Hyperactivation of B cells from Immunodeficient Patients

poster abstractChronic granulomatous disease (CGD) is an inherited immunodeficiency associated with defects in NADPH oxidase, an enzyme that produces oxygen radicals necessary to kill bacterial and fungal pathogens. NADPH oxidase, made up of six subunits, is located in endosomal and plasma membranes of immune cells. Although best studied in macrophages and neutrophils, the oxidase is expressed in B cells where we have shown its link to adaptive immunity and antigen presentation. Here, NADPH oxidase function was disrupted by mutations or gene knockdown in human B cells, and the role of the oxidase in innate immunity specifically Toll-like receptor (TLR) signaling tested. TLR7 and 9, which recognize viral single-stranded RNA and unmethylated CpG DNA respectively, potentially share an endosomal compartment with the oxidase in B cells. In this project, B cells were stimulated for 24 hours with TLR7 and 9 ligands along with a costimulator PMA. TLR7 signaling was significantly enhanced in oxidase deficient B cell lines compared with their respective control cells as evidenced by increased IL-6 secretion detected by an ELISA. CGD patients are incapable of producing oxygen radicals rendering them immunodeficient in terms of pathogen infection. Yet these patients also develop many autoimmune disorders associated with hyperactivation of the immune system. Thus, our studies on TLR activation using CGD cell lines may explain in part the development of autoimmunity in individuals with CGD. Additional studies are underway to examine the regulation of TLR including receptor expression levels and the subcellular localization of the NADPH oxidase in these B cells from CGD patients. This work has not yet been published and was supported by NIH 3R01AI079065-03S1

LAMP-2C inhibits MHC class II presentation of cytoplasmic antigens by disrupting chaperone-mediated autophagy

Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags

Hyper-responsive Toll-like receptor 7 and 9 activation in NADPH oxidase-deficient B lymphoblasts

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients

Spatial variability in airborne bacterial communities across land-use types and their relationship to the bacterial communities of potential source environments

Although bacteria are ubiquitous in the near-surface atmosphere and they can have important effects on human health, airborne bacteria have received relatively little attention and their spatial dynamics remain poorly understood. Owing to differences in meteorological conditions and the potential sources of airborne bacteria, we would expect the atmosphere over different land-use types to harbor distinct bacterial communities. To test this hypothesis, we sampled the near-surface atmosphere above three distinct land-use types (agricultural fields, suburban areas and forests) across northern Colorado, USA, sampling five sites per land-use type. Microbial abundances were stable across land-use types, with ∼105–106 bacterial cells per m3 of air, but the concentrations of biological ice nuclei, determined using a droplet freezing assay, were on average two and eight times higher in samples from agricultural areas than in the other two land-use types. Likewise, the composition of the airborne bacterial communities, assessed via bar-coded pyrosequencing, was significantly related to land-use type and these differences were likely driven by shifts in the sources of bacteria to the atmosphere across the land-uses, not local meteorological conditions. A meta-analysis of previously published data shows that atmospheric bacterial communities differ from those in potential source environments (leaf surfaces and soils), and we demonstrate that we may be able to use this information to determine the relative inputs of bacteria from these source environments to the atmosphere. This work furthers our understanding of bacterial diversity in the atmosphere, the terrestrial controls on this diversity and potential approaches for source tracking of airborne bacteria