N. Tony Eissa

Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells

The variable efficacy of Bacille Calmette Guerin (BCG) vaccination against tuberculosis has prompted efforts to improve the vaccine. In this study, we used autophagy to enhance vaccine efficacy against tuberculosis in a mouse model. We examined the effect of autophagy on the processing of the immunodominant mycobacterial antigen Ag85B by antigen presenting cells (APCs), macrophages and dendritic cells (DCs). We found that rapamycin-induced autophagy enhanced Ag85B presentation by APCs infected with wild-type Mycobacterium tuberculosis H37Rv, H37Rv-derived ΔfbpA attenuated candidate vaccine or BCG. Furthermore, rapamycin enhanced localization of mycobacteria with autophagosomes and lysosomes. Rapamycin-enhanced antigen presentation was attenuated when autophagy was suppressed by 3-methyladenine or by small interfering RNA against beclin-1. Notably, mice immunized with rapamycin-treated DCs infected with either ΔfbpA or BCG showed enhanced T helper type 1–mediated protection when challenged with virulent Mycobacterium tuberculosis. Finally, overexpression of Ag85B in BCG induced autophagy in APCs and enhanced immunogenicity in mice, suggesting that vaccine efficacy can be enhanced by augmenting autophagy-mediated antigen presentation.

Molecular mechanisms of increased nitric oxide (NO) in asthma: Evidence for transcriptional and post-translational regulation of NO synthesis

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l-arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Inducible Nitric-oxide Synthase Is Regulated by the Proteasome Degradation Pathway

Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis froml-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, andN-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited byN ω-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitorstrans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH4Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-κB inhibitor IκB-α, thus preventing activation of NF-κB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS.

Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll‐like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysac‐charide (LPS) induces C2C12 myotube atrophy via up‐regulating autophagosome formation and the expression of ubiquitin ligase atrogin‐1/MAFbx and MuRF1. TLR4‐mediated activation of p38 MAPK is necessary and sufficient for the up‐regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up‐regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3‐methyladenine completely abolishes LPS‐induced muscle proteolysis, while proteasome inhibitor lacta‐cystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS‐induced muscle proteolysis. Thus, TLR4 mediates LPS‐induced muscle catabolism via coordinate activation of the ubiq‐uitin‐proteasome and the autophagy‐lysosomal pathways.—Doyle, A., Zhang, G., Abdel Fattah, E. A., Eissa, N. T., Li, Y.‐P. Toll‐like receptor 4 mediates lipopolysac‐charide‐induced muscle catabolism via coordinate activation of ubiquitin‐proteasome and autophagy‐lysosome pathways. FASEB J. 25, 99–110 (2011). www.fasebj.org

Autophagy Regulates Phagocytosis by Modulating the Expression of Scavenger Receptors

Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7⁻/⁻) mice. Atg7⁻/⁻ macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7⁻/⁻ mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7⁻/⁻ macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7⁻/⁻ macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.

A hepatic protein, fetuin-A, occupies a protective role in lethal systemic inflammation.

BACKGROUND A liver-derived protein, fetuin-A, was first purified from calf fetal serum in 1944, but its potential role in lethal systemic inflammation was previously unknown. This study aims to delineate the molecular mechanisms underlying the regulation of hepatic fetuin-A expression during lethal systemic inflammation (LSI), and investigated whether alterations of fetuin-A levels affect animal survival, and influence systemic accumulation of a late mediator, HMGB1. METHODS AND FINDINGS LSI was induced by endotoxemia or cecal ligation and puncture (CLP) in fetuin-A knock-out or wild-type mice, and animal survival rates were compared. Murine peritoneal macrophages were challenged with exogenous (endotoxin) or endogenous (IFN-γ) stimuli in the absence or presence of fetuin-A, and HMGB1 expression and release was assessed. Circulating fetuin-A levels were decreased in a time-dependent manner, starting between 26 h, reaching a nadir around 24-48 h, and returning towards base-line approximately 72 h post onset of endotoxemia or sepsis. These dynamic changes were mirrored by an early cytokine IFN-γ-mediated inhibition (up to 50-70%) of hepatic fetuin-A expression. Disruption of fetuin-A expression rendered animals more susceptible to LSI, whereas supplementation of fetuin-A (20-100 mg/kg) dose-dependently increased animal survival rates. The protection was associated with a significant reduction in systemic HMGB1 accumulation in vivo, and parallel inhibition of IFN-γ- or LPS-induced HMGB1 release in vitro. CONCLUSIONS These experimental data suggest that fetuin-A is protective against lethal systemic inflammation partly by inhibiting active HMGB1 release.

Functional Variant in the Autophagy-Related 5 Gene Promotor is Associated with Childhood Asthma

Rationale and Objective Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. Methods Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. Measurements and Main Results We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. Conclusion Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.

A critical role for CHIP in the aggresome pathway.

ABSTRACT Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed “physiologic aggresome,” is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIP-mediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTRΔF508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.

Transient Aggregation of Ubiquitinated Proteins Is a Cytosolic Unfolded Protein Response to Inflammation and Endoplasmic Reticulum Stress

Background: ALIS are transient aggregation of ubiquitinated proteins. Results: p62, NF-κB and mTOR were required for ALIS formation. Lysosomal activity is responsible for ALIS clearance. Conclusion: There is a cross talk between UPR in the ER and cytosolic ALIS. Significance: There is an adaptive mechanism for cellular responses against inflammation and ER stress. Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins in the cytosol. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that occurs under these conditions. This response was aided by rapid transcriptional up-regulation of polyubiqutin-binding protein p62. NF-κB and mTOR activation were also required for ALIS formation. Importantly, we show a cross talk between UPR in the ER and cytosolic ALIS. Down-regulation of ER UPR in XBP1 deficient cells increases cyotosolic ALIS formation. Furthermore, lysosomal activity but not macroautophagy is responsible for ALIS clearance. This study reveals the underlying regulatory mechanisms of ALIS formation and clearance, and provides a previously unrecognized common adaptive mechanism for cellular responses against inflammation and ER stress.

Regulation of inducible nitric oxide synthase by rapid cellular turnover and cotranslational down-regulation by dimerization inhibitors

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of many disorders. iNOS is notably distinguished from constitutive NOSs by its production of large amounts of NO for a prolonged period; hence, it was termed the high-output NOS. Understanding how cells regulate iNOS is a prerequisite for strategies aimed at modulating NO synthesis. iNOS is thought to be regulated primarily at the transcriptional level in response to cytokines and inflammatory mediators. In this study, we report a posttranslational regulatory mechanism for control of iNOS expression through a rapid cellular rate of turnover. Unexpectedly, iNOS cellular half-life was found to be relatively short. In primary bronchial epithelial cells, iNOS half-life was 1.6 ± 0.3 h. A similar half-life was found for iNOS in several cell lines. This fast rate of turnover is in sharp contrast to that reported for the constitutive NOS isoforms. iNOS half-life was not affected by intracellular depletion of tetrahydrobiopterin, a critical cofactor required for iNOS activity. Further, iNOS monomers and dimers had a similar half-life. Importantly, we discovered a previously unrecognized cotranslational down-regulation mechanism by which the newly discovered pyrimidineimidazole-based allosteric dimerization inhibitors of iNOS lead to reduced iNOS expression. This study provides insights into the cellular posttranslational mechanisms of iNOS and has important implications for design of selective iNOS inhibitors and their use in therapeutic strategies.