Tanja Dzopalic

Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture

BACKGROUND The role of cytokines in pathogenesis of periapical lesions is not well understood. The aim of this study was to study the correlation between proinflammatory and immunoregulatory cytokines in periapical lesions and their relationship with cellular composition and clinical presentation. METHODS Inflammatory cells were isolated from 67 human periapical lesions and cultivated for 24 h. The levels of proinflammatory cytokines: interleukin-1 beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and immunoregulatory cytokines: transforming growth factor-beta (TGF-beta) and IL-10 were determined in culture supernatants using a fluorescent bead immunoassay or ELISA. The phenotype of cells was analysed by immunocytochemistry. RESULTS Inflammatory cells from symptomatic lesions which contained higher proportion of granulocytes, secreted higher levels of IL-1, IL-6 and IL-8 compared with asymptomatic lesions. Large-size lesions contained lower percentages of mononuclear phagocytes, higher percentages of CD8(+) T cells and produced higher levels of TNF-alpha, IL-6 and IL-10 compared with small-size lesions. There were negative correlations between the concentrations of TGF-beta and proinflammatory cytokines. TGF-beta, added to cultures, downregulated the levels of proinflammatory cytokines more strongly than IL-10, independently of clinical presentation of the lesions. By contrast, exogenous IL-10 was mainly immunosuppressive in cultures of asymptomatic lesions. CONCLUSION Symptomatic lesions are characterized by higher production of proinflammatory cytokines. Immunoregulatory cytokines are more important for suppression of inflammation in asymptomatic lesions and in this context the effect of TGF-beta is more potent and different from IL-10.

The response of human dendritic cells to co-ligation of pattern-recognition receptors

Dendritic cells (DCs) are key antigen-presenting cells that express a wide variety of pattern-recognition receptors (PRRs). Triggering of a single PRR, especially Toll-like receptors (TLRs) and C-type lectins, induces maturation of DCs, but cooperativity between multiple PRRs is needed in order to achieve an effective immune response. In this review, we summarize the published data related to the effect of individual and joint PRR agonists on DCs and Langerhans-like cells derived from monocytes (MoDCs and MoLCs, respectively). Our results demonstrate that MoDCs co-stimulated with TLR3/TLR7 and TLR3/Dectin-1 ligands induced superior T helper (Th)1 and Th17 immune responses, compared to effects of single agonists. The opposite outcome was observed after co-ligation of TLR3 and Langerin on MoLCs. These findings may be relevant to improve strategy for tumor immunotherapy.

Loxoribine, a selective Toll-like receptor 7 agonist, induces maturation of human monocyte-derived dendritic cells and stimulates their Th-1- and Th-17-polarizing capability.

Recently, a guanosine analog, 7-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), has been identified as a selective Toll-like receptor (TLR)7 agonist. Bearing in mind the controversy regarding the expression of TLR7 by human myeloid dendritic cells (DCs) and its significance for functions of these cells, the goal of this study was to investigate the effect of loxoribine on differentiation, maturation and functions of human monocyte-derived (Mo)DCs. Immature MoDCs were obtained by cultivation of monocytes for 6 days with recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. These cells were stimulated with loxoribine (250 μM) for an additional 48 h. Phenotypic properties of MoDCs were determined by flow cytometry, cytokine production was assayed by ELISA, whereas their allostimulatory capability was tested using a mixed leukocyte reaction. We showed that loxoribine up-regulated the expression of TLR7, CD40, CD54, CD80, CD83 and CCR7 and stimulated the production of IL-12, IL-23, IL-27 and IL-10 by MoDCs, whereas the level of interferon (IFN)-β was not modulated. Allogeneic CD4(+)T cells in co-culture with loxoribine-treated MoDCs proliferated more strongly, at lower DC/CD4(+)T-cell ratio (1:80), and secreted significantly higher levels of IL-17 and IFN-γ compared to the cultures with control MoDCs. The stimulatory effect of loxoribine on T helper (Th)1 polarization capability of MoDCs was further potentiated by ligation of CD40. In conclusion, our results show that loxoribine stimulated differentiation, maturation, allostimulatory as well as Th1 and Th17 polarization capability of human MoDCs and suggests that these effects might be associated with up-regulation of TLR7 expression, but not increased IFN-β production.

