Adaptive immune defects against glycoantigens in chronic granulomatous disease via dysregulated nitric oxide production.

Chronic granulomatous disease (CGD) is a primary immunodeficiency defined by mutations in the NADPH oxidase complex leading to reduced superoxide production, increased susceptibility to infection, chronic inflammation, and recurring abscess and granuloma formation. Here, we found that CGD mice were hyperresponsive to abscess-inducing T-cell-dependent carbohydrate antigens (glycoantigens) due to a ten-fold increase in NO production within APCs, which is known to be necessary for glycoantigen presentation on MHC class II. CGD mice exhibited increased Th1 pro-inflammatory T-cell responses in vitro and in vivo, characterized by more severe abscess pathology. This phenotype was also seen in WT animals following adoptive transfer of neutrophil-depleted APCs from CGD animals, demonstrating that this phenotype was independent of neutrophil and T-cell defects. Finally, pharmacological attenuation of NO production to WT levels in vivo reduced abscess incidence and severity in CGD without overt increases in inflammation or the ability to clear infection, suggesting a potential new treatment option for early stage CGD-associated infections.

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.

Carbohydrate oxidation acidifies endosomes, regulating antigen processing and TLR9 signaling.

Phagocytes kill encapsulated microbes through oxidative cleavage of surface carbohydrates, releasing glycan fragments and microbial contents that serve as ligands for immune receptors, which tailor the immune response against the offending pathogen. The glycan fragments serve as MHC class II (MHC II) ligands and innate receptor agonists, whereas microbial proteins serve as substrates for proteolytic cleavage and MHC II presentation, and released nucleic acids activate innate pattern-recognition receptors (e.g., TLR9). In the current study, confocal microscopy of live macrophages and dendritic cells revealed that endocytosis of carbohydrates lead to vesicular acidification independent of proton pump activity. Acidification was dependent on NO-mediated oxidation in the presence of the ingested carbohydrate and was sufficient to negatively regulate T cell-dependent polysaccharide Ag cleavage, promote acid-dependent protein Ag processing, and facilitate CpG-mediated TLR9 signaling. Our findings lead to a model in which oxidation of carbohydrates from encapsulated microbes facilitates adaptive immune responses against microbial protein and carbohydrate Ags through promoting Ag processing for MHC II-mediated presentation as well as innate responses against released microbial DNA via TLR9 signaling.

Type I Streptococcus pneumoniae carbohydrate utilizes a nitric oxide and MHC II-dependent pathway for antigen presentation.

Some pathogenic bacteria form thick capsules that both block immune responses through inhibition of complement deposition and phagocytosis and stimulate a weak response resulting from a lack of T-cell involvement. Contrary to this model, capsular polysaccharides from 23 serotypes of Streptococcus pneumoniae have been successfully used in a multivalent vaccine in the absence of a carrier protein. Furthermore, type I pneumococcal polysaccharide (Sp1) has been shown to activate T cells in vivo and in vitro via an uncharacterized mechanism. In the present report, we demonstrate that Sp1 utilizes the major histocompatibility complex (MHC) class II pathway in antigen-presenting cells (APCs) for processing and presentation. APCs internalize and process Sp1 through a nitric oxide-dependent mechanism and, once inside the cell, it associates with MHC II proteins in an H-2M-dependent manner that leads to in vivo T-cell activation. These results establish that Sp1 activates T cells which can lead to abscess formation in mice through an H-2M-dependent polysaccharide antigen presentation pathway in APCs, potentially contributing to pneumococcal polysaccharide vaccine efficacy through the recruitment of T-cell help.