Lipopolysaccharide Receptor Research Articles

IL-6 Mediates a Switch in Free Fatty Acid Metabolism from the Liver to the Bone Marrow to Support HSC Expansion during Infection

The hematopoietic stem cell (HSC) pool is tightly controlled in both normal and stressed conditions. Under normal conditions, HSCs reside quiescently in their hypoxic niche with minimal mitochondrial activity, instead favouring glycolysis to meet their energy needs. During acute infection HSCs switch to oxidative phosphorylation over glycolysis due to increased energy requirements. This metabolic switch enables HSCs to expand and differentiate into downstream progenitors to increase the immune cell pool. We recently showed that fatty acid (FA) uptake and metabolism play a central role in providing HSCs with sufficient energy for their expansion during acute infection. However, how infection drives changes in available FAs for HSC expansion is not known. This study investigated the role of liver in regulating lipid availability for HSC expansion. We have previously shown that circulating free fatty acids (FFAs) within the serum of mice increased in response to infection using both lipopolysaccharide (LPS) (6 hours) and S. Typhimurium (72 hours) models. This correlated with an expansion of bone marrow HSCs 16 hours after LPS and 72 hours after S. Typhimurium confirmed using flow cytometry. To investigate real-time fatty-acid uptake by hematopoietic cells in response to infection, an in vivo transplant model was used. CD45.1 lineage negative, CD117-positive cells tagged with firefly luciferase (LK+FF) were transplanted into CD45.2 mice. After transplantation was confirmed, mice were injected with LPS for 16 hours. Mice were then subsequently injected with a luciferin molecule conjugated to a long-chain FFA (FFA-luc), which is visible via bioluminescent imaging if the FFA-luc is taken up into the transplanted LK+FF engrafted cells. Live animal imaging using this method confirmed that long-chain FFA is taken up by hematopoietic cells in response to LPS in vivo. As the liver is the master regulator of FA in the serum we isolated livers from control and LPS (6 hour) treated mice. RNAseq revealed down-regulation of genes involved in FA uptake and metabolism as well genes involved in ketogenesis and up-regulation in genes involved in glucose uptake. Further kinetic studies showed that FA uptake and metabolism genes were downregulated within 2 hours of LPS treatment. To understand if this was a direct LPS effect on hepatocytes which express the receptor for LPS (TLR4) or mediated through unknown factors, we isolated primary hepatocytes and treated them with LPS or media conditioned with serum from control or LPS treated mice. Only the media conditioned with serum from LPS treated mice downregulated genes associated with FA uptake and metabolism. To identify the factors driving this metabolic switch in hepatocytes, a cytokine array of serum from control or LPS treated mice (90 minutes) was performed. This proteome cytokine array revealed a significant upregulation of several cytokines and chemokines, including IL-6, TNFα and CXCL1 and CXCL2. Primary mouse hepatocytes were then treated with factors identified in the array for 2 and 4 hours. RT-qPCR confirmed that IL-6 downregulated FA uptake and metabolism genes in primary hepatocytes. To understand if this was a LPS specific response or induce by infection mice were treated with S. Typhimurium for various time points and livers extracted. RT-qPCR confirmed that S. Typhimurium treated mice down-regulated genes associated with FA uptake and metabolism. To study the role of IL-6 in regulating liver FA metabolism in response to LPS, mice were treated with IL-6 neutralising antibody (MP5-20F3). Pre-treatment of mice with MP5-20F3 inhibited LPS induced downregulation of liver FA uptake and metabolic genes, which lead to reversing LPS-induced increase in serum FA. In conclusion we have shown a role for IL-6 in switching fatty acid metabolism from the liver to the bone marrow to allow HSC expansion during infection.

Assessing cell chimerism in an acute graft-versus-host disease model established in TLR4 knockout mice.

Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. To elucidate the role of Toll-like receptor 4 (TLR4), the major receptor for bacterial lipopolysaccharide, in the development of GVHD, we constructed a GVHD model in TLR4 knockout (TLR4-/-) mice and monitored the cell chimerism. In this study, we used polymerase chain reaction to identify whether TLR4 knockout (TLR4-/-) mice were established. Before transplantation, we pretreated mice with irradiation so as to obtain an appropriate irradiation dose. Flow cytometry was applied to measure the chimerism status, the distributions of antigen-presenting cells (APCs), and T-cells in TLR4+/+ and TLR4-/- recipient mice. The general condition of TLR4-/- recipients was better than that of TLR4+/+ recipients, and the TLR4-/- recipient mice showed less severe GVHD manifestations than the TLR4+/+ recipient mice. Most of the APCs and T-cells in the host mouse spleen were derived from donor cells, and CD4+ T-cells, including memory T-cells, were in the majority in host mice. In general, our data show that TLR4 deletion attenuated GVHD development, which suggests that TLR4 could be used as a novel target and therapeutic paradigm in GVHD therapies.

Essential oil extracted from Quzhou Aurantii Fructus prevents acute liver failure through inhibiting lipopolysaccharide-mediated inflammatory response

Quzhou Aurantii Fructus (QAF) has a long history as a folk medicine and food for the treatment of liver diseases. While our earlier study provided evidence of hepatoprotective properties contained within the flavonoids and limonins constituents in QAF, the potential preventative effects afforded by essential oil components present within QAF remains enigmatic. In this study, we prepared Quzhou Aurantii Fructus essential oil (QAFEO) and confirmed its anti-inflammatory effects on liver inflammation through experimentation on lipopolysaccharide and D-galactosamine (LPS/D-GalN) induced acute liver failure (ALF) mouse models. Using RNA-sequence (RNA-seq) analysis, we found that QAFEO prevented ALF by systematically blunting the pathways involved in response to LPS and toll-like receptor signaling pathways. QAFEO effectively suppressed the phosphorylation of tank-binding kinase 1 (TBK1), TGF-beta activated kinase 1 (TAK1), interferon regulatory factor 3 (IRF3), and the activation of mitogen activated kinase-like protein (MAPK) and nuclear factor-kappa B (NF-κB) pathways in vivo and in vitro. Importantly, QAFEO substantially reduced myeloid differentiation primary response gene 88 (MyD88)- toll-like receptor 4 (TLR4) interaction levels. Moreover, 8 compounds from QAFEO could directly bind to REAL, TAK1, MyD88, TBK1, and IRF3. Taken together, the results of our study support the notion that QAFEO exerts a hepatoprotective effect through inhibiting LPS-mediated inflammatory response.Graphical abstract

The Role of Caspase-11 and Pyroptosis in the Regulation of Inflammation in Peri-Implantitis.

Peri-implantitis is an important cause of oral implant failure. In the past, TLR4 and TLR2 in the Toll-like family were generally considered as the key immune recognition receptors regulating peri-implantitis. However, under the guidance of this theory, there are still some unexplainable peri-implantitis symptoms. With the discovery of novel intracellular LPS receptor Caspase-11, a new understanding of inflammatory signaling and immune regulation in the development of peri-implantitis has been gained. However, the regulatory role of Caspase-11 in peri-implantitis and its crosstalk with the TLR4 pathway remain unclear. The therapeutic effect of drugs targeting Caspase-11 on peri-implantitis is still in its early stages. In view of this situation, this paper reviews the possible role of Caspase-11 in peri-implant inflammation, elaborated the entry process of LPS and the activation mechanism of Caspase-11, and analyzes the differences in Caspase-11 between commonly studied animals, mice and humans. The current research hotspots and challenges are also analyzed to provide new insights and ideas for researchers.

