Mycobacterium tuberculosis Synergizes with ATP To Induce Release of Microvesicles and Exosomes Containing Major Histocompatibility Complex Class II Molecules Capable of Antigen Presentation

Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-A^b complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses.Exosomes are 50- to 80-nm membrane vesicles that are released by many cell types, including reticulocytes, B cells, and dendritic cells (DCs) (16, 17, 33-35, 40, 42, 49, 53). Invagination of the limiting membrane of late endosomes leads to the formation of intraluminal vesicles in multivesicular endosomes. The intraluminal vesicles are secreted as exosomes upon the fusion of multivesicular endosomes with the plasma membrane.Exosomes from B cells contain major histocompatibility complex class II (MHC-II) molecules and can stimulate CD4⁺ T-cell responses in vitro (40), although they may be more capable of activating primed T cells than naïve CD4⁺ T cells (27). The activation of naïve CD4⁺ T cells by DC exosomes occurs via an indirect pathway in which the exosomes and their constituent peptide-MHC-II molecules are presented in the context of intact antigen (Ag)-presenting cells (APCs) (e.g., DCs that may be MHC-II negative but must bear the costimulatory molecules CD80 and CD86 48]). The presence of ICAM-1 on exosomes is important for naïve T-cell priming (43).While the shedding of exosomes can be constitutive (27, 40), it can also be significantly enhanced by the stimulation of certain receptors, e.g., Toll-like receptors (TLRs) and the P2X7 purinergic receptor (P2X7R), which trigger inflammatory responses (37, 38). P2X7R can be activated by ATP, which is released into the extracellular milieu following cell death or injury (50). P2X7R signaling induces the assembly of inflammasome signaling complexes (10), which drive the proteolytic activation of caspase-1 and the maturation of interleukin 1b (IL-1b). Another P2X7R-induced response is the rapid extracellular release of MHC-II molecules (38), which was previously observed within 15 min of the addition of ATP and resulted in the release of ∼15% of the total MHC-II pool in macrophages within 90 min (38). Released MHC-II molecules were contained in two membrane fractions: larger (100- to 1,000-nm) plasma membrane-derived microvesicles and smaller (50- to 80-nm) exosomes. The ATP-stimulated release of MHC-II was markedly reduced in macrophages isolated from NLRP3 knockout or ASC knockout mice. Thus, P2X7R activation of the NLRP3 inflammasome induces the biogenesis and release of MHC-II-containing membranes. The precedent of synergy between lipopolysaccharide (LPS) and ATP suggests that MHC-II shedding might be enhanced in the context of bacterial infection, but this hypothesis has not been explored.Mycobacterium tuberculosis is a major human pathogen that infects one-third of the world population. M. tuberculosis and the related organism Mycobacterium bovis strain BCG infect host cells and regulate host cell functions by signaling through innate immune receptors, including TLR2. Cells infected with M. tuberculosis also secrete exosomes containing mycobacterial molecules that function as PAMPs (pathogen-associated molecular patterns) (2-5, 42) and can stimulate proinflammatory responses via TLR2, TLR4, and MyD88 (4, 5). The dissemination of PAMPs by exosomes released from infected cells may induce innate immune responses by a greater number of cells than are directly infected, magnifying host responses. M. tuberculosis and other mycobacteria can activate the ASC/NLRP3/caspase-1 inflammasome in macrophages via a mechanism dependent on the mycobacterial RD1 locus (encoding components of the ESX-1 secretion system, including the ESAT-6 protein) (9, 20, 26). The ability of M. tuberculosis to stimulate inflammasome activity is dependent on increased K⁺ efflux and occurs in macrophages from P2X7 receptor knockout mice (20). Thus, P2X7 receptor activation and M. tuberculosis infection may elicit similar signaling pathways that converge on the NLRP3 inflammasome and, possibly, on the inflammasome-dependent release of MHC-II membranes.In the current study, we demonstrate that infection of macrophages with mycobacteria elicits the shedding of MHC-II-containing membranes. Furthermore, M. tuberculosis increases the ATP-triggered release of exosomes and microvesicles containing MHC-II. In addition, we demonstrate that MHC-II in membranes released from mycobacterium-infected macrophages can present Ag to T cells. These findings suggest that exosomes and microvesicles from mycobacterium-infected cells may broadcast the stimulation of both innate and adaptive immune receptors beyond the directly infected host cells, contributing to the genesis of CD4 T-cell responses to mycobacterial pathogens such as M. tuberculosis.