Véronique Provost

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5‐oxo‐eicosatetraenoate‐induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells.

Introduction Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with TH2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT1 and CysLT2 receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the TH2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells. Methods A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD4. The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively. Results IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD4 weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD4 had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT1 but not CysLT2. CysLT1 expression was increased by IL-13 but not by IL-4 and/or LTD4. Importantly, the upregulation of CysLT1 by IL-13 preceded eotaxin-3 release. Conclusions These results demonstrate a stepwise cooperation between IL-13 and LTD4. IL-13 upregulates CysLT1 expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.

2-Arachidonoyl-glycerol- and arachidonic acid-stimulated neutrophils release antimicrobial effectors against E. coli, S. aureus, HSV-1, and RSV

The endocannabinoid 2‐AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB4 biosynthesis, independently of CB activation. In this study, we show that 2‐AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2‐AG and AA indeed inhibited the infectivity of HSV‐1 and RSV. Additionally, the supernatants of 2‐AG‐ and AA‐stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α‐defensins, as well as a limited amount (nanograms) of LL‐37. All the effects of AA and 2‐AG mentioned above were prevented by inhibiting LTB4 biosynthesis or by blocking BLT1. Importantly, neither CB2 receptor agonists nor antagonists could mimic nor prevent the effects of 2‐AG, respectively. In fact, qPCR data show that contaminating eosinophils express ∼100‐fold more CB2 receptor mRNA than purified neutrophils, suggesting that CB2 receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB2 expression by freshly isolated human neutrophils. The rapid conversion of 2‐AG to AA and their subsequent metabolism into LTB4 promote 2‐AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT1. Considering that nanomolar concentrations of AA or 2‐AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2‐AG and AA as regulators of host defense in vivo.

Regulation of Eosinophil and Group 2 Innate Lymphoid Cell Trafficking in Asthma

Asthma is an inflammatory disease usually characterized by increased Type 2 cytokines and by an infiltration of eosinophils to the airways. While the production of Type 2 cytokines has been associated with TH2 lymphocytes, increasing evidence indicates that group 2 innate lymphoid cells (ILC2) play an important role in the production of the Type 2 cytokines interleukin (IL)-5 and IL-13, which likely amplifies the recruitment of eosinophils from the blood to the airways. In that regard, recent asthma treatments have been focusing on blocking Type 2 cytokines, notably IL-4, IL-5, and IL-13. These treatments mainly result in decreased blood or sputum eosinophil counts as well as decreased asthma symptoms. This supports that therapies blocking eosinophil recruitment and activation are valuable tools in the management of asthma and its severity. Herein, we review the mechanisms involved in eosinophil and ILC2 recruitment to the airways, with an emphasis on eotaxins, other chemokines as well as their receptors. We also discuss the involvement of other chemoattractants, notably the bioactive lipids 5-oxo-eicosatetraenoic acid, prostaglandin D2, and 2-arachidonoyl-glycerol. Given that eosinophil biology differs between human and mice, we also highlight and discuss their responsiveness toward the different eosinophil chemoattractants.

Comparison of eight 15-lipoxygenase (LO) inhibitors on the biosynthesis of 15-LO metabolites by human neutrophils and eosinophils

Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 μM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO.

Leukotriene B₄ Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B₄ In Vitro and In Cellulo

LTB4 is an inflammatory lipid mediator mainly biosynthesized by leukocytes. Since its implication in inflammatory diseases is well recognized, many tools to regulate its biosynthesis have been developed and showed promising results in vitro and in vivo, but mixed results in clinical trials. Recently, the mTOR pathway component p70S6 kinase 1 (p70S6K1) has been linked to LTC4 synthase and the biosynthesis of cysteinyl-leukotrienes. In this respect, we investigated if p70S6K1 could also play a role in LTB4 biosynthesis. We thus evaluated the impact of the p70S6K1 inhibitors PF-4708671 and LY2584702 on LTB4 biosynthesis in human neutrophils. At a concentration of 10 μM, both compounds inhibited S6 phosphorylation, although neither one inhibited the thapsigargin-induced LTB4 biosynthesis, as assessed by the sum of LTB4, 20-OH-LTB4, and 20-COOH-LTB4. However, PF-4708671, but not LY2584702, inhibited the ω-oxidation of LTB4 into 20-OH-LTB4 by intact neutrophils and by recombinant CYP4F3A, leading to increased LTB4 levels. This was true for both endogenously biosynthesized and exogenously added LTB4. In contrast to that of 17-octadecynoic acid, the inhibitory effect of PF-4708671 was easily removed by washing the neutrophils, indicating that PF-4708671 was a reversible CYP4F3A inhibitor. At optimal concentration, PF-4708671 increased the half-life of LTB4 in our neutrophil suspensions by 7.5 fold, compared to 5 fold for 17-octadecynoic acid. Finally, Michaelis-Menten and Lineweaver-Burk plots indicate that PF-4708671 is a mixed inhibitor of CYP4F3A. In conclusion, we show that PF-4708671 inhibits CYP4F3A and prevents the ω-oxidation of LTB4 in cellulo, which might result in increased LTB4 levels in vivo.