Plasmid-based system for high-level gene expression and antisense gene knockdown in Bartonella henselae.

Six broad-host-range plasmid vectors were developed to study gene expression in Bartonella henselae. The vectors were used to express a beta-galactosidase reporter gene in B. henselae and to generate antisense RNA for gene knockdown. When applied to ompR, a putative transcription response regulator of B. henselae, this antisense RNA gene knockdown strategy reduced bacterial invasion of human endothelial cells by over 60%.

CB(1) and CB(2) cannabinoid receptors mediate different aspects of delta-9-tetrahydrocannabinol (THC)-induced T helper cell shift following immune activation by Legionella pneumophila infection.

Legionella pneumophila infection of mice induces proinflammatory cytokines and Th1 immunity as well as rapid increases in serum levels of IL-12 and IFNgamma and splenic IL-12Rbeta2 expression. Delta-9-tetrahydrocannabinol (THC) treatment prior to infection causes a shift from Th1 to Th2 immunity and here we demonstrate that CB(1) and CB(2) cannabinoid receptors mediate different aspects of the shift. Using cannabinoid receptor antagonists and cannabinoid receptor gene deficient mice (CB(1) (-/-) and CB(2) (-/-)), we showed that both CB(1) and CB(2) receptors were involved in the THC-induced attenuation of serum IL-12 and IFNgamma. IFNgamma production is dependent upon signaling through IL-12Rbeta2 (beta2) and THC treatment suppressed splenic beta2 message; moreover, this effect was CB(1) but not CB(2)-dependent from studies with receptor antagonists and CB1(-/-) and CB2(-/-) mice. Furthermore, observed increases in IL-4 induced by THC, were not involved in the drug effect on beta2 from studies with IL-4 deficient mice. The GATA-3 transcription factor is necessary for IL-4 production and is selectively expressed in Th2 cells. GATA-3 message levels were elevated in spleens of THC-treated and L. pneumophila-infected mice and the effect was shown to be CB(2) but not CB(1)-dependent. Furthermore, GATA-3 regulatory factors were modulated in that Notch ligand Delta4 mRNA was decreased and Jagged1 increased by THC also in a CB2-dependent manner and splenic NFkappaB p65 was increased. Together, these results indicate that CB(1) and CB(2) mediate the THC-induced shift in T helper activity in L. pneumophila-infected mice, with CB(1) involved in suppressing IL-12Rbeta2 and CB(2) involved in enhancing GATA-3.

Suppression of dendritic cell activation by anthrax lethal toxin and edema toxin depends on multiple factors including cell source, stimulus used, and function tested.

Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET), and they suppress the function of LPS-stimulated dendritic cells (DCs). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). LT, not ET, was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF-alpha in cells from BALB/c and B6 mice but increased IL-1beta in LPS-stimulated cultures. ET also suppressed IL-12 and TNF-alpha, but increased IL-6 and IL-1beta in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET generally decreased marker expression across all groups. We conclude that the suppression of cytokine production by anthrax toxins is dependent on variables, including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the stimuli or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.

Legionella pneumophila infection up-regulates dendritic cell Toll-like receptor 2 (TLR2)/TLR4 expression and key maturation markers.

Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of Legionella pneumophila infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live L. pneumophila resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.

Role of Toll-like receptor 9 in Legionella pneumophila-induced interleukin-12 p40 production in bone marrow-derived dendritic cells and macrophages from permissive and nonpermissive mice.

The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.

Cannabinoid treatment suppresses the T-helper cell-polarizing function of mouse dendritic cells stimulated with Legionella pneumophila infection.

Marijuana cannabinoids, such as delta-9-tetrahydrocannabinoid (THC), suppress type 1 T-helper 1 (Th1) immunity in a variety of models, including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, bone marrow-derived dendritic cells (DCs) were purified from BALB/c mice and studied following infection and drug treatment. DCs infected in vitro with Lp were able to protect mice when injected prior to a lethal Lp infection; however, the immunization potential of the Lp-loaded cells along with Th1 cytokine production was attenuated by THC treatment at the time of in vitro infection. In addition, THC-treated and Lp-loaded DCs were poorly stimulated in culture-primed splenic CD4(+) T cells to produce interferon-gamma; however, this stimulating deficiency was reversed by adding recombinant interleukin (IL)-12p40 protein to the cultures. Moreover, THC treatment inhibited the expression of DC maturation markers, such as major histocompatibility complex class II and costimulatory molecules CD86 and CD40 as determined by flow cytometry and suppressed the Notch ligand, Del-ta4, as determined by reverse transcription-polymerase chain reaction. However, THC treatment did not affect other DC functions, such as intracellular killing of Lp, determined by colony-forming unit counts of bacteria, and Lp-induced apoptosis, determined by annexin V staining. In conclusion, the data suggest that THC inhibits Th1 activation by targeting essential DC functions, such as IL-12p40 secretion, maturation, and expression of costimulatory and polarizing molecules.

Role of cannabinoid receptors in Delta-9-tetrahydrocannabinol suppression of IL-12p40 in mouse bone marrow-derived dendritic cells infected with Legionella pneumophila.

