Differential gene expression changes in children with severe dengue virus infections

Background

The host response to dengue virus infection is characterized by the production of numerous cytokines, but the overall picture appears to be complex. It has been suggested that a balance may be involved between protective and pathologic immune responses. This study aimed to define differential immune responses in association with clinical outcomes by gene expression profiling of a selected panel of inflammatory genes in whole blood samples from children with severe dengue infections.

Methodology/Principal Findings

Whole blood mRNA from 56 Indonesian children with severe dengue virus infections was analyzed during early admission and at day −1, 0, 1, and 5–8 after defervescence. Levels were related to baseline levels collected at a 1-month follow-up visit. Processing of mRNA was performed in a single reaction by multiplex ligation-dependent probe amplification, measuring mRNA levels from genes encoding 36 inflammatory proteins and 14 Toll-like receptor (TLR)-associated molecules. The inflammatory gene profiles showed up-regulation during infection of eight genes, including IFNG and IL12A, which indicated an antiviral response. On the contrary, genes associated with the nuclear factor (NF)-κB pathway were down-regulated, including NFKB1, NFKB2, TNFR1, IL1B, IL8, and TNFA. Many of these NF-κB pathway–related genes, but not IFNG or IL12A, correlated with adverse clinical events such as development of pleural effusion and hemorrhagic manifestations. The TLR profile showed that TLRs were differentially activated during severe dengue infections: increased expression of TLR7 and TLR4R3 was found together with a decreased expression of TLR1, TLR2, TLR4R4, and TLR4 co-factor CD14.

Conclusions/Significance

These data show that different immunological pathways are differently expressed and associated with different clinical outcomes in children with severe dengue infections.     

Inherited Inflammatory Response Genes Are Associated with B-Cell Non-Hodgkin’s Lymphoma Risk and Survival

Background

Malignant B-cell clones are affected by both acquired genetic alterations and by inherited genetic variations changing the inflammatory tumour microenvironment.

Methods

We investigated 50 inflammatory response gene polymorphisms in 355 B-cell non-Hodgkin’s lymphoma (B-NHL) samples encompassing 216 diffuse large B cell lymphoma (DLBCL) and 139 follicular lymphoma (FL) and 307 controls. The effect of single genes and haplotypes were investigated and gene-expression analysis was applied for selected genes. Since interaction between risk genes can have a large impact on phenotype, two-way gene-gene interaction analysis was included.

Results

We found inherited SNPs in genes critical for inflammatory pathways; TLR9, IL4, TAP2, IL2RA, FCGR2A, TNFA, IL10RB, GALNT12, IL12A and IL1B were significantly associated with disease risk and SELE, IL1RN, TNFA, TAP2, MBL2, IL5, CX3CR1, CHI3L1 and IL12A were, associated with overall survival (OS) in specific diagnostic entities of B-NHL. We discovered noteworthy interactions between DLBCL risk alleles on IL10 and IL4RA and FL risk alleles on IL4RA and IL4. In relation to OS, a highly significant interaction was observed in DLBCL for IL4RA (rs1805010) * IL10 (rs1800890) (HR = 0.11 (0.02–0.50)). Finally, we explored the expression of risk genes from the gene-gene interaction analysis in normal B-cell subtypes showing a different expression of IL4RA, IL10, IL10RB genes supporting a pathogenetic effect of these interactions in the germinal center.

Conclusions

The present findings support the importance of inflammatory genes in B-cell lymphomas. We found association between polymorphic sites in inflammatory response genes and risk as well as outcome in B-NHL and suggest an effect of gene-gene interactions during the stepwise oncogenesis.     

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IκBα and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam₃CSK₄, but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.     

