The effect of synthetic muramyl dipeptide on glycosaminoglycan synthesis in cultured rat costal chondrocytes was examined. Muramyl dipeptide alone had no effect on the glycosaminoglycan synthesis of rat chondrocytes, whereas Escherichia coli lipopolysaccharide and interleukin 1 alpha inhibited glycosaminoglycan synthesis in a dose dependent manner. Muramyl dipeptide, when added to chondrocyte cultures in the presence of lipopolysaccharide, enhanced the lipopolysaccharide induced inhibition of glycosaminoglycan synthesis in a dose dependent manner. Adjuvant active analogues of muramyl dipeptide, but not adjuvant inactive analogues, also enhanced the lipopolysaccharide induced inhibition of glycosaminoglycan synthesis. In combination with muramyl dipeptide, to inhibit glycosaminoglycan synthesis, lipopolysaccharide could be replaced with the synthetic lipid A, an active principle of lipopolysaccharide. These results show that the muramyl dipeptide portion of bacterial peptidoglycan enhances the susceptibility of rat chondrocytes to the lipid A portion of bacterial lipopolysaccharide, and therefore the interaction between chondrocytes and bacterial cell wall components might be involved in damaging the cartilage in inflammatory joint diseases.
Lysates obtained from amoebocytes of Limulus polyphemus, the horseshoe crab, showed gel formation after the addition of bacterial endotoxin. In contrast to living gram-negative bacteria, viable gram-positive microorganisms did not cause gelation of lysate. Nevertheless, peptidoglycan isolated from the cell walls of various gram-positive organisms did induce the reaction. However, the activity of peptidoglycan was 1,000 to 400,000 times less than that of Escherichia coli lipopolysaccharide. After exposure to lysozyme, peptidoglycan no longer gelled amoebocyte lysate, therefore apparently excluding endotoxin contamination. Gelation of amoebocyte lysate by endotoxin or peptidoglycan was inhibited by different concentrations of sodium polystyrolsulfonate. Whereas these studies confirm the specificity of the Limulus test for bacterial endotoxins, they also indicate that other substances of bacterial origin should be investigated for their ability to gel amoebocyte lysate.
Activation of the alternate complement pathway in human serum by several bacterial components was compared. Peptidoglycan from group A streptococcal cell walls was the most active material, on a weight basis, followed by cell walls, protoplast membranes, and whole cells. The group-specific carbohydrate was inactive. Treatment of peptidoglycan with low concentrations of lysozyme or short periods of sonic treatment enhanced complement activation. High concentrations of lysozyme or extended sonic treatment of peptidoglycan destroyed or greatly reduced the capacity to activate complement. Lysozyme treatment of group A streptococcal cell walls or lipopolysaccharide had no measurable effect. Activation of the alternate complement pathway by group D streptococcal cell walls was destroyed by lysozyme. Activity of peptidoglycan was not inhibited by N-acetyl glucosamine, N-acetyl muramic acid, or D-alanine-D-alanine. Conversion of C3 and factored B by peptidoglycan was shown to occur by immunoelectrophoresis and crossed immunoelectrophoresis.
Lipopolysaccharide (LPS) preparations are known to often contain substances which activate cells through Toll-like receptor 2 (TLR2), and it is suspected that bacterial lipoproteins are responsible for this activation. We compared the mode of action of the TLR2-stimulatory substances with that of a synthetic bacterial lipopeptide (tripalmitoyl-Cys-Ser-Ser-Asn-Ala [Pam3CSSNA]), as well as with that of peptidoglycan. Six out of eight LPS preparations tested induced NF-κB-dependent reporter activity in 293 cells expressing CD14 and TLR2. Phenol extract (PEX) prepared from Escherichia coli LPS by modified phenol extraction induced reporter activity in 293 cells expressing TLR2, and this activity was enhanced by coexpression of CD14, whereas the activity of Pam3CSSNA was not dependent on CD14. The activity of PEX, but not that of Pam3CSSNA or peptidoglycan, was also enhanced by LPS binding protein or serum and blocked by polymyxin B. In addition, the activity of PEX was inhibited by a lipid A precursor (compound 406) in 293 cells expressing CD14 and TLR2. These results indicate that E. coli LPS preparations contain LPS-mimetic TLR2-stimulatory substances which differ from bacterial lipopeptides or peptidoglycan.
