Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors.

The NLRP3 inflammasome is a multimeric protein complex that initiates an inflammatory form of cell death and triggers the release of proinflammatory cytokines IL-1ß and IL-18. The NLRP3 inflammasome has been implicated in a wide range of diseases, including Alzheimer's disease, Prion diseases, type 2 diabetes, and some infectious diseases. It has been found that a variety of stimuli including danger-associated molecular patterns (DAMPs, such as silica and uric acid crystals) and pathogen-associated molecular patterns (PAMPs) can activate NLRP3 inflammasome, but the specific regulatory mechanisms of NLRP3 inflammasome activation remain unclear. Understanding the mechanisms of NLRP3 activation will enable the development of its specific inhibitors to treat NLRP3-related diseases. In this review, we summarize current understanding of the regulatory mechanisms of NLRP3 inflammasome activation as well as inhibitors that specifically and directly target NLRP3.

Inflammasome-independent role of NLRP12 in suppressing colonic inflammation regulated by Blimp-1.

NLRP12 is a member of the Nod-like receptor (NLR). Previous studies have reported enhanced colitis-associated inflammatory responses in NLRP12-deficient mice. In this study, we sought to investigate the role of NLRP12 in DSS-stimulated proinflammatory response in dendritic cells and mice colitis, and the molecular mechanisms involved in the development of the inflammation. Our results showed that down-regulation of NLRP12 is required for DSS-induced release of proinflammatory cytokines IL-1ß and TNF-α; that PR domain zinc finger protein 1 (also known as Blimp-1) induces NLRP12 down-regulation during DSS-induced proinflammatory response and colitis; and that TLR4 is implicated in the up-regulation of Blimp-1 that led to the down-regulation of NLRP12 expression in DSS-induced colitis. Taken together, the results suggest that the TLR4-Blimp-1 axis promotes DSS induced experimental colitis through the down-regulation of NLRP12.

Cellular Prion Protein Promotes Neuronal Differentiation of Adipose-Derived Stem Cells by Upregulating miRNA-124.

The cellular prion protein (PrP(C)) is a highly conserved glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is also the source of pathogenic agent of scrapie prion protein (PrP(Sc)). Numerous researches have suggested putative physiological roles for PrP(C), including protection from ischemic and excitotoxic lesions, and participation in cell signaling and differentiation. Here, we demonstrated that PrP(C) positively regulates neuronal differentiation of mouse adipose-derived stem cells (ADSCs). The small C-terminal domain phosphatase 1 (SCP1) expression was knocked down by gene silencing. The mRNA expression of miRNA-124 and PrP(C) was measured with quantitative PCR. Western blot analysis was used to detect the protein levels of nestin, ßIII-tubulin, and SCP1, and dual-luciferase reporter assay was performed to test the target of miRNA-124. The expression level of PrP(C) was found to increase steadily during neuron-like differentiation process, and PrP(C) knockout resulted in the reduction of neuron-like cell markers. We further showed that miRNA-124 could directly target SCP1-3'-untranslated region to decrease small C-terminal domain phosphatase 1 (SCP1) SCP1, and that miRNA-124 expression is regulated by PrP(C). Our results suggest that PrP(C) may play a key role in the neuronal differentiation of ADSC through modulating miRNA-124-SCP1 axis. To date, this is the first time strong evidence for the involvement of PrP(C) in the neuronal differentiation of ADSC is reported.

NALP3 inflammasome activation in protein misfolding diseases.

Protein-misfolding diseases, such as Alzheimer's disease, type 2 diabetes, Prion diseases, and Parkinson's disease, are characterized by inflammatory reactions. In all these diseases, IL-1ß (Interlukine-1ß) has been shown to be an important regulator, and the misfolded proteins are proved to be triggers of the release of IL-1ß. Recently, several reports demonstrated that the inflammasome activation is involved in the progress of the misfolded protein diseases, and that the inflammasome can recognize pathogenic proteins leading to the release of IL-1ß. In this review, we discuss the role of inflammasome in the pathogenesis of misfolded protein diseases and the potential of inflammasome-targeting therapeutic interventions in the management of these diseases.

