Lipopolysaccharide Induces Alveolar Macrophage Necrosis via CD14 and the P2X7 Receptor Leading to Interleukin-1α Release.

Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca(2+) influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration.

Recruitment of pro-IL-1α to mitochondrial cardiolipin, via shared LC3 binding domain, inhibits mitophagy and drives maximal NLRP3 activation.

The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1ß release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1ß production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.

Innate Immune Dysregulation in the Development of Cardiovascular Disease in Lupus.

PURPOSE OF REVIEW: The systemic inflammatory nature of systemic lupus erythematosus (SLE) is patent not only in the diverse clinical manifestations of the disease but also in the increased risk of premature cardiovascular diseases (CVD). In this review, we discuss the latest findings on the key factors of the innate immune system known to play critical roles in the pathogenesis of accelerated CVD in patients with SLE and discuss the potential that immunometabolism may play a key role in this respect. RECENT FINDINGS: Recent studies exploring the association between SLE and premature CVD clearly showed that alterations of specific immune functions play a pivotal role in the increased cardiovascular morbidity and mortality in the SLE patients. Novel molecular factors such as type I interferons (IFN), dysregulated neutrophil function, and changes to cellular metabolism and metabolites are emerging as important regulators of systemic immune dysfunction and as strong risk factors for premature CVD in SLE. Although corticosteroids and cytotoxic agents can be used to effectively manage and control various lupus-related complications, to date, no drug has been proven to prevent the development of premature atherosclerosis in SLE. However, as new mechanisms underlying this complication of SLE are uncovered, such as the role of metabolism and neutrophil-driven inflammation, new avenues for therapeutic intervention are being discovered.

Ogg1-Dependent DNA Repair Regulates NLRP3 Inflammasome and Prevents Atherosclerosis.

RATIONALE: Activation of NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome-mediating interleukin (IL)-1ß secretion has emerged as an important component of inflammatory processes in atherosclerosis. Mitochondrial DNA (mtDNA) damage is detrimental in atherosclerosis, and mitochondria are central regulators of the nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3 inflammasome. Human atherosclerotic plaques express increased mtDNA damage. The major DNA glycosylase, 8-oxoguanine glycosylase (OGG1), is responsible for removing the most abundant form of oxidative DNA damage. OBJECTIVE: To test the role of OGG1 in the development of atherosclerosis in mouse. METHODS AND RESULTS: We observed that Ogg1 expression decreases over time in atherosclerotic lesion macrophages of low-density lipoprotein receptor (Ldlr) knockout mice fed a Western diet. Ogg1(-/-)Ldlr(-/-) mice fed a Western diet resulted in an increase in plaque size and lipid content. We found increased oxidized mtDNA, inflammasome activation, and apoptosis in atherosclerotic lesions and also higher serum IL-1ß and IL-18 in Ogg1(-/-)Ldlr(-/-) mice than in Ldlr(-/-). Transplantation with Ogg1(-/-) bone marrow into Ldlr(-/-) mice led to larger atherosclerotic lesions and increased IL-1ß production. However, transplantation of Ogg1(-/-)Nlrp3(-/-) bone marrow reversed the Ogg1(-/-) phenotype of increased plaque size. Ogg1(-/-) macrophages showed increased oxidized mtDNA and had greater amounts of cytosolic mtDNA and cytochrome c, increased apoptosis, and more IL-1ß secretion. Finally, we found that proatherogenic miR-33 can directly inhibit human OGG1 expression and indirectly suppress both mouse and human OGG1 via AMP-activated protein kinase. CONCLUSIONS: OGG1 plays a protective role in atherogenesis by preventing excessive inflammasome activation. Our study provides insight into a new target for therapeutic intervention based on a link between oxidative mtDNA damage, OGG1, and atherosclerosis via NLRP3 inflammasome.

Alternatively spliced myeloid differentiation protein-2 inhibits TLR4-mediated lung inflammation.

We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. In this study, we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intratracheally an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid. Compared to adenovirus serotype 5 containing an empty vector lacking a transgene control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cytokines and chemokines, such as IL-6, keratinocyte chemoattractant, and MIP-2. Bronchoalveolar lavage fluid from Ad-MD-2s mice transferred into lungs of naive mice before intratracheal LPS challenge diminished proinflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on HDM-induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and HDM-triggered allergic lung inflammation.

