- Email: [email protected]
Tissue specificity and species superiority of cathelicidin gene expression in Chinese indigenous Min pigs
Livestock Science 161 (2014) 36–40
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Tissue specificity and species superiority of cathelicidin gene expression in Chinese indigenous Min pigs Q.Q. Ma a, W.J. Jiao a, Z.Y. Wang a, C.X. Wu a, A.S. Shan a,n, Y.B. Wang b, J.M. Cai b a b
Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China Breeding Pig Farm, Lanxi County, Heilongjiang Province 151531, China
a r t i c l e in f o
abstract
Article history: Received 12 September 2013 Received in revised form 2 January 2014 Accepted 4 January 2014
Antimicrobial peptides (AMPs) are components of innate immunity, forming the first-line of defense used by many organisms against the invading pathogens. Min pigs are a Chinese indigenous breed with low mortality and strong disease resistance. In this study, the mRNA expressions of four cathelicidins (PMAP-23, PMAP-37, PR-39, and protegrin-1), the largest family of AMPs in pigs, were determined by real-time PCR in 14-day-old Min pigs and Landrace pigs were chosen as the control. The results showed that expression of four cathelicidin mRNAs in most tissues were higher in Min pigs than those of Landrace pigs, which may partly explain the higher immunity and disease resistance of Min pigs. The cathelicidin molecules were generally expressed at high levels in thymus, spleen, liver, and heart, and at low levels in ileum, jejunum, tongue, and lymph node in both breeds. The peptide pairs with significant correlation in one breed were generally not correlated with these in the other breed, suggesting differential synergistic or antagonistic regulation of cathelicidins in Min pigs and Landrace pigs. The high expression of cathelicidins might be one of the mechanisms by which the Min pigs display strong disease resistance. & 2014 Elsevier B.V. All rights reserved.
Keywords: Chinese Min pigs Antimicrobial peptide Cathelicidin Gene expression
1. Introduction Antimicrobial peptides (AMPs) are a unique and diverse group of molecules produced by all classes of life, considered to be part of the host innate immunity (Peters et al., 2010). There is a growing body of evidence that their role in defense against microbes is as important to the host as antibodies, immune cells, and phagocytes (Hancock and Scott, 2000; Tang et al., 2009). Cathelicidins are the largest family of AMPs in pigs (Lehrer and Ganz, 2002). Cathelicidins show a marked structural diversity and include broad-spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacterial species, certain fungi, parasites or enveloped viruses, significant proinflammatory property, and effective defense against invasive bacterial infection (Nizet and Gallo, 2003). Cathelicidins have been defined to play multiple roles in immunity contributing to both resolution of infections and
n
Corresponding author. Tel./fax: þ 86 451 55190685. E-mail address: [email protected] (A.S. Shan).
1871-1413/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2014.01.004
inflammation (Choi and Mookherjee, 2012). Cathelicidin AMP restricted the development of lesions produced by Leishmania and was crucial for the local control of cutaneous lesion development and parasite growth and metastasis (Kulkarni et al., 2011). Cathelicidin peptide displays chemotactic activity for eosinophils and neutrophils, and may play a role in inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (Tjabringa et al., 2006). Cathelin-related AMP differentially regulates B- and T-cell function and implicates mCRAMP in the regulation of adaptive immune responses (Kin et al., 2011). Anti-inflammatory mediators such as IL-10, TNFAIP3, NF-κB inhibitor NFκBIA are enhanced by cathelicidin peptides (Brown et al., 2011). They share a fairly conserved N-terminal prosequence of approximately 100 residues named cathelin domain (Zanetti, 2004). Based on primary amino-acid composition, porcine cathelicidins can be assigned to three subgroups: linear proline-rich (PR-39), disulfide-rich (protegrins), and a highly arginine/histidine-containing subgroup (PMAPs) (Lehrer and Ganz, 2002a, 2002b; Sang and Blecha, 2009; Zanetti, 2005).
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
Chinese Min pigs, a special Chinese indigenous pig breed, are distributed in northeast area with cold climate and characteristic of strong disease resistance with low mortality (Xu, 1989). But the mechanism is not yet elucidated. Previous studies proved that higher immunity and disease resistance of Tibetan (Qi et al., 2009) and Meishan (Chen et al., 2010) pigs was correlated with higher expression of AMPs. Here we hypothesize that the trait of Min pigs may be related to the gene expression of AMPs. Therefore, mRNA gene expressions of cathelicidins were determined in various tissues for 14-day-old Min pigs and Landrace pigs. Here we choose typical cathelicidin members PMAP-23, PMAP-37, protegrin-1, and PR-39 and these AMPs all exert strong antimicrobial activity against bacteria with MICs generally ranged from 0.1 mM to 16 μM (Agerberth et al., 1991; Steinberg et al., 1997; Tossi et al., 1995; Zanetti et al., 1994). In addition to its antibacterial activity, these AMPs have several other important functions and they may be potent anti-inflammatory drug to prevent bacteria infections, wound healing, and other inflammatory responses (Korthuis et al., 1999; Steinberg et al., 1997; Tang et al., 2012a, 2012b; Yao et al., 2008; Zhang et al., 2000). We expect that the differential tissue distribution is identified between the two breeds of pigs. 2. Materials and methods 2.1. Animals Three 14-day-old healthy Landrace pigs and three 14-dayold healthy Min pigs (Wang et al., 2004) were obtained from Breeding Pig Farm of Lanxi County, China. The pigs were fed in an environment with the room temperature about 25 1C and natural lighting. The pigs were fasted 12 h and killed by exsanguination. Then the spleen, liver, lung, tongue, duodenum, lymph node, thymus, trachea, kidney, brain, ileum, jejunum, and heart were removed, rapidly frozen in liquid nitrogen within 30 min of slaughter, and stored at 80 1C until RNA extraction. All the animal experiments were done according to the guidelines for animal experiments at the National Institute of Animal Health. 2.2. Total RNA extraction and reverse transcription Total RNA was extracted by using E.Z.N.A.TM Total RNA Kit I (Omega) according to the manufacturer0 s instructions. The purity and concentration of total RNA were calculated by a spectrophotometer at 260 and 280 nm and the ratio of OD260 and OD280 ranged from 1.8 to 2.0. Then total RNA was reverse-transcribed using Superscript system according to the manufacture0 s directions (PrimeScriptTM RT reagent Kit, TaKaRa). The RT products (cDNA) were stored at 20 1C for relative quantitative real-time PCR. 2.3. Relative quantitative real-time PCR Relative quantitative real-time PCR analysis was used to test relative levels of transcript abundance for each AMP in different tissues. The cDNA obtained served as a template for the PCR amplification of reference gene β-actin and four cathelicidin genes. The gene-specific primers were designed