A-246 Desialylation Profiles of Monocytes and Macrophages Upon LPS Stimulation

Abstract Background Cell surface expresses a dense layer of glycans terminated with sialic acids (Sias), known as sialylation, which often represent different cellular statuses. Meanwhile, the removal of Sias from the glycans catalyzed by sialidase (desialylation) is also involved in many biological processes. Previous studies have established that lipopolysaccharide (LPS) can induce the expression of endogenous sialidases in monocytes and macrophages, leading to desialylation of the LPS receptor TLR4. This desialylation of TLR4 has been shown to impact the LPS/TLR4 signaling pathway. In this study, we conduct a profiling of desialylation in THP-1 monocytes and THP-1 macrophages in response to LPS stimulation. This will provide insights on the intricate connection between sialylation, desialylation, and LPS-induced signaling. Methods The activity of sialidases was assessed by utilizing 4-methylumbelliferyl-N-acetylneuraminic acid (4-MU-NANA) and ganglioside GM3 as substrates. The total sialic acid in the THP1 was quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Flow cytometry was used to analyze the exposure of galactose on THP-1 monocytes and macrophages after LPS stimulation (100 ng/mL LPS for 24 h), using PNA-FITC staining. The expression level of Neu1 and Neu3 sialidases in cell lysates of THP-1 monocytes and macrophages were assessed by western blot technique after LPS stimulation. ELISA was used to measure the levels of sialidases in the cell supernatant of THP-1 monocytes and macrophages upon LPS stimulation. Result We found a significant reduction in total sialic acid levels in both THP-1 monocytes and macrophages upon LPS stimulation. This was further confirmed by the significant increase in total sialidase activities which measured by both 4-MU-Neu5Ac and GM3 substrates in THP-1 monocytes and macrophages upon LPS stimulation. Surprisingly, we also found that sialidase activities significantly increased in the cell supernatant of both THP-1 monocytes and macrophages, indicting sialidases secretion upon LPS stimulation. Western blot analysis further confirmed the high expression and secretion of both Neu1 and Neu3 sialidases from THP-1 monocytes and macrophages after LPS stimulation. Conclusion The present study conducted the first systematic profiling of desialylation and sialidase expression in monocytes and macrophages upon LPS stimulation. This results demonstrate that endogenous sialidase expression and secretion play a role in the LPS/TLR4 signaling pathway and subsequent cellular processes and functions in the context of inflammation and immune response.

67 Infant Health Status Impacts the Neutrophil Phenotypes and Polyunsaturated Fatty Acids Composition in Human Milk

Abstract Background The abundance of human milk leukocytes, including neutrophils, changes in response to the health status of mothers and their infants. Elevated leukocyte counts occur during infant infection and return to baseline levels upon recovery. However, it is unclear how infant health status can affect the neutrophil phenotypes and the concentration of fatty acids in their mothers’ human milk. Objectives This study investigates the association between infant health status and human milk neutrophil counts and activation state, and fatty acid levels, in human milk. Design/Methods This is a prospective cohort study of 50 healthy breastfeeding mothers recruited from St. Michael’s hospital, in Toronto, Ontario, who were followed up from 2-4 weeks until 4 months postpartum. Human milk samples were collected from participants and data regarding infant health status were collected from self-reported questionnaires completed by participants at both timepoints, then imputed based on whether infants had taken antibiotics, had been hospitalized, or had been diagnosed with a medical condition, at least 2 weeks prior to samples collection. Neutrophils were quantified using flow cytometry and labelled with a panel of antibodies to detect specific cluster of differentiation (CD) biomarkers. Fatty acids were identified and quantified using a gas chromatography-flame ionization detector (GC-FID). Linear mixed-effects models were used to evaluate the correlation between infant health status and changes in neutrophil counts, along with their expressed CD markers, and fatty acids composition in human milk during lactation. Results The CD markers were classified into 4 categories based on their function: degranulation/activation markers (CD63, CD64, and CD66a), an immunoregulation marker (CD16), adhesion markers (CD11b, CD18), and a lipopolysaccharide receptor (CD14). Human milk from mothers whose infants had a health condition had neutrophils which significantly expressed elevated levels of the CD64 biomarker (β: 85.65, p=0.009 in adjusted models), compared to human milk from mothers of healthy infants. However, there were no significant associations between infant health status, absolute hmPMN counts, andother CD biomarkers (p>0.05). There were also elevated levels of arachidonic acid (β: 0.12, p=0.013) and C22:5n-6 (β: 0.06, p=0.015) fatty acids in the human milk of mothers whose infants had a health condition, compared with human milk from mothers of healthy infants, after adjusting for maternal age, post-pregnancy body mass index, infant sex, and infant feeding pattern. Conclusion This study demonstrates that infant health status is associated with changes in human milk immunological components, suggesting an inflammatory protective response mechanism in the mammary glands triggered by infants infection. How these alterations can affect infant health outcomes in the short and longer terms need critical consideration.

