Gene organization and polymorphism of the swine major histocompatibility complex

The recent availability of the full‐length sequence of one haplotype of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), and significant progress in the studies on gene expression and polymorphisms led to major advances in deciphering its role in resistance to diseases in animals. The present status of the genomic organization and polymorphism of the SLA complex is presented in this Review. Additionally, a comparative analysis with mammalian MHC has also been provided. The sequenced SLA‐H01 haplotype harbors 152 loci including genuine SLA genes, non‐MHC genes and pseudogenes. Although the numbers of expressed SLA genes could vary across haplotypes, three SLA class Ia, three SLA class Ib, four SLA class IIa and four SLA class IIb genes are currently expressed. Except for the class I genes, which have no clear orthologs, the gene organization of the loci was highly conserved between humans and pigs. Moreover, the human leukocyte antigen (HLA) complex lies on a single chromosomal segment, whereas a centromere at the class II and III junction splits the SLA complex into two segments, without disturbing gene organization or impeding functionality. Over 400 SLA class I and II allele sequences available in databases have been recently clustered and assigned to a specific SLA locus according to a newly defined nomenclature system.     

Structure and expression of class I MHC genes in the miniature swine

The genome of the miniature swine, unlike other species, contains a relatively small class I MHC gene family, consisting of only seven members. This provides an excellent system in which to identify and characterize the regulatory mechanisms which operate to both coordinately and differentially regulate the expression of a multi-gene family. The structure of class I SLA genes, like other class I genes, consists of eight exons encoding a leader sequence, three extracytoplasmic domains, a transmembrane domain and intracytoplasmic domains. Despite the common structure, two sub-families of class I genes can be distinguished within the SLA family. One, containing the closely related PD1 and PD14 genes, encodes the classical transplantation antigens. Another contains the highly divergent PD6; the functions of the products of this subfamily, if any, are not known. The class I SLA genes share some common regulatory mechanisms, as evidenced by the fact that all three genes analyzed are transcribed in mouse L cells. Furthermore, interferon treatment of transfected mouse L cells enhances expression of all three genes. Both PD1 and PD6 are transcribed in vivo, where the highest levels of expression are observed in lymphoid tissues. Superimposed on the common patterns of class I gene expression are distinct ones, as evidenced by the findings that PD1 is preferentially expressed in B cells, whereas PD6 is preferentially expressed in T cells. These differences may reflect the extensive divergence of the 5' flanking sequences of these genes. Future studies will be aimed at elucidating the precise molecular interactions and mechanisms which give rise to the observed differential expression.     

Investigation of the relationship between SLA-1 and SLA-3 gene expression and susceptibility to Escherichia coli F18 in post-weaning pigs

Porcine post-weaning diarrhea and edema disease are principally caused by Escherichia coli strains that produce F18 adhesin. FUT1 genotyping and receptor binding studies divided piglets into E. coli F18-resistant and -sensitive groups, and the roles of SLA-1 and SLA-3 were investigated. SLA-1 and SLA-3 expression was detected in 11 pig tissues, with higher levels of SLA-1 in lung, immune tissues and gastrointestinal tract, and higher levels of SLA-3 also in lung and lymphoid tissues. Both genes were expressed higher in F18-resistant piglets, and their expression was positively correlated in different tissues; a negative correlation was observed in some tissues of F18-sensitive group, particularly in lung and lymphatic samples. Gene ontology and pathway analyses showed that SLA-1 and SLA-3 were involved in 37 biological processes, including nine pathways related to immune functions. These observations help to elucidate the relationship between SLA class I genes and E. coli F18-related porcine gastrointestinal tract diseases.     

High transfection efficiency of porcine peripheral blood T cells via nucleofection