37
using Primer Express Software (PE Applied Biosystems, CA) according to the sequences deposited in Genbank: PMAP-23 (L26053.1), PMAP-37 (L39641.1), PR-39 (L23825.1), and protegrin-1 (X79868.1). The sequences of these primers were as follows: PMAP-23 (forward 50 -AATCTCTACCGCCTCCTG-30 , reverse 50 -CGTTCTCCTTGAAGTCACAC-30 ), PMAP-37 (forward 50 -CTTAGCCGACTGCGTGAT-30 , reverse 50 -TAGCCTGAATCTTAGGACTGAA-30 ), PR-39 (forward 50 -GAACCCATCCATTCACTCAC-30 , reverse 50 -TTATCAGCCACTCCATCACC-30 ), protegrin-1 (forward 50 -ACGGGCGGGTGAAACAGT-30 , reverse 50 -TCCGACACAGACGCAGAAC-30 ), and β-actin (forward 50 -ATGCTTCTAGGCGGACTGT-30 , reverse 50 -CCATCCAACCGACTGCT-30 ). All primers were purchased from Takara (Dalian, China). The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System (Applied Biosystems) with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR profile was: a pre-run at 95 1C for 30 s, followed by 40 cycles with a 5 s denaturation step at 95 1C, a 34 s annealing step at 60 1C and a 15 s extension step at 95 1C, followed by a final elongation at 72 1C for 5 min. All reactions were performed in triplicate and samples were distributed in 96-well plates. The specificity of each product was confirmed by analyzing the melt curve and single product specific melting temperatures (Tm) were produced as follows: PMAP-23, 88.9 1C; PMAP-37, 80.3 1C; PR-39, 86.8 1C; protegrin-1, 85.9 1C; and β-actin, 80.6 1C. The mean quantity values were calculated from the cycle threshold (Ct) values using the SDS 1.3.1 software by the double-standard curve method (Applied Biosystems). The sample without template was included as control in each assay and relative gene expression to the housekeeping gene β-actin was performed in order to correct for the variance in amounts of RNA input in the reactions. 2.4. Statistical analysis The relative gene expressions compared to the housekeeping gene β-actin were carried out using SPSS software (SPSS Inc., Chicago, IL) for analysis of one-way ANOVA among different tissues, followed by a T-test between two breed of pigs. Correlation analysis was performed for four cathelicidin gene expressions. Data were presented as mean 7 SEM. The results were considered significantly different at Po0.05. 3. Results The results indicated that no non-specific products or primer–dimers were generated during the PCR amplification cycles. Real-time PCR analysis revealed expression of cathelicidins mRNA in all tested tissues of 14-day-old Landrace and Min pigs. 3.1. Expression of PMAP-23 mRNA in tissues In Landrace pigs (Fig. 1), PMAP-23 was expressed at high levels in spleen, liver, and thymus, and at low levels in tongue, lymph node, trachea, small intestine (duodenum, jejunum, and ileum), and lung. In spleen, liver, and thymus, expression of PMAP-23 mRNA were 56-fold, 43-fold and 189-fold higher (Po0.05) respectively than those in the ileum (control tissue) of Landrace pigs.
38
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
400
2000 Landrace pigs Min pigs
Landrace pigs Min pigs
300
1000
PMAP-37/actin
PMAP-23/actin
1500
500 100
200 100
50
3.3. Expression of PR-39 mRNA in tissues In Landrace pigs (Fig. 3), PR-39 was expressed at high levels in thymus, liver, spleen, heart, and brain, and at low levels in ileum, lung, lymph node, trachea, and tongue. Expression of PR-39 mRNA in thymus, liver, spleen, and brain were 92-fold, 44-fold, 35-fold, and 36-fold higher (Po0.05) respectively than expression in the control tissue. Expression of PR-39 mRNA in tongue was 85-fold lower (P o0.05) than that in the control tissue. In Min pigs (Fig. 3), PR-39 was expressed at high levels in heart, liver, thymus, and brain, and at low levels in duodenum, lymph node, and tongue. Expression of PR-39 mRNA in heart, liver, thymus, and brain were 309-fold, 91-fold, 47fold, and 74-fold higher (Po0.05) than expression in the control tissue. Expression of PR-39 mRNA in tongue was 65fold lower (Po0.05) than that in the control tissue.
brain
tongue
heart
trachea
liver
lung
thymus
lymph node
kidney
spleen
6000 4000 2000 300 200 100
brain
tongue
trachea
heart
lung
liver
thymus
lymph node
kidney
spleen
jejunum
0 duodenum
In Landrace pigs (Fig. 2), PMAP-37 was expressed at high levels in spleen, brain, and heart, and at low levels in lung, ileum, and lymph node. In spleen, brain, and heart, the expression of PMAP-37 mRNA was 31-fold, 26-fold, and 25-fold higher (Po0.05) than those in the control tissue of Landrace pigs. In Min pigs (Fig. 2), PMAP-37 was expressed at high levels in liver, thymus, and heart, and at low levels in jejunum and ileum. The expression of PMAP-37 mRNA in liver, thymus, and heart were 59-fold, 35-fold, and 68-fold higher (Po0.05) than expression in the control tissue.
Landrace pigs Min pigs
8000
ileum
3.2. Expression of PMAP-37 mRNA in tissues
Fig. 2. Tissue distribution of PMAP-37 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative PMAP-37/β-actin levels plus SEM (n¼3).
PR-39/actin
In Min pigs (Fig. 1), PMAP-23 was also expressed at high levels in spleen, liver, thymus, and heart, and at low levels in tongue, lymph node, duodenum, ileum, and jejunum. In addition, PMAP-23 was also expressed at high levels in heart. The expression of PMAP-23 mRNA in spleen, liver, thymus, and heart were 22-fold, 70-fold, 91-fold, and 23-fold higher (P o0.05) than expression in the control tissue (the ileum of Min pigs).
duodenum
ileum
brain
tongue
heart
trachea
liver
lung
thymus
lymph node
kidney
spleen
duodenum
ileum
jejunum
Fig. 1. Tissue distribution of PMAP-23 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative PMAP-23/β-actin levels plus SEM (n¼3).
jejunum
0
0
Fig. 3. Tissue distribution of PR-39 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative PR-39/β-actin levels plus SEM (n¼ 3).
3.4. Expression of protegrin-1 mRNA in tissues In Landrace pigs (Fig. 4), protegrin-1 was expressed at high levels in liver, thymus, and spleen, and at low levels in trachea, ileum, and lymph node. Expression of protegrin-1 mRNA in liver and thymus were 113-fold and 73-fold higher (Po0.05) respectively than expression in the control tissue. In Min pigs (Fig. 4), protegrin-1 was expressed at high levels in liver, thymus, and heart, and at low levels in jejunum and ileum. Expression of protegrin-1 mRNA in liver, thymus, and heart were 223-fold (Po0.05), 30fold, and 31-fold higher than expression in ileum. 3.5. Comparison of gene expression between the two breeds The comparisons for gene expression of cathelicidins between Min and Landrace pigs were exhibited in Figs. 1–4. Overall, expressions of four cathelicidin mRNAs in most tissues were higher in Min pigs than those of Landrace pigs.
