Zlatka Georgieva Dobreva

The inhibition of JNK and p38 MAPKs downregulates IL-10 and differentially affects c-Jun gene expression in human monocytes

Interleukin-10 is the most important anti-inflammatory cytokine that controls the progress of the immune response. The molecular mechanisms driving the IL10 gene regulation are not well understood. To gain insight into this process we studied the IL-10 expression on mRNA and protein levels, together with c-Jun, FOXP3 and RelA transcription factors gene expression in human monocytes. We investigated also, the involvement of JNK and p38 transduction pathways in IL-10, c-Jun, FOXP3 and RelA gene expression. The quantity determination of IL-10 was performed by ELISA. qRT-PCR was performed for the detection of mRNA transcripts. The pharmacological inhibitors SP600125 and SB202190 were used to explore JNK and p38 MAPKs involvement in IL10, c-Jun, FOXP3 and RelA gene expression. The measurement of IL-10 mRNA synthesis, triggered by lipopolysaccharide (LPS) or C3 binding glycoprotein (C3bgp) showed that stimulation with both inducers led to similar high level of IL-10 mRNA synthesis, whereas C3bgp was the stronger inducer of IL-10 production than LPS. JNK and p38 inhibition significantly decreased IL-10 expression in stimulated cells. C3bgp and LPS induced comparatively low expression of FOXP3, RelA and c-Jun mRNA in monocytes. The inhibition of p38 MAPK in stimulated monocytes resulted in significant enhancement of c-Jun mRNA synthesis suggesting the functional relation between p38 MAPK and c-Jun gene expression. We concluded that the IL10 gene transcription did not associate with enhancement of c-Jun, RelA and FOXP3 gene expression and strictly depended on the JNK and p38 MAPKs activation in stimulated human monocytes.

Preliminary studies on the immunomodulatory effect of the C3 binding glycoprotein isolated from Cuscuta europea.

This study investigates the immunomodulatory effect of a C3 binding glycoprotein (C3bgp), isolated from the parasitic plant Cuscuta europea. When BALB/c mice, immunized with sheep red blood cells (SRBC), were given a single intraperitoneal injection of C3bgp a dose-dependent immunostimulation was observed. The stimulation was assessed by an increase in the number of haemolytic plaque forming cells (PFC) and haemaglutination titres. The induction was time dependent in respect to the administration of both the C3bgp and SRBC. When C3bgp was applied 24 h before SRBC at a dose of 30 microg per mouse (1.2 mg/kg), a well expressed immunostimulation was found. It was also found that giving C3bgp to mice, which had previously been treated with the immunosuppressive drug cyclophosphamide (CY), produced an increase in PFC. The immune response was also restored in vitro experiments were performed using human whole blood cultures stimulated with 30 microg/ml C3bgp in the presence or absence of egg albumin (OVA) as antigen for 72 to 168 h. In C3bgp stimulated cultures it was found that after 120 h there was a high expression of the CD 19+ subset of the activation antigen CD25 (IL-2R) as assessed by flow cytometric phenotype analysis. Supernatants from cultures with different stimuli were assayed by a solid phase ELISA for the determination of OVA-specific IgM at 120, 144 and 168 h. It was found that C3bgp application alone, failed to enhance OVA specific IgM, but significantly high levels of IgM in cultures containing C3bgp and OVA, were detected. Overall it has been shown that the C3 binding glycoprotein, as obtained from the parasitic plant Cuscuta europea, has strong immunostimulatory properties both in vivo and in vitro.

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1.

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30-90 microg/ml) of SQGD for different time (3-12h) intervals at 37°C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30μg/ml) stimulation for 3h at 37°C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3-6h, which was found decrease at 12h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30-90 microg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25mg/kgbwt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH. Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.

Colorectal cancer severity and survival in correlation with tumour necrosis factor-alpha

Colorectal cancer (CRC) development is strongly associated with innate immune mechanisms and intestinal inflammation. The aim of the study was to investigate the pre-operative serum levels of TNF-α and its correlation with cancer progression and survival in CRC patients taking into account the genotype of –308G/A promoter polymorphism in TNF-α gene (rs1800629). TNF-α –308G/A genotypes of 119 CRC cases and 177 no CRC controls were determined by restriction fragment length polymorphism assay (RFLP-PCR). TNF-α serum levels were measured by enzyme-linked immunosorbent assay (ELISA). Although no significant differences in allele and genotype frequencies between CRC and controls were observed, it should be noted that the minor allele-A and its homozygous genotype were overrepresented among CRC. In addition, allele-A was more frequent in early CRC patients compared to advanced cases. TNF-α serum level was significantly higher in CRC patients than in controls (36.1 ± 8.4 pg/mL vs. 18.66 ± 11 pg/mL; p = 0.0000001). In the subgroup analysis by tumour–node–metastasis stages, the highest TNF-α level was found in stage IV (42.7 ± 12.5 pg/mL) and was significantly elevated compared to earlier stages of CRC and controls. The survival rate of CRC patients with low TNF-α serum level, estimated as median survival, was significantly higher than that of patients with high levels of TNF-α (38.4 vs. 7.761 months; log rank test p = 0.00015) In conclusion, we can affirm that TNF-α affects tumour development along with disease progression which has an impact on the survival of CRC.

The Immunomodulatory Activity of C3 Binding Glycoprotein (C3bgp) is Mediated by the Complement Receptor Type III and Mitogen-Activated Protein Kinase Signal Transduction Pathways

C3 binding glycoprotein (C3bgp) is immunomodulating molecule isolated from the plant Cuscuta europea. When neutrophils were incubated with C3bgp the subsequent binding of anti-CD11b mAb became significantly higher. C3bgp induced moderate TNF-α production in human PBMC and primary monocytes. This production was significantly inhibited by the specific inhibitors of JNK and p38 MAPKs. The inhibition of JNK reduced PBMC viability. We concluded that: (i) C3bgp utilized CD11b polypeptide chain of CR3 and mediated a part of its immunomodulatory properties by activation of JNK and p38 and (ii) PBMC viability at in vitro conditions depends of JNK signal transduction pathway activation.

Proinflammatory and anti-inflammatory cytokines in adolescents from Southeast Bulgarian cities with different levels of air pollution

Epidemiological studies demonstrated that the exposure of different air pollutants including particulate matter (PM) has been related to adverse effect on immune system. Current study was designed to investigate cytokines in blood plasma of adolescent persons continuously exposed to different degrees of ambient air pollutions. Tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-12p40, and IL-10 were chosen as cytokines of proinflammatory and anti-inflammatory immune response. The peripheral venous blood was taken from adolescents living in the cities of Stara Zagora region, Southeast Bulgaria, that is, in Stara Zagora, Kazanlak, and Chirpan. The quantity of cytokines in plasma samples was determined by enzyme-linked immunosorbent assay. Results demonstrated that youths living in Stara Zagora showed significantly smaller quantity of TNF-α, compared with adolescents from Kazanlak and Chirpan. Moreover, adolescents living in Stara Zagora showed significantly higher quantity of IL-10 than students from Kazanlak and Chirpan. Analysis of the data of air quality gives reason to assert that PM10 and PM2.5 have been the main atmospheric pollutants around the monitoring points. The complex air quality assessment based on these criteria determined that the highest air pollution was in the city of Stara Zagora, followed by Chirpan and the relatively unpolluted town was Kazanlak. We concluded that air pollutants, mostly PM2.5, can modulate cytokine production and can change the balance between proinflammatory TNF-α and anti-inflammatory IL-10 production. Increased levels of IL-10 combined with decreased level of TNF-α in adolescents living in Stara Zagora can serve as a biomarker for suppression of T helper 1 (Th1) cell-mediated immunity and exacerbation of Th2 humoral immune response and could be a prerequisite for the development of allergic and autoimmune diseases.

Monocytes expression of IL-12 related and IL-10 genes in association with development of colorectal cancer

The main regulator of anti-tumor immune response is the activity of monocytes, suggesting that the produced cytokines may have a prognostic role. This study investigates gene expression of interleukin (IL)-12-related cytokine and IL-10 in stimulated monocytes from colorectal cancer (CRC) patients. Relative quantification of IL-12A, IL-12B, IL-23A and IL-10 mRNA transcripts was performed on the third hours after stimulation by real-time qPCR. We also explored an inhibitor of JNK signaling pathway activation for the observed cytokine gene expression. A strong downregulation of IL-12B mRNA expression in CRC monocytes compared to healthy donors was observed. The rate of transcription of IL-12B in stimulated monocytes was associated with the stage of CRC. The expression of IL-12A gene in stimulated monocytes from patients with advanced was lower than early cancer. Moreover, we observed stage dependent JNK inhibition mediated reduction in IL-12A expression. The hyporesponsiveness was strongly expressed in monocytes from advanced then early stages of CRC. Expression of IL-10 mRNA was almost equally in CRC monocytes from early stages and healthy donors. We demonstrated that altered gene expression profiles of IL-12A, IL-12B, IL-23A at mRNA level in CRC monocytes was associated with tumor development and can be attributed to anticancer immune response.

Two glycine containing 2-chloroethylnitrosoureas—a comparative study on some physicochemical properties, in vivo antimelanomic effects and immunomodulatory properties

Physicochemical properties such as alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of the spin labeled (nitroxyl free radical containing) glycine nitrosourea (SLCNUgly) and its nonlabeled analogue (ChCNUgly), synthesized in our laboratory are studied and compared to those of antitumour drug 3-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU). We have demonstrated that introducing of glycine moiety in the nitrosourea structure in practice does not affect either alkylating or carbamoylating activity. On the other hand replacement of cyclohexyl moiety in ChCNUgly structure with nitroxyl free radical leads to a decrease in carbamoylating activity and an increase in alkylating activity. Compound ChCNUgly showed in vivo a higher antimelanomic activity against B16 melanoma in comparison with CCNU and SLCNUgly. It completely inhibited B16 melanoma growth (TGI=100%) at a dose 64.0 mg/kg. Moreover, we established that joint i.p. application in normal mice of SLCNUgly plus a new immunostimulator (C3bgp) formerly isolated in our laboratory led to a 75% restoration in immune function with respect to antibody production measured by Jerne hemolytic plaque assay. In contrast, no immunostimulation was found after joint application of C3bgp plus ChCNUgly or CCNU at the same experimental conditions. Based on these preliminary results, a possibility for developing of new combination immunochemotherapy schemes for treatment of human cancers is discussed.

The influence of JNK and P38 MAPK inhibition on IL-12P40 and IL-23 production depending on IL12B promoter polymorphism

The interleukin-12p40 gene (IL12B) encodes the p40 polypeptide chain, which, together with p19, composes IL-23. A bi-allelic promoter polymorphism (IL12Bpro) located at −2703 bp of the transcription initiation site has been reported to show associations with IL-12p40 production. To elucidate the dependence of IL-12p40 and IL-23 production on IL12Bpro polymorphism in relation to MAPK signal transduction pathways, we examined the effect of JNK and p38 inhibition on the secretion of these cytokines by stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with 1.1 and 1.2/2.2 IL12Bpro genotypes. Stimulation with LPS and C3bgp resulted in approximately equal IL-12p40 production from PBMC with the 1.1 and 1.2/2.2 genotypes. The inhibition of JNK and p38 before stimulation significantly upregulated IL-12p40 production by PBMC with the 1.1 genotype, but did not influence IL-12p40 production from PBMC with the 1.2/2.2 genotype. Cultures of PBMC with the 1.1 genotype produced significantly more IL-12p40 than PBMC with the 1.2/2.2 genotype after stimulation with PHA. Inhibition of p38 kinase upregulated p40 production only in cultures with the 1.1 genotype. Decreased IL-23 production was observed in C3bgp-stimulated cultures after the inhibition of p38 regardless of the genotype of the tested cells. We concluded that IL-12p40 and IL-23 expression, which is mediated by the p38 and JNK intracellular signaling pathways, is influenced by IL12Bpro polymorphism.

Suppression of IL-12p40-related regulatory cytokines by suberoylanilide hydroxamic acid an inhibitor of histone deacetylases

Abstract Small molecule inhibitors of histone deacetylases (HDACs) are a new class drugs used in clinical trials for the treatment of various malignancies. Emerging evidence suggest that HDAC inhibitors may also have anti-inflammatory properties, although the molecular mechanisms remain poorly defined. Our study investigates the effect of the HDACs inhibitor suberoylanilide hydroxamic acid (SAHA) on the expression of IL-12p40-related cytokines. For this purpose, human peripheral blood mononuclear cells (PBMC) were stimulated with LPS and C3bgp with or without SAHA. IL-12p40, IL-12p35 and IL-23p19 mRNA was determined at 6 h by qRT-PCR. Cytokine levels were determined in culture supernatants at 6 and 24 h, by ELISA. SAHA significantly inhibited IL-12p40 and IL-23p19 mRNA synthesis and did not change IL-12p35 mRNA transcription. Early at 6 h, we detected significantly decreased IL-12p40 and IL-23, but not IL-12p70 protein production in cultures treated with SAHA. Results also showed that the suppression of IL-12p40-related cytokines was clearly defined at 24 h. However, this suppression was less pronounced regarding IL-12p70. The present study showed that SAHA suppressed the gene expression of IL-23p19 stronger than the expression of IL-12p35, as well as the synthesis of IL-23 compared to that of IL-12p70. We suggest that this inhibitory effect of SAHA may be beneficial during treatment of inflammatory and autoimmune diseases mediated by Th17 immune response.