Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

Targeting inflammation and pro-resolving mediators with Anetholea anisita extract to improve scalp condition.

OBJECTIVE: A critical and often-overlooked factor that may give rise to dandruff and oily hair is the intrinsic quality of the scalp stratum corneum (SC), which is often unbalanced and susceptible to external aggressions. Addressing the inflammation element of unhealthy scalp plays an important role in promoting healthy-looking and feeling hair. Although specialized pro-resolving lipid mediators (SPMs) have been studied in the skin to end the inflammation process and promote tissue regeneration, no studies have been provided in the scalp. This study aims to investigate SPMs expression and its role in improving scalp integrity and consequently improving hair appearance using an Anetholea anisita extract. METHODS: The effect of Anetholea anisita extract was investigated in vitro on human follicle dermal papilla cells (HFDPC), evaluating its antioxidant and anti-inflammatory properties by fluorescence staining and ELISA, respectively. Ex vivo measurement of the volume of human scalp sebaceous glands was performed using X-ray microtomography (micro-CT). The extract was then clinically tested on a population of dandruff sufferers presenting oily hair. Volunteers' sebum was collected on the scalp and analysed by LC-MS/MS or ELISA to identify SPMs and pro-inflammatory markers. Scalp integrity was assessed by measuring the pH and the TEWL. Sebum production, dandruff and hair gloss were also evaluated. RESULT: Anetholea anisita extract reduced IL-8 and reactive oxygen species (ROS) generation in HFDPC. Interestingly, this extract also decreased the volume of sebaceous glands as revealed by micro-CT. This result was confirmed in vivo by a decrease in sebum production in volunteers. Moreover, SPMs were analysed and detected in the scalp for the first time. An increase in Lipoxin B4 (LxB4) and Resolvin D1 and D2 (RvD1 and RvD2) was observed after Anetholea anisita treatment as well as decrease in pro-inflammatory sebum mediators expression such as PGE2, LTB4 and IL-8. Consequently, the scalp barrier was reinforced as observed through improved transepidermal water loss (TEWL) and skin surface pH, reducing dandruff and improving hair health. CONCLUSION: The present results suggest the potential of cosmetic applications of Anetholea anisita extract to improve scalp health by targeting inflammation pathways to decrease dandruff and improve hair condition.

OBJECTIF: Un facteur important et peu étudié pouvant mener à l'apparition des pellicules ou des cheveux gras est la qualité intrinsèque du stratum corneum (SC) du cuir chevelu, souvent déséquilibré et susceptible aux agressions. L'inflammation joue un rôle clé dans l'état de santé du cuir chevelu et par conséquent du cheveu. Les médiateurs lipidiques pro-résolution (SPMs) ont été étudiés dans la peau pour mettre fin au processus inflammatoire et promouvoir la régénération des tissus. Cependant, aucune étude n'avait été réalisée sur le cuir chevelu. Cette étude vise donc à étudier l'expression des SPMs et leurs rôles dans l'amélioration de l'intégrité du cuir chevelu et de l'apparence des cheveux en utilisant un extrait de Anetholea anisita. MÉTHODES: Les propriétés antioxydantes et anti-inflammatoires de l'Anetholea anisita ont été étudiées in vitro sur les cellules papillaires folliculaires dermiques humaines (HFDPC) par fluorescence et ELISA. La mesure ex vivo du volume des glandes sébacées du cuir chevelu humain a été réalisée par microtomographie à rayons X (micro-CT). L'extrait a ensuite été cliniquement testé sur des volontaires présentant des pellicules et des cheveux gras. Le sébum des volontaires a été prélevé sur le cuir chevelu et analysé par LC-MS/MS ou ELISA pour identifier les SPMs et les marqueurs pro-inflammatoires. L'intégrité du cuir chevelu a ensuite été évaluée en mesurant le pH et la perte en eau transépidermique. La production de sébum, les pellicules et la brillance des cheveux ont également été évalués. RÉSULTATS: L'extrait d'Anetholea anisita a réduit la production d'IL-8 et d'espèces réactives oxygénées sur HFDPC. Cet extrait a également diminué le volume des glandes sébacées. Ce résultat a été confirmé in vivo avec une diminution de la production de sébum chez les volontaires. De plus, les SPMs ont été analysés et détectés pour la première fois sur le cuir chevelu. Une augmentation de la Lipoxine B4 (LxB4) ainsi que des Resolvines D1 et D2 (RvD1 et RvD2) a été observée après le traitement par Anetholea anisita en plus d'une diminution de l'expression des médiateurs pro-inflammatoires tels que PGE2, LTB4 et IL-8. Par conséquent, la barrière du cuir chevelu a été renforcée comme observé avec une diminution de la PIE et un ajustement pH de la surface du scalp, réduisant les pellicules et améliorant la santé des cheveux. CONCLUSION: Les résultats obtenus montrent qu'un extrait d'Anetholea anisita permet d'améliorer la santé du cuir chevelu en ciblant les voies de l'inflammation et de la résolution permettant ainsi de renforcer la barrière du cuir chevelu, pour diminuer les pellicules et améliorer l'état des cheveux.

CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids.

Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.

The Natural Combination Medicine Traumeel (Tr14) Improves Resolution of Inflammation by Promoting the Biosynthesis of Specialized Pro-Resolving Mediators.

The resolution of inflammation is an integral part of the acute inflammatory response and eventually leads to the return to homeostasis. It is supported by specialized pro-resolving mediators (SPMs) that act as immunoresolvents via specific G-protein-coupled receptors. In contrast to classical non-steroidal anti-inflammatory drugs (NSAIDs) that suppress the formation of pro-inflammatory lipid mediators such as prostaglandins, novel pharmacotherapeutic concepts propose to foster the biosynthesis of beneficial SPMs. Here, we demonstrate that the natural combination medicine Traumeel (Tr14) improves resolution of inflammation by promoting SPM formation. Tr14 enhanced the biosynthesis of 12-/15-lipoxygenase (LOX) products and of SPMs in zymosan-induced mouse peritonitis as well as in human monocyte-derived macrophages challenged with Staphylococcus aureus. Importantly, in the peritonitis model, Tr14 supported the recruitment of innate leukocytes and the efferocytotic capacity of macrophages, and positively influenced the inflammation resolution index. Taken together, we suggest that based on these properties Tr14 may possess therapeutic potential as an enhancer for the resolution of inflammatory processes.

Molecular and cellular profiles of the resolution phase in a damage-associated molecular pattern (DAMP)-mediated peritonitis model and revelation of leukocyte persistence in peritoneal tissues.

Models of microbe-elicited peritonitis have been invaluable to identify mechanisms underlying inflammation resolution, but whether resolution mechanisms differ from an inflammatory agent to another has not been determined. Thus, we analyzed the cellular and molecular components of the resolution phase of non-microbe-induced inflammation. In thioglycollate (TG)-induced peritonitis, resolution started at 12 h (Tmax) and displayed a 22 h resolution interval (Ri). During resolution, lipoxin A4, resolvin (Rv) D1 and RvD2, protectin D1 (PD1), and maresin 1 (MaR1) were transiently produced while RvD5 was continually generated. In addition, docosahexaenoic acid (DHA)-derived mediators were produced to a higher extent than in microbial peritonitis. We also investigated leukocyte infiltration and clearance in peritoneal tissues surrounding the inflammatory site. In the omentum, resolution parameters, neutrophil apoptosis, and efferocytosis were similar to those of the peritoneal cavity. However, we noticed long-term persistence of M2-polarized macrophages and B-lymphocytes in the omentum after TG administration, whereas zymosan injection caused M1/M2-macrophage and T-lymphocyte persistence regardless of the magnitude of the inflammatory response. Our study indicates that some aspects of resolution are shaped in a stimulus-specific manner, and it ultimately argues that the tissues surrounding the inflammatory site must also be considered to address the inflammatory response globally.

LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites.

Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography-tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6-155pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators.

Investigation of fatty acid elongation and desaturation steps in Fusarium lateritium by quantitative two-dimensional deuterium NMR spectroscopy in chiral oriented media.

The origin of hydrogen atoms during fatty acid biosynthesis in Fusarium lateritium has been quantified by isotope tracking close to natural abundance. Methyl linoleate was isolated from F. lateritium grown in natural abundance medium or in medium slightly enriched with labeled water, glucose, or acetate, and the (2)H incorporation was determined by quantitative (2)H-{(1)H} NMR in isotropic and chiral oriented solvents. Thus, the individual ((2)H/(1)H)(i) ratio at each pro-R and pro-S hydrogen position of the CH(2) groups along the chain can be analyzed. These values allow the isotope redistribution coefficients (a(ij)) that characterize the specific source of each hydrogen atom to be related to the nonexchangeable hydrogen atoms in glucose and to the medium water. In turn, these can be related to the stereoselectivity that operates during the introduction or removal of hydrogens along the fatty acid chain. First, at even CH(2) the pro-S hydrogen comes only from water by protonation, whereas the pro-R hydrogen is introduced partly via acetate but principally from water. Second, the nonexchangeable hydrogens of glucose (positions H-6,6 and H-1) are shown to be introduced to the odd CH(2) via the NAD(P)H pool used by both reductases involved in the elongation steps of the fatty acid chain. Third, it is proved that hydrogens removed at sites 9,10 and 12,13 during desaturation by Delta(9)- and Delta(12)-desaturases are pro-R, and that during these desaturation steps alpha-secondary kinetic isotope effects occur at the 9 and 12 positions and not at the 10 and 13 positions.

