Luigi Franchi

Sensing and reacting to microbes through the inflammasomes

Inflammasomes are multiprotein complexes that activate caspase-1, which leads to maturation of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18 and the induction of pyroptosis. Members of the Nod-like receptor (NLR) family, including NLRP1, NLRP3 and NLRC4, and the cytosolic receptor AIM2 are critical components of inflammasomes and link microbial and endogenous danger signals to the activation of caspase-1. In response to microbial infection, activation of the inflammasomes contributes to host protection by inducing immune responses that limit microbial invasion, but deregulated activation of inflammasomes is associated with autoinflammatory syndromes and other pathologies. Thus, understanding inflammasome pathways may provide insight into the mechanisms of host defense against microbes and the development of inflammatory disorders.

Cutting Edge: TNF-α Mediates Sensitization to ATP and Silica via the NLRP3 Inflammasome in the Absence of Microbial Stimulation

The Nlrp3 inflammasome is critical for the activation of caspase-1 in response to danger signals and particulate matter. However, its role in sterile inflammation remains unclear because prestimulation of phagocytic cells with microbial molecules is required for caspase-1 activation. We show here that exposure of macrophages and dendritic cells to TNF-α promotes ATP- or silica-mediated caspase-1 activation and IL-1β secretion in the absence of microbial stimulation. The effect of TNF-α was abolished in macrophages deficient in TNF receptor I and II, Nlrp3, or ASC, whereas that induced by TLR ligands required MyD88/Trif. In addition to TNF-α, IL-1α and IL-1β promoted caspase-1 activation via Nlrp3 in response to ATP. Remarkably, macrophages tolerized to TNF-α, but not to LPS, retained full sensitivity to ATP stimulation via Nlrp3. These results provide a mechanism by which danger signals and particulate matter mediate inflammation via the Nlrp3 inflammasome in the absence of microbial infection.

Activation of the Nlrp3 Inflammasome by Streptococcus pyogenes Requires Streptolysin O and NF-κB Activation but Proceeds Independently of TLR Signaling and P2X7 Receptor

Macrophages play a crucial role in the innate immune response against the human pathogen Streptococcus pyogenes, yet the innate immune response against the bacterium is poorly characterized. In the present study, we show that caspase-1 activation and IL-1β secretion were induced by live, but not killed, S. pyogenes, and required expression of the pore-forming toxin streptolysin O. Using macrophages deficient in inflammasome components, we found that both NLR family pyrin domain-containing 3 (Nlrp3) and apoptosis-associated speck-like protein (Asc) were crucial for caspase-1 activation and IL-1β secretion, but dispensable for pro-IL-1β induction, in response to S. pyogenes infection. Conversely, macrophages deficient in the essential TLR adaptors Myd88 and Trif showed normal activation of caspase-1, but impaired induction of pro-IL-1β and secretion of IL-1β. Notably, activation of caspase-1 by TLR2 and TLR4 ligands in the presence of streptolysin O required Myd88/Trif, whereas that induced by S. pyogenes was blocked by inhibition of NF-κB. Unlike activation of the Nlrp3 inflammasome by TLR ligands, the induction of caspase-1 activation by S. pyogenes did not require exogenous ATP or the P2X7R. In vivo experiments revealed that Nlrp3 was critical for the production of IL-1β but was not important for survival in a mouse model of S. pyogenes peritoneal infection. These results indicate that caspase-1 activation in response to S. pyogenes infection requires NF-κB and the virulence factor streptolysin O, but proceeds independently of P2X7R and TLR signaling.

Acetylation Suppresses the Proapoptotic Activity of GD3 Ganglioside

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.

Calnexin suppresses GD3 synthase-induced apoptosis

An accelerated activity of the GD3 synthase (ST8), with consequent GD3 accumulation, is part of the response to environmental stressors in different cell types. Depending on specific, yet largely undefined, cellular settings, this can be followed by adaptation or apoptosis, the latter mostly due to GD3‐induced mitochondrial damage. Here we show that subcellular localization of ST8 could significantly affect the biological outcome of GD3 accumulation. Binding to the molecular chaperone calnexin causes the retention of ST8 within the endoplasmic reticulum (ER) and prevents its relocalization to the Golgi. Calnexin‐dependent ER retention does not affect the activity of ST8; yet the de novo synthesized GD3 largely fails to reach the mitochondria. Accordingly, overexpression of calnexin suppresses the pro‐apoptotic activity of ST8, while the loss of calnexin sensitizes the cells to ST8‐induced apoptosis. Reconstitution of calnexin confers protection to deficient cells. Thus, calnexin controls the biological outcome of GD3 accumulation and reveals a novel role in the stress response.

