LPS-stimulated IL-6 Production Research Articles

Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice.

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.

Investigation of Association of Complement 5 Genetic Polymorphisms with Sepsis and Sepsis-Induced Inflammatory Responses.

BackgroundComplement 5 (C5) and C5a production play a pivotal role in the pathophysiology of sepsis. Strong evidence demonstrates an association of C5 gene polymorphisms with various inflammatory diseases. However, no current studies have explored the clinical relevance of C5 polymorphisms in sepsis.MethodsTwo C5 gene polymorphisms, rs17611 and rs2269067, were identified by genotyping in 636 sepsis patients and 753 controls in a Han Chinese population. C5 gene expression was detected via quantitative real-time PCR. C5a and proinflammatory cytokine production was measured by enzyme-linked immunosorbent assay. An Annexin V apoptosis assay was performed to assess cell apoptosis.ResultsOur results showed significantly lower frequencies of rs2269067 GC/CC genotypes or C allele in sepsis patients than healthy controls. The frequencies of rs17611 CC/CT genotypes or C allele were significantly overrepresented in both the septic shock and non-survivor subgroups. Patients with this sepsis-associated high-risk rs17611 C allele exhibited a significant increase in C5a, TNF-α and IL-6 production. However, no significant difference in C5a and downstream proinflammatory cytokine production was observed among patients with different rs2269067 genotypes. In addition, in vitro experiments showed an effect of recombinant C5a on enhancing LPS-stimulated IL-1β, IL-6 and TNF-α production and cell apoptosis in THP-1 monocytes.ConclusionThe rs2269067 polymorphism conferred protection against sepsis susceptibility. The rs17611 polymorphism was associated with increased C5a production, which ultimately potentiated the secretion of downstream proinflammatory cytokines and conferred susceptibility to sepsis progression and poor prognosis.

Carboxylesterase-1 Assisted Targeting of HDAC Inhibitors to Mononuclear Myeloid Cells in Inflammatory Bowel Disease.

Background and AimsHistone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].MethodsCES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn’s disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.Results CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.ConclusionsWe demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Novel Sca-1+ macrophages modulate the pathogenic progress of endotoxemia

Macrophages are important innate immune cells that play crucial roles in inflammatory responses. Accumulating evidence has demonstrated macrophage heterogeneity based on biomarkers, functions, and localization. Here, we report a novel stem cell antigen-1 (Sca-1)-positive macrophage population induced in the pathological conditions caused by lipopolysaccharide (LPS). Sca-1 is only upregulated in macrophages but not in monocytes and neutrophils upon LPS injection. Sca-1+ macrophages develop from resident peritoneal macrophages. LPS-induced Sca-1+ macrophage generation was partly blocked by anti–IFN–γ antibody, suggesting a role of IFN-γ in the process. LPS-stimulated production of IL-6, TNF-α, and CCL2 is significantly lower in Sca-1+ macrophages compared to their counterpart Sca-1- macrophages. Depletion of Sca-1+ macrophages using anti-Sca-1 antibody significantly increased survival rate and reduced lung and kidney damage in an LPS-induced sepsis model. Taken together, we discovered a novel population of Sca-1+ macrophages in LPS-induced septic conditions.

A wheat germ-derived peptide YDWPGGRN facilitates skin wound-healing processes

Wheat germ derivatives have been shown to inhibit inflammation-related diseases. In this study, a small peptide (YDWPGGRN) isolated from wheat germ was used to study its anti-inflammatory activity and its application in skin wound healing. Both the in vitro and in vivo results clearly showed that YDWPGGRN significantly inhibited the LPS-stimulated NO, IL-1β, IL-6 and TNF-α production but promoted the release of an anti-inflammatory cytokine, IL-10. In addition, YDWPGGRN directly enhanced the proliferation and migration of HaCaT cells and L929 cells. Furthermore, the results demonstrated that YDWPGGRN was able to stimulate angiogenesis and collagen production in wound areas, consequently accelerating the skin wound-healing processes in a rat model with a full thickness dermal wound. The current findings suggest that YDWPGGRN promotes wound healing by anti-inflammatory reactions and enhances the proliferation and migration of keratinocytes and fibroblasts; therefore, it may be applicable for skin wound therapeutics.

