Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells

Conjugated linoleic acid (CLA) isomers may have a role in preventing atherosclerosis through the modulation of inflammation, particularly of the endothelium. However, whether low concentrations of CLAs are able to affect basal unstimulated endothelial cell (EC) responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the basal inflammatory responses by ECs. EA.hy926 cells (HUVEC lineage) were cultured under standard conditions and exposed to individual CLAs for 48 h. Both CLAs were incorporated into ECs in a dose-dependent manner. CLA9,11 (1 μM) significantly decreased concentrations of MCP-1 (p < 0.05), IL-6 (p < 0.05), IL-8 (p < 0.01) and RANTES (p < 0.05) in the culture medium. CLA10,12 (10 μM) decreased the concentrations of MCP-1 (p < 0.05) and RANTES (p < 0.05) but increased the concentration of IL-6 (p < 0.001). At 10 μM both CLAs increased the relative expression of the NFκβ subunit 1 gene (p < 0.01 and p < 0.05, respectively), while decreasing the relative expression of PPARα (p < 0.0001), COX-2 (p < 0.0001) and IL-6 (p < 0.0001) genes. CLA10,12 increased the relative expression of the gene encoding IκK-β at 10 μM compared with CLA9,11 (p < 0.05) and increased the relative expression of the gene encoding IκBα at 1 and 10 μM compared with linoleic acid (both p < 0.05). Neither CLA affected the adhesion of monocytes to ECs. These results suggest that low concentrations of both CLA9,11 and CLA10,12 have modest anti-inflammatory effects in ECs. Thus, CLAs may influence endothelial function and the risk of vascular disease. Nevertheless, at these low CLA concentrations some pro-inflammatory genes are upregulated while others are downregulated, suggesting complex effects of CLAs on inflammatory pathways.     

Conjugated Linoleic Acids and CLA-Containing Phospholipids Inhibit NO Formation in Aortic Endothelial Cells

This study examined the effects of five CLA isomers and the non-conjugated LA on nitric oxide (NO) production, an important modulator of vasodilation, inflammation, and cytotoxicity. Bovine aortic endothelial cells (BAECs) were pretreated with pure CLAs (c9, c11-, c9, t11-, t9, t11-, t10, c12-, c11, c13-CLA) and the non-conjugated c9, c12-analog, then stimulated by the ionophore A23187 followed by fluorescence monitoring of NO production. CLAs (5 μM) decreased NO formation in the range of 20–40% relative to non-fatty acid-treated controls with the t9, t11- and t10, c12-CLAs being the most effective. The inhibitory effect of either of these CLAs was not time dependent over a 120 min time interval. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) play crucial roles in membrane structure and cell signaling. Since CLAs are usually esterified in these phospholipids (PLs), the effects of three CLA-containing PLs and 2-linoleoyl-PC on NO production by A23187-stimulated BAECs were also examined. c9, t11-CLA-PC and 2-linoleoyl-PC dose-dependently inhibited NO production over a 60–1,000 μM concentration range whereas c9, c11-CLA-PC and c9, t11-CLA-PE were ineffective. Both c9, t11-CLA-PC and linoleoyl-PC exhibited a time-dependent decrease in NO production from 5 to 120 min. The results of the present study suggest that the influence of several C18 polyunsaturated fatty acids incorporated into cellular phospholipids on the A23187-induced formation of NO by BAECs is strongly dependent on the positional and geometric nature of the double bonds.     