Incomplete Knockdown of MyD88 Inhibits LPS-Induced Lung Injury and Lung Fibrosis in a Mouse Model.

Acute lung injury (ALI) is a life-threatening disorder stemmed mainly from an uncontrolled inflammatory response. Lipopolysaccharide (LPS) is commonly used to induce ALI animal models. Toll-like receptor 4 (TLR4) is the main receptor for LPS, and myeloid differentiation factor 88 (MyD88) is a key adaptor protein molecule in the Toll-like receptor (TLR) signaling pathway. Thus, MyD88 knockdown heterozygous mice (MyD88+/-) were used to investigate the effect of incomplete knockout of the MyD88 gene on indirect LPS-induced ALI through intraperitoneal injection of LPS. The LPS-induced ALI significantly upregulated MyD88 expression, and heterozygous mice with incomplete knockout of the MyD88 gene (MyD88+/-) ameliorated LPS-induced histopathological injury and collagen fiber deposition. Heterozygous mice with incomplete knockout of the MyD88 gene (MyD88+/-) inhibited LPS-induced nuclear factor-κB (NF-κB) pathway activation, but TLR-4 expression tended to be upregulated. Incomplete knockdown of the MyD88 gene also downregulated LPS-induced expression of IL1-β, IL-6, TNF-α, TGF-β, SMAD2, and α-SMA. The transcriptome sequencing also revealed significant changes in LPS-regulated genes (such as IL-17 signaling pathway genes) after the incomplete knockdown of MyD88. In conclusion, this paper clarified that LPS activates the downstream NF-κB pathway depending on the MyD88 signaling pathway, which induces the secretion of inflammatory cytokines such as IL-1β/IL-6/TNF-α and ultimately triggers ALI. Incomplete knockdown of the MyD88 reverses LPS-induced lung fibrosis, which confirmed the vital role of MyD88 in LPS-induced ALI.

NLRP11 is a pattern recognition receptor for bacterial lipopolysaccharide in the cytosol of human macrophages.

Endotoxin-bacterial lipopolysaccharide (LPS)-is a driver of lethal infection sepsis through excessive activation of innate immune responses. When delivered to the cytosol of macrophages, cytosolic LPS (cLPS) induces the assembly of an inflammasome that contains caspases-4/5 in humans or caspase-11 in mice. Whereas activation of all other inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic pattern recognition receptor protein, whether pattern recognition receptors for cLPS exist has remained unclear, because caspase-4, caspase-5, and caspase-11 bind and activate LPS directly in vitro. Here, we show that the primate-specific protein NLRP11 is a pattern recognition receptor for cLPS that is required for efficient activation of the caspase-4 inflammasome in human macrophages. In human macrophages, NLRP11 is required for efficient activation of caspase-4 during infection with intracellular Gram-negative bacteria or upon electroporation of LPS. NLRP11 could bind LPS and separately caspase-4, forming a high-molecular weight complex with caspase-4 in HEK293T cells. NLRP11 is present in humans and other primates but absent in mice, likely explaining why it has been overlooked in screens looking for innate immune signaling molecules, most of which have been carried out in mice. Our results demonstrate that NLRP11 is a component of the caspase-4 inflammasome activation pathway in human macrophages.

Detection of mcr-1-harbouring Escherichia coli by quantum dot labelling of synthetic small peptides mimicking lipopolysaccharide receptors

Overuse of antibiotics and the emergence of multidrug-resistant bacteria has made colistin the last line of defence against complex infections. In previous studies, MCR-1-mediated colistin resistance was mainly detected through PCR or antimicrobial susceptibility testing. However, intuitive detection methods for phenotype are rarely reported. In this study, two small peptide antibodies were constructed for immunofluorescence detection of mcr-1-harbouring Escherichia coli: one was a small peptide labelled with a quantum dot antibody; and the other was a small peptide labelled with a fluorescein isothiocyanate (FITC) antibody. Whether using FITC or quantum dots, colistin-resistant bacteria in the sample could be qualitatively detected. The assembled antibodies achieved the desired goals in terms of sensitivity, specificity, precision and repeatability. The non-specific problem of sandwich antigen recognition of lipid A binding to small peptides in modified lipopolysaccharide (LPS) was resolved, and this relatively developed immunofluorescence technique standardised the detection process. Together, in addition to PCR, both fluorescent antibodies can be used for immunofluorescent detection of mcr-1-harbouring E. coli.

#5664 PERITONEAL DIFFUSE PODOPLANIN EXPRESSION IS ASSOCIATED WITH SEVERE ARTERIOLOPATHY IN CHILDREN ON PERITONEAL DIALYSIS

Abstract Background and Aims Podoplanin is a transmembrane glycoprotein binding chemokines and modulating inflammatory reactions. In healthy peritoneum, it is expressed in mesothelial cells and in lymphatic endothelium, expression beyond these structures (diffuse podoplanin staining, DPS) has been observed in adult patients with encapsulating peritoneal sclerosis (EPS). Method Parietal peritoneal tissues from 71 children on PD fluids with low glucose degradation product (GDP) content underwent digital histomorphometry. DPS and pathomechanisms of peritoneal membrane transformation and vasculopathy were hierarchically clustered. A panel of 13 peritoneal cell markers was selected and tissue sections were stained with metal conjugated antibodies and acquired using imaging mass cytometry (IMC; Hyperion Imaging System®). After single-cell segmentation, spillover correction, and normalization, cell types were identified using manual gating to train a random forest classifier for final cell type identifications using published workflows. Single-cell analysis was used to characterize peritoneal cells with positive podoplanin signals. Results DPS was present in 22% of the PD patients, all devoid of any clinical, radiological or histological feature of EPS. DPS positive patients were younger, had higher dialytic glucose exposure (median 103 vs. 154 g/m²/day, p = 0.002), and lower peritoneal arteriolar lumen to vessel (L/V) ratio, i.e. exhibited more arteriolar lumen narrowing. PD duration [median 20 (14.5, 48) months] and peritonitis incidence were similar as in non-DPS patients. In multivariable analysis, glucose exposure, PD duration, epithelial-to-mesenchymal transformed (EMT) cells and lower patient body surface area (BSA) independently predicted the presence of DPS. DPS and lower mesothelial surface coverage predicted lower arteriolar L/V ratio. In subgroups matched for BSA, PD duration and dialytic glucose exposure, DPS patients had a lower L/V ratio, (0.44 ± 0.13 vs. 0.31 ± 0.18, p = 0.003) and higher submesothelial CD45 leucocyte, CD68 macrophage and EMT cell count. Submesothelial lipopolysaccharide and hyaluronic acid receptor CD44 were increased in DPS positive patients, hyaluronan synthase 1 and 2 were similar. Among DPS positive patients, DPS intensity grade was higher in those with history of bacterial or fungal peritonitis (7 out of 15). Two patients had three and four peritonitis episodes respectively, their DPS pattern did not differ from patients with history of only one peritonitis episode. Hierarchical clustering demonstrated highest similarity of DPS positive areas with CD68 positive areas. In IMC analysis of DPS positive areas, highest podoplanin signals were derived from M2 classified macrophages (CD68+CD163+) and fibroblastic cells [αSMA positive/non-endothelial/non-macrophage (αSMA+PROX1-CD31-CD68-CD163-)], signals derived from M1 classified macrophages (CD68+CD163-) were lowest. Conclusion Peritoneal DPS in pediatric patients on low-GDP PD is associated with lower BSA, higher dialytic glucose exposure, history of infectious peritonitis and peritoneal inflammatory and EMT cell invasion, and predicts obliterating peritoneal vasculopathy. We identified M2 macrophages as major source of extra-lymphatic peritoneal podoplanin accumulation, suggesting their role in severe peritoneal transformation.