Differences in T-helper polarizing capability between human monocyte-derived dendritic cells and monocyte-derived Langerhans'-like cells

Langerhans’ cells (LCs) represent a specific subset of dendritic cells (DCs) which are important for detecting and processing pathogens that penetrate the skin and epithelial barriers. The aim of our study was to explain what makes their in vitro counterparts – monocyte‐derived Langerhans’‐like cells (MoLCs) – unique compared with monocyte‐derived dendritic cells (MoDCs). Immature MoDCs were generated by incubating peripheral blood monocytes with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4. The addition of transforming growth factor‐β (TGF‐β) to this cytokine cocktail resulted in the generation of MoLCs. MoLCs showed a lower expression of CD83, CD86, HLA‐DR and CCR7 compared with MoDCs, regardless of their maturational status. Both immature and mature MoLCs secreted higher quantities of IL‐23 compared with MoDCs and this finding correlated with a higher secretion of IL‐17 in co‐culture of MoLCs with allogeneic CD4+ T cells. Mature MoLCs, which produced higher levels of IL‐12 and lower levels of IL‐10 compared with mature MoDCs, were more potent at inducing interferon‐γ (IFN‐γ) production by CD4+ T cells in the co‐culture system. In conclusion, the finding that mature MoLCs stimulate stronger T‐helper 1 and T‐helper 17 immune responses than mature MoDCs, makes them better candidates for use in the preparation of anti‐tumour DC vaccines.

Signaling through Toll-like receptor 3 and Dectin-1 potentiates the capability of human monocyte-derived dendritic cells to promote T-helper 1 and T-helper 17 immune responses

BACKGROUND AIMS Recent studies have shown that the ligation of Toll-like receptor 3 (TLR3) or Dectin-1 on human monocyte-derived dendritic cells (MoDC) elicits their maturation, but with a different outcome on immunomodulation. Therefore the aim of this work was to study the response of MoDC to the combined effect of polyinosinic:polycytydilic acid [Poly (I:C)] and curdlan, selective TLR3 and Dectin-1 agonists, respectively. METHODS Immature MoDC, generated from human monocytes, were treated with Poly (I:C), curdlan or their combination for 2 days. Phenotypic characteristics of MoDC were determined by flow cytometry, and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) and FlowCytomix, while the stimulatory capability of MoDC was tested using a mixed leukocyte reaction assay. RESULTS The combination of Poly (I:C) and curdlan induced phenotypic maturation of MoDC with the capability to stimulate an alloreactive response. Such treated MoDC up-regulated the production of interleukin (IL)-12, IL-23 and IL-10, compared with the effect of Poly (I:C) alone. Curdlan-treated MoDC stimulated the production of IL-17 by alloreactive CD4 (+) T cells more strongly than Poly (I:C)-treated MoDC. The opposite effect was observed for interferon(IFN)-γ production. When combined, these agonists primed MoDC to increase further the production of IFN-γ by CD4 (+) T cells in co-culture, especially those of naive (CD45RA (+)) phenotype, and IL-17 by memory (CD45RO (+)) CD4 (+) T cells. CONCLUSIONS Ligation of TLR3 and Dectin-1 receptor up-regulates T-helper (Th) 1 and Th17 immune responses compared with single agonists. These findings may have therapeutic implications for the use of MoDC in immunotherapy.

