LPS-induced Inflammatory Gene Expression Research Articles

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our invitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

FRI011 Exploring The Contribution Of PARP1-mediated Site-specific ADP-Ribosylation Of C/EBPb In Macrophages To Diet-induced Obesity

Abstract Disclosure: A.L. Whitaker: None. R. Gupte: None. T.S. Nandu: None. Inflammation is an important component of the pathological effects of obesity, which are mediated in part by adipose-associated macrophages (TAMs). Signal-regulated post-translational modifications in the TAMs help modulate the transcriptional programs that mediate biological outcomes. ADP-ribosylation is a posttranslational modification that regulates various cellular processes, including macrophage activation. ADP-ribosylation is the covalent attachment of ADP-ribose units on proteins by poly(ADP-ribose) polymerase (PARP) enzymes, mainly PARP1 in the nucleus. We have shown that ADP-ribosylation of key transcription factors (e.g., C/EBPβ and STAT1α) by PARP1 regulates adipogenesis, as well as pro-inflammatory responses in macrophages. Here we aim to investigate the role of PARP1-mediated ADP-ribosylation of C/EBPβ in inflammation and obesity using monocyte-specific mouse models. We have found that knockout (KO) of PARP1 in the monocyte/macrophage lineage (i.e., using a LysM-Cre driver) results in dramatic weight gain in mice with a nearly 2-fold increase in fat body mass in animals fed a high fat diet. Further analysis revealed decreases in adipose-associated macrophages and adipose hypertrophy, as well as increased hepatic lipid accumulation. Our findings exemplify the critical role that PARP1 in macrophages plays in maintaining homeostasis in key metabolic tissues. Moreover, our results indicate that regulation by PARP1 in macrophages contributes to resistance of diet-induced obesity through tissue-collaborative mechanisms. Our ongoing experiments suggest that PARP1 regulates LPS-induced inflammatory gene expression in macrophages. PARP1 controls LPS-mediated gene expression, in part, by ADP-ribosylating two sites (Glu 135 and Glu 139) in the regulatory domain of C/EBPβ; mutation of these sites inhibits PARP1-mediated PARylation of C/EBPβ and LPS-mediated gene expression through C/EBPβ. Additionally, treatment with a PARP inhibitor decreases LPS-mediated gene expression associated with upstream C/EBPβ enhancers. Moreover, treatment with a PARP inhibitor also decreases histone H3 lysine 27 acetylation (H3K27ac) at these C/EBPβ enhancers. To investigate the physiological impact of loss of C/EBPβ ADP-ribosylation in macrophages, we have generated a knock-in mouse model for inducible monocyte-specific expression of a C/EBPβ ADP-ribosylation site mutant (CebpbE135A/E139A). This is the first physiological model for the study of site-specific ADP-ribosylation in vivo. Using this model, we are investigating the impact of site-specific ADP-ribosylation of C/EBPβ in mice fed a high fat diet. With these studies, we hope to reveal the specific roles of PARP1-mediated ADP-ribosylation in diet-induced obesity. Presentation: Friday, June 16, 2023

Glycolipid-enriched fraction of Osmanthus fragrans inhibits LPS-induced expression of inflammatory genes, COX-2, E-selectin, and Interleukin-8.

