tlr8 in the liver (P < 0.05). Finally, lysine restriction markedly enhanced extracellular signal regulated kinases 1/2 (ERK1/2) phosphorylation in the kidney and liver and nuclear transcription factor kappa B (NF-κB) was activated in the liver and spleen in response to dietary lysine restriction. In conclusion, lysine restriction affected inflammatory responses in the kidney, liver, and spleen via mediating serum antibody volume, inflammatory cytokines, Tlrs system, and ERK1/2 and NF-κB signals in piglets.


Lysine restriction influenced anti-inflammatory cytokines.
Dietary lysine restriction decreased the mRNA level of IL-4 in the kidney (P < 0.05) and mRNA level of IL-10 in the liver (P < 0.05). While the IL-4 mRNA level in the spleen were markedly higher in the lysine-restricted group compared to the control group (P < 0.05) (Fig. 2).

Effects of lysine restriction on toll-like receptors (Tlrs) system. Tlrs are widely demonstrated to
involve in the activation of inflammatory response. Thus, expressions of Tlr3, 4, 7, 8, 9, and Myd88 were determined in the kidney, liver, and spleen (Fig. 3). Lysine restriction increased the mRNA level of Tlr8 in the kidney (P < 0.05) but decreased the mRNA level of Tlr8 in the liver (P < 0.05). Furthermore, lysine restriction exhibited little effect on expression of other Tlrs and myeloid differentiation 88 (Myd88) in the kidney, liver, and spleen.
Lysine restriction induced the abundance of extracellular signal regulated kinases 1/2 (ERK1/2) and nuclear transcription factor kappa B (NF-κB) proteins. ERK1/2 signal was markedly activated in the kidney and liver and NF-κB signal was upregulated in the liver and spleen of lysine restricted piglets evidenced by the enhanced phosphorylation ratio of ERK1/2 and NF-κB (P < 0.01) (Fig. 4).

Discussion
Lysine is the first limiting amino acid for piglets and is one of the building blocks for the synthesis of proteins 8 . For this reason, inadequate lysine intake can limit the synthesis of inflammatory-related proteins (including cytokines) 9 . Numerous studies have demonstrated that the intake of amino acid affect the inflammatory responses of animals 7,10 . What's more, it was reported that the deficiency of dietary lysine also impaired animal immune responses 9,11 .  The serum antibody volume has been widely used to evaluated the humoral immunity 12 . IgG and IgM, two major serum immunoglobulins, are key components humoral immunity in all mammals 13 and protect the extravascular compartment against pathogenic virus and microorganisms 9 . In this study, dietary lysine restriction decreased the serum concentration of IgG and IgM. Pro-inflammatory cytokines (including IL-6 and IL-8)  serve as an important role in mediating inflammatory and immune responses [14][15][16][17] . IL-4 is involved in all major aspects of inflammatory responses 18 . IL-10, an anti-inflammatory cytokine, down-regulates macrophage activity in swine 19,20 . In this study, the mRNA abundance of IL-8 in the kidney and IL-6 in the spleen were significantly . The ration of phosphorylated JNK abundance to total JNK in the kidney (A), liver (D), and spleen (G) of piglets fed a basal diet (100% lysine) or a lysine-restricted diet containing 70% lysine of the basal diet. The ration of phosphorylated ERK1/2 abundance to total ERK1/2 in the kidney (B), liver (E), and spleen (H) of piglets fed a basal diet (100% lysine) or a lysine-restricted diet containing 70% lysine of the basal diet. The ration of phosphorylated NF-κB abundance to NF-κB in the kidney (C), liver (F), and spleen (I) of piglets fed a basal diet (100% lysine) or a lysine-restricted diet containing 70% lysine of the basal diet. Each image shows three samples from 100% Lysine group and three samples from 70% Lysine group. *Different from control, P < 0.01. higher in the lysine-restricted group compared with the control group. We also found that lysine restriction markedly decreased the abundance of IL-4 in the kidney and IL-10 in the liver, but significantly increased the abundance of IL-4 in the spleen. Tlrs are a family of pathogen recognition receptors which promote innate immunity 14 . Tlrs activate the expression of pro-inflammatory, such as IL-6 and TNF-α 21 . Myd88 plays an important role in the Tlr signaling pathway 22 . Dietary arginine supplementation has effects on the activation of Tlrs 23 . In the present study, lysine restriction influenced the expression of Tlr8 in the kidney and liver of piglets. These results showed that lysine restriction affect inflammatory response via mediating serum antibody volume, inflammatory cytokines, and Tlrs. Notably, the current results showed a tissue-dependent of gene expressions, which might be caused by different functions of these tissues. For example, liver mainly contributes to metabolism and kidney involves in excretion and re-absorption. Similarly, we also noticed that expressions of Tlr system varied from different sections of intestine (duodenum, jejunum, and ileum) 24 .
NF-κB pathway plays an important role in inflammation by mediating synthesis of pro-inflammatory (i.e. IL-6 and IL-8) 25 . Mitogen-activated protein kinase (MAPK) pathway involves in nuclear translocation of NF-κB and contributes to the production of inflammatory cytokines 26,27 . ERKs and c-Jun N-terminal protein kinase (JNK) are members of MAPK family, which is associated with inflammation 28 . Amino acids have been demonstrated to activate NF-κB and MAPK signaling pathways to regulate expression of pro-inflammatory cytokines and inflammation 23 . Similarly, in this study, lysine restriction activated ERK1/2 and NF-κB signals, which might further involve in immune and inflammatory responses. Our previous study has revealed that lysine deficiency induced apoptosis 5 , which is highly associated with inflammatory response 29 . Thus, it is not surprising to uncover that lysine restriction induces inflammatory response.
Taken together, this study indicated that lysine restriction can induce inflammatory via mediating serum concentration of IgG and IgM, the expression of inflammatory cytokines, Tlrs, and ERK1/2 and NF-κB signals in the kidney, liver, and spleen of piglets.