The purpose of this study was to identify the functional significance of the binding of soluble CD14 (sCD14) to bacterial peptidoglycan (PGN) and to compare the structural requirements of sCD14 for the binding to PGN and lipopolysaccharide (LPS) and for sCD14-mediated enhancement of PGN- and LPS-induced cell responses. sCD14 did not facilitate the responses of membrane CD14 (mCD14)-negative pre-B 70Z/3 cells to PGN, although it facilitated the responses of these cells to LPS and although mCD14 facilitated the responses of 70Z/3 cells to PGN. sCD14 enhanced mCD14-mediated cell activation by both PGN and LPS, but only the responses to LPS, and not to PGN, were enhanced by LPS-binding protein. Four 4- or 5-amino-acid-long sequences within the 65-amino-acid N-terminal region of sCD14 were needed for binding to both PGN and LPS and for enhancement of cell activation by both PGN and LPS. However, deletions of individual sequences had different effects on the ability of sCD14 to bind to PGN and to LPS and on the ability to enhance the responses to PGN and to LPS. Thus, there are different structural requirements of sCD14 for binding to PGN and to LPS and for the enhancement of PGN- and LPS-induced cell activation.
We have previously reported the cloning and characterization of CARD6, a caspase recruitment domain (CARD)-containing protein that is structurally related to the interferon (IFN)-inducible GTPases. CARD6 associates with microtubules and with receptor-interacting protein 2 (RIP2). RIP2 mediates NF-κB activation induced by the intracellular nucleotide-binding oligomerization domain (NOD) receptors that sense bacterial peptidoglycan. Here we report that the expression of CARD6 and RIP2 in bone marrow-derived macrophages is rapidly induced by beta IFN and gamma IFN. This IFN-induced upregulation of CARD6 is suppressed by lipopolysaccharide (LPS), in contrast to LPS's enhancement of IFN-induced RIP2 upregulation. We generated CARD6-deficient (CARD6−/−) mice and carried out extensive analyses of signaling pathways mediating innate and adaptive immune responses, including the NOD pathways, but did not detect any abnormalities. Moreover, CARD6−/− mice were just as susceptible as wild-type mice to infection by Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Candida albicans, lymphocytic choriomeningitis virus, or mouse adenovirus type 1. Thus, although structural and in vitro analyses strongly suggest an important role for CARD6 in immune defense, the physiological function of CARD6 remains obscure.
In this study, the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated. Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 46 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferative and to produce leukocyte migration inhibitory factor. Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations acted as mitogens. Cell walls and peptidoglycans had a modulating effect on purified protein derivative-induced and protein A-induced proliferation. In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response. However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation. Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells. Solubilization had no effect on immunomodulating capacity.
In this study the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated. Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 45 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferate and to produce leukocyte-inhibitory factor. Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations were acting as mitogens. Cell walls and peptidoglycans had a modulating effect on purified protein derivative- or protein A-induced proliferation. In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response. However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation. Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells. Solubilization had no effect on the immunomodulating capacity.
The objective of this study was to determine if inflammatory tolerance and enhancement of innate immune function could be induced by the gram-positive cell wall component peptidoglycan (PGN). Male mice (C57BL6/J or C3H/HeJ, 8-12 weeks of age) were given intraperitoneal injections of 1mg PGN on 2 consecutive days. The mice were then challenged with lipopolysaccharide (LPS) or live Pseudomonas aeruginosa (1 × 108 colony-forming units) 2 days after the second pretreatment. Mice pretreated with PGN had diminished plasma concentrations of TNFα and IFNγ and elevated concentrations of IL-10 in response to a subsequent LPS or Pseudomonas challenge when compared to untreated controls. Bacterial clearance was improved in mice pretreated with PGN, and mortality in response to a subsequent Pseudomonas challenge was significantly attenuated. PGN pretreatment of LPS-unresponsive mice (C3H/HeJ) verified that the effect of PGN pretreatment was not due to any LPS contamination. We have previously demonstrated that PGN pretreatment induced resistance to a Gram-positive bacterial challenge. The present study extends those results by showing that exposure to the Gram-positive bacterial cell wall component peptidoglycan also induces cross-tolerance to LPS and non-specifically enhances innate immune function in that PGN-pretreated mice had increased resistance to Gram-negative bacterial challenge.
