IL-1β Production In Macrophages Research Articles

Abstract 142: Rac2 Modulates Macrophage IL-1β Production, Atherosclerotic Plaque Inflammation, and Calcific Atherosclerosis

Ischemic heart disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaques is a well-described pathologic finding that has predictive value in terms of atherosclerotic burden, cardiovascular event risk, and mortality. Inflammation is critical to atherogenesis, but inflammatory mechanisms that drive atherosclerotic calcification are minimally understood. The Rho/Rac family of low molecular weight GTPases regulates the activation, recruitment, and differentiation of monocytes. Racs compete for similar regulatory elements, indicating that targeted modulation of specific family members may influence the role of other members in biologic processes. The hypothesis is that Rac2 can modulate atherosclerotic plaque inflammation and calcium composition via consequent effects on the activation of Rac1. Rac2 gene deletion results in elevated activation (GTP-binding) of Rac1 in macrophages. Increased activation of Rac1 is associated with higher macrophage IL-1β production in response to LPS-coupled cholesterol crystal exposure. In fact, macrophage IL-1β production is dependent on Rac1. When subjected to a high fat diet for 14 weeks, Rac2 -/-ApoE -/- mice demonstrate similar weight gain, serum cholesterol levels, circulating inflammatory monocyte levels, and aortic atherosclerotic lipid burden relative to Rac2 +/+ApoE -/- mice. Atherosclerotic plaques from Rac2 -/-ApoE -/- mice contain increased inflammatory cellular infiltration by histology and relative RNA expression. Ex vivo microCT imaging reveals calcified aortic plaques, and quantification of calcification by near-infrared imaging demonstrates increased calcium composition in plaques from Rac2 -/-ApoE -/- mice relative to Rac2 +/+ApoE -/- mice. Calcified aortic plaques are associated with an increase in IL-1β mRNA expression in the aortic tissue. In summary, Rac2 is a critical modulator of signaling mechanisms that regulate the activation state of Rac1, resulting in enhanced macrophage IL-1β expression in vitro and inflammatory calcification of atherosclerotic plaques in vivo. Targeting the balance of Rac regulation has therapeutic potential in modulation of atherosclerotic plaque composition.

Omega-3 free fatty acids suppress macrophage inflammasome activation by inhibiting NF-κB activation and enhancing autophagy.

The omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR) 4 (also known as GPR120), a G-protein coupled receptor (GPR) known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

Mo1716 Dual Regulation of Macrophage IL-1β Production by TRIF Signaling: Implication of Host Inflammatory Response to Commensal Bacteria

Mo1716 Dual Regulation of Macrophage IL-1β Production by TRIF Signaling: Implication of Host Inflammatory Response to Commensal Bacteria

Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP(L) is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1β production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP(L) interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP(L) in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.

284: Caspase-8-independent role of c-FLIP in Inflammasome Activation

Cellular FLICE inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage development. Recent studies reveal a selective role of caspase-8 in non-canonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Lipopolysaccharides (LPS)-, R848-, and heat-killed Listeria monocytogenes (HKLM)-induced IL-1β production in macrophages was attenuated in the absence of c-FLIP. Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP-deficient cells. In contrast, the production of TNF- α was not affected by deficiency in c-FLIP. c-FLIP interacted with NLRP3 or procaspase-1. Proximity ligation assay revealed that c-FLIP is required for the full NLRP3 inflammasome assembly, and c-FLIP associates with NLRP3 inflammasome components in situ . c-FLIP-deficiency also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic- or FasL-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a caspase-8-independent role of c-FLIP in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes. The aim of this study was to investigate how T-helper (Th) 17 cells and interleukin (IL) −17 contribute to root resorption during orthodontic tooth movement. Experiment 1: Rheumatoid arthritis (RA) model mice and wild type mice were subjected to the orthodontic force (OF) to induce root resorption (heavy OF) of the upper first molars. The double-immunofluorescence analysis for IL-17/CD4 was performed to detect Th17 cells in the periodontal ligament (PDL). Experiment 2: Atopic dermatitis (AD) model mice and wild type mice were subjected to the heavy OF to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and receptor activator of nuclear factor kappaB ligand (RANKL) proteins were determined in the PDL by an immunohistochemical analysis. Moreover, the fluorescent localization immunoassay was performed to detect Th17 cells. Furthermore, the effects of the compression force on co-cultures of CD4 + cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin (OPG). Results: Experiment 1: The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. Experiment 2: The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double-immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6 and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. Th17-producing IL-17 may be associated with the deterioration of orthodontic root resorption.

