Monocyte Cytokine Release Research Articles

CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome.

Chimeric antigen receptor (CAR) T cell expansion and persistence represent key factors to achieve complete responses and prevent relapses. These features are typical of early memory T cells, which can be highly enriched through optimized manufacturing protocols. Here, we investigated the efficacy and safety profiles of CAR T cell products generated from preselected naive/stem memory T cells (TN/SCM), as compared with unselected T cells (TBULK). Notwithstanding their reduced effector signature in vitro, limiting CAR TN/SCM doses showed superior antitumor activity and the unique ability to counteract leukemia rechallenge in hematopoietic stem/precursor cell–humanized mice, featuring increased expansion rates and persistence together with an ameliorated exhaustion and memory phenotype. Most relevantly, CAR TN/SCM proved to be intrinsically less prone to inducing severe cytokine release syndrome, independently of the costimulatory endodomain employed. This safer profile was associated with milder T cell activation, which translated into reduced monocyte activation and cytokine release. These data suggest that CAR TN/SCM are endowed with a wider therapeutic index compared with CAR TBULK.

412-P: Acute Hyperglycemia-Induced Endoplasmic Reticulum and Oxidative Stress Increases Monocyte Cholesterol Content and Cytokine Secretion

Background: Cardiovascular disease (CVD) is an inflammatory disorder enhanced by diabetes. The mechanisms by which recurrent acute hyperglycemia promotes inflammation and CVD are unclear. We showed that acute hyperglycemia mediates increased monocyte cholesterol uptake and cytokine release, but the underlying signaling pathways are unknown. We hypothesized that acute hyperglycemia-induced oxidative and ER stress in circulating monocytes increases cholesterol uptake and a pro-inflammatory phenotype. Methods: Circulating monocytes from 35 healthy subjects were isolated and exposed to euglycemic (5mM) or hyperglycemic (16.7 mM) conditions with or without inhibitors of ER stress (4 phenyl butyric acid) or oxidative stress (N-Acetyl cysteine) for 2 or 6 hrs to assess changes in LDL or oxLDL uptake and cytokine (TNF-α, IL1-β) release. Results: We found that acute hyperglycemia-induced ER and oxidative stress increased total and free cholesterol content via enhanced LDL (18% and 26%, p<0.05) and oxLDL (56% and 30%, p<0.05) uptake as a result of enhanced CD36 and LDL receptor expression. Conversely, inhibiting monocyte oxidative stress or ER stress attenuated monocyte LDL and oxLDL uptake. Finally, blocking monocyte ER stress decreased acute hyperglycemia-induced monocyte TNF-α (87% and 40%, p<0.05) and IL1-β (41% and 44%, p<0.05) secretion after 2 and 6 hrs. Conclusion: Recurrent acute hyperglycemia promotes inflammation by increasing monocyte ER and oxidative stress, which accelerates cholesterol uptake, cholesterol deposition, and cytokine secretion. Future studies are needed to test if acute hyperglycemia-induced monocyte cholesterol deposition is a mechanism to contribute to increased CVD in diabetes. Disclosure R.Zhang: Research Support; American Heart Association. J.Oh: None. G.A.Kutz: None. M.Bambouskova: None. A.S.Dusso: None. C.Bernal-mizrachi: None.

Dose and Dose Rate-Dependent Effects of Low-Dose Irradiation on Inflammatory Parameters in ApoE-Deficient and Wild Type Mice.

Anti-inflammatory low-dose therapy is well established, whereas the immunomodulatory impact of doses below 0.1 Gy is much less clear. In this study, we investigated dose, dose rate and time-dependent effects in a dose range of 0.005 to 2 Gy on immune parameters after whole body irradiation (IR) using a pro-inflammatory (ApoE−/−) and a wild type mouse model. Long-term effects on spleen function (proliferation, monocyte expression) were analyzed 3 months, and short-term effects on immune plasma parameters (IL6, IL10, IL12p70, KC, MCP1, INFγ, TGFβ, fibrinogen, sICAM, sVCAM, sE-selectin/CD62) were analyzed 1, 7 and 28 days after Co60 γ-irradiation (IR) at low dose rate (LDR, 0.001 Gy/day) and at high dose rate (HDR). In vitro measurements of murine monocyte (WEHI-274.1) adhesion and cytokine release (KC, MCP1, IL6, TGFβ) after low-dose IR (150 kV X-ray unit) of murine endothelial cell (EC) lines (H5V, mlEND1, bEND3) supplement the data. RT-PCR revealed significant reduction of Ki67 and CD68 expression in the spleen of ApoE−/− mice after 0.025 to 2 Gy exposure at HDR, but only after 2 Gy at LDR. Plasma levels in wild type mice, showed non-linear time-dependent induction of proinflammatory cytokines and reduction of TGFβ at doses as low as 0.005 Gy at both dose rates, whereas sICAM and fibrinogen levels changed in a dose rate-specific manner. In ApoE−/− mice, levels of sICAM increased and fibrinogen decreased at both dose rates, whereas TGFβ increased mainly at HDR. Non-irradiated plasma samples revealed significant age-related enhancement of cytokines and adhesion molecules except for sICAM. In vitro data indicate that endothelial cells may contribute to systemic IR effects and confirm changes of adhesion properties suggested by altered sICAM plasma levels. The differential immunomodulatory effects shown here provide insights in inflammatory changes occurring at doses far below standard anti-inflammatory therapy and are of particular importance after diagnostic and chronic environmental exposures.

CXCL12-PLGA/Pluronic Nanoparticle Internalization Abrogates CXCR4-Mediated Cell Migration.

Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as well as several processes involving cell migration. Among them, the role of CXCL12/CXCR4 signaling in cancer and metastasis is well known, and CXCR4 has been often targeted with small molecule-antagonists or short CXCL12-derived peptides to limit the pathological processes of cell migration and invasion. To reduce CXCR4-mediated chemotaxis, we adopted a different approach. We manufactured poly(lactic acid-co-glycolic acid) (PLGA)/Pluronic F127 nanoparticles through microfluidics-assisted nanoprecipitation and functionalized them with streptavidin to docking a biotinylated CXCL12 to be exposed on the nanoparticle surface. Our results show that CXCL12-decorated nanoparticles are non-toxic and do not induce inflammatory cytokine release in THP-1 monocytes cultured in fetal bovine and human serum-supplemented media. The cell internalization of our chemokine receptor-targeting particles increases in accordance with CXCR4 expression in FBS/medium. We demonstrated that CXCL12-decorated nanoparticles do not induce cell migration on their own, but their pre-incubation with THP-1 significantly decreases CXCR4+-cell migration, thereby antagonizing the chemotactic action of CXCL12. The use of biodegradable and immune-compatible chemokine-mimetic nanoparticles to reduce cell migration opens the way to novel antagonists with potential application in cancer treatments and inflammation.

The indirubin derivative 6-bromoindirubin-3′-glycerol-oxime ether (6BIGOE) potently modulates inflammatory cytokine and prostaglandin release from human monocytes through GSK-3 interference

Indirubin is a natural bis-indole alkaloid contained as active ingredient in the traditional Chinese remedy Danggui Longhui Wan. Indirubin and its 3′-oxime derivatives exhibit anti-cancer and anti-inflammatory properties and they inhibit glycogen synthase kinase (GSK)-3 in cell-free assays where 6-bromoindirubin-3′-oxime (6BIO) is among the most potent analogs. Here, we reveal 6-bromoindirubin-3′-glycerol-oxime ether (6BIGOE) as highly potent derivative able to inhibit pro-inflammatory cytokine, chemokine and prostaglandin (PG) release in human primary monocytes while increasing anti-inflammatory interleukin (IL)-10 levels. 6BIGOE suppressed lipopolysaccharide (LPS)-induced IL-1β and PGE2 release with IC50 of 0.008 and 0.02 µM, respectively, being ≥ 12-fold more potent than 6BIO. The effects of 6BIGOE are mediated via intracellular inhibition of GSK-3, where 6BIGOE again surpassed the effectiveness of 6BIO despite the higher potency of the latter in cell-free GSK-3 activity assays. Side-by-side comparison of 6BIGOE (0.1 µM) with the selective GSK-3 inhibitor SB216763 (5 µM) revealed congruent properties such as enrichment of β-catenin and suppression of cyclooxygenase (COX)-2 protein levels due to GSK–3 inhibition. Metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry showed that 6BIGOE selectively decreases pro-inflammatory COX-derived product formation without marked modulation of other lipid mediators. In summary, 6BIGOE is a highly potent indirubin derivative in the cellular context that favorably modulates pro- and anti-inflammatory cytokines as well as COX-2-derived PG via interference with GSK-3.

Cannabidiol differentially regulates basal and LPS-induced inflammatory responses in macrophages, lung epithelial cells, and fibroblasts

IntroductionCannabidiol (CBD) containing products are available in a plethora of flavors in oral, sublingual, and inhalable forms. Immunotoxicological effects of CBD containing liquids were assessed by hypothesizing that CBD regulates oxidative stress and lipopolysaccharide (LPS) induced inflammatory responses in macrophages, epithelial cells, and fibroblasts. MethodsEpithelial cells (BEAS-2B and NHBE), macrophages (U937), and lung fibroblast cells (HFL-1) were treated with varying CBD concentrations or exposed to CBD aerosols. Generated reactive oxygen species (ROS) and inflammatory mediators were measured. Furthermore, monocytes and epithelial cells were stimulated with LPS in combination with CBD or dexamethasone to understand the anti-inflammatory effects of CBD. ResultsCBD showed differential effects on IL-8 and MCP-1, and acellular and cellular ROS levels. CBD significantly attenuated LPS-induced NF-κB activity, IL-8, and MCP-1 release from macrophages. Cytokine array data depicted a differential cytokine response due to CBD. Inflammatory mediators, IL-8, serpin E1, CXCL1, IL-6, MIF, IFN-γ, MCP-1, RANTES, and TNF-α were induced, whereas MCP-1/CCL2, CCL5, eotaxin, and IL-2 were reduced. CBD and dexamethasone treatments reduced the IL-8 level induced by LPS when the cells were treated individually, but showed antagonistic effects when used in combination via MCPIP (monocytic chemotactic protein-induced protein). ConclusionCBD differentially regulated basal pro-inflammatory response and attenuated both LPS-induced cytokine release and NF-κB activity in monocytes, similar to dexamethasone. Thus, CBD has a differential inflammatory response and acts as an anti-inflammatory agent in pro-inflammatory conditions but acts as an antagonist with steroids, overriding the anti-inflammatory potential of steroids when used in combination.

The Role of Distinctive Sphingolipids in the Inflammatory and Apoptotic Effects of Electronegative LDL on Monocytes.

Electronegative low-density lipoprotein (LDL(−)) is a minor LDL subfraction that is present in blood with inflammatory and apoptotic effects. We aimed to evaluate the role of sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) in the LDL(−)-induced effect on monocytes. Total LDL was subfractioned into native LDL and LDL(−) by anion-exchange chromatography and their sphingolipid content evaluated by mass spectrometry. LDL subfractions were incubated with monocytes in the presence or absence of enzyme inhibitors: chlorpromazine (CPZ), d-erythro-2-(N-myristoyl amino)-1-phenyl-1-propanol (MAPP), and N,N-dimethylsphingosine (DMS), which inhibit Cer, Sph, and S1P generation, respectively. After incubation, we evaluated cytokine release by enzyme-linked immunosorbent assay (ELISA) and apoptosis by flow cytometry. LDL(−) had an increased content in Cer and Sph compared to LDL(+). LDL(−)-induced cytokine release from cultured monocytes was inhibited by CPZ and MAPP, whereas DMS had no effect. LDL(−) promoted monocyte apoptosis, which was inhibited by CPZ, but increased with the addition of DMS. LDL enriched with Sph increased cytokine release in monocytes, and when enriched with Cer, reproduced both the apoptotic and inflammatory effects of LDL(−). These observations indicate that Cer content contributes to the inflammatory and apoptotic effects of LDL(−) on monocytes, whereas Sph plays a more important role in LDL(−)-induced inflammation, and S1P counteracts apoptosis.

