Matthew T. Sorbara

NOD proteins: regulators of inflammation in health and disease

Entry of bacteria into host cells is an important virulence mechanism. Through peptidoglycan recognition, the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2 enable detection of intracellular bacteria and promote their clearance through initiation of a pro-inflammatory transcriptional programme and other host defence pathways, including autophagy. Recent findings have expanded the scope of the cellular compartments monitored by NOD1 and NOD2 and have elucidated the signalling pathways that are triggered downstream of NOD activation. In vivo, NOD1 and NOD2 have complex roles, both during bacterial infection and at homeostasis. The association of alleles that encode constitutively active or constitutively inactive forms of NOD2 with different diseases highlights this complexity and indicates that a balanced level of NOD signalling is crucial for the maintenance of immune homeostasis.

Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program

Autophagy, which targets cellular constituents for degradation, is normally inhibited in metabolically replete cells by the metabolic checkpoint kinase mTOR. Although autophagic degradation of invasive bacteria has emerged as a critical host defense mechanism, the signals that induce autophagy upon bacterial infection remain unclear. We find that infection of epithelial cells with Shigella and Salmonella triggers acute intracellular amino acid (AA) starvation due to host membrane damage. Pathogen-induced AA starvation caused downregulation of mTOR activity, resulting in the induction of autophagy. In Salmonella-infected cells, membrane integrity and cytosolic AA levels rapidly normalized, favoring mTOR reactivation at the surface of the Salmonella-containing vacuole and bacterial escape from autophagy. In addition, bacteria-induced AA starvation activated the GCN2 kinase, eukaryotic initiation factor 2α, and the transcription factor ATF3-dependent integrated stress response and transcriptional reprogramming. Thus, AA starvation induced by bacterial pathogens is sensed by the host to trigger protective innate immune and stress responses.