mice; peptidoglycan; tolerance; bacterial infection; TLR4
Symptoms of bacterial infection include decreases in body mass (cachexia), induction of depressive-like hedonic tone (anhedonia), decreases in food intake (anorexia), and increases in body temperature (fever). Recognition of bacteria by the innate immune system triggers the release of proinflammatory cytokines which induce these sickness behaviors via central and peripheral substrates. In Siberian hamsters, exposure to short day lengths decreases both the production of proinflammatory cytokines and the magnitude of the symptoms of infection. Short-day attenuation of sickness behaviors may arise solely from decreased production of cytokines; alternatively, substrates responsible for the generation of sickness behaviors may be less responsive to cytokines in short days. To discriminate among these hypotheses, Siberian hamsters were treated with either bacterial lipopolysaccharide (LPS; 25 μg) or recombinant mouse IL-1β (rIL-1β; 100 ng) following 11 weeks of exposure to long (15 h light/day) and short (9 h light/day) photoperiods. Replicating earlier work, the magnitude and/or duration of LPS-induced anorexia, anhedonia, cachexia, and fever were greater in long-day relative to short-day hamsters. A comparable short-day attenuation of sickness behaviors and fever was obtained in response to rIL-1β treatment, despite treatment with identical concentrations of cytokine. These data suggest that hamsters subjected to immunoenhancing short days exhibit diminished symptoms of infection not solely because infections elicit lower levels of cytokine production, but also because the substrates upon which cytokines act become relatively refractory.
seasonality; sickness behaviors; depression; anhedonia; food intake; body mass; fever; LPS; IL-β
Seasonal changes in day length enhance or suppress aspects of immune function in mammals. Following adaptation to short, winter-like short photoperiods, cytokine and behavioral responses to lipopolysaccharide (LPS)-induced simulated infections are attenuated in LPS-naive Siberian hamsters. This experiment examined whether diminished initial responses to LPS in short days are accompanied by decrements in the development of innate immunological memory that leads to endotoxin tolerance. Male hamsters exposed to short days (9h-light/day; SD) or kept in their natal long-day photoperiod (15h-light/day; LD) for 12–13 weeks were injected with bacterial LPS (625 µg/kg, i.p.) or sterile saline. Ten days later all hamsters were challenged with LPS (625 ug/kg, i.p.), and behavioral sickness responses (anorexia and reductions in nest building) were assessed. In LD hamsters, behavioral responses to the second LPS injection were markedly attenuated but still evident, indicative of partial tolerance. SD hamsters, in contrast, failed to exhibit anorexic or thermoregulatory responses to the second LPS injection, indicative of complete behavioral tolerance to LPS. Thus despite engaging greater naive responses to LPS, LD hamsters exhibited incomplete LPS tolerance relative to SD hamsters. The expression of behavioral tolerance to endotoxin is relatively diminished during the breeding season, a time of year when naive responses to endotoxin are at their greatest. During winter, enhancements in behavioral endotoxin tolerance may conserve energy and facilitate survival in the face of energetically-challenging conditions.
seasonality; sickness behavior; endotoxin tolerance; photoperiodism; Siberian hamster
The effects of bacterial cell walls, peptidoglycans, and a water-soluble polymer of peptidoglycan subunits on dengue virus type 2 replication in cultured mouse peritoneal macrophages were studied. Pretreatment of macrophage cultures with all of test cell walls isolated from seven bacterial species for 3 days significantly enhanced the virus production in the cultures. Peptidoglycans prepared from four of the above cell walls also exerted the virus production-enhancing effects in a similar manner as the walls. A water-soluble polymer of peptidoglycan subunits which was prepared by treatment of Staphylococcus epidermidis wall peptidoglycan with an interpeptide bridge-splitting enzyme (endopeptidase) also definitely enhanced the virus production in macrophage cultures, although its activity was weaker than that of the original wall and peptidoglycan. Macrophage cultures from athymic nude mice, when treated with cell walls and peptidoglycans of S. epidermidis and Lactobacillus plantarum for 3 days, also showed an increased ability to support dengue virus type 2 replication. The infectious center assay demonstrated that the virus replication enhancement by S. epidermidis cell wall and peptidoglycan was primarily due to an increase in the number of virus-infected cells. This finding did not seem to be in conflict with the observation that macrophages treated with the above cell wall or peptidoglycan phagocytized more latex particles than did untreated macrophages. The conclusions based on the above experiments are that the treatment of mouse peritoneal macrophage cultures with bacterial cell walls and their components increases the take of dengue virus type 2 by macrophages and thus raises the virus production in the macrophage cultures.
Sickness behaviour is an adaptive behavioural response to the activation of the innate immune system. It is mediated by brain cytokine production and action, especially interleukin-6 (IL-6). Polyunsaturated fatty acids (PUFA) are essential fatty acids that are highly incorporated in brain cells membranes and display immunomodulating properties. We hypothesized that a decrease in n-3 PUFA brain level by dietary means impacts on lipopolysaccharide (LPS)-induced IL-6 production and sickness behaviour. Our results show that mice exposed throughout life to a diet containing n-3 PUFA (n-3/n-6 diet) display a decrease in social interaction that does not occur in mice submitted to a diet devoid of n-3 PUFA (n-6 diet). LPS induced high IL-6 plasma levels as well as expression of IL-6 mRNA in the hippocampus and cFos mRNA in the brainstem of mice fed either diet, indicating intact immune-to-brain communication. However, STAT3 and STAT1 activation, a hallmark of IL-6 signalling pathway, was lower in the hippocampus of LPS-treated n-6 mice as compared to n-3/n-6 mice. In addition, LPS did not reduce social interaction in IL-6 knock-out (IL-6 KO) mice and failed to induce STAT3 activation in the brain of IL-6 KO mice. Altogether, these findings point to alteration in brain STAT3 as a key mechanism for the lack of effect of LPS on social interaction in mice fed with the n-6 PUFA diet. The relative deficiency of Western diets in n-3 PUFA could impact on behavioural aspects of the host response to infection.
docosahexaenoic acid; arachidonic acid; STAT3; social interaction; LPS
To search for the induction of the expression of antimicrobial peptides in corneal fibroblasts treated with bacterial components.
RT‐PCR was performed to search for mRNAs expression of antimicrobial peptides and toll‐like receptors (TLRs) in murine primary cultures of corneal fibroblast (PCCF) treated with lipopolysaccharide (LPS) from Escherichia coli, peptidoglycan from Staphylococcus aureus, and cytosine‐phosphorous‐guanine oligonucleotide (CpG‐ODN). Cellular activation was blocked with anti‐TRL antibodies.
LPS did not induce expression of antimicrobial peptide in corneal fibroblasts. Cathelin related antimicrobial peptide (CRAMP) and α‐defensin 3 were overexpressed in a time and dose dependent manner in corneal fibroblasts treated with peptidoglycan and with CpG‐ODN, respectively. CRAMP expression was blocked when PCCF were treated with anti‐TLR‐2 antibodies. α‐Defensin 3 was not expressed in NIH murine corneal fibroblasts (which do not express the TLR‐9 molecule) treated with CpG‐ODN.
Results suggest that corneal fibroblasts, which are the second cellular barrier of the cornea, can play an important part in the innate immunity of the eye via TLR stimulation.