Association analysis of single nucleotide polymorphisms of proinflammatory cytokine and their receptors genes with rheumatoid arthritis in northwest Chinese Han population

ObjectiveTo analyze the relationship of genetic polymorphisms in IL1β, IL6, TNF-α genes and their receptors genes with rheumatoid arthritis (RA) for northwest Han Chinese. This study also explores whether there are gene–gene interactions among these genetic polymorphisms.MethodsA total of 452 patients with RA and 373 matched healthy controls were enrolled to carry out a case-control study for 16 SNPs of IL1B-511 C > T, IL1B-31 T > C, IL1B+3954 C > T, IL1RN T > C, IL6-597 G > A, IL6-572 G > C, IL6-174 G > C, IL6R-183 G > A, IL6R exon2 T > A, IL6R exon1 A > C, TNFA-863 C > A, TNFA-857 C > T, TNFA-308 G > A, TNFA-238 G > A, TNFR1-383 A > C and TNFR2 T676G T > G from seven genes. Genotyping for the SNPs was conducted on the RotorGene 6000 PCR platform using in-house high resolution melting (HRM) approaches. Detection correctness was validated through direct sequencing. Generalized multifactor dimensionality reduction (GMDR) analysis was applied to discover likely gene–gene interaction model among the SNPs.ResultsThe results showed that the genotype distributions of TNFA-308, TNFA-857 and TNFA-863 are significantly different between case and control groups (P = 0.016, P = 0.048 and P = 0.016, respectively). Carriers of TNFA-857 mutant allele conferred risk to RA (OR = 1.525, 95% CI = 1.157–2.009) while those of TNFA-308 and TNFA-863 mutant alleles conferred protection to RA (OR = 0.459, 95% CI = 0.286–0.739; OR = 0.490, 95% CI = 0.329–0.732). GMDR analysis for the SNPs indicated that gene–gene interaction existed among IL1B-31, IL1RN, IL6-572, IL6R-183, IL6R-exon1 and TNFA-857. Thirteen of all genotypes of the six SNPs combination were discovered to have significant distribution difference between RA group and the control.ConclusionsThis study demonstrated that PCR-HRM assay is a highly efficient SNP genotyping method especially for the detection of large-scale samples. The SNPs of TNFA-308 and TNFA-863 are closely associated with RA susceptibility and that gene–gene interactions may occur among the six SNPs of IL1B-31, IL1RN, IL6-572, IL6R-183, IL6R-exon1 and TNFA-857 in RA patients from northwest Chinese Han population, especially these SNPs’ combination genotypes CT/TT/CC/GG/AC/CC, CT/TT/GC/AA/AC/CT and CT/CT/CC/GA/AC/CC to show high risk of RA susceptibility in our study.     

BCL-3 Attenuation of TNFA Expression Involves an Incoherent Feed-Forward Loop Regulated by Chromatin Structure

Induction of genes is rarely an isolated event; more typically occurring as part of a web of parallel interactions, or motifs, which act to refine and control gene expression. Here, we define an Incoherent Feed-forward Loop motif in which TNFα-induced NF-κB signalling activates expression of the TNFA gene itself and also controls synthesis of the negative regulator BCL-3. While sharing a common inductive signal, the two genes have distinct temporal expression profiles. Notably, while the TNFA gene promoter is primed to respond immediately to activated NF-κB in the nucleus, induction of BCL3 expression only occurs after a time delay of about 1h. We show that this time delay is defined by remodelling of the BCL3 gene promoter, which is required to activate gene expression, and characterise the chromatin delayed induction of BCL3 expression using mathematical models. The models show how a delay in inhibitor production effectively uncouples the rate of response to inflammatory cues from the final magnitude of inhibition. Hence, within this regulatory motif, a delayed (incoherent) feed-forward loop together with differential rates of TNFA (fast) and BCL3 (slow) mRNA turnover provide robust, pulsatile expression of TNFα . We propose that the structure of the BCL-3-dependent regulatory motif has a beneficial role in modulating expression dynamics and the inflammatory response while minimising the risk of pathological hyper-inflammation.     

Downregulation of TLR4 and 7 mRNA Expression Levels in Broiler’s Spleen Caused by Diets Supplemented with Nickel Chloride

Toll-like receptors (TLRs) are important immune receptors in discriminating self from nonself and in initiating the innate and adaptive immune response. TLR4 and TLR7 have been proven to be highly expressed in chicken’s spleen. Thus, this study was to evaluate the TLR4 and TLR7 messenger RNA (mRNA) expression levels in the spleen of broilers fed diets supplemented with nickel chloride (NiCl₂) using the methods of quantitative real-time PCR (qRT-PCR). Two hundred forty-one-day-old avian broilers were equally divided into 4 groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600, and 900 mg/kg of NiCl₂ for 42 days. Results showed that TLR4 and TLR7 mRNA expression levels in the spleen were lower (P?<?0.05 or P?<?0.01) in the 300, 600, and 900 mg/kg groups than those in the control group. It was concluded that dietary NiCl₂ in excess of 300 mg/kg could lower TLR4 and TLR7 mRNA expression levels in the spleen of broilers, implying that NiCl₂ could impair the innate and adaptive immunity in spleen by injuring immunocytes and/or decreasing the content of cytokines through TLRs.     

Interleukin-6 and Lipopolysaccharide Modulate Hepcidin mRNA Expression by Hepg2 Cells

Iron homeostasis is controlled by hepcidin (Hpc) as well as other ways. Hpc expression is regulated by iron (Fe) storage and by inflammation, but the joint effect of both stimuli remains unclear. We studied the modulatory role of inflammatory agents (IL6 and LPS) over Hpc and DMT1 mRNA expression in HepG2 cells preloaded with Fe. HepG2 cells were preloaded with different Fe concentrations (holo-Tf or Fe-NTA) and then incubated with IL6 or LPS. We measured intracellular Fe levels by AAS with graphite furnace, transferrin receptor (TfR) by ELISA and mRNA relative abundance of Hpc and DMT1 by qRT-PCR. The maximum effect on Fe uptake was observed in cells incubated with 30?ng/ml IL6 (p?<?0.01) and 500?ng/ml LPS (p?<?0.05). In HepG2 cells preloaded with holo-Tf or Fe-NTA and challenged with IL6 and LPS, we observed a decreased: (a) Hpc mRNA relative abundance (two-way ANOVA: p?<?0.05 and p?<?0.001, respectively), (b) DMT1 mRNA relative abundance and TfR1 protein levels (two-way ANOVA: p?<?0.001), and (c) intracellular Fe concentration (two-way ANOVA: p?<?0.001 and p?<?0.01, respectively) compared to control cells incubated only with Fe (holo-Tf or Fe-NTA). Our results support the idea that Fe storage and inflammation act together to regulate Fe homeostasis and suggest a negative regulation in this hepatic cellular model to prevent excessive increases in Hpc.     

Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis

Helicobacter pylori (H. pylori)-induced immune responses in the gastric mucosa are skewed toward T helper (Th) 1 phenotype, which is characterized by predominant production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by helper T cells. Toll-like receptors (TLRs) play an essential role in mucosal defense against microbes through the recognition of bacterial molecules. Among the members of the TLR family, TLR9 recognizes bacterial unmethylated CpG DNA sites, and signal transduction of TLR9 induces production of a variety of cytokines, including type-I IFN (IFN-α/β). We investigated the expression and role of TLR9 in H. pylori-induced gastritis in mice. Expression of TLR9 mRNA in the gastric tissue increased after infection with H. pylori. TLR9 was mainly expressed in the macrophages, dendritic cells, and CD3⁺ cells in the gastric mucosa. Neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA were higher in TLR9 knockout (KO) mice than in wild-type mice at 2 and 4 months after H. pylori inoculation. These differences in inflammatory parameters between H. pylori-infected wild-type and TLR9 KO mice disappeared 6 months after H. pylori inoculation. Expression of interleukin-4 mRNA, typical Th2 cytokine, in the gastric tissue did not differ between H. pylori-infected wild-type and TLR9 KO mice. Expression level of IFN-α/β mRNA in the TLR9 KO mice was lower than that in wild-type mice by 4 months after inoculation. Administration of IFN-α reduced H. pylori infection-induced increase in neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA in TLR9 KO mice. Our findings suggest that TLR9 signaling plays important roles in the suppression of H. pylori-induced gastritis in the early phase via downregulation of Th1-type cytokines modulated by IFN-α.     

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis

Toll‐like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT‐PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti‐TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre‐incubation of PBMCs from healthy donors with anti‐TLR2 led to reduced expression of IFN‐γ and IL‐12p70, but not of IL‐4 and IL‐10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.     