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
80000
protegrin-1/actin
(Po0.01), and 0.67 (P¼0.01) respectively. Another two pairs of peptides (PMAP-23 and PR-39, and PR-39 and protegrin-1) were significantly related in Landrace pigs and the Pearson correlation coefficients were 0.89 (Po0.01) and 0.72 (P¼0.01) respectively. For PMAP-23 and protegrin-1, they were significantly correlated in both Landrace and Min pigs and the coefficient were 0.66 (P¼0.01) and 0.64 (P¼ 0.02) respectively.
Landrace pigs Min pigs
60000
39
40000 20000 3000
1500
4. Discussion brain
tongue
trachea
lung
heart
liver
thymus
lymph node
kidney
spleen
duodenum
ileum
jejunum
0
Fig. 4. Tissue distribution of protegrin-1 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative protegrin-1/β-actin levels plus SEM (n¼ 3).
1
Coefficients
0.75
Landrace pigs
*
*
*
* *
*
Min pigs
*
0.5
0.25
0 A
B
C
D
E
F
-0.25 Fig. 5. Correlation analysis of peptides in Landrace and Min pigs. Y-axis represents Person correlation coefficients of cathelicidin gene expression. (A) PMAP-23 and PMAP-37; (B) PMAP-23 and PR-39; (C) PMAP-23 and protegrin-1; (D) PMAP-37 and PR-39; (E) PMAP-37 and protegrin-1; (F) PR-39 and protegrin-1. Column with a mark n means significant difference at Po0.05.
Obviously, PMAP-23 expression values of lung, trachea, brain, and heart for Min pigs were higher than those of Landrace pigs. For PMAP-37, values of liver, lymph node, thymus, kidney, ileum, and heart for Min pigs were higher than those of Landrace pigs. For protegrin-1, expression values of liver, lymph node, trachea, and heart for Min pigs were higher than those of Landrace pigs. No significant increases for PMAP-23, PMAP-37, and protegrin-1 were observed for Landrace pigs compared with Min pigs. For PR-39, expression values of lung, trachea, and heart for Min pigs were higher than those of Landrace pigs. mRNA expression in just a few tissues such as spleen, duodenum, and thymus for Min pigs were lower than those for Landrace pigs. 3.6. The expression correlations among cathelicidin genes The correlation analysis among cathelicidin genes expression in two pig breeds were carried out using SPSS software. As shown in Fig. 5, three pairs of peptides (PMAP23 and 37, PMAP-37 and PR-39, and PMAP-37 and protegrin-1) were significantly related in Min pigs and the Pearson correlation coefficients were 0.66 (P¼0.02), 0.86
In the present study, we detected mRNA expression abundance of cathelicidin members in Min and Landrace pigs. The cathelicidin family of host defense peptides includes a group of cationic and usually amphipathic peptides that display a variety of activities related to host defense function (Fu et al., 2008; Tomasinsig and Zanetti, 2005). The results showed that the expression of cathelicidin genes was detected in all tested tissues. It was previously reported that the expression of cathelicidins was not limited to lymphoid organs or leukocytes and had a broad distribution including skin, epithelial and brain tissue, which was consistent with this study (Brandenburg et al., 2012; Huttner and Bevins, 1999). Porcine cathelicidins were originally cloned from myeloid cells (PMAP-23 and 37) or neutrophils (PR-39 and protegrin-1) (Agerberth et al., 1991; Kokryakov et al., 1993; Tossi et al., 1995; Zanetti et al., 1994), and these cells play an important role in body immunity. In this study, mRNAs of these peptides were highly expressed at spleen and thymus, which are also importantly central immune organs. This was in agreement with previous reports (Brown and Hancock, 2006; Ganz, 2003). The thymus has a great effect upon the immunity of young animals (Qi et al., 2009). The expression of these peptides in other organs (such as brain, liver, and heart) that are exposed to the microbial environment may facilitate defense against opportunistic infections or have other functions beyond their antimicrobial activity (Chen et al., 2010). For cathelicidins, the expression pattern was different from defensins. Chen et al. (2010) reported that main expression sites for three pig β-defensins (pBD-1, 2 and 3) were tongue and oral mucosa in corssbred and Meishan pigs (the exception was pBD-2 of crossbred pig expressed in kidney and liver). In addition, high-level mRNA expressions of pBD-1 and 3 of crossbred pigs and Tibetan pigs were observed in tongue, oral epithelium and skin while pBD-2 was also in kidney and liver (Qi et al., 2009). But in this study, the cathelicidin molecules were highly expressed in thymus, spleen, liver, and heart while expressed at low levels in ileum, jejunum, tongue, and lymph node for Landrace and Min pigs. Although pig ages and breeds were different, this suggests differential tissue specificity and gene regulation of two important AMP families, cathelicidins and defensins. Especially in the intestine constitutively high levels of AMPs are unwanted because they could destroy the balance of the commensal intestinal microflora (Finlay and Hancock, 2004; Qi et al., 2009). It was reported that expressions of AMPs of Tibetan pigs and Meishan pigs in most tissues were higher than those of crossbred pigs and the higher expression of AMPs provide a direct evidence for
40
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
higher immunity and disease resistance (Chen et al., 2010; Qi et al., 2009). Similarly, in the present study mRNA abundance were higher in Min pigs than those of Landrace pigs in most tissues. The correlation analysis showed that significantly correlated gene pairs in Min pigs were generally not related in Landrace pigs. This hints that the two breeds may form differential synergistic or antagonistic regulation or different cathelicidin genes up-regulation or down-regulation mechanism in the long-term evolution. It was known that the cathelicidins constitutively stored as pro-peptides in peripheral PMN granules, where few or no transcripts are expressed (Zhang et al., 2000). We suppose that the cathelicidins may constitutively express in the tissues such as thymus and spleen and inducibly express in the tissues such as tongue and small intestine. In summary, we determined gene expression for cathelicidins in Min pigs and Landrace pigs and found characteristic expression patterns for four genes. The cathelicidin molecules were expressed at high levels in thymus, spleen, liver, and heart, and at low levels in ileum, jejunum, tongue, and lymph node in two breeds. In most tissues, cathelicidins had higher expression level in Min pigs than those in Landrace pigs. The high expression and wide abundance of cathelicidins may partly explain the strong disease resistance of Min pigs. Conflict of interest None. Acknowledgments This work was supported by grants from the National Key Technology R&D Program (2013BAD10B03), the China Postdoctoral Science Foundation (2012M510082 and 2013T60342), and the Open Projects of Key Laboratory of Feed Science, College of Heilongjiang Province. References Agerberth, B., Lee, J.Y., Bergman, T., Carlquist, M., Boman, H.G., Mutt, V., Jörnvall, H., 1991. Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline–arginine-rich antibacterial peptides. Eur. J. Biochem. 202, 849–854. Brandenburg, L.O., Merres, J., Albrecht, L.J., Varoga, D., Pufe, T., 2012. Antimicrobial peptides: multifunctional drugs for different applications. Polymers 2012 (4), 539–560. Brown, K.L., Hancock, R.E.W., 2006. Cationic host defense (antimicrobial) peptides. Curr. Opin. Immunol. 18, 24–30. Brown, K.L., Poon, G.F., Birkenhead, D., PRena, O.M., Falsafi, R., Dahlgren, C., Karlsson, A., Bylund, J., Hancock, R.E., Johnson, P., 2011. Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses. J. Immunol. 186, 5497–5505. Chen, J., Qi, S., Guo, R., Yu, B., Yu., B., Chen, D., 2010. Different messenger RNA expression for the antimicrobial peptides b-defensins between Meishan and crossbred pigs. Mol. Biol. Rep. 37, 1633–1639. Choi, K.Y.G., Mookherjee, N., 2012. Multiple immune-modulatory functions of cathelicidin host defense peptides. Front. Immunol. 3, 149. Finlay, B.B., Hancock, R.E., 2004. Can innate immunity be enhanced to treat microbial infections? Nat. Rev. Microbiol. 2, 497–504. Fu, J., Wenzel, S.C., Perlovan, O., Wang, J.P., Gross, F., Tang, Z.R., Yin, Y.L., Stewart, A.F., Muller, R., Zhang, Y.M., 2008. Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res. 36, 113–114.
