Anasuya Sarkar

Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria

Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

Inflammasome-Dependent Release of the Alarmin HMGB1 in Endotoxemia

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

ASC directs NF-κB activation by regulating receptor interacting protein-2 (RIP2) caspase-1 interactions

Receptor interacting protein-2 (RIP2) is a caspase recruitment domain (CARD)-containing kinase that interacts with caspase-1 and plays an important role in NF-κB activation. Apoptosis-associated speck-like protein containing a CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of apoptosis and caspase-1 activation. Although RIP2 has also been linked to caspase-1 activation, RIP2 knockout animals fail to show a defect in caspase-1-mediated processing of proIL-1β to its active form. Therefore, RIP2 function in binding to caspase-1 remains poorly understood. We hypothesized that caspase-1 may serve as a scaffolding molecule that promotes RIP2 interaction with IκB kinase-γ thus inducing NF-κB activation. We further hypothesized that ASC, which also interacts with caspase-1 via its CARD, may interfere with the caspase-1 RIP2 interaction. In HEK293 cells, ASC induced prominent activation of caspase-1 and proIL-1β processing. RIP2 transient transfection induced transcription of an NF-κB reporter gene. This RIP2-induced NF-κB activity and caspase-1 binding was inhibited in a dose-dependent fashion by ASC. Consistent with a role for caspase-1 as a scaffold for RIP2, caspase-1 knockout macrophages were suppressed in their ability to activate NF-κB, and septic caspase-1 knockout animals produced less IL-6, a functional marker of NF-κB activity. Lastly, THP-1 cells treated with small interfering RNA for ASC decreased their caspase-1 activity while enhancing their NF-κB signal. These data suggest that ASC may direct caspase-1 away from RIP2-mediated NF-κB activation, toward caspase-1-mediated processing of proIL-1β by interfering with the RIP2 caspase-1 interaction.

Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.

Inflammasome mRNA expression in human monocytes during early septic shock.

RATIONALE Monocytes are central to the initiation of the inflammatory response in sepsis, with caspase-1 activation playing a key role. Monocyte deactivation during sepsis has been linked to poor outcomes. OBJECTIVES Given the importance of caspase-1 in the immune response, we investigated whether monocytes from patients early in septic shock demonstrate alterations in mRNAs for caspase-1-related molecules. METHODS Patients with septic shock (n = 26; age >18 years), critically ill intensive care unit patients (n = 20), and healthy volunteers (n = 22) were enrolled in a prospective cohort study in a university intensive care unit. Demographic, biological, physiologic, and plasma cytokine measurements were obtained. Monocytes were assayed for ex vivo tumor necrosis factor-alpha production, and fresh monocyte mRNA was analyzed by quantitative reverse-transcription polymerase chain reaction for Toll-like receptors, NOD-LRR proteins, cytokines, and nuclear factor-kappaB-related genes. MEASUREMENTS AND MAIN RESULTS Relative copy numbers for the inflammasome mRNAs for ASC, caspase-1, NALP1, and Pypaf-7 were significantly lower in patients with septic shock compared with critically ill control subjects. NALP1 mRNA levels were linked to survival in patients with sepsis (P = 0.0068) and correlated with SAPS II scores (r = -0.63). CONCLUSIONS These data suggest that monocyte deactivation occurs during the earliest stages of the systemic inflammatory response and that changes in inflammasome mRNA expression are part of this process.

Bacterial peptide recognition and immune activation facilitated by human peptide transporter PEPT2.

Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di- and tripeptides across epithelial tissues, including the lung. Using computational tools, we initially established that bacterial dipeptides, particularly gamma-D-glutamyl-meso-diaminopimelic acid (gamma-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that gamma-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 microM, whereas muramyl dipeptide was not transported. Exposure to gamma-iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in an NOD1- and RIP2-dependent manner, resulting in release of IL-6 and IL-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung.

T cell-intrinsic ASC critically promotes TH17-mediated experimental autoimmune encephalomyelitis

Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17–producing helper T (TH17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell–intrinsic inflammasome that drives IL-1β production during TH17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3–dependent caspase-8 activation. IL-1R was detected on TH17 cells but not on type 1 helper T (TH1) cells, and ATP-treated TH17 cells showed enhanced survival compared with ATP-treated TH1 cells, suggesting autocrine action of TH17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a TH17 cell–intrinsic ASC–NLRP3–caspase-8 inflammasome during inflammation of the central nervous system.

P2X7 receptor and macrophage function

Macrophages are unique innate immune cells that play an integral role in the defense of the host by virtue of their ability to recognize, engulf, and kill pathogens while sending out danger signals via cytokines to recruit and activate inflammatory cells. It is becoming increasingly clear that purinergic signaling events are essential components of the macrophage response to pathogen challenges and disorders such as sepsis may be, at least in part, regulated by these important sensors. The activation of the P2X7 receptor is a powerful event in the regulation of the caspase-1 inflammasome. We provide evidence that the inflammasome activation requires “priming” of macrophages prior to ATP activation of the P2X7R. Inhibition of the inflammasome activation by the tyrosine kinase inhibitor, AG126, suggests regulation by phosphorylation. Finally, the P2X7R may also be activated by other elements of the host response such as the antimicrobial peptide LL-37, which adds a new, physiologically relevant agonist to the P2X7R pathway. Therapeutic approaches to inflammation and sepsis will certainly be enhanced by an increased understanding of how purinergic receptors modulate the inflammasomes.

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.

Thrombospondin-1 Contributes to Mortality in Murine Sepsis through Effects on Innate Immunity

Background Thrombospondin-1 (TSP-1) is involved in many biological processes, including immune and tissue injury response, but its role in sepsis is unknown. Cell surface expression of TSP-1 on platelets is increased in sepsis and could activate the anti-inflammatory cytokine transforming growth factor beta (TGFβ1) affecting outcome. Because of these observations we sought to determine the importance of TSP-1 in sepsis. Methodology/Principal Findings We performed studies on TSP-1 null and wild type (WT) C57BL/6J mice to determine the importance of TSP-1 in sepsis. We utilized the cecal ligation puncture (CLP) and intraperitoneal E.coli injection (IP E.coli) models of peritoneal sepsis. Additionally, bone-marrow-derived macrophages (BMMs) were used to determine phagocytic activity. TSP-1−/− animals experienced lower mortality than WT mice after CLP. Tissue and peritoneal lavage TGFβ1 levels were unchanged between animals of each genotype. In addition, there is no difference between the levels of major innate cytokines between the two groups of animals. PLF from WT mice contained a greater bacterial load than TSP-1−/− mice after CLP. The survival advantage for TSP-1−/− animals persisted when IP E.coli injections were performed. TSP-1−/− BMMs had increased phagocytic capacity compared to WT. Conclusions TSP-1 deficiency was protective in two murine models of peritoneal sepsis, independent of TGFβ1 activation. Our studies suggest TSP-1 expression is associated with decreased phagocytosis and possibly bacterial clearance, leading to increased peritoneal inflammation and mortality in WT mice. These data support the contention that TSP-1 should be more fully explored in the human condition.