Mitochondrial ROS fuel the inflammasome

IL-1β and IL-18 are pro-inflammatory cytokines that play a critical role in the response to a diverse array of injuries and infections. Accordingly, the production of these potent cytokines is tightly regulated at transcriptional and post-translational levels through control of both maturation and secretion. The maturation of these cytokines is regulated by caspase-1 inflammasomes. Several members of the Nod-like receptor (NLR) family of intracellular sensors, including NLRP3, NLRC4 and NLRP1, play critical roles in inflammasome regulation. However, the nature of the physiological cues that trigger inflammasome activation remain incompletely understood. A recent study by Zhou et al. examined this important question in the context of activation of the NLRP3 inflammasome 1. Zhou et al. demonstrated that generation of mitochondrial reactive oxygen species (ROS) led to NLRP3 inflammasome activation, and treatment of macrophages with NLRP3 activators resulted in the recruitment of NLRP3 to mitochondria-associated ER membranes (MAMs) where the protein recruited the ASC adaptor critical for inflammasome formation (Figure 1). Furthermore, autophagy limited NLRP3 inflammasome activation by targeting ROS-producing mitochondria (Figure 1). Therefore, this study provides a fundamental link between inflammasome activation and mitochondrial function. Figure 1 Mitochondrial ROS promote NLRP3 inflammasome formation. Treatment of macrophages with MSU, alum or nigericin leads to the production of mitochondrial ROS. In (1), this triggers the induction of mitophagy and the formation of an LC3+ autophagosome to remove ... NLRP3 can be activated by host-derived danger signals, such as monosodium urate (MSU) crystals or extracellular ATP, bacterial or viral infection, as well as environmental stimuli including asbestos and UVB radiation 2. Several models of NLRP3 activation have been proposed. In the case of large particles or crystalline structures, such as asbestos, MSU and amyloid-β fibers, uptake may result in “frustrated phagocytosis” 3 or damage to phagosome and lysosome vesicles, leading to release of cathepsin B and NLRP3 activation 4. A role for K+ efflux in inflammasome activation has also been proposed. Interfering with K+ efflux through the addition of extracellular K+ or chemical inhibitors, such as glyenclamide, blocks NLRP3 inflammasome activation in response to several stimuli, including extracellular ATP, MSU and nigericin 5. Finally, despite the diversity of signals recognized by NLRP3, the generation of ROS appears to be a common cellular response critical for NLRP3 activation since ROS scavengers attenuate NLRP3 activation 4. However, the source of ROS and the mechanism by which NLRP3 senses ROS generation are currently unclear. Indeed, while several studies have suggested a role for NADPH oxidases in NLRP3 activation 4, macrophages lacking functional NOX1, NOX2 and NOX4 respond normally to NLRP3 stimulation 1. Oxidative phosphorylation generates the largest source of ATP in eukaryotic cells and occurs through the reduction of O2 to H2O at the mitochondria. The reduction of O2 by a series of protein complexes (I to IV) is used to generate an H+ gradient that powers ATP synthesis by complex V. This process leads to the formation of several types of ROS intermediates, including O2−, H2O2 and hydroxyl radical. Therefore, mitochondria constitute a major source of cellular ROS. Zhou et al. hypothesized that mitochondrial ROS may be involved in NLRP3 activation. To investigate a role for mitochondrial ROS in NLRP3 activation, the authors first inhibited complex I function with rotenone. As previously described 6, complex I inhibition led to increased mitochondrial ROS production. Interestingly, this correlated with increased IL-1β secretion. The increased production of IL-1β was specific to and dependent on NLRP3 inflammasome activation since macrophages lacking NLRC4/IPAF responded similarly to wild-type cells, and enhanced IL-1β production was lost in NLRP3−/− macrophages. Having established a role for mitochondrial ROS in NLRP3 inflammasome activation, the authors examined how NLRP3 senses ROS. The subcellular localization of NLRP3 was examined using Flag-tagged versions of NLRP3 and the adaptor ASC. In unstimulated conditions, NLRP3 localized to the endoplasmic reticulum, as determined by colocalization with the ER marker calreticulin, while the ASC adaptor was primarily cytosolic. Following treatment with MSU, alum or nigericin, both NLRP3 and ASC localized to MAMs. ASC recruitment to MAMs depended on NLRP3 and did not occur in cells silenced for NLRP3. MAMs are important for mitochondrial function, as they contribute to phospholipid transfer and synthesis, steroidogenesis and regulate mitochondrial Ca2+ levels 7. To directly test the hypothesis that NLRP3 inflammasome activation depends on ROS production from respiring mitochondria, Zhou et al. inhibited mitochondrial respiration by knocking down expression of voltage-dependent anion channels (VDAC). Interestingly, in cells lacking VDACs, IL-1β production and caspase-1 activation were abrograted in response to NLRP3 activators. Similarly, cells overexpressing Bcl-2, which inhibits VDAC function, also displayed impaired production of IL-1β in response to MSU, alum or nigericin. Previous studies have shown that macrophages lacking the autophagy proteins ATG16L1 or ATG7 produce elevated levels of IL-1β, suggesting that autophagy may regulate inflammasome activation 8. Furthermore, autophagy plays an important role in clearing damaged ROS-producing mitochondria 9. Therefore, Zhou et al. investigated a role for autophagy in attenuating NLRP3 activation. Inhibition of complex I function with rotenone led to the targeting of mitochondria by autophagy. Interestingly, inhibition of autophagy resulted in accumulation of damaged, ROS-producing mitochondria. In cells displaying impaired autophagy, either following treatment with 3-methyladenine or by knocking down ATG5 or Beclin-1, an NLRP3-dependent, NLRC4-independent enhancement of IL-1β was observed. The findings of Zhou et al. are compatible with those of Nakahira et al. who also recently examined the interplay of autophagy, mitochondria and NLRP3 inflammasome activation 10. Nakahira et al. also found that autophagy limited caspase-1 activation by clearing ROS-producing mitochondria. Furthermore, the authors also demonstrated the necessity of respiring mitochondria for NLRP3 activation, in this case by depleting cells of mitochondria DNA (mtDNA) through ethidium bromide treatment. In Nakahira et al.s model, treatment of macrophages with LPS plus ATP leads to the release of mtDNA. The release of mtDNA requires ROS formation and is enhanced in cells lacking autophagy. Furthermore, transfection of mtDNA enhanced the response to LPS and ATP, and cytosolic mtDNA levels correlated with IL-1β production. Notably, NLRP3 was required for mtDNA release following LPS+ATP stimulation and the loss of mitochondrial membrane potential, but not mitochondrial ROS production. Taken together, the results by Zhou et al., associated with those by Nakahira et al., suggest a complex model, in which NLRP3 activators trigger mitochondrial ROS production that is limited by autophagic clearance of damaged mitochondria. ROS production leads to the relocation of NLRP3 to MAMs, where ASC is recruited, thereby promoting NLRP3 inflammasome activation. In addition, the NLRP3 inflammasome would promote the release of caspase-1-activating mtDNA. The link proposed between NLRP3 activation, mitochondrial ROS generation, mtDNA release and regulation of mitophagy holds great promise for future research in the field of inflammasome activation, and also opens up new questions. It will be interesting to dissect the role that NLRP3-driven mtDNA release has in caspase-1 activation and IL-1β secretion, and whether this release functions as a positive feedback through the NLRP3 inflammasome or another cytosolic, AIM2-independent sensor. It will also be important to determine whether mitochondrial ROS are ubiquitously induced by NLRP3 activators, and elucidate the pathways leading to mitochondrial stress in each case. The relative contribution of NADPH-triggered versus mitochondria-triggered ROS in NLRP3-dependent induction of the inflammasome will also need to be carefully evaluated. Nevertheless, the study by Zhou et al. adds to the emerging picture that the mitochondrion not only serves as critical organelle involved in cell metabolism, but also plays essential roles in controlling innate immune pathways.