Materials and Methods
Animals and Experimental Design. This study was conducted in accordance with the guidelines of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. All experimental protocols were approved by animal ethical committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. 38 male piglets (about 35-day old, 9.62 ± 0.30 kg) were randomly divided into 2 groups: a control group and a lysine-restricted group. Piglets in the control group were received the basal diet according to the NRC (2012) ( Table 2), whereas piglets in the lysine-restricted group were fed a lysine-restricted diet containing 70% lysine of the control group. Piglets were individually housed in cages and had ad libitum access to drinking water and feed for 21 days. Then 7 animals were sampled randomly from each group. Blood samples from the overnight fasting piglets were collected in plastic uncoated tubes. Sera were obtained by centrifugation at 3000 rpm for 20 min and stored at −20 °C until analysis for IgG and IgM. After blood sampling, the piglets were sacrificed for kidney, liver, and spleen collection.

Real-Time Quantitative RT-PCR.
Total RNA was isolated from liquid nitrogen-frozen kidney, liver, and spleen using TRIZOL reagent (Invitrogen, USA) and then treated with DNase I (Invitrogen, USA) according to the instructions of the manufacturer. Synthesis of the first strand (cDNA) was performed with PrimeScript Enzyme Mix 1, RT Primer Mix, and 5 × PrimerScript Buffer 2. The reverse transcription was conducted at 37 °C for 15 m, 85 °C for 5 s. Primers (Table 3) used in this study were presented in the previous study [23][24][25] . β-actin was used as a housekeeping gene to normalize target gene transcript levels. Real-time PCR was performed according to our previous study 3 . Briefly, 1 μl cDNA template was added to a total volume of 10 μl containing 5 μl SYBR Green mix, 0. Western blot analysis. The expression of protein in the kidney, liver and spleen was determined by Western blot analysis as described previously. Briefly, about 50 µg of total protein obtained from samples were extracted by a reducing SDS-PAGE electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes and blocked with 5% nonfat milk in tris-Tween-buffered saline buffer (20 mM tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1.5 hour. Then the primary antibodies were incubated overnight at 4°C and the horseradish peroxidase-conjugated secondary antibodies were subsequently incubated for 1.5 hour at room temperature before development of the blot using the Alpha Imager 2200 software (Alpha Innotech Corporation, CA, USA). We quantified the resultant signals and normalize the data to the abundance of β-actin according to our previous reports. Statistical analysis. All data were analyzed between two groups using the student's T test (SPSS 16.0 software). Data are expressed as the mean ± SEN. Differences of p < 0.05 are considered significant.  Table 3. Primers used for quantitative reverse transcription PCR.