Reducing Stress PERKs up Anti-tumor Immunity.

Myeloid-derived suppressor cells (MDSCs) can promote tumor progression. In this issue of Immunity, Mohamed et al. show that the unfolded protein response sensor, PERK, enhances MDSC-mediated immunosuppression through the NRF2 transcription factor, preventing oxidative damage, mitochondrial DNA release, and DNA sensor-STING-dependent type I interferon production.

Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage.

Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Anti-infective Activity of 2-Cyano-3-Acrylamide Inhibitors with Improved Drug-Like Properties against Two Intracellular Pathogens.

Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection.

Membrane damage during Listeria monocytogenes infection triggers a caspase-7 dependent cytoprotective response.

The cysteine protease caspase-7 has an established role in the execution of apoptotic cell death, but recent findings also suggest involvement of caspase-7 during the host response to microbial infection. Caspase-7 can be cleaved by the inflammatory caspase, caspase-1, and has been implicated in processing and activation of microbial virulence factors. Thus, caspase-7 function during microbial infection may be complex, and its role in infection and immunity has yet to be fully elucidated. Here we demonstrate that caspase-7 is cleaved during cytosolic infection with the intracellular bacterial pathogen, Listeria monocytogenes. Cleavage of caspase-7 during L. monocytogenes infection did not require caspase-1 or key adaptors of the primary pathways of innate immune signaling in this infection, ASC, RIP2 and MyD88. Caspase-7 protected infected macrophages against plasma membrane damage attributable to the bacterial pore-forming toxin Listeriolysin O (LLO). LLO-mediated membrane damage could itself trigger caspase-7 cleavage, independently of infection or overt cell death. We also detected caspase-7 cleavage upon treatment with other bacterial pore-forming toxins, but not in response to detergents. Taken together, our results support a model where cleavage of caspase-7 is a consequence of toxin-mediated membrane damage, a common occurrence during infection. We propose that host activation of caspase-7 in response to pore formation represents an adaptive mechanism by which host cells can protect membrane integrity during infection.

Regulation of bacterial pathogenesis by intestinal short-chain Fatty acids.

The human gut microbiota is inextricably linked to health and disease. One important function of the commensal organisms living in the intestine is to provide colonization resistance against invading enteric pathogens. Because of the complex nature of the interaction between the microbiota and its host, multiple mechanisms likely contribute to resistance. In this review, we dissect the biological role of short-chain fatty acids (SCFA), which are fermentation end products of the intestinal microbiota, in host-pathogen interactions. SCFA exert an extensive influence on host physiology through nutritional, regulatory, and immunomodulatory functions and can also affect bacterial fitness as a form of acid stress. Moreover, SCFA act as a signal for virulence gene regulation in common enteric pathogens. Taken together, these studies highlight the importance of the chemical environment where the biology of the host, the microbiota, and the pathogen intersects, which provides a basis for designing effective infection prevention and control.

Non-canonical activation of IRE1α during Candida albicans infection enhances macrophage fungicidal activity.

Infection by intracellular pathogens can trigger activation of the IRE1α branch of the unfolded protein response (UPR), which then modulates innate immunity and infection outcomes during bacterial or viral infection. However, the mechanisms by which infection activates IRE1α have not been fully elucidated. While recognition of microbe-associated molecular patterns can activate IRE1α, it is unclear whether this depends on the canonical role of IRE1α in detecting misfolded proteins. Here, we report that Candida albicans infection of macrophages results in IRE1α activation through C-type lectin receptor signaling, reinforcing a role for IRE1α as a central regulator of host responses to infection by a broad range of pathogens. However, IRE1α activation was not preceded by protein misfolding in response to either C. albicans infection or lipopolysaccharide treatment, implicating a non-canonical mode of IRE1α activation after recognition of microbial patterns. Investigation of the phenotypic consequences of IRE1α activation in macrophage antimicrobial responses revealed that IRE1α activity enhances the fungicidal activity of macrophages. Macrophages lacking IRE1α activity displayed inefficient phagolysosomal fusion, enabling C. albicans to evade fungal killing and escape the phagosome. Together, these data provide mechanistic insight for the non-canonical activation of IRE1α during infection, and reveal central roles for IRE1α in macrophage antifungal responses.

Fatty acids regulate stress resistance and virulence factor production for Listeria monocytogenes.

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.

A complex lipoate utilization pathway in Listeria monocytogenes.

