Julie S. Lefebvre

Arachidonic Acid Regulates the Translocation of 5-Lipoxygenase to the Nuclear Membranes in Human Neutrophils

Elevation of the intracellular cAMP concentration in agonist-activated human neutrophils (PMN) leads to the concomitant inhibitions of arachidonic acid (AA) release, 5-lipoxygenase (5-LO) translocation, and leukotriene (LT) biosynthesis. We report herein that exogenous AA completely prevents cAMP-dependent inhibition of 5-LO translocation and LT biosynthesis in agonist-activated PMN. Moreover, the group IVA phospholipase A2 inhibitor pyrrophenone and the MEK inhibitor U-0126 inhibited AA release and 5-LO translocation in activated PMN, and these effects were also prevented by exogenous AA, demonstrating a functional link between AA release and 5-LO translocation. Polyunsaturated fatty acids of the C18 and C20 series containing at least three double bonds located from carbon 9 (or closer to the carboxyl group) were equally effective as AA in restoring 5-LO translocation in pyrrophenone-treated agonist-activated PMN. Importantly, experiments with the 5-LO-activating protein inhibitor MK-0591 and the intracellular Ca2+ chelator BAPTA-AM demonstrated that the AA-regulated 5-LO translocation is FLAP- and Ca2+-dependent. Finally, the redox and competitive 5-LO inhibitors L-685,015, L-739,010, and L-702,539 (but not cyclooxygenase inhibitors) efficiently substituted for AA to reverse the pyrrophenone inhibition of 5-LO translocation, indicating that the site of regulation of 5-LO translocation by AA is at or in the vicinity of the catalytic site. This report demonstrates that AA regulates the translocation of 5-LO in human PMN and unravels a novel mechanism of the cAMP-mediated inhibition of LT biosynthesis.

Extra Domain A of Fibronectin Primes Leukotriene Biosynthesis and Stimulates Neutrophil Migration Through Activation of Toll-like Receptor 4

OBJECTIVE There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling. METHODS The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo. RESULTS Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 α inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4). CONCLUSION These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.

The aged microenvironment contributes to the age-related functional defects of CD4 T cells in mice

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T‐cell function, and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T‐cell response. In this study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T‐cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T‐cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naïve CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations.

The Endocannabinoid 2-Arachidonoyl-Glycerol Activates Human Neutrophils: Critical Role of Its Hydrolysis and De Novo Leukotriene B4 Biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B4 biosynthesis, or LTB4 receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB4 biosynthesis, and an autocrine activation loop involving LTB4 receptor 1.

Toll-like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4 and platelet-activating factor

Activation of toll‐like receptors (TLRs) and polymorphonuclear leukocyte (PMN) accumulation at infection sites are critical events of host defense. The involvement of leukotriene (LT) B4 and platelet‐activating factor (PAF) in TLR ligand‐induced activation of inflammatory cell functions is essentially unknown. Using an in vitro model of human PMN migration through human endothelial cell monolayers’ we demonstrate that prototypic ligands of TLR1/2, 2/6, 3, 4, 5, and 7/8 promote PMN migration, an effect markedly inhibited by 3 LTB4 receptor antagonists (70–80% inhibition at 100 nM compared to vehicle‐treated cells), 3 PAF receptor antagonists (20–50% inhibition at 10 nM), 3 LT biosynthesis inhibitors (75–85% inhibition at 100 nM), and 1 cytosolic phospholipase A2α (cPLA2α) inhibitor (90% inhibition at 1 μM). Accordingly, selected TLR ligands caused Ser505‐phosphorylation of cPLA2α and measurable LTB4 and PAF biosynthesis in the transmigration assay. As negative controls, interleukin‐8‐ and formyl‐methionyl‐leucyl‐phenylalanine‐elicited migration in vitro was not inhibited either by an LTB4 receptor antagonist or by the cPLA2α inhibitor. Finally, LTB4 and PAF receptor antagonists inhibited (up to ~65% at optimal doses) TLR ligand‐induced PMN infiltration in the mouse air‐pouch model. These studies unravel the critical involvement of de novo LTB4 and PAF biosynthesis in PMN migration elicited by TLR ligands.—Lefebvre, J. S., Marleau, S., Milot, V., Lévesque, T., Picard, S., Flamand, N., Borgeat, P. Toll‐like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4 and platelet‐activating factor. FASEB J. 24, 637–647 (2010). www.fasebj.org

Aging of the CD4 T Cell Compartment.

