Toll-like receptor expression in feline lymphoid tissues

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.     

Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues

We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT.     

Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets

Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.     

Expression of Toll-like receptor 9 (TLR9) in the lungs and lymphoid tissue of pigs

The pattern of distribution of Toll-like receptor 9 (TLR9) in different tissues varies between species. The aim of the present study was to describe the distribution of TLR9 expression in selected tissues and organs of healthy pigs at 3 weeks and 3 months of age. Representative formalin-fixed samples of lung, thymus and secondary lymphoid tissues were evaluated by immunohistochemistry. TLR9 positive staining was observed in epithelial cells, vascular endothelium and myoepithelial-like cells, as well as in cells of the alveolar septa of the lung. Antigen presenting cells of perifollicular zones (interdigitating, macrophage and dendritic-like cells) of the Peyer's patches, lymph nodes, spleen and thymus were also immunoreactive for TLR9. No differences were seen in TLR9 protein expression in tissues from the two age groups.     

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.     

Morphologic studies of intrapulmonary airway mucosa-associated lymphoid tissues in swine

Morphologic structures of the lymphoid tissues associated with the mucosa of the respiratory tract in swine aged 11 to 13 weeks were studied by dissecting, light, and transmission electron microscopy. Tissues along the respiratory tract were collected for further examination. Light microscopic examination showed that the lymphoid tissues were associated with bronchi (8.38%), bronchioles (81.63%), and respiratory bronchioles (9.98%). The lymphoid tissues were identified at different levels of the airways and were designated intrapulmonary airway mucosa-associated lymphoid tissues. Anatomically, the lymphoid tissues were found mostly in the bronchiolar bifurcations, with an arteriole frequently nearby. Histologically, the lymphoid tissues contained a single dome-shaped lymphoid follicle, which bulged out from the mucosal surface into the airway, where it was covered along the mucosal side by a layer of specialized epithelium. Ultrastructurally, the mucosal surface of the specialized epithelial cells was equipped with microfolds or cytoplasmic projections instead of cilia. The cytoplasm was elongated, and therefore much thinner than that of ordinary epithelial cells. Many lymphocytes were found scattered in the mucosal epithelium between or beneath the specialized epithelial cells. This structure was similar to that seen in the Peyer's patches of the small intestine in swine and other animals.     

Distribution of immunoglobulin-bearing cells in the gut-associated lymphoid tissues of the turkey: effect of antibiotics

Distribution of immunoglobulin (Ig)-bearing cells in the gut-associated lymphoid tissues of antibiotic treated and untreated control turkeys (Meleagris gallopavo) was compared. Antibiotic treatment was similar to a regimen used in commercial turkey production, which included preincubation dipping of fertile eggs in gentamicin solution, injection of turkeys with gentamicin at hatching, and inclusion of chlortetracycline in the diet. Tissues were examined from turkeys at 3, 7, 14, and 21 days of age with a direct immunofluorescence procedure. Cell distribution in control turkeys was as follows: In the bursa of Fabricius, IgA-carrying cells predominated at 3 days of age, but at later intervals, the 3 classes of Ig-bearing cells were in equal numbers. In the cecal tonsils, IgM- and IgA-bearing cells were in larger numbers at 3 days of age, whereas, the IgG-bearing cells were sparsely distributed. By 7 days of age, IgM cells became more numerous in the cecal tonsils and remained numerous until 21 days of age. At 3 days of age, IgA cells predominated in the small intestines and IgM cells predominated in the large intestine. At 7 and 14 days of age, IgM cells were more numerous in the small and large intestines, but by 21 days of age, IgA cell population equaled that of IgM. The IgG cells were generally sparse in the intestines. Antibiotic treatment often resulted in lower numbers of Ig-positive cells, especially those bearing IgM and IgA. Normal development of the bursa of Fabricius was also retarded in this group.     

Interleukin 4 increases CCR9 expression and homing of lymphocytes to gut-associated lymphoid tissue in chickens

The effects of in vitro and in vivo IL-4 supplementation on thymocyte and splenocyte CCR9 mRNA amount and migration were studied. Thymocytes, splenocytes, splenocytes+thymocytes (2:1), and splenocytes+bursocyte cells (2:1) were supplemented with either 0 or 5 ng/ml IL-4 for 5d. CCR9 mRNA was undetectable in all experimental groups supplemented with 0 ng/ml IL-4. IL-4 treatment (5 ng/ml) upregulated (P=0.01) CCR9 mRNA only in the splenocyte+thymocyte cell culture. IL-4-mediated CCR9 mRNA induction in the splenocyte+thymocyte cell culture was dependent on the in vitro dose of IL-4 supplementation. IL-4-treated splenocyte+thymocyte cells when injected in vivo preferentially migrated to cecal tonsils. In vivo supplementation of IL-4 was achieved through in ovo injection of recombinant chicken IL-4 plasmid. Cecal tonsils in chicks hatched from IL-4-plasmid-injected eggs weighed more, had a higher amount of CCR9 mRNA, and had a higher percentage of CD8(+) cells than cecal tonsils from chicks hatched from PBS-injected eggs. It could be concluded that IL-4 induces CCR9 mRNA in thymocytes and splenocytes and directs the migration of cells to gut-associated lymphoid tissue.     

Effect of vitamin E supplementation on lymphocyte distribution in gut-associated lymphoid tissues obtained from weaned piglets

Fifteen piglets were used to determine the effect of vitamin E supplementation on the number of CD4-immunoreactive (CD4+) T-lymphocytes, CD8-immunoreactive (CD8+) T-lymphocytes and IgA-immunoreactive (IgA+) B-lymphocytes per follicle in the Peyer's patch of distal ileum and the mesenteric lymph nodes of weaned piglets. Piglets, following a 3-day adaptation period after weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), vitamin E supplementation of 100 mg/kg of diet and vitamin E supplementation of 300 mg/kg of diet. Supplementation of vitamin E lasted for a period of 36 days. The basal diet contained 80 mg alpha-tocopherol/kg of diet. All piglets were killed at day 39 after weaning and samples of the distal ileum and adjacent mesenteric lymph nodes were collected. The number of cells for each lymphocyte subset was counted in the Peyer's patch and the mesenteric lymph nodes follicles, in cryostat sections processed for immunohistochemistry. Results showed that vitamin E supplementation (300 mg/kg diet) of piglets caused an increase (P < 0.05) in the number of IgA+ B-lymphocytes in the Peyer's patch, but not in the mesenteric lymph nodes, compared with the corresponding values in control animals. Vitamin E supplementation had no effect (P > 0.05) on the number of CD4+ and CD8+ T-lymphocytes in the follicles of the Peyer's patch and the adjacent mesenteric lymph nodes. Thus, vitamin E had relatively minor effects on distribution of the major immunocompetent cells in the gut. The numbers of CD4+ and CD8+ T-lymphocytes as well as IgA+ B-lymphocytes per follicle were higher by 26-77% (P < 0.05) in the mesenteric lymph nodes than the corresponding values in the Peyer's patch.     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

Polymorphism of Toll-like receptor 9 (TLR9) gene in sheep

Bacterial and synthetic DNA containing unmethylated CpG dinucleotides in particular sequence contexts, activates the vertebrate immune system through Toll-like receptor 9 (TLR9). In this study, we use PCR-single-strand conformational polymorphism (PCR-SSCP) analysis to investigate genetic variation in a key region of the ovine TLR9 gene. Three novel SSCP patterns, representing three different sequences, were identified. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology to the TLR9 sequences from a variety of species, suggesting that these sequences represent allelic variants of the ovine TLR9 gene. Four single nucleotide polymorphisms (SNPs) were detected in the region amplified and two of them were non-synonymous substitutions that would result in amino acid changes. Variation detected here might have an impact on the structure and/or function of TLR9 and hence affect the immune response to pathogens.     

Molecular cloning and functional analysis of duck Toll-like receptor 5

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.     

Toll-like receptor genes are differentially expressed at the sites of infection during the progression of Johne's disease in outbred sheep

Toll-like receptors (TLR) are engaged by ligands on microbial pathogens to initiate innate and adaptive immune responses. Little is known about TLR involvement during infection with Mycobacterium avium subsp. paratuberculosis (M. ptb), the cause of Johne's disease in ruminants, although there is a profound immunopathological response in affected animals. We have analyzed the expression of 10 TLR genes relative to validated reference genes at predilection sites in ileum, jejunum and associated lymph nodes as well as in peripheral blood, to determine if TLR expression is altered in response to infection with M. ptb in outbred sheep. Previously unexposed animals from two flocks and animals from three naturally infected flocks were used with restricted maximum likelihood linear mixed modeling applied to determine significant differences. These were related to the pathologies observed at different stages of infection in exposed sheep, after allowing for other sources of variation. In most cases there were differences in TLR expression between early paucibacillary and multibacillary groups when compared to uninfected sheep, with most TLRs for the paucibacillary group having lower expression levels than the multibacillary group. Increased expression of TLR1-5, and 8 was observed in ileum or jejunum, and TLR1-4, 6, and 8 in mesenteric lymph nodes. There was a trend for increased expression of TLR1, 2, and 6-8 in PBMCs of exposed compared to non-exposed animals. Further study of TLR expression in Johne's disease in ruminants is warranted as these observed differences may help explain pathogenesis and may be useful in the future diagnosis of M. ptb infection.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

胰岛素受体mRNA在新生犊牛组织中的表达

应用半定量RT-PCR方法检测了新生犊牛中枢神经系统和外周组织中胰岛素受体（insulin receptor，InsR）基因的表达。结果表明，InsR基因在肝、皮下脂肪、半腱肌、胰、肾皮质、脾、心、肺、下丘脑、肠系膜淋巴结、主动脉、十二指肠、结肠、垂体、大脑皮质、小脑皮质中都有表达。其中，肝、半腱肌、下丘脑、胰、主动脉、垂体中InsR基因的表达量显著多于其他组织（P〈0．05）。InsR基因在各组织中的广泛分布表明胰岛素在体内具有广泛的生理功能。     