Ganz, T., 2003. The role of antimicrobial peptides in innate immunity. Integr. Comp. Biol. 43, 300–304. Hancock, R.E.W., Scott, M.G., 2000. The role of antimicrobial peptides in animal defenses. Proc. Natl. Acad. Sci. USA 97, 8856–8861. Huttner, K.M., Bevins, C.L., 1999. Antimicrobial peptides as mediators of epithelial host defense. Pediatr. Res. 45, 785–794. Kin, N.W., Chen, Y., Stefanov, E.K., Gallo, R.L., Kearney, J.F., 2011. Cathelinrelated antimicrobial peptide differentially regulates T- and B-cell function. Eur. J. Immunol. 41, 3006–3016. Kokryakov, V.N., Harwig, S.S., Panyutich, E.A., Shevchenko, A.A., Aleshina, G.M., Shamova, O.V., Korneva, H.A., Lehrer, R.I., 1993. Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins. FEBS Lett. 327, 231–236. Korthuis, R.J., Gute, D.C., Blecha, F., Ross, C.R., 1999. PR-39, a proline/ arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction. Am. J. Physiol. 277, H1007–H1013. Kulkarni, M.M., Barbi, J., McMaster, W.R., Gallo, R.L., Satoskar, A.R., McGwire, B.S., 2011. Mammalian antimicrobial peptide influences control of cutaneous Leishmania infection. Cell. Microbiol. 13, 913–923. Lehrer, R.I., Ganz, T., 2002. Cathelicidins: a family of endogenous antimicrobial peptides. Curr. Opin. Hematol. 9, 18–22. Nizet, V., Gallo, R.L., 2003. Cathelicidins and innate defense against invasive bacterial infection. Scand. J. Infect. Dis. 35, 670–676. Peters, B.M., Shirtliff, M.E., Jabra-Rizk, M.A., 2010. Antimicrobial peptides: primeval molecules or future drugs? Plos Pathog. 6, e1001067. Qi, S., Chen., J., Guo, R., Yu, B., Chen, D., 2009. β-defensins gene expression in tissues of the crossbred and Tibetan pigs. Livest. Sci. 123, 161–168. Sang, Y., Blecha, F., 2009. Porcine host defense peptides: expanding repertoire and functions. Dev. Comp. Immunol. 33, 334–343. Steinberg, D.A., Hurst, M.A., Fujii, C.A., Kung, A.H., Ho, J.F., Cheng, F.C., Loury, D.J., Fiddes, J.C., 1997. Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity. Antimicrob. Agents Chemother. 41, 1738–1742. Tang, X., Fatufe, A.A., Yin, Y., Tang, Z., Wang, S, Liu, Z., Xin, W., Li, T., 2012. Dietary supplementation with recombinant lactoferrantmpinlactoferricin improves growth performance and affects serum parameters in piglets. J. Anim. Vet. Adv. 11, 2548–2555. Tang, X.S., Shao, H., Li, T.J., Tang, Z.R., Huang, R.L., Wang, S.P., Kong, X.F., Wu, X., Yin, Y.L., 2012. Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets. Appl. Biochem. Biotechnol. 168, 887–898. Tang, Z.R., Yin, Y.L., Zhang, Y.M., Huang, R.L., Sun, Z.H., Li, T.J., Chu, W.Y., Kong, X.F., Li, L.L., Geng, M.M., Tu, Q., 2009. Effects of dietary supplementation with an expressed fusion peptide bovine lactoferricin-lactoferrampin on performance, immune function and intestinal mucosal morphology in piglets weaned at age 21 d. Br. J. Nutr. 101, 998–1005. Tjabringa, G.S., Ninaber, D.K., Drijfhout, J.W., Rabe, K.F., Hiemstra, P.S., 2006. Human cathelicidin LL-37 is a chemoattractant for eosinophils and neutrophils that acts via formyl-peptide receptors. Int. Arch. Allergy Immunol. 140, 103–112. Tomasinsig, L., Zanetti, M., 2005. The Cathelicidins – structure, function and evolution. Curr. Protein Pept. Sci. 6, 23–34. Tossi, A., Scocchi, M., Zanettp, M., Storici, P., Gennaro, R., 1995. PMAP-37, a novel antibacterial peptide from pig myeloid cells cDNA cloning, chemical synthesis and activity. Eur. J. Biochem. 228, 941–946. Wang, Y.Z., Xu, Z.R., Lin, W.X., Huang, H.Q., Wang, Z.Q., 2004. Developmental gene expression of antimicrobial peptide PR-39 and effect of zinc oxide on gene regulation of PR-39 in piglets. Asian-Aust. J. Anim. Sci. 17, 1635–1640. Xu, Z.Y., 1989. Breed Characteristics of Indigenous Pig Breeds in China. Zhejiang Scientific and Technical Press. Hangzhou, Zhejiang Province, China. Yao, K., Yin, Y.L., Chu, W.Y., Liu, Z.Q., Dun, D., Li, T.J., Huang, R.L., Zhang, J.S., Tan, B., Wang, W.C., Wu, G.Y., 2008. Dietary arginine supplementation increases mTOR signaling activity in skeletal muscle of neonatal pigs. J. Nutr. 138, 867–872. Zanetti, M., 2004. Cathelicidins, multifunctional peptides of the innate immunity. J. Leukoc. Biol. 75, 39–48. Zanetti, M., 2005. The role of cathelicidins in the innate host defenses of mammals. Curr. Issues Mol. Biol. 7, 179–196. Zanetti, M., Storici, P., Tossi, A., Scocchi, M., Gennaro, R., 1994. Molecular cloning and chemical synthesis of a novel antimicrobial peptide derived from pig myeloid cells. J. Biol. Chem. 269, 7855–7858. Zhang, G., Ross, C.R., Blecha, F., 2000. Porcine antimicrobial peptides: new prospects for ancient molecules of host defense. Vet. Res. 31, 277–296l.