α-Ketoglutarate-Dependent KDM6 Histone Demethylases and Interferon-Stimulated Gene Expression in Lupus.

OBJECTIVE: We aimed to investigate the hypothesis that interferon (IFN)-stimulated gene (ISG) expression in systemic lupus erythematosus (SLE) monocytes is linked to changes in metabolic reprogramming and epigenetic regulation of ISG expression. METHODS: Monocytes from healthy volunteers and patients with SLE at baseline or following IFNα treatment were analyzed by extracellular flux analysis, proteomics, metabolomics, chromatin immunoprecipitation, and gene expression. The histone demethylases KDM6A/B were inhibited using glycogen synthase kinase J4 (GSK-J4). GSK-J4 was tested in pristane and resiquimod (R848) models of IFN-driven SLE. RESULTS: SLE monocytes had enhanced rates of glycolysis and oxidative phosphorylation compared to healthy control monocytes, as well as increased levels of isocitrate dehydrogenase and its product, α-ketoglutarate (α-KG). Because α-KG is a required cofactor for histone demethylases KDM6A and KDM6B, we hypothesized that IFNα may be driving "trained immune" responses through altering histone methylation. IFNα priming (day 1) resulted in a sustained increase in the expression of ISGs in primed cells (day 5) and enhanced expression on restimulation with IFNα. Importantly, decreased H3K27 trimethylation was observed at the promoters of ISGs following IFNα priming. Finally, GSK-J4 (KDM6A/B inhibitor) resulted in decreased ISG expression in SLE patient monocytes, as well as reduced autoantibody production, ISG expression, and kidney pathology in R848-treated BALB/c mice. CONCLUSION: Our study suggests long-term IFNα exposure alters the epigenetic regulation of ISG expression in SLE monocytes via changes in immunometabolism, a mechanism reflecting trained immunity to type I IFN. Importantly, it opens the possibility that targeting histone-modifying enzymes, such as KDM6A/B, may reduce IFN responses in SLE.

eNAMPT/TLR4 inflammatory cascade activation is a key contributor to SLE Lung vasculitis and alveolar hemorrhage.

Rationale: Effective therapies to reduce the severity and high mortality of pulmonary vasculitis and diffuse alveolar hemorrhage (DAH) in patients with systemic lupus erythematosus (SLE) is a serious unmet need. We explored whether biologic neutralization of eNAMPT (extracellular nicotinamide phosphoribosyl-transferase), a novel DAMP and Toll-like receptor 4 ligand, represents a viable therapeutic strategy in lupus vasculitis. Methods: Serum was collected from SLE subjects (n = 37) for eNAMPT protein measurements. In the preclinical pristane-induced murine model of lung vasculitis/hemorrhage, C57BL/6 J mice (n = 5-10/group) were treated with PBS, IgG (1 mg/kg), or the eNAMPT-neutralizing ALT-100 mAb (1 mg/kg, IP or subcutaneously (SQ). Lung injury evaluation (Day 10) included histology/immuno-histochemistry, BAL protein/cellularity, tissue biochemistry, RNA sequencing, and plasma biomarker assessment. Results: SLE subjects showed highly significant increases in blood NAMPT mRNA expression and eNAMPT protein levels compared to healthy controls. Preclinical pristane-exposed mice studies showed significantly increased NAMPT lung tissue expression and increased plasma eNAMPT levels accompanied by marked increases in alveolar hemorrhage and lung inflammation (BAL protein, PMNs, activated monocytes). In contrast, ALT-100 mAb-treated mice showed significant attenuation of inflammatory lung injury, alveolar hemorrhage, BAL protein, tissue leukocytes, and plasma inflammatory cytokines (eNAMPT, IL-6, IL-8). Lung RNA sequencing showed pristane-induced activation of inflammatory genes/pathways including NFkB, cytokine/chemokine, IL-1ß, and MMP signaling pathways, each rectified in ALT-100 mAb-treated mice. Conclusions: These findings highlight the role of eNAMPT/TLR4-mediated inflammatory signaling in the pathobiology of SLE pulmonary vasculitis and alveolar hemorrhage. Biologic neutralization of this novel DAMP appears to serve as a viable strategy to reduce the severity of SLE lung vasculitis.