Higher morbidity and mortality following infections, particularly influenza, is observed in the elderly population. Because of this, people over 65 years old are often targeted for preventive immunization. Many vaccines, however, are not as effective in generating protective antibodies in older individuals. CD4+ T cells, through their B cell helper functions, play a central role in the humoral response. Aging has deleterious effects on the immune system, and understanding how aging impairs CD4+ T cell functions is of critical importance to design new immunization and treatment strategies targeted to the elderly population. In this paper, we review some of the qualitative and quantitative changes in the CD4+ T cell compartment that arise with aging. We also summarize the age-related intrinsic defects that impact naïve, memory and regulatory CD4+ T cell functions.

Immunity to the Conserved Influenza Nucleoprotein Reduces Susceptibility to Secondary Bacterial Infections

Influenza causes >250,000 deaths annually in the industrialized world, and bacterial infections frequently cause secondary illnesses during influenza outbreaks, including pneumonia, bronchitis, sinusitis, and otitis media. In this study, we demonstrate that cross-reactive immunity to mismatched influenza strains can reduce susceptibility to secondary bacterial infections, even though this fails to prevent influenza infection. Specifically, infecting mice with H3N2 influenza before challenging with mismatched H1N1 influenza reduces susceptibility to either Gram-positive Streptococcus pneumoniae or Gram-negative Klebsiella pneumoniae. Vaccinating mice with the highly conserved nucleoprotein of influenza also reduces H1N1-induced susceptibility to lethal bacterial infections. Both T cells and Abs contribute to defense against influenza-induced bacterial diseases; influenza cross-reactive T cells reduce viral titers, whereas Abs to nucleoprotein suppress induction of inflammation in the lung. These findings suggest that nonneutralizing influenza vaccines that fail to prevent influenza infection may nevertheless protect the public from secondary bacterial diseases when neutralizing vaccines are not available.

Involvement of endogenous leukotriene B4 and platelet-activating factor in polymorphonuclear leucocyte recruitment to dermal inflammatory sites in rats.

A critical role for leukotriene B4 (LTB4) and/or platelet‐activating factor (PAF) in regulating polymorphonuclear cell (PMN) trafficking to inflammatory sites has been reported in a number of experimental inflammatory models. In vitro, newly synthesized LTB4 and PAF were shown to act in an autocrine/paracrine or intracrine fashion to enhance intracellular arachidonic acid availability and leukotriene biosynthesis. This suggested potentially cooperative effects of these lipid mediators in regulating PMN extravasation. The present study aimed to elucidate whether endogenous LTB4 and PAF may both act to regulate plasma extravasation and PMN trafficking to inflammatory sites in experimental inflammation. With this aim, we have used selective and potent PAF and LTB4 receptor antagonist pretreatments in dermal and pulmonary inflammation models in rats. Our results show additive inhibitory effects of dual LTB4 and PAF receptor blockade in either PAF‐ or LTB4‐elicited cutaneous PMN accumulation compared to single‐drug administration. Furthermore, the combined administration of the drugs inhibited the PMN accumulation induced by the chemically unrelated soluble agonists tumour necrosis factor‐α and C5a. Finally, in a model of pulmonary inflammation induced by the intravenous injection of Sephadex beads, lung neutrophilia was reduced by 63% following the administration of LTB4 and PAF antagonists, in contrast with the lack of effect of single drug administration. Our results strongly support a role of both endogenous LTB4 and PAF in regulating PMN trafficking to inflammatory sites in various experimental conditions.

Vaccine strategies to enhance immune responses in the aged

The elderly population is more susceptible to infections with higher risks of morbidity and mortality. This is caused by the accumulation of immune defects with aging. The best way to protect people against infections is vaccination. Unfortunately, the same immune defects that render the elderly susceptible to infectious diseases also prevent the development of protective immunity following immunization. A good example of this is the influenza vaccine that only protects between 40 and 60% of the vaccinees over 65 years. In the past decade, tremendous efforts have been put toward improving the influenza vaccine for the elderly. We therefore use this example to present various strategies employed to overcome these age-associated immune defects and hence make vaccines more efficacious for the aged.

Age-related impairment of humoral response to influenza is associated with changes in antigen specific T follicular helper cell responses

T follicular helper (TFH) cell responses are essential for generation of protective humoral immunity during influenza infection. Aging has a profound impact on CD4+ T cell function and humoral immunity, yet the impact of aging on antigen specific TFH responses remains unclear. Influenza specific TFH cells are generated in similar numbers in young and aged animals during infection, but TFH cells from aged mice exhibit significant differences, including reduced expression of ICOS and elevated production of IL-10 and IFNγ, which potentially impairs interaction with cognate B cells. Also, more influenza specific T cells in aged mice have a regulatory phenotype, which could contribute to the impaired TFH function. Adoptive transfer studies with young T cells demonstrated that TGF-β1 in the aged environment can drive increased regulatory T cell accumulation. Aging and the aged environment thus impact antigen specific TFH cell function and formation, which contribute to reduced protective humoral responses.