Different signaling pathways involved in the anti-inflammatory effects of unfractionated heparin on lipopolysaccharide-stimulated human endothelial cells

BackgroundThere is a complex interplay between inflammatory response and coagulation in sepsis. Heparin is used as a recognized anticoagulant and possesses multiple biological properties that possibly affect sepsis. This study aimed to determine the possible signaling pathways involved in the anti-inflammatory effects of unfractionated heparin (UFH) on lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells (HPMECs).MethodsHPMECs were transfected with siRNA targeting IκB-α. Cells were treated with UFH (0.01 U/ml~ 10 U/ml) 15 min before adding LPS (10 μg/ml). We detected the markers of systemic inflammatory response. Release of interleukin (IL)-6, IL-8 were evaluated at 3 h by ELISA and at 1 h by qRT-PCR. After 1 h, nuclear factor-κB (NF-κB) as well as phosphorylated inhibitor κB-α (IκB-α), signal transducer and activator of transcription-3 (STAT3) and ERK1/2, JNK, p38 mitogen-activated protein kinase (MAPK) expressions were evaluated by Western blot. DNA binding was conducted to further prove the activation of NF-κB pathway.ResultsIn HPMECs, UFH obviously inhibited LPS-stimulated production of IL-6 and IL-8, especially in 10 U/ml. UFH inhibited LPS-induced phosphorylation of IκB-α, ERK1/2, JNK, p38 MAPK and STAT3. UFH also suppressed LPS-stimulated nuclear translocation of NF-κB. Importantly, transfection with siRNA targeting IκB-α induced more obvious inflammatory response. UFH suppressed cytokines production and phosphorylation of different signaling pathways in IκB-α silencing cells.ConclusionThese results demonstrate that UFH exerts the anti-inflammatory effects on LPS-stimulated HPMECs by different signaling pathways.

Combined Ibrutinib and Venetoclax Changes Myeloid Phenotype and Improves Immune Function in CLL Patients

Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through recognition and phagocytosis of pathogens, and mediation of acute inflammation through secretion of pro-inflammatory cytokines. Furthermore, monocytes constitute an important link between innate and adaptive immunity through antigen presentation to lymphocytes. Patients with chronic lymphocytic leukemia (CLL) have an increased risk of infection, despite often displaying normal neutrophil and monocyte counts. CLL-cells depend on interactions with the immune microenvironment (IME) for proliferation and survival, while inducing changes in surrounding immune cells. However, changes in myeloid cell function in patients with CLL remain sparsely documented. Since the btk-inhibitor ibrutinib impairs B-cell receptor signaling and disrupts CLL-IME interactions (Niemann et al, CCR, 2016), we investigated the impact of ibrutinib and venetoclax on monocyte- and neutrophil phenotype and function in CLL patients. Methods: Nine patients treated with ibrutinib 420 mg daily for 8 weeks followed by addition of venetoclax with an initial 5-week ramp up period were included. Blood samples were taken at baseline, after 8 weeks ibrutinib monotherapy, and after another 8 weeks of ibrutinib and venetoclax combination therapy. Immune phenotype was assessed in whole blood by an 8-tube, 10 color flow cytometry panel with custom designed lyophilized antibodies (Duraclone). Immune function was characterized using TruCulture, a whole blood-ligand stimulation assay applying the toll like receptor (TLR) ligands heat killed candida albicans (TLR 2,4,6), lipopolysaccharide (LPS; TLR4), resiquimod (single-stranded RNA-virus analog, TLR7,8), and Poly:IC (double-stranded RNA-virus analog, TLR3), after which the cytokine response was measured. Informed consent from patients and approval from the ethics committee was obtained. Results: Monocyte and neutrophil counts, as wells as distribution of mature and immature neutrophils, were within normal range at baseline (n=9) and remained unchanged throughout treatment. At baseline, expression of HLA-DR on monocytes was suppressed, but increased significantly upon combination treatment with ibrutinib and venetoclax (p=0.04). HLA-DR expression on neutrophils was high at baseline, remained unchanged upon ibrutinib treatment (n=8), but declined after addition of venetoclax (n=7) (p<0.01). LPS-stimulated TNF-α and IL-6 production was suppressed at baseline, IL-6 levels increased significantly upon ibrutinib monotherapy, and levels of both TNF-α and IL-6 almost normalized upon addition of venetoclax to ibrutinib (p=0.02 for TNF-α, p=0.009 for IL-6). Conclusion: Here we show for the first time that myeloid leukocytes in CLL patients exhibit an altered HLA-DR expression at baseline, which is paralleled by an impaired cytokine response to LPS stimulation. Following treatment with ibrutinib and venetoclax, HLA-DR expression increased on monocytes and decreased on neutrophils, both representing normalization. In parallel, LPS-induced TNF-α and IL-6 production improved, likely reflecting an improved monocytic function, as monocytes respond strongly to whole-blood LPS-stimulation. Increased monocyte HLA-DR expression has previously been linked to improved cytokine response (Belge et al, J Immunol, 2002). The contribution of neutrophils to the improved cytokine response is unclear, since the function of HLA-DR expressing neutrophils in CLL is largely unknown. We propose a model where ibrutinib and venetoclax eradicate CLL-cells, disrupt tumor-IME interactions, and maybe directly impact microenvironmental cell signaling, thereby restoring neutrophil and monocyte function and improving immune function. Further investigation of myeloid function in CLL at baseline and upon different treatment regimens is warranted to guide personalized treatment based on modulation of infection-risk. Disclosures Svanberg: Novo Nordisk Foundation: Research Funding; Abbvie: Other: Travel grant. Kater:Genentech: Research Funding; Roche: Other: Travel funding, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Niemann:Acerta: Consultancy, Research Funding; Roche: Other: Travel grant; Astra Zeneca: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding; CSL Behring: Consultancy; Janssen: Consultancy, Other: Travel grant, Research Funding; Gilead: Other: Travel grant; Sunesis: Consultancy; Abbvie: Consultancy, Other: Travel grant, Research Funding.

Fish oil supplementation with various lipid emulsions suppresses in vitro cytokine release in home parenteral nutrition patients: a crossover study

Long-chain n-3 polyunsaturated fatty acids modulate immune cell functions. The primary objective of this study was to evaluate the impact of different lipid emulsions (LEs) with supplemented doses of fish oil (FO) on serum cytokine concentration and in vitro cytokine production in patients with intestinal failure on home parenteral nutrition (HPNPs). We hypothesized that FO supplementation would diminish lipopolysaccharide (LPS)-stimulated cytokine production. Twelve HPNPs receiving Smoflipid for at least 3 months were given FO (Omegaven) for a further 4 weeks. After this cycle, the patients were randomized to subsequently receive 1 cycle with Lipoplus and 1 cycle with ClinOleic for 6 weeks or vice versa plus 4 weeks of added Omegaven after each cycle in a crossover design. Comparison of the baseline LE regimens showed lower LPS-stimulated production of IL-1β in the HPNPs on Lipoplus than on the Smoflipid and ClinOleic regimens, as well as lower IL-8 compared to the Smoflipid regimen. Omegaven reduced IL-8 concentration in serum under the Lipoplus regimen and diminished LPS-stimulated production of IL-1β under the Smoflipid and ClinOleic. IL-6 and TNF-α production was depressed only in those on Smoflipid. Irrespective of the LE used, the HPNPs compared to the healthy controls showed higher IL-6, IL-8, and TNF-α concentrations in serum and LPS-stimulated production of IL-6 as well as lower n-6/n-3 long-chain polyunsaturated fatty acids in the erythrocyte phospholipids. LPS-stimulated production of IL-6 correlated negatively with the parenteral dose of eicosapentaenoic acid + docosahexaenoic acid. In conclusion, FO-supplemented parenteral nutrition suppresses in vitro cytokine production.