Toll-like receptor 2 deficiency shifts PrP106-126-induced microglial activation from a neurotoxic to a neuroprotective phenotype.

Prion diseases are fatal neurodegenerative diseases characterized by spongiform change, neuronal loss, and gliosis involving microglial activation in the central nervous system. Microglial activation is thought to play a key role in the pathogenesis of prion disease; however, the molecular mechanisms underlying prion-induced microglial activation are not well understood. The present study underlines the importance of toll-like receptor (TLR)-2 in mediating PrP106-126-induced microglial activation. We found that PrP106-126 induced expression of proinflammatory molecules and TLR2 in microglial cells; however, functional blocking antibodies against TLR2 suppressed PrP106-126-induced expression of proinflammatory molecules. PrP106-126-induced expression of proinflammatory molecules was also reduced in microglial cells isolated from TLR2-/- mice compared to those isolated from wild-type mice. Consistent with the importance of nuclear factor kappa B (NF-κB) mediating TLR functions, NF-κB inhibition also inhibited PrP106-126-induced expression of proinflammatory molecules. To better understand the effect of TLR2 deficiency on active microglial cells, we studied the expression of Arg1 and Mrc1 and anti-inflammatory cytokines, which indicated that TLR2 deficiency in microglial cells results in a shift from neurotoxic to neuroprotective phenotype. Taken together, our results indicate that the TLR2 signaling pathway mediates PrP106-126-induced microglial activation and potentially reveal new therapeutic strategies for prion diseases that modulate TLR2 signaling.

PrP106-126 and Aß 1-42 peptides induce BV-2 microglia chemotaxis and proliferation.

Transmissible spongiform encephalopathies (TSEs) and Alzheimer's disease (AD) belong to a growing family of neurodegenerative disorders that is characterized by the generation of toxic protein aggregates in affected brains (PrP(Sc) and Aß in TSEs and AD, respectively). To better understand how protein aggregates can modulate microglial processes in these diseases, we treated BV-2 microglia with PrP(106-126) or Aß1-42 peptides individually at three different concentrations (25-100 µM PrP(106-12) and 2.5-10 µM Aß1-42) or with a mixture of PrP(106-126) and Aß1-42 peptides at specified concentrations for 6-24 h. BV-2 microglia chemotaxis, proliferation, and monocyte chemoattractant protein-1 and transforming growth factor-ß1 (TGF-ß1) secretion were measured and compared between treatments. The results demonstrate that PrP(106-126) and Aß1-42 peptides induce increases in all four parameters from 6 to 12 h. However, the measured indices plateaued beyond 12 h in BV-2 cells treated >50 µM PrP or >5 µM Aß1-42, with the exception of TGF-ß1 secretion, which continued to increase gradually. Overall, the results of this study indicate that these two peptides may mutually inhibit microglial chemotaxis and proliferation simultaneously via changes induced at the protein level.

Inhibition of phagocytosis reduced the classical activation of BV2 microglia induced by amyloidogenic fragments of beta-amyloid and prion proteins.