Delta-9-tetrahydrocannabinol (THC) injection suppresses serum interleukin-12 (IL-12) levels in Legionella pneumophila-infected mice. Dendritic cells are a major producer of IL-12 and mouse, bone marrow-derived dendritic cell cultures produced high levels of the IL-12p40 following L. pneumophila infection. Treatment with THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoid, 2-arachidonoyolglycerol, less potently suppressed cytokine production. Dendritic cells expressed mRNA for cannabinoid receptor 1 (CB(1)), cannabinoid CB(2) receptor, and vanilloid receptor 1 (TRPV1) and the addition of the G(i) inhibitor, pertussis toxin, completely attenuated suppression induced by 3 and 6 muM THC but not by 10 muM THC. Furthermore, THC suppression was partially attenuated in dendritic cells from cannabinoid CB(1) receptor and CB(2) receptor knockout mice and in dendritic cells co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist, capsazepine. These results suggest that THC-induced suppression of serum IL-12 is partly due to a suppression of IL-12 production by dendritic cells and that G(i) signaling and cannabinoid receptors, but not TRPV1, are involved in this suppressive effect.

Epigallocatechin gallate modulates cytokine production by bone marrow-derived dendritic cells stimulated with lipopolysaccharide or muramyldipeptide, or infected with Legionella pneumophila.

The primary polyphenol in green tea extract is the catechin epigallocatechin gallate (EGCG). Various studies have shown significant suppressive effects of catechin on mammalian cells, either tumor or normal cells, including lymphoid cells. Previous studies from this laboratory reported that EGCG has marked suppressive activity on murine macrophages infected with the intracellular bacterium Legionella pneumophila (Lp), an effect mediated by enhanced production of both tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma). In the present study, primary murine bone marrow (BM)-derived dendritic cells (DCs), a phagocytic monocytic cell essential for innate immunity to intracellular microorganisms, such as Lp, were stimulated in vitro with the microbial stimulant lipopolysaccharide (LPS) from gram-negative bacteria, the cell wall component from gram-positive bacteria muramyldipeptide (MDP) or infected with Lp. Production of the T helper cell (Th1)-activating cytokine, interleukin-12 (IL-12) and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha),produced mainly by phagocytic cells and important for antimicrobial immunity, was determined in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Treatment of the cells with EGCG inhibited, in a dose-dependent manner, production of IL-12. In contrast, enhanced production of TNF-alpha occurred in a dose-dependent manner in the DC cultures stimulated with either soluble bacterial product or infected with Lp. Thus, the results of this study show that the EGCG catechin has a marked effect in modulating production of these immunoregulatory cytokines in stimulated DCs, which are important for antimicrobial immunity, especially innate immunity. Further studies are necessary to characterize the physiologic function of the effect of EGCG on TNF-alpha and IL-12 during Lp infection, and the mechanisms involved.

The THC-induced suppression of Th1 polarization in response to Legionella pneumophila infection is not mediated by increases in corticosterone and PGE2.

T helper cell type 1 (Th1)-polarizing cytokines are induced by Legionella pneumophila infection and are suppressed by pretreatment with marijuana cannabinoids (CB). Glucocorticoids and prostaglandin E2(PGE2) are also reported to suppress Th1 polarization and are induced by Delta9-tetrahydrocannabinol (THC), so their role in the suppression of polarizing cytokines was examined. Injection of L. pneumophila or THC alone into BALB/c mice induced a rapid and transient rise in serum corticosterone (CS), and the injection of both agents significantly augmented the CS response, demonstrating that THC increased CS in Legionella-infected mice. Pretreatment with the CB receptor 1 (CB1) antagonist SR141716A had no effect on the THC-induced CS response, but CB2 antagonist (SR144528) treatment increased the CS response. To see if increased CS contributed to the down-regulation of Th1 cytokines, mice were pretreated with the steroid antagonist RU486 before THC injection and Legionella infection. The results showed that RU486 did not attenuate the THC-induced suppression of serum interleukin (IL)-12 or interferon-gamma (IFN-gamma). In addition to CS, THC injection increased urinary PGE2 metabolites, and the CB1 antagonist attenuated this increase. Although L. pneumophila infection increased urinary PGE2, THC pretreatment did not enhance this response; in addition, treatment with the cyclooxygenase inhibitor, indomethacin, did not block the THC-induced suppression of IL-12 and IFN-gamma. These results suggest that the elevation of CS and PGE2 does not account for the THC-induced attenuation of the Th1 cytokine response, and it is concluded that other suppressive mediators are induced by THC or that the drug acts directly on immune cells to suppress cytokine production.

Cannabinoid receptors and T helper cells.

We have reported that injection of marijuana cannabinoids, such as Delta(9)-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) gamma production and enhancement of IL-4 production suggesting THC-induced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL-12Rbeta2 or GATA3 gene products. The results showed that THC suppressed IL-12Rbeta2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rbeta2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rbeta2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells.

The cannabinoid system and immune modulation.

Studies on the effects of marijuana smoking have evolved into the discovery and description of the endocannabinoid system. To date, this system is composed of two receptors, CB1 and CB2, and endogenous ligands including anandamide, 2-arachidonoyl glycerol, and others. CB1 receptors and ligands are found in the brain as well as immune and other peripheral tissues. Conversely, CB2 receptors and ligands are found primarily in the periphery, especially in immune cells. Cannabinoid receptors are G protein-coupled receptors, and they have been linked to signaling pathways and gene activities in common with this receptor family. In addition, cannabinoids have been shown to modulate a variety of immune cell functions in humans and animals and more recently, have been shown to modulate T helper cell development, chemotaxis, and tumor development. Many of these drug effects occur through cannabinoid receptor signaling mechanisms and the modulation of cytokines and other gene products. It appears the immunocannabinoid system is involved in regulating the brain-immune axis and might be exploited in future therapies for chronic diseases and immune deficiency.