CRAMP; α‐defensin 3; corneal fibroblast; toll‐like receptors; antimicrobial peptides
Bacterial infections play an important role in the multifactorial etiology of rheumatoid arthritis. The arthropathic properties of Gram-positive bacteria have been associated with peptidoglycan–polysaccharide complexes (PG-PS), which are major structural components of bacterial cell walls. There is little agreement as to the identity of cellular receptors that mediate innate immune responses to PG-PS. A glycosylphosphatidylinositol-linked cell surface protein, CD14, the lipopolysaccharide receptor, has been proposed as a PG-PS receptor, but contradictory data have been reported. Here, we examined the inflammatory and pathogenic responses to PG-PS in CD14 knockout mice in order to examine the role for CD14 in PG-PS-induced signaling. We found that PG-PS-induced responses in vitro, including transient increase in intracellular calcium, activation of nuclear factor-κB, and secretion of the cytokines tumor necrosis factor-α and interleukin-6, were all strongly inhibited in CD14 knockout macrophages. In vivo, the incidence and severity of PG-PS induced acute polyarthritis were significantly reduced in CD14 knockout mice as compared with their wild-type counterparts. Consistent with these findings, CD14 knockout mice had significantly inhibited inflammatory cell infiltration and synovial hyperplasia, and reduced expression of inflammatory cytokines in PG-PS arthritic joints. These results support an essential role for CD14 in the innate immune responses to PG-PS and indicate an important role for CD14 in PG-PS induced arthropathy.
bacterial; cell surface molecules; rheumatoid arthritis; transcription factors
Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis.
Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.
Two common pathogens known to cause bone infection, Salmonella and Staphylococcus aureus, were investigated to determine their abilities to induce chemokine expression in cultured mouse and human osteoblasts. While these cells are responsible for bone formation, we were surprised to find that they could respond to bacterial infection by upregulating expression of the chemokine CXCL10 (IP-10). However, there were significant differences in the abilities of the gram-negative bacterium Salmonella and the gram-positive bacterium S. aureus to induce expression of CXCL10. Reverse transcription-PCR and enzyme-linked immunosorbent assay analyses showed high levels of Salmonella-induced CXCL10 mRNA and protein expression, respectively, whereas the osteoblast response to S. aureus was significantly less. Consistent with these findings, Salmonella-derived lipopolysaccharide (LPS), but not S. aureus-derived peptidoglycan, could induce expression of CXCL10. An antibody against toll-like receptor 4 (TLR4) could block the LPS-induced CXCL10 production, demonstrating the functional expression of TLR4 by osteoblasts. Despite the inducible nature of TLR2 mRNA expression by bacterium-infected osteoblasts, peptidoglycan failed to stimulate CXCL10 secretion. Immunofluorescent staining of bacterium-infected calvaria (i.e., skull bone) demonstrated the presence of CXCL10 in osteoblasts. The fact that osteoblasts did not express CXCR3 mRNA, whereas T lymphocytes can express high levels of this receptor, suggests that osteoblast-derived CXCL10 may recruit T lymphocytes to the sites of bone infections.
Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05), and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05). Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.
During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-α and IL-1β. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 μg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-α, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.
Nuclear factor κB (NF-κB) plays a pivotal role in sepsis. Activation of NF-κB is initiated by the signal-induced ubiquitylation and subsequent degradation of inhibitors of kappa B (IκBs) primarily via activation of the IκB kinase (IKK). This study was designed to investigate the effects of IKK inhibition on sepsis-associated multiple organ dysfunction and/or injury (MOD) and to elucidate underlying signaling mechanisms in two different in vivo models: male C57BL/6 mice were subjected to either bacterial cell wall components [lipopolysaccharide and peptidoglycan (LPS/PepG)] or underwent cecal ligation and puncture (CLP) to induce sepsis-associated MOD. At 1 hour after LPS/PepG or CLP, mice were treated with the IKK inhibitor IKK 16 (1 mg/kg body weight). At 24 hours, parameters of organ dysfunction and/or injury were assessed in both models. Mice developed a significant impairment in systolic contractility (echocardiography), and significant increases in serum creatinine, serum alanine aminotransferase and lung myeloperoxidase activity, thus indicating cardiac dysfunction, renal dysfunction, hepatocellular injury and lung inflammation, respectively. Treatment with IKK 16 attenuated the impairment in systolic contractility, renal dysfunction, hepatocellular injury and lung inflammation in LPS/PepG-induced MOD and in polymicrobial sepsis. Compared with mice that were injected with LPS/PepG or underwent CLP, immunoblot analyses of heart and liver tissues from mice that were injected with LPS/PepG or underwent CLP and were also treated with IKK 16 revealed: (1) significant attenuation of the increased phosphorylation of IκBα; (2) significant attenuation of the increased nuclear translocation of the NF-κB subunit p65; (3) significant attenuation of the increase in inducible nitric oxide synthase (iNOS) expression; and (4) a significant increase in the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Here, we report for the first time that delayed IKK inhibition reduces MOD in experimental sepsis. We suggest that this protective effect is (at least in part) attributable to inhibition of inflammation through NF-κB, the subsequent decrease in iNOS expression and the activation of the Akt-eNOS survival pathway.
We investigated the induction of interleukin-8 (IL-8) by bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) in the bladder cancer cell lines T24, 5637, UM-UC-3, and HT1197. T24 and 5637 cells strongly induced IL-8 after stimulation with LPS or PGN in a dose- and time-dependent manner, whereas UM-UC-3 and HT1197 cells did so very weakly. The expression of CD14 at the mRNA, total cellular protein, and cell surface protein levels differed among these cell lines, but the expression levels of Toll-like receptors 2 and 4 (TLR2 and TLR4) were not significantly different. The CD14 expression levels were found to correlate with the inducibility of IL-8 by LPS or PGN. Treatment of T24 and 5637 cells with phosphatidylinositol-specific phospholipase C to eliminate CD14 from the cell surface dramatically suppressed the induction of IL-8. On the other hand, UM-UC-3 cells transfected with CD14 cDNA expressed membrane-anchored CD14 and showed more efficent induction of IL-8 by LPS stimulation than untransfected controls. These results suggest that the presence of the membrane-anchored, but not the soluble, form of CD14 is a strong factor in IL-8 induction in bladder epithelial cells in response to bacterial components. The presence of the membrane-anchored form of CD14 may thus be a determinant for the inflammatory response of uroepithelial cells.
Toll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion.
Middle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis.
Among the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20±60.94 ng/mL, 21.59±7.96 ng/mL and 11.55±16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile).
TLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.
Otitis media; Toll-like receptor; Mutation
Activation of prophenoloxidase and synthesis of antimicrobial peptides (AMPs) are two important innate immune mechanisms in insects. In the current study, we investigated immune responses activated by three major bacterial components, lipopolysaccharide (LPS) (including rough mutants of LPS), lipoteichoic acid (LTA), and peptidoglycan (PG), in the larvae of a lepidopteran insect, Manduca sexta. We found that two DAP (Diaminopimelic acid)-type PGs from Escherichia coli and Bacillus subtilis were much more potent than LPS and LTA from the respective bacteria as well as a Lysine-type PG in activation of prophenoloxidase in M. sexta larval plasma in vitro. Transcription levels of AMP genes, such as Attacin, Lebocin and Moricin genes, in the hemocytes and fat body of larvae were significantly induced by smooth LPS (TLR4grade) and rough mutants of LPS (TLRgrade™), synthetic lipid A, LTA, and PG. LPS from E. coli and LTA from B. subtilis activated AMP expression to significantly higher levels than PGs from the respective bacterial strains, and smooth LPS were more potent than lipid A and rough mutants of LPS in activation of AMP expression. Our results demonstrated for the first time that LTA can activate AMP expression, and different moieties of LPS may synergistically activate AMP expression in M. sexta.
Lipopolysaccharide; Lipoteichoic acid; Peptidoglycan; Phenoloxidase; Antimicrobial peptide; Manduca sexta
Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNγ). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNγ resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNγ also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNγ treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi.