Dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice

To investigate the effect of dietary lipids with different fatty acid compositions upon the in vivo cytokine response to bacterial lipopolysaccharide (LPS), mice were fed for 5 weeks on a low-fat diet or on one of four high-fat diets that contained 20%, by weight, of coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO). The mice were injected intraperitoneally with a non-lethal dose of Escherichia coli LPS (100 micrograms/20 g body weight) and killed 90 or 180 min later. Plasma tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6 and IL-10 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Plasma TNF-alpha and IL-10 concentrations were higher 90 min postinjection than after 180 min, whereas plasma IL-1beta and IL-6 concentrations were higher 180 min postinjection than after 90 min. Peak plasma TNF-alpha, IL-1beta and IL-6 concentrations were lower in the CO- and FO-fed mice than in those fed the SO diet. Peak plasma IL-10 concentrations were higher in CO-fed mice than in those fed some of the other diets. These observations suggest that, relative to the n-6 polyunsaturated fatty acid-rich SO diet, CO and FO diminish production of proinflammatory cytokines in vivo. This indicates that these fatty acids might be useful therapies in acute and chronic inflammatory diseases. The enhanced production of IL-10 following CO feeding appears to be an additional antiinflammatory effect of this oil, which could give added benefit in various clinical conditions.     

Modification of the inflammatory response to allergen challenge after exposure to bacterial lipopolysaccharide

The potential role of respiratory infections in altering the development of atopy and asthma is complex. Infections have been suggested to be effective in preventing the induction of T-helper 2-polarized allergen-specific immunity in early life, but also to exacerbate asthma in older, sensitized individuals. The mechanism(s) underlying these effects are poorly defined. The aim of this work was to determine the influence of lipopolysaccharide (LPS) exposure on the development of sensitization to allergen and the response to allergen challenge in vivo. Piebald-Virol-Glaxo rats were exposed to a single aerosol of LPS 1 d before or 1, 2, 4, 6, 8, or 10 d after sensitization with ovalbumin (OVA). On Day 11 animals were exposed to 1% OVA and responses to allergen were measured 24 h later, monitoring inflammatory cell influx and microvascular leakage into bronchoalveolar lavage (BAL) fluid as well as pulmonary responses to methacholine using the forced oscillation technique. Histologic analysis was included to complement the BAL results. Single aerosol exposure to LPS 1 d before and up to 4 d after intraperitoneal injection of OVA protected against the development of OVA-specific immunoglobulin (Ig) E. LPS exposure 6, 8, or 10 d after sensitization further exacerbated the OVA-induced cellular influx, resulting in neutrophilia and increased Evans Blue dye leakage with no effect on serum IgE levels. In addition, LPS abolished the OVA-induced hyperresponsiveness in sensitized animals when given 18 h after OVA challenge. This study demonstrates that exposure to LPS can modify the development of allergic inflammation in vivo by two independent mechanisms. Exposure early in the sensitization process, up to Day 6 after exposure to allergen, prevented allergen sensitization. Exposure to LPS after allergen challenge in sensitized animals abolished the hyperresponsiveness and modified the inflammatory cell influx characteristic of late-phase response to allergen.     

Regulation of the immune response to bacterial lipopolysaccharide by adherent cells.

Immune response to bacterial lipopolysaccharide is usually short lived, but it often reappears without additional stimulus in a cyclic fashion. Activated adherent cells, presumably macrophages, were found to have a role in the reduction of the immune response to Escherichia coli O127 lipopolysaccharide. The suppressive activity of the adherent cells was abrogated before renewal of the responsiveness.     

The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection

The Drosophila host defense against gram-negative bacteria is mediated by the Imd pathway upon sensing of peptidoglycan by the peptidoglycan recognition protein (PGRP)-LC. Here we report a functional analysis of PGRP-LB, a catalytic member of the PGRP family. We show that PGRP-LB is a secreted protein regulated by the Imd pathway. Biochemical studies demonstrate that PGRP-LB is an amidase that specifically degrades gram-negative bacteria peptidoglycan. In agreement with its amidase activity, PGRP-LB downregulates the Imd pathway. Hence, activation of PGRP-LB by the Imd pathway provides a negative feedback regulation to tightly adjust immune activation to infection. Our study also reveals that PGRP-LB controls the immune reactivity of flies to the presence of ingested bacteria in the gut. Our work highlights the key role of PGRPs that encode both sensors and scavengers of peptidoglycan, which modulate the level of the host immune response to the presence of infectious microorganisms.     

Chronic stress modulates the immune response to a pneumococcal pneumonia vaccine

OBJECTIVE: Influenza and pneumonia account for significant morbidity and mortality, particularly in older individuals. Previous studies have shown that spousal caregivers of patients with dementia have poorer antibody and virus specific T cell responses to an influenza virus vaccine relative to noncaregiving control subjects. This study tested the hypothesis that stress can also significantly inhibit the IgG antibody response to a pneumococcal bacterial vaccine. METHOD: We measured antibody titers of current caregivers, former caregivers, and control subjects after vaccination with a pneumococcal bacterial vaccine. RESULTS: Caregivers showed deficits relative to controls and former caregivers in their antibody responses to vaccination. Although the groups did not differ before vaccination or in the rise in antibody 2 weeks or 1 month after vaccination, current caregivers had lower antibody titers 3 and 6 months after vaccination than either former caregivers or controls. CONCLUSIONS: These data, the first evidence that chronic stress can inhibit the stability of the IgG antibody response to a bacterial vaccine for pneumonia, provide additional evidence of health risks associated with dementia caregiving.     

Meta-tyrosine modulates the immune response induced by bacterial endotoxins

Sepsis is characterized by an early pro-inflammatory phase followed by compensatory anti-inflammatory mechanisms that lead to a late generalized immunosuppression, period where most deaths occur. Immunotherapy approaches to recover the immunocompetence in sepsis are similar to those used in cancer. Meta-tyrosine (m-Tyr) is a product of oxidative stress present in circulation during the sepsis and cancer-associated pro-inflammatory stages. In this work, considering its potential participation in pro-inflammatory processes, we evaluate the effect of m-Tyr during LPS induced immunosuppression phase in a murine model. In addition, we examine the effect of m-Tyr in a vaccination strategy using a weakly immunogenic tumor model. Our results showed that m-Tyr could prevent the establishment of immunosuppression and rescue the host from an installed immunosuppression induced by LPS. These effects were parallel to the ability of m-Tyr to improve the pro-inflammatory effects induced by LPS and inhibit the anti-inflammatory action of dexamethasone. Also, m-Tyr treatment prevents both the reduction of splenic lymphocytes and the increase of the expression of programmed death ligand-1 in splenic myeloid cells associated with immunosuppression. Besides, treatment with m-Tyr increased the protective effect of an anti-tumor vaccine, suggesting that m-Tyr could improve the immune response. In summary, we suggest that m-Tyr can modulate critical immunological indicators through the inflammatory context, which could improve the management of diseases, such as sepsis and cancer, in which immunosuppression is a significant clinical problem.     

Concurrent lipopolysaccharide enhances chemotactic response of human polymorphonuclear leukocytes to bacterial chemotaxin

Polymorphonuclear neutrophil (PMN) function is thought to be critical in resistance to infectious agents and this implies that the PMN must be able to migrate into, and to function in, environments that may have high levels of bacterial lipopolysaccharide (LPS). Therefore, we have evaluated the effect of LPS on the in vitro migration of PMNs. Our data reveal that the human PMN is resistant to the deleterious effects of high levels of LPS, that in high concentrations LPS is, itself, a direct chemoattractant for PMNs, and that PMN migration toward a bacterial chemotaxin is enhanced if LPS is also present. Such capabilities suggest that the PMN may be uniquely qualified to migrate into microenvironments that are rich in LPS.     