Neutrophils Contribute to ER Stress in Lung Epithelial Cells in the Pristane-Induced Diffuse Alveolar Hemorrhage Mouse Model.

Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH.

Tumor necrosis factor-a augments lipopolysaccharide-induced suppressor of cytokine signalling 3 (SOCS-3) protein expression by preventing the degradation.

The regulatory role of tumour necrosis factor-a (TNF-a) on the expression of suppressor of cytokine signalling 3 (SOCS-3) in response to lipopolysaccharide (LPS) was examined using peritoneal macrophages from TNF-a-deficient mice. The LPS-induced SOCS-3 expression was markedly augmented in macrophages from wild-type mice whereas such augmentation was not seen in the cells from TNF-a-deficient mice. However, there was no significant difference in the level of SOCS-3 messenger RNA expression between macrophages from wild-type mice and those from TNF-a-deficient mice. The addition of exogenous TNF-a augmented the LPS-induced SOCS-3 expression in macrophages from TNF-a-deficient mice. The pulse chase analysis suggested augmented degradation of LPS-induced SOCS-3 protein in macrophages from TNF-a-deficient mice. Moreover, MG 132, a 26S proteasome inhibitor, sustained the LPS-induced SOCS-3 expression in those cells. The tyrosine phosphorylation of SOCS-3 was definitely induced in LPS-stimulated macrophages from TNF-a-deficient mice but not wild-type mice. A tyrosine phosphatase inhibitor enhanced the tyrosine phosphorylation of SOCS-3 in wild-type mice and accelerated the degradation. Therefore, it was suggested that TNF-a prevented the degradation of SOCS-3 protein via inhibition of the tyrosine phosphorylation in LPS-stimulated macrophages.

Seladin-1 is a novel lipopolysaccharide (LPS)-responsive gene and inhibits the tumour necrosis factor-alpha production and osteoclast formation in response to LPS.

Selective Alzheimer disease indicator-1 (seladin-1) is a broadly expressed oxidoreductase and is related to Alzheimer disease, cholesterol metabolism and carcinogenesis. The effect of lipopolysaccharide (LPS) on the expression of seladin-1 was examined using RAW 264.7 macrophage-like cells and murine peritoneal macrophages. Lipopolysaccharide induced the expression of seladin-1 protein and messenger RNA in those macrophages. The seladin-1 expression was also augmented by a series of Toll-like receptor ligands. The LPS augmented the expression of seladin-1 via reactive oxygen species generation and p38 activation. Seladin-1 inhibited LPS-induced activation of p38 but not nuclear factor-kappaB and inhibited the production of tumour necrosis factor-alpha in response to LPS. Moreover, seladin-1 inhibited LPS-induced osteoclast formation and enhanced LPS-induced alkaline phosphatase activity. Therefore, it was suggested that seladin-1 might be an LPS-responsible gene product and regulate the LPS-induced inflammatory response negatively.

Retinoblastoma protein-interacting zinc finger 1, a tumor suppressor, augments lipopolysaccharide-induced proinflammatory cytokine production via enhancing nuclear factor-kappaB activation.

The involvement of retinoblastoma protein-interacting zinc finger 1 (RIZ1), a tumor suppressor, in lipopolysaccharide (LPS)-induced inflammatory responses was investigated by using RAW 264.7 macrophage-like cells. LPS significantly augmented the expression of RIZ1 and the augmentation was mediated by the activation of nuclear factor (NF)-kappaB and Akt. The silencing of RIZ1 with the siRNA led to the inactivation of NF-kappaB in response to LPS. Moreover, the RIZ1 silencing caused the down-regulation of p53 activation and a p53 pharmacological inhibitor attenuated the RIZ1 expression. LPS-induced tumor necrosis factor-alpha and interleukin-6 production was prevented by RIZ1 siRNA or a p53 pharmacological inhibitor. Therefore, RIZ1 was suggested to augment LPS-induced NF-kappaB activation in collaboration with p53 and enhance the production of proinflammatory cytokines in response to LPS.

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands.

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5(+) IgM(+) WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-kappaB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.

Retinoblastoma protein-interacting zinc finger 1 (RIZ1) regulates the proliferation of monocytic leukemia cells via activation of p53.

The role of retinoblastoma protein-interacting zinc finger 1 (RIZ1) on the cell growth of mouse and human monocytic leukemia cells was examined. RIZ1 expression was induced in response to tumor necrosis factor (TNF)-α. The expression was dependent on the nuclear factor-κB and AKT signaling. Further, RIZ1 expression led to the augmentation of p53 expression and the silencing of RIZ1 prevented it. On the other hand, a p53 inhibitor enhanced the TNF-α-induced RIZ1 expression. Silencing of RIZ1 augmented the proliferative activity of TNF-α-treated cells. Therefore, it is suggested that RIZ1 negatively regulated the cell proliferation of monocytic leukemia cells via activation of p53.

Oxidative DNA Damage Accelerates Skin Inflammation in Pristane-Induced Lupus Model.

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease in which type I interferons (IFN) play a key role. The IFN response can be triggered when oxidized DNA engages the cytosolic DNA sensing platform cGAS-STING, but the repair mechanisms that modulate this process and govern disease progression are unclear. To gain insight into this biology, we interrogated the role of oxyguanine glycosylase 1 (OGG1), which repairs oxidized guanine 8-Oxo-2'-deoxyguanosine (8-OH-dG), in the pristane-induced mouse model of SLE. Ogg1-/- mice showed increased influx of Ly6Chi monocytes into the peritoneal cavity and enhanced IFN-driven gene expression in response to short-term exposure to pristane. Loss of Ogg1 was associated with increased auto-antibodies (anti-dsDNA and anti-RNP), higher total IgG, and expression of interferon stimulated genes (ISG) to longer exposure to pristane, accompanied by aggravated skin pathology such as hair loss, thicker epidermis, and increased deposition of IgG in skin lesions. Supporting a role for type I IFNs in this model, skin lesions of Ogg1-/- mice had significantly higher expression of type I IFN genes (Isg15, Irf9, and Ifnb). In keeping with loss of Ogg1 resulting in dysregulated IFN responses, enhanced basal and cGAMP-dependent Ifnb expression was observed in BMDMs from Ogg1-/- mice. Use of the STING inhibitor, H151, reduced both basal and cGAMP-driven increases, indicating that OGG1 regulates Ifnb expression through the cGAS-STING pathway. Finally, in support for a role for OGG1 in the pathology of cutaneous disease, reduced OGG1 expression in monocytes associated with skin involvement in SLE patients and the expression of OGG1 was significantly lower in lesional skin compared with non-lesional skin in patients with Discoid Lupus. Taken together, these data support an important role for OGG1 in protecting against IFN production and SLE skin disease.

Astrocyte elevated gene-1 (AEG-1) is induced by lipopolysaccharide as toll-like receptor 4 (TLR4) ligand and regulates TLR4 signalling.

Astrocyte elevated gene-1 (AEG-1) is induced by human immunodeficiency virus 1 (HIV-1) infection and involved in tumour progression, migration and invasion as a nuclear factor-kappaB (NF-kappaB) -dependent gene. The involvement of AEG-1 on lipopolysaccharide (LPS) -induced proinflammatory cytokine production was examined. AEG-1 was induced via NF-kappaB activation in LPS-stimulated U937 human promonocytic cells. AEG-1 induced by LPS subsequently regulated NF-kappaB activation. The prevention of AEG-1 expression inhibited LPS-induced tumour necrosis factor-alpha and prostaglandin E(2) production. The AEG-1 activation was not induced by toll-like receptor ligands other than LPS. Therefore, AEG-1 was suggested to be a LPS-responsive gene and involved in LPS-induced inflammatory response.

Low susceptibility of NC/Nga mice to tumor necrosis factor-alpha-mediated lethality and hepatocellular damage with D-galactosamine sensitization.