Osmanthus fragrans Lour. is a small ornamental tree native to the Southeastern parts of China. It is mainly cultivated because of its characteristic fragrance, and used in the food and perfume industry. Besides, its flowers are used in traditional Chinese medicine to treat a variety of diseases including those related to inflammation. The aim of the study was to investigate in more detail the anti-inflammatory properties of O. fragrans flowers, and to characterize their active principles and mechanisms of action. O. fragrans flowers were successively extracted with n-hexane, dichloromethane and methanol. The extracts were further fractionated by chromatographic separation. COX-2 mRNA expression in PMA-differentiated, LPS-stimulated THP-1cells was used as lead assay for activity-guided fractionation. The most potent fraction was chemically analyzed by LC-HRMS. The pharmacological activity was also evaluated in other inflammation-related in-vitro models, such as analysis of IL-8 secretion and E-selectin expression in HUVECtert cells and selective inhibition of COX-isoenzymes. n-Hexane and dichloromethane extracts of O. fragrans flowers significantly inhibited COX-2 (PTGS2) mRNA expression. Additionally, both extracts inhibited COX-2 enzyme activity, whereas COX-1 enzyme activity was affected to a significantly lower extent. Fractionation of the extracts led to a highly active, glycolipid-containing fraction. In total, 10 glycolipids were tentatively annotated by LC-HRMS. This fraction also inhibited LPS-induced COX-2 mRNA expression, IL-8 secretion and E-selectin expression. The effects were limited to LPS-induced inflammation and not observed when inflammatory genes were induced by TNF-α, IL-1β or FSL-1. Since all these inducers of inflammation act via different receptors, it is likely that the fraction interferes with the binding of LPS to the TLR4-receptor, which mediates pro-inflammatory effects of LPS. Taken together, the results demonstrate the anti-inflammatory potential of O. fragrans flower extracts in general, and of the glycolipid-enriched fraction in particular. The effects of glycolipid-enriched fraction are potentially mediated via the inhibition of the TLR4 receptor complex.

Inhibitory Effects of Ginsenoside Compound K on Lipopolysaccharide-Stimulated Inflammatory Responses in Macrophages by Regulating Sirtuin 1 and Histone Deacetylase 4

Inflammation, an innate immune response mediated by macrophages, has been a hallmark leading to the pathophysiology of diseases. In this study, we examined the inhibitory effects of ginsenoside compound K (CK) on lipopolysaccharide (LPS)-induced inflammation and metabolic alteration in RAW 264.7 macrophages by regulating sirtuin 1 (SIRT1) and histone deacetylase 4 (HDAC4). LPS suppressed SIRT1 while promoting HDAC4 expression, accompanied by increases in cellular reactive oxygen species accumulation and pro-inflammatory gene expression; however, the addition of CK elicited the opposite effects. CK ameliorated the LPS-induced increase in glycolytic genes and abrogated the LPS-altered genes engaged in the NAD+ salvage pathway. LPS decreased basal, maximal, and non-mitochondrial respiration, reducing ATP production and proton leak in macrophages, which were abolished by CK. SIRT1 inhibition augmented Hdac4 expression along with increased LPS-induced inflammatory and glycolytic gene expression, while decreasing genes that regulate mitochondrial biogenesis; however, its activation resulted in the opposite effects. Inhibition of HDAC4 enhanced Sirt1 expression and attenuated the LPS-induced inflammatory gene expression. In conclusion, CK exerted anti-inflammatory and antioxidant properties with the potential to counteract the alterations of energy metabolism, including glycolysis and mitochondrial respiration, through activating SIRT1 and repressing HDAC4 in LPS-stimulated macrophages.

Exosome-Based Delivery of Super-Repressor IκBα Alleviates Alcohol-Associated Liver Injury in Mice.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) instigates nuclear factor-κB (NF-κB)-mediated inflammatory responses in alcohol-associated liver diseases (ALD). Here, we utilized a novel optogenetically engineered exosome technology called 'exosomes for protein loading via optically reversible protein-protein interactions (EXPLOR)' to efficiently deliver the super-repressor IκB-loaded exosomes (Exo-srIκB) to the liver and examined its therapeutic potential in acute-on-chronic alcohol-associated liver injury. We detected enhanced uptake of DiI-labeled Exo-srIκB by LPS-treated inflammatory KCs, which suppressed LPS-induced inflammatory gene expression levels. In animal experiments, a single intravenous injection of Exo-srIκB prior to alcohol binge drinking significantly attenuated alcohol-associated hepatic steatosis and infiltration of neutrophils and macrophages but not a liver injury. Notably, three consecutive days of Exo-srIκB injection remarkably reduced alcohol-associated liver injury, steatosis, apoptosis of hepatocytes, fibrosis-related gene expression levels in hepatic stellate cells, infiltration of neutrophils and macrophages, and inflammatory gene expression levels in hepatocytes and KCs. In particular, the above effects occurred with inhibition of nuclear translocation of NF-κB in liver tissues, and these beneficial effects of Exo-srIκB on ALD were shown regardless of doses. Our results suggest an exosome-based modulation of NF-κB activity in KCs by Exo-srIκB as a novel and efficient therapeutic approach in ALD.

