Cholera toxin impairs the differentiation of monocytes into dendritic cells, inducing professional antigen-presenting myeloid cells

Cholera toxin (CT) is a potent adjuvant for mucosal vaccination; however, its mechanism of action has not been clarified completely. It is well established that peripheral monocytes differentiate into dendritic cells (DCs) both in vitro and in vivo and that monocytes are the in vivo precursors of mucosal CD103⁻ proinflammatory DCs. In this study, we asked whether CT had any effects on the differentiation of monocytes into DCs. We found that CT-treated monocytes, in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), failed to differentiate into classical DCs (CD14^low CD1a^high) and acquired a macrophage-like phenotype (CD14^high CD1a^low). Cells differentiated in the presence of CT expressed high levels of major histocompatibility complex class I (MHC-I) and MHC-II and CD80 and CD86 costimulatory molecules and produced larger amounts of IL-1β, IL-6, and IL-10 but smaller amounts of tumor necrosis factor alpha (TNF-α) and IL-12 than did monocytes differentiated into DCs in the absence of CT. The enzymatic activity of CT was found to be important for the skewing of monocytes toward a macrophage-like phenotype (Ma-DCs) with enhanced antigen-presenting functions. Indeed, treatment of monocytes with scalar doses of forskolin (FSK), an activator of adenylate cyclase, induced them to differentiate in a dose-dependent manner into a population with phenotype and functions similar to those found after CT treatment. Monocytes differentiated in the presence of CT induced the differentiation of naïve T lymphocytes toward a Th2 phenotype. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells (APCs) following systemic immunization.Adjuvant design has been mainly empirical, and the mechanism of action of the most efficacious molecules has remained obscure. It is important to investigate the mechanisms of action of already known adjuvants to facilitate the design of new effective molecules with high potency and decreased side effects. The bacterial enterotoxin cholera toxin (CT) from Vibrio cholerae is extraordinarily effective as a mucosal and systemic adjuvant, yet its capacity to amplify the immune response has not been completely clarified (12, 24, 25). It is a holotoxin, composed of an enzymatically active A subunit, noncovalently linked to a pentameric B subunit, which binds to the ganglioside GM1 on host cell membranes. Once internalized, the A subunit ADP-ribosylates the α subunit of the GTP-binding regulatory protein Gs, thereby inducing permanent adenylate cyclase activation, resulting in an increase of intracellular cyclic AMP (cAMP) (55). The mechanism of the adjuvanticity of CT may be a complex phenomenon resulting from the interaction of the toxin with different cell types present in the architecture of the mucosa. Dissecting the effect of CT on different cells types could help in understanding the contribution of each to its adjuvant activity.Given the crucial role of dendritic cells (DCs) and other professional antigen-presenting cells (APCs) (monocytes/macrophages and B cells) in the induction of adaptive immunity (26), the potentiation of APC function can be a major aspect of adjuvant action. Cholera toxin upregulates expression of the B7-2 (CD86) costimulatory molecule and stimulates antigen presentation through enhancement of major histocompatibility complex class II (MHC-II) expression and interleukin 1β (IL-1β) production (7, 11, 37, 54). CT also interacts with lymphocytes and promotes B cell isotype-switch differentiation toward IgG1 and IgA in mice (2, 54) and enhances the antigen-presenting function of human B cells (38). We and others showed that CT, by inducing maturation of human DCs, polarizes a mixed Th1/Th2 T cell response, with a strong bias toward Th2 cells (15, 16).The capacity of CT to interact with DCs or monocytes as innate immune cells or as precursors of DCs may represent a crucial step for its adjuvant mechanism. DCs represent heterogeneous populations that comprise distinct subtypes that vary in hematopoietic origin, life cycle, and function (4, 52). It is well established that peripheral monocytes differentiate into DCs both in vitro and in vivo (19, 20, 43-45, 48) and that monocytes are the in vivo precursors of mucosal CD103⁻ proinflammatory DCs (5, 27, 58). More important, the role of monocytes as precursors of DCs in mediating the in vivo adjuvant effects has been described (29, 60). Therefore, in this study, we asked whether CT affected the differentiation of monocytes into DCs. By using human DCs generated from monocytes (48), we found that CT interferes with the differentiation of monocytes into DCs, giving rise to a distinct population (Ma-DCs), which displays an activated macrophage-like phenotype, induces a strong allogeneic and antigen-specific response, and promotes the polarization of naive CD4⁺ T lymphocytes toward a Th2 profile. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells following systemic immunization.     

Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin,but Not Their Nontoxic Counterparts,Improve the Antigen-Presenting Cell Function of Human B Lymphocytes

B lymphocytes play an important role in the immune response induced by mucosal adjuvants. In this study we investigated the in vitro antigen-presenting cell (APC) properties of human B cells upon treatment with cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) and nontoxic counterparts of these toxins, such as the B subunit of CT (CT-B) and the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Furthermore, forskolin (FSK), a direct activator of adenylate cyclase, and cyclic AMP (cAMP) analogues were used to investigate the role of the increase in intracellular cAMP caused by the A subunit of CT and LT. B lymphocytes were cultured with adjuvants and polyclonal stimuli necessary for activation of B cells in the absence of CD4 T cells. Data indicated that treatment with CT, LT, FSK, or cAMP analogues, but not treatment with CT-B or LTK63, upregulated surface activation markers on B cells, such as CD86 and HLA-DR, and induced inhibition of the proliferation of B cells at early time points, while it increased cell death in long-term cultures. Importantly, B cells treated with CT, LT, or FSK were able to induce pronounced proliferation of both CD4⁺ and CD8⁺ allogeneic T cells compared with untreated B cells and B cells treated with CT-B and LTK63. Finally, only treatment with toxins or FSK induced antigen-specific T-cell proliferation in Mycobacterium tuberculosis purified protein derivative or tetanus toxoid responder donors. Taken together, these results indicated that the in vitro effects of CT and LT on human B cells are mediated by cAMP.The development of effective mucosal vaccines has been hindered by the lack of useful adjuvants and our limited knowledge of their modes of action. Cholera toxin (CT) from Vibrio cholerae and Escherichia coli heat-labile enterotoxin (LT) are potent immunological adjuvants, as indicated by mouse vaccine studies, although their mechanisms of action are not fully understood. These toxins are holotoxins composed of an enzymatically active A subunit that is noncovalently linked to a pentamer of B subunits binding a variety of galactose-containing molecules present in the plasma membranes of eukaryotic cells. CT binds mostly to the ganglioside GM1, which is believed to be the major toxin receptor, whereas LT binds not only to GM1 but also to other glycosphingolipids. Once internalized, the A subunit ADP ribosylates the α subunit of the GTP-binding regulatory protein Gs, thereby inducing permanent adenylate cyclase activation, resulting in an increase in the level of intracellular cyclic AMP (cAMP) (reviewed in reference 34).The potentiation of antigen-presenting cell (APC) function is a major aspect of adjuvant action, and it has been shown that CT and LT induce maturation of both murine dendritic cells (DC) (26, 36) and human DC (5, 14, 15). Several studies demonstrated the ability of these toxins to promote B-cell isotype switch differentiation in mice (19, 27) and upregulation of activation markers in both murine and human B cells (2-4). While these toxins are potent adjuvants, their toxicity makes them unsuitable for human use. For this reason, a number of investigators have tried to develop nontoxic derivatives of CT and LT that retain adjuvanticity either by removing the A domain or by rendering it enzymatically inactive by site-directed mutagenesis (34). Although the current data suggest that the enzymatic activity of CT and LT holotoxins is responsible for the most potent adjuvant activity, a number of reports proposed that there are multiple immune modulating pathways that are triggered by CT and LT, including mechanisms independent of ADP ribosyltransferase activity (11, 13, 30, 33, 42). Numerous studies have suggested that engagement of the ganglioside GM1, the major receptor for CT and LT, is required for the ability of these molecules to modulate immune responses (22, 31). Recently, workers demonstrated that in the absence of the toxic A subunit, the B subunit of CT (CT-B) induces intracellular signaling associated with the in vitro activation of murine B cells and macrophages (37).The majority of these studies have been performed with murine cells and have confirmed the in vivo adjuvanticity of nontoxic compounds, such as CT-B and LTK63, a mutant of LT lacking the ADP ribosyltransferase enzymatic activity, when they were mucosally delivered into animals, even if the immune responses observed in the in vivo studies were usually weaker than those induced by the wild-type toxins (6, 11, 20, 36, 40, 41). In order to develop a mucosal adjuvant for human vaccine, the mechanism(s) of action of potential nontoxic adjuvants should be investigated in vitro by using human APC. It has been shown that the B-cell antigen-presenting functions may be important for the induction of optimal vaccine-induced responses (10, 35). Moreover, B cells are present in mucosa-associated lymphoid tissues (8), and their function in these sites is related not only to immunoglobulin (Ig) production but also to their antigen-presenting properties (24). To elucidate the mechanisms by which enterotoxins modulate antigen-presenting properties, we decided to carry out a comprehensive and comparative analysis of the effects of the toxins and their nontoxic derivatives on the APC function of human B cells. Here we present evidence that CT and LT, as well as forskolin (FSK) and cAMP analogues, but not CT-B and LTK63, increase the activation of human B cells and induce improvement in their APC capability, indicating that the presence of the enzymatic subunit is critical for their adjuvanticity.     