Although a complete pathway of lipoic acid metabolism has been established in Escherichia coli, lipoic acid metabolism in other bacteria is more complex and incompletely understood. Listeria monocytogenes has been shown to utilize two lipoate-protein ligases for lipoic acid scavenging, whereas only one of the ligases can function in utilization of host-derived lipoic acid-modified peptides. We report that lipoic acid scavenging requires not only ligation of lipoic acid but also a lipoyl relay pathway in which an amidotransferase transfers lipoyl groups to the enzyme complexes that require the cofactor for activity. In addition, we provide evidence for a new lipoamidase activity that could allow utilization of lipoyl peptides by lipoate-protein ligase. These data support a model of an expanded, three-enzyme pathway for lipoic acid scavenging that seems widespread in the Firmicutes phylum of bacteria.

A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.

Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

Branched-chain fatty acids promote Listeria monocytogenes intracellular infection and virulence.

Anteiso-branched-chain fatty acids (BCFA) represent the dominant group of membrane fatty acids and have been established as crucial determinants in resistance against environmental stresses in Listeria monocytogenes, a facultative intracellular pathogen. Here, we investigate the role of anteiso-BCFA in L. monocytogenes virulence by using mutants deficient in branched-chain alpha-keto acid dehydrogenase (BKD), an enzyme complex involved in the synthesis of BCFA. In tissue culture models of infection, anteiso-BCFA contributed to intracellular growth and survival in macrophages and significantly enhanced plaque formation upon prolonged infection in L2 fibroblasts. The intracellular defects observed could be attributed partially to insufficient listeriolysin O (LLO) production, indicating a requirement for anteiso-BCFA in regulating virulence factor production. In a murine model of infection, the BKD-deficient mutant was highly attenuated, further emphasizing the importance of BKD-mediated metabolism in L. monocytogenes virulence. This study demonstrates an underappreciated role for BCFA in bacterial pathogenesis, which may provide insight into the development and application of antimicrobial agents.

Innate recognition of intracellular bacteria.

The molecular repertoire for innate recognition of bacterial pathogens has expanded rapidly in the past decade. These immunosensors include Toll-like receptors and the more recently defined NOD-like receptors (NLRs): NODs, NALPs, NAIP and IPAF. Toll-like receptors signal from the cell surface or endosome upon ligand binding, whereas NLRs are activated by characteristic bacterially derived molecules, such as peptidoglycan, RNA, toxins and flagellin, in the cytosol. Studies using animal and culture models of bacterial infection indicate a pro-inflammatory role for NLRs, mediated by signaling through nuclear transcription factor kappaB and activation of caspase-1 by the inflammasome. These data also support a synergistic role for extracellular and intracellular bacterial sensing in regulating inflammation. In humans, NLR mutations are often associated with autoinflammatory syndromes, suggesting a complex role for cytosolic surveillance in systemic innate immunity.

XIAP regulates cytosol-specific innate immunity to Listeria infection.

The inhibitor of apoptosis protein (IAP) family has been implicated in immune regulation, but the mechanisms by which IAP proteins contribute to immunity are incompletely understood. We show here that X-linked IAP (XIAP) is required for innate immune control of Listeria monocytogenes infection. Mice deficient in XIAP had a higher bacterial burden 48 h after infection than wild-type littermates, and exhibited substantially decreased survival. XIAP enhanced NF-kappaB activation upon L. monocytogenes infection of activated macrophages, and prolonged phosphorylation of Jun N-terminal kinase (JNK) specifically in response to cytosolic bacteria. Additionally, XIAP promoted maximal production of pro-inflammatory cytokines upon bacterial infection in vitro or in vivo, or in response to combined treatment with NOD2 and TLR2 ligands. Together, our data suggest that XIAP regulates innate immune responses to L. monocytogenes infection by potentiating synergy between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) through activation of JNK- and NF-kappaB-dependent signaling.

The Mitochondrial Fission Regulator DRP1 Controls Post-Transcriptional Regulation of TNF-α.

The mitochondrial network plays a critical role in the regulation of innate immune signaling and subsequent production of proinflammatory cytokines such as IFN-ß and IL-1ß. Dynamin-related protein 1 (DRP1) promotes mitochondrial fission and quality control to maintain cellular homeostasis during infection. However, mechanisms by which DRP1 and mitochondrial dynamics control innate immune signaling and the proinflammatory response are incompletely understood. Here we show that macrophage DRP1 is a positive regulator of TNF-α production during sterile inflammation or bacterial infection. Silencing macrophage DRP1 decreased mitochondrial fragmentation and TNF-α production upon stimulation with lipopolysaccharide (LPS) or methicillin-resistant Staphylococcus aureus (MRSA) infection. The defect in TNF-α induction could not be attributed to changes in gene expression. Instead, DRP1 was required for post-transcriptional control of TNF-α. In contrast, silencing DRP1 enhanced IL-6 and IL-1ß production, indicating a distinct mechanism for DRP1-dependent TNF-α regulation. Our results highlight DRP1 as a key player in the macrophage pro-inflammatory response and point to its involvement in post-transcriptional control of TNF-α production.