Combined analysis of C-18 unsaturated fatty acids using natural abundance deuterium 2D NMR spectroscopy in chiral oriented solvents.

The quantitative determination of isotopic (2H/1H)i ratios at natural abundance using the SNIF-NMR protocol is a well-known method for understanding the enzymatic biosynthesis of metabolites. However, this approach is not always successful for analyzing large solutes and, specifically, is inadequate for prochiral molecules such as complete essential unsaturated fatty acids. To overcome these analytical limitations, we use the natural abundance deuterium 2D NMR (NAD 2D NMR) spectroscopy on solutes embedded in polypeptide chiral liquid crystals. This approach, recently explored for measuring (2H/1H)i ratios of small analytes (Lesot, P.; Aroulanda, C.; Billault, I. Anal. Chem. 2004, 76, 2827-2835), is a powerful way to separate the 2H signals of all nonequivalent enantioisotopomers on the basis both of the 2H quadrupolar interactions and of the 2H chemical shift. Two significant advances over our previous work are presented here and allow the complete isotopic analysis of four mono- and polyunsaturated fatty acid methyl esters: methyl oleate (1), methyl linoleate (2), methyl linolenate (3), and methyl vernoleate (4). The first consists of using NMR spectrometers operating at higher magnetic field strength (14.1 T) and equipped with a selective cryoprobe optimized for deuterium nuclei. The second is the development of Q-COSY Fz 2D NMR experiments able to produce phased 2H 2D maps after a double Fourier transformation. This combination of modern hardware and efficient NMR sequences provides a unique tool to analyze the (2H/1H)i ratios of large prochiral molecules (C-18) dissolved in organic solutions of poly(gamma-benzyl-L-glutamate) and requires smaller amounts of solute than previous study on fatty acids. For each compound (1-4), all 2H quadrupolar doublets visible in the 2D spectra have been assigned on the basis of 2H chemical shifts, isotopic data obtained from isotropic quantitative NAD NMR, and by an interspectral comparison of the anisotropic NAD spectra of four fatty acids. The NMR results are discussed in terms of (2H/1H)i isotopic distribution and molecular orientation in the mesophase. For the first time, we show that the investigation of natural isotopic fractionation of complete fatty acids is possible without the need of chemical modifications, hence providing an alternative method to probe the mechanisms of enzymes implied in the biosynthetic pathway of unsaturated fatty acids.

Assignment of absolute configuration of natural abundance deuterium signals associated with (R)- and (S)-enantioisotopomers in a fatty acid aligned in a chiral liquid crystal: enantioselective synthesis and NMR analysis.

Previous experimental natural abundance deuterium (NAD) NMR results have shown an odd/even-related alternation in the ((2)H/(1)H) ratio of the methylene groups of fatty acids (ChemBioChem 2001, 2, 425) and, by NAD NMR in CLC, a marked difference between enantiotopic deuterons for each methylenic site (Anal. Chem. 2004, 76, 2827). However, to date, the assignment of the absolute configuration for each deuterium has not been possible. To investigate further the origin of these effects, the assignment of NAD quadrupolar doublets observed in chiral oriented solvent is required. Here we describe the assignment of R- and S-isomers resulting from the isotopic substitution in positions 4 and 5 in the aliphatic chain of 1,1'-bis(thiophenyl)hexane 1 (BTPH) derived from natural linoleic acid of plant origin. This was achieved using an optimized synthetic strategy to obtain separately four regio- and stereoselectively deuterated enantiomers of BTPH. By reference to the deuterium spectra of these isotopically labeled reference compounds, we demonstrate that, on both 4 and 5 positions of BTPH, the isotopic enantiomers of S configuration are depleted relative to those of R configuration. This finding effectively explains the observed low ((2)H/(1)H) ratio in NAD of some ethylenic sites of unsaturated fatty acids.