Increasing both CoCrMo‐alloy particle size and surface irregularity induces increased macrophage inflammasome activation in vitro potentially through lysosomal destabilization mechanisms

Recent investigations indicate that innate immune "danger-signaling" pathways mediate metal implant debris induced-inflammatory responses, for example, NALP3 inflammasome. How the physical characteristics of particles (size, shape, and chemical composition) affect this inflammatory reactivity remains controversial. We examined the role of Cobalt-Chromium-Molybdenum (CoCrMo) alloy particle shape and size on human macrophage phagocytosis, lysosomal destabilization, and inflammasome activation. Round/smooth versus irregularly shaped/rough CoCrMo-alloy particles of ∼1 and 6-7 µm diameter were investigated for differential lysosomal damage and inflammasome activation in human monocytes/macrophages. While spherical/smooth 1 µm CoCrMo-alloy particles did not measurably affect macrophage IL-1β production, irregular 1 µm CoCrMo-alloy particles induced significant IL-1β increases over controls. Both round/smooth particles and irregular CoCrMo-alloy particles that were 6-7 µm in size induced >10-fold increases in IL-1β production compared to similarly shaped smaller particles (p < 0.05). Larger irregular particles induced a greater degree of intracellular lysosomal damage and a >3-fold increase in IL-1β versus similarly sized round/smooth particles (at an equal dose, particles/cell). CoCrMo-alloy particle-size-induced IL-1β production was dependent on the lysosomal protease Cathepsin B, further supporting lysosomal destabilization as causative in inflammation. Phagocytosable larger/irregular shaped particles (6 µm) demonstrated the greatest lysosomal destabilization (observed immunofluorescently) and inflammatory reactivity when compared on an equal dose basis (particles/cell) to smaller/spherical 1 µm particles in vitro.

Ovarian Cancer-Derived Exosomal Fibronectin Induces Pro-Inflammatory IL-1beta

The tumour microenvironment is characterized by pro-inflammatory profiles, including interleukin-1β (IL-1β). This profile is generated by infiltrating macrophages following interactions with tumours or their components. The objectives of this study were to identify whether tumour exosomes could induce macrophage IL-1β production and the mechanism involved. Exosomes were isolated from ovarian cancer patients and ovarian tumour cells by chromatography and ultracentrifugation. Specific exosomal proteins were defined by mass spectrometry (MS) and confirmed by Western immunoblotting. Using macrophage-like THP-1 cells, induction of IL-1β release was investigated by ELISA. RGD peptides were used to block fibronectin binding by THP-1 α5β1 integrin. Exosomes isolated from ovarian cancer patients and from ovarian cancer cells were demonstrated, by MS and immunoblotting, to express fibronectin. Incubation of THP-1 cells with these exosomes induced pro-inflammatory cytokines, in particular IL-1β. Blocking of THP-1 binding of exosomal fibronectin with RGD peptides abrogated exosome-mediated IL-1β production and down-stream phosphorylation of Akt and c-Jun. Although cancer patients generally exhibit increased levels of IL-1β, the underlying mechanism is unclear. Here, tumour-derived exosomes are demonstrated to induce pro-inflammatory cytokine in macrophages including IL-1β, whose induction is mediated by fibronectin.

Activating mutations in β-catenin in colon cancer cells alter their interaction with macrophages; the role of snail.

BackgroundTumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment.Principal FindingsWe showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1β. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for β-catenin (HCT116WT/MT), harboring one wild type (WT) and one mutant (MT) allele, but isogenic lines that carry only the WT (HCT116WT) or MT β-catenin allele (HCT116MT) have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT β-catenin allele, but not in HCT116WT cells. Consistent with this observation, macrophages and IL1β failed to stabilize Snail in HCT116WT cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4) or HCT116 cells with silenced Snail failed to stimulate IL1β production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor.SignificanceOur data demonstrate that oncogenic β-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages.

Inducible microRNA-223 down-regulation promotes TLR-triggered IL-6 and IL-1β production in macrophages by targeting STAT3.

MicroRNAs are small non-coding RNA molecules that regulate gene expression by either translational inhibition or mRNA degradation. MicroRNAs play pivotal roles in the regulation of both innate and adaptive immune responses, including TLR-triggered inflammatory response. Here we reported that the expression of microRNA-223 (miR-223) was significantly decreased in murine macrophages during activation by lipopolysaccharide (LPS) or poly (I∶C) stimulation. The inducible miR-223 down-regulation resulted in the activation of signal transducer and activator of transcription 3 (STAT3), which is directly targeted by miR-223, thus promoting the production of pro-inflammatory cytokines IL-6 and IL-1β, but not TNF-α. Interestingly, IL-6 was found to be a main factor in inducing the decrease in miR-223 expression after LPS stimulation, which formed a positive feedback loop to regulate IL-6 and IL-1β. Herein, our findings provide a new explanation characterizing the molecular mechanism responsible for the regulation of IL-6 production after TLR-triggered macrophage activation.

Non-canonical inflammasome activation targets caspase-11

Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11(-/-) mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1(-/-) mice lack both caspase-11 and caspase-1. Interestingly, Casp11(-/-) macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1(-/-)Casp11(129mt/129mt) macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.

