Lipopolysaccharide Activation Research Articles

Rhizoctonia bataticola lectin (RBL) induces phenotypic and functional characteristics of macrophages in THP-1 cells and human monocytes

We have previously reported that a fungal lectin, Rhizoctonia bataticola lectin (RBL), stimulates proliferation and secretion of Th1/Th2 cytokines in human peripheral blood mononuclear cells (PBMC). In the present study, we evaluated the ability of RBL to differentiate human monocytes to macrophages. RBL induced morphological changes indicative of differentiation in primary monocytes and THP-1 cells. Stimulation with RBL resulted in significant up-regulation of differentiation markers – CD54, HLA-DR, CD11b and CD11c and secretion of proinflammatory cytokines – IL-1β, TNF-α and IL-6. Functionally, RBL profoundly increased phagocytic activity in monocytes. In THP-1 cells, RBL-induced phagocytosis was higher compared to the effect induced by combination of phorbol-12-myristate-13-acetate (PMA) and lipopolysaccharide (LPS). RBL induced a significant increase in matrix metalloproteinase-9 (MMP-9) activity in comparison with a combined treatment of PMA+LPS. Mechanistic studies revealed the involvement of the NF-κB pathway in RBL-induced differentiation of monocytes. The data suggest that RBL mimics the combined action of PMA and LPS to induce morphological and functional differentiation in human monocytes and monocytic cell line – THP-1 to macrophages. Human monocytes differentiated to macrophages with RBL have the potential as an in vitro model to study macrophage biology.

Anamorsin Overexpression Leads to Dysregulation of Lipopolysaccharide-Stimulated B Cell Proliferation through Ras Signaling

Introduction: Anamorsin (AM, also called CIAPIN1) was originally isolated as a molecule that conferred resistance to apoptosis caused by growth factor deprivation. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and small compared to wild type (WT) embryos. It suggests that AM is indispensable for embryo growth and hematopoiesis. To determine which signaling pathways AM utilizes for these functions, we analyzed murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. P38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggested that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells. However, functions of AM still remain not fully understood. In order to elucidate functions of AM, we generated AM transgenic (Tg) mice under control of CAG promoter. Since our previous study showed AM overexpression in approximately 30% of B cell type malignant lymphoma (DLBCL and FL) cases, we focused on AM overexpressed B cells from AM Tg mice in this study.Methods and Results: At first, we compared the number of B cells in the peripheral blood and spleen size between AM Tg mice and WT mice and found no difference. Next, we analyzed the lipopolysaccharide (LPS) stimulated B cells. B cells were selected from spleen by using anti-B220 antibody. The purified B cells were exposed to LPS for three days and measured. Unexpectedly, LPS-stimulated proliferation of B cells from AM Tg mice was decreased compared to WT mice. Since we initially confirmed that AM overexpression did not affect TLR4 expression on B cells, we examined TLR4 signaling pathway activation status by detecting phosphorylation of the signal transduction molecules using Western-blotting, and found that phosphorylation of Erk1/2 and IKBα were decreased in LPS-stimulated B cells from AM Tg mice. Next, we performed cDNA microarray analysis to reveal the mechanisms of inhibition of the LPS signaling pathways by comparing the differential gene expression profiles in B cells of AM Tg and WT mice with or without LPS stimulation. We extracted 2375 genes from the data sets that met the following criteria: genes with > 2.0 fold change between LPS- vs LPS+ in AM Tg or WT mice, and secondly with >1.5 fold difference in the rate of gene expression change with LPS stimulation between AM Tg and WT mice, and performed upstream regulator analysis to predict the upstream regulators on the cascade of LPS signaling pathway with Ingenuity Pathway Analysis software. The data suggested that Ras activation was decreased in LPS-stimulated AM Tg B cells compared to WT B cells. Then, we confirmed the impairment of LPS induced Ras activation in AM Tg B cells using Ras activation assay kit. Furthermore, we found that AM mRNA level was increased (1.8-4.0 fold) in WT B cells at 4 hours after LPS stimulation. From these data, it was shown that AM overexpression in AM Tg B cells inhibited Ras signaling pathways and retarded the cell proliferation of LPS-stimulated B cells.Conclusion and Discussion: In our previous study, we showed that AM negatively regulates novel PKCs and p38MAPK by using MEFcells generated from AM KO mice, while in this study it was shown that overexpressed AM negatively regulates Ras signaling pathways by using AM Tg B cells. Although there are no reports other than ours that show the relationships between the signaling molecules and AM, our present data showed the novel role of AM that regulates the signaling molecules in the different manner. LPS increased AM expression via Ras activation in B cells, and AM overexpression inhibited Ras, which suggested AM might induce a negative feedback loop that attenuates Ras activation in LPS stimulated B cells. Our previous data showed that low international prognostic index (IPI) DLBCL patients with AM overexpression had a poor prognosis, especially in the patients who received chemotherapy without rituximab. In those patients, AM overexpression might contribute at least in part to chemotherapy resistance possibly through negative regulation of Ras. In summary, we showed that overexpressed AM is one of negative regulators of LPS induced signaling pathways in B cells. DisclosuresNo relevant conflicts of interest to declare.