Linagliptin Inhibits Lipopolysaccharide-Induced Inflammation Concentration-Dependently And -Independently.

PurposeDipeptidyl peptidase-4 inhibitors, including linagliptin, prevent inflammation. However, the in vitro effects of linagliptin are unclear. Moreover, although linagliptin inhibits lipopolysaccharide (LPS)-induced inflammation, the anti-inflammatory effects of linagliptin in this context are not concentration-dependent. In the absence of LPS-binding protein (LBP), the pro-inflammatory effects of LPS involve pathways other than the Toll-like receptor (TLR) 4 pathway. Here, we aimed to determine the anti-inflammatory mechanisms of linagliptin in an experimental model in which LBP was added to the medium.MethodsHuman U937 monocytes were cultured at 1 × 106 cells/mL in Roswell Park Memorial Institute medium and differentiated into macrophages using phorbol myristate acetate. All processes were carried out in medium containing 10% fetal bovine serum (FBS). After 48 hrs of culture, we replaced the medium and pretreated the cells with 100, 250, 500, or 2500 nM linagliptin for 1 hr. We exchanged the medium again, and the cells were treated with 1 ng/mL LPS with or without 100, 250, 500, or 2500 nM linagliptin. Interleukin (IL)-6 and LBP in the supernatant, nuclear factor (NF)-κB/p65 in the nucleus, and reactive oxygen species (ROS) in the cells, as important markers of the mechanism of inflammation induction by LPS, were measured using enzyme-linked immunosorbent assay kits.ResultsLinagliptin significantly prevented LPS-stimulated IL-6 production and intranuclear NF-κB/p65 levels in a concentration-dependent manner. LPS-induced intracellular ROS levels were significantly decreased by linagliptin at all concentrations. LBP levels were markedly higher in FBS-containing medium than in medium without FBS. However, LBP levels did not change following administration of linagliptin and/or LPS.ConclusionConcentration-dependent and -independent inflammatory suppression was observed following linagliptin treatment in the context of LPS-induced pro-inflammatory responses. Thus, our findings suggested that linagliptin induced two different mechanisms to repress inflammation, i.e., TLR4-dependent and -independent mechanisms.

The Effects of Noninvasive Vagus Nerve Stimulation on Fatigue and Immune Responses in Patients With Primary Sjögren’s Syndrome

Primary Sjögren's syndrome (pSS) sufferers have rated chronic fatigue as the most important symptom needing improvement. Emerging data suggest that stimulation of the vagus nerve can modulate immunological responses. The gammaCore device (electroCore), developed to stimulate the cervical vagus nerve noninvasively, was used to assess the effects of vagus nerve activation on immune responses and clinical symptoms of pSS. Fifteen female pSS subjects used the nVNS device twice daily a 26-day period. At baseline, blood was drawn before and after application of the gammaCore device for 90 sec over each carotid artery. The following fatigue-related outcome measures were collected at baseline, day 7 and day 28: EULAR patient reported outcome index, profile of fatigue (Pro-F), visual analogue scale of abnormal fatigue, and Epworth sleepiness scale (ESS). Whole blood samples were stimulated with 2 ng/mL lipopolysaccharide (LPS) and the supernatant levels of IFNγ, IL12-p70, TNFα, MIP-1α, IFNα, IL-10, IL-1β, IL-6, and IP-10 were measured at 24 hours. In addition, clinical hematology and flow cytometric profiles of whole blood immune cells were analyzed. Pro-F and ESS scores were significantly reduced across all three visits. LPS-stimulated production of IL-6, IL-1β, IP-10, MIP-1α, and TNFα were significantly reduced over the study period. Patterns of NK- and T-cell subsets also altered significantly over the study period. Interestingly, lymphocyte counts at baseline visit correlated to the reduction in fatigue score. The vagus nerve may play a role in the regulation of fatigue and immune responses in pSS and nVNS may reduce clinical symptoms of fatigue and sleepiness. However, a sham-controlled follow-up study with a larger sample size is required to confirm the findings.

