Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.—Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance

Bicarbonate-stimulated membrane reorganization in stallion spermatozoa

"Bicarbonate-stimulated membrane reorganization in stallion spermatozoa", published in: Frontiers Cell and Developmental Biology https://www.frontiersin.org/articles/10.3389/fcell.2021.772254 This study aimed to investigate and clarify several aspects of stallion sperm capacitation occurring in the presence of bicarbonate, calcium and albumin. Specifically, re-organization of the plasma membrane after the initiation of sperm capacitation was investigated using lipidomics, cryo-electron microscopy, flow cytometry and complementary biochemical strategies. To this end, phosphatidylserine (PS) and phosphatidylethanolamine exposure in viable sperm was monitored with flow cytometry and PS exposure with live imaging. The current data-package contains the raw data files of the lipidomics analysis of these stallion sperm cells. The data is contained in primary Thermo-Fisher Scientific (Waltham, MA, USA) raw LC/MS-data files. The raw data-files were converted to mz(X)ML files by msConvert (part of ProteoWizard v3.0.913) (also contained in this data-package) and processed with the R package xcms v2.99.3. After deisotoping, annotation of lipids was performed by matching measured MS-1 m/z values with theoretical m/z values as described in Molenaar et al., 2019 (doi:10.1093/gigascience/giz061)

Acute joint inflammation induces a sharp increase in the number of synovial fluid EVs and modifies their phospholipid profile

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8hr post-injection, which started to decline at 24h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0h), SF-EVs collected at 5, 24, and 48h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5h and 24h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation

Extracellular vesicles in joint disease and therapy

The use of extracellular vesicles (EVs) as a potential therapy is currently explored for different disease areas. When it comes to the treatment of joint diseases this approach is still in its infancy. As in joint diseases both inflammation and the associated articular tissue destruction are important factors, both the immune-suppressive and the regenerative properties of EVs are potentially advantageous characteristics for future therapy. There is, however, only limited knowledge on the basic features, such as numerical profile and function, of EVs in joint articular tissues in general and their linking medium, the synovial fluid, in particular. Further insight is urgently needed in order to appreciate the full potential of EVs and to exploit these in EV-mediated therapies. Physiologic joint homeostasis is a prerequisite for proper functioning of joints and we postulate that EVs play a key role in the regulation of joint homeostasis and hence can have an important function in re-establishing disturbed joint homeostasis, and, in parallel, in the regeneration of articular tissues. In this mini-review EVs in the joint are explained from a historical perspective in both health and disease, including the potential niche for EVs in articular tissue regeneration. Furthermore, the translational potential of equine models for human joint biology is discussed. Finally, the use of MSC-derived EVs that is recently gaining ground is highlighted and recommendations are given for further EV research in this field

Mass Spectrometric Detection of Cholesterol Oxidation in Bovine Sperm

We report on the presence and formation of cholesterol oxidation products (oxysterols) in bovine sperm. Although cholesterol is the most abundant molecule in the membrane of mammalian cells and is easily oxidized, this is the first report on cholesterol oxidation in sperm membranes as investigated by state-of-the-art liquid chromatographic and mass spectrometric methods. First, oxysterols are already present in fresh semen samples, showing that lipid peroxidation is part of normal sperm physiology. After chromatographic separation (by high-performance liquid chromatography), the detected oxysterol species were identified with atmospheric pressure chemical ionization mass spectrometry in multiple-reaction-monitoring mode that enabled detection in a broad and linear concentration range (0.05–100 pmol for each oxysterol species detected). Second, exposure of living sperm cells to oxidative stress does not result in the same level and composition of oxysterol species compared with oxidative stress imposed on reconstituted vesicles from protein-free sperm lipid extracts. This suggests that living sperm cells protect themselves against elevated oxysterol formation. Third, sperm capacitation induces the formation of oxysterols, and these formed oxysterols are almost completely depleted from the sperm surface by albumin. Fourth, and most importantly, capacitation after freezing/thawing of sperm fails to induce both the formation of oxysterols and the subsequent albumin-dependent depletion of oxysterols from the sperm surface. The possible physiological relevance of capacitationdependent oxysterol formation and depletion at the sperm surface as well as the omission of this after freezing/thawing semen is discussed.Supported by the research program Biology of Reproductive Cells of the Graduate School of Animal Sciences from the Faculty of Veterinary Medicine of Utrecht University. P.F.N.S. is supported by Portuguese Foundation for Science and Technology, Ministry for Science, Technology, and Higher Education grant SFRH/2888/2000; R.A.v.G. was financed by ZonMw grant 903-44-156; A.B. was financed by the High Potentials program of Utrecht University; and N.G.-G. was funded by Spanish Ministry of Education and Science grant EX 2005- 0460.info:eu-repo/semantics/publishedVersio

