LPS-induced TNF-α Production Research Articles

Sparassis crispa Intake Improves the Reduced Lipopolysaccharide-Induced TNF-α Production That Occurs upon Exhaustive Exercise in Mice.

Our previous study showed that lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production is inhibited by acute exhaustive exercise in mice, leading to transient immunodepression after exercise. Sparassis crispa (SC), an edible mushroom, has immunopotentiative properties. This study aimed to clarify the effects of SC intake on reduced LPS-induced TNF-α production upon exhaustive exercise in mice. Male C3H/HeN mice were randomly divided into three groups: normal chow intake + resting sedentary, normal chow intake + acute exhaustive treadmill running exercise, and SC intake (chow containing 5% SC powder for 8 weeks) + the exhaustive exercise groups. Each group was injected with LPS immediately after the exhaustive exercise or rest. Plasma and tissue TNF-α levels were significantly decreased by exhaustive exercise. However, this reduction of the TNF-α level was partially attenuated in the plasma and small intestine by SC intake. Although levels of TLR4 and MyD88 protein expression were significantly decreased in tissues by exhaustive exercise, the reduction of TLR4 and MyD88 levels in the small intestine was partially attenuated by SC intake. These results suggest that SC intake attenuates exhaustive exercise-induced reduction of TNF-α production via the retention of TLR4 and MyD88 expression in the small intestine.

Functional circuitry of neuro-immune communication in the mesenteric lymph node and spleen

The peripheral nervous system is an active participant in immune responses capable of blocking aberrant activation of a variety of immune cells. As one of these neuro-immune circuits, the cholinergic anti-inflammatory pathway has been well established to reduce the severity of several immunopathologies. While the activation of this pathway by vagal nerve stimulation requires sympathetic innervation of the spleen, the neuro-immune circuitry remains highly controversial. Neuro-immune pathways in other lymphoid tissues such as mesenteric lymph nodes (MLN) that are critical to the surveillance of the small intestine and proximal colon have not been assessed. Using conditionally expressed Channelrhodopsin, selective stimulation of sympathetic post-ganglionic neurons in the superior mesenteric ganglion (SMG) prevented macrophage activation and LPS-induced TNFα production in the spleen and MLN, but not in the inguinal LN. Site selective stimulation of the SMG induced the release of norepinephrine, resulting in β2AR dependent acetylcholine release in the MLN and spleen. VNS-evoked release of norepinephrine and acetylcholine in the MLN and spleen was significantly reduced using selective optogenetic blockade applied at the SMG. Additionally, this optogenetic blockade restored LPS-induced TNFα production, despite VNS. These studies identify the superior mesenteric ganglion as a critical node in a neuro-immune circuit that can inhibit immune function in the MLN and the spleen.

Quercetin Inhibits Inflammatory Response Induced by LPS from Porphyromonas gingivalis in Human Gingival Fibroblasts via Suppressing NF-κB Signaling Pathway.

Quercetin, a natural flavonol existing in many food resources, has been reported to be an effective antimicrobial and anti-inflammatory agent for restricting the inflammation in periodontitis. In this study, we aimed to investigate the anti-inflammatory effects of quercetin on Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide- (LPS-) stimulated human gingival fibroblasts (HGFs). HGFs were pretreated with quercetin prior to LPS stimulation. Cell viability was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), along with chemokine interleukin-8 (IL-8), were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IκBα, p65 subunit of nuclear factor-kappa B (NF-κB), peroxisome proliferator-activated receptor-γ (PPAR-γ), liver X receptor α (LXRα), and Toll-like receptor 4 (TLR4) were measured by real-time quantitative PCR (RT-qPCR). The protein levels of IκBα, p-IκBα, p65, p-p65, PPAR-γ, LXRα, and TLR4 were characterized by Western blotting. Our results demonstrated that quercetin inhibited the LPS-induced production of IL-1β, IL-6, IL-8, and TNF-α in a dose-dependent manner. It also suppressed LPS-induced NF-κB activation mediated by TLR4. Moreover, the anti-inflammatory effects of quercetin were reversed by the PPAR-γ antagonist of GW9662. In conclusion, these results suggested that quercetin attenuated the production of IL-1β, IL-6, IL-8, and TNF-α in P. gingivalis LPS-treated HGFs by activating PPAR-γ which subsequently suppressed the activation of NF-κB.