The inflammatory responses in Alzheimer's disease and prion diseases are dominated by microglia activation. Three different phenotypes of microglial activation, namely classical activation, alternative activation, and acquired deactivation, have been described. In this study, we investigated the effect of amyloidogenic fragments of amyloid ß and prion proteins (Aß1-42 and PrP106-126) on various forms of microglial activation. We first examined the effect of Aß1-42 and PrP106-126 stimulation on the mRNA expression levels of several markers of microglial activation, as well as the effect of cytochalasin D, a phagocytosis inhibitor, on microglial activation in Aß1-42- and PrP106-126-stimulated BV2 microglia. results showed that Aß1-42 and PrP106-126 induced the classical activation of BV2 microglia, decreased the expression level of alternative expression markers, and had no effect on the expression of acquired deactivation markers. Cytochalasin D treatment significantly reduced Aß1-42- and PrP106-126-induced up-regulation of proinflammatory factors, but did not change the expression profile of the markers of alternative activation or acquired deactivation in BV2 cells which were exposed to Aß1-42 and PrP106-126. Our results suggested that microglia interact with amyloidogenic peptides in the extracellular milieu-stimulated microglial classical activation and reduce its alternative activation, and that the uptake of amyloidogenic peptides from the extracellular milieu amplifies the classical microglial activation.

the AIM2 inflammasome is involved in macrophage activation during infection with virulent Mycobacterium bovis strain.

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, infects host macrophages and triggers production of the proinflammatory cytokine interleukin 1ß (IL-1ß). The mechanism by which macrophages become activated and secrete IL-1ß in tuberculosis has not yet been elucidated. METHODS: In this study, we investigated the role of the absence in melanoma 2 (AIM2) inflammasome in IL-1ß release from macrophages infected with pathogenic M. bovis strain. RESULTS: We found that the AIM2 inflammasome activation is involved in the production of IL-1ß in primary and immortalized mouse macrophage upon M. bovis infection; that the activation process requires cytoplasmic potassium efflux, mycobacterial internalization, but not reactive oxygen species (ROS) or IFN-ß release; that the AIM2 inflammasome contributes to the synthesis of proinflammatory and chemotatic factors in M. bovis-infected macrophages; and that the activation of the AIM2 inflammasome is due, at least in part, to mycobacterial translocation into the cytosol. CONCLUSIONS: We conclude that the AIM2 inflammasome is involved in macrophage activation during infection with virulent M. bovis strain. To our knowledge, this is the first evidences for the involvement of the AIM2 inflammasome in M. bovis infection.

PP2 and piceatannol inhibit PrP106-126-induced iNOS activation mediated by CD36 in BV2 microglia.

Prion diseases are a group of transmissible fatal neurodegenerative disorders of humans and animals, including bovine spongiform encephalopathy, scrapie, and Creutzfeldt-Jakob disease. Microglia, the resident macrophages of the central nervous system, are exquisitely sensitive to pathological tissue alterations, altering their morphology and phenotype to adopt a so-called activated state and perform immunological functions in response to pathophysiological brain insults. Although recent findings have provided valuable insights into the role microglia play in the proinflammatory events observed in prion, the intracellular signaling molecules responsible for the initiation of these responses remain to be elucidated. It seems that microglial activation involve PrP106-126 binding and the activation of cell surface immune and adhesion molecules such as CD36 and integrins, with the subsequent recruitment of Src family tyrosine kinases such as Fyn, Lyn, and Syk kinases. In the present study, we show that CD36 is involved in PrP106-126-induced microglial activation and that PP2 and piceatannol (Pic) can abrogate neurotoxic prion peptides-induced inducible nitric oxide synthase activation in microglia. These findings unveil a previously unrecognized role of PP2 and Pic as Src family kinase Fyn and the tyrosine kinase Syk inhibitor involved in neurotoxic prion peptides-microglia interactions, thus providing new insights into mechanisms underlying the activation of microglia by neurotoxic prion peptides.

Inhibition of phagocytosis and lysosomal acidification suppresses neurotoxic prion peptide-induced NALP3 inflammasome activation in BV2 microglia.

Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein. In a previous study, we showed that neurotoxic prion peptide (PrP106-126) induced NALP3 inflammasome activation in mouse primary and immortalized microglia. In the present work, we examined the relevance of phagocytosis and lysosomal acidification to the activation of the NALP3 inflammasome in PrP106-126-stimulated microglia. Our results showed that the inhibition of phagocytosis or lysosomal acidification significantly reduced IL-1ß and IL-18 production, downregulated NALP3 and ASC expression, and decreased the expression of proinflammatory factors. We concluded that phagocytosis and lysosomal acidification are necessary for PrP106-126-induced NALP3 activation in BV2 cells.