An integral biochemical analysis of the main constituents of articular cartilage, subchondral and trabecular bone

AbstractObjectiveIn articular joints, the forces generated by locomotion are absorbed by the whole of cartilage, subchondral bone and underlying trabecular bone. The objective of this study is to test the hypothesis that regional differences in joint loading are related to clear and interrelated differences in the composition of the extracellular matrix (ECM) of all three weight-bearing constituents.MethodCartilage, subchondral- and trabecular bone samples from two differently loaded sites (site 1, dorsal joint margin; site 2, central area) of the proximal articular surface of 30 macroscopically normal equine first phalanxes were collected. Collagen content, cross-linking (pentosidine, hydroxylysylpyridinoline (HP), lysylpyridinoline (LP)) hydroxylation, and denaturation, as well as glycosaminoglycan (GAG) and DNA content were measured in all three tissues. In addition, bone mineral density (BMD), the percentage of ash and the mineral composition (calcium, magnesium and phosphorus) were determined in the bony samples.ResultsFor pentosidine cross-links there was an expected correlation with age. Denatured collagen content was significantly higher in cartilage at site 1 than at site 2 and was higher in trabecular bone compared to subchondral bone, with no site differences. There were significant site differences in hydroxylysine (Hyl) concentration and HP cross-links in cartilage that were paralleled in one or both of the bony layers. In subchondral bone there was a positive correlation between total (HP+LP) cross-links and Ca content. For Ca and other minerals there were corresponding site differences in both bony layers.ConclusionsIt is concluded that there are distinct differences in distribution of the major biochemical components over both sites in all three layers. These differences show similar patterns in cartilage, subchondral bone and trabecular bone, stressing the functional unity of these tissues. Overall, differences could be interpreted as adaptations to a considerably higher cumulative loading over time at site 2, requiring stiffer tissue. Turnover is higher in trabecular bone than in subchondral bone. In cartilage, the dorsal site 1 appears to suffer more tissue damage

A Comprehensive Functional Characterization of Escherichia coli Lipid Genes

Summary: Lipid membranes are the border between living cells and their environments. The membrane’s lipid composition defines fluidity, thickness, and protein activity and is controlled by the intricate actions of lipid gene-encoded enzymes. However, a comprehensive analysis of each protein’s contribution to the lipidome is lacking. Here, we present such a comprehensive and functional overview of lipid genes in Escherichia coli by individual overexpression or deletion of these genes. We developed a high-throughput lipidomic platform, combining growth analysis, one-step lipid extraction, rapid LC-MS, and bioinformatic analysis into one streamlined procedure. This allowed the processing of more than 300 samples per day and revealed interesting functions of known enzymes and distinct effects of individual proteins on the phospholipidome. Our data demonstrate the plasticity of the phospholipidome and unexpected relations between lipid classes and cell growth. Modeling of lipidomic responses to short-chain alcohols provides a rationale for targeted membrane engineering. : Jeucken et al. analyzed lipidomes of E. coli strains with knockout or overexpression of known lipid-related genes. They demonstrate relationships between lipid species and classes and investigate their link to cell growth. The high-throughput lipidomic method is then used to model lipidomic changes to exogenous alcohols. Keywords: lipidomics, systems biology, Escherichia coli, lipids, phospholipids, lipid genes, lipid network, high-throughput, LC-MS, bioinformatic

Lipidome profiling of neutrophil-derived extracellular vesicles unveils their contribution to the ensemble of synovial fluid-derived extracellular vesicles during joint inflammation

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.</p

PMAP-36 reduces the innate immune response induced by Bordetella bronchiseptica-derived outer membrane vesicles

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines

Proteome and phospholipidome interrelationship of synovial fluid-derived extracellular vesicles in equine osteoarthritis: An exploratory ‘multi-omics’ study to identify composite biomarkers

Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p < 0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite diagnostic biomarkers of disease