Lysophosphatidic acid up-regulates IL-10 production to inhibit TNF-α synthesis in Mϕs stimulated with LPS

Bacterial LPS strongly induces pro-inflammatory responses of Mϕs after binding to CD14 protein and the TLR4/MD-2 receptor complex. The LPS-triggered signaling can be modulated by extracellular lysophosphatidic acid (LPA), which is of substantial importance for Mϕ functioning under specific pathophysiological conditions, such as atherosclerosis. The molecular mechanisms of the crosstalk between the LPS- and LPA-induced signaling, and the LPA receptors involved, are poorly known. In this report, we show that LPA strongly inhibits the LPS-induced TNF-α production at the mRNA and protein levels in primary Mϕs and Mϕ-like J774 cells. The decreased TNF-α production in LPA/LPS-stimulated cells is to high extent independent of NF-κB but is preceded by enhanced expression and secretion of the anti-inflammatory cytokine IL-10. The IL-10 elevation and TNF-α reduction are both abrogated upon depletion of the LPA5 and LPA6 receptors in J774 cells and can be linked with LPA-mediated activation of p38. We propose that the binding of LPA to LPA5 and LPA6 fine-tunes the LPS-induced inflammatory response by activating p38, and up-regulating IL-10 and down-regulating TNF-α production.

Canine IL4-10 fusion protein provides disease modifying activity in a canine model of OA; an exploratory study.

ObjectiveAn ideal disease modifying osteoarthritis drug (DMOAD) has chondroprotective, anti-inflammatory, and analgesic effects. This study describes the production and characterization of a canine IL4-10 fusion protein (IL4-10 FP) and evaluates its in vivo DMOAD activity in a canine model of osteoarthritis (OA).DesignThe canine Groove model was used as an in vivo model of degenerative knee OA. Six weeks after OA induction dogs were intra-articularly injected weekly, for ten weeks, with either IL4-10 FP or phosphate buffered saline (PBS). In addition to the use of human IL4-10 FP, canine IL4-10 FP was developed and characterized in vitro, and tested in vivo. Force plate analysis (FPA) was performed to analyze joint loading as a proxy measure for pain. After ten weeks dogs were euthanized and cartilage and synovial tissue samples were analyzed by histochemistry (OARSI scores) and biochemistry (cartilage proteoglycan turnover).ResultsRepetitive intra-articular injections with human IL4-10 FP led to antibody formation, that blocked its functional activity. Therefore, a canine IL4-10 FP was developed, which completely inhibited LPS-induced TNFα production by canine blood cells, and increased proteoglycan synthesis of canine cartilage in vitro (p = 0.043). In vivo, canine IL4-10 FP restored the, by OA impaired, joint loading (p = 0.002) and increased cartilage proteoglycan content (p = 0.029).ConclusionsThis first study on the potential DMOAD activity upon prolonged repeated treatment with IL4-10 FP demonstrates that a species-specific variant has anti-inflammatory and chondroprotective effects in vitro and chondroprotective and analgesic effects in vivo. These data warrant further research on the DMOAD potential of the IL4-10 FP.

In vitro anti-inflammatory activity of ginger (Zingiber officinale Rosc.) rhizome, callus and callus treated with some elicitors