Expression and distribution of laminin receptor precursor/laminin receptor in rabbit tissues.

The 37/67-kDa laminin receptor precursor (LRP)/laminin receptor (LR) is a cell surface receptor for cellular prion proteins and misfolded pathological prions. Previous research has shown that blocking or decreasing LRP/LP levels by anti-LRP/LR antibodies or small interfering RNAs (siRNAs) can prolong the incubation phase of experimental prion infection. This study aimed to investigate potential mechanisms contributing to prion resistance/susceptibility by using the rabbit, a species unsusceptible to prion infection, as a model. We investigated the expression level and distribution of LRP/LR in rabbit tissues by real-time polymerase chain reaction and by immunochemical analysis with a monoclonal anti-67 kDa LR antibody. Our results showed LRP/LR mRNA expression in all the tissues examined. Very low LRP/LR expression levels were observed in central nervous system (CNS) tissues, whereas high expression levels were observed in reproductive and digestive tissues, which differed from the expression patterns that have been reported for prion-susceptible animals. The immunochemical staining results were generally consistent with the mRNA findings, although no LR protein was detected in CNS tissues. Our findings provide a basis for further studies on prion resistance in rabbits and other animal species.

Cellular prion protein participates in the regulation of inflammatory response and apoptosis in BV2 microglia during infection with Mycobacterium bovis.

The cellular prion protein (PrP(C)) is a glycoprotein anchored by glycosylphosphatidylinositol to the cell surface and is abundantly expressed in the central nervous system. A previous study has shown that PrP(C) contributes to the establishment of infections with intracellular bacteria in macrophages. In the present work, we investigated the role of PrP(C) in the response of BV2 microglia to Mycobacterium bovis infection. For this purpose, we examined the mRNA expression of prion protein gene (PRNP) upon M. bovis infection and analyzed the effect of siRNA-mediated disruption of PRNP on different parameters of microglial activation and apoptosis in M. bovis-infected microglia. We found that M. bovis infection induced a gradual increase in PRNP mRNA level and that siRNA-mediated silencing of PRNP in M. bovis-infected microglia reduced M. bovis-induced upregulation of pro-inflammatory factors, increased the rate of apoptosis in infected microglia, promoted the intrinsic apoptotic pathway, and downregulated the extrinsic apoptotic pathway. We conclude that PrP(C) participates in the regulation of the response of microglia to M. bovis infection through the upregulation of pro-inflammatory cytokines and the modulation of apoptosis by interference with the intrinsic apoptotic pathway.

Prion protein participates in the regulation of classical and alternative activation of BV2 microglia.

The cellular prion protein (PrP(C) ) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is abundantly expressed in the central nervous system. Numerous studies have suggested a protective function for PrP(C) , including protection from ischemic and excitotoxic lesions and several apoptotic insults, and recent reports have shown that PrP(C) has a context-dependent neuroprotective function. In this study, we investigated the effect of PPNP down-regulation on various forms of microglial activation. We first examined the mRNA expression of PRNP upon exposure to IFN-γ, IL-4, or IL-10 in BV2 microglia. We then analyzed the effect of si-RNA-mediated disruption of PRNP on different parameters of microglial activation in IFN-γ-, IL-4-, or IL-10-stimulated microglia. The results showed that PRNP mRNA expression was invariably down-regulated in microglia upon exposure to IFN-γ, IL-4, or IL-10. PRNP silencing prior to cytokines treatment reduced the responsiveness of microglia to INF-γ treatment, significantly altered IL-4-induced microglial activation phenotype, and had no effect on IL-10-induced microglial activation. Together, these results support a role of PrP(C) in the modulation of the shift of microglia from a quiescent state to an activated phenotype and in the regulation of the microglial response during classical and alternative activation.