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Tissue specificity and species superiority of cathelicidin gene expression in Chinese indigenous Min pigs Q.Q. Ma a, W.J. Jiao a, Z.Y. Wang a, C.X. Wu a, A.S. Shan a,n, Y.B. Wang b, J.M. Cai b a b
Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China Breeding Pig Farm, Lanxi County, Heilongjiang Province 151531, China
a r t i c l e in f o
abstract
Article history: Received 12 September 2013 Received in revised form 2 January 2014 Accepted 4 January 2014
Antimicrobial peptides (AMPs) are components of innate immunity, forming the first-line of defense used by many organisms against the invading pathogens. Min pigs are a Chinese indigenous breed with low mortality and strong disease resistance. In this study, the mRNA expressions of four cathelicidins (PMAP-23, PMAP-37, PR-39, and protegrin-1), the largest family of AMPs in pigs, were determined by real-time PCR in 14-day-old Min pigs and Landrace pigs were chosen as the control. The results showed that expression of four cathelicidin mRNAs in most tissues were higher in Min pigs than those of Landrace pigs, which may partly explain the higher immunity and disease resistance of Min pigs. The cathelicidin molecules were generally expressed at high levels in thymus, spleen, liver, and heart, and at low levels in ileum, jejunum, tongue, and lymph node in both breeds. The peptide pairs with significant correlation in one breed were generally not correlated with these in the other breed, suggesting differential synergistic or antagonistic regulation of cathelicidins in Min pigs and Landrace pigs. The high expression of cathelicidins might be one of the mechanisms by which the Min pigs display strong disease resistance. & 2014 Elsevier B.V. All rights reserved.
Keywords: Chinese Min pigs Antimicrobial peptide Cathelicidin Gene expression
1. Introduction Antimicrobial peptides (AMPs) are a unique and diverse group of molecules produced by all classes of life, considered to be part of the host innate immunity (Peters et al., 2010). There is a growing body of evidence that their role in defense against microbes is as important to the host as antibodies, immune cells, and phagocytes (Hancock and Scott, 2000; Tang et al., 2009). Cathelicidins are the largest family of AMPs in pigs (Lehrer and Ganz, 2002). Cathelicidins show a marked structural diversity and include broad-spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacterial species, certain fungi, parasites or enveloped viruses, significant proinflammatory property, and effective defense against invasive bacterial infection (Nizet and Gallo, 2003). Cathelicidins have been defined to play multiple roles in immunity contributing to both resolution of infections and
n
Corresponding author. Tel./fax: þ 86 451 55190685. E-mail address: [email protected] (A.S. Shan).
1871-1413/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2014.01.004
inflammation (Choi and Mookherjee, 2012). Cathelicidin AMP restricted the development of lesions produced by Leishmania and was crucial for the local control of cutaneous lesion development and parasite growth and metastasis (Kulkarni et al., 2011). Cathelicidin peptide displays chemotactic activity for eosinophils and neutrophils, and may play a role in inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (Tjabringa et al., 2006). Cathelin-related AMP differentially regulates B- and T-cell function and implicates mCRAMP in the regulation of adaptive immune responses (Kin et al., 2011). Anti-inflammatory mediators such as IL-10, TNFAIP3, NF-κB inhibitor NFκBIA are enhanced by cathelicidin peptides (Brown et al., 2011). They share a fairly conserved N-terminal prosequence of approximately 100 residues named cathelin domain (Zanetti, 2004). Based on primary amino-acid composition, porcine cathelicidins can be assigned to three subgroups: linear proline-rich (PR-39), disulfide-rich (protegrins), and a highly arginine/histidine-containing subgroup (PMAPs) (Lehrer and Ganz, 2002a, 2002b; Sang and Blecha, 2009; Zanetti, 2005).
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
Chinese Min pigs, a special Chinese indigenous pig breed, are distributed in northeast area with cold climate and characteristic of strong disease resistance with low mortality (Xu, 1989). But the mechanism is not yet elucidated. Previous studies proved that higher immunity and disease resistance of Tibetan (Qi et al., 2009) and Meishan (Chen et al., 2010) pigs was correlated with higher expression of AMPs. Here we hypothesize that the trait of Min pigs may be related to the gene expression of AMPs. Therefore, mRNA gene expressions of cathelicidins were determined in various tissues for 14-day-old Min pigs and Landrace pigs. Here we choose typical cathelicidin members PMAP-23, PMAP-37, protegrin-1, and PR-39 and these AMPs all exert strong antimicrobial activity against bacteria with MICs generally ranged from 0.1 mM to 16 μM (Agerberth et al., 1991; Steinberg et al., 1997; Tossi et al., 1995; Zanetti et al., 1994). In addition to its antibacterial activity, these AMPs have several other important functions and they may be potent anti-inflammatory drug to prevent bacteria infections, wound healing, and other inflammatory responses (Korthuis et al., 1999; Steinberg et al., 1997; Tang et al., 2012a, 2012b; Yao et al., 2008; Zhang et al., 2000). We expect that the differential tissue distribution is identified between the two breeds of pigs. 2. Materials and methods 2.1. Animals Three 14-day-old healthy Landrace pigs and three 14-dayold healthy Min pigs (Wang et al., 2004) were obtained from Breeding Pig Farm of Lanxi County, China. The pigs were fed in an environment with the room temperature about 25 1C and natural lighting. The pigs were fasted 12 h and killed by exsanguination. Then the spleen, liver, lung, tongue, duodenum, lymph node, thymus, trachea, kidney, brain, ileum, jejunum, and heart were removed, rapidly frozen in liquid nitrogen within 30 min of slaughter, and stored at 80 1C until RNA extraction. All the animal experiments were done according to the guidelines for animal experiments at the National Institute of Animal Health. 2.2. Total RNA extraction and reverse transcription Total RNA was extracted by using E.Z.N.A.TM Total RNA Kit I (Omega) according to the manufacturer0 s instructions. The purity and concentration of total RNA were calculated by a spectrophotometer at 260 and 280 nm and the ratio of OD260 and OD280 ranged from 1.8 to 2.0. Then total RNA was reverse-transcribed using Superscript system according to the manufacture0 s directions (PrimeScriptTM RT reagent Kit, TaKaRa). The RT products (cDNA) were stored at 20 1C for relative quantitative real-time PCR. 2.3. Relative quantitative real-time PCR Relative quantitative real-time PCR analysis was used to test relative levels of transcript abundance for each AMP in different tissues. The cDNA obtained served as a template for the PCR amplification of reference gene β-actin and four cathelicidin genes. The gene-specific primers were designed
37
