In vitro Th1 cytokine-independent Th2 suppressive effects of bifidobacteria

A comparison between 17 strains of lactic acid bacteria and 15 strains of bifidobacteria indicated that bifidobacteria induced significantly lower levels of interleukin-12 (IL-12) in murine splenic cells. The present study aims to evaluate the effect and mechanism of Bifidobacterium longum BB536, a probiotic strain, in suppressing antigen-induced Th2 immune response in vitro. BB536 suppressed immunoglobulin (Ig) E and IL-4 production by ovalbumin-sensitized splenic cells, but induction of Th1-inducing cytokine production, such as IL-12 and gamma interferon (IFN-gamma) tended to be lower compared with lactic acid bacteria. Neutralization with antibodies to IL-12, IFN-gamma, IL-10 and transforming growth factor beta indicated negative involvement of Th1-inducing cytokines and regulatory cytokines in the suppression of Th2 immune response by BB536, especially when treated at higher doses of BB536 (>10 microg cells/ml). Furthermore, BB536 induced the maturation of immature bone marrow-derived dendritic cells (BM-DCs), and suppressed antigen-induced IL-4 production mediated by BM-DCs. These results suggested that BB536 suppressed Th2 immune responses, partially independent of Th1-inducing cytokines and independent of regulatory cytokines, mediated by antigen-presenting cells such as dendritic cells.     

Bacterial adhesion, intracellular survival and cytokine induction upon stimulation of mononuclear cells with planktonic or biofilm phase Staphylococcus epidermidis

Staphylococcus epidermidis is a leading cause of hospital-acquired and biofilm-associated infections. Interactions of peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages with planktonic or biofilm phase S.?epidermidis cells were studied. Biofilm phase bacteria exhibited higher attachment, as well as, a 10-fold higher intracellular survival in monocyte-derived macrophages than their planktonic counterparts. Stimulation of PBMCs and monocyte-derived macrophages was performed with live or formalin-fixed bacterial cells. Supernatant concentration of selected cytokines was measured by Luminex(?) xMAP(?) technology at different time points. As compared to planktonic phase, biofilm phase bacteria elicited lower amounts of proinflammatory cytokines and Th1 response cytokines, such as TNFα, IL-12p40, IL-12p70 and IFN-γ, whereas they enhanced production of IL-8, GM-CSF and IL-13. This phenomenon was independent of formalin pretreatment. Taken together, these results may contribute to interpretation of observed silent course of biofilm-associated infections.     

Kinetic study of cytokines production by human peripheral blood mononuclear cells in response to Brucella DNA

In spite of reports on cytokines induction by the Brucella DNA in murine model, there is no comparison between pathogenic and appropriate vaccine strains in human. We investigated the cytokines profile of human peripheral blood mononuclear cells (PBMCs) induced by DNA extracted from pathogenic isolates of Brucella melitensis and B. abortus as well as Rev1 and S19; the appropriate vaccine strains. It was observed that despite differential induction of Interleukin(IL)-12 and IL-10 production, identical IL-12/IL-10 concentration ratio was obtained by all Brucella strains DNAs that was 2 after 24h and 4 after 5 days of incubation. In addition, IL-2 and Interferon(IFN)-gamma production were profoundly increased compared to the medium at day 3 and 5 respectively but IFN-alpha was not induced. Therefore, Brucella strains DNAs are Th1 inducing component with similar pattern in human PBMCs.     

Coagulase-negative staphylococci isolated from two cases of toxic shock syndrome lack superantigenic activity, but induce cytokine production

Abstract Two strains of Staphylococcus epidermidis isolated from patients with toxic shock symptoms have been reported to carry genes related to S. aureus enterotoxins B and C by dot-blot hybridisation, although the corresponding superantigenic toxins were not detected immunologically. We here show that these strains produce no superantigens capable of stimulating proliferation of human mononuclear leukocytes or rabbit splenocytes, and that no DNA homologous to the seb or sec genes can be detected by PCR. However, stimulation of human monocytes by whole killed bacteria induced dose-dependent production of the cytokines TNFα, IL-1 β and IL-6, which may be responsible for the clinical symptoms in these patients.     

Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway.

Monocytes and macrophages play a significant role in host's defense system, since they produce a number of cytokines in response to microbial infections. We have studied IL-1 beta, IL-18, IFN-alpha/beta, and TNF-alpha gene expression and protein production in human primary monocytes and GM-CSF-differentiated macrophages during influenza A and Sendai virus infections. Virus-infected monocytes released only small amounts of IL-1 beta or IL-18 protein, whereas 7- and 14-day-old GM-CSF-differentiated macrophages readily produced these cytokines. Constitutive expression of proIL-18 was seen in monocytes and macrophages, and the expression of it was enhanced during monocyte/macrophage differentiation. Expression of IL-18 mRNA was clearly induced only by Sendai virus, whereas both influenza A and Sendai viruses induced IL-1 beta mRNA expression. Since caspase-1 is known to cleave proIL-1 beta and proIL-18 into their mature, active forms, we analyzed the effect of a specific caspase-1 inhibitor on virus-induced IL-1 beta and IL-18 production. The release of IL-1 beta and IL-18, but not that of IFN-alpha/beta or TNF-alpha, was clearly blocked by the inhibitor. Our results suggest that the cellular differentiation is a crucial factor that affects the capacity of monocytes/macrophages to produce IL-1 beta and IL-18 in response to virus infections. Furthermore, the virus-induced activation of caspase-1 is required for the efficient production of biologically active IL-1 beta and IL-18.     