Antibody-mediated inhibition of integrin α5ß1 blocks neurotoxic prion peptide PrP106-126-induced activation of BV2 microglia.

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The identification of cell surface molecules that mediate the prion protein (PrP) synthetic peptide interaction with microglia is of great significance as it represents potential target molecules to modulate the events leading to the pathophysiology of prion diseases. Here, we carried out in vitro experiments to investigate the involvement of α5ß1 integrin in neurotoxic prion peptide PrP(106-126)-induced activation of BV2 microglia. The results showed that the exposure to PrP(106-126) upregulated the mRNA expression of proinflammatory factors (IL-1 ß, IL-6, and iNOS) and NALP3 inflammasome components (NALP3 and ASC), increased the release of iNOS and its product nitric oxide, and stimulated NF-κB activation. Blockade of α5ß1 integrin with monoclonal antibody BMC5 prior to PrP(106-126) treatment abrogated the upregulation of the mRNA expression of IL-1 ß, IL-6, iNOS, and ASC, but had no effect on the mRNA expression of NALP3, blocked the release of iNOS and nitric oxide, and inhibited NF-κB activation. These results suggest that α5ß1 integrin is involved in the PrP(106-126)-induced microglial activation through the participation in the activation of NF-κB and NALP3/ASC inflammasome. Our study unveils a previously unidentified role of α5ß1 integrin as an intermediate signaling molecule in neurotoxic prion peptides-microglia interactions and identifies a potential molecular target for the modulation of prion-induced microglial activation.

The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation.

BACKGROUND: Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrPSc. In prion-infected brains, activated microglia are often present in the vicinity of PrPSc aggregates, and microglial activation is thought to play a key role in the pathogenesis of prion diseases. Although interleukin (IL)-1ß release by prion-induced microglia has been widely reported, the mechanism by which primed microglia become activated and secrete IL-1ß in prion diseases has not yet been elucidated. In this study, we investigated the role of the NACHT, LRR and PYD domains-containing protein (NALP)3 inflammasome in IL-1ß release from lipopolysaccharide (LPS)-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126). METHODS: The inflammasome components NALP3 and apoptosis-associated speck-like protein (ASC) were knocked down by gene silencing. IL-1ß production was assessed using ELISA. The mRNA expression of NALP3, ASC, and pro-inflammatory factors was measured by quantitative PCR. Western blot analysis was used to detect the protein level of NALP3, ASC, caspase-1 and nuclear factor-κB. RESULTS: We found that that PrP106-126-induced IL-1ß release depends on NALP3 inflammasome activation, that inflammasome activation is required for the synthesis of pro-inflammatory and chemotactic factors by PrP106-126-activated microglia, that inhibition of NF-κB activation abrogated PrP106-126-induced NALP3 upregulation, and that potassium efflux and production of reactive oxygen species were implicated in PrP106-126-induced NALP3 inflammasome activation in microglia. CONCLUSIONS: We conclude that the NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. To our knowledge, this is the first time that strong evidence for the involvement of NALP3 inflammasome in prion-associated inflammation has been found.

CD36 participates in PrP(106-126)-induced activation of microglia.

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP) fragment 106-126 (PrP(106-126)). We first examined the time course of CD36 mRNA expression upon exposure to PrP(106-126) in BV2 microglia. We then analyzed different parameters of microglial activation in PrP(106-126)-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb). The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP(106-126). The results showed that PrP(106-126) treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α), increased iNOS expression and nitric oxide (NO) production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP(106-126)-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP(106-126)-induced iNOS stimulation, downregulated IL-1ß and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP(106-126)-treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP(106-126). Together, these results suggest that CD36 is involved in PrP(106-126)-induced microglial activation and that the participation of CD36 in the interaction between PrP(106-126) and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides and open perspectives for new therapeutic strategies for prion diseases by modulation of CD36 signaling.

