Dragana Vucevic

Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.

Adult mesenchymal stem cells (MSCs) have recently become a potent tool in regenerative medicine. Due to certain shortcomings of obtaining bone marrow MSCs, alternate sources of MSCs have been sought. In this work, we studied MSCs from dental pulp (DP-MSCs) and dental follicle (DF-MSCs), isolated from the same tooth/donor, to define differences in their phenotypic properties, differentiation potential, and immunomodulatory activities. Both cell types showed colony-forming ability and expressed typical MSCs markers, but differed in the levels of their expression. DF-MSCs proliferated faster, contained cells larger in diameter, exhibited a higher potential to form adipocytes and a lower potential to form chondrocytes and osteoblasts, compared with DP-MSCs. In contrast to DF-MSCs, DP-MSCs produced the transforming growth factor (TGF)-β and suppressed proliferation of peripheral blood mononuclear cells, which could be neutralized with anti-TGF-β antibody. The treatment with toll-like receptor 3 (TLR3) agonist augmented the suppressive potential of both cell types and potentiated TGF-β and interleukin-6 secretions by these cells. TLR4 agonist augmented the suppressive potential of DF-MSCs and increased TGF-β production, but abrogated the immunosuppressive activity of DP-MSCs by inhibiting TGF-β production and the expression of indolamine-2,3-dioxygenase-1. Some of these effects correlated with the higher expression of TLR3 and TLR4 by DP-MSCs compared with DF-MSCs. When transplanted in imunocompetent xenogenic host, both cell types induced formation of granulomatous tissue. In conclusion, our results suggest that dental MSCs are functionally different and each of these functions should be further explored in vivo before their specific biomedical applications.

Proinflammatory and immunoregulatory mechanisms in periapical lesions

Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high production of IL-17, positive correlation between IL-17 and IFN-gamma, but not between IL-17 and IL-23 production. Most IL-17(+) cells coexpressed IFN-gamma. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-gamma which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most B-type lesions higher levels of IL-5 and TGF-beta were observed, as well as positive correlation between the production of TGF-beta and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2 and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the production of IFN-gamma. IL-10 and TGF-beta inhibited the production of both Th1 and Th17 cytokines. Dendritic cells (DCs) from periapical lesions, composed of immature (CD83(-)), and mature (CD83(+)) myeloid type DCs and plasmacytoid (BDCA2(+)) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and TNF-alpha than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas the secretion of IFN-gamma was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1) Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2 and immunoregulatory cytokines are more significant for advanced types of lesions with the predominance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating inflammation.

Relationship between microstructure, cytotoxicity and corrosion properties of a Cu-Al-Ni shape memory alloy.

Cu-Al-Ni shape memory alloys (SMAs) have been investigated as materials for medical devices, but their biomedical application is still limited. The aim of this work was to compare the microstructure, corrosion and cytotoxicity in vitro of a Cu-Al-Ni SMA. Rapidly solidified (RS) thin ribbons, manufactured via melt spinning, were used for the tests. The control alloy was a permanent mould casting of the same composition, but without shape memory effect. The results show that RS ribbons are significantly more resistant to corrosion compared with the control alloy, as judged by the lesser release of Cu and Ni into the conditioning medium. These results correlate with the finding that RS ribbons were not cytotoxic to L929 mouse fibroblasts and rat thymocytes. In addition, the RS ribbon conditioning medium inhibited cellular proliferation and IL-2 production by activated rat splenocytes to a much lesser extent. The inhibitory effects were almost completely abolished by conditioning the RS ribbons in culture medium for 4 weeks. Microstructural analysis showed that RS ribbons are martensitic, with boron particles as a minor phase. In contrast, the control Cu-Al-Ni alloy had a complex multiphase microstructure. Examination of the alloy surfaces after conditioning by energy dispersive X-ray and Auger electron spectroscopy showed the formation of Cu and Al oxide layers and confirmed that the metals in RS ribbons are less susceptible to oxidation and corrosion compared with the control alloy. In conclusion, these results suggest that rapid solidification significantly improves the corrosion stability and biocompatibility in vitro of Cu-Al-Ni SMA ribbons.

