Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and non-hematopoietic 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 on exosomes inducing these host responses were not defined. The present study used LC-MS/MS to identify 41 mycobacterial proteins present on 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 (CFP) 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 on 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.

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-γ 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.

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 lead to direct ferroportin transcriptional downregulation, whereas TLR4 ligands, such as lipopolysaccharide, induce hepcidin expression. Infection with 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.

IntroductionCD1d-restricted natural killer T (NKT) cells represent a unique lineage of T cells that shares properties with both natural killer (NK) cells and memory T cells. NKT cells rapidly produce an array of cytokines on activation and play critical roles in the regulation of a variety of immune responses, including control of autoimmune diabetes, antitumor immunity, and protection from infectious diseases. 1 To date, 2 NKT-cell subsets have been defined. Type I NKT cells, also referred to as invariant NKT (iNKT) cells, express an invariant T-cell receptor ␣ (TCR␣) chain (V␣14J␣18 in mice and V␣24-J␣18 in humans) that pairs with a limited repertoire of TCR␤ chains (V␤8, V␤7, or V␤2 in mice, and V␤11 in humans). 2 iNKT cells can be identified using CD1d tetramer loaded with the glycosphingolipid antigen ␣-galactosylceramide (␣GalCer). 3 Type II NKT cells represent the second subset of NKT cells; they exhibit diverse TCR␣ and TCR␤ chain usage and do not bind to CD1d/␣GalCer tetramers. 4 This study focuses on iNKT cells, because the various stages of iNKT-cell maturation and differentiation have been clearly defined.Like conventional T cells, iNKT cells originate from thymic CD4 ϩ CD8 ϩ double-positive (DP) progenitors. 5 However, the iNKTcell lineage deviates from conventional T cells at the DP stage, and their positive selection is distinct from that of conventional T cells. 6,7 Rare DP-precursor cells that express a rearranged V␣14J␣18 TCR␣ chain are positively selected by CD1d-expressing DP thymocytes that provide unique costimulatory signals to iNKT-cell precursors through homotypic interactions with signaling lymphocytic activation molecules (SLAM) family receptors. These interactions led to the recruitment of SLAMassociated protein (SAP) and the Src kinase Fyn, as well as downstream activation of nuclear factor-B (NF-B). [8][9][10][11][12] After positive selection, iNKT-cell precursors down-regulate their expression of CD24 and transition through several maturation stages that can be defined based on the cell-surface expression of CD44 and NK1.1. 13 Stage I iNKT cells display an NK1.1 Ϫ CD44 low phenotype and undergo several rounds of cell division. This expansion is accompanied by the up-regulation of CD44 (NK1.1 Ϫ CD44 high , stage II iNKT cells). Some of these NK1.1 Ϫ CD44 high iNKT cells continue to differentiate into mature NK1.1 ϩ CD44 high (stage III) iNKT cells in the thymus, while others exit the thymus and mature into NK1.1 ϩ iNKT cells in the periphery. 1,13,14 iNKT cells can also be subdivided into CD4 ϩ and CD4 Ϫ CD8 Ϫ (double-negative [DN]) subsets. The earliest iNKT cells are CD4 ϩ , with the DN subset diverging at the immature NK1.1 Ϫ stage in the thymus. 13,14 Recent studies have shown that the transcription factor Th-Pok is required for the repression of CD8 expression and the functional maturation of iNKT cells. 15,16 The unique developmental program of iNKT cells is controlled by several transcription factors/molecules that are distinct from those required for the development of conve...