Augmentation of Human Monocyte Responses to Lipopolysaccharide by the Protein S and Mer/Tyro3 Receptor Tyrosine Kinase Axis.

Resolution of the inflammatory response requires coordinated regulation of pro- and anti-inflammatory mediator production, together with clearance of recruited inflammatory cells. Many different receptors have been implicated in phagocytosis of apoptotic cells (efferocytosis), including Mer, a receptor tyrosine kinase that can mediate recognition and subsequent internalization of apoptotic cells. In this manuscript, we examine the expression and function of the Tyro3/Axl/Mer (TAM) family of receptors by human monocytes. We demonstrate that the Mer ligand, protein S, binds to the surface of viable monocytes via phosphatidylserine-dependent and -independent mechanisms. Importantly, we have identified a novel role for receptor tyrosine kinase signaling in the augmentation of monocyte cytokine release in response to LPS. We propose that low-level phosphatidylserine exposure on the plasma membrane of viable monocytes allows protein S binding that leads to TAM-dependent augmentation of proinflammatory cytokine production. Our findings identify a potentially important role for TAM-mediated signaling during the initiation phase of inflammation.

Abstract A30: Chronic lymphocytic leukemia-derived extracellular vesicles mediate NFkB signaling and pro-inflammatory cytokine release in monocytes

Abstract The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Extracellular vesicles (EV) released by CLL cells are taken up by non-malignant cells in the microenvironment and mediate major disease-related changes in recipient cells. In the current study, we characterized CLL EVs, especially focusing on vesicle-incorporated RNA transcripts, and defined the role of CLL EVs in changing the myeloid tumor microenvironment. EVs were isolated from blood plasma of CLL patients and healthy donors as well as from supernatant of the CLL cell line MEC-1 by a serial centrifugation protocol. Characterization of EVs by electron microscopy, Nanoparticle Tracking Analysis (NTA) and Western blotting revealed vesicles 30 to 350 nm in size, which are positive for various EV marker proteins such as Rab5a and Hsp70. Quantification of blood plasma-derived EVs indicated an enrichment of B-cell derived EVs in plasma of CLL patients compared to healthy donors, although absolute EV counts were not altered in CLL. Focusing on RNA analysis, an enrichment of small RNAs in EVs was observed. Subsequent small RNA sequencing revealed a unique microRNA signature of MEC-1 EVs, with CLL-relevant miRNAs such as miR-21, miR-155 and miR-146a being the most abundant miRNAs. Moreover, full length and 5'end fragment forms of Y RNAs, another class of small non-coding RNAs, were enriched in MEC-1 EVs and CLL plasma EVs. Further evaluating the functional relevance of CLL EVs within the tumor microenvironment, a rapid uptake of CLL cell-derived EVs by human monocytes and macrophages was observed. Uptake of CLL EVs in monocytes induced NFkB signaling and the release of multiple pro-inflammatory cytokines such as CCL2, CCL3, IL-6 and IL-8, which are also upregulated in plasma of CLL patients. In conclusion, tumor-derived EVs harbor a distinct set of non-coding RNAs. The uptake of EVs in recipient cells and the concomitant transfer of incorporated RNAs mediate substantial phenotypic changes in target cells. In the current study, this is exemplified for monocytes, which present several disease-relevant alterations upon EV uptake, including cellular activation and secretion of pro-inflammatory cytokines. Citation Format: Franziska Haderk, Laura Llao Cid, Etienne Moussay, Jerome Paggetti, Karolin Willmund, Jana Seiler, Sven Diederichs, Maria Goebel, Jan Duerig, Thorsten Zenz, Stephan Stilgenbauer, Marc Zapatka, Peter Lichter, Martina Seiffert. Chronic lymphocytic leukemia-derived extracellular vesicles mediate NFkB signaling and pro-inflammatory cytokine release in monocytes. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A30.