Retraction of "Development of Novel Adenosine Monophosphate-Activated Protein Kinase Activators".

In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)gamma-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidinedione-based focused compound library to identify novel agents with these dual pharmacological activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homologue of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase, respectively) and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human macrophages were used to screen this compound library, which led to the identification of compound 53 (N-{4-[3-(1-methyl-cyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoro-methyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore, compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells, indicating that it is not a cell line-specific event.

Associations of postpartum sleep, stress, and depressive symptoms with LPS-stimulated cytokine production among African American and White women

BackgroundPostpartum is a period of unique psychosocial stress characterized by sleep disturbance, risk for depressed mood, and heightened parenting stress. However, data on effects of these exposures on inflammatory immune function are limited. MethodsThis study examined associations among sleep, psychosocial stress (i.e., parenting stress, general perceived stress), mood (i.e., depressive symptoms), serum cytokine levels, and LPS-stimulated proinflammatory cytokine production among 69 women (32 African American, 37 White) assessed at 7–10weeks postpartum. ResultsNo associations between behavioral measures and serum cytokine levels were observed among women of either race. In African American women, but not Whites, poorer sleep quality, greater parenting stress, and greater depressive symptoms were associated with greater LPS-stimulated IL-6 and IL-8 production (ps≤0.05). Also in African Americans, greater general perceived stress was associated with greater IL-8 production, and greater depressive symptoms with greater stimulated TNF-α production (ps≤0.05). Simple mediation models highlighted the bidirectional relationship between stress and sleep in relation to inflammation among African American women. ConclusionsSignificant effects of both stress/distress and poor sleep quality on proinflammatory cytokine production during postpartum were observed uniquely among African American women. These data are consistent with an allostatic load model which predicts that conditions of chronic stress impart vulnerability to dysregulated responses to novel stressor exposures. The bidirectional nature of the stress-sleep relationship has clinical relevance. Studies examining whether interventions focused on one or both of these psychological factors during postpartum is beneficial for inflammatory profiles would be informative. In addition, examination of these models in relation to maternal health at postpartum, including delivery related wounds and other infections, is warranted.

Immunological effects of behavioral activation with exercise in major depression: an exploratory randomized controlled trial

Major depression (MD) is associated with peripheral inflammation and increased cardiovascular risk. Regular physical exercise can have anti-inflammatory effects. The present study examined whether behavioral activation with exercise affects inflammatory processes in MD. Ninety-eight patients with MD were randomly assigned to cognitive-behavioral therapy (CBT) emphasizing exercise during behavioral activation (CBT-E), CBT with pleasurable low-energy activities as an active control condition (CBT-C) or a passive waiting list control group (WL). Plasma levels of C-reactive protein (CRP), interleukin (IL)-6, IL-10, lipopolysaccharide (LPS)-stimulated IL-6 production, and blood immune cell counts were analyzed at baseline and weeks 8 (post-behavioral activation) and 16 (post-treatment). Thirty non-depressed age- and sex-matched controls were included to examine potential immunological alterations in MD at baseline. Patients with MD exhibited higher levels of CRP, higher neutrophil and monocyte counts, lower IL-10 levels and reduced LPS-stimulated IL-6 production compared to controls (P<0.001−0.045). Multilevel modeling indicated that CBT-E was associated with increased anti-inflammatory IL-10 at weeks 8 and 16 compared to CBT-C and WL (P=0.004−0.018). CBT-E did not significantly affect other immunological makers in the total sample. A subgroup analysis including patients with potentially higher cardiovascular risk (CRP ⩾1 μg ml−1) indicated that CRP was reduced in CBT-E compared to CBT-C (P<0.007) and marginally reduced compared to WL (P<0.085) after week 16. The present findings provide new insights into immunological effects of behavioral treatments against depression. Behavioral activation in conjunction with exercise may have the potential to reverse, in part, immunological alterations in MD.