This study evaluated the effect of ginger rhizome and its callus as well as callus elicited by yeast extract, glycine and salicylic acid on the production of pro-inflammatory (TNF-α, IL-1 and IL-6) and anti-inflammatory (IL-10 and TGF-β) cytokines in vitro. Petroleum ether (PE) and chloroform; methanol (1:1) (CM) extracts of rhizome and callus were shown to significantly (P < 0.05) suppress in a dose-dependent manner the LPS-induced production of TNF-α, IL-1 and IL-6. Both callus extracts showed significantly (P < 0.05) higher ability than the rhizome extracts. CM extract of ginger callus treated with elicitors showed significant (p < 0.05) capacity to inhibit IL-1, IL-6 and TNF-α secretion at highest concentration used (100 μg/mL) when compared to control (untreated callus). Elicitors improved significantly (P < 0.05) the callus capacity to produce the IL-10 and TGF-β anti-inflammatory cytokines. HPLC analysis showed that 6-gingerol and 6-shogaol were found in both extracts of rhizome, but were not detected in the callus extracts. Furthermore, gallic acid was found only in CM extracts of rhizome (34.05 ± 0.39 μg/mg) and callus (17.88 ± 0.01 μg/mg). Yeast extract, salicylic acid and glycine elicitors enhanced significantly (p < 0.05) the production of gallic acid in callus CM extract where the highest content was obtained from callus elicited with 100 mg/L of yeast extract followed by callus elicited with 50 mg/L of salicylic acid and 200 mg/L of glycine, respectively. Therefore, ginger callus could be included in nutraceutical formulations where it could provide valuable protection against inflammatory diseases. Key words: Anti-inflammatory, ginger, callus, elicitors.

Methamphetamine exacerbates neuroinflammatory response to lipopolysaccharide by activating dopamine D1-like receptors

Methamphetamine (METH) is a highly addictive and widely abused drug worldwide. Although much research is on the drug's direct effects, METH may also alter host immunity. The mechanism by which METH influences immunity remains elusive. Here, C57BL6/J mice were intraperitoneally injected with 5 mg/kg METH four times at two-hour intervals. The microglial inhibitor minocycline or dopamine D1-like receptor antagonist SCH-23390 was also applied prior to METH injection. Twenty-four hours following the first METH injection, mice were challenged by lipopolysaccharide (LPS) at a dose of 330 μg/kg, and the hippocampus (Hip), caudate putamen (CPU), nucleus accumbens (NAc) and prefrontal cortex (PFC) were collected 4 h after LPS administration. IL-6 and TNF-α levels were detected by ELISA. The activation of D1-like receptors and microglial marker Iba1 were examined by immunohistochemical staining and Western blot. Finally, we examined the phosphorylation of ERK1/2 and CREB. We found that METH exposure increased LPS-induced IL-6 and TNF-α production in the Hip, CPU and NAc regions. METH also augmented microglia activation and D1/5DR expression in response to LPS. Moreover, administering SCH-23390 significantly reduced IL-6 and TNF-α production and Iba1 expression following LPS challenge. Similar inhibitory effects were also observed by minocycline administration. Moreover, phosphorylation of ERK1/2 and CREB was increased after METH and LPS exposure but decreased by SCH-23390. These data illustrate that METH exacerbates neuroinflammation response in LPS-stimulated mouse brains through dopamine D1-like receptors, microglia, and relevant signaling proteins, which may have therapeutic implications.

NAD metabolites interfere with proliferation and functional properties of THP-1 cells.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

Genipin protects rats against lipopolysaccharide-induced acute lung injury by reinforcing autophagy

Although the protective effects of genipin against acute lung injury (ALI) have been described previously, the associated mechanism remains unclear. We have previously reported that genipin exerts its pharmacological effects by regulating autophagy. Here, we hypothesized that the up-regulation of autophagy may contribute to the protective effects exhibited by genipin against ALI. In the present study, ALI was induced by intratracheal LPS administration in rats. Genipin treatment significantly reduced LPS-induced lung injury as evidenced by improved histopathology, decreased lung edema, total cells, and protein concentration in the bronchoalveolar lavage fluid (BALF). This protection was inhibited by 3-methyladenine (3-MA), an inhibitor of autophagy. Genipin treatment reduced the expression of P62 and increased the expression of Beclin-1 and LC3II, indicating increased autophagy. Genipin treatment also alleviated LPS-induced cell apoptosis (down-regulation of Bax, up-regulation of Bcl-2, and decreased number of terminal deoxynucleotidyl transferase dUTP nick end label-positive cells) and oxidative stress (increased SOD and decreased MDA content) in the lung. Furthermore, genipin attenuated LPS-induced production of TNF-α, IL-1β, and IL-6 in the lung and BALF. These protective effects induced by genipin were reversed by 3-MA treatment, indicating that autophagy was involved in the protective effects exerted by genipin against inflammation and apoptosis in ALI. In A549 cells incubated with LPS for 6 h, genipin treatment increased the number of GFP-LC3 punctae. 3-MA prevented the protective effects of genipin against mitochondrial dysfunction and cell death. These findings suggest that genipin protects against apoptosis and inflammation in LPS-induced ALI by promoting autophagy.

