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The ocular cytokine network plays pivotal roles in terms of the initiation and progression of retinal degeneration. Several types of immunocompetent cells such as microglia participate in inflammation, and a temporal transition in the molecular events of inflammation has been hypothesized. We previously found that the Csf2 gene was induced in the early phase of retinal degeneration. CSF2 participates in the transcriptional activation of several cytokines expressed by microglia; however, whether CSF2 is essential in this context is not known. In this work, we approach this question by using anti-CSF2 neutralizing bntibody and the protein synthesis inhibitor cycloheximide (CHX). We first revealed that CSF2 positively regulated the cytokine induction cascade using a CSF2-neutralizing antibody (anti-CSF2) to treat the microglial cell line that were activated by lipopolysaccharide (LPS). LPS or Lipid A stimulation in the presence of the protein synthesis inhibitor cycloheximide (CHX) led to cytokine superinduction, but suppression of the expression of a few cytokines was also noted in MG5 cells. To examine transitions of the molecular events within LPS-activated microglia, we next performed proteome analysis of MG5 cells stimulated with LPS for 0, 4, and 9 h. The Database for Annotation, Visualization, and Integrated Discovery analysis of differentially expressed proteins showed that various mRNA-modifying molecules were induced after LPS stimulation, in addition to molecules involved in inflammation. However, the numbers of common proteins founded in the comparison between the induced proteins of 4 and 9 h were only one-third and one-half of induced proteins at 4 and 9 h, respectively, suggesting dynamic transition of the induced proteins. LPS-induced mRNA-modifying proteins were almost completely suppressed by CHX, as expected, suggesting that transient induction of transcription-editing proteins plays an important role in terms of the phenotype of inflammation that develops in microglia after LPS stimulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: We aim to establish an LPS-induced human aortic endothelial cells (HAECs) inflammatory injury model and explore the optimal conditions for inducing its injury. We expect to provide modeling references for the related experiments of vascular inflammatory diseases., Methods: HAECs were cultured in vitro and treated with different concentrations of lipopolysaccharide (LPS) (0.1, 1, 10, 50, 100 μg/mL) for 6, 12, and 24 h to establish the HAECs inflammatory injury model. The cell viability was determined by CCK-8 assay; the expression levels of inflammatory cytokines in the cells were detected by RT-PCR；the apoptosis rate of the cells was detected by flow cytometry., Results: ① Within 24 h of LPS treatment, the cell viability of the 0.1 and 1 μg/mL groups showed an overall increasing trend with time, while the cell viability of the 10, 50, and 100 μg/mL groups increased first and then decreased with time, and the cell viability of 50 and 100 μg/mL groups was significantly lower than the normal control group at 24 h (P<0.01). ② RT-PCR results showed that after 50 and 100 μg/mL LPS for 24 h, the inflammatory cytokines all showed an apparent upward trend compared with the normal control group (P<0.05), which was more significant in the 100 μg/mL group. ③ After 100 μg/mL LPS for 24 h, the apoptotic necrosis rate of HAECs was higher than the normal control group (P<0.01)., Conclusions: This experiment successfully established a HAECs injury model, indicating that the optimal conditions for inducing injury are an LPS concentration of 100 μg/mL and a treatment time of 24 h., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Maternal immune activation results in altered levels of cytokines in the maternal-fetal system, which has a negative impact on fetal development, including the gonadotropin-releasing hormone (GnRH) system, which is crucial for the reproduction. Suppression of GnRH-neuron migration may be associated with cytokine imbalances, and primarily with proinflammatory cytokine interleukin (IL)-6. This study aimed to determine the effects of IL-6 and monoclonal antibody to IL-6 or IL-6R or polyclonal IgG on the formation of migration route of GnRH-neurons in ex vivo and in vivo rodent models on day 11.5 of embryonic development. The increased level of IL-6 in mouse nasal explants suppressed peripherin-positive fiber outgrowth, while this led to an increase in the number of GnRH-neurons in the nose and olfactory bulbs and a decrease in their number in the fetal brain. This effect is likely to be realized via IL-6 receptors along the olfactory nerves. The suppressive effect of IL-6 was diminished by monoclonal antibodies to IL-6 or its receptors and by IgG.

Effects of Holstein genotype on innate immune response were assessed with ex-vivo lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation of whole blood from unselected (UH, n = 10) and contemporary (CH, n = 11) Holsteins that differ in production by more than 4,500 kg/lactation. Blood was collected at -14, 7, 28, and 49 days in milk (DIM), mixed with a pathogen-associated molecular pattern (PAMP) molecule (0.01 or 1.0 µg LPS or 10 or 100 µg LTA per mL blood) and incubated (4 h, 37 °C). Plasma cytokines were quantified by ELISA, log 10 -transformed and analyzed by repeated measures with DIM as the repeated effect. Cytokine responses increased with PAMP dose and decreased as DIM increased. There was a genotype by LPS dose interaction for IL-1β as response to the low dose was greater in UH but did not differ between genotypes for the high dose. The IL-1β response was greater while the IL-6 response to LTA tended to be greater in UH than in CH cows. The more negative energy balance of CH cows did not impact genotype difference in cytokine responses. Results indicate selection since the mid-1960s has decreased ex-vivo, whole blood cytokine response of CH cows to LPS and to LTA., (Copyright © 2022. Published by Elsevier B.V.)

We investigated the immune response mechanisms under systemic and local inflammation using mouse models whereby lipopolysaccharide (LPS) was administered intraperitoneally to induce systemic inflammation, and epicutaneous sensitization with ovalbumin was used to induce local inflammation. LPS increased the immune cell infiltration in the cardiac muscle near the aorta, alveoli, hepatic sinusoid, renal interstitium, and the submucosal layer of the duodenum. Similarly, ovalbumin increased the abundance of macrophages in the skin. Both LPS and ovalbumin induced NF-κB p65 and IκBα phosphorylation, as well as the expression of NF-κB target genes ( TLR4 , IL6 , and TNF ). Additionally, both LPS and ovalbumin led to an increase in the absolute IL-1β, IL-6, and TNFα serum levels and cytokine-related janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) phosphorylation. Moreover, the activated JAK2/STAT3 signaling increased the number of Ki67-positive cells (proliferating cells) and development pathway target gene expression (regeneration) in the inflammation models. In conclusion, LPS and ovalbumin increase immune cell infiltration in tissues, NF-κB activation, cytokine levels in serum, cytokine-stimulated JAK2/STAT3 signaling, and tissue regeneration.α ). Additionally, both LPS and ovalbumin led to an increase in the absolute IL-1β, IL-6, and TNFα serum levels and cytokine-related janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) phosphorylation. Moreover, the activated JAK2/STAT3 signaling increased the number of Ki67-positive cells (proliferating cells) and development pathway target gene expression (regeneration) in the inflammation models. In conclusion, LPS and ovalbumin increase immune cell infiltration in tissues, NF-κB activation, cytokine levels in serum, cytokine-stimulated JAK2/STAT3 signaling, and tissue regeneration.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes ( NFκB , iNOS , and IL-1β ), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

Background: Noninfectious complications are the greatest cause of morbidity and mortality in common variable immunodeficiency (CVID), but their pathogenesis remains poorly defined., Objective: Using high-throughput approaches, we aimed to identify, correlate, and determine the significance of immunologic features of CVID with noninfectious complications (CVIDc)., Methods: We simultaneously applied proteomics, RNA sequencing, and mass cytometry to a large cohort with primary antibody deficiency., Results: CVIDc is differentiated from uncomplicated CVID, other forms of primary antibody deficiency, and healthy controls by a distinct plasma proteomic profile. In addition to confirming previously reported elevations of 4-1BB, IL-6, IL-18, and IFN-γ, we found elevations of colony-stimulating factor 1, IL-12p40, IL-18R, oncostatin M, TNF, and vascular endothelial growth factor A to differentiate CVIDc. This cytokine dysregulation correlated with deficiency of LPS-specific antibodies and increased soluble CD14, suggesting microbial translocation. Indicating potential significance of reduced LPS-specific antibodies and resultant microbial-induced inflammation, CVIDc had altered LPS-induced gene expression matching plasma proteomics and corresponding with increased CD14 + CD16 - monocytes, memory T cells, and tissue inflammation ameliorated by T-cell-targeted therapy. Unsupervised machine learning accurately differentiated subjects with CVIDc and supported cytokine dysregulation, antibody deficit, and T-cell activation as defining and convergent features., Conclusions: Our data expand understanding of CVIDc proteomics, establish its link with deficiency of IgA and LPS-specific antibodies, and implicate altered LPS-induced gene expression and elevated monocytes and T cells in this cytokine dysregulation. This work indicates that CVIDc results when insufficient antibody neutralization of pathogen-associated molecular patterns, like LPS, occurs in those with a heightened response to these inflammatory mediators, suggesting a 2-hit model of pathogenesis requiring further exploration., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Macrophages (MΦ) are known to exhibit distinct responses to viral and bacterial infection, but how they react when exposed to the pathogens in succession is less well understood. Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by an LPS-induced challenge on cytokine production, signal transduction and metabolic pathways in human GM (M1-like)- and M (M2-like)-MΦ. We found that infection of both subsets with RV resulted in a low TNF-α and a high interferon (IFN, type I and type III) release whereby M-MΦ produced far more IFNs than GM-MΦ. Thus, TNF-α production in contrast to IFN production is not a dominant feature of RV infection in these cells. Upon addition of LPS to RV-infected MΦ compared to the addition of LPS to the uninfected cells the TNF-α response only slightly increased, whereas the IFN-response of both subtypes was greatly enhanced. The subset specific cytokine expression pattern remained unchanged under these assay conditions. The priming effect of RV was also observed when replacing RV by IFN-β one putative priming stimulus induced by RV. Small amounts of IFN-β were sufficient for phosphorylation of Stat1 and to induce IFN-production in response to LPS. Analysis of signal transduction pathways activated by successive exposure of MΦ to RV and LPS revealed an increased phosphorylation of NFκB (M-MΦ), but different to uninfected MΦ a reduced phosphorylation of ERK1/2 (both subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show that RV infection and exogenously added IFN-β can prime MΦ to produce high amounts of IFNs in response to LPS and that changes in glycolysis and signal transduction are associated with the priming effect. These findings will help to understand to what extent MΦ defense to viral infection is modulated by a following exposure to a bacterial infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schilling, Grahnert, Pfeiffer, Koehl, Claus and Hauschildt.)

