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Ubiquitination by SAG regulates macrophage survival/death and immune response during infection
Authors:S C Chang  J L Ding
Affiliation:1.Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
Abstract:The checkpoint between the life and death of macrophages is crucial for the host''s frontline immune defense during acute phase infection. However, the mechanism as to how the immune cell equilibrates between apoptosis and immune response is unclear. Using in vitro and ex vivo approaches, we showed that macrophage survival is synchronized by SAG (sensitive to apoptosis gene), which is a key member of the ubiquitin–proteasome system (UPS). When challenged by pathogen-associated molecular patterns (PAMPs), we observed a reciprocal expression profile of pro- and antiapoptotic factors in macrophages. However, SAG knockdown disrupted this balance. Further analysis revealed that ubiquitination of Bax and SARM (sterile α- and HEAT/armadillo-motif-containing protein) by SAG-UPS confers survival advantage to infected macrophages. SAG knockdown caused the accumulation of proapoptotic Bax and SARM, imbalance of Bcl-2/Bax in the mitochondria, induction of cytosolic cytochrome c and activation of caspase-9 and -3, all of which led to disequilibrium between life and death of macrophages. In contrast, SAG-overexpressing macrophages challenged with PAMPs exhibited upregulation of protumorigenic cytokines (IL-1β, IL-6 and TNF-α), and downregulation of antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10). This suggests that SAG-dependent UPS is a key switch between immune defense and apoptosis or immune overactivation and tumorigenesis. Altogether, our results indicate that SAG-UPS facilitates a timely and appropriate level of immune response, prompting future development of potential immunomodulators of SAG-UPS.In an infection, the pattern recognition receptors (PRRs) of the macrophages recognize pathogen pattern-associated molecular patterns (PAMPs), leading to phagocytosis of the pathogen, release of cytokines and secretion of antimicrobial peptides. When overwhelmed by pathogens, macrophages may undergo apoptosis, which produces microbicidal reactive oxygen species.1 Apoptotic death of macrophages is a strategic sacrifice, representing a severe terminal stage of cellular defense against microbial invasion.The mitochondria has a decisive role in cell death or survival by controlling apoptosis signals via recruitment of pro- and antiapoptosis factors.2 Although it is known that many pathogens regulate apoptosis in the host,3 the mechanisms underlying how the host immune cell equilibrates its own death and survival to elicit an optimal immune response is poorly understood. This prompted us to investigate how signaling proteins might regulate the checkpoint between apoptosis or immune response. In this regard, we noted several lines of evidence indicating a hitherto undiscovered phenomenon on the control of host cell death or survival versus immune defense. Firstly, it is proposed that the ubiquitin–proteasome system (UPS)-mediated degradation of Bcl-2 family proteins regulates apoptotic cell death.4 Secondly, the sensitive to apoptosis gene (SAG), a key component of UPS, is strongly induced during early infection,5 suggesting its role in frontline defense. Thirdly, SARM (sterile α- and HEAT/armadillo-motif-containing protein), an evolutionarily conserved mitochondria-associated protein,6, 7 which downregulates TLR-TRIF signaling,8 exerts a strong proapoptotic killing of infection-activated T cells during the pathogen-clearance phase.9 As SAG confers survival to cancer cells,10, 11 we hypothesize that SAG and SARM have opposing roles in modulating apoptosis and immune response. Thus, we investigated the dynamic expression profiles of SAG and Bcl-2 (antiapoptotic) and Bax and SARM (proapoptotic) in macrophages challenged with bacterial and viral PAMPs.We demonstrated that SAG responds dynamically to PAMP stimulation. SAG knockdown abrogates ubiquitination and stabilizes the proapoptotic Bax and SARM proteins, leading to their accumulation in the mitochondria and resulting in intrinsic apoptosis. SAG overexpression in macrophages downregulated the antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10), but upregulated the protumorigenic cytokines (IL-1β, IL-6 and TNF-α), indicating cellular sensitization to SAG activation. Taken together, we propose that the crossroad between macrophage survival/death and immune response is synchronized to a large extent by SAG-UPS.
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