Astragaloside IV Alleviates Lipopolysaccharide-Induced Acute Kidney Injury Through Down-Regulating Cytokines, CCR5 and p-ERK, and Elevating Anti-Oxidative Ability

BackgroundAstragaloside IV (AS-IV) has been shown to prevent ischemia-induced acute kidney injury (AKI) in rat models of ischemia and reperfusion. However, the effects of AS-IV on AKI during sepsis and endotoxinemia is unclear. The current study aimed to investigate the effects and molecular mechanisms of AS-IV on lipopolysaccharide (LPS)-induced AKI.Material/MethodsAdult male CD-1 mice were randomly assigned into 6 groups (n=8/group): control group: mice were intraperitoneally (i.p.) injected with normal saline; LPS group (10 mg/kg, i.p.); low-dose AS-IV (25 mg/kg; gavage for 7 days) + LPS (i.p., 1 hour after last gavage) group; medial-dose AS-IV (50 mg/kg) + LPS group; high-dose AS-IV (100 mg/kg) + LPS group; high-dose AS-IV alone (100 mg/kg; gavage for 7 days) group. Blood samples were collected at 24 hours after LPS injection, and plasma uric acid and BUN were measured with colorimetric detection kits. The concentration of plasma tumor necrosis factor (TNF)-α and interleukin 1β, renal p-extracellular signal-regulated kinases, and urinary albumin were evaluated by ELISA. The expression of CCR5 in renal tissue was evaluated by PCR and Western blotting. Concentrations of glutathione (GSH) and reactive oxygen species (ROS) in renal tissue were also measured.ResultsAS-IV decreased LPS-stimulated production of blood TNF-α and IL-6, LPS-induced the expression of CCR5, and activation of ERK in the kidneys in a rodent model of endotoxinemia. AS-IV attenuated LPS-caused decreased GSH and increased ROS. It also attenuated LPS-induced increases in plasma uric acid, BUN, and urinary albumin.ConclusionsAS-IV protects against AKI during bacterial endotoxinemia by attenuating expression of cytokines, CCR5, and p-ERK, and elevating anti-oxidative ability.

In vivo and in vitro anti-inflammatory effects of ethanol fraction from Periploca forrestii Schltr.

To determine the anti-inflflammatory effects of an ethanol fraction of Periploca forrestii Schltr. (EFPF) and to investigate the potential mechanisms underlying in vivo and in vitro models. The antiinflflammatory effects of EFPF were evaluated using the xylene-induced mouse ear edema and carrageenan-induced rat paw edema models in vivo. In vitro, RAW264.7 cells were exposed to 0-800 μg/mL EFPF and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then cells were treated with different concentrations of EFPF (100-400 μg/mL) and stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 24 h. The supernatant was analyzed for nitric oxide (NO) using the Griess reagent, and the levels of inflflammatory mediators and cytokines were determined using enzyme-linked immunosorbent assays for prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin (IL) 6, and IL-10. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor κB (NF-κB), and mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK were examined by Western blot. Compared with the control group, EFPF signifificantly reduced mouse ear edema and rat paw edema rate (P<0.05 or P<0.01). Compared with the LPS group, EFPF signifificantly inhibited the LPS-stimulated production of NO, PGE2, TNF-α and IL-6 (P<0.05 or P<0.01), and increased the IL-10 production (P<0.05). EFPF also signifificantly inhibited LPS-induced protein expressions of iNOS and COX-2, suppressed the phosphorylation and degradation of inhibitor of NF-κB-α, decreased p65 level, and inhibited the phosphorylation of p38, ERK1/2 and JNK P<0.05 or P<0.01). EFPF exerted anti-inflflammatory effect by reducing protein expressions of iNOS and COX-2 and the production of the inflflammation factors, including TNF-α, IL-6, NO and PGE2, mainly through inhibition of LPS-mediated stimulation of NF-κB and MAPK signaling pathways.