Distinct characteristics of murine STAT4 activation in response to IL-12 and IFN-alpha

The role of type I IFN in Th1 development, STAT4 activation, and IFN-gamma production in murine T cells has remained unresolved despite extensive examination. Initial studies indicated that IFN-alpha induced Th1 development and IFN-gamma production in human, but not murine, T cells, suggesting species-specific differences in signaling. Later studies suggested that IFN-alpha also induced Th1 development in mice, similar to IL-12. More recent studies have questioned whether IFN-alpha actually induces Th1 development even in the human system. In the present study, we compared the capacity of IL-12 and IFN-alpha to induce Th1 differentiation, STAT4 phosphorylation, and IFN-gamma production in murine T cells. First, we show that IFN-alpha, in contrast to IL-12, cannot induce Th1 development. However, in differentiated Th1 cells, IFN-alpha can induce transient, but not sustained, STAT4 phosphorylation and, in synergy with IL-18, can induce transient, but not sustained, IFN-gamma production in Th1 cells, in contrast to the sustained actions of IL-12. Furthermore, loss of STAT1 increases IFN-alpha-induced STAT4 phosphorylation, but does not generate levels of STAT4 activation or IFN-gamma production achieved by IL-12 or convert transient STAT4 activation into a sustained response. Our findings agree with recent observations in human T cells that IFN-alpha-induced STAT4 activation is transient and unable to induce Th1 development, and indicate that IFN-alpha may act similarly in human and murine T cells.     

Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food

AIMS: To develop a multiplex PCR that allows the identification of bacteria belonging to the Staphylococcus genus and in particular to the species Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus isolated from food manufacturing plants. METHODS AND RESULTS: Five primer pairs were used in the multiplex PCR, one specific to the Staphylococcus genus and four specific to S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. All the 31 Staphylococcus reference strains yielded a specific PCR product with the genus-specific primers. Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus gave a specific PCR fragment with the corresponding species-specific primers. No amplification with the Kocuria, Macrococcus and Micrococcus strains was observed in our conditions. This multiplex PCR was performed on 30 strains of Gram-positive cocci isolated from different workshops and fermented sausages. Among them, 28 belonged to the Staphylococcus genus and 14 were identified to S. saprophyticus, four to S. xylosus, two to S. aureus and one to S. epidermidis. CONCLUSIONS: This multiplex PCR provided reliable and repeatable PCR results. It allowed the identification of a major part of the isolates, highlighting the predominance of the S. saprophyticus species in the workshops studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This tool is a useful way to screen the strains isolated from foodstuff and food environment and to monitor these species during the food processing.     

Comparative study of the binding of human blood plasma fibronectin with clinical strains of staphylococci

The interaction of 62 S. aureus clinical strains and, respectively, 20 and 17 isolated S. epidermidis and S. saprophyticus strains with human blood plasma fibronectin (FN) has been studied. The specific interaction of FN with bacteria has been evaluated simultaneously by the binding of 125I with FN (method 1), the FN-mediated agglutination of staphylococci (method 2) and the character of colonies formed in 0.15% agar medium containing FN (method 3). The data obtained in this investigation indicated that all S. aureus strains under study react with FN to a different extent. When evaluating the binding of FN with bacteria, the most pronounced correlation was observed between methods 1 and 3. None of the methods used in this investigation has revealed interaction between FN and S. epidermidis and S. saprophyticus strains under study. The authors suggest that a preliminary inference on the capacity of the isolated clinical strains of staphylococci for reaction with FN may be made from the character of colonies formed in 0.15% agar medium containing FN.     

Role of Th1 and Th2 cytokines in BCG-induced IFN-gamma production: cytokine promotion and simulation of BCG effect

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-gamma, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-alpha, or IFN-alpha, except IL-10) by neutralizing antibodies leads to reduced IFN-gamma production (19-82% inhibition in mouse and 44-77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-gamma production, whereas in human cytokines IL-2, IL-12, TNF-alpha, and IFN-alpha exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-gamma production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.     