Glutathione Trisulfide Prevents Lipopolysaccharide-induced Inflammatory Gene Expression in Retinal Pigment Epithelial Cells

ABSTRACT We investigated the effects of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammatory gene expression in immortalized ARPE-19, and primary human and mouse retinal pigment epithelial (RPE) cells. Sulfane sulfur molecules were significantly increased in GSSSG-treated ARPE-19 cells. GSSSG prevented the LPS-induced upregulation of interleukin (IL)-1β, IL-6, and C-C motif chemokine ligand 2 (CCL2) in ARPE-19/primary RPE cells. Moreover, GSSSG prevented the activation of the nuclear factor-kappa B p65 subunit, and promoted the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LPS-treated ARPE-19 cells. ERK1/2 inhibition prevented the GSSSG-mediated inhibition of LPS-induced IL-6 and CCL2 upregulation. Additionally, ERK1/2 activation prevented the upregulation of these genes in the absence of GSSSG. Knockdown of HMOX1 or NRF2, known as anti-oxidative genes, did not affect the activity of GSSSG in the context of LPS stimulation. These findings suggest that GSSSG attenuates LPS-induced inflammatory gene expression via ERK signaling hyperactivation, independently of the NRF2/HMOX1 pathway.

Effect of replacement of dietary fish oil with four vegetable oils on prostaglandin E2 synthetic pathway and expression of inflammatory genes in marine fish Larimichthys crocea

As a lipid mediator with important immune function, prostaglandin E2 (PGE2) has been widely studied in mammals, whereas its synthetic pathway and immune function in fish have yet to be fully studied. To investigate the regulation of PGE2 synthetic pathway and inflammatory genes expression by dietary different oils and the underlying relationship, a 10-week feeding experiment and an immune challenge were carried out in marine fish Larimichthys crocea. Replacement of dietary fish oil (FO) with four vegetable oils (VO), including soybean oil, linseed oil, palm oil, and olive oil, all reduced PGE2 levels, and the decrease of arachidonic acid (ARA, substrate for PGE2) could account for this decline. Meanwhile, the expression of PGE2 synthesis related genes was basically upregulated, which seemed to be a feedback regulation, but it cannot compensate the deficiency of ARA. In addition, mRNA expression of inflammatory genes, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)α and interferon (IFN)γ was all upregulated in four VO groups compared with FO group, which was the opposite of PGE2 levels. To verify the inflammatory regulation of PGE2, an immune challenge was conducted, and PGE2 alleviated LPS-induced expression of inflammatory genes, including IL-6, TNFα and IFNγ, and the similar downregulation of toll-like receptor (TLR) genes expression revealed that TLR signaling pathway participated in the anti-inflammatory regulation of PGE2. In conclusion, replacement of dietary FO with four VO (lack of ARA) reduced the levels of PGE2 that could alleviate LPS-induced inflammatory genes expression via TLR signaling pathway, which could be one of the reasons that VO induced inflammation in marine fish.

Differential Immune Activation in Fetal Macrophage Populations

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity.

Nanoparticle drug delivery characterization for fluticasone propionate and in vitro testing 1.

Glucocorticoids, such as fluticasone propionate (FP), are used for the treatment of inflammation and alleviation of nasal symptoms and allergies, and as an antipruritic. However, both short- and long-term therapeutic use of glucocorticoids can lead to muscle weakness and atrophy. In the present study, we evaluated the feasibility of the nanodelivery of FP with poly(dl-lactide-co-glycolide) (PLGA) and tested in vitro function. FP-loaded PLGA nanoparticles were prepared via nanoprecipitation and morphological characteristics were studied via scanning electron microscopy. FP-loaded nanoparticles demonstrated an encapsulation efficiency of 68.6% ± 0.5% with a drug loading capacity of 4.6% ± 0.04%, were 128.8 ± 0.6 nm in diameter with a polydispersity index of 0.07 ± 0.008, and displayed a zeta potential of -19.4 ± 0.7. A sustained in vitro drug release pattern was observed for up to 7 days. The use of fluticasone nanoparticle decreased lipopolysaccharide (LPS)-induced lactate dehydrogenase release compared with LPS alone in C2C12 treated cells. FP also decreased expression of LPS-induced inflammatory genes in C2C12 treated cells as compared with LPS alone. Taken together, the present study demonstrates in vitro feasibility of PLGA-FP nanoparticle delivery to the skeletal muscle cells, which may be beneficial for treating inflammation.

Glucosamine improves survival in a mouse model of sepsis and attenuates sepsis-induced lung injury and inflammation

The aim of the current study was to investigate the effects of glucosamine (GlcN) on septic lethality and sepsis-induced inflammation using animal models of mice and zebrafish. GlcN pretreatment improved survival in the cecal ligation and puncture (CLP)-induced sepsis mouse model and attenuated lipopolysaccharide (LPS)-induced septic lung injury and systemic inflammation. GlcN suppressed LPS-induced M1-specific but not M2-specific gene expression. Furthermore, increased expressions of inflammatory genes in visceral tissue of LPS-injected zebrafish were suppressed by GlcN. GlcN suppressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and NF-κB in lung tissue. LPS triggered a reduction in O-GlcNAc levels in nucleocytoplasmic proteins of lung, liver, and spleen after 1 day, which returned to normal levels at day 3. GlcN inhibited LPS-induced O-GlcNAc down-regulation in mouse lung and visceral tissue of zebrafish. Furthermore, the O-GlcNAcase (OGA) level was increased by LPS, which were suppressed by GlcN in mouse and zebrafish. OGA inhibitors suppressed LPS-induced expression of inflammatory genes in RAW264.7 cells and the visceral tissue of zebrafish. Stable knockdown of Oga via short hairpin RNA led to increased inducible nitric oxide synthase (iNOS) expression in response to LPS with or without GlcN in RAW264.7 cells. Overall, our results demonstrate a protective effect of GlcN on sepsis potentially through modulation of O-GlcNAcylation of nucleocytoplasmic proteins.

PO-385 Genetical inhibition of IRE1α in macrophages suppresses hepatocellular carcinoma growth