The susceptibility of NC/Nga mice to tumor necrosis factor (TNF)-alpha was examined by using sensitization with d-galactosamine (d-GalN). Administration of TNF-alpha and d-GalN killed none of the NC/Nga mice, whereas it killed all of the BALB/c mice. Treatment with TNF-alpha and d-GalN caused few hepatic lesions in NC/Nga mice but massive hepatocellular apoptosis in BALB/c mice. Unlike BALB/c mice, there was no elevation in caspase 3 and 8 activities in the livers of NC/Nga mice receiving TNF-alpha and d-GalN. On the other hand, administration of anti-Fas antibody definitely killed both NC/Nga and BALB/c mice via activation of caspases 3 and 8. Treatment with TNF-alpha and d-GalN led to translocation of nuclear factor (NF)-kappaB in NC/Nga and BALB/c mice. However, NF-kappaB translocation was sustained in NC/Nga mice, although it disappeared in BALB/c mice 7 h after the treatment. NF-kappaB inhibitors activated caspases 3 and 8, and enhanced TNF-alpha-mediated lethality in NC/Nga. Taken together, the low susceptibility of NC/Nga mice to TNF-alpha-mediated lethality was suggested to be responsible for the sustained NF-kappaB activation.

Involvement of interleukin-1 receptor-associated kinase (IRAK)-M in toll-like receptor (TLR) 7-mediated tolerance in RAW 264.7 macrophage-like cells.

The effect of toll-like receptor (TLR) 7 ligand pretreatment on the production of tumor necrosis factor (TNF)-alpha in response to TLR7 or TLR2 ligand was examined in order to establish a new TLR-mediated tolerance. RAW 264.7 macrophage-like cells were treated with imiquimod R837 as a TLR7 ligand for 18h, washed and incubated in fresh culture medium 6h. The second challenge with imiquimod R837 as a TLR7 ligand or Pam3CysSK4 as a TLR2 ligand resulted in reduced TNF-alpha production in TLR7 ligand-pretreated cells. There was impaired activation of NF-kappaB, p38 and stress-activated protein kinase (SAPK) in the tolerant cells. The expression of IRAK-M as a negative regulator of TLR signaling was markedly augmented in the tolerant cells while the interleukin-1 receptor-associated kinase (IRAK)-1 functioned normally. The involvement of IRAK-M in the TLR7-mediated tolerance is discussed.

The inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha and nitric oxide production by Clostridium perfringens alpha-toxin and its relation to alpha-toxin-induced intracellular ceramide generation.

The effect of Clostridium perfringens alpha-toxin on production of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The pretreatment of wild type alpha-toxin, but not the inactive mutant, significantly decreased LPS-induced TNF-alpha and NO production. alpha-Toxin inhibited the expression of TNF-alpha and an inducible type of NO synthase protein and mRNA. Furthermore, it inhibited the phosphorylation of IkappaB-alpha and p65 NF-kappaB subunit, and the NF-kappaB luciferase reporter gene activity in LPS-stimulated cells. The pretreatment of alpha-toxin increased the level of intracellular ceramide. Taken together, Clostridium perfringens alpha-toxin pretreatment was suggested to inhibit LPS-induced TNF-alpha and NO production through the inhibition of NF-kappaB activation. The relationship between alpha-toxin-induced intracellular ceramide generation and the NF-kappaB inhibition is discussed.

Autophagy Limits Inflammasome During Chlamydia pneumoniae Infection.

Autophagy can either antagonize or promote intracellular bacterial growth, depending on the pathogen. Here, we investigated the role of autophagy during a pulmonary infection with the obligate intracellular pathogen, Chlamydia pneumoniae (CP). In mouse embryonic fibroblasts (MEFs) or macrophages, deficiency of autophagy pathway components led to enhanced CP replication, suggesting that autophagy exerts a bactericidal role. However, in vivo, mice with myeloid-specific deletion of the autophagic protein ATG16L1 suffered increased mortality during CP infection, neutrophilia, and increased inflammasome activation despite no change in bacterial burden. Induction of autophagy led to reduced CP replication in vitro, but impaired survival in CP-infected mice, associated with an initial reduction in IL-1ß production, followed by enhanced neutrophil recruitment, defective CP clearance, and later inflammasome activation and IL-1ß production, which drove the resulting mortality. Taken together, our data suggest that a delicate interplay exists between autophagy and inflammasome activation in determining the outcome of CP infection, perturbation of which can result in inflammatory pathology or unrestricted bacterial growth.

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation.

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.