Macronutrient deprivation modulates antigen trafficking and immune recognition through HSC70 accessibility.

B lymphocytes exploit macroautophagy to capture cytoplasmic and nuclear proteins within autophagosomes. Fusion of autophagosomes with lysosomes and endosomes facilitates content proteolysis, with the resulting peptides selectively binding MHC class II (MHC II) molecules, which are displayed for recognition by T lymphocytes. Nutrient deprivation or stress amplified this pathway, favoring increased MHC II presentation of cytoplasmic Ags targeted to autophagosomes. By contrast, this stress diminished MHC II presentation of membrane Ags including the BCR and cytoplasmic proteins that use the chaperone-mediated autophagy pathway. Whereas intracellular protease activity increased with nutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of macronutrients such as high molecular mass proteins restored endocytosis and Ag presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Altering cellular levels of the cytosolic chaperone HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and Ag presentation. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating membrane trafficking pathways during cellular stress

A central role for HSC70 in regulating antigen trafficking and MHC class II presentation

Cells rely on multiple intracellular trafficking pathways to capture antigens for proteolysis. The resulting peptides bind to MHC class II molecules to promote CD4(+) T cell recognition. Endocytosis enhances the capture of extracellular and cell surface bound antigens for processing and presentation, while autophagy pathways shunt cytoplasmic and nuclear antigens for presentation in the context of MHC class II molecules. Understanding how physiological changes and cellular stress alter antigen trafficking and the repertoire of peptides presented by class II molecules remains challenging, yet important in devising novel approaches to boost immune responses to pathogens and tumors. An abundant, constitutively expressed cytoplasmic chaperone, HSC70 plays a central role in modulating antigen transport within cells to control MHC class II presentation during nutrient stress. HSC70 may serve as a molecular switch to modulate endocytic and autophagy pathways, impacting the source of antigens delivered for MHC class II presentation during cellular stress

A role for HSC70 in regulating antigen trafficking and presentation during macronutrient deprivation

Indiana University-Purdue University Indianapolis (IUPUI)Globally, protein malnutrition remains problematic, adversely affecting several systems including the immune system. Although poorly understood, protein restriction severely disrupts host immunity and responses to infection. Induction of high-affinity, long-lasting immunity depends upon interactions between B and T lymphocytes. B lymphocytes exploit several pathways including endocytosis, macroautophagy, and chaperone-mediated autophagy to capture and deliver antigens to the endosomal network. Within the endosomal network antigens are processed and loaded onto major histocompatibility complex (MHC) class II molecules for display and recognition by T lymphocytes. To examine the effect of macronutrient malnutrition on MHC class II antigen presentation, we grew B lymphocytes in media containing amino acids, sugars and vitamins but lacking serum, which contains several types of macronutrients. Our studies show macronutrient stress amplified macroautophagy, favoring MHC class II presentation of cytoplasmic antigens targeted to autophagosomes. By contrast, macronutrient stress diminished MHC class II presentation of membrane antigens including the B cell receptor (BCR) and cytoplasmic proteins that utilize the chaperone-mediated autophagy pathway. The BCR plays a critical role in MHC class II antigen presentation, as it captures exogenous antigens leading to internalization and degradation within the endosomal network. While intracellular protease activity increased with macronutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of high molecular mass macronutrients restored endocytosis and antigen presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Cytosolic chaperone HSC70 has been shown to play a role in endocytosis, macroautophagy, chaperone-mediated autophagy and proteolysis by the proteasome, potentially connecting distinct routes of antigen presentation. Here, altering the abundance of HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and antigen presentation suggesting macronutrient deprivation alters the availability of HSC70. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating distinct membrane trafficking pathways during cellular stress. These results offer a new explanation for impaired immune responses in protein malnourished individuals

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

Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α

A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1β, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity

Profiling neural editomes reveals a molecular mechanism to regulate RNA editing during development

Adenosine (A) to inosine (I) RNA editing contributes to transcript diversity and modulates gene expression in a dynamic, cell type-specific manner. During mammalian brain development, editing of specific adenosines increases, whereas the expression of A-to-I editing enzymes remains unchanged, suggesting molecular mechanisms that mediate spatiotemporal regulation of RNA editing exist. Herein, by using a combination of biochemical and genomic approaches, we uncover a molecular mechanism that regulates RNA editing in a neural- and development-specific manner. Comparing editomes during development led to the identification of neural transcripts that were edited only in one life stage. The stage-specific editing is largely regulated by differential gene expression during neural development. Proper expression of nearly one-third of the neurodevelopmentally regulated genes is dependent on adr-2, the sole A-to-I editing enzyme in C. elegans However, we also identified a subset of neural transcripts that are edited and expressed throughout development. Despite a neural-specific down-regulation of adr-2 during development, the majority of these sites show increased editing in adult neural cells. Biochemical data suggest that ADR-1, a deaminase-deficient member of the adenosine deaminase acting on RNA (ADAR) family, is competing with ADR-2 for binding to specific transcripts early in development. Our data suggest a model in which during neural development, ADR-2 levels overcome ADR-1 repression, resulting in increased ADR-2 binding and editing of specific transcripts. Together, our findings reveal tissue- and development-specific regulation of RNA editing and identify a molecular mechanism that regulates ADAR substrate recognition and editing efficiency

Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing

A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes

A role for NADPH oxidase in antigen presentation

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-). This radical is an important precursor of hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC) crosspresentation by major histocompatibility complex class I molecules (MHC-I) through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II)