Preliminary evaluation of selected inflammatory cytokine gene expression in lymphocytes isolated from whole human blood infected with trans-anethole-treated Staphylococcus aureus Newman strain

In our previous study based on a whole-blood model of sepsis infected with trans-anethole (TA)-treated Staphylococcus aureus, we have found that innate immune response was more effective in comparison to non-treated cells. Due to the previous observation, in the current preliminary study, a primary adaptive immune response was analysed. This study was conducted to evaluate the expression of selected cytokine (IL1B, IL2, IL6, IL10, TNF, TGFB1, IFNG) and Toll-like receptor (TLR2) genes in lymphocytes isolated from whole human blood infected with S. aureus Newman strain treated with TA. The lymphocytes were isolated by density gradient centrifugation from blood samples infected with S. aureus, as well as from non-infected samples. Gene expression was measured using quantitative real-time PCR. The lymphocytes isolated from the blood infected with TA-treated staphylococcal cells demonstrated significantly greater IL10, IL1B, IL6, TNF and TLR2 expression. Hence, it is possible that the previously observed changes in the surface structure of TA-treated S. aureus Newman strain may significantly increase the relative expression of IL10, IL1B, IL6, TNF and TLR2 genes in lymphocytes; however, further studies are needed.     

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum

Background  

Inflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1β (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period.     

Synergistic effect of genetic polymorphisms in TLR6 and TLR10 genes on the risk of pulmonary tuberculosis in a Moldavian population

Polymorphisms in genes that control immune function and regulation may influence susceptibility to pulmonary tuberculosis (TB). In this study, 14 polymorphisms in 12 key genes involved in the immune response (VDR, MR1, TLR1, TLR2, TLR10, SLC11A1, IL1B, IL10, IFNG, TNF, IRAK1, and FOXP3) were tested for their association with pulmonary TB in 271 patients with TB and 251 community-matched controls from the Republic of Moldova. In addition, gene–gene interactions involved in TB susceptibility were analyzed for a total of 43 genetic loci. Single nucleotide polymorphism (SNP) analysis revealed a nominal association between TNF rs1800629 and pulmonary TB (Fisher exact test P = 0.01843). In the pairwise interaction analysis, the combination of the genotypes TLR6 rs5743810 GA and TLR10 rs11096957 GT was significantly associated with an increased genetic risk of pulmonary TB (OR = 2.48, 95% CI = 1.62–3.85; Fisher exact test P value = 1.5 × 10⁻⁵, significant after Bonferroni correction). In conclusion, the TLR6 rs5743810 and TLR10 rs11096957 two-locus interaction confers a significantly higher risk for pulmonary TB; due to its high frequency in the population, this SNP combination may serve as a novel biomarker for predicting TB susceptibility.     

Induction of toll-like receptor (TLR) 2, and MyD88-dependent TLR- signaling in response to ligand stimulation and bacterial infections in the Indian major carp,mrigal (<Emphasis Type="Italic">Cirrhinus mrigala)</Emphasis>

Toll-like receptor 2 (TLR2) is a member of TLR family. It recognizes a wide range of bacteria and their products, and is involved in inducing innate immune responses. In this article, we reported inductive expression of TLR2 and myeloid differentiation primary response gene 88 (MyD88)-dependent signaling in the Indian major carp, mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) genes by quantitative real-time PCR (qRT-PCR) revealed constitutive expression of these genes in all embryonic developmental stages, indicating their involvement in embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by qRT-PCR showed their wide distribution in various organs and tissues. Highest expression of TLR2 was in gill, MyD88 in liver and TRAF6 was in kidney. Inductive expression of TLR2, MyD88 and TRAF6 genes were observed following peptidoglycan (PGN)-treatment, and Streptococcus uberis and Aeromonas hydrophila infections. Expression of interleukin (IL)-8 and TNF-α in various organs were significantly enhanced by PGN-treatment and bacterial infections, and were closely associated with TLR2 induction. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish, and indicated it’s important role in immune surveillance of various organs during pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish, and will be helpful in developing preventive measures against infectious diseases in fish.     