Metformin treatment of juvenile mice potentiates innate immune response to bacterial lipopolysaccharide in males

Objective: Metformin is a widely used drug, with a good safety profile, for treatment of type 2 diabetes. Recently, it has been found to also exhibit immunomodulatory potential. We aim to preliminarily explore the potential role of metformin in regulating innate immunity using UM-HET3 mice. Methods: In this study, we treated juvenile UM-HET3 mice with metformin and investigated the alteration of the innate immune response. Peripheral blood of HET3 mice was treated with ex-vivo lipopolysaccharide (LPS) stimulation to induce a leukocyte inflammatory response, which was detected by analyzing the expression of LPS receptors TLR4 and CD14 on leukocytes with flow cytometry. Furthermore, cytokine release induced by LPS was measured in isolated cell culture supernatants with ELISA. Results: In this study LPS treatment triggered a downregulation of TLR4 as well as an upregulation of CD14 expressed on the cell surface. Besides, it enhanced the secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. The results showed that metformin treatment led to a significant ( p < .05) increase in the expression of CD14 and pro-inflammatory cytokines, including IL-1β and IL-6, as well as increased proportion of TLR4 downregulation on the cell surface in response to LPS, specifically in males. Conclusions: This study indicates that treating juvenile mice with metformin enhances the innate immune response to bacterial endotoxin in males, but not in females.

Importance of circulating monocyte subpopulations and Fcγ receptors in immune thrombocytopenia.

Monocytes have been linked to the pathogenesis of immune thrombocytopenia (ITP) because of their role in autoantibody-mediated platelet phagocytosis. However, monocytes constitute unique populations with major differences in expression for surface Fcγ receptors (FcγRs). Thus, we evaluated monocytes in whole blood samples from patients with newly diagnosed and chronic ITP. Monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 (lipopolysaccharide receptor) and of CD16 (low-affinity Fcγ receptor III) into classical (CLM), intermediate (INTM) and nonclassical (non-CLM) monocytes. We also examined the expression of FcγRI/CD64 and FcγRIII/CD16 by monocyte subpopulations. Newly diagnosed patients showed a decrease in non-CLM, expressed as a relative percentage of total monocytes compared with controls and chronic ITP patients. Both non-CLM and INTM of newly diagnosed patients closely correlated with platelet count. These monocyte subpopulations showed significantly enhanced CD64 expression in newly diagnosed patients. On the contrary, patients with chronic ITP presented higher non-CLM in percentage than controls and concomitant lower CLM and total monocytes, in percentage and number. The expression of CD64 was increased by all monocyte subpopulations, CLM, INTM, and non-CLM in chronic patients. In conclusion, differences in monocyte subpopulations, together with enhanced expression of FcγRI/CD64 are evident in patients with ITP.

Skewing of UV-induced cell death pathways using TLR4 ligands

Abstract Toll-like receptor 4 (TLR4) is a canonical innate immune receptor for the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). TLR4 deficiency has been shown to contribute to UV-induced carcinogenesis in epidermal cells. UV-induced cellular damage results in apoptosis which is characterized by a non-inflammatory cell death through the cleavage and activation of caspase-3. Utilizing bacterial enzyme combinational chemistry (BECC) we generated hypoacylated lipid A moieties from Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium which have moderately attenuated immune-stimulatory capacity. Continuously exposing TLR4 to these innate immune ligands leads to tolerization and decreased expression of the receptor in mammalian cells. We hypothesize that skewing cell death away from apoptosis using lipid A mimetics to lower TLR4 expression prior to UV exposure will reduce carcinogenic potential. To analyze the roll of TLR4 in UV-induced carcinogenesis, wildtype and TLR4-deficient bone marrow-derived macrophage (BMDM) cell lines were tolerized with hexa-acylated or penta-acylated lipid A from multiple Gram-negative species and exposed to either 10 or 25 mJ/cm 2doses of UVB radiation. UV-irradiated BMDM cells show evidence of apoptosis including cleavage of DNA creating a ladder, whereas UV-irradiated BMDM cells tolerized with lipid A mimetics show a lack of apoptosis. The data suggests that tolerization of TLR4 using canonical ligands may be a useful tool in promoting DNA repair and maintaining immune responses following UV-induced damage. The research was supported by a Fisher College of Science and Mathematics Undergraduate Research Grant.