Direct Isomerization of Linoleic Acid to Conjugated Linoleic Acids (CLA) using Gold Catalysts

Supported gold catalysts, e.g., Au on Al₂O₃, Fe₂O₃, CeO₂, MnO₂, TiO₂, ZrO₂, activated carbon, titanium silicalite TS‐1, were prepared and used for the isomerization of linoleic acid (cis‐9,cis‐12‐octadecadienoic acid) to conjugated linoleic acids (CLA) in the presence of hydrogen at 165 °C in a batch reactor. The best results were obtained using a catalyst with 2 wt % Au on TS‐1, which exhibits a high selectivity (78 %) towards CLA. The two biologically active target CLA isomers, i.e., cis‐9,trans‐11‐CLA and trans‐10,cis‐12‐CLA, were the main products. During the isomerization of linoleic acid to CLA, consecutive reactions also took place. These were the hydrogenation of linoleic acid and CLA to monounsaturated octadecenoic acids and the further hydrogenation of monounsaturated acids to stearic acid. Thus, gold catalysts are capable of isomerizing linoleic acid to CLA and hydrogenating their double bonds to an extent that depends on the Au catalyst used.     

Differential Effect of Dietary Supplementation with a Soybean Oil Enriched in Oleic Acid versus Linoleic Acid on Plasma Lipids and Atherosclerosis in LDLR-Deficient Mice

Both monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) play important roles in lipid metabolism, and diets enriched with either of these two fatty acids are associated with decreased cardiovascular risk. Conventional soybean oil (CSO), a common food ingredient, predominantly contains linoleic acid (LA; C18:2), a n-6 PUFA. Recently, a modified soybean oil (MSO) enriched in oleic acid (C18:1), a n-9 MUFA, has been developed, because of its improved chemical stability to oxidation. However, the effect of the different dietary soybean oils on cardiovascular disease remains unknown. To test whether diets rich in CSO versus MSO would attenuate atherosclerosis development, LDL receptor knock-out (LDLR-KO) mice were fed a Western diet enriched in saturated fatty acids (control), or a Western diet supplemented with 5% (w/w) LA-rich CSO or high-oleic MSO for 12 weeks. Both soybean oils contained a similar amount of linolenic acid (C18:3 n-3). The CSO diet decreased plasma lipid levels and the cholesterol content of VLDL and LDL by approximately 18% (p < 0.05), likely from increased hepatic levels of PUFA, which favorably regulated genes involved in cholesterol metabolism. The MSO diet, but not the CSO diet, suppressed atherosclerotic plaque size compared to the Western control diet (Control Western diet: 6.5 ± 0.9%; CSO diet: 6.4 ± 0.7%; MSO diet: 4.0 ± 0.5%) (p < 0.05), independent of plasma lipid level changes. The MSO diet also decreased the ratio of n-6/n-3 PUFA in the liver (Control Western diet: 4.5 ± 0.2; CSO diet: 6.1 ± 0.2; MSO diet: 2.9 ± 0.2) (p < 0.05), which correlated with favorable hepatic gene expression changes in lipid metabolism and markers of systemic inflammation. In conclusion, supplementation of the Western diet with MSO, but not CSO, reduced atherosclerosis development in LDLR-KO mice independent of changes in plasma lipids.     

Soluble Endoglin Stimulates Inflammatory and Angiogenic Responses in Microglia That Are Associated with Endothelial Dysfunction

Increased soluble endoglin (sENG) has been observed in human brain arteriovenous malformations (bAVMs). In addition, the overexpression of sENG in concurrence with vascular endothelial growth factor (VEGF)-A has been shown to induce dysplastic vessel formation in mouse brains. However, the underlying mechanism of sENG-induced vascular malformations is not clear. The evidence suggests the role of sENG as a pro-inflammatory modulator, and increased microglial accumulation and inflammation have been observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that the presence of sENG along with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, we observed increased microglial activation around dysplastic vessels with the expression of NLRP3, an inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased levels of angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs but it decreased the level of IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to express angiogenic/inflammatory molecules which may be involved in EC dysfunction. Our study corroborates the contribution of microglia to the pathology of sENG-associated vascular malformations.     

Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage,Endothelial Cells and Animal Models of Cardiovascular Disease

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.     

Modeling In Vitro Osteoarthritis Phenotypes in a Vascularized Bone Model Based on a Bone-Marrow Derived Mesenchymal Cell Line and Endothelial Cells

The subchondral bone and its associated vasculature play an important role in the onset of osteoarthritis (OA). Integration of different aspects of the OA environment into multi-cellular and complex human, in vitro models is therefore needed to properly represent the pathology. In this study, we exploited a mesenchymal stromal cell line/endothelial cell co-culture to produce an in vitro human model of vascularized osteogenic tissue. A cocktail of inflammatory cytokines, or conditioned medium from mechanically-induced OA engineered microcartilage, was administered to this vascularized bone model to mimic the inflamed OA environment, hypothesizing that these treatments could induce the onset of specific pathological traits. Exposure to the inflammatory factors led to increased network formation by endothelial cells, reminiscent of the abnormal angiogenesis found in OA subchondral bone, demineralization of the constructs, and increased collagen production, signs of OA related bone sclerosis. Furthermore, inflammation led to augmented expression of osteogenic (alkaline phosphatase (ALP) and osteocalcin (OCN)) and angiogenic (vascular endothelial growth factor (VEGF)) genes. The treatment, with a conditioned medium from the mechanically-induced OA engineered microcartilage, also caused increased demineralization and expression of ALP, OCN, ADAMTS5, and VEGF; however, changes in network formation by endothelial cells were not observed in this second case, suggesting a possible different mechanism of action in inducing OA-like phenotypes. We propose that this vascularized bone model could represent a first step for the in vitro study of bone changes under OA mimicking conditions and possibly serve as a tool in testing anti-OA drugs.     

Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells

Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1–MRE and Nrf2–ARE pathways, whereas MT-2A expression requires only activation of the MTF-1–MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism.     

Fatty-Acid-Binding Protein 4 as a Novel Contributor to Mononuclear Cell Activation and Endothelial Cell Dysfunction in Atherosclerosis

Background—Elevated circulating fatty-acid-binding protein 4 (FABP4) levels may be linked with cardiovascular events. This study aimed to investigate the mechanistic role of FABP4 in atherosclerosis. Methods—We recruited 22 patients with angiographically proven coronary artery disease (CAD) and 40 control subjects. Mononuclear cells (MNCs) and human coronary endothelial cells (HCAECs) were used for in vitro study. Results—Patients with CAD were predominantly male with an enhanced prevalence of hypertension, diabetes, and smoking history. FABP4 concentrations were up-regulated in culture supernatants of MNCs from CAD patients, which were positively correlated with the patients’ age, waist–hip ratio, body mass index, serum creatinine, type 2 diabetes, and the presence of hypertension. The adhesiveness of HCAECs to monocytic cells can be activated by FABP4, which was reversed by an FABP4 antibody. FABP4 blockade attenuated the oxidized low-density lipoprotein (oxLDL)-induced expression of ICAM-1, VCAM-1, and P-selectin. FABP4 impaired the tube formation and migration via the ERK/JNK/STAT-1 signaling pathway. FABP4 suppressed phosphorylation of eNOS and expression of SDF-1 protein, both of which can be reversed by treatment with VEGF. Blockade of FABP4 also improved the oxLDL-impaired cell function. Conclusion—We discovered a novel pathogenic role of FABP4 in MNC activation and endothelial dysfunction in atherosclerosis. FABP4 may be a therapeutic target for modulating atherosclerosis.     

Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1β)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.     

