Exosomal Hsp70 Induces a Pro-Inflammatory Response to Foreign Particles Including Mycobacteria

© 2010 Anand et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins. Methodology/Principal Findings: We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-kB activation and TNFa release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages. Conclusions/Significance: Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.E.A. was supported by Fundação para a Ciência e a Tecnologia (FCT) Grant PIC/IC/82859/2007 and PTDC/SAU-MII/098024/2008. P.K.A was supported by a post-doctoral research grant from Alexander von Humboldt foundation, Germany and Marie-Curie fellowship from European Union, FP6 programme MIF1-CT- 2006-039351. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Role of the Nlrp3 Inflammasome in Microbial Infection

The intracellular Nod-like receptor Nlrp3 has emerged as the most versatile innate immune receptor because of its broad specificity in mediating immune response to a wide range of microbial or danger signals. Nlrp3 mediates assembly of the inflammasome complex in the presence of microbial components leading to the activation of caspase-1 and the processing and release of the pro-inflammatory cytokines IL-1β and IL-18. In this review, we give an update on the recent literature examining the role of Nlrp3 inflammasome in response to fungal, bacterial, and viral infections

Reactive oxygen species regulate caspase-11 expression and activation of the non-canonical NLRP3 inflammasome during enteric pathogen infection

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3-and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway

NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-kappa B (NF-kappa B), type I interferon and inflammasome signalling(1). Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis(2-4), but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-kappa B pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-kappa B-and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens

Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation

Cellular lipids determine membrane integrity and fluidity and are being increasingly recognized to influence immune responses. Cellular cholesterol requirements are fulfilled through biosynthesis and uptake programs. In an intricate pathway involving the lysosomal cholesterol transporter NPC1, the sterol gets unequally distributed across intracellular compartments. By using pharmacological and genetic approaches targeting NPC1, we reveal that blockade of cholesterol trafficking through the late endosome–lysosome pathway blunts NLRP3 inflammasome activation. Altered cholesterol localization at the plasma membrane (PM) in Npc1−/− cells abrogated AKT–mTOR signaling by TLR4. However, the inability to activate the NLRP3 inflammasome was traced to perturbed cholesterol trafficking to the ER but not the PM. Accordingly, acute cholesterol depletion in the ER membranes by statins abrogated casp-1 activation and IL-1β secretion and ablated NLRP3 inflammasome assembly. By contrast, assembly and activation of the AIM2 inflammasome progressed unrestricted. Together, this study reveals ER sterol levels as a metabolic rheostat for the activation of the NLRP3 inflammasome

Lipids regulate P2X7-receptor-dependent actin assembly by phagosomes via ADP translocation and ATP synthesis in the phagosome lumen

Latex bead phagosomes isolated from J774 macrophages polymerize actin. We show here that five lipids phosphatidylinositol-4-phosphate, phosphatidylinositol-(4,5)bisphosphate, sphingosine-1-phosphate (S1P), ceramide-1-phosphate and phosphatidic acid - stim. - Fundacao para a Ciencia e a tecnologia (FCT). - We would like to thank Sabrina Marion and Simi Antony for their technical support. We greatly appreciate the suggestions by Jens Reich and Thomas Dandekar. Thanks also to Christopher Bleck for preparing Fig. 4 and to Luis Mayorga, Sabrina Marion and Maxi

The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis

NLRP12 is a member of the intracellular Nod-like receptor (NLR) family that has been suggested to downregulate the production of inflammatory cytokines, but its physiological role in regulating inflammation has not been characterized. We analyzed mice deficient in Nlrp12 to study its role in inflammatory diseases such as colitis and colorectal tumorigenesis. We show that Nlrp12-deficient mice are highly susceptible to colon inflammation and tumorigenesis, which is associated with increased production of inflammatory cytokines, chemokines, and tumorigenic factors. Enhanced colon inflammation and colorectal tumor development in Nlrp12-deficient mice are due to a failure to dampen NF-kappa B and ERK activation in macrophages. These results reveal a critical role for NLRP12 in maintaining intestinal homeostasis and providing protection against colorectal tumorigenesis

FADD and Caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes

The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. In this study, we show that loss of FADD or caspase-8 in a RIP3-deficient background, but not RIP3 deficiency alone, hampered transcriptional priming and posttranslational activation of the canonical and noncanonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli but not the Nlrc4 inflammasome. In addition, FADD deletion prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8-deficient mice had impaired IL-1 beta production when challenged with LPS or infected with the enteropathogen Citrobacter rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and noncanonical Nlrp3 inflammasome priming and activation

Enhanced inflammasome activation in <i>Nod2^−/−</i> and <i>Rip2^−/−</i> BMDMs.

<p>BMDM were generated from WT, <i>Nod2^−/−</i> and <i>Rip2^−/−</i> mice and infected with 20MOI of <i>Citrobacter rodentium</i> for 18 h. <b>(A–F)</b> Combined supernatant and lysates were examined by Western blot for caspase-1 cleavage (casp-1p20) visually (A,D) and by densitometry (B,E), or (C,F) supernatants were examined for IL-18 secretion by ELISA. <b>(G)</b> BMDM were infected with <i>C. rodentium</i> and assayed for intracellular growth at the indicated times post-infection. (A–F) Data are representative of five independent experiments with n = 2–3 wells per experiment. (G) Data are representative of two independent experiments with n = 2–3 wells per experiment. (B,C,E,F,G) Data are shown as the mean ± SEM. (*, p<0.05; **, p<0.01; ***, p<0.001).</p