Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1ß (IL-1ß) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1ß secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1ß secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1ß secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1ß secretion through the NLRP3 inflammasome.

Cyclooxygenase-2 regulates NLRP3 inflammasome-derived IL-1ß production.

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a reactive oxygen species-sensitive multiprotein complex that regulates IL-1ß maturation via caspase-1. It also plays an important role in the pathogenesis of inflammation-related disease. Cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli and contributes to the pathogenesis of inflammation-related diseases. However, there is currently little known about the relationship between COX-2 and the NLRP3 inflammasome. Here, we describe a novel role for COX-2 in regulating the activation of the NLRP3 inflammasome. NLRP3 inflammasome-derived IL-1ß secretion and pyroptosis in macrophages were reduced by pharmaceutical inhibition or genetic knockdown of COX-2. COX-2 catalyzes the synthesis of prostaglandin E2 and increases IL-1ß secretion. Conversely, pharmaceutical inhibition or genetic knockdown of prostaglandin E2 receptor 3 reduced IL-1ß secretion. The underlying mechanisms for the COX-2-mediated increase in NLRP3 inflammasome activation were determined to be the following: (1) enhancement of lipopolysaccharide-induced proIL-1ß and NLRP3 expression by increasing NF-κB activation and (2) enhancement of the caspase-1 activation by increasing damaged mitochondria, mitochondrial reactive oxygen species production and release of mitochondrial DNA into cytosol. Furthermore, inhibition of COX-2 in mice in vivo with celecoxib reduced serum levels of IL-1ß and caspase-1 activity in the spleen and liver in response to lipopolysaccharide (LPS) challenge. These findings provide new insights into how COX-2 regulates the activation of the NLRP3 inflammasome and suggest that it may be a new potential therapeutic target in NLRP3 inflammasome-related diseases.

Polyenylpyrrole derivatives inhibit NLRP3 inflammasome activation and inflammatory mediator expression by reducing reactive oxygen species production and mitogen-activated protein kinase activation.

Two polyenylpyrroles from a soil ascomycete Gymnoascus reessii were previously identified as hit compounds in screening for cytotoxicity against lung cancer cells. These compounds and various analogs, which have been previously synthesized and tested for anti-lung cancer cell activity, were tested for anti-inflammatory activity. After preliminary screening for cytotoxicity for RAW 264.7 murine macrophage cells, the non-toxic compounds were tested for anti-inflammatory activity using lipopolysaccharide (LPS)-activated RAW 264.7 cells. Compounds 1h, 1i, and 1n reduced LPS-induced nitric oxide (NO) production, with respective ED50 values of 15 ± 2, 16 ± 2, and 17 ± 2 µM. They also reduced expression of inducible NO synthase and interleukin-6 (IL-6) without affecting cyclooxygenase-2 expression. Compound 1h also reduced secretion of IL-6 and tumor necrosis factor-α by LPS-activated J774A.1 murine macrophage cells, primary mice peritoneal macrophages, and JAWSII murine bone marrow-derived dendritic cells and reduced NLRP3 inflammasome-mediated interleukin-1ß (IL-1ß) secretion by LPS + adenosine triphosphate-activated J774A.1 and JAWSII cells. The underlying mechanisms for the anti-inflammatory activity of compound 1h were found to be a decrease in LPS-induced reactive oxygen species (ROS) production, mitogen-activated protein kinase phosphorylation, and NF-κB activation and a decrease in ATP-induced ROS production and PKC-α phosphorylation. These results provide promising insights into the anti-inflammatory activity of these conjugated polyenes and a molecular rationale for future therapeutic intervention in inflammation-related diseases. They also show how compound 1h regulates inflammation and suggest it may be a new source for the development of anti-inflammatory agents to ameliorate inflammation- and NLRP3 inflammasome-related diseases.

Lipopolysaccharide/adenosine triphosphate-mediated signal transduction in the regulation of NLRP3 protein expression and caspase-1-mediated interleukin-1ß secretion.