Distinct biological activity of lipopolysaccharides with different lipid A acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter.

Correlation between the chemical structure of lipidA from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipidA. LipidA of Y. pestis double ΔlpxM/ΔlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipidA from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipidA fatty acids with shorter acyl chains (C10-C12) than those in lipidA from LPS of Y. pestis or E. coli (C12-C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipidA.

LPS promote the odontoblastic differentiation of human dental pulp stem cells via MAPK signaling pathway.

Human dental pulp stem cells (hDPSCs) show significant potential for exploitation in novel regeneration strategies, although lack of understanding of their responses to bacterial challenge constrains their application. The present study aimed to investigate whether lipopolysaccharide (LPS), the major pathogenic factor of Gram-negative bacteria, regulates the differentiation of hDPSCs and which intracellular signaling pathways may be involved. LPS treatment significantly promoted the differentiation of hDPSCs demonstrable by increased mineralized nodule formation and mRNA expression of several odontoblastic markers in a dose-dependent manner. While inhibition of TLR4, p38, and ERK signaling markedly antagonized LPS-mediated differentiation of hDPSCs. The inhibition of JNK and NF-κB signaling had no detectable effect on LPS activation of hDPSCs. LPS stimulation resulted in phosphorylation of NF-κB p65, IκB-α, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) in DPSCs in a time-dependent manner, which was markedly suppressed by their specific inhibitors, respectively. Data demonstrated that LPS promoted odontoblastic differentiation of hDPSCs via TLR4, ERK, and P38 MAPK signaling pathways, but not NF-κB signaling.

Differential partial activation phenotype and production of tumour necrosis factor-α by conventional dendritic cells in response to lipopolysaccharide in HIV+ viraemic subjects and HIV+ controllers.

HIV(+) subjects are reported to have increased soluble CD14 (sCD14) in plasma, an indicator of microbial translocation. We evaluated if microbial translocation has a differential impact on the activation and function of conventional dendritic cells (cDC) from viraemic HIV(+) subjects and HIV(+) controllers (CTs). The HIV(+) subjects were classified into two groups according to their plasma viral load (pVL): CT and viraemic. Subjects without HIV were included as controls (HIV(-) ). The frequencies and phenotypes of cDC from these subjects were evaluated by multi-parameter flow cytometry. In addition, peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) or single-stranded RNA40 (ssRNA40), the phenotype of the cDC and the intracellular production of tumour necrosis factor (TNF)-α by the cDC were evaluated by flow cytometry. We observed a partial activation phenotype for the cDC in the viraemic subjects and CTs ex vivo and after LPS activation, which showed differences in the expression of CD40 and CD86. Furthermore, in response to LPS the cDC from the viraemic subjects produced more TNF-α compared to the cDC from CTs. Interestingly, the percentage of TNF-α(+) cDC was found to be correlated positively with the pVL. The partial activation of cDC and the over-production of TNF-α in response to LPS in viraemic HIV(+) subjects might be related to the increased chronic activation observed in these subjects. In contrast, cDC from CTs seem to have a regulated response to LPS, indicating that they respond differently to chronic immune activation. These results may have implications in the development of HIV therapies and vaccines using DC.

Endotoxemia, nutrition, and cardiometabolic disorders.

