Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells.

Upon infection, pathogen and host compete for the same iron pool, because this trace metal is a crucial micronutrient for all living cells. Iron dysregulation in the host is strongly associated with poor outcomes in several infectious diseases, including tuberculosis, AIDS, and malaria, and inefficient iron scavenging by pathogens severely affects their virulence. Hepcidin is the master regulator of iron homeostasis in vertebrates, responsible for diminishing iron export from macrophages during iron overload or infection. Hepcidin regulation in hepatocytes is well characterized and mostly dependent on interleukin-6 signaling during inflammation, although in myeloid cells, hepcidin induction and the mechanisms leading to intracellular iron regulation remain elusive. Here we show that activation of different Toll-like receptors (TLRs) by their respective ligands leads to increased iron sequestration in macrophages. By measuring the transcriptional levels of iron-related proteins (eg, hepcidin, ferroportin, and ferritin), we observed that TLR signaling can induce intracellular iron sequestration in macrophages through 2 independent but redundant mechanisms. Interestingly, TLR2 ligands or infection with Listeria monocytogenes lead to direct ferroportin transcriptional downregulation, whereas TLR4 ligands, such as lipopolysaccharide, induce hepcidin expression. Infection with Mycobacterium bovis Bacillus Calmette-Guerin promotes intracellular iron sequestration through both hepcidin upregulation and ferroportin downregulation. This is the first study in which TLR1-9-mediated iron homeostasis in human macrophages was evaluated, and the outcome of this study elucidates the mechanism of iron dysregulation in macrophages during infection.

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages.

Exosomes are small 30-100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC-MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis-infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP-treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis-infected cells. The exosomes from CFP-treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.

Adjunct immunotherapy with Ag85 complex proteins based subunit vaccine in a murine model of Mycobacterium tuberculosis infection.

This study was designed to evaluate the immunotherapeutic potential of Mycobacterium tuberculosis Ag85AB emulsified with unmethylated CpG motif-containing oligonucleotide (CpG-ODN) and dimethyldioctadecylammonium bromide (DDA) adjuvants (Ag85AB-CpG-DDA) in conjunction with antituberculous drugs. Ag85 complex proteins of M. tuberculosis purified from total culture filtrate and purified proteins were emulsified with CpG-ODN and DDA adjuvants. Mice were infected with M. tuberculosis H37 Rv and left for 30 days to establish infection. These mice were named 'tuberculous mice'. Tuberculous mice were treated with Ag85AB-CpG-DDA alone or in conjunction with antituberculous drugs. Treatment of tuberculous mice with Ag85AB-CpG-DDA in conjunction with antituberculous drugs reduced significant bacilli burden in lung and spleen. Moreover, treatment of tuberculous mice with Ag85AB-CpG-DDA induced higher production of type-I cytokines, generated more CD44-positive T cells and suppresses secretion of IL-4 as compared with untreated animals. In conclusion, this study shows that Ag85AB-CpG-DDA formulation may act as a potential future therapeutic regimen in conjunction with antituberculous drugs.

Is intranasal vaccination a feasible solution for tuberculosis?

Bacille Calmette-Guerin (BCG) vaccine has been the only licensed tuberculosis (TB) vaccine administered to humans and, until today, more than 3 billion people have received BCG. However, despite the use of BCG, TB remains a global epidemic with a third of the world population being infected. Regardless of the protection induced by BCG in childhood TB, BCG vaccination fails to protect against pulmonary TB in adults, which represents more than 85% of the total TB burden. Therefore, the development of safe and efficacious TB vaccines that can confer potent protection in the lung mucosa has remained a major challenge to TB vaccinologists. Intranasal vaccination by different antigen formulations has shown promising results in the augmentation of immunity and the combat of the pathogens at the site of infection. This article will focus on the potential of intranasal vaccination and mucosal adjuvants for the development of new-generation TB vaccines.

How could we have better vaccines against tuberculosis?