Indoprofen is a non-steroidal anti-inflammatory drug, and has provided insights into treatment of spinal muscular atrophies; however, the treatment effect of indoprofen on sepsis and the precise underlying mechanism remain to be elucidated. This study was carried out to examine the inhibitory effect of indoprofen on high mobility group box 1 (HMGB1)-mediated inflammatory responses in vivo and in vitro. Intraperitoneal injection of indoprofen (20 or 40 mg/kg) at 8 h post-sepsis markedly improved the survival of BALB/c mice and ameliorated multiple-organ injury by blocking the inflammatory responses. In addition, indoprofen partially reduced the HMGB1 level in the serum and in the lung, as well as ameliorated pulmonary edema. Mechanistically, indoprofen potently inhibited the release of HMGB1 following stimulation by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C), and suppressed recombinant human HMGB1(rhHMGB1)-induced inflammatory responses. It was also found that indoprofen has both cyclooxygenase 2-dependent and -independent inhibitory effects on the proinflammatory effect of HMGB1 in THP-1 cells. Further, the drug reduced rhHMGB1-induced cell surface levels of toll-like receptor 2, toll-like receptor 4, and receptor of advanced glycation end-products in a concentration-dependent manner. Collectively, these data demonstrated that the anti-inflammatory effect of indoprofen in sepsis was associated with HMGB1-mediated inflammatory responses, thus offering a favorable mechanistic basis to support the therapeutic potential of indoprofen for the treatment of lethal sepsis or other inflammatory diseases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study was designed to determine the effect of acute caffeine (CAF) administration, which exerts a broad spectrum of anti-inflammatory activity, on the synthesis of pro-inflammatory cytokines and their receptors in the hypothalamus and choroid plexus (ChP) during acute inflammation caused by the injection of bacterial endotoxin-lipopolysaccharide (LPS). The experiment was performed on 24 female sheep randomly divided into four groups: control; LPS treated (iv.; 400 ng/kg of body mass (bm.)); CAF treated (iv.; 30 mg/kg of bm.); and LPS and CAF treated. The animals were euthanized 3 h after the treatment. It was found that acute administration of CAF suppressed the synthesis of interleukin (IL-1β) and tumor necrosis factor (TNF)α, but did not influence IL-6, in the hypothalamus during LPS-induced inflammation. The injection of CAF reduced the LPS-induced expression of TNF mRNA in the ChP. CAF lowered the gene expression of IL-6 cytokine family signal transducer ( IL6ST ) and TNF receptor superfamily member 1A ( TNFRSF1 ) in the hypothalamus and IL-1 type II receptor ( IL1R2 ) in the ChP. Our study on the sheep model suggests that CAF may attenuate the inflammatory response at the hypothalamic level and partly influence the inflammatory signal generated by the ChP cells. This suggests the potential of CAF to suppress neuroinflammatory processes induced by peripheral immune/inflammatory challenges.

Peritonitis caused by LPS is a severe clinical challenge, which causes organ damage and death. However, the mechanism of LPS-induced peritonitis has not been fully revealed yet. Here, we investigated the transcriptome profile of the peritoneal tissue of LPS-induced peritonitis in mice. A model of LPS-induced peritonitis in mice was established (LPS 10 mg/kg, i.p.), and the influence of TAK 242 (TLR4 inhibitor) on the level of inflammatory cytokines in mouse peritoneal lavage fluid was investigated by using an ELISA test. Next, the peritoneal tissues of the three groups of mice (Control, LPS, and LPS+TAK 242) ( n = 6) were isolated and subjected to RNA-seq, followed by a series of bioinformatics analyses, including differentially expressed genes (DEGs), enrichment pathway, protein-protein interaction, and transcription factor pathway. Then, qPCR verified-hub genes that may interact with TAK 242 were obtained. Subsequently, the three-dimensional structure of hub proteins was obtained by using homology modeling and molecular dynamics optimization (300 ns). Finally, the virtual docking between TAK 242 and hub proteins was analyzed. Our results showed that TAK 242 significantly inhibited the production of inflammatory cytokines in the peritoneal lavage fluid of mice with peritonitis, including IL-6, IFN-γ, IL-1β, NO, and TNF-α. Compared with the Control group, LPS treatment induced 4201 DEGs (2442 down-regulated DEGs and 1759 up-regulated DEGs). Compared with the LPS group, 30 DEGs were affected by TAK 242 (8 down-regulated DEGs and 22 up-regulated DEGs). A total of 10 TAK 242-triggered hub genes were obtained, and the possible docking modes between TAK 242 and hub proteins were acquired. Overall, our data demonstrated that a large number of DEGs were affected in LPS-triggered peritonitis mice. Moreover, the TLR4 inhibitor TAK 242 is capable of suppressing the inflammatory response of LPS-induced peritonitis. Our work provides clues for understanding the pathogenesis of LPS-induced peritonitis in mice.

Objective: To investigate the expression of Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and inflammation in astrocytes under lipopolysaccharide ( LPS ) combined with hypoxia. Methods: Primary cultured astrocytes and neurons in vitro were divided into four groups: normoxia group; hypoxia group; LPS group; LPS plus hypoxia group (each group is provided with 3 duplicate holes). After treated with LPS(100 ng/ml), hypoxia group and LPS plus hypoxia group were placed in hypoxia cell incubator with 0.3% O 2 , and normoxia group and LPS group were placed in normal cell incubator for 24 h. Primary astrocytes were divided four groups as above for 6 h,12 h and 24 h. The expression of BNIP3 in astrocytes was detected by Western blot. The expressions of tumor necrosis factor-α(TNF-ɑ), interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA in astrocytes were detected by RT-PCR. The levels of TNF-ɑ, IL-1β and IL-6 in cultured medium were detected by ELISA. Results: Compared with the normoxia group, the expressions of inflammatory cytokines TNF-ɑ, IL-1β and IL-6 mRNA had no change in hypoxia group and were increased in LPS group and LPS plus hypoxia group ( P ＜0.01). Compared with the LPS group, the expressions of inflammatory cytokines IL-1β and IL-6 mRNA were increased in LPS plus hypoxia group ( P ＜0.05, P ＜0.01). Compared with the normoxia group, the levels of inflammatory cytokines had no change in hypoxia group and the levels of TNF-ɑ and IL-6 were increased in LPS group and LPS plus hypoxia group ( P ＜0.01), the level of IL-1β had no change in LPS group and LPS plus hypoxia group. Compared with the LPS group, the levels of TNF-ɑ and IL-6 had no more change in LPS plus hypoxia group. BNIP3 was expressed in primary neurons and astrocytes in vitro . Compared with astrocytes in the normoxia group, the expression of BNIP3 in LPS group had no change and was increased markedly in hypoxia group and LPS plus hypoxia group ( P ＜0.01). Compared with neurons in the normoxia group, the expression of BNIP3 in LPS group had no change and was increased in hypoxia group and LPS plus hypoxia group ( P ＜0.05, P ＜0.01). Compared with neurons in the hypoxia group, the expression of BNIP3 in astrocytes of hypoxia group was increased ( P ＜0.01). Compared with the normoxia group at the same time point, the expression of BNIP3 in LPS group had no change and was increased in hypoxia group and LPS plus hypoxia group ( P ＜0.05, P ＜0.01). Compared with the hypoxia group at the same time point, the expression of BNIP3 was increased markedly in LPS plus hypoxia group at 6 h and 12 h ( P ＜0.01). Conclusion: The combination of hypoxia with LPS augmented inflammation in astrocyte and LPS enhanced the expression of BNIP3 in astrocyte under hypoxia, suggesting BNIP3 might be involved in regulating astrocyte inflammation.

Acute lung injury is a fatal condition characterized by excessive inflammation responses. Salidroside, the active constituent of Rhodiola rosea, possesses properties including anti-oxidation, anti-aging, anti-inflammatory, anti-hypoxia, and anti-cancer activities. In the present study, Salidroside attenuated acute lung injury via inhibition of inflammatory cytokine production. Rats pre-treated with Salidroside showed attenuated lipopolysaccharide (LPS)-induced pathological damage and suppressed tumor necrosis factor-alpha (TNFα) and interleukin 6 (IL-6) secretion in the lung. Furthermore, flow cytometry showed that Salidroside reduced the production of TNFα and IL-6 in NR8383 alveolar macrophages. These findings suggest that Salidroside may attenuate LPS-induced acute lung injury., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

LRRK2 protein is expressed prominently in immune cells, cell types whose contribution to LRRK2-associated genetic Parkinson's disease (PD) is increasingly being recognised. We investigated the effect of inflammatory stimuli using RAW264.7 murine macrophage cells as model systems. A detailed time course of TLR2 and TLR4 stimulation was investigated through measuring LRRK2 phosphorylation at its specific phospho-sites, and Rab8 and Rab10 phosphorylation together with cytokine release following treatment with LPS and zymosan. LRRK2 phosphorylation at Ser935, Ser955 and Ser973 was increased significantly over untreated conditions at 4-24h in both WT-LRRK2 and T1348N-LRRK2 cell lines to similar extents although levels of Ser910 phosphorylation were maintained at higher levels throughout. Importantly we demonstrate that LPS stimulation significantly decreased phospho-Rab10 but not phospho-Rab8 levels over 4-24h in both WT-LRRK2 and T1348N-LRRK2 cell lines. The dephosphorylation of Rab10 was not attributed to its specific phosphatase, PPM1H as the levels remained unaltered with LPS treatment. MAPK phosphorylation occurred prior to LRRK2 phosphorylation which was validated by blocking TLR4 and TLR2 receptors with TAK242 or Sparstolonin B respectively. A significant decrease in basal level of TNFα release was noted in both T1348N-LRRK2 and KO-LRRK2 cell lines at 48h compared to WT-LRRK2 cell line, however LPS and zymosan treatment did not cause any significant alteration in the TNFα and IL-6 release between the three cell lines. In contrast, LPS and zymosan caused significantly lower IL-10 release in T1348N-LRRK2 and KO-LRRK2 cell lines. A significant decrease in phospho-Rab10 levels was also confirmed in human IPS-derived macrophages with TLR4 activation. Our data demonstrates for the first time that LRRK2-dependent Rab10 phosphorylation is modulated by LPS stimulation, and that cytokine release may be influenced by the status of LRRK2. These data provide further insights into the function of LRRK2 in immune response, and has relevance for understanding cellular dysfunctions when developing LRRK2-based inhibitors for clinical treatment., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1β, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.

Illicit drug use can cause a variety of effects including alterations in the immune system. The aim of this study was to investigate the effects of illicit drugs on circulating lipopolysaccharide (LPS), systemic inflammation and oxidative stress markers in drug users. We evaluated the levels of soluble CD14 (sCD14), LPS, inflammatory (TNF-α and IL-6) and regulatory (IL-10) cytokines, as well as C-reactive protein (CRP), lipid peroxidation (TBARS) and total thiols in the peripheral blood of 81 men included in groups of cannabis (n = 21), cocaine (n = 12), cannabis-plus-cocaine users (n = 27), and non-drug users (n = 21). The use of cannabis plus cocaine leads to higher systemic levels of LPS, CRP, IL-6 and higher IL-6/IL-10 ratio, characterizing a proinflammatory profile. In contrast, a regulatory profile as viewed by lower systemic TNF-α and IL-6 levels and lower TNF-α/IL-10 ratio were observed in cannabis users compared to the control group. Moreover, cocaine users presented a lower content of non-enzymatic antioxidant thiol compared to control group, cannabis group and cannabis plus cocaine group. In conclusion, our results indicate that the use of cannabis contributes to an anti-inflammatory/or regulatory profile while the concomitant cannabis plus cocaine consumption coexists with increased circulating amounts of LPS and proinflammatory status., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The family of human β-defensins consists of small cysteine-rich peptides, which are receiving significant attention due to their antimicrobial activity. The N-terminal cysteine motif of β-defensin is considered to contribute to its biological activity. Human β-defensin 118 (DEFB 118) is a particular anion β-defensin expressed predominantly in the male reproductive tract, but its physiological activity has not yet been revealed. In order to verify the potential role of the N-terminal domain of DEFB118 peptide in the regulation of infection, the truncated β-defensin core region of DEFB118 peptide was expressed with IMPACT-pTWIN1 system in Escherichia coli. Herein, the purified homogeneous DEFB118 peptide was identified by mass spectrometry and circular dichroism spectroscopy. The in vitro experiments revealed that DEFB118 peptide exhibited prominent LPS-binding potency (K D : 2.94 nM). Moreover, the DEFB118 core peptide significantly inhibited the mRNA level of LPS-induced inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in RAW264.7 cells, and correspondingly decreased secretion of IL-6 and TNF-α. We concluded that strong binding of DEFB118 to LPS might prevent LPS from binding to its receptor, and hence inhibited cytokines secretion. The results of this study may be a benefit to elucidate the immune protection of DEFB118 in the male reproductive tract., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thymic stromal lymphopoietin (TSLP) is a member of the IL-2 cytokine family, which is known to activate type 2 innate lymphoid cells, mast cells, and Th2 cells; this activation results in allergic inflammation and host defense against parasites. TSLP has also been shown to promote Th17-mediated immune responses, such as those observed in the development of rheumatoid arthritis; however, its role in osteoclastogenesis remains poorly understood. Here, we investigated the functional involvement of TSLP in RANKL-induced osteoclast differentiation from murine bone marrow-derived macrophages (BMMs). Both RANK - and RANK + macrophages expressed TSLP receptor (TSLPR), while RANK + osteoclast precursors maintained TSLPR expression after RANKL stimulation. TSLP stimulation led to inhibition of RANK-induced osteoclast differentiation in wild-type BMMs, but not Tslpr -/- BMMs; TSLP stimulation also led to suppression of osteoclastogenic gene expression (Nfatc1, Acp5, Mmp9, and Ctsk). These inhibitory effects of TSLP were significantly reduced following STAT1 inhibition. Finally, we found that LPS stimulation induced TSLP production in murine calvarial osteoblasts, but not BMMs. Together, these observations suggest that TSLP acts directly on osteoclast precursors to suppress osteoclastogenesis. Osteoblasts, along with other TSLP-producing cells, may therefore contribute to the inhibition of osteoclastogenesis under inflammatory conditions., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Jiao Mei Gu (JMG), Cayratia albifolia C.L.Li, is a type of Dong plant widely growing in Dong autonomous counties, Hunan province, China. As a type of traditional herbal medicine, the root of JMG plant has been used to treat inflammatory-related diseases such as arthritis because of its prominent anti-inflammatory effects in Dong medicine., Aim of the Study: This work investigated the anti-inflammatory effects and mechanisms of the water extract from the root of JMG on lipopolysaccharide (LPS)-induced inflammatory models., Methods: Endotoxemia was induced in C57BL/6 mice by intraperitoneal injection of LPS (20 mg/kg), meanwhile intraperitoneal administration of safe doses of JMG. The survival curve of mice was determined. Serum inflammatory cytokines were detected by the Bio-Plex Mouse Cytokine 23-Plex Panel Kit and enzyme-linked immunosorbent assay (ELISA) at 6 h after drug treatment. Hematoxylin-eosin (HE) staining of important organs was completed at 24 h after treatment. The mechanism of inflammatory action was investigated in vitro on LPS-stimulated macrophages. Macrophage inflammation was then induced using 10 μg/mL LPS. The anti-inflammatory effect of JMG was investigated by the quantitative polymerase chain reaction (qPCR) and ELISA. The anti-inflammatory mechanism was determined using western blotting, the electrophoretic mobility shift assay, and immunocytochemistry. Finally, the antimicrobial activity of JMG was verified by survival experiments in vivo and by bacterial culture experiments in vitro., Results: A 200 mg/kg water extract of JMG was safe for mice and had a significant protective effect on LPS-induced sepsis. Organ damage of heart, liver, lung and kidney was also significantly reduced at 24 h in the JMG group, when compared with the LPS group. The serum MIP-1α (CCL-3), MIP-1β (CCL-4), IL-1β, and TNFα cytokines were significantly decreased at 6 h in the JMG group, when compared with the LPS group. In a similar manner, 0.2μg/ml JMG significantly reduced mRNA and protein levels of MIP-1α (CCL-3), MIP-1β (CCL-4), IL-1β, and TNFα in LPS-stimulated macrophage. JMG treatment inhibited the phosphorylation of NF-κB p65 and reduced nuclear transduction, thus reducing transcriptional activity. At the same time, we showed that JMG had no protective effect on Escherichia coli-induced sepsis, as well as no antimicrobial activity., Conclusions: Our results showed that a water-soluble extract of JMG inhibited LPS-induced inflammation via attenuating the NF-κB signaling pathway, which provides an important rationale for the treatment of inflammation-related diseases., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Abnormal neuroinflammation ignited by overproduction of chemokines and cytokines via microglial cells can induce the occurrence and development of neurodegenerative disorders. The aim of this study is to investigate the effects of dexamethasone sodium phosphate (Dex-SP) on chemokine and cytokine secretion in lipopolysaccharide (LPS)-activated microglial cells. LPS markedly enhanced the secretion of pro-inflammatory factors such as regulated on activation, normal T cell expressed and secreted (RANTES), transforming growth factor beta-β1 (TGF-β1) and nitric oxide (NO), but decreased the production of macrophage inflammatory protein-1α (MIP-1α) and interleukin 10 (IL-10) in BV-2 microglial cells. Furthermore, LPS increased BV-2 microglial cell migration. However, Dex-SP treatment had the opposite effect, dampening the secretion of RANTES, TGF-β1, and NO, while increasing the production of MIP-1α and IL-10 and blocking migration of LPS-stimulated BV-2 microglial cells. Furthermore, Dex-SP markedly suppressed the LPS-induced degradation of IRAK-1 and IRAK-4, and blocked the activation in TRAF6, p-TAK1, and p-JNK in BV-2 microglial cells. These results showed that Dex-SP inhibited the neuroinflammatory response and migration in LPS-activated BV-2 microglia by inhibiting the secretion of RANTES, TGF-β1, and NO and increasing the production of MIP-1α and IL-10. The molecular mechanism of Dex-SP may be associated with inhibition of TRAF6/TAK-1/JNK signaling pathways mediated by IRAK-1 and IRAK-4.

The neutrophil granule protein lactoferrin is cleaved and accumulates in efferocytic macrophages as inflammation is resolved. Two peptides present within a resolution-associated 17 kDa fragment of lactoferrin promote the termination of inflammation in vivo by enhancing murine macrophage reprogramming. Here, we report that these two bioactive tripeptides, phenylalanine-lysine-aspartic acid and phenylalanine-lysine-glutamic acid (FKD and FKE, respectively), inhibit ERK and cJun activation following human macrophage exposure to LPS. In addition, these peptides at low concentrations (1-10 μM) modulate human macrophage reprogramming to an anti-inflammatory/pro-resolving phenotype. This was reflected by inhibition of LPS-induced TNF-α and IL-6 secretion and increased IL-10 levels. Moreover, we found naturally occurring FKE analogs (FKECH and FKECHLA) can recapitulate the activity of the short peptide in regulating macrophage cytokine secretion, whereas a reversed EKF peptide was inert in this respect. Curiously, FKD and FKE also regulated cytokine production by bone marrow-derived mouse macrophages, but in a very different fashion than their effect on human macrophages. Thus, lactoferrin peptides limit pro-inflammatory signaling and cytokine production by LPS-activated human macrophages and thereby enhance the resolution of inflammation.

Caldecrin was previously isolated as a serum calcium-decreasing factor from the pancreas and is known to suppress receptor activator of nuclear factor-κB ligand (RANKL)-induced calcium oscillation pathways in osteoclasts. Here, we explored the effects of caldecrin on lipopolysaccharide (LPS)-Toll-like receptor-4 (TLR-4) signaling pathways in macrophages. Caldecrin inhibited the LPS-induced gene expression of pro-inflammatory cytokines and M1 macrophage polarization in mouse bone marrow macrophages and the RAW264.7 mouse macrophage cell line. Next, we focused on triggering receptor expressed in myeloid cells-2 (TREM-2) as a co-receptor common to RANKL receptor and TLR-4, and established Trem2-KO RAW264.7 cells, in which Trem2 gene was deleted using the CRISPR/Cas9 system. Caldecrin-mediated alterations in pro-inflammatory cytokine expression and M1 macrophage polarization were not observed in Trem2-KO RAW264.7 cells. These results suggest that caldecrin is not only an inhibitor of osteoclast activation but also a negative regulator of LPS-induced inflammatory responses, functioning via TREM-2., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We investigated cellular functional and targeted immune cytokine responses of farmed Chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells (PBMCs) in vitro to LPS from Escherichia coli (E. coli) serotypes O111: B4 and O55: B5, and a phorbol ester phorbol 12-myristate 13-acetate (PMA). Bacterial LPS and PMA significantly (p < 0.05) induced reactive oxygen species (ROS) production in O. tshawytscha PBMCs, and enhanced by interferon (IFN)-inducible cytokine production. Cellular phagocytosis was significantly enhanced with PMA and E. coli serotype O111: B4 LPS after 1 and 2 h respectively. At the molecular level, LPS and PMA significantly (p < 0.05) upregulated pro-inflammatory cytokine gene transcripts for IFNγ, TNF-α, and anti-inflammatory IL-10, 24 h post-stimulation. This response is postulated to be mediated via the MyD88 and TRIF pathways in TLR4, or synergistic TLR1 and TLR2 receptors. This is the first report of LPS induced immune related in vitro responses in farmed O. tshawytscha PBMCs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Fibrosis in the lungs usually occurs in the initial phase of acute respiratory distress syndrome (ARDS), which exacerbates poor prognosis among patients. MicroRNAs (miRs) have the ability to modulate the expression profiles of many genes, thus essentially altering cell phenotypes. We hypothesize that miRs may be involved in the development of lung fibrosis in mice. In this study, mice were treated with lipopolysaccharide (LPS) to establish the lung fibrosis animal model. Hematoxylin and eosin (H&E) staining and western blot (WB) were performed to confirm the successful establishment of the model. Quantitative PCR (qPCR) and WB were utilized to monitor the expression of miRs and proteins. A dual-luciferase reporter assay was used to detect the interaction between miR and genes. We observed miR-506 downregulation in lung tissues during lung fibrosis after ARDS rat modeling by LPS exposure. We also observed that its expression level was similar to that observed in TGF-β1-induced human MRC-5 cells. The proportion of apoptotic cells decreased, while levels of inflammatory cytokines were upregulated in lung tissues during lung fibrosis and in fibroblasts after TGF-β1 treatment. In order to elucidate the possible role of miR-506, it was overexpressed in mice with ARDS. It was revealed that miR-506 significantly ameliorated the degree and spread of pulmonary damage stimulated by LPS. miR-506 also induced apoptosis in vivo and in vitro, while also ameliorating the inflammatory response. Notably, p65, a subunit of NF-κB, acts as a target of miR-506. p65 expression was downregulated in TGF-β1-treated MRC-5 cells upon transfection with miR-506 mimic. Indeed, the 3'-UTR of human p65 contained functional human miR-506-responsive sequences. LPS induction and TGF-β1 stimulation in mice led to p65 upregulation. In addition, p65 knockdown in the ARDS mouse model partially ameliorated the severity of lung lesions, induced apoptosis and reduced inflammation in lung tissue. Our findings revealed that miR-506 could be an important modulator of apoptosis and inflammation and a regulator of lung fibrosis.

The production of inflammatory cytokines is closely related to pathogen-associated molecular pattern (PAMP)-triggered activation of the Toll-like receptor (TLR), intracellular signal transduction pathways such as MAPK and NF-κB, and histone modifications. Histone methylation, a type of histone modifications, is mainly accomplished by a class of SET family proteins containing highly conserved SET domains. In the present study, we found that SET domain-containing protein 4 (SETD4) regulated inflammatory cytokines in response to TLR agonists. LPS stimulation led to the enhanced SETD4 expression, while the increased IL-6 and TNF-α release from LPS-stimulated RAW264.7 cells was attenuated by depletion of SETD4 using RNA interference. The results were further confirmed in BMDMs and pMφ isolated from SETD4-deficient mice where SETD4 -/- macrophages treated with LPS, BLP or Poly(I:C) showed down-regulated IL-6 and TNF-α mRNA and protein levels when compared with SETD4 +/+ macrophages. Moreover, the mRNA levels of all NF-κB-dependent genes including IL-1β, IL-10, NFKBA, DUSP1, CCL2, CCL5, and CXCL10 in SETD4 -/- macrophages were substantially reduced. To further clarify the regulatory mechanism(s) by which SETD4 modulates inflammatory cytokines, we examined the effect of SETD4 on the activation of MAPK and NF-κB signalling pathways, and found that knockout of SETD4 had no effect on phosphorylation of p38, ERK, JNK, p65, and IκBα. Notably, SETD4 translocated quickly from the cytosol to the nucleus upon LPS stimulation, suggesting that SETD4 may exert its regulatory function downstream of the MAPK and NF-κB pathways. To characterize this, we performed an in vitro HMTase assay to measure histone methyltransferase (HMTase) activity of SETD4. H3K4me1 and H3K4me2 levels were enhanced dramatically with the supplementation of SETD4, whereas both H3K4me1 and H3K4me2 were strongly attenuated in SETD4 -/- BMDMs. Moreover, the LPS-stimulated recruitment of H3K4me1 and H3K4me2 at both TNF-α and IL-6 promoters was severely impaired in SETD4 -/- BMDMs. Collectively, these results demonstrate that SETD4 positively regulates IL-6 and TNF-α expression in TLR agonist-stimulated macrophages by directly activating H3K4 methylation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Perturbation of the circadian rhythm damages the biological characteristics of cells and leads to their dysfunction. Rev-erbα, an important gene in the transcription-translation loop of circadian rhythm, is involved in regulating the balance between pro-inflammation and anti-inflammation. The disruption of this balance in human endometrial stroma cells (hESCs) destroys their biological behavior function in maintaining the menstrual cycle and embryonic implantation. Whether pharmacological modulation of Rev-erbα affects the inflammation of hESCs remains unclear. In this study, we treated hESCs with lipopolysaccharide (LPS) and found that LPS treatment increased the mRNA levels of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8, IL-18, and TNFα, and the secretion of IL-6. SR9009, a Rev-erbα agonist, significantly alleviated the LPS-induced production of pro-inflammatory cytokines in hESCs. Meanwhile, knockdown of Rev-erbα increased the expressions of IL-1β, IL-6, and IL-8, accompanied by an increased mRNA level of the core clock gene Bmal1. Western blot analysis showed that SR9009 inhibited the expression of toll-like receptor 4 (TLR4) and the activation of NF-κB induced by LPS. All these findings suggested that pharmacological activation of Rev-erbα attenuated the LPS-induced inflammatory response of hESCs by suppressing TLR4-regulated NF-κB activation. This study may provide a strategy for preventing inflammation-related endometrial dysfunction and infertility or recurrent implantation failure., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Lipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process., Methods: THP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay., Results: In THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1 cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NFκB) complex in nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced p65-p52 nuclear translocation and NFκB activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout., Conclusion: IGF2BP1 promotes LPS-induced NFκB signalling and transcriptional activation in human macrophages and monocytes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Context: Pulegone, a key compound in Schizonepeta essential oil, has been identified as an anti-inflammatory. However, its underlying molecular mechanisms on NLR family pyrin domain containing 3 (NLRP3) inflammasome have not been elucidated. Objective: Here, the modulatory effects of pulegone on NLRP3 inflammasome were investigated. Materials and methods: The C57BL/6J mice were randomly divided into five groups: Normal, Lipopolysaccharides (LPS), Dexamethasone (DEX, 5 mg/kg), Pulegone (0.095 and 0.190 g/kg) groups. All mice were challenged by LPS except for the Normal group. Results: A reduced expression of Interleukin-18 (IL-18), Interleukin-1β (IL-1β), Interleukin-5 (IL-5), Tumor necrosis factor-α (TNF-α), Interferon-gamma (IFN-γ), Monocyte chemoattratctant protein-1 (MCP-1), Macrophage inflammatory protein-1β (MIP-1β), Monocyte colony stimulating factor (M-CSF) and Granulocyte-macrophage colony stimulating factor (GM-CSF) in serum were detected in the pulegone groups as compared to the LPS group. In addition, a reduced mRNA and protein expression production of ASC, NLRP3, and Caspase-1 were detected in lungs after pulegone administration. Histological analysis results indicated that the histological changes of lungs caused by LPS were ameliorated by pulegone. Immunohistochemical study showed a decreased positive cell numbers of P2X7R in Pulegone (0.095 and 0.190 g/kg) groups. Conclusion: Pulegone exerts anti-inflammatory effects on LPS-induced sepsis mice via inhibition of the NLRP3 expression.

This experiment was conducted to study the effects of orally administered carvacrol essential oils on immune response and inflammation-related genes expression in broilers challenged by lipopolysaccharide (LPS). Eighty 28-day-old (1.28 ± 0.15 kg) ROSS 308 broilers were assigned to a 2 × 2 factorial arrangement of treatments (20 pens of 1 chick/trt). Factors were carvacrol essential oil (orally administered or non-orally administered) and LPS (challenged or non-challenged). Individually housed broilers were randomly assigned (n = 20 broilers per treatment: 10 males and 10 females) to four treatments: (1) basic diet (CTR), (2) basic diet + carvacrol (CAR), (3) basic diet + LPS-challenge (LPS), (4) basic diet + carvacrol + LPS-challenge (CAR+LPS). All were fed with the same diet. The experimental period was for 15 d, after which injecting LPS significantly up-regulated the gene expression levels of TNF-α (P < 0.05), IL-1β (P < 0.05), IL-6 (P < 0.05), IL-8 (P < 0.05), TLR2 (P < 0.05), TLR4 (P < 0.05), NF-κB p65 (P < 0.05), AVBD-9 (P < 0.05), and SIgA(P < 0.05) compared with the CTR group; the broilers were challenged by LPS after oral administration of carvacrol, they had significant lower on the gene expression levels of TNF-α (P < 0.05), IL-1β (P < 0.05), IL-6 (P < 0.05), TLR4 (P < 0.05), NF-κB p65 (P < 0.05), and AVBD-9 (P < 0.05) than the LPS group. In conclusion, the broilers orally administrated carvacrol essential oils inhibited the secretion of inflammatory cytokines caused by LPS, affected the pathway of TLRs/NF-κB, and showed an anti-inflammatory function., (© 2018 Poultry Science Association Inc.)

Seasonal changes in day length enhance and suppress immune function in a trait-specific manner. In Siberian hamsters (Phodopus sungorus) winter-like short days (SDs) increase blood leukocyte concentrations and adaptive T cell dependent immune responses, but attenuate innate inflammatory responses to simulated infections. Thyroid hormone (TH) signaling also changes seasonally and has been implicated in modulation of the reproductive axis by day length. Immunologically, TH administration in long days (LD) enhances adaptive immune responses in male Siberian hamsters, mimicking effects of SDs. This experiment tested the hypothesis that T 3 is also sufficient to mimic the effects of SD on innate immune responses. Adult male hamsters housed in LDs were pretreated with triiodothyronine (T 3 ; 1 μg, s.c.) or saline (VEH) daily for 6 weeks; additional positive controls were housed in SD and received VEH, after which cytokine, behavioral, and physiological responses to simulated bacterial infection (lipopolysaccharide; LPS) were evaluated. SD pretreatment inhibited proinflammatory cytokine mRNA expression (i.e. interleukin 1β, nuclear factor kappa-light-chain-enhancer of activated B cells). In addition, the magnitude and persistence of anorexic and cachectic responses to LPS were also lower in SD hamsters, and LPS-induced inhibition of nest building behavior was absent in SD. T 3 treatments failed to affect behavioral (food intake, nest building) or somatic (body mass) responses to LPS in LD hamsters, but one CNS cytokine response to LPS (e.g., hypothalamic TNFα) was augmented by T 3 . Together these data implicate thyroid hormone signaling in select aspects of innate immune responses to seasonal changes in day length., (Copyright © 2019. Published by Elsevier Inc.)

The present study examined the content of cytokines (IL-1β, IL-2, IL-6, IL-10) in the brain structures (the hypothalamus, striatum, frontal cortex, and hippocampus) in two rat lines selected for differences in fear-induced aggression at 2, 4, and 24 h after a peripheral injection of saline or lipopolysaccharide (LPS, 250 μg/kg). LPS stimulation elevated cytokine activity above baseline levels in both aggressive and nonaggressive rats, but the pattern, time course of cytokine changes, and their regional characteristics varied according to the animal aggressiveness. After LPS administration, aggressive rats showed increased levels of IL-1β in the hypothalamus at 2 and 4 h and in the frontal cortex at 4 and 24 h compared to LPS-treated nonaggressive line. IL-2 was increased in the frontal cortex and striatum of aggressive rats within 24 h, while IL-6 elevation in the hypothalamus was found at 4 h and in the frontal cortex at 2 and 4 h. In the hippocampus, the levels of IL-1β, IL-2, and IL-6 were lower in LPS-treated aggressive rats than in nonaggressive animals. The levels of anti-inflammatory cytokine IL-10 were also decreased in all brain structures of aggressive rats receiving LPS. The results indicate that genetic predisposition to increased aggression is associated with a time and region-dependent changes in the levels of pro- and anti-inflammatory cytokines., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Contaminated marine bathing water has been reported to adversely affect human health. Our data demonstrated a correlation between total endotoxin (lipopolysaccharide; LPS) levels and degree of contamination of marine bathing waters. To assess the potential health implications of LPS present in marine bathing waters, the inflammation-inducing potency of water samples collected at different time points at multiple sampling sites were assessed using a cell culture-based assay. The numbers of fecal indicator bacteria (FIB) were also examined in the same samples. Water samples were used to stimulate two cell culture models: (1) a novel non-transformed continuously growing murine cell line Max Plank Institute (MPI) characteristic of alveolar macrophages and (2) human MonoMac 6 monocyte cell line. The inflammatory potential of the samples was assessed by measuring the release of inflammatory cytokines. The presence of high levels of LPS in contaminated bathing water led to induction of inflammatory response from our in vitro cell-based bioassays suggesting its potential health impact. This finding introduces an in vitro culture assay that reflects the level of LPS in water samples. These observations further promote previous finding that LPS is a reliable surrogate biomarker for fecal contamination of bathing water.

Acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) are clinical conditions caused by trauma, lung infection or sepsis. ALI/ARDS is associated with massive recruitment of neutrophils into the lung with release of reactive oxygen species and excessive inflammatory response that damage alveolar tissue. Here we report the successful use of a potent recombinant chemotaxis inhibitory protein (rCHIPS) derived from Staphylococcus aureus in reducing the severity of ALI/ARDS. Treatment with rCHIPS reduces pulmonary inflammation and permeability in mice after intranasal administration of lipopolysaccharide (LPS). rCHIPS treatment significantly reduces lung myeloperoxidase (MPO) activity, pro-inflammatory cytokines, broncho-alveolar lavage (BAL) fluid protein content as well as histopathological changes. In addition, treatment with rCHIPS significantly diminishes neutrophils and leukocytes recruitment into lung tissue after LPS administration and hence protects mice from reactive oxygen species mediated lung injury. Our finding reveals potential therapeutic benefits of using rCHIPS for the treatment of ALI/ARDS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The FDA-approved small-molecule drug ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL). Ibrutinib inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. However, the potential regulation of neuroinflammatory responses in the brain by ibrutinib has not been comprehensively examined., Methods: BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO), followed by lipopolysaccharide (LPS; 1 μg/ml) or PBS. RT-PCR, immunocytochemistry, and subcellular fractionation were performed to examine the effects of ibrutinib on neuroinflammatory responses. In addition, wild-type mice were sequentially injected with ibrutinib (10 mg/kg, i.p.) or vehicle (10% DMSO, i.p.), followed by LPS (10 mg/kg, i.p.) or PBS, and microglial and astrocyte activations were assessed using immunohistochemistry., Results: Ibrutinib significantly reduced LPS-induced increases in proinflammatory cytokine levels in BV2 microglial and primary microglial cells but not in primary astrocytes. Ibrutinib regulated TLR4 signaling to alter LPS-induced proinflammatory cytokine levels. In addition, ibrutinib significantly decreased LPS-induced increases in p-AKT and p-STAT3 levels, suggesting that ibrutinib attenuates LPS-induced neuroinflammatory responses by inhibiting AKT/STAT3 signaling pathways. Interestingly, ibrutinib also reduced LPS-induced BV2 microglial cell migration by inhibiting AKT signaling. Moreover, ibrutinib-injected wild-type mice exhibited significantly reduced microglial/astrocyte activation and COX-2 and IL-1β proinflammatory cytokine levels., Conclusions: Our data provide insights on the mechanisms of a potential therapeutic strategy for neuroinflammation-related diseases.

Lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg-LPS) is a key bacterial structure involved in the maintenance of a chronic pro-inflammatory environment during periodontitis. Similar to other gram-negative LPS, Pg-LPS induces the release of pro-inflammatory cytokines through interaction with Toll-Like Receptor 4 (TLR4) and is able to stimulate negative TLR4 regulatory pathways, such as those involving microRNA (miRNA). In this work, we employed CyP, an LPS with TLR4-MD2 antagonist activity obtained from the cyanobacterium Oscillatoria planktothrix FP1 , to study the effects on pro-inflammatory cytokine production and miRNA expression in human monocytic THP-1 cells stimulated with Pg-LPS or E. coli LPS (Ec-LPS). Results showed that CyP inhibited TNF-α, IL-1β and IL-8 expression more efficiently when co-incubated with Pg-LPS rather than with Ec-LPS. The inhibition of pro-inflammatory cytokine production was maintained even when CyP was added 2 h after LPS. The analysis of the effects of CyP on miRNA expression showed that, although being an antagonist, CyP did not inhibit miR-146a induced by Pg-LPS or Ec-LPS, whereas it significantly inhibited miR-155 only in the cultures stimulated with Ec-LPS. These results suggest that CyP may modulate the pro-inflammatory response induced by Pg-LPS, not only by blocking TLR4-MD2 complex, but also by preserving miR-146a expression.

BACKGROUND The aim of this study was to evaluate the protective effects of sodium tanshinone IIA sulfonate (STS) on hemodynamic parameters, cytokine release, and multiple organ damage in an animal model of lipopolysaccharide (LPS)-induced endotoxemia. MATERIAL AND METHODS Twenty-four rabbits were randomly divided into 3 groups: control (n=8), LPS (n=8), and STS pretreatment + LPS (n=8) groups. With arterial invasive monitoring, hemodynamic variables were observed at 30 min before and at 0, 10, 20, 30, 60, 120, 180, 240, and 300 min after LPS injection. Circulatory inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), and relevant biochemical markers, including arterial partial pressure of oxygen (PaO2), plasma cardiac troponin I (cTnI), alanine aminotransferase (ALT), and creatinine (Cr), were measured at each time point. At the end of the experiment, all rabbits were sacrificed; histopathological examination of the heart, lung, liver, and kidney tissue was performed and organ injury was semi-quantitatively scored for each organ. RESULTS Mean arterial pressure (MAP) and heart rate (HR) significantly decreased within 30 min and again after 120 min following LPS injection. However, STS pretreatment gradually normalized MAP and HR after 120 min following LPS injection. In addition, STS ameliorated LPS-induced decrease of PaO2, LPS-induced increase of TNF-α, cTnI, and ALT, and enhanced LPS-induced increase of IL-10. Moreover, STS reduced heart, lung, and liver histopathologic injury. CONCLUSIONS STS can significantly stabilize LPS-induced hemodynamic deterioration, regulate inflammatory cytokine secretion, and protect heart, lung, and liver in rabbits.

NF-κB pathway and p38MAPK (p38mitogen-activated protein kinase) pathway have been shown to play a key role in neuroinflammation, however, the phosphorylation modification is an important process that affects the activation of above pathways. Dual-specificity tyrosine-phosphorylation-regulated kinase 2(Dyrk2), as a phosphokinase that can phosphorylate signal molecules, has been demonstrated to regulate Type I Interferon(TIF) by promoting ser527 phosphorylation of TBK1. Therefore, to investigate the role of Dyrk2 in neuroinflammation, we analyzed the effect of Dyrk2 on LPS-induced the activation of microglia. Here, we found Dyrk2 expressed in BV2 cells, and LPS induced different expression trend of Dyrk2 in the cytoplasm and nucleus. In addition, we revealed that Dyrk2 interacted with Akt, p38MAPK and NF-κB subunit p65, however, in the nucleus of BV2 cells, Dyrk2 selectively interacted with p38MAPK instead of with p65. Although the overexpression of Dyrk2 increased the expression level of phospho-p65, phospho-Akt and phospho-p38MAPK in LPS-stimulated BV2 cells, less TNF-α and IL-1β were detected. Probably, the inhibitory effect of Dyrk2 on the release of TNF-α and IL-1β was associated with the induction of phospho-Akt. In conclusion, these data suggested Dyrk2 involved in regulating LPS-induced the release of proinflammatory cytokines through its phosphokinase function., (Copyright © 2017 Elsevier B.V. All rights reserved.)

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3' untranslated region (UTR) but not TRAF6 3'-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adenine-thymine (AT)-rich interactive domain 5a (Arid5a) is an RNA-binding protein found in the cytoplasm and nucleus of normally growing cells. Although Arid5a is known to play an important role in immune regulation, whether and how Arid5a subcellular localization impacts immune regulation has remained unclear. In this study, we generated Arid5a transgenic (TG) mice to address this question. While ectopic Arid5a overexpression did not affect expression of inflammatory cytokines under unstimulated conditions, significantly higher levels of inflammatory cytokines, such as IL-6, were produced in response to lipopolysaccharide (LPS) stimulation. Consistent with this, TG mice were more sensitive to LPS treatment than wild-type mice. We also found that Arid5a is imported into the nucleus via a classical importin-α/β1-mediated pathway. On stimulation, nuclear Arid5a levels were decreased, while there was a concomitant increase in cytoplasmic Arid5a. Arid5a is associated with up-frameshift protein 1, and its nuclear export is regulated by a nuclear export receptor, chromosomal region maintenance 1. Taken together, these data indicate that Arid5a is a dynamic protein that translocates to the cytoplasm from the nucleus so as to properly exert its dual function in mRNA stabilization and transcriptional regulation during inflammatory conditions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Inflammation of the uterus and oviduct is associated with reduced reproductive performance in humans and domestic animals. Toll-like receptors are expressed in various immune and non-immune cells and play a crucial role in innate immunity. Toll-like receptor - 4 (TLR4) can detect lipopolysaccharide (LPS) from Gram-negative bacteria leading to the secretion of pro-inflammatory cytokines, chemokines, antimicrobial peptides and other inflammatory mediators. To investigate the effects of a local inflammation on the expression levels of TLR4 and pro-inflammatory cytokines, 12 female rabbits received an intracervical infusion with either saline solution endotoxin-free (carrier, 2 mL; n = 6) or LPS (500 μg diluted in 2 mL of saline solution; n = 6). Blood samples were performed at 0, 30, 60 and 90 min and 2,4,6 and 24 h after treatment to evaluate interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) plasma concentrations. Animals were sacrificed 24 h post-treatment. The uterus and oviducts were immediately collected. The gene expression and protein levels of TLR4 and pro-inflammatory cytokines were detected by quantitative real-time PCR (qRT-PCR) and immuno-histochemical assay, respectively. Our study showed that the intracervical administration of LPS induced local inflammation given that the animals showed no clinical signs, the histological samples revealed signs of inflammation and plasma levels of pro-inflammatory cytokines were unchanged compared to the control. LPS produced an increase in the TLR4 mRNA expression levels in the uterus with respect to the control (P < 0.05). In LPS-treated rabbits the gene expression of IL-1β was higher in the uterus and oviducts and TNF-α only in the oviduct (P < 0.05) as compared to the control. The immuno-histochemical assay showed that TLR4, IL-1β and TNF-α were expressed in the reproductive tissues of the rabbit. Moreover, after the LPS stimulation the stromal cells of the uterus exhibited a higher staining for TLR4 (P < 0.05) and the epithelial cells of the oviduct for TNF-α and IL-1β (P < 0.05) with respect to the control. These results suggest that (1) TRL4, IL-1β and TNF-α are expressed in uterus and oviducts of the doe, and (2) LPS up-regulates the gene and protein expression of TLR4 and pro-inflammatory cytokines in uterus and oviducts. Therefore, the rabbit could be a useful animal model for studying the local mechanisms involved in reproductive dysfunctions caused by subclinical infections., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Extensive research links depression and inflammation, with emerging evidence suggesting some differences between males and females in these associations. However, relatively few studies have examined stimulated inflammatory responses (ex vivo) in depression. The present research investigated the associations between depressive symptoms, basal inflammation, and LPS-stimulated production of pro- (IL-1β, IL-6, IL-8, TNF-α) and an anti-inflammatory cytokine (IL-10), with a focus on the extent to which gender moderates these relationships. As part of a larger study, 162 socio-economically and racially diverse subjects (ages 25-65, 67% women) completed extensive self-report measures, including depressive symptoms. Whole blood was quantified for basal inflammation, or incubated with 1μg/mL lipopolysaccharide (LPS) for 2h (at 37°C, 5% CO 2 ) to quantify inflammatory responses to bacterial challenge. We examined the associations between depression and inflammatory markers in regression analyses, controlling for age, BMI, race/ethnicity, income, education, and use of medications. No main effects were observed between depressive symptoms and basal or stimulated levels of inflammation. Moderation analyses revealed a significant interaction between depressive symptoms and gender for stimulated TNF-α, stimulated IL-6 (p<0.05), and a marginally significant interaction for stimulated IL-10 (p=0.07). For men, higher depressive symptoms were associated with significantly higher production of TNF-α (p<0.05) and marginally higher IL-6 (p=0.07), but not with the anti-inflammatory cytokine IL-10. For women, higher depressive symptoms were associated with significantly lower production of TNF-α and IL-10 (ps<0.05), and marginally lower IL-6 (p=0.06). These findings provide evidence for gender differences in the association of depressive symptoms with inflammatory response patterns, and highlight the utility of assessing ex vivo immune responses in blood. Implications for health are discussed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aims: Acute kidney injury (AKI) is a frequent and serious complication of sepsis; however, there is no effective treatment for it. FangJiFuling (FF) decoction is widely used to treat acute glomerulonephritis and nephritic syndrome in the clinical setting., Methods: On the basis of its anti-inflammatory properties, the renoprotective effect of FF on a mouse model of lipopolysaccharide (LPS)-induced AKI was investigated. Major compounds were identified in FF with high-performance liquid chromatography. A bioinformatics analysis tool was used to predict target genes. Quantitative real-time PCR and western blot analyses were performed to validate the targets. Furthermore, the expression of a target gene was silenced by small interfering RNA-mediated knockdown in vitro., Results: Bioinformatics analysis indicated that inflammation, apoptosis, and cell junction were closely related to the renoprotective effects of FF. Validation was confirmed by an in vivo test. A reduction of inflammatory cell infiltration and inflammatory cytokine mRNA expression (iNOS, NF-κB, MCP-1, and TNF-α) following the administration of FF (50 mg/kg) was observed in LPS-treated renal tissue. In addition, FF treatment suppressed mitochondrial-mediated apoptosis by regulating the Bax/Bcl-2 ratio in LPS-induced renal injury. Silencing Cx43, a cell-to-cell junction protein, was found to enhance the protective effect of FF against LPS-induced renal injury., Conclusion: Our study suggests that FF exhibits a renoprotective effect against LPS-induced inflammatory and apoptotic responses. In addition, Cx43 might be involved in these processes. These findings indicate the potential role of FF as a natural renoprotective product., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: The purpose of the present study was to investigate the effects of Onjisaponin B (OB) in lipopolysaccharide (LPS)-induced cognitive deficits., Methods: The rats were divided into four groups: sham group, LPS group (the model group), LPS + OB (1 mg/kg) group and LPS + OB (2 mg/kg) group. OB was treated three days before surgery and thereafter continuously for 7 days. Three days later, rats were intracerebroventricularly injected with LPS. The levels of inflammatory cytokines and the capability of free radical scavenging in serum and hippocampus were determined after the LPS challenge. PC12 cells were divided into control group, LPS group (the model group), LPS + OB (10 µM) group, LPS + OB (20 µM) group, LPS + OB (40 µM) group, LPS + OB (2 mg/kg) + nicotinamide group. The cell viability was measured by MTT assay. The protein expressions of Sirt1, p-AMPK, AMPK, Nrf-2, HO-1, Bcl-2, Bax, caspase-9, caspase-3, p-IκBα, IκBα, p-NF-κBp65 and NF-κBp65 were detected by western blot analysis., Results: As a result, OB administration effectively relived the cognitive impairment, reduced the contents of IL-1β, IL-6, TNF-α, MDA and restored SOD activities of SOD in serum and hippocampus of LPS-induced rats. Furthermore, OB treatment improved cell viability, ameliorated the alterations of IL-1β, IL-6, TNF-α, MDA and SOD in the supernatant of LPS-induced PC12 cells. Of note, the expressions of Sirt1, Nrf-2, HO-1, Bcl-2 and p-AMPK were downregulated, while Bax, caspase-9, caspase-3 and the phosphorylations of IκBα and NF-κBp65 in the LPS-stimulated hippocampus and PC12 cells were increased attributed to the LPS stimulation. Nevertheless, the conditions were significantly attenuated by OB treatment. In the LPS-induced PC12 cell, nicotinamide, a SIRT1 inhibitor, abrogated the beneficial effects of OB, as indicated by the antioxidant, anti-inflammatory and anti-apoptosis signaling., Conclusion: Based on the above evidence, our results demonstrated that OB was a potential therapeutic candidate for LPS-induced cognitive deficits., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Objective: The objective of the present study was to determine the effects of concurrent LPS and cytokine priming, reflective of the in vivo milieu, on macrophage production of key periodontitis associated cytokines TNF, IL-1β and IL-6., Design: THP-1 cells were pre-treated with combinations of Porphyromonas gingivalis and Escherichia coli lipopolysaccharide (LPS), concurrently with polarising cytokines IFNγ and IL-4, or PMA as a non-polarised control. Production of key periodontitis associated cytokines in response to subsequent LPS challenge were measured by enzyme - linked immunosorbent assay., Results: Compared with cells incubated with IFNγ or IL-4 alone in the "polarisation" phase, macrophages that were incubated with LPS during the first 24h displayed a down-regulation of TNF and IL-1β production upon secondary LPS treatment in the "activation" phase. In all three macrophage populations (M0, M1 and M2), pre-treatment with P. gingivalis LPS during the polarisation process led to a significant decrease in TNF production in response to subsequent activation by LPS (p=0.007, p=0.002 and p=0.004, respectively). Pre-treatment with E. coli LPS also led to a significant down-regulation in TNF production in all three macrophage populations (p<0.001). Furthermore, the presence of E. coli LPS during polarisation also led to the down-regulation of IL-1β in the M1 population (p<0.001), whereas there was no measurable effect on IL-1β production in M0 or M2 macrophages. There was no significant effect on IL-6 production., Conclusions: Macrophages become refractory to further LPS challenge, whereby production of key periodontitis associated cytokines TNF and IL-1β is reduced after exposure to LPS during the polarisation phase, even in the presence of inflammatory polarising cytokines. This diminished cytokine response may lead to the reduced ability to clear infection and transition to chronic inflammation seen in periodontitis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Probucol 4,4'- (Isopropylidenedithio)bis(2,6-di-tert-butylphenol) is a synthetic molecule clinically used for prevention and treatment of hypercholesterolemia and atherosclerosis. Recent studies have shown that the beneficial effects of probucol mainly derive from its anti-inflammatory and antioxidant properties. Gram-negative bacteria are common infectious agents and their wall components, e.g. lipopolysaccharide (LPS), are important elicitors of inflammation. LPS is sensed by tissue resident cells and it triggers a Toll-like receptor 4/MyD88-dependent signaling cascade resulting in endothelial activation, leukocyte recruitment and nociception. Therefore the present study aimed to investigate the anti-inflammatory and analgesic effects of probucol in models of LPS-induced acute inflammation. Probucol at 0.3-30mg/kg was administrated to male Swiss mice per oral 1h before intraplantar or intraperitoneal lipopolysaccharide stimulus. Probucol at 3mg/kg reduced lipopolysaccharide-induced mechanical and thermal hyperalgesia. These effects were accompanied by reduced leukocyte influx and cytokine production in both paw skin and peritoneum exudate. Unexpectedly, probucol did not alter lipopolysaccharide-induced tissue oxidative stress at anti-inflammatory /analgesic dose. On the other hand, probucol inhibited lipopolysaccharide-induced nuclear factor kappa B (NF-кB) activation in paw tissue as well as NF-кB activity in cultured macrophages in vitro, reinforcing the inhibitory effect of probucol over the NF-кB signaling pathway. In this sense, we propose that probucol acts on resident immune cells, such as macrophages, targeting the NF-кB pathway. As a result, it prevents the amplification and persistence of the inflammatory response by attenuating NF-кB-dependent cytokine production and leukocyte recruitment explaining its analgesic effects as well., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The golden apple snail Pomacea canaliculata is an invasive pest originating from South America. It has already been found in Asia, the southern United States and more recently in the EU. Aiming to target the immune system of the snail as a way to control its spreading, we have developed organ-specific transcriptomes and looked for molecules controlling replication and differentiation of snail hemocytes. The prokineticin domain-containing protein Astakine 1 is the only cytokine known thus far capable of regulating invertebrate hematopoiesis, and we analyzed the transcriptomes looking for molecules containing a prokineticin domain. We have identified a prokineticin-like protein (PlP), that we called Pc-plp and we analyzed by real-time PCR (qPCR) its expression. In control snails, highest levels of Pc-plp were detected in the digestive gland, the ampulla (i.e., a hemocyte reservoir) and the pericardial fluid (i.e., the hematopoietic district). We tested Pc-plp expression after triggering hematopoiesis via multiple hemolymph withdrawals, or during bacterial challenge through LPS injection. In both cases a reduction of Pc-plp mRNA was observed. The multiple hemolymph withdrawals caused a significant decrease of Pc-plp mRNA in pericardial fluid and circulating hemocytes, while the LPS injection promoted the Pc-plp mRNA drop in anterior kidney, mantle and gills, organs that may act as immune barrier in molluscs. Our data indicate an important role for prokineticin domain-containing proteins as immunomodulators also in gastropods and their dynamic expression may serve as a biosensor to gauge the effectiveness of immunological interventions aimed at curtailing the spreading of the gastropod pest P. canaliculata., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The effect of enrofloxacin on cytokine secretion by porcine peripheral blood mononuclear cells (PBMCs) was studied. Twenty 8-20-week-old pigs were randomly divided into two groups: control (C, n = 10) and experimental (E, n = 10) were used. Pigs from group E received enrofloxacin at therapeutic dose for 5 consecutive days. Blood samples were collected at 0 (before antibiotic administration), 2, 4 (during antibiotic therapy) 6, 9, 14 21, 35, 49, and 63 d of study (after treatment). PBMCs of pigs from both groups were incubated with or without lipopolysaccharide (LPS). Ex vivo production on interleukin (IL)-4, IL-6, IL-10, INF-γ, and TNF-α were analyzed using ELISA assay. Intramuscular administration of enrofloxacin to healthy pigs for 5 consecutive days induced a transitory reduction of the ex vivo response of PBMCs to LPS in terms of IL-6 and TNF-α secretion. The level of IL-6 returned to day 0 level shortly after end of treatment, while the TNF-α production remained reduced 10 d after the end of treatment. Our results indicate that enrofloxacin given in vivo in therapeutic doses has an immunomodulatory effect through its capacity to inhibit ex vivo secretion of IL-6 and TNF-α by porcine PBMC after LPS stimulation.

M1 and M2 macrophages are the key players in innate immunity, and are associated with tissue homeostasis and diseases. Although M2 macrophages are known to depend on fatty acid oxidation (FAO) for their activation, how metabolic pathways affect the production of each cytokine induced by pathogen or bacterial components is unclear. Here, we examined the role of the glycolytic pathway in M2 polarized human macrophages in cytokine production induced by lipopolysaccharide (LPS) stimulation. Human monocytes were isolated from peripheral blood by positive selection for CD14 expression and cultured with macrophage colony-stimulating factor (M-CSF), to obtain M-CSF-induced macrophages (M-MΦ). LPS-induced cytokine production by M-MΦ in the presence or absence of metabolic inhibitors was evaluated. M-MΦ showed a M2 macrophage phenotype with a high IL-10 production level. Glycolytic pathway inhibitors reduced IL-6 production by M-MΦ. Meanwhile, an FAO inhibitor suppressed IL-10 production, while it did not suppress IL-6 production. Interestingly, glycolytic pathway inhibitors downregulated extracellular signal-regulated kinase (ERK) phosphorylation, but FAO inhibitor did not. Nuclear factor kappa B (NF-κB) and the other mitogen-activated protein kinases (MAPKs), p38 and c-jun N-terminal kinase (JNK), were not affected by these metabolic inhibitors. These results suggest that M2 polarized human macrophages use the glycolytic pathway in addition to FAO for cytokine production. Furthermore, ERK may be the key molecule that links metabolic pathways to cytokine production, especially the glycolytic pathway., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Emerging evidence suggests toll-like receptor 4 (TLR4) signaling contributes to cancer development and progression. However, the consequences of signaling via the TLR4 pathway in esophageal squamous cell carcinoma (ESCC) are still unclear. Here, we investigated the impact of Lipopolysaccharide (LPS)-induced TLR4 signaling on ESCC cell proliferation, inflammatory cytokines expression, and downstream molecular mechanisms. Seventy-eight ESCC and 26 normal esophageal specimens were analyzed by immunohistochemistry, and two cell lines (Eca-109 and TE-1) were used for in vitro studies. LPS, a natural agonist of TLR4, was used to activate TLR4 signaling. The effects of LPS-TLR4 signaling on cell proliferation and inflammatory cytokines regulation were examined. Specific inhibitors of mitogen-activated protein kinase (MAPK) (extracellular regulated protein kinase [ERK] and p38) signaling pathways were used to investigate the role of each pathway in LPS-TLR4 signaling. TLR4 protein was increased in ESCC tumor tissues compared with the adjacent normal tissues. TLR4 over-expression was significantly correlated with tumor differentiation grade, lymph node metastasis, and UICC stage. LPS-induced activation of TLR4 signaling promoted cancer cell proliferation, increased production of proinflammatory or immunosuppressive cytokines TNF-α, TGF-β and inhibited the anti-inflammatory cytokine IL-10. LPS-TLR4 signaling was associated with the activation of ERK and p38 MAPK signaling pathways. Further inactivation of the two pathways by specific inhibitors attenuated cell proliferation and inflammatory cytokines expression induced by LPS. Our results indicate that LPS-TLR4 signaling in cancer cells contributes to the progression of human ESCC., (© 2016 International Society for Diseases of the Esophagus.)

Considerable recent attention has focused on the rapid antidepressant effects observed in treatment resistant patients produced by the NMDA receptor antagonist, ketamine. Surprisingly, the effects of ketamine in the context of stressor exposure, as well as the consequences of its chronic use are unclear. Thus, we assessed the impact of acute and repeated ketamine treatment together with acute [restraint or lipopolysaccharide (LPS)] or chronic (unpredictable different psychogenic challenges) stressor exposure. Importantly, acute ketamine treatment did provoke an antidepressant-like effect in a forced swim test (FST) and this effect lasted for 8 days following repeated exposure to the drug. Although acute restraint and LPS individually provoked the expected elevation of plasma corticosterone and brain-region specific monoamine variations, ketamine had no influence on corticosterone and had, at best, sparse effects on the monoamine changes. Similarly, ketamine did not appreciably influence the stressor induced neurochemical and sucrose preference alterations, it did however, dose-dependently reverse the LPS induced elevation of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Likewise, repeated ketamine administration increased adult hippocampal neurogenesis. These data indicate that repeated ketamine administration had greater behavioral consequences than acute treatment and that the drug might be imparting antidepressant effects through its effects on neuroplasticity and inflammatory processes rather than the typical neurochemical/hormonal factors affected by stressors. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'., (Copyright © 2016 Elsevier Ltd. All rights reserved.)