IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity

Background

Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity.

Methods and Findings

Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity.

Conclusions

This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.     

Association of PARP-1, NF-κB,NF-κBIA and IL-6, IL-1β and TNF-α with Graves Disease and Graves Ophthalmopathy

Background

Graves Disease (GD) is an autoimmune disorder affected by an interaction of multiple genes such as Nuclear Factor-κB (NF-κB), Nuclear Factor-κB Inhibitor (NF-κBIA), Poly (ADP-ribose) polymerase-1 (PARP-1) and cytokines like Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) and mostly accompanied by an ocular disorder, Graves Ophthalmopathy (GO). We hypothesize that there is a relationship between GD, GO, polymorphisms of inflammatory related genes and their association with cytokines, which may play important roles in autoimmune and inflammatory processes.

Subjects and methods

To confirm our hypothesis, we studied the polymorphisms and cytokine levels of 120 patients with GD and GO using PCR-RFLP and ELISA methods, respectively.

Results

We found that patients with GG genotype and carriers of G allele of PARP-1 G1672A polymorphism are at risk in the group having GD (p = 0.0007) while having GA genotype may be protective against the disease. PARP-1 C410T polymorphism was found to be associated with GO by increasing the risk by 1.7 times (p = 0.004). Another risk factor for development of GO was the polymorphism of del/ins of NFkB1 gene (p = 0.032) that increases the risk by 39%. Levels of cytokines were also elevated in patients with GD, but no association was found between levels of cytokines and the development of GO as there was no change in levels of cytokines.

Conclusions

We suggest that, PARP-1 and NFkB1 gene polymorphisms may be risk factors for developing Graves Disease and Ophthalmopathy.     

Immunohistochemistry of IL-1β, IL-6 and TNF-α in spleens of mice treated with gold nanoparticles

Gold nanoparticles (AuNPs) possess considerable biocompatibility and therefore gaining more attention for their biomedical applications. Previous studies have shown the transient increase in pro-inflammatory cytokines expression in different organs of rats and mice exposed to AuNPs. Structural changes in the spleen of mice treated with AuNPs have also been reported. This investigation was aimed to study the immunostaining of IL-1β, IL-6 and TNF-α in mice treated with different sizes of AuNPs. The animals were divided into 7 groups of 4 animals in each group. One group received saline and served as control. Two sets of three groups were treated with 5 nm, 20 nm and 50 nm diameter AuNPs. One set was sacrificed on day 1 and the other on day 7 following the AuNPs injections. Spleens were dissected out and promptly fixed in formalin for 3 days and then processed for IL-1β, IL-6 and TNF-α immunostaining using target-specific antibodies. The immunoreactivities of IL-1β and IL-6 were increased with the increase of AuNP size. The immunostaining of IL-1β in spleen of 20 nm AuNP treated mice was subsequently decreased on day 7 whereas it persisted in 50 nm AuNP group. The increase in the immunoreactivity of IL-6 on day 1 was decreased on day 7 in the spleens of mice treated with 20 nm or 50 nm AuNPs. The immunostaining of TNF-α was found to be negative in all the treatment groups. In conclusion, the size of AuNPs plays an important role in the expression of proinflammatory cytokines in mouse spleen; small size (5 nm) AuNPs caused minimal effect, whereas larger (50 nm) AuNPs produced intense immunostaining.     

Effects of Dietary Manganese on Cu, Fe, Zn, Ca, Se, IL-1β, and IL-2 Changes of Immune Organs in Cocks

Manganese (Mn) is an essential trace element required for normal development and bodily function. However, little is known about the effect of excessive amounts of Mn in immune organs of poultry. The aim of this study was to investigate the effect of dietary Mn on the content of trace elements, such as copper (Cu), iron (Fe), zinc (Zn), calcium (Ca), and selenium (Se), and the mRNA level of IL-1β and IL-2 in immune organs (spleen, thymus, and bursa of Fabricius) and the content of IL-1β and IL-2 in serum of poultry. Fifty-day-old male Hyline cocks were fed either a commercial diet or a Mn-supplemented diet containing 600, 900, and 1,800 mg/kg. The immune organs were collected at 30, 60, and 90 days, respectively, and the content of trace elements and the mRNA level of IL-1β and IL-2 were examined; the serum were collected and the IL-1β and IL-2 contents detected. The results showed that Mn content in immune organs increased and Fe, Zn, and Ca contents decreased; however, Cu and Se contents showed no difference. IL-1β and IL-2 mRNA levels and IL-1β and IL-2 contents decreased. The present study demonstrates that excess exposure to Mn results in metal accumulations in immune organs. Manganism can disturb the balance of trace elements in immune organs and induce immune suppression in the molecular level; therefore, the immune function of cocks are also suppressed after manganism.     

Combined Analysis of IFN-γ, IL-2, IL-5, IL-10, IL-1RA and MCP-1 in QFT Supernatant Is Useful for Distinguishing Active Tuberculosis from Latent Infection

The QuantiFERON®-TB Gold In-Tube test (QFT), an interferon-γ release assay, is used to diagnose Mycobacterium tuberculosis, but its inaccuracy in distinguishing active tuberculosis from latent infection is a major concern. There is thus a need for an easy and accurate tool for achieving that goal in daily clinical settings. This study aimed to identify candidate cytokines for specifically differentiating active tuberculosis from latent infection. Our study population consisted of 31 active TB (tuberculosis) patients, 29 LTBI (latent tuberculosis infection) patients and 10 healthy control subjects. We assayed for 27 cytokines in QFT supernatants of both specific antigen-stimulated blood samples (TBAg) and negative-control samples (Nil). We analyzed their specificities and sensitivities by creating receiver operating characteristic (ROC) curves and measuring the area under those curves (AUCs). In TBAg–Nil supernatants, IL-10, IFN-γ, MCP-1 and IL-1RA showed high AUCs of 0.8120, 0.7842, 0.7419 and 0.7375, respectively. Compared with each cytokine alone, combined assay for these top four cytokines showed positive rates in diagnosing active TB, and GDA analysis revealed that MCP-1 and IL-5 are potent in distinguishing active TB from LTBI, with Wilk’s lambda = 0.718 (p < 0.001). Furthermore, utilizing the unique characteristic of IL-2 that its TBAg–Nil supernatant levels are higher in LTBI compared to active TB, the difference between IFN-γ and IL-2 showed a large AUC of 0.8910. In summary, besides IFN-γ, IL-2, IL-5, IL-10, IL-1RA and MCP-1 in QFT supernatants may be useful for distinguishing active TB from LTBI. Those cytokines may also help us understand the difference in pathogenesis between active TB and LTBI.     

Assessment of IL-10, IL-1ß and TNF-α gene polymorphisms in patients with peri-implantitis and healthy controls

Molecular Biology Reports - Peri-implantitis (PI) is a multifactorial condition caused by the interactions of pathogens and the host immune response. Previous studies have demonstrated a...     

Adipophilin affects the expression of TNF-α, MCP-1, and IL-6 in THP-1 macrophages

Macrophages accumulated in the arterial intima play an important role in the development of atherosclerosis by producing a large number of proinflammatory cytokines which accelerate the disease. Recent studies show that adipophilin might be involved in inflammatory processes in macrophages. In this study, we observe the effect of adipophilin on proinflammatory cytokine expression and secretion in THP-1 macrophages. SiRNA and adipophilin gene overexpression mediated by an pEGFP-C3 vector were used to observe the effect of adipophilin on proinflammatory cytokines in THP-1 macrophages in vitro. Realtime PCR and enzyme-linked immunosorbent assay (ELISA) were applied to detect the production of tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). It was found that acetylated low-density lipoprotein (AcLDL), pioglitazone [a peroxisome proliferator-activated receptor γ (PPARγ) agonist] increased adipophilin expression in macrophages, while glucose had no such affect. It was also shown that adipophilin augments TNF-α, MCP-1, and IL-6 expression in AcLDL induced macrophages. Our results suggest that adipophilin augment inflammation in macrophages, which might be one role of adipophilin in atherosclerosis.     