Differentiation of monocytes into CD1a- dendritic cells correlates with disease progression in HIV-infected patients

Monocyte differentiation into dendritic cells (DCs) depends on microenvironmental conditions. In this study, the capacity of human monocytes to differentiate into mature DCs and their ability to induce an antiviral immune response was investigated in HIV-infected patients. In healthy subjects, monocytes differentiate into CD1a+ DCs in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 and matured in the presence of lipopolysaccharide. Here, we found that in 30% and 45% of HIV-infected white and African subjects, respectively, monocytes gave rise to a homogeneous CD1a* DC population. In the patients who gave rise only to the CD1a* DCs, this population spontaneously produced IL-10 but not IL-12, and induced a T helper 2-like immune response when cultured with human T cells isolated from cord blood mononuclear cells. In patients with monocytes differentiated into CD1a* DCs, a high percentage of HIV-specific CD4 T cells producing IL-4 were seen in the peripheral blood. Furthermore, differentiation of monocytes into DCs with CD1a* phenotype correlated with low CD4 T-cell counts and high viral loads in HIV-infected subjects. These results suggest that the differentiation of monocytes into CD1a* DCs may be a phenotypic marker associated with progression of the disease.     

The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo

ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.     

Anergic T cells act as suppressor cells in vitro and in vivo

The potential suppressive effects of allospecific anergic T cells were investigated both in vitro and in vivo. Allospecific T cells were rendered unresponsive in vitro using immobilized anti-CD3 mAb. These anergic T cells profoundly inhibited proliferation of responsive T cells in an antigen-specific manner. The observed inhibition did not appear to be due to the release of inhibitory cytokines in that secretion of IL-2, IFN-gamma, IL-4, IL-10 and TGF-beta was greatly reduced following the induction of anergy, and neutralizing mAb specific for IL-4, IL-10 and TGF-beta failed to reverse the inhibition. Furthermore, the suppression mediated by anergic T cells required cell to cell contact. In vivo, adoptive transfer of anergic T cells into recipients of allogeneic skin grafts led to prolonged skin graft survival. Consistent with the lack of inhibitory cytokine production by the anergic cells, prolongation of skin allograft rejection was not influenced by the simultaneous administration of a neutralizing anti-IL-4 antibody. These results indicate that anergic T cells can function as antigen-specific suppressor cells both in vitro and in vivo.     

Antihypertensive Efficacy of Candesartan in Comparison to Losartan: The CLAIM Study

An 8-week, multicenter, double-blind, randomized, parallel-group, forced-titration study was conducted to evaluate the antihypertensive efficacy of candesartan vs. losartan in 654 hypertensive patients with a diastolic blood pressure between 95 and 114 mm Hg from 72 sites throughout the U.S. Eligible patients were randomized to candesartan cilexetil 16 mg once daily, or losartan 50 mg once daily. Two weeks following randomization, patients doubled the respective doses of their angiotensin receptor blockers for an additional 6 weeks. At week 8, candesartan cilexetil lowered trough systolic/diastolic blood pressure by a significantly greater amount than did losartan (13.3/10.9 mm Hg with candesartan cilexetil vs. 9.8/8.7 mm Hg with losartan; p < 0.001). At the same period, candesartan cilexetil also lowered peak blood pressure by a significantly greater amount than did losartan (15.2 to 11.6 mm Hg with candesartan cilexetil vs. 12.6 to 10.1 mm Hg with losartan; p < 0.05). There were statistically significantly ( p < 0.05) higher proportions of responders and controlled patients in the candesartan cilexetil group (62.4% and 56.0%, respectively) than in the losartan group (54.0% and 46.9%, respectively). Both treatment regimens were well tolerated; 1.8% in the candesartan cilexetil group and 1.6% in the losartan group withdrew because of adverse events. In conclusion, this forced-titration study confirms that candesartan cilexetil is more effective than losartan in lowering blood pressure when both are administered once daily at maximum doses. Both drugs were well tolerated.     

Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response

Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens.We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors.