Jargalsaikhan Dagvadorj

Oxidized Mitochondrial DNA Activates the NLRP3 Inflammasome during Apoptosis

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

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.

Ogg1-Dependent DNA Repair Regulates NLRP3 Inflammasome and Prevents Atherosclerosis

RATIONALEnActivation of NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome-mediating interleukin (IL)-1β 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.nnnOBJECTIVEnTo test the role of OGG1 in the development of atherosclerosis in mouse.nnnMETHODS AND RESULTSnWe 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-1β 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-1β 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-1β 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.nnnCONCLUSIONSnOGG1 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.

The NLRP3 Inflammasome Is Required for the Development of Hypoxemia in LPS/Mechanical Ventilation Acute Lung Injury

IL-1β is a potent proinflammatory cytokine that is implicated in the pathogenesis of acute respiratory distress syndrome. We hypothesized that LPS and mechanical ventilation (MV) together could lead to IL-1β secretion and the development of acute lung injury (ALI), and that this process would be dependent on caspase-1 and the nucleotide binding domain and leucine-rich repeat (NLR) pyrin domain containing 3 (NLRP3) inflammasome activation. The objectives of this study were to determine the specific role of IL-1β, caspase-1, and the NLRP3 inflammasome in a two-hit model of ALI due to LPS plus MV. We used a two-hit murine model of ALI in which both inhaled LPS and MV were required for the development of hypoxemia, pulmonary neutrophil infiltration, and alveolar leakage. Nlrp3-deficent and Casp1-deficient mice had significantly diminished IL-1β levels in bronchoalveolar lavage fluid, and were specifically protected from hypoxemia, despite similar alveolar neutrophil infiltration and leakage. The IL-1 receptor antagonist, Anakinra, significantly improved the specific development of hypoxemia without significant effects on neutrophil infiltration or alveolar leakage. MV resulted in increased bronchoalveolar lavage extracellular ATP and alveolar macrophage apoptosis as triggers of NLRP3 inflammasome activation. NLRP3 inflammasome activation and IL-1β production play a key role in ALI caused by the combination of LPS and MV, particularly in the hypoxemia associated with acute respiratory distress syndrome. Blocking IL-1 signaling in this model specifically ameliorates hypoxemia, without affecting neutrophil infiltration and alveolar leakage, disassociating these readouts of ALI. MV causes alveolar macrophage apoptosis, a key step in the activation of NLRP3 inflammasome and production of IL-1β.

Myeloid Differentiation Factor-2 Interacts with Lyn Kinase and Is Tyrosine Phosphorylated Following Lipopolysaccharide-Induced Activation of the TLR4 Signaling Pathway

Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrated that myeloid differentiation factor-2 (MD-2) is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific; it is blocked by the tyrosine kinase inhibitor, herbimycin A, as well as by an inhibitor of endocytosis, cytochalasin D, suggesting that MD-2 phosphorylation occurs during trafficking of MD-2 and not on the cell surface. Furthermore, we identified two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine had reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD-2 coprecipitated and colocalized with Lyn kinase, most likely in the endoplasmic reticulum. A Lyn-binding peptide inhibitor abolished MD-2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phosphorylation. Our study demonstrated that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response.

IL-1 Signaling Is Critically Required in Stromal Cells in Kawasaki Disease Vasculitis Mouse Model

Objective—Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1&bgr; signaling are required for the Lactobacillus casei cell wall extract–induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c+ dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract–induced KD mouse model of vasculitis. Approach and Results—Using mouse knockout models and antibody depletion, we found that both IL-1&agr; and IL-1&bgr; were required for Lactobacillus casei cell wall extract–induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells. Conclusions—Our results suggest that IL-1&agr; and IL-1&bgr;, as well as CD11c+ dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1&agr; and IL-1&bgr; and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

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.

IL-1 Signaling Is Critically Required in Stromal Cells in Kawasaki Disease Vasculitis Mouse Model Role of Both IL-1α and IL-1β

Objective—Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1&bgr; signaling are required for the Lactobacillus casei cell wall extract–induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c+ dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract–induced KD mouse model of vasculitis. Approach and Results—Using mouse knockout models and antibody depletion, we found that both IL-1&agr; and IL-1&bgr; were required for Lactobacillus casei cell wall extract–induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells. Conclusions—Our results suggest that IL-1&agr; and IL-1&bgr;, as well as CD11c+ dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1&agr; and IL-1&bgr; and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

TGF-β1 inhibits the production of IFN in response to CpG DNA via ubiquitination of TNF receptor-associated factor (TRAF) 6.

The effect of TGF-β1 on CpG DNA-induced type I IFN production was examined by reconstituting a series of signaling molecules in TLR 3 signaling. TGF-β1 inhibited CpG DNA-induced IFN-α4 productivity in HeLa cells. Transfection of IFN regulatory factor (IRF)7 but not TNF receptor-associated factor (TRAF)6 and TRAF3 into cells triggered IFN-α4 productivity, and TGF-β1 inhibited IRF7-mediated type I IFN production in the presence of TRAF6. TGF-β1 induced ubiquitination of TRAF6, although CpG DNA did not induce it. Moreover, TGF-β1 accelerated the ubiquitination of TRAF6 in the presence of CpG DNA. TGF-β1 ubiquitinated TRAF6 at K63 but not K48. TGF-β1 also induced ubiquitination of IRF7. Further, TGF-β1 did not impair the interaction of IRF7 and TRAF6. CpG DNA induced the phosphorylation of IRF7 in the presence of TRAF6, whereas TGF-β1 inhibited the IRF7 phosphorylation. Blocking of TRAF6 ubiquitination abolished the inhibition of CpG DNA-induced type I IFN production by TGF-β. Taken together, TGF-β was suggested to inhibit CpG DNA-induced type I IFN production transcriptionally via ubiquitination of TRAF6.

Chlamydia pneumoniae Hijacks a Host Autoregulatory IL-1β Loop to Drive Foam Cell Formation and Accelerate Atherosclerosis

Pathogen burden accelerates atherosclerosis, but the mechanisms remain unresolved. Activation of the NLRP3 inflammasome is linked to atherogenesis. Here we investigated whether Chlamydia pneumoniae (C.pn) infection engages NLRP3 in promoting atherosclerosis. C.pn potentiated hyperlipidemia-induced inflammasome activity in cultured macrophages and in foam cells in atherosclerotic lesions of Ldlr-/- mice. C.pn-induced acceleration of atherosclerosis was significantly dependent on NLRP3 and caspase-1. We discovered that C.pn-induced extracellular IL-1β triggers a negative feedback loop to inhibit GPR109a and ABCA1 expression and cholesterol efflux, leading to accumulation of intracellular cholesterol and foam cell formation. Gpr109a and Abca1 were both upregulated in plaque lesions inxa0Nlrp3-/- mice in both hyperlipidemic and C.pn infection models. Mature IL-1β and cholesterol mayxa0compete for access to the ABCA1 transporter to be exported from macrophages. C.pn exploits this metabolic-immune crosstalk, which can be modulated by NLRP3 inhibitors to alleviate atherosclerosis.