IL-1α and IL-1β recruit different myeloid cells and promote different stages of sterile inflammation

The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1α and IL-1β in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1α, and not IL-1β, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1α in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1β correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1α or mature IL-1β can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1α, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1β promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation.     

Serum TNF-α, sTNFR1, IL-6, IL-8 and IL-10 levels in Weil's syndrome

Studies on cytokine levels in Weil's syndrome are lacking. In this study, TNF-α, sTNFR1, IL-6, IL-8 and IL-10 levels were measured in 44 serum samples of patients diagnosed with Leptospira interrogans serovar icterohaemorrhagiae infection. TNF-α levels linked with pulmonary hemorrhagic implications, while elevated sTNFR1 and IL-10 levels linked with fatal cases. IL-6 and IL-8 did not seem to affect the outcome of the disease. Immune response pattern in Weil's syndrome bears resemblance to other patterns described for hemorrhagic fevers. IL-10/TNF-α ratio is proposed as a marker for prognosis.     

Lack of association or interactions between the IL-4, IL-4Rα and IL-13 genes, and rheumatoid arthritis

Introduction  

A feature of rheumatoid arthritis (RA) is an imbalance between proinflammatory and anti-inflammatory cytokines. Several recent studies have implicated polymorphism in the IL-4 signalling pathway in the development of erosive RA. The aim of the present study was to investigate the role of polymorphism in the IL-4, IL-4Rα and IL-13 genes in RA, including an examination of epistasis.     

Effects of Sodium Selenite on Aflatoxin B1-Induced Decrease of Ileac T cell and the mRNA Contents of IL-2, IL-6, and TNF-α in Broilers

The aim of this work was to assess the protective effect of sodium selenite on the ileum mucosal immunologic toxicity induced by aflatoxin B₁ (AFB₁). One hundred and eighty one-day-old healthy male avian broilers were divided into four groups of three replicates and 15 birds per replicate and fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg Se (+Se group), and 0.3 mg/kg AFB₁?+?0.4 mg/kg Se (AFB₁+Se group), respectively. The ileac T-cell subsets were determined by the methods of flow cytometry (FCM), and the mRNA contents of interleukin-2 (IL-2), interleukin-6(IL-6), and tumor necrosis factor-alpha (TNF-α) by quantitative real-time PCR. Compared with those in control group, the percentages of CD₃ ⁺, CD₃ ⁺CD₄ ⁺, CD₃ ⁺CD₈ ⁺ intraepithelial lymphocytes (IELs) and LPLs, the CD4⁺/CD8⁺ ratio of IELs, and the mRNA contents of IL-2, IL-6, and TNF-α were decreased in AFB₁ group. However, compared with those in AFB₁ group, these parameters of AFB₁+Se group were increased to be close to those in control group. It was concluded that 0.3 mg/kg AFB₁ could reduce the cellular immune function of the ileum mucosa, but 0.4 mg/kg supplemented dietary selenium showed protective effects on AFB₁-induced immunologic injury.     

Genome-Wide Association Study of Genetic Variants in LPS-Stimulated IL-6, IL-8, IL-10, IL-1ra and TNF-α Cytokine Response in a Danish Cohort

Background

Cytokine response plays a vital role in various human lipopolysaccharide (LPS) infectious and inflammatory diseases. This study aimed to find genetic variants that might affect the levels of LPS-induced interleukin (IL)-6, IL-8, IL-10, IL-1ra and tumor necrosis factor (TNF)-α cytokine production.

Methods

We performed an initial genome-wide association study using Affymetrix Human Mapping 500 K GeneChip® to screen 130 healthy individuals of Danish descent. The levels of IL-6, IL-8, IL-10, IL-1ra and TNF-α in 24-hour LPS-stimulated whole blood samples were compared within different genotypes. The 152 most significant SNPs were replicated using Illumina Golden Gate® GeneChip in an independent cohort of 186 Danish individuals. Next, 9 of the most statistical significant SNPs were replicated using PCR-based genotyping in an independent cohort of 400 Danish individuals. All results were analyzed in a combined study among the 716 Danish individuals.