Evaluation of the Immunomodulatory Activities of Royal Jelly Components In Vitro

In this work the effect of different components isolated from royal jelly (RJ) was studied using an in vitro rat T-cell proliferation assay. We found that lower concentrations of MEL 174 (final water extract of RJ) and MEL 147 (3-10-dihydroxydecanoic acid) stimulated T-cell proliferation, triggered by concanavalin A (Con-A) and the process was followed by an increase in the production of interleukin-2 (IL-2). Higher concentrations of MEL 174, MEL 247 (dry powder of RJ) and MEL 138 (trans-10-hydroxydec-2-enoic acid) inhibited T-cell proliferation. The inhibition of T-cell proliferation in the presence of MEL 174 was followed by a decrease in IL-2 production, which was partly abrogated by exogenous IL-2, a decrease in nitric oxide (NO) production and increased apoptosis. In conclusion, our results showed the complexity of biological activity of RJ and suggest that its water extract possesses the most potent immunomodulatory activity in vitro.

Regulatory T-cells in Periapical Lesions

CD4+CD25hiFoxp3+ regulatory T-cells (Tregs) are of crucial importance in regulating the immune response, including the control of any defense against infection. Their presence in periapical lesions has not been demonstrated, as yet. We hypothesized that Tregs infiltrate periapical lesions, where they inhibit T-cell proliferation. The aim of this study was to characterize Tregs in periapical lesions by confocal microscopy, flow cytometry, and functional assays. We showed that CD4+CD25hiFoxp3+ cells in periapical lesions expressed IL-10 and TGF-β. Their frequency was significantly higher than in peripheral blood and correlated with the levels of TGF-β and IL-10 in culture supernatants of periapical lesion mononuclear cells. Tregs inhibited the proliferation of responder T-cells in vitro, at least in part, by stimulating the production of IL-10. These findings suggest that CD4+CD25hiFoxp3+ cells in periapical lesions may play regulatory roles in controlling local immune/inflammatory processes.

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.

Immunomodulatory effect of xylazine, an α2 adrenergic agonist, on rat spleen cells in culture

Abstract Xylazine is an adrenergic α2 agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 μM) and inhibitory (50–500 μM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine, another α2 adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an α2 adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro- l -arginine methyl ester ( l -NAME), an inhibitor of nitric oxide (NO) synthase, enhanced proliferation of splenocytes to Con A, partly abrogated the inhibitory effect of xylazine in the proliferation assay, and, only at high concentration (1000 μM), partly suppressed apoptosis of lymphocytes. The enhancing effect of l -NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through α2 adrenoreceptors.

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.

Comparative effects of aspirin and NO-releasing aspirins on differentiation, maturation and function of human monocyte-derived dendritic cells in vitro.

Acetylsalicilyc acid (aspirin, ASA) is a well known anti-inflammatory drug with immunomodulatory properties. NO-releasing aspirins (NO-ASA) are new compounds with anti-inflammatory properties. We studied the effects of ASA and two NO-ASA (NCX 4016 and NCX 4040) on human monocyte-derived dendritic cells (MoDC). Immature MoDC were generated in vitro from monocytes in the presence of recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Mature MoDC were obtained by adding lipopolysaccharide (LPS) in cultures of immature MoDC. As we found that ASA at 4-8 mM, NCX 4016 at 400-800 microM and NCX 4040 at 4-8 microM stimulated apoptosis of monocytes and immature MoDC, sub-apoptotic concentrations of ASA (2 mM), NCX 4016 (200 microM) and NCX 4040 (2 microM) were used in experiments. Examined substances were added at the beginning of MoDC cultivation. MoDC differentiated in the presence of examined compounds had lower expression of HLA-DR, CD80, CD40 and CD54, decreased allostimulatory activity and lower production of IL-12 p40. ASA and NCX 4016 decreased production of IL-10, whereas NCX 4040 had the opposite effect. ASA inhibited the expression of CD1a and prevented downregulation of CD14, NCX 4016 stimulated the differentiation of CD1a+CD14+ and CD1a(-)CD14+ cells, whereas NCX 4040 decreased the proportion of CD1a+CD14(-) and increased the frequency of CD1a+CD14+ cells, compared to control. Maturation, both in ASA and NO-ASA treated MoDC was characterized by decreased allostimulatory activity, lower expression of CD83, HLA-DR, costimulatory molecules and CD54 and decreased production of IL-10 and IL-12 p40. In conclusion, we confirmed that ASA impairs differentiation, maturation and function of MoDC and found that NCX 4016 and NCX 4040 exerted similar, but not identical effects at about 10- and 1000-fold lower concentrations, respectively, compared to ASA.

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.