Effect of silicon-rich water intake on the systemic and peritoneal inflammation of rats with chronic low levels of aluminum ingestion

BACKGROUND AND OBJECTIVES Study evaluated effect of silicon-rich water intake on systemic inflammation and functional characteristics of peritoneal macrophages (PMs) of rats that were chronically exposed to dietary aluminum. METHODS One month-old female Wistar Albino rats were administered aluminum chloride dissolved in distilled water (1.6mg/kg body weight in 0.5mL) by gavage for 90days. The rats were then given standard (6mg/L) or silicon-rich water (19mg/L silicon) (n=7/group). Control rats underwent sham gavage and received standard or silicon-rich water (n=7/group). Blood was assessed for cytokine levels. Unstimulated and lipopolysaccharide (LPS)-stimulated PMs were assessed in terms of phagocytic activity and cytokine secretion in vitro. RESULTS Chronic exposition to dietary aluminum and silicon-rich drinking water did not change serum TNF-α levels. Aluminum increased serum IL-2 and this was reversed by silicon-rich water. The aluminum-exposed rats had higher serum sICAM-1 than sham-gavaged, unrelated to type of water. LPS-stimulated PMs from aluminum-intoxicated animals exhibited low phagocytic activity and release of TNF-α, this was significantly improved by silicon-rich water intake. In the presence of silicon-rich water, LPS-stimulated and unstimulated PMs from aluminum-exposed rats produced significantly more IL-10. CONCLUSIONS Chronic ingestion of aluminum, increases systemic and peritoneal inflammation and PM dysfunction. The presence of high levels of the natural aluminum antagonist silicon in the drinking water restored IL-10 and TNF-α PM secretion, preventing prolonged inflammation. Thus, silicon intake can decrease the immunotoxicity of aluminum.

Dose-dependent response of dendritic cells to 7-thia-8-oxo-guanosine and its modulation by polyinosinic:polycytidylic acid.

Targeting the endosomal Toll-like receptors (TLRs) by specific agonists seems to be a promising tool for stimulation of the immunogenicity of dendritic cells (DCs). Since the functional outcome upon the engagement of TLRs may be different, the aim of our study was to examine if and how different concentrations of 7-thia-8-oxo-guanosine (7-TOG), a selective TLR7 agonist, influence differentiation, maturation and functions of human monocyte-derived DCs (MoDCs) and if its effects on MoDCs could be modulated by co-ligation of TLR3. Immature MoDCs were treated with different concentrations of 7-TOG (25, 100 and 250 μmol/L) alone, or together with polyinosinic:polycytidylic acid, Poly (I:C) (10 ng/mL), a selective TLR3 agonist, for an additional 48 h. We showed that the highest concentration of 7-TOG stimulated the differentiation, maturation and allostimulatory capability of MoDCs. These changes were accompanied by an increased production of interleukin 12 (IL-12) and induction of T helper (Th)1 and Th17 immune responses. Both Th responses were significantly augmented by additional stimulation of MoDCs with Poly (I:C). The treatment of MoDCs with the intermediate concentration of 7-TOG resulted in the up-regulation of co-stimulatory molecule (CD86) and increased production of IL-1β and IL-6 by MoDCs, followed by the stimulation of the Th17 immune response. The lowest concentration of 7-TOG down-regulated the expression of CD40 on MoDCs and potentiated the Th2 immune response. The Th2 response was not significantly modulated by additional treatment of MoDCs with Poly (I:C), but this combination of TLR3/TLR7 agonists also stimulated both Th1 and Th17 responses. In conclusion, our results show that 7-TOG influences the phenotype and functions of MoDCs in a dose-dependent manner and suggests that fine-tuned signaling through TLR7 may be modified by the engagement of TLR3, resulting in a different outcome of immune response.

The response of peripheral blood mononuclear cells to shape memory alloys

The aim of this work was to study the response of human peripheral blood mononuclear cells (PB-MNCs) to Cu-Al-Ni and Ni-Ti shape memory alloys (SMAs). The alloys were prepared as rapidly solidified thin ribbons via melt spinning. It has been shown that the ribbons were non cytotoxic for PB-MNCs as determined by the viability and apoptosis assay. In 18 cultures out of 20 cultures of individual donor PB-MNCs cultivated with Cu-Al-Ni SMAs, no significant changes in the production of Th1 (IFN-γ), Th2 (IL-5 and IL-10) and proinflammatory (IL-1s, TNF-α, IL-6 and IL-8) cytokines were detected compared to controls. In two cultures out of 20, a significant increase in the production of Th1 and proinflammatory cytokines was observed. Microstructural analyses confirmed the formation of thin oxide on the surface of both SMA ribbons.