Circulating lipopolysaccharides (LPSs), associated with both infection and inflammation, may arise from the gastrointestinal tract microbiota, and the levels may be affected by daily nutrition. We investigated whether nutrient intake affects the association of serum LPS activity with prevalent obesity, metabolic syndrome (MetS), diabetes, and coronary heart disease (CHD) and with the risk of incident CHD events. The nutrition cohort (n = 2,452, mean age ± SD, 52.2 ± 10.1 years) of the FINRISK 1997 Study was followed up for 10 years. Information on macronutrient intake at baseline was collected from 24-h dietary recall. Serum endotoxin activities were determined by the Limulus amebocyte lysate assay. LPS activity was associated directly with the total energy intake and indirectly with carbohydrate intake in lean, healthy subjects. High LPS was significantly associated with prevalent obesity, MetS, diabetes, and CHD events, independently of established risk factors, CRP, and total energy or nutrient intake. The ORs (95 % CI) were 1.49 (1.21-1.85, p < 0.001, Q2-4 vs. Q1) for obesity, 2.56 (1.97-3.32, p < 0.001, Q2-4 vs. Q1) for MetS, 1.94 (1.06-3.52, p = 0.031, Q2-4 vs. Q1) for CHD, and 1.01 (1.00-1.01, p = 0.032, LPS unit) for diabetes. In the follow-up, high LPS was significantly associated with the risk of CHD events with a hazard ratio of 1.88 (1.13-3.12, p = 0.013, Q2-4 vs. Q1). This association was independent of baseline established risk factors, diet, obesity, MetS, and diabetes. A high serum LPS activity is strongly associated with cardiometabolic disorders, which supports the role of bacterial infections and immune response in their etiology.

Abstract 771: TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis

Abstract Background: Paclitaxel (PXL) is a widely-used drug for breast cancer therapy. Resistance, however, occurs frequently and the evasion mechanisms remain unclear. PXL is known to activate Toll-like Receptor-4 (TLR4), a major signaling receptor expressed on immune cells responding to lipopolysaccharide (LPS). LPS activation of TLR4 in the immune cells substantially enhances their migratory, invasive, proliferative, and pro-survival functions as necessary for the body defense. Since PXL is a functional LPS mimetic, we hypothesized that acquisition of such phenotype by TLR4-expressing cancer cells promotes tumor growth, metastasis, and resistance to therapy. Methods: We tested this hypothesis in two breast cancer luciferase-tagged models genetically modified to either suppress TLR4 expression in a positive line, MDA-MB-231, or overexpress it in a negative line, HCC1806. Modified cell lines and their controls were orthotopically implanted in mice followed by measuring tumor growth prior and post-PXL treatment. Incidence and burden of lymph node (LN) and lung metastases were quantified by measuring luciferase activity in respective organs. TLR4-induced inflammation was assessed by measuring cytokines in blood, tumor lysates, and metastatic organs using RT-qPCR and ELISA. Cell composition of spleens and bone marrow from control and treated mice were analyzed by FACS. TLR4-induced changes in tumor vasculature were determined by immunostaining for blood and lymphatic vessel markers. Results: TLR4 expressed in tumor cells significantly increased rate of recurrence and metastasis, an event augmented by PXL treatment. Local inflammation was also enhanced by the PXL•TLR4 axis as illustrated by a 276-fold increase in IL-6 in lysates of TLR4+ tumors from treated mice as compared with samples of isogenic tumors with depleted TLR4. Activation of this pathway also increased cytokine levels in the blood and distant organs as indicted by 3-6 fold increase in IL-4 and IL-10 in LNs and lungs. These pro-inflammatory systemic changes promoted generation of myeloid progenitors in bone marrow and spleen evident by 4-5 fold increase in Ly6C+ cell population in TLR4-overexpressing tumors compared with those lacking TLR4. Importantly, activation of the PXL•TLR4 axis induced intratumoral formation of lymphatic vessels in HCC1806 tumors absent in all other experimental groups. Conclusion: These data imply that PXL therapy activates TLR4 often overexpressed in breast cancer. Activation of this pathway substantially increases local tumor and systemic inflammation leading to generation of myeloid progenitors that can promote metastasis by inducing angiogenesis and lymphatic vessel formation, particularly inside the tumor. These findings suggest that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway may improve current anti-cancer treatments. Citation Format: Sandeep Rajput, Lisa Volk-Draper, Kelly Hall, Sophia Ran. TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2014-771

Metabotropic glutamate receptor 5 modulates calcium oscillation and innate immune response induced by lipopolysaccharide in microglial cell

Microglia, the primary immune cells in the brain, have been implicated as the predominant cells governing inflammation-mediated neuronal damage. In response to immunological challenges such as lipopolysaccharide (LPS), microglia are activated and subsequently inflammatory process is initiated as evidenced by the release of pro-inflammatory chemokines and cytokines. Here we show that Group I metabotropic glutamate receptor 5 (mGluR5) is involved in LPS-induced microglia activation. LPS triggered a similar pattern of [Ca2+]i oscillation in N9, Toll-like receptor 4 (TLR4)-mutant EOC 20, TLR4-wild-type and TLR4-deficient primary mouse microglia, suggesting that LPS-induced [Ca2+]i oscillation is independent of TLR4. The characteristics of [Ca2+]i oscillation induced by LPS are consistent with those observed in mGluR5 activation. In addition, mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) abolished LPS-induced [Ca2+]i oscillation. Immunocytochemistry demonstrated that LPS colocalizes with mGluR5 in microglia and the direct binding of LPS and mGluR5 was further validated by antibody-based fluorescence resonance energy transfer (FRET) technology. Activation of mGluR5 using a selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) significantly expanded LPS-induced nuclear factor-kappa B (NF-κB) activity and CHPG alone increased NF-κB activity as well. But, mGluR5 antagonist MTEP attenuated the actions of LPS, CHPG and the additive effect of LPS and CHPG in microglia. LPS induced tumor necrosis factor-α (TNF-α) secretion in N9 microglia, but not in TLR4-mutant EOC 20 and TLR4-deficient primary mouse microglia. CHPG reduced LPS-caused TNF-α production, but MTEP increased LPS-induced TNF-α production and blocked the effect of CHPG in N9 microglia. These data demonstrate that mGluR5 and TLR4 are two critical receptors that mediate microglia activation in response to LPS, suggesting that mGluR5 may represent a novel target for modulating microglia-dependent neuroinflammation.

Metformin Suppresses Lipopolysaccharide (LPS)-induced Inflammatory Response in Murine Macrophages via Activating Transcription Factor-3 (ATF-3) Induction

Metformin, a well known antidiabetic agent that improves peripheral insulin sensitivity, also elicits anti-inflammatory actions, but its mechanism is unclear. Here, we investigated the mechanism responsible for the anti-inflammatory effect of metformin action in lipopolysaccharide (LPS)-stimulated murine macrophages. Metformin inhibited LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a concentration-dependent manner and in parallel induction of activating transcription factor-3 (ATF-3), a transcription factor and member of the cAMP-responsive element-binding protein family. ATF-3 knockdown abolished the inhibitory effects of metformin on LPS-induced proinflammatory cytokine production accompanied with reversal of metformin-induced suppression of mitogen-activated protein kinase (MAPK) phosphorylation. Conversely, AMP-activated protein kinase (AMPK) phosphorylation and NF-κB suppression by metformin were unaffected by ATF-3 knockdown. ChIP-PCR analysis revealed that LPS-induced NF-κB enrichments on the promoters of IL-6 and TNF-α were replaced by ATF-3 upon metformin treatment. AMPK knockdown blunted all the effects of metformin (ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation), suggesting that AMPK activation by metformin is required for and precedes ATF-3 induction. Oral administration of metformin to either mice with LPS-induced endotoxemia or ob/ob mice lowered the plasma and tissue levels of TNF-α and IL-6 and increased ATF-3 expression in spleen and lungs. These results suggest that metformin exhibits anti-inflammatory action in macrophages at least in part via pathways involving AMPK activation and ATF-3 induction.

Rcor2 underexpression in senescent mice: a target for inflammaging?