Peptidoglycan: a critical activator of the mammalian immune system during infection and homeostasis

Summary:  Peptidoglycan is a conserved structural component of the bacterial cell wall with molecular motifs unique to bacteria. The mammalian immune system takes advantage of these properties and has evolved to recognize this microbial associated molecular pattern. Mammals have four secreted peptidoglycan recognition proteins, PGLYRP‐1‐4, as well as two intracellular sensors of peptidoglycan, Nod1 and Nod2. Recognition of peptidoglycan is important in initiating and shaping the immune response under both homeostatic and infection conditions. During infection, peptidoglycan recognition drives both cell‐autonomous and whole‐organism defense responses. Here, we examine recent advances in the understanding of how peptidoglycan recognition shapes mammalian immune responses in these diverse contexts.

Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures

Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)‐dependent cytosolic escape of Listeria triggered a transient amino‐acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro‐autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3‐phosphate (PI3P) levels, causing pre‐autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co‐infection experiments, wild‐type Listeria protected PlcA/B‐deficient bacteria from autophagy‐mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense.

Emerging themes in bacterial autophagy

The role of autophagy in the control of intracellular bacterial pathogens, also known as xenophagy, is well documented. Here, we highlight recent advances in the field of xenophagy. We review the importance of bacterial targeting by ubiquitination, diacylglycerol (DAG) or proteins such as Nod1, Nod2, NDP52, p62, NBR1, optineurin, LRSAM1 and parkin in the process of xenophagy. The importance of metabolic sensors, such as mTOR and AMPK, in xenophagy induction is also discussed. We also review the in vitro and in vivo evidence that demonstrate a global role for xenophagy in the control of bacterial growth. Finally, the mechanisms evolved by bacteria to escape xenophagy are presented.

Bacterial autophagy: the trigger, the target and the timing.

Autophagy is a vital process through which cellular material and dysfunctional organelles are degraded and recycled, and it is inhibited by the metabolic checkpoint kinase MTOR. Autophagy also targets intracellular bacteria (a process termed xenophagy) for lysosomal degradation, thereby playing a key role in innate immunity. In the past few years, the identification of molecules, such as CALCOCO2/NDP52, SQSTM1/p62 and ubiquitin, implicated in the specific targeting of intracellular bacteria, received considerable attention. However, it remains unclear how xenophagy is initiated, since this process commonly occurs in metabolically replete cells. In a recent study, we demonstrated that infection with Shigella and Salmonella triggered an early state of intracellular amino acid (AA) starvation causing MTOR dissociation from endomembranes, downregulation of MTOR activity and activation of the EIF2AK4/GCN2-EIF2S1/eIF2α/ATF3 signaling axis. We also observed that AA starvation was caused by host membrane damage, which appeared to be transient in the case of Salmonella and sustained in Shigella-infected cells, thus highlighting the existence of key timing disparities in xenophagy triggering, depending on the bacterial pathogen. Together, our findings demonstrate that xenophagy is only one arm of a more general metabolic switch geared toward AA starvation in bacteria-infected cells.

Intracellular Bacterial Pathogens Trigger the Formation of U Small Nuclear RNA Bodies (U Bodies) through Metabolic Stress Induction.

Background: The impact of metabolic stress on host response to bacterial infection remains poorly characterized. Results: Intracellular bacteria (Shigella, Salmonella, and Listeria) induce cytoplasmic U bodies through metabolic stress. Conclusion: Bacterial infection and metabolic stress affect the splicing machinery. Significance: Regulation of U snRNA maturation is a novel checkpoint in innate immunity. Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense.

Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes

Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general.

Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.