BACKGROUND: Tuberculosis (TB), an infirmity that mainly affects the respiratory system, is the world's second deadliest infectious disease, with > 9 million new cases diagnosed in 2006. One-third of the world's population is now infected with the TB bacillus. According to the WHO, an estimated 1.7 million people died from TB in 2006. More precisely, every 15 seconds, one person dies due to TB worldwide. OBJECTIVE: To review some of the key advances in the field of TB immunology and to discuss potential means for the development of new generation vaccines against TB disease. METHODS: Systematic review of the published literature in various journals. RESULTS/CONCLUSION: The current TB vaccine Bacillus Calmette-Guérin, developed > 85 years ago, reduces the risk of severe forms of TB in early childhood but is not very effective in preventing pulmonary TB in adolescents and adults, the populations with the highest rates of TB disease. TB is changing and evolving, making the development of new vaccines more crucial to controlling the pandemic. Rigorous research using cutting edge vaccine technology is occurring worldwide to combat TB, and various vaccination strategies, especially prime-boost, have been pursued by many scientists.

Exosomes derived from M. Bovis BCG infected macrophages activate antigen-specific CD4+ and CD8+ T cells in vitro and in vivo.

Activation of both CD4(+) and CD8(+) T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in naïve macrophages. In the present study we demonstrate that exosomes stimulate both CD4(+) and CD8(+) splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4(+) and CD8(+) T cells. The isolated T cells also produced IFN-gamma upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate.

Enhanced immunoprotective potential of Mycobacterium tuberculosis Ag85 complex protein based vaccine against airway Mycobacterium tuberculosis challenge following intranasal administration.

This study examined the role of intranasal vaccination with Mycobacterium tuberculosis antigen85 complex proteins formulated in dimethyldioctadecylammonium bromide against airway Mycobacterium tuberculosis challenge in mice. Intranasal vaccination with antigen85A and antigen85B induced a significantly higher level of interferon-gamma, interleukin-12 and interleukin-4 in cervical lymph nodes together with IgA and IgG, predominantly IgG2a isotype in nasal secretion over subcutaneous vaccination. Further, intranasal vaccination with antigen85A and antigen85B imparted protection comparable with that obtained from intranasal or subcutaneous Mycobacterium bovis bacillus Calmette-Guerin immunization. These results suggest that mucosal vaccination via the intranasal route is of importance in the development of vaccine for tuberculosis.

Protective efficacy of intranasal vaccination with Mycobacterium bovis BCG against airway Mycobacterium tuberculosis challenge in mice.

The effect of route of immunization on the protective efficacy of BCG against tuberculosis has been investigated. Immunoprotection was monitored by evaluating the bacterial burden in the lungs and spleen of mice challenged with Mycobacterium tuberculosis H(37)Rv after BCG immunization by intranasal (i.n.) and subcutaneous (s.c.) routes. Our results showed that as compared to s.c. BCG immunization, intranasal BCG vaccination induces significantly higher immune responses at local level (mediastinal lymph nodes, cervical lymph nodes and lung). Further, i.n. BCG vaccination induced significantly higher reduction in bacterial load in the lungs over s.c. BCG vaccination, whereas, the bacilli load in the spleen was comparable in both the groups. Hence, intranasal vaccination with BCG holds promise for pulmonary tuberculosis.

Comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis.

Activation of mucosal immunity in the respiratory tract is crucial for protection against respiratory infections. Whether the intranasal route of vaccination imparts better protection against pulmonary tuberculosis than that of subcutaneous vaccination remains a debatable issue. In this study, we have investigated the effect of the routes of immunization on the induction of immunoprotection against experimental tuberculosis employing mycobacterial culture filtrate proteins complexed with dimethyldioctadecylammonium bromide. Vaccination via intranasal and subcutaneous routes triggered immune activation in the spleen and cervical lymph node, while the former route of vaccination lead to higher antigen-specific lymphocyte proliferation, interferon-gamma, interleukin-12 and interleukin-4 responses in cervical lymph node and induction of antigen-specific IgA responses at mucosal level of the respiratory tract. Mice vaccinated via the intranasal route were found to be better protected against experimental tuberculosis particularly in lung compared to subcutaneous-immunized mice. These results emphasize the importance of the intranasal route vaccination in tuberculosis.