using Primer Express Software (PE Applied Biosystems, CA) according to the sequences deposited in Genbank: PMAP-23 (L26053.1), PMAP-37 (L39641.1), PR-39 (L23825.1), and protegrin-1 (X79868.1). The sequences of these primers were as follows: PMAP-23 (forward 50 -AATCTCTACCGCCTCCTG-30 , reverse 50 -CGTTCTCCTTGAAGTCACAC-30 ), PMAP-37 (forward 50 -CTTAGCCGACTGCGTGAT-30 , reverse 50 -TAGCCTGAATCTTAGGACTGAA-30 ), PR-39 (forward 50 -GAACCCATCCATTCACTCAC-30 , reverse 50 -TTATCAGCCACTCCATCACC-30 ), protegrin-1 (forward 50 -ACGGGCGGGTGAAACAGT-30 , reverse 50 -TCCGACACAGACGCAGAAC-30 ), and β-actin (forward 50 -ATGCTTCTAGGCGGACTGT-30 , reverse 50 -CCATCCAACCGACTGCT-30 ). All primers were purchased from Takara (Dalian, China). The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System (Applied Biosystems) with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR profile was: a pre-run at 95 1C for 30 s, followed by 40 cycles with a 5 s denaturation step at 95 1C, a 34 s annealing step at 60 1C and a 15 s extension step at 95 1C, followed by a final elongation at 72 1C for 5 min. All reactions were performed in triplicate and samples were distributed in 96-well plates. The specificity of each product was confirmed by analyzing the melt curve and single product specific melting temperatures (Tm) were produced as follows: PMAP-23, 88.9 1C; PMAP-37, 80.3 1C; PR-39, 86.8 1C; protegrin-1, 85.9 1C; and β-actin, 80.6 1C. The mean quantity values were calculated from the cycle threshold (Ct) values using the SDS 1.3.1 software by the double-standard curve method (Applied Biosystems). The sample without template was included as control in each assay and relative gene expression to the housekeeping gene β-actin was performed in order to correct for the variance in amounts of RNA input in the reactions. 2.4. Statistical analysis The relative gene expressions compared to the housekeeping gene β-actin were carried out using SPSS software (SPSS Inc., Chicago, IL) for analysis of one-way ANOVA among different tissues, followed by a T-test between two breed of pigs. Correlation analysis was performed for four cathelicidin gene expressions. Data were presented as mean 7 SEM. The results were considered significantly different at Po0.05. 3. Results The results indicated that no non-specific products or primer–dimers were generated during the PCR amplification cycles. Real-time PCR analysis revealed expression of cathelicidins mRNA in all tested tissues of 14-day-old Landrace and Min pigs. 3.1. Expression of PMAP-23 mRNA in tissues In Landrace pigs (Fig. 1), PMAP-23 was expressed at high levels in spleen, liver, and thymus, and at low levels in tongue, lymph node, trachea, small intestine (duodenum, jejunum, and ileum), and lung. In spleen, liver, and thymus, expression of PMAP-23 mRNA were 56-fold, 43-fold and 189-fold higher (Po0.05) respectively than those in the ileum (control tissue) of Landrace pigs.
38
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
400
2000 Landrace pigs Min pigs
Landrace pigs Min pigs
300
1000
PMAP-37/actin
PMAP-23/actin
1500
500 100
200 100
50
3.3. Expression of PR-39 mRNA in tissues In Landrace pigs (Fig. 3), PR-39 was expressed at high levels in thymus, liver, spleen, heart, and brain, and at low levels in ileum, lung, lymph node, trachea, and tongue. Expression of PR-39 mRNA in thymus, liver, spleen, and brain were 92-fold, 44-fold, 35-fold, and 36-fold higher (Po0.05) respectively than expression in the control tissue. Expression of PR-39 mRNA in tongue was 85-fold lower (P o0.05) than that in the control tissue. In Min pigs (Fig. 3), PR-39 was expressed at high levels in heart, liver, thymus, and brain, and at low levels in duodenum, lymph node, and tongue. Expression of PR-39 mRNA in heart, liver, thymus, and brain were 309-fold, 91-fold, 47fold, and 74-fold higher (Po0.05) than expression in the control tissue. Expression of PR-39 mRNA in tongue was 65fold lower (Po0.05) than that in the control tissue.
brain
tongue
heart
trachea
liver
lung
thymus
lymph node
kidney
spleen
6000 4000 2000 300 200 100
brain
tongue
trachea
heart
lung
liver
thymus
lymph node
kidney
spleen
jejunum
0 duodenum
In Landrace pigs (Fig. 2), PMAP-37 was expressed at high levels in spleen, brain, and heart, and at low levels in lung, ileum, and lymph node. In spleen, brain, and heart, the expression of PMAP-37 mRNA was 31-fold, 26-fold, and 25-fold higher (Po0.05) than those in the control tissue of Landrace pigs. In Min pigs (Fig. 2), PMAP-37 was expressed at high levels in liver, thymus, and heart, and at low levels in jejunum and ileum. The expression of PMAP-37 mRNA in liver, thymus, and heart were 59-fold, 35-fold, and 68-fold higher (Po0.05) than expression in the control tissue.
Landrace pigs Min pigs
8000
ileum
3.2. Expression of PMAP-37 mRNA in tissues
Fig. 2. Tissue distribution of PMAP-37 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative PMAP-37/β-actin levels plus SEM (n¼3).
PR-39/actin
In Min pigs (Fig. 1), PMAP-23 was also expressed at high levels in spleen, liver, thymus, and heart, and at low levels in tongue, lymph node, duodenum, ileum, and jejunum. In addition, PMAP-23 was also expressed at high levels in heart. The expression of PMAP-23 mRNA in spleen, liver, thymus, and heart were 22-fold, 70-fold, 91-fold, and 23-fold higher (P o0.05) than expression in the control tissue (the ileum of Min pigs).
duodenum
ileum
brain
tongue
heart
trachea
liver
lung
thymus
lymph node
kidney
spleen
duodenum
ileum
jejunum
Fig. 1. Tissue distribution of PMAP-23 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative PMAP-23/β-actin levels plus SEM (n¼3).
jejunum
0
0
Fig. 3. Tissue distribution of PR-39 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative PR-39/β-actin levels plus SEM (n¼ 3).
3.4. Expression of protegrin-1 mRNA in tissues In Landrace pigs (Fig. 4), protegrin-1 was expressed at high levels in liver, thymus, and spleen, and at low levels in trachea, ileum, and lymph node. Expression of protegrin-1 mRNA in liver and thymus were 113-fold and 73-fold higher (Po0.05) respectively than expression in the control tissue. In Min pigs (Fig. 4), protegrin-1 was expressed at high levels in liver, thymus, and heart, and at low levels in jejunum and ileum. Expression of protegrin-1 mRNA in liver, thymus, and heart were 223-fold (Po0.05), 30fold, and 31-fold higher than expression in ileum. 3.5. Comparison of gene expression between the two breeds The comparisons for gene expression of cathelicidins between Min and Landrace pigs were exhibited in Figs. 1–4. Overall, expressions of four cathelicidin mRNAs in most tissues were higher in Min pigs than those of Landrace pigs.
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
80000
protegrin-1/actin
(Po0.01), and 0.67 (P¼0.01) respectively. Another two pairs of peptides (PMAP-23 and PR-39, and PR-39 and protegrin-1) were significantly related in Landrace pigs and the Pearson correlation coefficients were 0.89 (Po0.01) and 0.72 (P¼0.01) respectively. For PMAP-23 and protegrin-1, they were significantly correlated in both Landrace and Min pigs and the coefficient were 0.66 (P¼0.01) and 0.64 (P¼ 0.02) respectively.
Landrace pigs Min pigs
60000
39
40000 20000 3000
1500
4. Discussion brain
tongue
trachea
lung
heart
liver
thymus
lymph node
kidney
spleen
duodenum
ileum
jejunum
0
Fig. 4. Tissue distribution of protegrin-1 mRNA in Landrace pigs and Min pigs. The real-time PCR was carried out in ABI 7500 Fast Real-Time PCR System with SYBR Premix Ex TaqTM Kit (Takara) according to the manufacturer0 s instructions. The PCR products were normalized to β-actin and values are depicted as relative protegrin-1/β-actin levels plus SEM (n¼ 3).
1
Coefficients
0.75
Landrace pigs
*
*
*
* *
*
Min pigs
*
0.5
0.25
0 A
B
C
D
E
F
-0.25 Fig. 5. Correlation analysis of peptides in Landrace and Min pigs. Y-axis represents Person correlation coefficients of cathelicidin gene expression. (A) PMAP-23 and PMAP-37; (B) PMAP-23 and PR-39; (C) PMAP-23 and protegrin-1; (D) PMAP-37 and PR-39; (E) PMAP-37 and protegrin-1; (F) PR-39 and protegrin-1. Column with a mark n means significant difference at Po0.05.
Obviously, PMAP-23 expression values of lung, trachea, brain, and heart for Min pigs were higher than those of Landrace pigs. For PMAP-37, values of liver, lymph node, thymus, kidney, ileum, and heart for Min pigs were higher than those of Landrace pigs. For protegrin-1, expression values of liver, lymph node, trachea, and heart for Min pigs were higher than those of Landrace pigs. No significant increases for PMAP-23, PMAP-37, and protegrin-1 were observed for Landrace pigs compared with Min pigs. For PR-39, expression values of lung, trachea, and heart for Min pigs were higher than those of Landrace pigs. mRNA expression in just a few tissues such as spleen, duodenum, and thymus for Min pigs were lower than those for Landrace pigs. 3.6. The expression correlations among cathelicidin genes The correlation analysis among cathelicidin genes expression in two pig breeds were carried out using SPSS software. As shown in Fig. 5, three pairs of peptides (PMAP23 and 37, PMAP-37 and PR-39, and PMAP-37 and protegrin-1) were significantly related in Min pigs and the Pearson correlation coefficients were 0.66 (P¼0.02), 0.86
In the present study, we detected mRNA expression abundance of cathelicidin members in Min and Landrace pigs. The cathelicidin family of host defense peptides includes a group of cationic and usually amphipathic peptides that display a variety of activities related to host defense function (Fu et al., 2008; Tomasinsig and Zanetti, 2005). The results showed that the expression of cathelicidin genes was detected in all tested tissues. It was previously reported that the expression of cathelicidins was not limited to lymphoid organs or leukocytes and had a broad distribution including skin, epithelial and brain tissue, which was consistent with this study (Brandenburg et al., 2012; Huttner and Bevins, 1999). Porcine cathelicidins were originally cloned from myeloid cells (PMAP-23 and 37) or neutrophils (PR-39 and protegrin-1) (Agerberth et al., 1991; Kokryakov et al., 1993; Tossi et al., 1995; Zanetti et al., 1994), and these cells play an important role in body immunity. In this study, mRNAs of these peptides were highly expressed at spleen and thymus, which are also importantly central immune organs. This was in agreement with previous reports (Brown and Hancock, 2006; Ganz, 2003). The thymus has a great effect upon the immunity of young animals (Qi et al., 2009). The expression of these peptides in other organs (such as brain, liver, and heart) that are exposed to the microbial environment may facilitate defense against opportunistic infections or have other functions beyond their antimicrobial activity (Chen et al., 2010). For cathelicidins, the expression pattern was different from defensins. Chen et al. (2010) reported that main expression sites for three pig β-defensins (pBD-1, 2 and 3) were tongue and oral mucosa in corssbred and Meishan pigs (the exception was pBD-2 of crossbred pig expressed in kidney and liver). In addition, high-level mRNA expressions of pBD-1 and 3 of crossbred pigs and Tibetan pigs were observed in tongue, oral epithelium and skin while pBD-2 was also in kidney and liver (Qi et al., 2009). But in this study, the cathelicidin molecules were highly expressed in thymus, spleen, liver, and heart while expressed at low levels in ileum, jejunum, tongue, and lymph node for Landrace and Min pigs. Although pig ages and breeds were different, this suggests differential tissue specificity and gene regulation of two important AMP families, cathelicidins and defensins. Especially in the intestine constitutively high levels of AMPs are unwanted because they could destroy the balance of the commensal intestinal microflora (Finlay and Hancock, 2004; Qi et al., 2009). It was reported that expressions of AMPs of Tibetan pigs and Meishan pigs in most tissues were higher than those of crossbred pigs and the higher expression of AMPs provide a direct evidence for
40
Q.Q. Ma et al. / Livestock Science 161 (2014) 36–40
higher immunity and disease resistance (Chen et al., 2010; Qi et al., 2009). Similarly, in the present study mRNA abundance were higher in Min pigs than those of Landrace pigs in most tissues. The correlation analysis showed that significantly correlated gene pairs in Min pigs were generally not related in Landrace pigs. This hints that the two breeds may form differential synergistic or antagonistic regulation or different cathelicidin genes up-regulation or down-regulation mechanism in the long-term evolution. It was known that the cathelicidins constitutively stored as pro-peptides in peripheral PMN granules, where few or no transcripts are expressed (Zhang et al., 2000). We suppose that the cathelicidins may constitutively express in the tissues such as thymus and spleen and inducibly express in the tissues such as tongue and small intestine. In summary, we determined gene expression for cathelicidins in Min pigs and Landrace pigs and found characteristic expression patterns for four genes. The cathelicidin molecules were expressed at high levels in thymus, spleen, liver, and heart, and at low levels in ileum, jejunum, tongue, and lymph node in two breeds. In most tissues, cathelicidins had higher expression level in Min pigs than those in Landrace pigs. The high expression and wide abundance of cathelicidins may partly explain the strong disease resistance of Min pigs. Conflict of interest None. Acknowledgments This work was supported by grants from the National Key Technology R&D Program (2013BAD10B03), the China Postdoctoral Science Foundation (2012M510082 and 2013T60342), and the Open Projects of Key Laboratory of Feed Science, College of Heilongjiang Province. References Agerberth, B., Lee, J.Y., Bergman, T., Carlquist, M., Boman, H.G., Mutt, V., Jörnvall, H., 1991. Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline–arginine-rich antibacterial peptides. Eur. J. Biochem. 202, 849–854. Brandenburg, L.O., Merres, J., Albrecht, L.J., Varoga, D., Pufe, T., 2012. Antimicrobial peptides: multifunctional drugs for different applications. Polymers 2012 (4), 539–560. Brown, K.L., Hancock, R.E.W., 2006. Cationic host defense (antimicrobial) peptides. Curr. Opin. Immunol. 18, 24–30. Brown, K.L., Poon, G.F., Birkenhead, D., PRena, O.M., Falsafi, R., Dahlgren, C., Karlsson, A., Bylund, J., Hancock, R.E., Johnson, P., 2011. Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses. J. Immunol. 186, 5497–5505. Chen, J., Qi, S., Guo, R., Yu, B., Yu., B., Chen, D., 2010. Different messenger RNA expression for the antimicrobial peptides b-defensins between Meishan and crossbred pigs. Mol. Biol. Rep. 37, 1633–1639. Choi, K.Y.G., Mookherjee, N., 2012. Multiple immune-modulatory functions of cathelicidin host defense peptides. Front. Immunol. 3, 149. Finlay, B.B., Hancock, R.E., 2004. Can innate immunity be enhanced to treat microbial infections? Nat. Rev. Microbiol. 2, 497–504. Fu, J., Wenzel, S.C., Perlovan, O., Wang, J.P., Gross, F., Tang, Z.R., Yin, Y.L., Stewart, A.F., Muller, R., Zhang, Y.M., 2008. Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res. 36, 113–114.