Decreased Lymphangiogenic Activities and Genes Expression of Cord Blood Lymphatic Endothelial Progenitor Cells (VEGFR3+/Pod+/CD11b+ Cells) in Patient with Preeclampsia

The abnormal development or disruption of the lymphatic vasculature has been implicated in metabolic and hypertensive diseases. Recent evidence suggests that the offspring exposed to preeclampsia (PE) in utero are at higher risk of long-term health problems, such as cardiovascular and metabolic diseases in adulthood, owing to in utero fetal programming. We aimed to investigate lymphangiogenic activities in the lymphatic endothelial progenitor cells (LEPCs) of the offspring of PE. Human umbilical cord blood LEPCs from pregnant women with severe PE (n = 10) and gestationally matched normal pregnancies (n = 10) were purified with anti-vascular endothelial growth factor receptor 3 (VEGFR3)/podoplanin/CD11b microbeads using a magnetic cell sorter device. LEPCs from PE displayed significantly delayed differentiation and reduced formation of lymphatic endothelial cell (LEC) colonies compared with the LEPCs from normal pregnancies. LECs differentiated from PE-derived LEPCs exhibited decreased tube formation, migration, proliferation, adhesion, wound healing, and 3D-sprouting activities as well as increased lymphatic permeability through the disorganization of VE-cadherin junctions, compared with the normal pregnancy-derived LECs. In vivo, LEPCs from PE showed significantly reduced lymphatic vessel formation compared to the LEPCs of the normal pregnancy. Gene expression analysis revealed that compared to the normal pregnancy-derived LECs, the PE-derived LECs showed a significant decrease in the expression of pro-lymphangiogenic genes (GREM1, EPHB3, VEGFA, AMOT, THSD7A, ANGPTL4, SEMA5A, FGF2, and GBX2). Collectively, our findings demonstrate, for the first time, that LEPCs from PE have reduced lymphangiogenic activities in vitro and in vivo and show the decreased expression of pro-lymphangiogenic genes. This study opens a new avenue for investigation of the molecular mechanism of LEPC differentiation and lymphangiogenesis in the offspring of PE and subsequently may impact the treatment of long-term health problems such as cardiovascular and metabolic disorders of offspring with abnormal development of lymphatic vasculature.     

Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.     

The Caspase Pathway of Linoelaidic Acid (9t, 12t-C18:2)-Induced Apoptosis in Human Umbilical Vein Endothelial Cells

Trans fatty acids (TFA) are reported to contribute to inflammation and coronary heart disease. The study aim was to investigate the proapoptotic effects of two double bond TFA (TDTFA) on human umbilical vein endothelial cells (HUVEC). The HUVEC were grown in media supplied with linoelaidic acid (9t,12t-C18:2) at 50, 100, 200, 400 μmol/l for 24 or 48 h to examine the effects of TDTFA on the viability and apoptosis of these cells. Flow cytometry analysis and confocal scanning were used to measure apoptosis, cell binding of Annexin V and propidium iodide uptake. Colorimetric assay and RT-PCR were used to analyze enzyme activities and mRNA expression of caspase-3, -8 and -9 in HUVEC. Results showed that 9t,12t-C18:2 inhibited the viability of HUVEC in a dose-dependent and time-dependent manner. The percentages of 9t,12t-C18:2 induced apoptotic and necrotic cells significantly increased compared with that of the control. The activities and mRNA expression of caspase-8, -9 and -3 were significantly increased in 9t,12t-C18:2 treated cells compared to that of the control. Addition of specific inhibitors of caspase-8 (z-IETD-fmk) and caspase-9 (z-LEHD-fmk) to HUVEC was found to completely inhibit 9t,12t-C18:2-induced activation of caspase-3, and z-IETD-fmk inhibited the activation of caspase-9. Meanwhile, it was found that mRNA expression of Bid, Smac/DIABLO and the release of mitochondrial cytochrome c were significantly elevated by 9t,12t-C18:2 treatment. These results suggest that 9t,12t-C18:2 may induce apoptosis of HUVEC through activating caspase-8, -9 and -3. Both the death receptor pathway and the mitochondrial pathway may be involved in the apoptosis induced by 9t,12t-C18:2.     