Nucleic acids of Enterococcus faecalis strain EC-12 are potent Toll-like receptor 7 and 9 ligands inducing interleukin-12 production from murine splenocytes and murine macrophage cell line J774.1

In experiment 1 of this study, the interleukin-12 (IL-12)-inducing ability of six Enterococcus strains was evaluated in comparison with that of five Lactobacillus strains using murine splenocytes. At the same time, the involvement of Toll-like receptor (TLR) ligands in IL-12-inducing ability was assessed using splenocytes from TLR2-, TLR4- and MyD88-deficient mice. Most Enterococcus strains, especially Enterococcus faecalis strain EC-12, exerted higher IL-12-inducing ability compared with the Lactobacillus strains evaluated. Almost the same amount of IL-12 protein was produced by all lactic acid bacteria strains in splenocytes from TLR2- and TLR4-deficient mice, whereas splenocytes from MyD88-deficient mice showed no IL-12 production against all bacteria evaluated. In experiment 2, the role of TLR7, 8 and 9 ligands of E. faecalis strain EC-12 in the induction of IL-12 production was evaluated using murine macrophage cell line J774.1. A drastic decrease in IL-12-inducing ability was observed when heat-killed E. faecalis strain EC-12 was treated with nuclease, particularly RNase. In addition, less than one-tenth of IL-12 was produced by heat-killed E. faecalis strain EC-12 when both TLR7 and 9 were antagonized. These facts indicate that the nucleic acids of E. faecalis strain EC-12, particularly its RNA, are the potent TLR7 and 9 ligands that induce IL-12 production from antigen-presenting cells.     

Cytokine production by the murine macrophage cell line J774.1 after exposure to lactobacilli

Eleven strains of lactobacilli were tested for their ability to induce the murine macrophage-like cell line J774.1 to secrete cytokines. Some of the bacteria tested induce the production of interleukin(IL) 6, IL-12, and tumor necrosis factor a (TNF-alpha) by J774.1 cells. Seven strains also induced the production of IL-10. However, no IL-1beta was produced. Lactobacillus acidophilus TMC 0356 significantly induced the production of more IL-6, IL-10, IL-12, and TNF-alpha than the other bacteria tested (p < 0.0001; ANOVA). These results suggest that lactobacilli can activate macrophages to secrete both inflammatory and anti-inflammatory cytokines. Selected strains might be used to bring about pro or antiinflammatory immune reactions.     

The capacity of bovine endothelial cells to eliminate intracellular Staphylococcus aureus and Staphylococcus epidermidis is increased by the proinflammatory cytokines TNF-alpha and IL-1beta

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.     

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-alpha, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-gamma-inducing cytokines, IL-12 and IFN-alpha, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-gamma production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-alpha, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-alpha and IL-18 play a significant role in enhancing IFN-gamma synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.     

Regulation of Staphylococcus epidermidis-induced IFN-gamma in whole human blood: the role of endogenous IL-18, IL-12, IL-1, and TNF

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.     

IFN-alpha and IL-12 induce IL-18 receptor gene expression in human NK and T cells

IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-alpha or IL-12 to induce IFN-gamma. Here we show that in human NK and T cells IFN-alpha and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-alpha enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-alpha or IL-12 enhanced IL-18-induced NF-kappaB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-alpha and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.     

Activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection,hierarchical production of innate cytokines and chemokines,and the synergistic effect of lipopolysaccharide

Dengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (M phi). We have shown in the present study that DV can productively infect primary human MO/M phi regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/M phi secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-alpha), interleukin-1 beta (IL-1 beta), IL-8, IL-12, MIP-1 alpha, and RANTES but not IL-6, IL-15, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/M phi cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/M phi was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-alpha could be seen in DV-infected MO/M phi. The secretion of innate cytokines and chemokines by differentiated MO/M phi suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-alpha production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.     

IL-27 activates human monocytes via STAT1 and suppresses IL-10 production but the inflammatory functions of IL-27 are abrogated by TLRs and p38

IL-27 is a member of the IL-12 family of cytokines that activates the Jak-STAT signaling pathway in a context-dependent manner and has pleiotropic effects on acquired immunity. IL-27 has the capacity to promote early stages of Th1 generation, but recent evidence has suggested a predominant suppressive effect on Th1, Th2, and Th17 differentiation. Although modest suppressive effects of IL-27 on myeloid lineage cells have been observed, there is limited knowledge about the role of IL-27 in the regulation of innate immunity. In this study we report that although in resting murine macrophages IL-27 had minimal if any effects, in resting human monocytes IL-27 had profound proinflammatory functions. IL-27 activated a STAT1-dominant pattern of signaling in human monocytes with the consequent activation of STAT1-dependent inflammatory target genes. IL-27 primed monocytes for augmented responses to TLR stimulation in a STAT1-dependent manner, altered IL-10 signaling, and attenuated IL-10-induced gene expression. Strikingly, IL-27 strongly suppressed TLR-induced IL-10 production in human monocytes. However, the proinflammatory effects of IL-27 on human monocytes were rapidly abrogated by LPS via a p38-mediated mechanism that inhibited IL-27 signaling. Our findings identify a predominantly proinflammatory function for IL-27 in human monocytes and suggest a mechanism by which the activating effects of IL-27 on innate immunity are attenuated as an immune response proceeds and IL-27 transitions to predominantly suppressive effects on acquired immunity.