Transgenic technology is an effective approach to assess the roles of specific genes in the activation and differentiation of T cells and modify T cell qualities. However, porcine T cell transfection is poorly documented. Here, we developed a non-virus-based method for the transfection of resting and ConA-stimulated porcine peripheral blood T cells using "Nucleofection" gene transfer technology; both plasmid DNA- and mRNA-mediated nucleofection systems were developed. The results demonstrated for the first time that plasmid DNA encoding green fluorescent protein (GFP) and in vitro transcribed GFP mRNA could be delivered efficiently into resting and activated porcine T cells. For both methods, the onset of gene expression was rapid and occurred within 2h post-nucleofection. Optimised plasmid DNA-mediated nucleofection induced approximately 40% transgene expression with 51% cell viability in resting T cells and approximately 20% transgene expression with 53% cell viability in activated T cells at 24h post-gene delivery. However, optimised mRNA-based nucleofection resulted in higher transfection efficiencies and cell viability, with more than 50% transgene expression and 62% viability for resting T cells and approximately 40% transgene expression and 59% viability for activated T cells. Finally, we measured the impact of the developed nucleofection systems on T cell function by detecting the mRNA levels of the activation markers CD25, CD69 and the cytokine IFN-γ; cell proliferation of the nucleofected resting peripheral blood mononuclear cells (PBMC) after ConA stimulation was also examined. The nucleofected resting PBMCs proliferated normally and up-regulated CD25, CD69 and IFN-γ mRNA expression levels in a manner comparable to non-nucleofected cells. These results indicate that the developed nucleofection systems have no adverse effects on T cell function and can be utilised in swine immunological research.     

Generation of porcine induced pluripotent stem cells and evaluation of their major histocompatibility complex protein expression in vitro

Induced pluripotent stem cells (iPSCs) are thought to be highly beneficial in the field of regenerative medicine and are believed to overcome immunogenic barriers to cell transplantation. However, issues remain regarding their safety and efficiency for medical use. Furthermore, some recent reports have suggested that iPSCs could be targeted by the autologous immune system. To promote practical applications of iPSCs, in depth research using appropriate animal models is needed and porcine species appear to provide an ideal model. Recent studies have focused on the generation of porcine iPSC cells, but no investigations of their immunological properties have been conducted to date. In the present study, we generated putative iPSCs from porcine somatic cells and measured major histocompatibility complex (MHC) expression on the iPSCs and their derivatives. Compact colonies that expressed pluripotent markers appeared 11 days after viral infection. Embryonic bodies (EB) were produced and differentiated into three germ layers in vitro. Karyotyping and swine leukocyte antigen (SLA) typing showed that the iPSCs were identical to parental somatic cells. Porcine iPSCs expressed only low levels of MHC class I and moderately increased levels on their differentiated derivatives, whereas MHC class II was rarely expressed. In the presence of interferon-gamma (IFN-γ), the expression of MHC class I was elevated on differentiated iPSCs, and gradually decreased after withdrawal of the cytokine. Our data suggest that porcine iPSCs could be useful for preclinical studies of the efficiency and viability of iPSCs, and for devising strategies to rescue transplanted cells from the autologous immune system.     

猪圆环病毒2型感染对猪瘟弱毒疫苗接种猪免疫应答的影响

为探索猪圆环病毒2型(PCV2)感染对猪瘟(CSF)弱毒疫苗接种猪免疫应答的影响,将20头28 d断奶仔猪随机分为V-I、I-V、V和C4组,5头·组-1.V-I组在接种CSF弱毒疫苗后2d感染PCV2;I-V组在感染PCV2 后2d接种CSF弱毒疫苗;V组只接种CSF弱毒疫苗;C组为空白对照组.共免疫2次,间隔21 d.免疫后定期检测血清CSFV特异性抗体水平、外周血淋巴细胞(PBLC)增殖活性和PBLC中IFN-γ、IL-2、IL-4和IL-10 mRNA的表达水平.结果显示:在CSF弱毒疫苗免疫前或免疫后感染PCV2,均会导致CSFV抗体水平低下,血清抗体阳转率下降,PBLC增殖活性降低.初次免疫后,V-I与I-V组的PBLC内IFN-γ、IL-2、IL-4和IL-10 mRNA的表达量严重不足,其中V-I组的表达量最低.加强免疫后V-I与I-V组的PBLC内IFN-γ与IL-10的表达失衡,IL-2和IL-4的表达缺乏,其中I-V组的细胞因子表达失衡和缺乏更严重.上述研究表明,CSF弱毒疫苗免疫前或免疫后感染PCV2均会影响机体的体液和细胞免疫应答水平,导致PBLC内细胞因子的表达严重抑制和紊乱.     

IFN-γ renders human intestinal epithelial cells responsive to lipopolysaccharide of Vibrio cholerae by down-regulation of DMBT1

Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). However, inflammatory cytokines such as interferon-γ (IFN-γ) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-γ-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-γ-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-γ-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-γ enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-γ indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-γ and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-γ is known to increase IFN-β expression in the IECs, we examined if IFN-β functioned similar to IFN-γ. Although IFN-β alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-γ primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.     