Chemical Structures of 4-Oxo-Flavonoids in Relation to Inhibition of Oxidized Low-Density Lipoprotein (LDL)-Induced Vascular Endothelial Dysfunction

Vascular endothelial dysfunction induced by oxidative stress has been demonstrated to be the initiation step of atherosclerosis (AS), and flavonoids may play an important role in AS prevention and therapy. Twenty-three flavonoids categorized into flavones, flavonols, isoflavones, and flavanones, all with 4-oxo-pyronenucleus, were examined for what structural characteristics are required for the inhibitory effects on endothelial dysfunction induced by oxidized low-density lipoprotein (oxLDL). Human vascular endothelial cells EA.hy926 were pretreated with different 4-oxo-flavonoids for 2 hs, and then exposed to oxLDL for another 24 hs. Cell viability and the level of malondialdehyde (MDA), nitric oxide (NO) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured, respectively. Then, correlation analysis and paired comparison were used to analyze the structure-activity relationships. Significant correlations were observed between the number of -OH moieties in total or in B-ring and the inhibitory effectson endothelial dysfunction. Furthermore, 3',4'-ortho-dihydroxyl on B-ring, 3-hydroxyl on C-ring and 2,3-double bondwere correlated closely to the inhibitory effects of flavonolson cell viability decrease and lipid peroxidation. 5,7-meta-dihydroxyl group on A-ring was crucial for the anti-inflammatory effects of flavones and isoflavones in endothelial cells. Moreover, the substituted position of B-ring on C3 rather than C2 was important for NO release. Additionally, hydroxylation at C6 position significantly attenuated the inhibitory effects of 4-oxo-flavonoids on endothelial dysfunction. Our findings indicated that the effective agents in inhibiting endothelial dysfunction include myricetin, quercetin, luteolin, apigenin, genistein and daidzein. Our work might provide some evidence for AS prevention and a strategy for the design of novel AS preventive agents.     

Neonate Plutella xylostella Responses to Surface Wax Components of a Resistant Cabbage (Brassica oleracea)

Behavior of neonate Plutella xylostella was observed and quantified during the first 5 min of contact with cabbage surface waxes and surface wax components deposited as a film (60 g/cm²) on glass. The time larvae spent biting was greater and the time walking was less on waxes extracted from the susceptible cabbage variety, Round-Up, than on an insect-resistant glossy-wax breeding line, NY 9472. The waxes of both cabbage types were characterized and some of the compounds present at higher concentrations in the glossy waxes were tested for their deterrent effects on larvae by adding them to the susceptible waxes. Adding a mixture of four n-alkane-1-ols or a mixture of - and -amyrins to wax from susceptible cabbage reduced the number of insects biting and, among those biting, reduced the time biting and increased the time walking in a dose-dependent manner. Among individual n-alkane-1-ols, adding C₂₄ or C₂₅ alcohols reduced the number of insects biting but only adding C₂₅ alcohol reduced the time spent biting among those insects that initiated biting. Adding a mixture of five n-alkanoic acids did not affect biting, but increased the time spent palpating and decreased walking time. Among individual n-alkanoic acids, only adding C₁₄ significantly increased the time palpating. If the observed responses were gustatory, the results indicate that some primary wax components, including specific long-chain alkyl components, have allelochemical activity influencing host acceptance behavior by a lepidopteran larva.     

Sphingosine-1-Phosphate Receptor Type 4 (S1P4) Is Differentially Regulated in Peritoneal B1 B Cells upon TLR4 Stimulation and Facilitates the Egress of Peritoneal B1a B Cells and Subsequent Accumulation of Splenic IRA B Cells under Inflammatory Conditions

Background: Gram-negative infections of the peritoneal cavity result in profound modifications of peritoneal B cell populations and induce the migration of peritoneal B cells to distant secondary lymphoid organs. However, mechanisms controlling the egress of peritoneal B cells from the peritoneal cavity and their subsequent trafficking remain incompletely understood. Sphingosine-1-phosphate (S1P)-mediated signaling controls migratory processes in numerous immune cells. The present work investigates the role of S1P-mediated signaling in peritoneal B cell trafficking under inflammatory conditions. Methods: Differential S1P receptor expression after peritoneal B cell activation was assessed semi‑quantitatively using RT-PCR in vitro. The functional implications of differential S1P₁ and S1P₄ expression were assessed by transwell migration in vitro, by adoptive peritoneal B cell transfer in a model of sterile lipopolysaccharide (LPS)‑induced peritonitis and in the polymicrobial colon ascendens stent peritonitis (CASP) model. Results: The two sphingosine-1-phosphate receptors (S1PRs) expressed in peritoneal B cell subsets S1P₁ and S1P₄ are differentially regulated upon stimulation with the TLR4 agonist LPS, but not upon PMA/ionomycin or B cell receptor (BCR) crosslinking. S1P₄ deficiency affects both the trafficking of activated peritoneal B cells to secondary lymphoid organs and the positioning of these cells within the functional compartments of the targeted organ. S1P₄ deficiency in LPS-activated peritoneal B cells results in significantly reduced numbers of splenic innate response activator B cells. Conclusions: The S1P-S1PR system is implicated in the trafficking of LPS-activated peritoneal B cells. Given the protective role of peritoneal B1a B cells in peritoneal sepsis, further experiments to investigate the impact of S1P₄-mediated signaling on the severity and mortality of peritoneal sepsis are warranted.     

T Cells in Multisystem Inflammatory Syndrome in Children (MIS-C) Have a Predominant CD4+ T Helper Response to SARS-CoV-2 Peptides and Numerous Virus-Specific CD4− CD8− Double-Negative T Cells

We studied SARS-CoV-2-specific T cell responses in 22 subacute MIS-C children enrolled in 2021 and 2022 using peptide pools derived from SARS-CoV-2 spike or nonspike proteins. CD4+ and CD8+ SARS-CoV-2-specific T cells were detected in 5 subjects, CD4+ T helper (Th) responses alone were detected in 12 subjects, and CD8+ cytotoxic T cell (CTL) responses alone were documented in 1 subject. Notably, a sizeable subpopulation of CD4− CD8− double-negative (DN) T cells out of total CD3+ T cells was observed in MIS-C (median: 14.5%; IQR 8.65–25.3) and recognized SARS-CoV-2 peptides. T cells bearing the Vβ21.3 T cell receptor (TcRs), previously reported as pathogenic in the context of MIS-C, were detected in high frequencies, namely, in 2.8% and 3.9% of the CD4+ and CD8+ T cells, respectively. However, Vβ21.3 CD8+ T cells that responded to SARS-CoV-2 peptides were detected in only a single subject, suggesting recognition of nonviral antigens in the majority of subjects. Subjects studied 6–14 months after MIS-C showed T cell epitope spreading, meaning the activation of T cells that recognize more SARS-CoV-2 peptides following the initial expansion of T cells that see immunodominant epitopes. For example, subjects that did not recognize nonspike proteins in the subacute phase of MIS-C showed good Th response to nonspike peptides, and/or CD8+ T cell responses not appreciable before arose over time and could be detected in the 6–14 months’ follow-up. The magnitude of the Th and CTL responses also increased over time. In summary, patients with MIS-C associated with acute lymphopenia, a classical feature of MIS-C, showed a physiological response to the virus with a prominent role for virus-specific DN T cells.     

Metalloendopeptidase ADAM-like Decysin 1 (ADAMDEC1) in Colonic Subepithelial PDGFRα+ Cells Is a New Marker for Inflammatory Bowel Disease

Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα⁺ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRα⁺ cells. ADAMDEC1 protein was mainly released from PDGFRα⁺ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRα⁺ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45⁻ PDGFRα⁺ cells in DSS-induced colitis mice, with only minimal expression in CD45⁺ CD64⁺ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRα⁺ cells, but not in CD64⁺ macrophages found in human colonic mucosal tissue affected by Crohn’s disease. In summary, PDGFRα⁺ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn’s disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn’s disease.