Tumor suppressor death-associated protein kinase is required for full IL-1β production

AbstractInterleukin-1β (IL-1β) is critical for inflammation and control of infection. The production of IL-1β depends on expression of pro-IL-1β and inflammasome component induced by inflammatory stimuli, followed by assembly of inflammasome to generate caspase-1 for cleavage of pro-IL-1β. Here we show that tumor suppressor death-associated protein kinase (DAPK) deficiency impaired IL-1β production in macrophages. Generation of tumor necrosis factor-α in macrophages, in contrast, was not affected by DAPK knockout. Two tiers of defects in IL-1β generation were found in DAPK-deficient macrophages: decreased pro-IL-1β induction by some stimuli and reduced caspase-1 activation by all inflammatory stimuli examined. With a normal NLRP3 induction in DAPK-deficient macrophages, the diminished caspase-1 generation is attributed to impaired inflammasome assembly. There is a direct binding of DAPK to NLRP3, suggesting an involvement of DAPK in inflammasome formation. We further illustrated that the formation of NLRP3 inflammasome in situ induced by inflammatory signals was impaired by DAPK deficiency. Taken together, our results identify DAPK as a molecule required for full production of IL-1β and functional assembly of the NLRP3 inflammasome. In addition, DAPK knockout reduced uric acid crystal-triggered peritonitis, suggesting that DAPK may serve as a target in the treatment of IL-1β-associated autoinflammatory diseases.

Uncaria rhynchophyllaInhibits the Production of Nitric Oxide and Interleukin-1β Through Blocking Nuclear Factor κB, Akt, and Mitogen-Activated Protein Kinase Activation in Macrophages

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

FoxO1 Links Insulin Resistance to Proinflammatory Cytokine IL-1β Production in Macrophages

OBJECTIVEMacrophages play an important role in the pathogenesis of insulin resistance via the production of proinflammatory cytokines. Our goal is to decipher the molecular linkage between proinflammatory cytokine production and insulin resistance in macrophages.RESEARCH DESIGN AND METHODSWe determined cytokine profiles in cultured macrophages and identified interleukin (IL)-1β gene as a potential target of FoxO1, a key transcription factor that mediates insulin action on gene expression. We studied the mechanism by which FoxO1 mediates insulin-dependent regulation of IL-1β expression in cultured macrophages and correlated FoxO1 activity in peritoneal macrophages with IL-1β production profiles in mice with low-grade inflammation or insulin resistance.RESULTSFoxO1 selectively promoted IL-1β production in cultured macrophages. This effect correlated with the ability of FoxO1 to bind and enhance IL-1β promoter activity. Mutations of the FoxO1 binding site within the IL-1β promoter abolished FoxO1 induction of IL-1β expression. Macrophages from insulin-resistant obese db/db mice or lipopolysaccharide-inflicted mice were associated with increased FoxO1 production, correlating with elevated levels of IL-1β mRNA in macrophages and IL-1β protein in plasma. In nonstimulated macrophages, FoxO1 remained inert with benign effects on IL-1β expression. In response to inflammatory stimuli, FoxO1 activity was augmented because of an impaired ability of insulin to phosphorylate FoxO1 and promote its nuclear exclusion. This effect along with nuclear factor-κB acted to stimulate IL-1β production in activated macrophages.CONCLUSIONSFoxO1 signaling through nuclear factor-κB plays an important role in coupling proinflammatory cytokine production to insulin resistance in obesity and diabetes.

Chlorophyllin suppresses interleukin-1 beta expression in lipopolysaccharide-activated RAW 264.7 cells

Our previous findings demonstrated that chlorophyllin (CHL) inhibits inducible nitric oxide gene expression in macrophages. In the present study, we show that CHL inhibited IL-1β production and its mRNA expression in a lipopolysaccharide (LPS)-stimulated murine macrophage cell-line, RAW 264.7. The inhibitory effect of CHL on IL-1β gene expression was further supported by an in vitro transfection assay using a pIL-1(870 bp)-CAT construct, where CHL inhibited the activation of the IL-1β promoter. Furthermore, CHL attenuated the activation of NF-κB, NF-IL6 and AP-1, which are known to be responsible for IL-1β gene expression, as determined by an electrophoretic mobility shift assay and an in vitro transfection assay using p(NF-κB)3-CAT, p(NF-IL6)3-CAT, and p(AP-1)3-CAT, respectively. However, it was evident that the inhibitory activity of CHL on IL-1β expression in the LPS-stimulated macrophages was independent of CRE/ATF. The immunoblot experiment demonstrated that CHL also caused a substantial decrease in the phosphorylation of p38 MAP kinase in LPS-stimulated RAW 264.7. These results suggest that CHL inhibits IL-1β production in macrophages stimulated with LPS at transcriptional level by blocking the phosphorylation of p38 and by suppressing the activation of transcription factors, NF-κB, NF-IL6, and AP-1.