IntroductionThe microenvironment of hepatocellular carcinoma (HCC) will be synchronised to tumor-promoting niche by the cytokine and growth factors produced by infiltrated inflammatory cells. Macrophages, in particular, have been regarded as the inflammatory effector in HCC. Inositol-requiring enzyme 1α (IRE1α) is an unfolded protein response sensor, that controls protein regulation, metabolism and inflammation. Inhibition of IRE1α has been reported to inhibit metabolic inflammation through macrophages modulation. Therefore, the aim of this study is to investigate the role of IRE1α in tumour associated macrophages of HCC.Material and methodsBone marrow isolated mononuclear cells were differentiated by either culturing with M-CSF or tumour supernatant for 7 days. Expressions of mRNAs was detected by quantitative real time PCR; protein expression was detected by immunoblotting assay; gene silencing was performed by RNA interference. HCC proliferation was detected by BrDU incorporation assay. The animal model was established by implanting subcutaneous grown tumour cube to left lobe of mouse liver. The hepatic tumour growth was monitored by in vivo luciferase imaging system.Results and discussionsWe observed pharmacological inhibition of IRE1α by genipin, an iridoid aglycone reduced expressions of inflammatory factors such as CCR2, TNFα, CCL5, iNOS and IL6 on tumour supernatant-cultured and M-CSF induced macrophages. LPS-induced inflammatory gene expression in macrophages was similarly suppressed by genipin treatment. The role of IRE1α was confirmed from the observation of suppression of all the inflammatory mediators in IRE1α silencing macrophages. Furthermore, co-incubation of HCC cells with IRE1α silencing macrophages inhibited HCC cells proliferation. Our further in vivo study showed suppression of orthotopic HCC tumour growth after genipin intervention. We observed reduced IRE1α expression in hepatic macrophages after genipin treatment. This was accompanied with consistent reduction of inflammatory markers on the sorted hepatic macrophages.ConclusionAltogether, our findings unveil the role of IRE1α in modulating inflammation in macrophages and pharmacological or genetical inactivation of IRE1α in macrophages suppressed HCC progression.

Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases.

We previously demonstrated that the organic extract of Spirulina platensis (SPE), an edible blue-green alga, possesses potent anti-inflammatory effects. In this study, we investigated if the regulation of histone deacetylases (HDACs) play a role in the anti-inflammatory effect of SPE in macrophages. Treatment of macrophages with SPE rapidly and dose-dependently reduced HDAC2, 3, and 4 proteins which preceded decreases in their mRNA levels. Degradation of HDAC4 protein was attenuated in the presence of inhibitors of calpain proteases, lysosomal acidification, and Ca2+/calmodulin-dependent protein kinase II, respectively, but not a proteasome inhibitor. Acetylated histone H3 was increased in SPE-treated macrophages to a similar level as macrophages treated with a pan-HDAC inhibitor, with concomitant inhibition of inflammatory gene expression upon LPS stimulation. Knockdown of HDAC3 increased basal and LPS-induced pro-inflammatory gene expression, while HDAC4 knockdown increased basal expression of interleukin-1β (IL-1β), but attenuated LPS-induced inflammatory gene expression. Chromatin immunoprecipitation showed that SPE decreased p65 binding and H3K9/K14 acetylation at the Il-1β and tumor necrosis factor α (Tnfα) promoters. Our results suggest that SPE increased global histone H3 acetylation by facilitating HDAC protein degradation, but decreases histone H3K9/K14 acetylation and p65 binding at the promoters of Il-1β and Tnfα to exert its anti-inflammatory effect.

A1.68 An Acetyl-Histone MiMetic blocks inflammatory activation of rheumatoid arthritis fibroblast-like synoviocytes

Background and ObjectivesGenetic backgrounds do not completely explain the etiology of rheumatoid arthritis (RA), and increasing attention has turned to the potential contributions of epigenetic mechanisms. Fibroblast-like synoviocytes (FLS) isolated...

The Crosstalk Between Nrf2 and AMPK Signal Pathways Is Important for the Anti-Inflammatory Effect of Berberine in LPS-Stimulated Macrophages and Endotoxin-Shocked Mice