Mycobacterium bovis BCG increase the selected determinants of monocyte/macrophage activity, which were diminished in response to gastric pathogen Helicobacter pylori

High antibiotic resistance of gastric pathogen Helicobacter pylori (Hp) and the ability to escape the host immune response prompt searching for therapeutic immunomodulators. Bacillus Calmette–Guerin (BCG) vaccine with Mycobacterium bovis (Mb) is a candidate for modulation the activity of immunocompetent cells, and onco-BCG formulation was successfully used in immunotherapy of bladder cancer. We determined the influence of onco-BCG on the phagocytic capacity of human THP-1 monocyte/macrophage cells, using the model of Escherichia coli bioparticles and Hp fluorescently labeled. Deposition of cell integrins CD11b, CD11d, CD18, membrane/soluble lipopolysaccharide (LPS) receptors, CD14 and sCD14, respectively, and the production of macrophage chemotactic protein (MCP)-1 were determined. Furthermore, a global DNA methylation, was also assessed. Human THP-1 monocytes/macrophages (TIB 202) primed or primed and restimulated with onco-BCG or Hp, were used for assessment of phagocytosis towards E. coli or Hp, surface (immunostaining) or soluble activity determinants, and global DNA methylation (ELISA). THP-1 monocytes/macrophages primed/restimulated with BCG showed increased phagocytosis capacity towards E. coli fluorescent particles, elevated expression of CD11b, CD11d, CD18, CD14, sCD14, increased MCP-1 secretion and DNA methylation. Preliminary results indicate that BCG mycobacteria may also induce the phagocytosis of H. pylori by THP-1 monocytes. Priming or priming and restimulation of monocytes/macrophages with BCG resulted in an increased activity of these cells, which was negatively modulated by Hp.

Water-soluble protein from walleye pollock (Gadus chalcogrammus) suppresses lipopolysaccharide-induced inflammation by attenuating TLR4–MyD88 expression in macrophages

Water-soluble protein (WSP) from fish meat is abundant in the waste effluent generated via the surimi manufacturing process. This study investigated the anti-inflammatory effects and mechanisms of fish WSP using primary macrophages (MΦ) and animal ingestion. MΦ were treated with digested-WSP (d-WSP, 500 µg/mL) with or without lipopolysaccharide (LPS) stimulation. For the ingestion study, male ICR mice (5 weeks old) were fed 4% WSP for 14 days following LPS administration (4 mg/kg body weight). d-WSP decreased the expression of Tlr4, an LPS receptor. Additionally, d-WSP significantly suppressed the secretion of inflammatory cytokines, phagocytic ability, and Myd88 and Il1b expressions of LPS-stimulated macrophages. Furthermore, the ingestion of 4% WSP attenuated not only LPS-induced IL-1β secretion in the blood but also Myd88 and Il1b expressions in the liver. Thus, fish WSP decreases the expressions of the genes involved in the TLR4–MyD88 pathway in MΦ and the liver, thereby suppressing inflammation.

Bacterial lipopolysaccharide hyperpolarizes the membrane potential and is antagonized by the K2p channel blocker doxapram

Exposure of Drosophila skeletal muscle to bacterial lipopolysaccharides (LPS) rapidly and transiently hyperpolarizes membrane potential. However, the mechanism responsible for hyperpolarization remains unclear. The resting membrane potential of the cells is maintained through multiple mechanisms. This study investigated the possibility of LPS activating calcium-activated potassium channels (KCa) and/or K2p channels. 2-Aminoethyl diphenylborinate (2-APB), blocks uptake of Ca2+ into the endoplasmic reticulum (ER); thus, limiting release from ryanodine-sensitive internal stores to reduce the function of KCa channels. Exposure to 2-APB produces waves of hyperpolarization even during desensitization of the response to LPS and in the presence of doxapram. This finding in this study suggests that doxapram blocked the acid-sensitive K2p tandem-pore channel subtype known in mammals. Doxapram blocked LPS-induced hyperpolarization and depolarized the muscles as well as induced motor neurons to produce evoked excitatory junction potentials (EJPs). This was induced by depolarizing motor neurons, similar to the increase in extracellular K+ concentration. The hyperpolarizing effect of LPS was not blocked by decreased extracellular Ca2+or the presence of Cd2+. LPS appears to transiently activate doxapram sensitive K2p channels independently of KCa channels in hyperpolarizing the muscle. Septicemia induced by gram-negative bacteria results in an increase in inflammatory cytokines, primarily induced by bacterial LPS. Currently, blockers of LPS receptors in mammals are unknown; further research on doxapram and other K2p channels is warranted. (220 words).

Tissue CD14+CD8+ T cells reprogrammed by myeloid cells and modulated by LPS.

The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours1-4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability.

Rhodobacter capsulatus PG Lipopolysaccharide Blocks the Effects of a Lipoteichoic Acid, a Toll-Like Receptor 2 Agonist.

Lipopolysaccharides (LPS) and lipoteichoic acids (LTA) are the major inducers of the inflammatory response of blood cells caused by Gram-negative and some Gram-positive bacteria. CD14 is a common receptor for LPS and LTA that transfers the ligands to TLR4 and TLR2, respectively. In this work, we have demonstrated that the non-toxic LPS from Rhodobacter capsulatus PG blocks the synthesis of pro-inflammatory cytokines during the activation of blood cells by Streptococcus pyogenes LTA through binding to the CD14 receptor, resulting in the signal transduction to TLR2/TLR6 being blocked. The LPS from Rhodobacter capsulatus PG can be considered a prototype for developing preparations to protect blood cells against the LTA of gram-positive bacteria.

A short history of innate immunity

Innate immunity refers to the mechanisms responsible for the first line of defense against pathogens, cancer cells and toxins. The innate immune system is also responsible for the initial activation of the body's specific immune response (adaptive immunity). Innate immunity was studied and further developed in parallel with adaptive immunity beginning in the first half of the 19th century and has been gaining increasing importance to our understanding of health and disease. In the present overview, we describe the main findings and ideas that contributed to the development of innate immunity as a continually expanding branch of modern immunology. We start with the toxicological studies by Von Haller and Magendie, in the late 18th and early 19th centuries, and continue with the discoveries in invertebrate immunity that supported the discovery and characterization of lipopolysaccharide (LPS) and pattern recognition receptors that led to the development of the pattern recognition and danger theory.

Scopolamine causes delirium-like brain network dysfunction and reversible cognitive impairment without neuronal loss.

Delirium is a severe acute neuropsychiatric syndrome that commonly occurs in the elderly and is considered an independent risk factor for later dementia. However, given its inherent complexity, few animal models of delirium have been established and the mechanism underlying the onset of delirium remains elusive. Here, we conducted a comparison of three mouse models of delirium induced by clinically relevant risk factors, including anesthesia with surgery (AS), systemic inflammation, and neurotransmission modulation. We found that both bacterial lipopolysaccharide (LPS) and cholinergic receptor antagonist scopolamine (Scop) induction reduced neuronal activities in the delirium-related brain network, with the latter presenting a similar pattern of reduction as found in delirium patients. Consistently, Scop injection resulted in reversible cognitive impairment with hyperactive behavior. No loss of cholinergic neurons was found with treatment, but hippocampal synaptic functions were affected. These findings provide further clues regarding the mechanism underlying delirium onset and demonstrate the successful application of the Scop injection model in mimicking delirium-like phenotypes in mice.