Correction of the Mutant SLA-1-632-TPK Gene from ToPigs Pig and Construction of SLA-1-TPK/pMD19-T Recombinant Plasmid

The swine leukocyte antigen (SLA) genes in pigs were the important immune gene group in antigen presentation, and studing on SLA could provide the references for the prevention of some infectious diseases. Earlier studies found that SLA-1-632-TPK gene in ToPigs pig had a deleted base in its coding sequence (a single base "C" was lost in 632 bp from the 5' end of the SLA-1-632-TPK gene), which lead to frameshift mutation. In order to correct the SLA-1-632-TPK gene, two pairs of gene-correction's primers were designed to correct the gene by the splicing overlap extension PCR (SOE-PCR) in template of recombinant plasmid of SLA-1-632-TPK/pMD18-T. Firstly,the 5'and 3'ends of SLA-1-632-TPK gene were amplified, respectively, then both of them were spliced and amplified to form an intact SLA-1-632-TPK gene. After detected by agarose electrophoresis, the interest of the product was further cloned into pMD19-T Simple vector. The positive clones were screened by colony PCR and then sequenced. The result showed that the 5'and 3' ends of the SLA-1-632-TPK gene were all amplified successfully by SOE-PCR, with the products of about 650 and 590 bp, which were consistent with the theoretical value of 648 and 585 bp, respectively. After spliced, the intact sequence of SLA-1-632-TPK gene was obtained with the product of about 1 200 bp, which was close to the theoretical value of 1 223 bp. The colony PCR result showed that the corrected gene was successfully inserted into pMD19-T Simple vector . After the sequence was analyzed by GENETYX version 9.0, it was shown that the nucleotide "C" in 632 bp numbered from the 5'end of the gene was added and the SLA-1-632-TPK gene was coded correctly. In this study, the SLA-1-632-TPK was corrected successfully, and the recombinant plasmid SLA-1-TPK/pMD19-T was constructed, which would lay a foundation to further study the protein expression and associated function of SLA-1-TPK.     

托佩克猪SLA-1-632-TPK基因的矫正及重组质粒SLA-1-TPK/pMD19-T的构建

猪白细胞抗原（swine leukocyte antigen,SLA）在猪免疫系统中起递呈抗原作用,对其展开研究可为猪相关传染病的预防提供依据。研究发现,托佩克猪SLA-1-632-TPK基因编码序列发生碱基缺失（距离SLA-1-632-TPK基因5'端632 bp处丢失1个碱基"C"）,导致移码突变。为矫正SLA-1-632-TPK基因,设计2对矫正引物,以原重组质粒SLA-1-632-TPK/pMD18-T为模板,利用剪切重叠延伸PCR（splicing overlap extention PCR,SOE-PCR）技术分别扩增SLA-1-632-TPK 5'和3'端,之后进行拼接,最后扩增全序列从而矫正目的基因,并进一步连接pMD19-T Simple载体,通过单菌落PCR筛选阳性克隆并测序,并通过GENETYX version 9.0软件对所测序列进行分析。结果显示,SOE-PCR成功扩增得到5'和3'端片段,大小约为650和590 bp,与理论设计值大小（648和585 bp）接近,经过拼接以后,得到全长约1 200 bp,与理论设计值1 223 bp接近。菌落PCR结果显示,矫正基因成功克隆入pMD19-T Simple载体。序列分析结果显示,托佩克猪SLA-1-632-TPK基因距离5'端632 bp处丢失的碱基"C"得到矫正并正确编码。本研究成功矫正了SLA-1-632-TPK基因,并构建其重组质粒SLA-1-TPK/pMD19-T,为下一步研究SLA-1-TPK蛋白表达和相关功能奠定基础。     

Production of IFN-γ in feline whole blood after incubation with potential T-cell epitopes of the nucleocapsid protein of feline coronavirus

Interferon gamma (IFN-γ) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-γ in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-γ concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-γ mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-γ in samples incubated with peptides decreased and the expression of IFN-γmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a "pre-activated" immune response). These cats responded to "avirulent" or "mixed" peptides by increasing the concentration of IFN-γ and the expression of IFN-γ mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-γ especially in cats with a "pre-activated" cell mediated response.     