TBK1 protects vacuolar integrity during intracellular bacterial infection.

TANK-binding kinase-1 (TBK1) is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella), more extensive bacterial proliferation was observed in tbk1(-/-) than tbk1(+/+) cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-)cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.

Homeostatic maintenance of pathogen-containing vacuoles requires TBK1-dependent regulation of aquaporin-1.

Membranes are an integral component of many cellular functions and serve as a barrier to keep pathogenic bacteria from entering the nutrient-rich host cytosol. TANK-binding-kinase-1 (TBK1), a kinase of the IkappaB kinase family, is required for maintaining integrity of pathogen-containing vacuoles (PCV) upon bacterial invasion of host cells. Here we investigate how vacuolar integrity is maintained during bacterial infection, even in the presence of bacterial membrane damaging agents. We found that Aquaporin-1 (AQP1), a water channel that regulates swelling of secretory vesicles, associated with PCV. AQP1 levels were elevated in TBK1-deficient cells, and overexpression of AQP1 in wild-type cells led to PCV destabilization, similar to that observed in tbk1(-/-) cells. Inhibition of physiological levels of AQP1 in multiple cell types also led to increased instability of PCV, demonstrating a need for tightly regulated AQP1 function to maintain vacuole homeostasis during bacterial infection. AQP1-dependent modulation of PCV was triggered by bacterially induced membrane damage and ion flux. These results highlight the contribution of water channels to promoting PCV membrane integrity, and reveal an unexpected role for TBK1 and AQP1 in restricting bacterial pathogens to the vacuolar compartment.

Metabolism of the Gram-Positive Bacterial Pathogen Listeria monocytogenes.

Bacterial metabolism represents the biochemical space that bacteria can manipulate to produce energy, reducing equivalents and building blocks for replication. Gram-positive pathogens, such as Listeria monocytogenes, show remarkable flexibility, which allows for exploitation of diverse biological niches from the soil to the intracytosolic space. Although the human host represents a potentially rich source for nutrient acquisition, competition for nutrients with the host and hostile host defenses can constrain bacterial metabolism by various mechanisms, including nutrient sequestration. Here, we review metabolism in the model Gram-positive bacterium, L. monocytogenes, and highlight pathways that enable the replication, survival, and virulence of this bacterial pathogen.

Perturbation of ubiquitin homeostasis promotes macrophage oxidative defenses.

The innate immune system senses microbial ligands through pattern recognition and triggers downstream signaling cascades to promote inflammation and immune defense mechanisms. Emerging evidence suggests that cells also recognize alterations in host processes induced by infection as triggers. Protein ubiquitination and deubiquitination are post-translational modification processes essential for signaling and maintenance of cellular homeostasis, and infections can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the impact of ubiquitin homeostasis alteration on macrophage function. Perturbation of ubiquitin homeostasis led to a rapid and transient burst of reactive oxygen species (ROS) that promoted macrophage inflammatory and anti-infective capacity. Moreover, we found that ROS production was dependent on the NOX2 phagocyte NADPH oxidase. Global alteration of the ubiquitin proteome also enhanced proinflammatory cytokine production in mice stimulated with a sub-lethal dose of LPS. Collectively, our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.

Glycolysis Is an Intrinsic Factor for Optimal Replication of a Norovirus.

The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics and energetic profiling of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus (MNV) infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in macrophages revealed that glycolysis is an important factor for optimal MNV infection, while inhibiting the PPP and OXPHOS showed a relatively minor impact of these pathways on MNV infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased viral protein production and viral RNA. The requirement of glycolysis was specific for MNV (but not astrovirus) infection, independent of the type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMP-activated protein kinase (AMPK), two master regulators of cellular metabolism, implicating Akt signaling in upregulating host metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates glycolysis as a virulence determinant. They further point to cellular metabolism as a novel therapeutic target for norovirus infections and improvements in current human norovirus culture systems.IMPORTANCE Viruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase glycolysis upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high-efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies.

Invasion of the Brain by Listeria monocytogenes Is Mediated by InlF and Host Cell Vimentin.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.IMPORTANCEListeria monocytogenes is an intracellular bacterial pathogen that is capable of invading numerous host cells during infection. L. monocytogenes can cross the blood-brain barrier, leading to life-threatening meningitis. Here we show that an L. monocytogenes surface protein, InlF, is necessary for optimal colonization of the brain in mice. Furthermore, in the absence of vimentin, a cytosolic intermediate filament protein that is also present on the surface of brain endothelial cells, colonization of the brain was significantly impaired. We further show that InlF binds vimentin to mediate invasion of host cells. This work identifies InlF as a bacterial surface protein with specific relevance for infection of the brain and underscores the significance of host cell surface vimentin interactions in microbial pathogenesis.