Genetic polymorphisms involved in the inflammatory response and lung cancer risk: A case-control study in Japan

Evidence is accumulating that chronic inflammation may have an important mechanism for the development and progression of lung cancer. Therefore, genetic polymorphisms in genes that involved in the inflammatory response may be associated with lung cancer risk. We evaluated the role of tumor necrosis factor α (TNFA) rs1799724, interleukin 1β (IL1B) rs16944, IL6 rs1800796, myeloperoxidase (MPO) rs2333227 and C-reactive protein (CRP) rs2794520 in a case-control study comprised of 462 lung cancer cases and 379 controls in a Japanese population. Unconditional logistic regression was used to assess the adjusted odds ratios (OR) and 95% confidence intervals (95% CI). CRP rs2794520 (OR = 1.64, 95% CI = 1.19–2.26) and IL6 rs1800796 (OR = 1.41, 95% CI = 1.02–1.96) were associated with lung cancer risk. In addition, we assessed interactions between the polymorphisms and smoking. The polymorphisms did not significantly modify the association between smoking and lung cancer. As TNFA triggers a cytokine cascade, the modifying effect of the TNFA rs1799724 genotypes on the association of any of the remaining polymorphisms with lung cancer risk was also examined. There was a significant interaction between TNFA rs1799724 and MPO rs2333227 (P_interaction = 0.058). Future studies involving larger control and case populations will undoubtedly lead to a more thorough understanding of the role of the polymorphisms involved in the inflammation pathway in lung cancer.     

Expression of TLR2/4 in the sperm‐storing oviduct of the Chinese soft‐shelled turtle Pelodiscus sinensis during hibernation season

The initiation of innate immunology system could play an important role in the aspect of protection for sperms long‐term storage when the sperms got into oviduct of turtles and come into contact with epithelium. The exploration of TLR2/4 distribution and expression in oviduct during hibernation could help make the storage mechanism understandable. The objective of this study was to examine the gene and protein expression profiles in Chinese soft‐shelled turtle during hibernation from November to April in the next year. The protein distribution of TLR2/4 was investigated in the magnum, isthmus, uterus, and vagina of the turtle oviduct using immunohistochemistry, and the gene expression of TLR2/4 was analyzed using quantitative real‐time PCR (qRT‐PCR). The results showed positive TLR2 protein expression primarily in the epithelium of the oviduct. TLR4 immunoreactivity was widely observed in almost every part of the oviduct, particularly in the epithelium and secretory gland membrane. Analysis of protein, mRNA expression revealed the decreased expression of TLR2/4 in the magnum compared with the isthmus, uterus, and vagina during hibernation. The protein and mRNA expression of TLR2 in the magnum, isthmus, uterus, and vagina was decreased in April compared with that in November. TLR4 protein and mRNA expression in the magnum, isthmus, uterus and vagina was decreased in November compared with that in April. These results indicated that TLR2/4 expression might protect the sperm from microbial infections. In contrast to the function of TLR2, which protects sperm during the early stages of hibernation, TLR4 might play a role in later stages of storage. The present study is the first to report the functions of TLR2/4 in reptiles.     

Expression profiles of Toll-like receptors 1, 2 and 5 in selected organs of commercial and indigenous chickens

Toll-like receptors (TLRs) are members of the cellular receptors that constitute a major component of the evolutionary conserved pattern recognition system (PRR). TLRs are expressed in a wide variety of tissues and cell types. In this study we compared the expression profiles of the chicken TLR1, TLR2 and TLR5 genes in a range of organs (lung, ovary, liver, thymus, duodenum, spleen and large intestine) in commercial Hy-Line (HL) and indigenous Green-legged Partridgelike (GP) chickens. The level of mRNA was determined with RT-qPCR using the TaqMan probes for target and reference (ACTB) genes. We determined that the tissue profiles differed with respect to each TLR and they were ranked as follows: spleen, lungs, large intestine (TLR1), large intestine, lungs, thymus/ovary (TLR2) and lungs, thymus, liver (TLR5). A differential expression between HL and GP chickens was determined for TLR1 and TLR5 genes in large intestine and thymus of HL (P?<?0.05) and GP (P?<?0.05) chickens. We conclude that the commercial chickens expressed higher levels of TLR1 mRNA in large intestine and TLR5 mRNA in thymus than indigenous chickens.