Calcium-Independent Phospholipase A2β Is Dispensable in Inflammasome Activation and Its Inhibition by Bromoenol Lactone

Calcium-independent phospholipase A2 (iPLA2) has been suggested to play an important role in the activation of caspase-1 induced by lipopolysaccharides (LPS). Here, we used pharmacological and genetic approaches to study the role of iPLA2 in the activation of caspase-1. Bromoenol lactone (BEL), an inhibitor that was originally used to support a role for iPLA2 in the secretion of IL-1β, prevented caspase-1 activation induced by LPS and ATP as described, and also activation triggered by Salmonella infection and cytosolic flagellin, which rely on the Nlrc4 inflammasome. Analysis of BEL enantiomers showed that the S-BEL form was more effective than R-BEL in inhibiting the inflammasome, suggesting a role for iPLA2β. However, caspase-1 activation and IL-1β secretion and their inhibition by BEL were unimpaired in macrophages deficient in iPLA2β. BEL was originally identified as an inhibitor of serine proteases. Consistent with the latter, the serine proteases inhibitors TPCK, TLCK and AAF-cmk prevented the activation of the Nlrc4 and Nlrp3 inflammasomes while pan-cathepsin inhibitors were ineffective. These results indicate that iPLA2β is not critical for caspase-1 activation as currently proposed. Instead, the results suggest that serine protease(s) targeted by BEL may play a critical role in the activation of the inflammasome triggered by microbial stimuli.

Ceramide catabolism critically controls survival of human dendritic cells

The regulation of dendritic cell (DC) survival is crucial for the modulation of adaptive immunity. Ceramide is a lipid mediator of the stress response, which accumulates intracellularly during DC differentiation. We found that ceramide levels are tightly regulated in human DCs and that the pharmacological inhibition of enzymes responsible for ceramide catabolism, such as ceramidases and sphingosine kinases, sensitizes DCs to ceramide‐induced cell death. It is important that inhibition of sphingosine kinases, during lipopolysaccharide stimulation, causes extensive ceramide accumulation and death of DCs. These data indicate that ceramide catabolism regulates urvival of human DCs and reveal novel potential targets for the pharmacological manipulation of the immune response.

An automated phenotype-based microscopy screen to identify pro-longevity interventions acting through mitochondria in C. elegans.

Mitochondria are multifunctional organelles that play a central role in cellular homeostasis. Severe mitochondrial dysfunction leads to life-threatening diseases in humans and accelerates the aging process. Surprisingly, moderate reduction of mitochondrial function in different species has anti-aging effects. High-throughput screenings in the nematode Caenorhabditis elegans lead to the identification of several pro-longevity genetic and pharmacological interventions. Large-scale screens, however, are manual, subjective, time consuming and costly. These limitations could be reduced by the identification of automatically quantifiable biomarkers of healthy aging. In this study we exploit the distinct and reproducible phenotypes described in C. elegans upon different levels of mitochondrial alteration to develop an automated high-content strategy to identify new potential pro-longevity interventions. Utilizing the microscopy platform Cellomics ArrayScan Reader, we optimize a workflow to automatically and reliably quantify the discrete phenotypic readouts associated with different degrees of silencing of mitochondrial respiratory chain regulatory proteins, and validate the approach with mitochondrial-targeting drugs known to extend lifespan in C. elegans. Finally, we report that a new mitochondrial ATPase modulator matches our screening phenotypic criteria and extends nematodes lifespan thus providing the proof of principle that our strategy could be exploited to identify novel mitochondrial-targeted drugs with pro-longevity activity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

Is it cytotoxicity or apoptosis which causes cell death in autoimmunity

Autoimmune diseases occur in 3–5% of the general population. They represent an heterogeneous group of diseases in which an adaptive immune response against self antigens causes damage to an organ or tissue. Although animal models and experimental data suggest that alterations in the selection, regulation or death of lymphocytes, which result in aberrant responses to self antigens, may play a major role in autoimmune pathogenesis, it is not often possible to determine whether a given autoimmune disease is due to a general abnormality in lymphocyte function or to an antigen-specific hyperreactivity. In fact, both genetic and environmental factors affect the susceptibility to autoimmunity and the severity of the disease [10, 34]. Cytotoxic responses mediated by lymphoid effectors imply recognition and subsequent killing of target cells. Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize target cells through different and specific recognition modules. However, the effector mechanisms adopted to eventually kill target cells are remarkably similar. In fact, both CTL and NK cells may use perforin/granzymeB and death ligand-receptor interactions to induce caspase-dependent apoptosis in target cells [50]. In addition to lymphocyte-mediated cytotoxicity, tissue damage may be greatly amplified by soluble factors and even by non-immunological mechanisms of cytotoxicity, which may also use similar molecular mechanisms and result in apoptotic cell death [13]. In this review we describe the main features of the apoptotic program of mammalian cells and mention selected potential targets for therapeutic interventions. We also focus our attention on the role of death receptor-induced apoptosis in some autoimmune diseases. In autoimmune lymphoproliferative response (ALPS), mutations in a single gene cause defective Fas-mediated apoptosis of lymphocytes, which in turn results in an abnormal immune response. In Crohn’s disease (CD), a defect in the NOD-2 gene is supposed to cause resistance of epithelial cells to TNF-induced apoptosis, resulting in a sustained inflammatory response. Moreover, we briefly dis-

593 Modulation of the F1F0-ATPase Induces Apoptosis of Mouse and Human Lamina Propria T-cells and Is Efficacious in Models of IBD

Background: There is an increasing recognition that targeting immune metabolism may represent a new approach for therapeutic intervention in autoimmune diseases such as inflammatory bowel disease (IBD). Small molecules that allosterically modulate the activity of the mitochondrial F1F0-ATPase induce apoptosis in susceptible cells and have shown robust efficacy in several models of immune-mediated disease. Chronically activated lymphocytes have a distinctive metabolic phenotype that sensitizes these cells to ATPase Modulatorinduced apoptosis compared to acutely activated cells. This may provide an advantage over current less specific immunosuppressive treatments. In IBD, lamina propria (LP) lymphocytes are chronically activated, overexpress anti-apoptotic proteins and are resistant to activationinduced cell death (AICD). Restoring AICD of lymphocytes by inhibiting ATPase activity is a potential therapeutic strategy. Here, three ATPase Modulators were evaluated in murine IBD models and in LP cells from IBD patient biopsies.Methods: A relapsing-remitting model of chronic murine IBD was induced by intracolonic administration of 4-5 escalating doses of TNBS. ATPase modulators were dosed orally after 2-3 TNBS challenges. Mucosal LP mononuclear cells were isolated from mice treated with vehicle or LYC-51194 and analyzed for viability and inflammatory molecules by flow cytometry and RT-PCR. LP cells isolated from IBD patient biopsies were treated with LYC-51194 or anti-Fas mAb and viability assessed by flow cytometry. Results: Bz-423, LYC-51661 and LYC-51194 inhibited the F1F0-ATPase and induced lymphocyte apoptosis in vitro. In a relapsing-remitting model of chronic murine IBD, ATPase modulators did not show any overt toxicity and produced benefits including attenuated weight loss, normalized histology, decreased inflammatory cytokine production (IFNγ, IL-6, TNF) and increased the percentage of cell death in LP T cells (vehicle 22±6 vs LYC-51194 53±10 p<0.0009). To translate these findings to human biology, LP cells isolated from IBD patient biopsies were treated with LYC-51194 or antiFas mAb. In agreement with previously published results, LP T cells from inflamed areas from CD subjects showed resistance to Fas-induced cell death (% cell death: vehicle 22.5±7.7 n=10 vs anti-Fas 22±12 n=7). However, similar to what was observed in rodent disease models, LYC-51194 treatment was able to induce apoptosis in LP T cells from inflamed IBD patient samples (% cell death: vehicle 22.5±7.7 n=10 vs LYC-51194 49.3±8.1 n=9; p= 0.0003). Conclusions: Taken together, these results suggest that induction of apoptosis by application of ATPase Modulator compounds may be a promising approach for treating IBD.