Results

Only one marker of the 500 K Gene Chip in the discovery study showed a significant association with LPS-induced IL-1ra cytokine levels after Bonferroni correction (P<10⁻⁷). However, this SNP was not associated with the IL-1ra cytokine levels in the replication dataset. No SNPs reached genome-wide significance for the five cytokine levels in the combined analysis of all three stages.

Conclusions

The associations between the genetic variants and the LPS-induced IL-6, IL-8, IL-10, IL-1ra and TNF-α cytokine levels were not significant in the meta-analysis. This present study does not support a strong genetic effect of LPS-stimulated cytokine production; however, the potential for type II errors should be considered.     

GSK-3 mediates the release of IL-1β, TNF-α and IL-10 from cortical glia

Neuroinflammation has been shown to contribute to neurodegenerative and psychiatric disorders such as Alzheimer's disease and major depression due to the inappropriate release of pro-inflammatory cytokines from activated microglia. The precise molecular events that mediate cytokine release from glia remain unknown but we suggest that the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) may be involved. The aim of this study therefore was to investigate the effect of lipopolysaccharide (LPS) on expression and activity of the GSK-3β isoform in glia, and to assess if GSK-3 mediates the LPS-induced change in inflammatory cytokine levels in culture medium from rat glial-enriched cortical cultures. GSK-3β was expressed in microglia and astrocytes, and stimulation of these cultures with LPS induced an increase in GSK-3β expression and activity, and in pro-inflammatory cytokine levels in culture media. We show that GSK-3 inhibition using a small molecule inhibitor SB216763 or the mood stabiliser lithium chloride reduced the LPS-induced elevated levels of pro-inflammatory cytokines present in culture media from rat glial-enriched cortical cultures. These results demonstrate a role for GSK-3 as a modulator of inflammatory cytokine levels in the brain, and contribute to a mechanistic insight into neurological disorders in which neuroinflammation is a characteristic feature.     

Genetic Variations of IL-12B,IL-12Rβ1, IL-12Rβ2 in Behcet's Disease and VKH Syndrome

Purpose

To investigate the associations of single nucleotide polymorphisms (SNPs) of three genes (IL-12B, IL-12Rβ1 and IL-12Rβ2) in Behcet''s disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population.

Methods

A total of 806 BD cases, 820 VKH patients, and 1600 healthy controls were involved in this study. The first investigation included 400 BD patients, 400 VKH cases, and 600 healthy individuals. A second confirmatory study included a separate set of 406 BD patients, 420 VKH cases and another 1000 normal controls. Genotyping was carried out by PCR-restriction fragment length polymorphism assay and results were validated by using direct sequencing. The χ2 test was performed to compare the allele and genotype frequencies between cases and healthy controls.

Results

This study comprised two phases. In the first phase study, a significantly increased frequency of the rs3212227/IL-12B genotype CC and C allele was found in BD patients as compared to controls (Bonferroni corrected p value (p_c) = 0.009, OR 1.8; p_c = 0.024, OR 1.3, respectively). Moreover, the frequency of the C allele of rs3212227/IL-12B was also significantly increased in VKH patients (p_c = 0.012, OR 1.3, 95% CI 1.1 to 1.6). No associations were found for the other seven tested SNPs either in BD or VKH disease. The second study as well as the combined data confirmed the significant association of rs3212227/IL-12B with BD (CC genotype: combined p_c = 6.3×10⁻⁷, OR = 1.8; C allele: combined p_c = 2.0×10⁻⁵, OR = 1.3, respectively) and the C allele frequency of rs3212227/IL-12B as the risk factor to VKH patients (combined p_c = 2.5×10⁻⁵, OR 1.3, 95% CI 1.2 to 1.5).

Conclusions

Our study revealed that the IL-12B gene is involved both in the susceptibility to BD as well as VKH syndrome.     

Brain IL-6- and PG-dependent actions of IL-1β and lipopolysaccharide in avian fever

There is no persuasive evidence of a correlation between proinflammatory cytokines and avian fever. In this study, for the first time, we use avian cytokines to investigate a role for proinflammatory cytokines in the central component of avian fever. IL-1β and IL-6 injected intracerebroventricularly into Pekin ducks (n = 8) initiated robust fevers of equal magnitude and duration, although there was a significant difference in the latency to a febrile response. In addition, the IL-1β-induced fever could be abolished with an intracerebroventricular injection of antibodies to avian IL-6 or an oral administration of a PG synthesis inhibitor. Our findings indicate the following sequence of events within the central component of the avian febrile mechanism: IL-1β gives rise to bioactive IL-6, which stimulates an accelerated synthesis of PGs, and these PGs then adjust the sensitivity of warm-sensitive neurons in the avian brain stem to mediate fever. Yet PGE? was not upregulated in the cerebrospinal fluid of ducks made febrile with LPS. We conclude that IL-1β and IL-6 may well mediate fever by instigating an accelerated synthesis of brain-derived PG, of a class other than PGE?, or that IL-6 serves as one of the terminal mediators of the avian febrile response.     

Modulation of Antigenic Phenotype in Cultured Human Osteoblast-like Cells by FGFb, TGFβ1, PDGF-BB, IL-2, IL-1β, LPS and IFNγ

Background/Aims Recent reports demonstrated that osteoblast-like cells can also exert activities directly associated with the immune system (cytokine synthesis, antigen presentation, phagocytosis and stimulation of T lymphocytes). The present study aimed to analyze the effect of Transforming growth factorβ1 (TGFβ1), Fibroblast growth factor basic (FGFb), Platelet-derived growth factor-BB (PDGF-BB), Interleukin-1β (IL-1β), Interleukin-2 (IL-2), Lipopolysaccharide (LPS) and Interferon-γ (IFNγ) on the expression on osteoblast-like cells of antigens involved in antigen presentation.Methods Flow cytometry was used to investigate whether the growth factors FGFb, TGFβ1, PDGF-BB, IL-2, IL-1β, LPS and IFNγ modulate the expression on cultured human osteoblast-like cells of different antigens involved in antigen-presentation and T cell activation.Results TGFβ1 treatment significantly reduced the expression of CD54 and CD86. IL-1β treatment significantly enhanced the expression of CD54, CD86 and HLA-DR. LPS and IFNγ treatments produced a major increase in CD54, CD80, CD86 and HLA-DR expression. Expression of these antigen-presenting molecules was not significantly modified by FGFb, PDGF-BB or IL-2 treatment.     

Short communication: Concentration of TGF-β1, IL-10 and IL-6 in boar seminal plasma and TGF-β1 level in different fractions of ejaculates

Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-β1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.     

Lipopolysaccharide from Rhodobacter capsulatus counteracts the effects of toxic lipopolysaccharides and inhibits the release of TNF-α, IL-6, and IL-1β in human whole blood

The outcome of pathological process during sepsis caused by Gram-negative bacteria depends on the reaction of human blood cells to bacterial structural components, lipopolysaccharides (LPS). A general inflammatory response develops due to the increased production of proinflammatory cytokines. One of the current methods of prevention of inflammatory response is the inhibition of LPS binding to cellular receptors. We have studied the efficacy of antagonistic properties of LPS from Rhodobacter capsulatus on the production of TNF-α, IL-6, and IL-1β cytokines induced by toxic LPS from Salmonella typhimurium and Escherichia coli in human whole blood. LPS from R. capsulatus in concentrations of 0.1 and 1 μg/mL did not induce synthesis of TNF-α, IL-6, or IL-1β. Measurements of cytokine levels showed that LPS from R. capsulatus exerted a clear protective effect against toxic LPS. In particular, LPS from R. capsulatus fullly inhibited the production of TNF-α and IL-1β and significantly decreased the IL-6 production induced by LPS from S. typhimurium. Additionally, LPS from R. capsulatus antagonized the effects of LPS from E. coli, fully inhibiting the TNF-α production and decreasing the IL-6 and IL-1β levels by 60% and 70%, respectively. Thus, LPS from R. capsulatus acts as a potent antagonist of cell activation induced by toxic LPS.