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells.

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed‐infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T‐cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi‐maturation of human monocytes‐derived DCs (moDCs), and elevated levels of IL‐10, IL‐27 and IL‐6 but not IL‐12p70 and TNF‐α. Consistently, HMOS‐conditioned human moDCs promoted Treg generation from naïve CD4+ T cells. Interestingly, HMOS limited LPS‐induced maturation of human moDCs, while maintained IL‐10 and IL‐27 secretion and reduced LPS‐induced production of IL‐12p70, IL‐6 and TNF‐α. Furthermore, HMOS+LPS‐stimulated DCs induced a higher frequency of Tregs and increased IL‐10 production, while a reduction in Tbet+Th1 frequency and IFN‐γ production was detected as compared to LPS‐DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC‐SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.

Effects of Ascaris and Trichuris antigens on cytokine production in porcine blood mononuclear and epithelial cells

Helminth parasites are highly prevalent in swine production, causing chronic infections and considerable morbidity due to growth retardation. Moreover, helminths actively modulate host immune responses to other pathogens and/or vaccines. Here, we investigated the modulatory effects of Ascaris suum adult body fluid (ABF) and Trichuris suis Soluble Products (TsSP) on the cytokine response in porcine peripheral blood mononuclear cells (PBMCs) and the intestinal epithelial cell line IPEC-J2. In PBMCs, TsSP induced the secretion of IL-6, IL-10 and IL-1β, but not TNF-α. Moreover, TsSP significantly enhanced the production of bacterial lipopolysaccharide (LPS)-induced IL-6 and IL-10 but suppressed the production of LPS-induced TNF-α. ABF did not induce cytokine secretion from PBMC, but suppressed LPS-induced secretion of TNF-α and IL-6. ABF did not have any effect on cytokine production in IPEC-J2 cells. In contrast, TsSP selectively induced the secretion of IL-6, and enhanced the IL-6 response induced by LPS. The IL-6 response appeared to be a conserved response to T. suis products, as significant secretion was also observed in alveolar macrophages. Thus, T. suis products have diverse modulatory effects on cytokine secretion in vitro, with IL-6 production a consistent feature of the innate host response.

E3 ubiquitin ligase tripartite motif 7 positively regulates the TLR4-mediated immune response via its E3 ligase domain in macrophages

Members of the tripartite motif (TRIM) family as E3 ubiquitin ligases have been regarded as critical regulators of innate immunity and antiviral response. However, the role of TRIM7 is still elusive. Here, we provide evidence for the importance of TRIM7 in regulation of the TLR4-mediated innate response. In detail, we find that TRIM7 is highly expressed in antigen-presenting cells like macrophages. Knockdown of TRIM7 clearly inhibits the LPS-induced production of IFN-β, TNF-α and IL-6 in macrophages. Conversely, forced expression of TRIM7 could exert an opposite effect on these pro-inflammatory cytokines. Further analysis indicates that such effect is mediated by the TLR4-associated signaling pathways including MAPKs, NF-κB and IRF3-involved pathways. Truncation of the E3 ligase domain on TRIM7 may reduce the production of pro-inflammatory cytokines, suggesting a critical role of this domain in the regulation of LPS-initiated innate response. Taken together, we report here that TRIM7 may facilitate the TLR4-mediated innate response via its E3 ligase domain in macrophages, which provides new insight into the mechanistic role of TRIM7 in innate immunity.

Docosahexaenoic Acid Inhibits Inflammation-Induced Osteoclast Formation and Bone Resorption in vivo Through GPR120 by Inhibiting TNF-α Production in Macrophages and Directly Inhibiting Osteoclast Formation.

Docosahexaenoic acid (DHA) is an n-3 fatty acid that is an important structural component of the cell membrane. DHA exerts potent anti-inflammatory effects through G protein-coupled receptor 120 (GPR120), which is a functional receptor for n-3 fatty acids. DHA also regulates osteoclast formation and function. However, no studies have investigated the effect of DHA on inflammation-induced osteoclast formation in vivo. In the present study, we investigated whether DHA influences osteoclast formation, bone resorption and the expression of osteoclast-associated cytokines during lipopolysaccharide (LPS)-induced inflammation in vivo, and then we elucidated the underlying mechanisms by using in vitro experiments. In vitro experiments revealed both receptor activator of NF-kB ligand (RANKL)- and tumor necrosis factor-α (TNF-α)-induced osteoclast formation was inhibited by DHA. Supracalvarial administration of LPS with or without DHA was carried out for 5 days and then the number of osteoclasts, ratio of bone resorption pits and the level of type I collagen C-terminal cross-linked telopeptide were measured. All measurements were significantly lower in LPS+DHA-co-administered mice than LPS-administered mice. However, this DHA-induced inhibition was not observed in LPS-, DHA-, and selective GPR120 antagonist AH7614-co-administered mice. Furthermore, the expression of RANKL and TNF-α mRNAs was lower in the LPS+DHA-co-administered group than in the LPS-administered group in vivo. TNF-α mRNA levels were decreased in macrophages co-treated with LPS+DHA compared with cells treated with LPS in vitro. In contrast, RANKL mRNA expression levels from osteoblasts co-treated with DHA and LPS in vitro were equal to that in cells treated with LPS alone. Finally, the inhibitory effects of DHA on osteoclast formation in vitro were not observed by using osteoclast precursors from GPR120-deficient mice, and inhibition of LPS-induced osteoclast formation and bone resorption by DHA in vivo was not observed in GPR120-deficient mice. These results suggest that DHA inhibits LPS-induced osteoclast formation and bone resorption in vivo via GPR120 by inhibiting LPS-induced TNF-α production in macrophages along with direct inhibition of osteoclast formation.

Sp2-Iminosugar glycolipids as inhibitors of lipopolysaccharide-mediated human dendritic cell activation in vitro and of acute inflammation in mice in vivo

Glycolipid mimetics consisting of a bicyclic polyhydroxypiperidine-cyclic carbamate core and a pseudoanomeric hydrophobic tail, termed sp2-iminosugar glycolipids (sp2-IGLs), target microglia during neuroinflammatory processes. Here we have synthesized and investigated new variants of sp2-IGLs for their ability to suppress the activation of human monocyte-derived dendritic cells (DCs) by lipopolysaccharide (LPS) signaling through Toll-like receptor 4. We report that the best lead was (1R)-1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO2-ONJ), able to inhibit LPS-induced TNFα production and maturation of DCs. Immunovisualization experiments, using a mannoside glycolipid conjugate (MGC) that also suppress LPS-mediated DC activation as control, evidenced a distinct mode of action for the sp2-IGLs: unlike MGCs, DSO2-ONJ did not elicit internalization of the LPS co-receptor CD14 or induce its co-localization with the Toll-like receptor 4. In a mouse model of LPS-induced acute inflammation, DSO2-ONJ demonstrated anti-inflammatory activity by inhibiting the production of the pro-inflammatory interleukin-6. The ensemble of the data highlights sp2-IGLs as a promising new class of molecules against inflammation by interfering in Toll-like receptor intracellular signaling.