The response of AMP-activated protein kinase (AMPK) to oxidative stress has been recently reported but the downstream signals of this response are largely unknown. Meanwhile, the upstream events for the activation of nuclear factor erythroid-2-related factor-2 (Nrf2), a critical transcriptional activator for antioxidative responses, remain unclear. In the present study, we investigated the relationship between AMPK and Nrf2 signal pathways in lipopolysaccharide (LPS)-triggered inflammatory system, in which berberine (BBR), a known AMPK activator, was used for inflammation suppression. In inflammatory macrophages, BBR attenuated LPS-induced expression of inflammatory genes (inducible nitric oxide synthase [iNOS], cyclooxygenase-2 [COX2], interleukin [IL]-6), and the generation of nitric oxide and reactive oxygen species, but increased the transcription of Nrf2-targeted antioxidative genes (NADPH quinone oxidoreductase-1 [NQO-1], heme oxygenase-1 [HO-1]), as well as the nuclear localization and phosphorylation of Nrf2 protein. Importantly, we found BBR-induced activation of Nrf2 is AMPK-dependent, as either pharmacologically or genetically inactivating AMPK blocked the activation of Nrf2. Consistent with in vitro experiments, BBR down-regulated the expression of proinflammatory genes but upregulated those of Nrf2-targeted genes in lungs of LPS-injected mice, and these effects were attenuated in Nrf2-deficient mice. Moreover, the effect of BBR on survival time extension and plasma redox regulation in endotoxin-shocked mice was largely weakened when Nrf2-depleted. Our results demonstrate convergence between AMPK and Nrf2 pathways and this intersection is essential for anti-inflammatory effect of BBR in LPS-stimulated macrophages and endotoxin-shocked mice. Uncovering this intersection is significant for understanding the relationship between energy homeostasis and antioxidative responses and may be beneficial for developing new therapeutic strategies against inflammatory diseases. Antioxid. Redox Signal. 20, 574-588.

FRI0039 An acetyl-histone mimetic blocks inflammatory activation of rheumatoid arthritis fibroblast-like synoviocytes

BackgroundGenetic backgrounds and environmental factors do not completely explain the etiology of disease and predisposition to rheumatoid arthritis (RA), and increasing attention has turned to the potential contributions of heritable...

Identification of 34 Novel Proinflammatory Proteins in a Genome-Wide Macrophage Functional Screen

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.

Pellino 2 Is critical for Toll-like Receptor/Interleukin-1 Receptor (TLR/IL-1R)-mediated Post-transcriptional Control

Interleukin 1 receptor-associated kinase 1(IRAK1), a key molecule in TLR/IL-1R-mediated signaling, is phosphorylated, ubiquitinated, and degraded upon ligand stimulation. We and others have recently identified Pellino proteins as novel RING E3 ubiquitin ligases involved in IRAK1 polyubiquitination and degradation. However, it remains unclear how each Pellino member distinctly regulates TLR/IL-1R signaling by modulating IRAK1 ubiquitination. In this study we examined the role of Pellino 2 in IL-1- and LPS-mediated signaling and gene expression by knocking down Pellino 2 in human 293-IL-1R cells and primary bone marrow macrophages. Pellino 2 (but not Pellino 1) knockdown abolished IL-1- and LPS-induced Lys-63-linked IRAK1 ubiquitination with reduced Lys-48-linked IRAK1 ubiquitination. Furthermore, Pellino 2 is required for TAK1-dependent NFκB activation. However, because of the retained TAK1-independent NFκB activation, the levels of IL-1- and LPS-induced NFκB activation were not substantially affected in Pellino 2 knockdown 293-IL-1R cells and primary macrophages, respectively. On the other hand, Pellino 2 knockdown reduced the IL-1- and LPS-induced inflammatory gene expression at late time points, which was accompanied by increased decay rates of the mRNAs of the inflammatory genes. Importantly, IL-1- and LPS-mediated JNK and ERK activation were substantially attenuated in Pellino 2 knock-down cells, implicating MAPK activation in TLR/IL-1R-induced mRNA stabilization. Taken together, this study demonstrated that Pellino 2 plays a critical role for TLR/IL-1R-mediated post-transcriptional control.