Prion peptide PrP106-126 induces inducible nitric oxide synthase and proinflammatory cytokine gene expression through the activation of NF-κB in macrophage cells.

The inflammatory response in prion diseases is dominated by microglia activation. The molecular mechanisms that lie behind this inflammatory process are not very well understood. In the present study, we examined the activat2ion of nuclear factor-kappa B (NF-κB) upon exposure to PrP106-126 and its role in PrP106-126-induced upregulation of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines (interleukin [IL]-1ß, tumor necrosis factor [TNF]-α, IL-6) in Ana-1 macrophages. The results showed that iNOS and proinflammatory cytokine release was significantly elevated in Ana-1 macrophages upon exposure to PrP106-126; that PrP106-126 treatment led to a significant NF-κB activation; that proinflammatory cytokines gene expression was elevated in macrophages upon exposure to PrP106-126; and that NF-κB inhibition significantly abrogated PrP106-126-induced upregulation of iNOS and inflammatory cytokine mRNA expression. These results suggest that treatment with neurotoxic prion peptides leads to the activation of transcription factor NF-κB, which in turn stimulates gene expression of iNOS and proinflammatory cytokines in Ana-1 macrophages.

Comparison of mRNA expression patterns of class B scavenger receptors in BV2 microglia upon exposure to amyloidogenic fragments of beta-amyloid and prion proteins.

The inflammatory responses in Alzheimer's disease and prion diseases are dominated by microglia activation. Scavenger receptors have been recently related to the innate immune activation of microglia initiated by endogenous ligands. In this study, we investigated mRNA expression patterns of B class scavenger receptors CD36 and scavenger receptor B1 (SR-B1) in BV2 microglia upon exposure to amyloid fibril Aß(1-42) and PrP(106-126), respectively. CD36 and SR-B1 showed similar mRNA expression patterns following each treatment. PrP(106-126) induced a rapid increase of CD36 and SR-B1 mRNA levels in the treated microglia, whereas Aß(1-42) induced a delayed but persistent increase in the mRNA expression of CD36 and SR-B1. These results suggest a possible involvement of CD36 and SR-B1 in microglial interaction with amyloidogenic fragments of beta-amyloid and prion proteins.

IFN-γ promotes THP-1 cell apoptosis during early infection with Mycobacterium bovis by activating different apoptotic signaling.

Macrophage apoptosis represents an important innate defense mechanism against intracellular mycobacterial infection. Previous publications have shown that interferon-γ (IFN-γ) is involved in apoptosis of immune cells infected with mycobacteria. In this study, the impact of IFN-γ treatment on phorbol-12-myristate-13-acetate-differentiated THP-1 cells infected with Mycobacterium bovis was investigated. The results showed that IFN-γ increased apoptosis of THP-1 cells infected with M. bovis at a low multiplicity of infection (MOI) in a time-dependent manner. The percentage of cells undergoing apoptosis in IFN-γ-treated THP-1 cells increased from 4.3% at 12 h to 36.5% at 72 h upon infection with an MOI of 10. Activation of caspases-3 and -8 increased 8.3- and 6.7-fold, respectively. Neutralizing endogenous tumor necrosis factor-α (TNF-α) significantly inhibited IFN-γ-induced apoptosis of M. bovis-infected THP-1 cells. No significant change in IFN-γ-induced apoptosis was observed in M. bovis-infected cells after the addition of c-Jun N-terminal kinase and NF-κB pathways' inhibitors. Translocation of apoptosis-inducing factor (AIF) to the nucleus of M. bovis-infected THP-1 cells was observed in 23.4% of IFN-γ-treated cells, compared with 11.0% in untreated cells. Taken together, these results suggest that IFN-γ promotes apoptosis of M. bovis-infected THP-1 cells during early infection through the TNF-α-mediated death receptor and the AIF apoptotic pathway.