P-selectin binds to bacterial lipopolysaccharide

Multiple organ failure associated with disseminated intravascular coagulation is a frequent complication in septic shock patients. Accumulation of platelets and neutrophils in the organs contributes to the manifestation of lipopolysaccharide (LPS)-induced organ failure. Although a direct interaction between LPS and platelets is well documented, the nature of the surface receptor for LPS on platelets is unknown. In this article we show that P-selectin is a receptor for LPS. The binding of LPS to P-selectin is independent of Ca²⁺ , and is blocked by antibodies to P-selectin, lipid A and fucoidan. Platelets pre-treated with thrombin showed fourfold higher binding of fluorescein isothiocyanate (FITC)-conjugated LPS compared to untreated platelets and the binding of FITC-conjugated LPS to platelets was blocked in the presence of anti-P-selectin antibodies. It is likely that the binding of LPS via P-selectin on activated platelets or epithelium could have a significant role in the pathophysiology of organ failure in septic shock.     

Drosophila miR-964 modulates Toll signaling pathway in response to bacterial infection

Recent studies suggest that microRNA (miRNA) plays important roles in the control of immune response and tolerance. We previously found that the expression level of antimicrobial peptide gene Drosomycin (Drs) is decreased in miR-964 overexpressing flies. Here, we further verified that miR-964 deficiency leads to hyper-activation of Drs. In addition, we employed three widely-used bioinformatic algorithms to screen potential miR-964 targets. Finally, we identified that miR-964 modulates Toll signaling pathway, at least in part, by repressing the expression of Drs. Taken together, our study identifies miR-964 as a modulator of Toll signaling and enriches the repertoire of immune-modulating miRNAs in Drosophila.     

Chronic effects of the novel glucocorticosteroid RPR 106541 administered to beagle dogs by inhalation

The preclinical safety of RPR 106541, a novel 17-thiosteroid, was evaluated in young adult and mature dogs by inhalation exposure for 26 weeks and 52 weeks, respectively. A dry powder formulation of RPR 106541 in lactose was administered to young adult dogs (approximately 6 months of age at initiation) at doses of 0 (air and placebo controls), 10, 100, or 1,000 microg/kg/d for 26 weeks. A solution-based aerosol formulation was administered to mature dogs (approximately 10 months at initiation) from a pressurized metered dose inhaler at 0 (air and placebo controls), 10, 50, and 150 microg/kg/d for 52 weeks. Clinical evidence of glucocorticosteroid-induced immunosuppression was observed by weeks 20-26 following relatively high dose exposures (100 microg/kg/d and 1,000 microg/kg/d) in young dogs receiving the dry powder formulation for 26 weeks. Classic glucocorticosteroid effects were observed, including adrenocortical atrophy, reduced bone mass with retention of epiphyseal growth plates in long bones, prominence of stromal adipose tissue in bone marrow, and atrophy of lymphoid tissues. Inhalation administration of RPR 106541 to sexually mature dogs facilitated more definitive characterization of endocrine affects of RPR 106541 as compared with administration in younger, sexually immature animals. Significant effects in female reproductive organs included absence of corpora lutea in association with atresia of vesicular follicles within the ovaries, endometrial hyperplasia, and lobular development of mammary tissue. Discordant development of mammary tissue, accumulation of secretory material within hyperplastic endometrial glands, and hypertrophy of uterine lining epithelium in absence of ovulation were consistent with a secondary progestin effect by a potent glucocorticosteroid.     

Pregnancy influences the plasma cytokine response to intraperitoneal administration of bacterial endotoxin in rats

Rats have an attenuated febrile response to intraperitoneal (i.p.) administration of exogenous pyrogen (e.g. bacterial endotoxin) near the term of pregnancy. To investigate possible mechanisms of this unique thermoregulatory response, the present experiments were carried out on 18 non-pregnant and 16 near-term pregnant Sprague-Dawley rats to test the hypothesis that pregnancy alters the balance of pyrogenic cytokines and antipyretic and/or cryogenic (antipyretic/cryogenic) cytokines in response to exogenous pyrogen. To test our hypothesis, we measured plasma levels of interleukin (IL)-1beta, IL-6, interleukin-1 receptor antagonist (IL-1ra) and tumour necrosis factor alpha(TNFalpha) at 2 and 4 h following i.p. administration of 160 microg kg(-1) E. coli lipopolysaccharide (LPS) (i.e. EC100 dose, or the smallest dose that elicits a maximal febrile response in non-pregnant rats) in non-pregnant as well as pregnant rats at day 20 of gestation (term approximately 21 days). In non-pregnant rats, E. coli LPS elicited statistically significant increases in plasma concentrations of IL-1beta, IL-6, IL-1ra and TNFalpha as compared to that observed following administration of vehicle. However in pregnant rats, E. coli LPS elicited statistically significant increases in antipyretic/cryogenic cytokines (IL-1ra and TNFalpha) but not in pyrogenic cytokines (IL-1beta and IL-6). Thus, a differential pyrogenic and antipyretic/cryogenic plasma cytokine response may mediate in part the attenuated febrile response to exogenous pyrogen observed in rats near the term of pregnancy.     

Separation of the mitogenic and antigenic responses to bacterial lipopolysaccharide.

An attempt was made to separate the antigenic and mitogenic properties of E. coli bacteria and bacterial lipopolysaccharide antigen inhibited the mitogenic response by the cultures but did not inhibit the induction of anti-LPS antibody or polyclonal antibody synthesis to SRBC. Dextran sulphate, acting as a B-cell mitogen, increased the mitogenic response in spleen cell cultures incubated with bacteria, but did not affect the production of anti-LPS antibody. Mild alkaline hydrolysis (0-1 N NaOH at 56 degrees) of LPS destroyed the mitogenic properties of the molecule, leaving the antigenic properties qualitatively intact. Harsher conditions of base hydrolysis destroyed both the mitogenic and antigenic properties of LPS, as determined by antigenicity in murine spleen cell cultures and haemagglutination inhibition tests.     

Ovalbumin-specific IgE modulates ovalbumin-specific T-cell response after repetitive oral antigen administration

BACKGROUND: Some patients outgrow their food allergies even though their serum antigen-specific IgE levels remain high. OBJECTIVE: To elucidate the role of T cells in outgrowing food allergies in the presence of antigen-specific IgE, we tracked antigen-specific T-cell responses after oral antigen administration. METHODS: Ovalbumin (OVA)-specific T-cell receptor (TCR) and OVA-specific IgE transgenic (Tg) mice (OVA-TCR/IgE-Tg) and OVA-specific TCR Tg (OVA-TCR-Tg) mice were fed with high doses of OVA or PBS every other day. After 7 administrations, OVA-specific proliferation and cytokine production of mononuclear cells of the spleen, mesenteric lymph nodes, and Peyer's patches and the number of splenic CD4 + CD25 + T cells were analyzed. RESULTS: Without OVA administration, the splenocytes from OVA-TCR/IgE-Tg mice exhibited a higher proliferative response and produced more IL-4 and IL-10 and less IFN-gamma than those from OVA-TCR-Tg mice. The proliferative responses of the splenocytes from either OVA-TCR/IgE-Tg mice or OVA-TCR-Tg mice fed with OVA were significantly reduced compared with those from PBS-fed mice. The number of OVA-specific TCR + T cells decreased in the spleen from OVA-fed mice, whereas the number of CD4 + CD25 + T cells increased. The suppressed proliferation of splenocytes of OVA-fed mice was partially resumed by neutralization of TGF-beta1, but not of IL-10. CONCLUSION: The presence of OVA-specific IgE modulated the OVA-specific responses of the splenocytes. Irrespective of the presence of OVA-specific IgE, repetitive oral administration of OVA induced tolerance, which seems to be composed of clonal deletion/anergy and TGF-beta1-mediated active suppression.     