BackgroundAging is characterized by a low-grade systemic inflammation that contributes to the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease (AD). However, little knowledge is currently available on the molecular processes leading to chronic neuroinflammation. In this context, recent studies have described the role of chromatin regulators in inflammation and longevity including the REST corepressor (Rcor)-2 factor, which seems to be involved in an inflammatory suppressive program.MethodsTo assess the impact of Rcor2 in age-related inflammation, gene expression levels were quantified in different tissues and ages of the spontaneous senescence-accelerated P8 mouse (P8) using the SAMR1 mouse (R1) as a control. Specific siRNA transfection in P8 and R1 astrocyte cultures was used to determine Rcor2 involvement in the modulation of neuroinflammation. The effect of lipopolysaccharide (LPS) treatment on Rcor2 levels and neuroinflammation was analyzed both in vivo and in vitro.ResultsP8 mice presented a dramatic decrease in Rcor2 gene expression compared with R1 controls in splenocytes, an alteration also observed in the brain cortex, hippocampus and primary astrocytes of these mice. Rcor2 reduction in astrocytes was accompanied by an increased basal expression of the interleukin (Il)-6 gene. Strikingly, intraperitoneal LPS injection in R1 mice downregulated Rcor2 in the hippocampus, with a concomitant upregulation of tumor necrosis factor (Tnf-α), Il1-β and Il6 genes. A negative correlation between Rcor2 and Il6 gene expression was also verified in LPS-treated C6 glioma cells. Knock down of Rcor2 by siRNA transfection (siRcor2) in R1 astrocytes upregulated Il6 gene expression while siRcor2 further increased Il6 expression in P8 astrocytes. Moreover, LPS activation provoked a further downregulation of Rcor2 and an amplified induction of Il6 in siRcor2-tranfected astrocytes.ConclusionsData presented here show interplay between Rcor2 downregulation and increased inflammation and suggest that Rcor2 may be a key regulator of inflammaging.

Lipopolysaccharide (LPS) stimulation of fungal secondary metabolism

We report on a preliminary investigation of the use the Gram-negative bacterial cell wall constituent lipopolysaccharide (LPS) as a natural chemical cue to stimulate and alter the expression of fungal secondary metabolism. Integrated high-throughput micro-cultivation and micro-analysis methods determined that 6 of 40 (15%) of fungi tested responded to an optimal exposure to LPS (0.6 ng/mL) by activating, enhancing or accelerating secondary metabolite production. To explore the possible mechanisms behind this effect, we employed light and fluorescent microscopy in conjunction with a nitric oxide (NO)-sensitive fluorescent dye and an NO scavenger to provide evidence that LPS stimulation of fungal secondary metabolism coincided with LPS activation of NO. Several case studies demonstrated that LPS stimulation can be scaled from single microplate well (1.5 mL) to preparative (>400 mL) scale cultures. For example, LPS treatment of Penicillium sp. (ACM-4616) enhanced pseurotin A and activated pseurotin A1 and pseurotin A2 biosynthesis, whereas LPS treatment of Aspergillus sp. (CMB-M81F) substantially accelerated and enhanced the biosynthesis of shornephine A and a series of biosynthetically related ardeemins and activated production of neoasterriquinone. As an indication of broader potential, we provide evidence that cultures of Penicillium sp. (CMB-TF0411), Aspergillus niger (ACM-4993F), Rhizopus oryzae (ACM-165F) and Thanatephorus cucumeris (ACM-194F) were responsive to LPS stimulation, the latter two examples being particular noteworthy as neither are known to produce secondary metabolites. Our results encourage the view that LPS stimulation can be used as a valuable tool to expand the molecular discovery potential of fungal strains that either have been exhaustively studied by or are unresponsive to traditional culture methodology.

Cysteine deleted protegrin-1 (CDP-1): Anti-bacterial activity, outer-membrane disruption and selectivity

BackgroundProtegin-1 (PG-1: RGGRLCYCRRRFCVCVGR-amide) assumes a rigid β-hairpin like structure that is stabilized by two disulfide bridges between Cys6–Cys15 and Cys8–Cys13. Previous studies, employing linear analogs of PG-1, with Cys to Ala mutations or modified Cys, have demonstrated that the disulfide bridges are critical for the broad spectrum and salt resistant antimicrobial activity of PG-1. MethodsIn order to understand structural and functional roles of disulfide bonds in protegrins, we have synthesized a Cys deleted variant of PG-1 or CDP-1, RGGRLYRRRFVVGR-amide, and two of its analogs, RR11, RLYRRRFVVGR-amide, and LR10, LYRRRFVVGR-amide, containing deletion of residues at the N-terminus. These peptides have been characterized for bactericidal activity and mode of action in lipopolysaccharide (LPS) using optical spectroscopy, ITC and NMR. ResultsAntibacterial activity, against Gram-negative and Gram-positive strains, of the three peptides follows the order: CDP-1>RR11>LR10. LR10 displays only limited activity toward Gram-negative strains. CDP-1 demonstrates efficient membrane permeabilization and high-affinity interactions with LPS. CDP-1 and RR11 both assume β-hairpin like compact structures in complex with LPS, whereas LR10 adopts an extended conformation in LPS. In zwitterionic DPC micelles CDP-1 and the truncated analog peptides do not adopt folded conformations. Major conclusionsDespite the absence of stabilizing disulfide bridges CDP-1 shows broad-spectrum antibacterial activity and assumes β-hairpin like structure in complex with LPS. The β-hairpin structure may be essential for outer membrane permeabilization and cell killing.

A GPBAR1 (TGR5) small molecule agonist shows specific inhibitory effects on myeloid cell activation in vitro and reduces experimental autoimmune encephalitis (EAE) in vivo.

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.

Leptin deficiency in vivo enhances the ability of splenic dendritic cells to activate T cells.

Leptin is a pleiotropic adipokine that is critical for regulating food intake and energy expenditure and also participates in functions of the immune system, including those of antigen-presenting cells. Here, we assess the effect of leptin deficiency on the function splenic dendritic cells (sDC). sDC from leptin-deficient mice (Lep(ob)) were evaluated ex vivo for phenotype, ability to respond to inflammatory stimuli, to acquire and process antigens and to activate T cells. The data show that Lep(ob) sDC express activation markers similar to controls and respond similarly to LPS activation or anti-CD40 cross-linking. In addition, antigen acquisition and processing by Lep(ob) sDC was similar to controls. However, Lep(ob) sDC elicited higher production of IFN-γ in mixed lymphocyte reactions and increased production of IL-2 by antigen-specific T-cell hybridoma relative to controls. To assess Lep(ob) sDC activation of T cells in vivo, Lep(ob) and control mice were infected systemically with Mycobacterium avium. Lep(ob) mice were significantly better at neutralizing the infection as measured by splenic bacterial load over time. This was mirrored with an increased percentage of activated T cells in M. avium-infected Lep(ob) mice. Thus, although no changes were detected in sDC phenotype, activation, antigen processing or presentation, these DC surprisingly presented an enhanced ability to activate T cells ex vivo and in vivo. These data demonstrate that leptin can modulate DC function and suggest that leptin may dampen T-cell responsiveness in the physiological setting.

Estrogen inhibits LPS-induced IL-6 production in macrophages partially via the nongenomic pathway

17β-estradiol (E2)-signaling is widely considered to be mediated through the transcription-regulating intracellular estrogen receptor (iER). In this study, using the cell-impermeable E2-BSA, we investigated the nongenomic effects of E2 on the IL-6 production, MAPK and transcription factor activation following LPS stimulation in mouse bone marrow-derived macrophages (BMMs). It was found that E2 normalized LPS-induced IL-6 production in BMMs. Although the increase in IL-6 production induced by LPS was also attenuated by E2-BSA treatment, the capacity of BMMs to produce the IL-6 cytokine remained higher than the control. In addition, the iER blocker, ICI 182780, did not abolish the total effects of E2 on LPS-stimulated IL-6 production capacity in BMMs. Furthermore, E2 and E2-BSA attenuated the LPS activation of p38 but not that of ERK1/2 and JNK. The p38 inhibitor, SB 203580, significantly reduced the LPS-induced IL-6 production. Moreover, E2 and E2-BSA inhibited LPS-induced activation of NF-κB. This inhibitory effect was associated with decreases in nuclear p65 protein levels. Taken together, these results indicate that E2 has an inhibitory effect on LPS-induced IL-6 production in BMMs through inhibition of p38 MAPK phosphorylation, and blockade of NF-κB activation. These effects are mediated at least in part via a nongenomic pathway.

Docosahexanoic acid diet supplementation attenuates the peripheral mononuclear cell inflammatory response to exercise following LPS activation

Exercise induces changes in circulating pro- and anti-inflammatory cytokines. The aim was to investigate the effect of docosahexaenoic acid (DHA) diet supplementation on the plasma cytokine levels and on the peripheral mononuclear (PBMCs) cells cytokine production after a training season or an acute bout of exercise. Fifteen male soccer players were randomly assigned to a placebo or an experimental group. The experimental group consumed an almond-based beverage enriched with DHA, whereas the placebo group consumed the same beverage without DHA. Three blood samples were taken: in basal conditions at the beginning of the nutritional intervention and after eight weeks of training season in basal and post-exercise conditions. The DHA content increased in erythrocytes after 8weeks of training and supplementation. Neither diet supplementation with DHA nor training season altered the basal plasma cytokines and growth factors. Only acute exercise significantly increased plasma IL6 in experimental and placebo groups. Lipopolysaccharide (LPS) activation induced the inflammatory response in PBMCs, with a significant production rate of TNFα, IL6 and IL8 mainly after acute exercise. DHA supplementation significantly reduced the rate of TNFα and IL6 production by stimulated PBMCs. Acute exercise increased the Toll-like receptor 4 (TLR4) protein levels in PBMCs, although the increase was only statistically significant in the placebo group. In conclusion, a training season does not induce significant changes in the circulating cytokine profile in well-trained soccer players. Exercise increases the PBMCs cell capabilities to produce cytokines after TLR4 stimulation with LPS and this rate of cytokine production is attenuated by diet DHA supplementation.

Reduced inflammatory phenotype in microglia derived from neonatal rat spinal cord versus brain.

Microglia are the primary immune cells of the central nervous system (CNS). Membrane bound sensors on their processes monitor the extracellular environment and respond to perturbations of the CNS such as injury or infection. Once activated, microglia play a crucial role in determining neuronal survival. Recent studies suggest that microglial functional response properties vary across different regions of the CNS. However, the activation profiles of microglia derived from the spinal cord have not been evaluated against brain microglia in vitro. Here, we studied the morphological properties and secretion of inflammatory and trophic effectors by microglia derived from the brain or spinal cord of neonatal rats under basal culture conditions and after activation with lipopolysaccharide (LPS). Our results demonstrate that spinal microglia assume a less inflammatory phenotype after LPS activation, with reduced release of the inflammatory effectors tumor necrosis factor alpha, interleukin-1 beta, and nitric oxide, a less amoeboid morphology, and reduced phagocytosis relative to brain-derived microglia. Phenotypic differences between brain and spinal microglia are an important consideration when evaluating anti-inflammatory or immunomodulatory therapies for brain versus spinal injury.

InsP4 negatively regulates dendritic cell cytokine secretion and migration to peripheral draining lymph nodes. (CAM1P.234)

Abstract Inositol 1,3,4,5-tetrakisphosphate (InsP4) is a novel second messenger that is critical for T and B cell development and activation. However, whether InsP4 has a similar regulatory role in dendritic cell (DC) function is not yet known. We show here that InsP4 differentially regulates ERK and Akt signaling in response to LPS activation of bone marrow derived DCs (BMDCs). InsP4 supports ERK activation but negatively regulates Akt and its down-stream signals. Interestingly, endogenous migratory DCs from InsP4 deficient mice and BMDCs migrated more efficiently to peripheral lymph nodes in vivo. InsP4 deficient BMDCs also have enhanced secretion of pro-inflammatory cytokines/chemokines and mediate stronger antigen presentation to antigen-specific T cells. Our data support an important role for InsP4 in DC migration and cytokine secretion, identifying InsP4 as a dual regulator of Akt and ERK signaling pathways that controls DC function.

β-Boomerang Antimicrobial and Antiendotoxic Peptides: Lipidation and Disulfide Bond Effects on Activity and Structure.

Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS)-mediated inflammations are among some of the most prominent health issues globally. Antimicrobial peptides (AMPs) are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt β-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the β-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, β-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules.

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001–0.01%), and BAK (0.001%–0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.