Distribution of immunoglobulin-bearing cells in the gut-associated lymphoid tissues of the turkey: effect of oral treatment with intestinal microflora

One-day-old turkeys (Meleagris gallopavo) were orally inoculated with the intestinal contents of an adult turkey, and the intestinal tissues were studied by immunofluorescence for immunoglobulin (Ig)-bearing cells at 3, 7, 14, and 21 days of age. Microflora inoculation increased numbers of Ig-bearing cells in the gut-associated lymphoid tissues; the most uniform effects were observed at 3 days of age. As the birds grew older, this uniformity in response to the microflora inoculation was not evident in all the tissues. In the bursa of Fabricius and the large intestine of the inoculated birds, IgM-bearing cells were more numerous throughout the study period. Compared with noninoculated control turkeys, IgA-bearing cells in the cecal tonsils, and IgG- and IgA-bearing cells in the small intestine were increased at all age intervals.     

Development of immune assay system for both CpG and non-CpG DNA from lactic acid bacteria using a transfectant of swine Toll-like receptor 9

Bacterial DNA is expected to be a potent immune stimulating agent to Toll‐like receptor (TLR) 9 expressed cells such as macrophages, monocytes, B lymphocytes, NK cells and dendritic cells. In the present study, we constructed a transfectant of swine TLR9 with mammalian cells. We demonstrated that the transfectant, recognizing both CpG and non‐CpG oligonucleotides from lactic acid bacteria, induced NF‐κB activation by gene reporter assay. These findings indicate that the swine TLR9 transfectant will be highly useful for the screening of immunostimulatory DNA from lactic acid bacteria.     

猪Toll样受体9基因的克隆、序列分析及其结构预测

本研究应用反转录-聚合酶链式反应(RT-PCR)扩增技术,从猪脾脏淋巴细胞中,克隆了猪Toll样受体9基因(pTLR9).基因序列分析表明,克隆的pTLR9基因ORF为3 093 bp,编码1 030个氨基酸,含18.5%的亮氨酸,含有24个氨基酸的信号肤序列,属于Ⅰ型跨膜受体,具有富含亮氨酸的重复序列(LRR)和Toll/IL-1R同源区结构域;与GenBank上登载的pTLR9参考序列(AY859728)的同源性为99.3%,与牛、马、羊和人的同源性较高,与家鼠、褐鼠的次之,TLR9的演化关系与亲缘关系密切.     

梅花鹿天然Toll样受体9基因克隆与序列分析

Trizol法提取梅花鹿肝脏总RNA,采用ORF两端兼并引物,RT-PCR方法克隆梅花鹿天然Toll样受体9基因（TLR9）并进行系统的生物信息学分析。RT-PCR结果显示,梅花鹿TLR9基因的mRNA长4 043bp,含有800bp左右的5’UTR,完整ORF编码1 081个氨基酸（GenBank序列号为：HQ260632）。同源性分析显示梅花鹿TLR9基因编码区与其他物种高度同源,但5’UTR区存在基因结构的变异。功能结构域分析显示不同物种间TLR9结构域相对保守但也存在细微差别,结构域的差别导致梅花鹿TLR9蛋白胞外区马蹄形弧顶内侧空间结构由人类TLR9蛋白的弧形改变为梅花鹿TLR9蛋白的三角形。进化分析显示TLR9基因分子进化关系与物种间真实进化相一致。     

Influence of age,sex and rearing systems on Toll-like receptor 7 (TLR7) expression pattern in gut,lung and lymphoid tissues of indigenous ducks

1. The objective of the experiment was to determine the influence of age, sex and rearing system on Toll-like receptor 7 (TLR7) gene expression in gut, lung and lymphoid tissues and physiological responses to stress in male and female indigenous ducks of Tamil Nadu, India.

2. A total of 36 ducks (12 males and 24 females) were obtained from local farmers and tissue samples of gut tissues (duodenum, jejunum, ileum and caecum), lymphoid organs (spleen and bursa) and lungs were collected in RNAlater solution followed by RNA extraction.

3. After normalisation to β-actin (endogenous control) qPCR analysis identified a significant effect of age, sex and rearing system on TLR7 expression in the ducks.

4. A significant up-regulation of TLR7 expression was observed in lungs, duodenum, jejunum, ileum and caecum of sexually mature (45 wk) compared with that of immature ducks (16 wk). Among sexes, male ducks had significantly higher TLR7 expression than female ducks.

5. Age and sex interactions were significant in lungs, duodenum, jejunum and caecum. Ducks reared in an extensive housing system showed significantly higher TLR7 expression in bursa, lungs, duodenum, ileum and caecum compared to intensively reared ducks. There were no effects of age, sex and rearing systems on TLR7 expression in the spleen.

6. The heterophil-to-lymphocyte ratio and serum corticosterone were higher in ducks reared on an intensive system compared with ducks from an extensive rearing system.