Construction and Expression of the Heavy Chain Tetramer Precursor Chain of SLA-1 in Hebao Pig

To construct tetramer precursor chain of swine lymphocyte antigen 1 (SLA-1) heavy chain in Hebao pig and study its protein expression in the pET-21a (+) vector,the SLA-1 complete genome sequence was referenced with the characteristics of the expression vector,and a pair of primers was designed to integrate the BirA substrate peptide (BSP) sequence at the C-terminus of the SLA-1-HB01 and the SLA-1-HB01-BSP was amplified by PCR. Then,the products were cloned into the pEASY T1 vector and the positive clones of SLA-1-HB01-BSP/pEASY T1 was selected. After the double digestion,the interest of the gene in positive clone was further ligated into the pET-21a (+) expression vector and transformed into E.coli BL21 to construct the recombinant strain of pET-21a (+)/SLA-1-HB01-BSP. After induction with IPTG,the target protein were detected by SDS-PAGE. Finally,the inclusion body of the SLA-1-HB01-BSP was isolated and detected to evaluate its purity. The PCR results showed that the length of SLA-1-HB01-BSP was about 898 bp,which was consistent with the theoretical value. The amplified fragment was successfully cloned into pEASY T1 vector, and the positive clones were identified by Nde Ⅰ and Xho Ⅰ digestion. The size of inserted fragment was 876 bp. The interest of gene was also inserted into pET-21a (+) and transformed into E.coil BL21 successfully. After induction,SDS-PAGE detection results showed that the target protein was 31.4 ku. Further detection showed that the target protein was mainly expressed as inclusion bodies,and the purity of the protein was about 80%. In this study,the recombinant tetramer precursor of SLA-1-HB01 heavy chain was constructed in pET-21a (+) expression line successfully, which would lay a foundation to detect the tetramer of SLA class Ⅰ molecular.     

All three classes of CpG ODNs up-regulate IP-10 gene in pigs

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR^@ green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-γ, IL-12 (P40), IL-6, IL-4 and TNF-α mRNA, C-class CpG ODN induced significant level of IFN-γ, IFN-α and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12 h post stimulation. These in vitro and in vivo observations suggest that interferon-γ inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.     

荷包猪SLA-1重链四聚体前体链的构建及表达研究

试验旨在构建荷包猪猪白细胞抗原1（swine lymphocyte antigen 1,SLA-1）重链四聚体前体链并研究其在表达载体pET-21a（+）中蛋白的表达情况。以SLA-1全基因序列为模板,结合表达载体的特点设计引物,利用PCR扩增技术在SLA-1-HB01胞外区序列C-末端加载上可生物素酰化序列（BirA substrate peptide,BSP）。将PCR扩增产物SLA-1-HB01-BSP克隆到pEASY T1载体上,筛选出阳性克隆菌SLA-1-HB01-BSP/pEASY T1,经双酶切后进一步与表达载体pET-21a（+）连接,再转化到E.coli BL21感受态细胞中构建pET-21a（+）/SLA-1-HB01-BSP重组表达菌,通过IPTG诱导蛋白表达,SDS-PAGE检测蛋白的大小及表达情况,提取包涵体检测蛋白的纯度。PCR扩增结果显示,SLA-1-HB01-BSP大小为898 bp,与理论值相符,扩增片段成功克隆至pEASY T1载体构建克隆菌,经Nde Ⅰ和Xho Ⅰ双酶切筛选及测序,成功获得阳性克隆菌株SLA-1-HB01-BSP/pEASY T1,插入的目的基因片段大小为876 bp。阳性克隆菌株与pET-21a（+）表达载体经Nde Ⅰ和Xho Ⅰ双酶切后进行连接,转化E coli BL21感受态细胞后获得pET-21a（+）/SLA-1-HB01-BSP重组表达菌,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小为31.4 ku。进一步检测分析发现,目的蛋白以包涵体形式存在,且蛋白纯度达到80%以上。本研究成功构建了荷包猪SLA-1-HB01重链四聚体前体链的pET-21a（+）重组表达体系,为下一步进行猪SLA Ⅰ类分子四聚体的检测奠定基础。     

Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares

Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was shown to have significantly inhibited E. coli induced proliferation and expression of IFN-γ in PBMCs. These results indicate that P(4) inhibits the immune response to E. coli in mares.     

Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection

The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8⁺ IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8^?IFN-γ⁺ phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8⁺IFN-γ⁺ as well as CD8^?IFN-γ⁺ cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.