Quercetin loaded nanoemulsion-based gel for rheumatoid arthritis: In vivo and in vitro studies

Current research reports the development, optimization and evaluation of Quercetin (QCT) loaded nanoemulsion (NE)-based gel for the effective rheumatoid arthritis (RA) management. The formulation of QCT- NE was developed using spontaneous emulsification techniques using the Box- Behnken experimental design. The cytotoxicity study and effect on TNF-α production were evaluated respectively on HIG-82 and RAW 264.7 cells. The study showed that QCT- NE has no toxic effect on synoviocytes and a strong inhibitory effect on LPS-induced TNF-α production. QCT- NE gel has confirmed adequate rheological behavior with a good texture profile and improved drug permeation compared to free QCT gel. In addition, the gel was found to be non-irritating and showed the inhibition of paw edema in rats induced by CFA over 24 h contrary to free QCT gel. In conclusion, the formulation of QCT- NE gel is an efficient topical treatment strategy for rheumatoid arthritis.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

Pharmacokinetic-Pharmacodynamic Modeling for Coptisine Challenge of Inflammation in LPS-Stimulated Rats

Pro-inflammatory factors are important indicators for assessing inflammation severity and drug efficacy. Coptisine has been reported to inhibit LPS-induced TNF-α and NO production. In this study, we aim to build a pharmacokinetic-pharmacodynamic model to quantify the coptisine time course and potency of its anti-inflammatory effect in LPS-stimulated rats. The plasma and lung coptisine concentrations, plasma and lung TNF-α concentrations, plasma NO concentration, and lung iNOS expression were measured in LPS-stimulated rats after intravenous injection of three coptisine doses. The coptisine disposition kinetics were described by a two-compartment model. The coptisine distribution process from the plasma to the lung was described by first-order dynamics. The dynamics of plasma TNF-α generation and elimination followed zero-order kinetics and the Michaelis-Menten equation. A first-order kinetic model described the TNF-α diffusion process from the plasma to the lung. A precursor-pool indirect response model was used to describe the iNOS and NO generation induced by TNF-α. The inhibition rates of TNF-α production by coptisine (54.73%, 26.49%, and 13.25%) calculated from the simulation model were close to the decline rates of the plasma TNF-α AUC (57.27%, 40.33%, and 24.98%, respectively). Coptisine suppressed plasma TNF-α generation in a linear manner, resulting in a cascading reduction of iNOS and NO. The early term TNF-α response to stimulation is a key factor in the subsequent inflammatory cascade. In conclusion, this comprehensive PK-PD model provided a rational explanation for the interlocking relationship among TNF-α, iNOS and NO production triggered by LPS and a quantitative evaluation method for inhibition of TNF-α production by coptisine.

Differential expression and regulation of the non-integrin 37/67-kDa laminin receptor on peripheral blood leukocytes of healthy individuals and patients with rheumatoid arthritis

The non-integrin 37/67-kDa laminin receptor (LAMR1) is a complex protein with diverse functions. LAMR1 is widely expressed in epithelial cells and recently it was reported on neutrophils and a subset of activated T cells. Ligation of LAMR1 on peripheral blood mononuclear cells (PBMC) downregulated LPS-induced TNFα production, suggesting immune functions. However, its expression on primary monocytes remain unknown. Interestingly, LAMR1 mRNA is downregulated in PBMC of patients with early rheumatoid arthritis (RA), and low gene expression is an independent predictor of poor response to anti-TNFα treatment, suggesting a role in RA pathogenesis. We found LAMR1 was constitutively expressed on all peripheral blood monocytes and a subset of B cells from healthy individuals and patients with RA and it was abundantly present in synovial tissue of patients with RA. On monocytes and synovial tissue lower levels of LAMR1 expression tended to correlate with increased disease activity scores. In vitro treatment of monocytes with IFNγ or IL-10 up-regulated surface LAMR1 in healthy individuals and patients with RA with greater effects observed in healthy individuals. Importantly, treatment with IFNγ significantly increased specific binding of monocytes to laminin-1. TNFα and IL-1β caused marginal downregulation of LAMR1 in patients but effects in controls were variable. Taken together, constitutively expressed LAMR1 on monocytes is differentially regulated by pro-inflammatory and immune-regulatory cytokines suggesting LAMR1 may regulate the threshold and amplitude of their activation and migration. Decreased levels in patients with RA may indicate loss of this potentially critical homeostatic regulation thereby contributing to the excessive inflammation.

P092 Circulating classical monocytes and intestinal macrophages exhibit reduced response to IL-10 in IBD

Mice in which the IL-10 receptor (IL-10R) is knocked out in macrophages (Mφs) alone develop bacterially driven colitis, demonstrating that IL-10 mediated control of these cells is essential to prevent intestinal inflammation. Humans who have loss-of-function IL-10R mutations develop severe early-onset IBD; these individuals may represent the end of a spectrum in which suboptimal control of Mφs by IL-10 leads to gut inflammation. Our aim was to investigate whether monocytes and monocyte-derived intestinal Mφs from adult-onset IBD patients exhibit a diminished response to IL-10. Blood monocyte subsets (CD14++CD16− classical; CD14++CD16+ intermediate; CD14+CD16++ non-classical) and monocyte-derived intestinal Mφs in IBD patients and controls were identified by flow cytometry. Inhibition of LPS-induced TNFα production by IL-10 was measured by intracellular cytokine staining. LPS-induced TNFα production by classical monocytes (78 ± 4.46% TNFα+) was significantly (p < 0.001) inhibited by IL-10 in healthy controls. A similar frequency (89 ± 2.39%) of intermediate monocytes produced TNFα. However, compared with classical monocytes, this response was significantly (p = 0.009) less well controlled by IL-10 despite higher IL-10R expression and similar IL-10-induced STAT3 phosphorylation. Fewer LPS-stimulated non-classical monocytes produced TNFα (33 ± 6.24%; p < 0.001), which was poorly inhibited by IL-10 due to poor IL-10-induced STAT3 phosphorylation as a consequence of low STAT3 availability. IL-10 was significantly less effective at inhibiting TNFα production by classical monocytes from IBD patients than from controls (p = 0.026) (Figure 1), despite increased expression of IL-10Rα and IL-10-induced STAT3 phosphorylation. The implications of a suboptimal response to IL-10 in classical monocytes was investigated in CD14+ monocyte-derived Mφs in the intestine. Two populations of CD45+HLA-DRhi cells were identified based on CD14 expression: CD14hi (P1) and CD14lo (P2). Both populations spontaneously produced TNFα, which was enhanced with LPS stimulation. Inhibition of LPS-induced TNFα by IL-10 was reduced in IBD patients compared with controls. Figure 1. Inhibition of LPS-induced TNFα production in classical monocyte in health and IBD. TNFα production by intermediate and non-classical monocytes is poorly controlled by IL-10 and these populations may contribute to inflammation in the IL-10-rich intestine. A lower responsive to IL-10 observed in both classical monocytes and monocyte-derived intestinal Mφs from IBD patients and may contribute to inflammation.

Functional Circuitry of Neuro-Immune Communication in the Mesenteric Lymph Node and Spleen

The peripheral nervous system is an active participant in immune responses capable of blocking aberrant activation of a variety of immune cells. As one of these neuro-immune circuits, the cholinergic anti-inflammatory pathway has been well established to reduce the severity of several immunopathologies. While the activation of this pathway by vagal nerve stimulation requires both sympathetic innervation of the spleen the neuro-immune circuitry remains highly controversial. Neuro-immune pathways in other lymphoid tissues such as mesenteric lymph nodes (MLN) that are critical to the surveillance of the small intestine and proximal colon have not been assessed. Using conditionally expressed Channelrhodopsin, selective stimulation of sympathetic post-ganglionic neurons in the superior mesenteric ganglion prevented LPS-induced TNFα production in the spleen and MLN but not the inguinal LN. Site selective stimulation of the SMG induced norepinephrine that resulted in β2 adrenergic receptors (β2AR) acetylcholine release in MLN and spleen. Using selective optogenetic blockade applied at the SMG, we determined that electrical VNS-evoked release of norepinephrine and acetylcholine in the MLN and spleen could be inhibited and restored LPS induced TNFα production. These studies identify the superior mesenteric ganglion as a critical node in the inhibition of immune function in the MLN and the spleen.