Differential binding of bacterial lipopolysaccharide to bovine peripheral-blood leukocytes

The response of mammalian monocytes and macrophages to bacterial lipopolysaccharide (LPS) may be influenced by serum factors that increase or decrease the propensity of LPS to bind to cell surfaces. We used fluorescence flow cytometric analysis to investigate the capability of bovine peripheral-blood leukocytes to bind LPS in the presence or absence of bovine serum. At all concentrations of FITC-LPS tested (LPS fromE. coli 0111:B4; 10 ng/ml, 100 ng/ml, 1 g/ml), monocytes, lymphocytes, and granulocytes bound more FITC-LPS in the presence of 10% bovine serum than in serum-free conditions (P<0.01). At the intermediate concentration tested (100 ng/ml), monocytes displayed a relative fluorescence intensity (RFI) of 2.27±1.24 units without serum and 17.48±8.05 with 10% serum. Values for granulocytes were similar to those of monocytes, 3.55±1.31 without and 19.24±6.93 with serum, and values for lymphocytes were 1.89±0.47 RFI units without serum and 6.27±2.61 RFI units with serum. At 10 ng/ml and 1 g/ml FITC-LPS the RFI of monocytes and granulocytes were also similar and not significantly different, and both bound significantly more LPS than lymphocytes (P<0.01). When 100 ng/ml FITC-LPS was coincubated with leukocytes, 10% serum, and a 100-fold excess of unlabeled LPS, the amount of FITC-LPS bound to monocytes was reduced from 23.99 to 7.23 RFI units (P<0.01), reduced from 22.00 to 7.30 RFI units with granulocytes (P<0.05), and reduced from 7.51 to 2.29 RFI units (P<0.10) with lymphocytes. These data demonstrate that factors in bovine serum significantly amplify the association of LPS with peripheral-blood leukocytes and that increased binding of LPS is greatest with monocytes and granulocytes.     

The antimicrobial antiproteinase elafin binds to lipopolysaccharide and modulates macrophage responses

Lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria represent a primary target for innate immune responses. We demonstrate here that the antimicrobial/anti-neutrophil elastase full-length elafin (FL-EL) is able to bind both smooth and rough forms of LPS. The N-terminus was shown to bind both forms of LPS more avidly. We demonstrate that the lipid A core-binding proteins polymyxin B (PB) and LPS-binding protein (LBP) compete with elafin for binding, and that LBP is able to displace prebound elafin from LPS. When PB, FL-EL, N-EL, and C-EL were pre-incubated with LPS before addition to immobilized LBP, PB was the most potent inhibitor of LPS transfer to LBP. These data prompted us to examine the biological consequences of elafin binding to LPS, using tumor necrosis factor (TNF)-alpha release by murine macrophages. In serum-containing conditions, N-EL had no effect, whereas both C-EL and FL-EL inhibited TNF-alpha production. In serum-free conditions, however, all moieties had a stimulatory activity on TNF-alpha release, with C-EL being the most potent at the highest concentration. The differential biological activity of elafin in different conditions suggests a role for this molecule in either LPS detoxification or activation of innate immune responses, depending on the external cellular environment.     

Enhancement of murine macrophage binding of and response to bacterial lipopolysaccharide (LPS) by LPS-binding protein.

We have studied the effects of highly purified rabbit lipopolysaccharide (LPS)-binding protein (LBP) on the ability of murine bone marrow-derived macrophages to respond to bacterial LPS. Macrophage responses studied include the secretion of tumor necrosis factor alpha, production of arginine-derived nitrite (NO2-), and killing of an intracellular pathogen, Leishmania enriettii. Macrophages from either CBA or LPS-hyporesponsive C3H/HeJ mice exhibited significantly greater sensitivity to LPS in the presence of LBP. Furthermore, both CBA and C3H/HeJ macrophages demonstrated an LBP-dependent enhancement of LPS binding. These results suggest that C3H/HeJ macrophages are capable of binding LPS-LBP complexes and support the hypothesis that hyporesponsiveness in this strain involves a step subsequent to LPS binding. Furthermore, these findings provide additional evidence of the important role played by the acute-phase plasma protein LBP in modifying host response to LPS.