Different genetic patterns in avian Toll-like receptor (TLR)5 genes

Toll-like receptors (TLRs) mediate immune response via recognition of pathogen-associated molecular patterns (PAMPs), thus play important roles in host defense. Polymorphisms of TLR5 may affect their recognition of bacterial flagellin, leading to varied host resistance to pathogenic infections. Here, we cloned TLR5 genes from Common Pheasant, Guinea fowl and 9 Chicken breeds and analyzed their sequences. The open reading frames of TLR5 were sequenced. Amino acid analysis indicated that TLR5 from Chicken breeds shared 99.4–99.9% homology. The amino acid homology of TLR5 ranged from 92.1 to 92.5% between Chickens and Guinea fowl, 95.7–96.1% between Chickens and Turkey, 94.3–94.7% between Chickens and Common Pheasant, and 79.9–80.1% between Chickens and Zebra-finch. Different genetic patterns were determined among Chickens, Common Pheasant, Guinea fowl, Turkey and Zebra-finch. It was found that there were 92 amino acid polymorphic sites, among which 5 sites in chicken TLR5, 63 sites in Guinea fowl TLR5 and 44 sites in Common Pheasant TLR5. Our data indicate that the positive Darwinian selection occurred in avian TLR5 genes since frequency of non-synonymous (d _N ) > frequency of synonymous (d _S ). These results also demonstrate that avian TLR5 genes are polymorphic among avian breeds, suggesting a varied resistance among breeds of avian. This information might be of help to improve the health of avian by breeding and vaccination.     

Genotype frequency and F ST analysis of polymorphisms in immunoregulatory genes in Chinese and Caucasian populations

Selection and genetic drift can create genetic differences between populations. Cytokines and chemokines play an important role in both hematopoietic development and the inflammatory response. We compared the genotype frequencies of 45 SNPs in 30 cytokine and chemokine genes in two healthy Chinese populations and one Caucasian population. Several SNPs in IL4 had substantial genetic differentiation between the Chinese and Caucasian populations (F _ST ~0.40), and displayed a strikingly different haplotype distribution. To further characterize common genetic variation in worldwide populations at the IL4 locus, we genotyped 9 SNPs at the IL4 gene in the Human Diversity Panel’s (N = 1056) individuals from 52 world geographic regions. We observed low haplotype diversity, yet strikingly different haplotype frequencies between non-African populations, which may indicate different selective pressures on the IL4 gene in different parts of the world. SNPs in CSF2, IL6, IL10, CTLA4, and CX3CR1 showed moderate genetic differentiation between the Chinese and Caucasian populations (0.15 < F _ST < 0.25). These results suggest that there is substantial genetic diversity in immune genes and exploration of SNP associations with immune-related diseases that vary in incidence across these two populations may be warranted.     

SNP/haplotype associations in cytokine and cytokine receptor genes and immunity to rubella vaccine

An effective immune response to vaccination is, in part, a complex interaction of alleles of multiple genes regulating cytokine networks. We conducted a genotyping study of Th1/Th2/inflammatory cytokines/cytokine receptors in healthy children (n = 738, 11–19 years) to determine associations between individual single-nucleotide polymorphisms (SNPs)/haplotypes and immune outcomes after two doses of rubella vaccine. SNPs (n = 501) were selected using the ldSelect-approach and genotyped using Illumina GoldenGate™ and TaqMan assays. Rubella-IgG levels were measured by immunoassay and secreted cytokines by ELISA. Linear regression and post hoc haplotype analyses were used to determine associations between single SNPs/haplotypes and immune outcomes. Increased carriage of minor alleles for the promoter SNPs (rs2844482 and rs2857708) of the TNFA gene were associated with dose-related increases in rubella antibodies. IL-6 secretion was co-directionally associated (p ≤ 0.01) with five intronic SNPs in the TNFRSF1B gene in an allele dose-related manner, while five promoter/intronic SNPs in the IL12B gene were associated with variations in IL-6 secretion. TNFA haplotype AAACGGGGC (t-statistic = 3.32) and IL12B promoter haplotype TAG (t-statistic = 2.66) were associated with higher levels of (p ≤ 0.01) rubella-IgG and IL-6 secretion, respectively. We identified individual SNPs/haplotypes in TNFA/TNFRSF1B and IL12B genes that appear to modulate immunity to rubella vaccination. Identification of such “genetic fingerprints” may predict the outcome of vaccine response and inform new vaccine strategies.     

Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes

Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. Polymorphisms in TLRs may influence their recognition of pathogen-derived molecules; swine TLRs are predicted to be associated with responses to infectious diseases such as pneumonia. In this study, we searched for single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR1, TLR2, TLR4, TLR5, and TLR6 genes in 96 pigs from 11 breeds and elucidated 21, 11, 7, 13, and 11 SNPs, respectively, which caused amino acid substitutions in the respective TLRs. Distribution of these nonsynonymous SNPs was biased; many were located in the leucine-rich repeats, particularly in TLR1. These data demonstrated that the heterogeneity of TLR genes was preserved in various porcine breeds despite intensive breeding that was carried out for livestock improvement. It suggests that the heterogeneity in TLR genes is advantageous in increasing the possibility of survival in porcine populations.Electronic SupplementaryMaterial Supplementary material is available for this article at     

Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression

Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45–55 days postpartum) were flow-cytometrically quantified. Gene expression of whole blood leukocytes was assessed by PAXgene analysis. Subclinical endometritis cows showed significantly higher number of blood mononuclear cells and neutrophils. Among mononuclear cells, numbers of B-cells, NK-cells, and CD172a-positive monocytes were significantly elevated. Compared with non-SCE cows, blood leukocytes of SCE cows significantly expressed higher copy numbers of CXCL8, TNF, and IL12. To test whether circulating plasma factors are responsible for these changes, leukocytes, polymorphonuclear cells, and monocyte subpopulations (classical, intermediate, nonclassical) of healthy cows were stimulated with plasma of SCE and non-SCE cows. Although gene expression of whole leukocytes and polymorphonuclear cells remained unaltered, plasma from SCE animals significantly elevated expressed messenger RNA copy numbers of CXCL8, CXCL1, and IL1B in intermediate monocytes. In conclusion, elevated number of selected mononuclear subpopulations in peripheral blood and enhanced expression of distinct genes encoding for inflammatory mediators in blood leukocytes reflect the subclinical uterine inflammatory process in cows. Whether the observed changes in the periphery of SCE cows are the consequence of the uterine inflammatory process, or whether they affect the pathogenesis of the disease is currently unknown.     

Gene conversion limits divergence of mammalian TLR1 and TLR6

Background  

Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and are important mediators of the innate immune system. TLR1 and TLR6 are paralogs and located in tandem on the same chromosome in mammals. They form heterodimers with TLR2 and bind lipopeptide components of gram-positive and gram-negative bacterial cell walls. To identify conserved stretches in TLR1 and TLR6, that may be important for their function, we compared their protein sequences in nine mammalian species(Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus; Erinaceus europaeus, Bos Taurus, Sus scrofa and Canis familiaris).     

Evaluation of the relative expression of genes associated with adherence after different hours of co-culture between Streptococcus uberis and MAC-T cells

Streptococcus uberis is an environmental pathogen associated with subclinical and clinical IMI in both lactating and non-lactating cows. RC19 strain was isolated from a cow with subclinical mastitis, qualitatively classified as moderate biofilm producer in Todd Hewitt medium (THB), and it showed a high value of the adhered bacteria (CFU/ml). Hence, the aims of this study were (a) to determine ability to adhere to and internalize into epithelial cells MAC-T for 1, 2 and 3 h, (b) to evaluate the relative expression of adherence-associated genes from co-cultures of S. uberis with MAC-T cells at 1, 2 and 3 h. We hypothesized that upon contact with bovine mammary epithelial cells, S. uberis upregulates adherence-associated genes encoding adhesins, which enable it a higher adherence to and/or internalization into host cells. Four to six genes increased their R with regard to the control after initial contact with MAC-T cells (group 1) at 1, 2 and 3 h. The highest value of R was observed at 2 h after co-culture between RC19 and MAC-T cells.     

Sequence analysis of three canine adipokine genes revealed an association between TNF polymorphisms and obesity in Labrador dogs

Obesity is an emerging health problem in purebred dogs. Due to their crucial role in energy homeostasis control, genes encoding adipokines are considered candidate genes, and their variants may be associated with predisposition to obesity. Searching for polymorphism was carried out in three adipokine genes (TNF, RETN and IL6). The study was performed on 260 dogs, including lean (n = 109), overweight (n = 88) and obese (n = 63) dogs. The largest cohort was represented by Labrador Retrievers (n = 136). Altogether, 24 novel polymorphisms were identified: 12 in TNF (including one missense SNP), eight in RETN (including one missense SNP) and four in IL6. Distributions of five common SNPs (two in TNF, two in RETN and one in IL6) were further analyzed with regard to body condition score. Two SNPs in the non‐coding parts of TNF (c.‐40A>C and c.233+14G>A) were associated with obesity in Labrador dogs. The obtained results showed that the studied adipokine genes are highly polymorphic and two polymorphisms in the TNF gene may be considered as markers predisposing Labrador dogs to obesity.     

Aged mice have increased inflammatory monocyte concentration and altered expression of cell-surface functional receptors

The expression of monocyte cell-surface receptors represents one index of immune dysfunction, which is common with aging. Although mouse models of aging are prevalent, monocyte subset assessment is rare. Our purpose was to compare cell receptor expression on classic (CD115⁺/Gr-1^high) and non-classic (CD115⁺/Gr-1^low) monocytes from 80- or 20-week-old CD-1 mice. Three-colour flow cytometry was used to determine the concentration of monocyte subsets and their respective cell-surface expression of TLR2, TLR4, CD80, CD86, MHC II and CD54. These receptors were selected because they have been previously associated with altered monocyte function. Data were analysed with independent t-tests; significance was set at P < 0.05. Old mice had a greater concentration of both classic (258%, P = 0.003) and non-classic (70%, P = 0.026) monocytes. The classic : non-classic monocyte ratio doubled in old as compared with that in young mice (P = 0.006), indicating a pro-inflammatory shift. TLR4 (↓27%, P = 0.001) and CD80 (↓37%, P = 0.004) were decreased on classic monocytes from old as compared with those from young mice. TLR2 (↑24%, P = 0.002) and MHCII (↓21%, P = 0.026) were altered on non-classic monocytes from old as compared with those from young mice. The increased classic : non-classic monocyte ratio combined with changes in the cell-surface receptor expression on both monocyte subsets is indicative of immune dysfunction, which may increase age-associated disease risk.     

Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults

Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3′ UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai–Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-γ (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94–5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17–9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-γ (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3′ UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29–0.90; p = 0.020) and reduced circulating IFN-γ (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3′ UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-γ levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-γ levels.     

Comparative <Emphasis Type="Italic">in vivo</Emphasis> infection models yield insights on early host immune response to Campylobacter in chickens

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian β-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic variations in the SPP1 promoter affect gene expression and the level of osteopontin secretion into bovine milk

Osteopontin (OPN) is now recognized as an important cytokine and extracellular integrin‐binding protein at the crossroads of inflammation and homeostasis. In a previous study, we found that OPN gene (SPP1) polymorphisms are associated with milk performance traits and somatic cell score (SCS), a parameter used to estimate the genetic value of udder health in dairy cattle. In this study, we assessed whether the genetic variations had an impact on SPP1 promoter activity, immune response and the level of OPN secreted into milk. The influence of DNA polymorphisms on the promoter activity of SPP1 was confirmed in vitro. To measure the impact of the genetic variations on OPN secretion into milk, we measured OPN levels in both plasma and milk throughout lactation. Cows were grouped by the OPN haplotypes associated with a high (H2 × H3) or low (H1 × H4) SCS. For both H2 × H3 and H1 × H4, the OPN level in plasma remained low throughout lactation, although the concentration in the milk of H1 × H4 cows increased more in late lactation. Moreover, the macrophages of H1 × H4 cows expressed a lower SPP1 and proinflammatory IL6 in response to infection. Regarding the immune cell response, cows with the genetic potential to secrete higher OPN levels during late lactation had macrophages expressing fewer proinflammatory cytokines, a situation that might explain the genetic association with low somatic cells. Although OPN's favorable roles during late lactation remain to be elucidated, the tissue remodeling properties associated with OPN may be beneficial for reducing the incidence of infection during the transition period in lactating cows.