Investigation of sanguinarine and chelerythrine effects on LPS-induced inflammatory gene expression in THP-1 cell line

Quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine have been used in folk medicine for their wide range of useful properties. One of their major effect is also anti-inflammatory activity, that is not clarified in detail.This study focused on the ability of these alkaloids to modulate the gene expression of pro-inflammatory tumour necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1, also known as CCL-2), interleukin (IL)-6, IL-1β and anti-inflammatory cytokines IL-1 receptor antagonist (IL-1RA) and IL-10. The effect of these alkaloids was compared with that of conventional drug prednisone.Human monocyte-derived macrophages were pre-treated with alkaloids or prednisone and inflammatory reaction was induced by lipopolysaccharide. Changes of gene expression at the transcriptional level of mentioned cytokines were measured.In our study mainly affected pro-inflammatory cytokines were CCL-2 and IL-6. Two hours after LPS stimulation, cells influenced by sanguinarine and chelerythrine significantly declined the CCL-2 expression by a factors of 3.5 (p<0.001) and 1.9 (p<0.01); for those treated with prednisone the factor was 5.3 (p<0.001). Eight hours after LPS induction, both alkaloids significantly diminished the CCL-2 expression. The lower expression was found for sanguinarine – lower by a factor of 4.3 than for cells treated with the vehicle (p<0.001).Two hours after LPS stimulation, cells treated with sanguinarine decreased the IL-6 mRNA level by a factor of 3.9 (p<0.001) compared with cells treated with the vehicle. Chelerythrine decreased the level of IL-6 mRNA by a factor of 1.6 (p<0.001). Sanguinarine decreased gene expression of CCL-2 and IL-6 more than chelerythrine and its effect was quite similar to prednisone. Four hours after LPS stimulation, cells pre-treated with sanguinarine exhibited significantly higher expression (a factor of 1.7, p<0.001) of IL-1RA than cells without sanguinarine treatment. Our results help to clarify possible mechanisms of action of these alkaloids in the course of inflammation.

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL-4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL-4-dependent E-cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL-4-induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1-deficient macrophages display rather enhanced IL-4-induced polyamine production, suggesting that an Arg1-independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS-induced expression of several proinflammatory genes was increased significantly in polyamine-depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL-4-induced expression of several AAM mediators, they inhibit the LPS-mediated expression of proinflammatory genes in CAMs.

A Novel Antiinflammatory Role for the Short-Chain Fatty Acids in Human Labor

Human parturition is an inflammatory process that can be activated prematurely by pathological stimuli. This study investigated the expression of G protein-coupled receptors GPR43 and GPR41 receptors in human uteroplacental tissues and the role of short-chain fatty acids (SCFA) in modulating inflammatory pathways in fetal membranes. Expression of GPR43 and GPR41 was investigated in uteroplacental tissues collected from women delivering at term or preterm after ethical approval and patient informed consent. The effect of SCFA on expression of inflammatory genes was assessed in amnion explants after culture with a mimetic of infection (lipopolysaccharide, LPS). Sodium propionate effect on LPS-induced neutrophil chemotaxis was evaluated by transwell assay. GPR43 and GPR41 mRNA expression was higher in myometrium and fetal membranes collected from women after the onset of labor. GPR43 protein expression localized to immune cells and vascular endothelium in the myometrium and epithelium of fetal membranes. Treatment with LPS significantly increased mRNA expression of GPR43 and inflammatory genes. Cotreatment with LPS and sodium propionate decreased LPS-induced expression of inflammatory genes including IL-6, IL-8, cyclooxygenase-2, IL-1α, intercellular adhesion molecule-1, and platelet endothelial cell adhesion molecule-1 but not IL-1β or lymphocyte function-associated antigen-1. Sodium propionate reduced LPS-induced neutrophil chemotaxis and protein secretion of the neutrophil chemoattractant IL-8. Finally, fetal membrane expression of GPR43 was significantly higher in women delivering preterm with evidence of infection. GPR43-SCFA interactions may represent novel pathways that regulate inflammatory processes involved in human labor. Suppression of inflammatory pathways by SCFA may be therapeutically beneficial for pregnant women at risk of pathogen-induced preterm delivery.