Sodium houttuyfonate inhibits LPS-induced inflammatory response via suppressing TLR4/NF-ĸB signaling pathway in bovine mammary epithelial cells.

Mastitis is an inflammatory reaction caused by microorganisms in the mammary gland which usually leads to the decrease of the dairy production. It vastly makes bad effect on the cattle industry all over the world. Nowadays, an increasing number of scientists keep a watchful eye on natural compounds to prevent mastitis. Sodium houttuyfonate (SH) has been reported to have antibacterial and anti-inflammatory effects. This experiment aimed to investigate the anti-inflammatory properties of SH on LPS-stimulated primary bovine mammary epithelial cells (bMEC). The effects of SH on TNF-α, IL-1β, and IL-6 production were detected by qRT-PCR. Western blot analysis was used for detecting the effects of SH on TLR4/NF-κB signal pathways. The results showed that SH significantly inhibited LPS-stimulated TNF-α, IL-1β and IL-6 production. Furthermore, SH significantly inhibited LPS-induced TLR4 expression and NF-ĸB activation. In summary, these results suggested that SH inhibited LPS-induced inflammatory response by inhibiting TLR4/NF-ĸB signaling pathway. SH is a potential agent for the treatment of mastitis.

Downregulated expression of miR-142-3p in macrophages contributes to increased IL-6 levels in aged mice.

Macrophages are innate immune cells that are important contributors to age-related functional impairment of the immune system. During the cell aging process, microRNAs are differentially expressed and participate in the regulation of aging-related immune responses. However, the role of aging-associated changes in miRNA expression in macrophages remains unclear. Here, we found that miR-142-3p expression is downregulated 50% in peritoneal macrophages from aged mice compared with young mice and is not upregulated by cell treatment with lipopolysaccharide (LPS), CpG, or polyinosinic-polycytidylic acid. Serum levels of miR-142-3p are also lower in aged mice than in young mice by q-PCR. Luciferase reporter analysis showed that IL-6 is a target of miR-142-3p in macrophages. In addition, the histone deacetylase inhibitor trichostatin A increased miR-142-3p expression by more than 3-fold in LPS-treated macrophages from aged mice compared with young mice, which in turn suppressed LPS-stimulated IL-6 production, suggesting that inhibition of miR-142-3p by histone deacetylation may be involved in the lack of response to LPS stimulation in macrophages of aged mice. These findings suggest that downregulation of miR-142-3p in macrophages of aged mice might contribute to IL-6-associated aging disorders and that epigenetic modification might be involved in age-related inflammatory diseases.

Abstract # 1820 Stimulated cytokine production is heightened among pregnant women carrying female versus male fetuses

Fetal sex plays a role in regulating maternal adaptation across pregnancy including glycemic control, blood pressure, and cortisol. However, data examining effects of fetal sex on maternal immune parameters is lacking, despite observed differences in maternal health responses and pregnancy outcomes in relation to fetal sex. In the current study, serum levels of IL-6, IL-8, and TNF-alpha as well as LPS-stimulated production of IL-6, IL-8, TNF-alpha, and IL-1 beta by PBMCs incubated for 24 h were assessed during each trimester of pregnancy in 80 women (46 with male and 34 with female fetuses). Linear mixed models showed that women carrying females versus males exhibited heightened stimulated production of IL-6 during each trimester (ps 0.85). In conclusion, in pregnant women, those carrying female versus male fetuses exhibit greater stimulated cytokine production. Heightened inflammation could affect maternal health and fetal development. Fetal sex should be considered as a factor in studies of maternal inflammation. Future investigations focused on women with health conditions (e.g., preeclampsia) and adverse pregnancy outcomes (e.g., preterm birth) would be informative.

Fetal sex is associated with maternal stimulated cytokine production, but not serum cytokine levels, in human pregnancy

Some studies suggest that fetal sex plays a role in maternal physiological processes during pregnancy including glycemic control, blood pressure, and cortisol regulation. However, data examining fetal sex-specific differences in maternal immune parameters is lacking. In the current study, serum levels of interleukin(IL)-6, IL-8, and tumor necrosis factor(TNF)-α as well as LPS-stimulated production of IL-6, IL-8, TNF-α, and IL-1β by PBMCs incubated for 24h were assessed in early, mid, and late pregnancy among 80 women (46 with male and 34 with female fetuses). Linear mixed models showed that women carrying females versus males exhibited greater stimulated production of IL-6 at each timepoint (ps⩽0.03), TNF-α in early pregnancy (p=0.04), and IL-1β in mid- and late pregnancy (ps⩽0.05). Despite changes in serum levels of IL-8 (p=0.002) and TNF-α (p<0.0001) across pregnancy, no differences in any serum cytokines were observed in relation to fetal sex (ps>0.85). In conclusion, in pregnant women, those carrying female versus male fetuses exhibited greater stimulated cytokine production across pregnancy. Differential inflammatory responses could affect maternal health and fetal development. Fetal sex should be considered as a factor in studies of maternal inflammation. These findings have relevance both clinically and conceptually. For example, maternal asthma is exacerbated among women carrying female versus male fetuses. In addition, data on associations between fetal sex and maternal immune function among women with health conditions (e.g., preeclampsia) and adverse pregnancy outcomes (e.g., preterm birth) would be informative.

Linagliptin inhibits lipopolysaccharide-stimulated interleukin-6 production, intranuclear p65 expression, and p38 mitogen-activated protein kinase phosphorylation in human umbilical vein endothelial cells

Linagliptin, the only bile-excreted dipeptidyl peptidase-4 (DPP-4) inhibitor, is a therapeutic drug for patients with diabetes receiving hemodialysis, for whom inflammation is a prognosis-related factor, because of its potential anti-inflammatory effects. Although the anti-inflammatory effects of linagliptin in vivo are reported, no study has described these effects in vitro. DPP-4 degrades glucagon-like peptide-1 (GLP-1), which is known to have anti-inflammatory properties. Since GLP-1 is a gut hormone secreted by intestinal L cells, in vivo examination of the GLP-1-independent anti-inflammatory effects of DPP-4 inhibitors is difficult. We evaluated the mitogen-activated protein kinase (MAPK)-dependent, GLP-1-independent, anti-inflammatory effects of linagliptin in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) which do not secrete GLP-1. Furthermore, to determine whether linagliptin has unique pharmacological actions compared with other DPP-4 inhibitors, we assessed the anti-inflammatory effects of sitagliptin (a DPP-4 inhibitor without xanthine-related skeletal system activity), caffeine (a phosphodiesterase inhibitor), loxoprofen, and diclofenac sodium. HUVECs were cultured for 24 h at densities of 1–2 × 105 cells/mL. We pretreated HUVECs with or without linagliptin (1, 5, 10, 50, and 100 nM), 150 nM sitagliptin, 50 nM caffeine, 17 μM loxoprofen, or 1.3 μM diclofenac sodium for 1 h prior to incubation with LPS. The concentration of LPS used (1 μg/mL) was sufficient to induce an inflammatory response in HUVECs. Five hours after incubation with LPS, culture media was evaluated for interleukin (IL)-6 expression. Intranuclear p65 (a subunit of nuclear factor kappa-B (NFκB)) levels were measured 5 h after treatment with LPS and 50 nM linagliptin, while phosphorylated p38 MAPK levels were measured in the cytosolic fractions obtained 30 min after treatment with LPS and 50 nM linagliptin. Linagliptin significantly inhibited LPS-stimulated IL-6 production, intranuclear p65 expression, and p38 MAPK phosphorylation. Treatment with sitagliptin, caffeine, loxoprofen, and diclofenac sodium significantly inhibited LPS-stimulated IL-6 production. The results of this study demonstrate the GLP-1-independent anti-inflammatory effects of linagliptin via MAPK-dependent mechanisms in vitro. Our findings suggest that linagliptin will play a crucial role in the treatment of hemodialysis (HD) patients with diabetes and chronic inflammation.