Ganz, T., 2003. The role of antimicrobial peptides in innate immunity. Integr. Comp. Biol. 43, 300–304. Hancock, R.E.W., Scott, M.G., 2000. The role of antimicrobial peptides in animal defenses. Proc. Natl. Acad. Sci. USA 97, 8856–8861. Huttner, K.M., Bevins, C.L., 1999. Antimicrobial peptides as mediators of epithelial host defense. Pediatr. Res. 45, 785–794. Kin, N.W., Chen, Y., Stefanov, E.K., Gallo, R.L., Kearney, J.F., 2011. Cathelinrelated antimicrobial peptide differentially regulates T- and B-cell function. Eur. J. Immunol. 41, 3006–3016. Kokryakov, V.N., Harwig, S.S., Panyutich, E.A., Shevchenko, A.A., Aleshina, G.M., Shamova, O.V., Korneva, H.A., Lehrer, R.I., 1993. Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins. FEBS Lett. 327, 231–236. Korthuis, R.J., Gute, D.C., Blecha, F., Ross, C.R., 1999. PR-39, a proline/ arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction. Am. J. Physiol. 277, H1007–H1013. Kulkarni, M.M., Barbi, J., McMaster, W.R., Gallo, R.L., Satoskar, A.R., McGwire, B.S., 2011. Mammalian antimicrobial peptide influences control of cutaneous Leishmania infection. Cell. Microbiol. 13, 913–923. Lehrer, R.I., Ganz, T., 2002. Cathelicidins: a family of endogenous antimicrobial peptides. Curr. Opin. Hematol. 9, 18–22. Nizet, V., Gallo, R.L., 2003. Cathelicidins and innate defense against invasive bacterial infection. Scand. J. Infect. Dis. 35, 670–676. Peters, B.M., Shirtliff, M.E., Jabra-Rizk, M.A., 2010. Antimicrobial peptides: primeval molecules or future drugs? Plos Pathog. 6, e1001067. Qi, S., Chen., J., Guo, R., Yu, B., Chen, D., 2009. β-defensins gene expression in tissues of the crossbred and Tibetan pigs. Livest. Sci. 123, 161–168. Sang, Y., Blecha, F., 2009. Porcine host defense peptides: expanding repertoire and functions. Dev. Comp. Immunol. 33, 334–343. Steinberg, D.A., Hurst, M.A., Fujii, C.A., Kung, A.H., Ho, J.F., Cheng, F.C., Loury, D.J., Fiddes, J.C., 1997. Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity. Antimicrob. Agents Chemother. 41, 1738–1742. Tang, X., Fatufe, A.A., Yin, Y., Tang, Z., Wang, S, Liu, Z., Xin, W., Li, T., 2012. Dietary supplementation with recombinant lactoferrantmpinlactoferricin improves growth performance and affects serum parameters in piglets. J. Anim. Vet. Adv. 11, 2548–2555. Tang, X.S., Shao, H., Li, T.J., Tang, Z.R., Huang, R.L., Wang, S.P., Kong, X.F., Wu, X., Yin, Y.L., 2012. Dietary supplementation with bovine lactoferrampin-lactoferricin produced by Pichia pastoris fed-batch fermentation affects intestinal microflora in weaned piglets. Appl. Biochem. Biotechnol. 168, 887–898. Tang, Z.R., Yin, Y.L., Zhang, Y.M., Huang, R.L., Sun, Z.H., Li, T.J., Chu, W.Y., Kong, X.F., Li, L.L., Geng, M.M., Tu, Q., 2009. Effects of dietary supplementation with an expressed fusion peptide bovine lactoferricin-lactoferrampin on performance, immune function and intestinal mucosal morphology in piglets weaned at age 21 d. Br. J. Nutr. 101, 998–1005. Tjabringa, G.S., Ninaber, D.K., Drijfhout, J.W., Rabe, K.F., Hiemstra, P.S., 2006. Human cathelicidin LL-37 is a chemoattractant for eosinophils and neutrophils that acts via formyl-peptide receptors. Int. Arch. Allergy Immunol. 140, 103–112. Tomasinsig, L., Zanetti, M., 2005. The Cathelicidins – structure, function and evolution. Curr. Protein Pept. Sci. 6, 23–34. Tossi, A., Scocchi, M., Zanettp, M., Storici, P., Gennaro, R., 1995. PMAP-37, a novel antibacterial peptide from pig myeloid cells cDNA cloning, chemical synthesis and activity. Eur. J. Biochem. 228, 941–946. Wang, Y.Z., Xu, Z.R., Lin, W.X., Huang, H.Q., Wang, Z.Q., 2004. Developmental gene expression of antimicrobial peptide PR-39 and effect of zinc oxide on gene regulation of PR-39 in piglets. Asian-Aust. J. Anim. Sci. 17, 1635–1640. Xu, Z.Y., 1989. Breed Characteristics of Indigenous Pig Breeds in China. Zhejiang Scientific and Technical Press. Hangzhou, Zhejiang Province, China. Yao, K., Yin, Y.L., Chu, W.Y., Liu, Z.Q., Dun, D., Li, T.J., Huang, R.L., Zhang, J.S., Tan, B., Wang, W.C., Wu, G.Y., 2008. Dietary arginine supplementation increases mTOR signaling activity in skeletal muscle of neonatal pigs. J. Nutr. 138, 867–872. Zanetti, M., 2004. Cathelicidins, multifunctional peptides of the innate immunity. J. Leukoc. Biol. 75, 39–48. Zanetti, M., 2005. The role of cathelicidins in the innate host defenses of mammals. Curr. Issues Mol. Biol. 7, 179–196. Zanetti, M., Storici, P., Tossi, A., Scocchi, M., Gennaro, R., 1994. Molecular cloning and chemical synthesis of a novel antimicrobial peptide derived from pig myeloid cells. J. Biol. Chem. 269, 7855–7858. Zhang, G., Ross, C.R., Blecha, F., 2000. Porcine antimicrobial peptides: new prospects for ancient molecules of host defense. Vet. Res. 31, 277–296l.