Inflammatory intracellular pathways activated by electronegative LDL in monocytes

AimsElectronegative LDL (LDL(−)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(−) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(−) leading to cytokine release in monocytes. Methods and resultsWe determined LDL(−)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(−) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(−). A specific ELISA showed that LDL(−) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(−). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(−)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively. ConclusionsLDL(−) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(−)-induced cytokine release in monocytes.

Chronic Lymphocytic Leukemia-Derived Extracellular Vesicles Mediate NFκB Signaling and Pro-Inflammatory Cytokine Release in Monocytes

Abstract Chronic lymphocytic leukemia (CLL)is a B-cell malignancy associated with an inflammatory milieu and impairedanti-tumor immunity. This study aimed at characterizing CLL-derived extracellularvesicles (EVs) and their role in the tumor microenvironment. EVs were isolated from blood plasma or culture supernatant of the CLL cell line MEC-1 by a serialcentrifugation protocol. Characterization of EVs by electron microscopy, Nanoparticle Tracking Analysis (NTA) and Western blotting revealed vesicles 30to 350 nm in size, which were positive for various EV marker proteins. Quantificationof EVs by NTA and ELISA indicated an enrichment of B-cell derived EVs in plasmaof CLL patients compared to healthy controls, although absolute EV counts were not different. As we observed an accumulation of small RNAs in EVs, small RNAsequencing was performed which revealed a unique microRNA signature of MEC-1 EVs, with CLL-relevant miRNAs such as miR-21, miR-155 and miR-146a being the mostabundant. Moreover, full length and 5′ end fragment forms of Y RNAs, another class of small non-coding RNAs, were enriched in MEC-1 and CLL plasma EVs. In vitro and in mice, CLL EVs were rapidly taken up by monocytes and macrophages which resulted in NFκB signaling and the release of multiple pro-inflammatory cytokines such as CCL2, CCL3, IL-6 andIL-8, proteins known to be elevated in CLL plasma. Moreover, several surface markers on monocytes were altered upon EV uptake, including an increase of the immune checkpoint molecule PD-L1 as well as downregulation of the chemokine receptorCCR2. Of interest, liposomes containing Y RNA transcripts induced a similar response in respective assays suggesting a novel role for these molecules in the tumor microenvironment of CLL.

The effect of hypolipidemic treatment on monocyte cytokine release in different age groups of patients with type 2 diabetes and atherogenic dyslipidemia.

Although both statins and fibrates have been found to reduce monocyte cytokine release, no study has investigated whether the effect of hypolipidaemic agents depends on age. This study retrospectively analysed the results of 65 patients with type 2 diabetes and atherogenic dyslipidaemia, complying with lifestyle intervention, and receiving metformin. These patients were then treated with simvastatin (40 mg daily), micronized fenofibrate (200 mg daily), or simvastatin plus fenofibrate. Tumour necrosis factor-alpha (TNF-alpha), inteleukin-1beta, interleukin-6, and monocyte chemoattractant protein-1 (MCP-1) release, as well as circulating levels of high-sensitivity C-reactive protein (hsCRP), were determined separately for patients aged between 20 and 50 years and between 51 and 75 years before the study and after 12 weeks of hypolipidaemic treatment. Older adults were characterised by higher monocyte release of TNF-alpha and interleukin-6, as well as higher circulating levels of hsCRP, than the younger subjects. The decrease in monocyte release of all investigated cytokines and in plasma hsCRP was similar in both age groups. In turn, the effect of fenofibrate, alone or in combination with simvastatin, on TNF-alpha, interleukin-6, and hsCRP, but not on interleukin-1beta and MCP-1, was stronger in patients aged between 50 and 75 years, and correlated with an improvement in insulin sensitivity only in this age group. Our results suggest that age may partially determine monocyte-suppressing and systemic anti-inflammatory effects of fenofibrate.