OBJECTIVE: Reactive oxygen species (ROS) plays a critical role in the regulation of NLRP3 inflammasome activation. However, the ROS-mediated signaling pathways controlling NLRP3 inflammasome activation are not well defined. METHODS: Using lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated murine macrophages as the testing model, cytokine release and protein expression were quantified by enzyme-linked immunosorbent assay and Western blot, respectively. ROS was scavenged by N-acetyl cysteine; NADPH oxidase, the major source of ROS, was inhibited by diphenyliodonium, apocynin or gp91-phox siRNA transfection; and protein kinase was inhibited by its specific inhibitor. RESULTS: LPS-induced NLRP3 protein expression was regulated through the NADPH oxidase/ROS/NF-κB-dependent, JAK2/PI3-kinase/AKT/NF-κB-dependent, and MAPK-dependent pathways, while ATP-induced caspase-1 activation was regulated through the NADPH oxidase/ROS-dependent pathway. CONCLUSIONS: These results demonstrate that ROS regulates not only the priming stage, but also the activation stage, of NLRP3 inflammasome activation in LPS + ATP-activated macrophages.

Structure and immunological characterization of the capsular polysaccharide of a pyrogenic liver abscess caused by Klebsiella pneumoniae: activation of macrophages through Toll-like receptor 4.

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-ß-D-Glc-(1→4)-[2,3-(S)-pyruvate]-ß-D-GlcA-(1→4)-α-L-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C(2)-OH or C(3)-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.

Composition, antioxidant and antimicrobial activities of the seed essential oil of Calocedrus formosana from Taiwan.

The hydrodistillated seed essential oil of Calocedrus formosana was analyzed to determine its composition and yield. Twenty-seven compounds were identified, the main ones being alpha-pinene (63.8%), totarol (9.9%) and ferruginol (8.9%). Monoterpene hydrocarbons (73.5%) and oxygenated diterpenes (18.8%) were the predominant groups of compounds. The seed essential oil exhibited excellent antioxidant, antimicrobial and anti-wood-decay fungal activities.

Composition and antimicrobial activities of the leaf essential oil of Machilus kusanoi from Taiwan.

The hydrodistillated leaf essential oil of Machilus kusanoi was analyzed to determine its composition and yield. Twenty-three compounds were identified, the main components being beta-caryophyllene (23.3%), beta-eudesmol (17.1%), alpha-terpineol (16.0%), n-dodecanal (14.2%), and n-decanal (10.4%). Sesquiterpene hydrocarbons (28.1%) and non-terpenoids (25.0%) were the predominant groups of compounds. The leaf oil exhibited excellent antimicrobial and anti-wood-decay fungal activities.

Composition and antimicrobial activity of the leaf and twig oils of Litsea mushaensis and L. linii from Taiwan.

The hydrodistilled essential oils of the leaves and twigs of Litsea mushaensis and L. linii were analyzed. Sixty-nine and ninety compounds were identified in the leaf and twig oils, respectively, of L. mushaensis. The main components of the leaf oil were beta-eudesmol (24.2%), tau-cadinol (10.2%), alpha-humulene (10.1%), alpha-pinene (9.7%), and trans-beta-ocimene (6.5%), whereas the main components of the twig oil were trans-beta-ocimene (19.5%), alpha-pinene (12.8%) and cis-beta-ocimene (7.7%). With L. linii, 72 and 78 compounds were respectively identified in the leaf and twig oils. The main components of the leaf oil were beta-selinene (15.7%), alpha-selinene (15.5%), beta-caryophyllene (12.2%), alpha-humulene (7.2%), and delta-cadinene (5.6%), and of the twig oil trans-beta-ocimene (20.8%), beta-selinene (11.4%), alpha-cadinol (6.0%), delta-cadinene (5.8%), tau-cadinol (5.4%) and beta-eudesmol (5.2%). L. mushaensis leaf oil was shown to have excellent antimicrobial and anti-wood-decay fungal activity, superior to the other oils.