Naturally Occurring Subclinical Endotoxemia in Humans Alters Adaptive and Innate Immune Functions through Reduced MAPK and Increased STAT1 Phosphorylation.

Multiple studies have shown correlates of immune activation with microbial translocation and plasma LPS during HIV infection. It is unclear whether this activation is due to LPS, residual viral replication, or both. Few studies have addressed the effects of persistent in vivo levels of LPS on specific immune functions in humans in the absence of chronic viral infection or pathological settings such as sepsis. We previously reported on a cohort of HIV-negative men with subclinical endotoxemia linked to alterations in CD4/CD8 T cell ratio and plasma cytokine levels. This HIV-negative cohort allowed us to assess cellular immune functions in the context of different subclinical plasma LPS levels ex vivo without confounding viral effects. By comparing two samples of differing plasma LPS levels from each individual, we now show that subclinical levels of plasma LPS in vivo significantly alter T cell proliferative capacity, monocyte cytokine release, and HLA-DR expression, and induce TLR cross-tolerance by decreased phosphorylation of MAPK pathway components. Using this human in vivo model of subclinical endotoxemia, we furthermore show that plasma LPS leads to constitutive activation of STAT1 through autocrine cytokine signaling, suggesting that subclinical endotoxemia in healthy individuals might lead to significant changes in immune function that have thus far not been appreciated.

Increased Acetylation of Histone H4 at Lysine 12 (H4K12) in Monocytes of Transgenic Alzheimer’s Mice and in Human Patients

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-β (Aβ) plaque formation, tau pathology, neurodegeneration and inflammatory processes. Monocytes are involved in inflammation in AD and are recruited to the diseased brain. Recently it has been shown that aberrant epigenetic processes including acetylation are associated with the development of AD. The aim of the present study was to examine acetylation of histone H4 at lysine 12 (H4K12) in monocytes in two transgenic AD mouse models (the triple transgenic 3xTg and a model overexpressing amyloid-precursor protein APP with the Swedish-Dutch-Iowa mutations), and to compare with monocytes isolated from human patients with mild cognitive impairment (MCI) and AD. Mouse and human monocytes were selectively isolated with a positive (PluriSelect) respectively with a negative selection method (Miltenyi). Histones were extracted and acetylation of H4K12 was analyzed by a quantification fluorometric kit. Moreover, monocyte cytokine release was measured and cell death analyzed by FACS using incorporation of 7-AAD. Our data show a significant increase of monocytic H4K12 acetylation in both transgenic AD mouse models early during development of the plaque deposition in the brain. In line with these data we found significantly elevated acetylation of H4K12 in human patients with MCI but not in patients with AD. Further we observed that the monocytes of AD mice and of AD patients were significantly more vulnerable to cell damage (as seen by 7-AAD incorporation in FACS analysis) and displayed an enhanced release of pro-inflammatory cytokines (MIP2 and TNFα). Our findings indicate that epigenetic changes in peripheral monocytes are an early event in AD-pathology. Thus H4K12 acetylation may be considered as a novel biomarker for early changes in AD development.

Comparison of the effects of hypolipidemic treatment on monocyte proinflammatory cytokine release in men and women with type 2 diabetes and atherogenic dyslipidemia.

Statins and fibrates reduce monocyte release of proinflammatory cytokines, but it remains unknown whether this effect is sex dependent. We retrospectively analysed age-, weight-, and lipid-matched populations of type 2 diabetic patients of both sexes, who, because of atherogenic dyslipidaemia, were treated with simvastatin (40 mg daily), fenofibrate (200 mg daily), or simvastatin plus fenofibrate. Monocyte release of tumour necrosis factor α (TNF-α), inteleukin-1β, interleukin-6, and monocyte chemoattractant protein-1 (MCP-1), as well as circulating levels of high-sensitivity C-reactive protein (hsCRP) and free fatty acids (FFA) were assessed separately for men and women before and after 12 weeks of treatment. Baseline monocyte release of TNF-α, interleukin-1β, interleukin-6, and MCP-1, as well as plasma hsCRP and FFA levels were comparable in both sexes. Simvastatin, fenofibrate, and simvastatin/fenofibrate combination therapy reduced monocyte release of TNF-α, inteleukin-1β, interleukin-6, and MCP-1, with no difference between the treatment groups. The impact of simvastatin and fenofibrate administered alone on monocyte cytokine release and systemic inflammation did not differ between the men and women. The effect of simvastatin/fenofibrate combination therapy on monocyte release of interleukin-6 and MCP-1 was more pronounced in the male population. The impact of simvastatin administered together with fenofibrate on TNF-α, interleukin-1β, and hsCRP was also stronger in the men than in the women, but the difference did not reach the level of significance. The obtained results suggest that sex differences determine the strength of the monocyte-suppressing effect of simvastatin/ /fenofibrate combination therapy in type 2 diabetic patients with atherogenic dyslipidaemia.

Cardiorenal Syndrome Type 1: Activation of Dual Apoptotic Pathways

Cardiorenal syndrome type 1 (CRS1) pathophysiology is complex, and immune-mediated damage, including alterations in the immune response with monocyte apoptosis and cytokine release, has been reported as a potential mechanism. In this study, we examined the putative role of renal tubular epithelial cell (RTC) apoptosis as a pathogenic mechanism in CRS1. In particular, we investigated the caspase pathways involved in induced apoptosis. We enrolled 29 patients with acute heart failure (AHF), 11 patients with CRS1, and 15 controls (CTR) without AHF or acute kidney injury (AKI). Patients who had AKI prior to the episode of AHF or who had any other potential causes of AKI were excluded. Plasma from different groups was incubated with RTCs for 24 h. Subsequently, cell apoptosis, DNA fragmentation, and caspase-3, -8, and -9 activities were investigated in RTCs incubated with AHF, CRS1, and CTR plasma. A p value <0.5 was considered statistically significant. A quantitative analysis of apoptosis showed significantly higher apoptosis rates in CRS1 patients compared to AHF patients and CTR (p < 0.01). This increase in apoptosis was strongly confirmed by caspase-3 levels (ρ = 0.73). Caspase-8 and -9 were significantly higher in CRS1 patients compared to AHF patients and CTR (p < 0.01). Furthermore, caspase-3 levels showed a significantly positive correlation with caspase-8 (ρ = 0.57) and -9 (ρ = 0.47; p < 0.001). This study demonstrated the significantly heightened presence of dual apoptotic disequilibrium in CRS1. Our findings indicated that apoptosis may have a central role in the mechanism of CRS1, and it could be a potential therapeutic target in this syndrome.

Electronegative LDL induces il-1b release through the activation of cd14 and tlr4 in human monocytes

Objectives: Secretion of the proinflammatory cytokine IL-1β requires the presence of proIL-1b and its processing by caspase-1, which is activated by the NLRP3 inflammasome pathway. Since caspase-1 is constitutively active in monocytes, Toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS), are sufficient to trigger IL-1β secretion by inducing proIL-1β expression. Electronegative LDL (LDL(-)) is a minor LDL subfraction that promotes inflammatory cytokine release in monocytes through CD14-TLR4 activation.

Electronegative LDL-induced cytokine release in human monocytes is promoted through activation of p38-mitogen activated protein kinase and nuclear factor kappa-b

electronegative LDL-induced cytokine release in human monocytes is promoted through activation of p38-mitogen activated protein kinase and nuclear factor kappa-b

C1q/TNF-related protein-3 (CTRP-3) attenuates lipopolysaccharide (LPS)-induced systemic inflammation and adipose tissue Erk-1/-2 phosphorylation in mice in vivo

BackgroundThe C1q/TNF-related proteins comprise a growing family of adiponectin paralogous proteins. CTRP-3 represents a novel adipokine with strong expression in adipose tissue and was shown to inhibit chemokine and cytokine release in adipocytes and monocytes in vitro. The aim of the study was to gain the proof of principle that CTRP-3 is a potent anti-inflammatory adipokine in vivo. MethodsC57BL/6N mice were treated intraperitoneally (i.p.) with bacterial lipopolysaccharide (LPS) for 2h. The effects of a 30min pre-treatment with CTRP-3 i.p. or intravenously (i.v.) on systemic and on epididymal, perirenal and subcutaneous adipose tissue inflammation was analyzed via real-time RT-PCR, ELISA and Western blot analysis. ResultsLPS (1μg i.p.) significantly increased serum IL-6 and MIP-2 levels as well as epididymal adipose tissue expression of IL-6 and MIP-2 in mice, whereas CTRP-3 (10μg i.p.) alone or PBS (i.p.) had no effect. Pre-treatment of mice by CTRP-3 i.p. prior to LPS application significantly attenuated LPS-induced cytokine levels but had no effect on adipose tissue cytokine mRNA expression. In contrast to i.p. application of CTRP-3, systemic i.v. application was not sufficient to inhibit LPS-induced cytokine levels or mRNA tissue expression. CTRP-3 given i.p. significantly attenuated LPS-induced phosphorylation of Erk-1/-2 in inguinal adipose tissue. ConclusionThe present study shows the proof of principle that the novel adipokine CTRP-3 is a potent inhibitor of LPS-induced systemic inflammation and LPS-induced signaling in adipose tissue in vivo.

Ceramide-enriched LDL induces cytokine release through TLR4 and CD14 in monocytes. Similarities with electronegative LDL

IntroductionIn vitro ceramide-enriched LDL (CER-LDL) reproduces most of the properties of electronegative LDL (LDL(−)), a heterogeneous subfraction of LDL found in plasma. LDL(−) comprises several modifications of LDL and has an increased content in ceramide (CER). It promotes cytokine release in monocytes through CD14 and TLR4. CER-LDL also induces cytokine release in these cells but the mechanism is unknown. AimTo evaluate TLR4 andCD14 as the putative receptors involved in cytokine release induced by CER-LDL. MethodsCER-LDL was obtained by incubating native LDL with CER-enriched liposomes. CER content in CER-LDL was assessed by thin layer chromatography of lipid extracts. CER-LDL and LDL(−) were incubated for 20h with human monocytes in the presence or absence of a TLR4 signaling inhibitor. Both LDLs were also incubated with two human monocytic cell lines, normal and THP1 overexpressing CD14 (THP1-CD14) cells. The release of IL-6, IL-10 and MCP-1 was evaluated by ELISA in culture medium. ResultsThe release of IL-6, IL-10 and MCP-1 induced by CER-LDL in monocytes was inhibited by VIPER (90% inhibition), a specific TLR4 inhibitor. The cytokine release induced by CER-LDL was negligible in THP1, cells presenting a very low CD14 expression. In contrast, the induction of cytokine release in THP1-CD14 was high and dependent on the CER content in LDL. ConclusionCER-LDL induces IL-6, IL-10 and MCP-1 release through the activation of CD14 and TLR4 in monocytes, reproducing the behavior of LDL(−). The increased content of CER in LDL(−) is then related to the inflammatory action of LDL(−).