Monocyte TNF-α Research Articles

In Vitro Anti-inflammatory Effects of Larch Turpentine, Turpentine Oil, Eucalyptus Oil, and Their Mixture as Contained in a Marketed Ointment.

An ointment containing larch turpentine, turpentine oil, and eucalyptus oil has been used for almost a century for the symptomatic treatment of mild, localized, purulent inflammations of the skin. Its clinical efficacy in the treatment of skin infections has been shown in clinical trials, but the mode of action of the active ingredients on inflammation is not known. We studied the anti-inflammatory properties of the active ingredients of the ointment and their mixture in a human monocyte cell model, in which the cells were stimulated with lipopolysaccharide and incubated with the test substances. The cytotoxic threshold of each test substance and the mixture was identified using the alamarBlue assay, and their anti-inflammatory activity was assessed by measuring the release of interleukins IL-1β, IL-6, IL-8, monocyte chemoattractant protein-1, prostaglandin E2, and TNF-α. Cell toxicity was observed at a mixture concentration of 10 µg/mL. All immunological assays were carried out at nontoxic concentrations. Larch turpentine decreased IL-1β, monocyte chemoattractant protein-1, and prostaglandin E2 release at a concentration of 3.9 µg/mL and TNF-α at concentrations > 1.95 µg/mL, whereas eucalyptus oil and turpentine oil had no relevant inhibitory effects. The mixture dose-dependently inhibited IL-1β, IL-6, monocyte chemoattractant protein-1, prostaglandin E2, and TNF-α release at concentrations > 1 µg/mL. IL-8 release was only marginally affected. The anti-inflammatory activity of the herbal ingredients and their mixture was confirmed in this model. This effect seems to be mediated mainly by larch turpentine, with turpentine oil and eucalyptus oil exerting an additive or possibly synergistic function.

Expression of miR-155 in monocytes of people with migraine: association with phenotype, disease severity and inflammatory profile

BackgroundmiR-155 is involved in the generation and maintenance of inflammation and pain, endothelial function and immune system homeostasis, all functions that are relevant for migraine. The present study aims to assess the levels of miR-155 in migraine subtypes (episodic and chronic) in comparison to age- and sex-matched healthy controls.MethodsThis is a cross-sectional, controlled, study involving three study groups: I) episodic migraine (n = 52, EM), II) chronic migraine with medication overuse (n = 44, CM-MO), and III) healthy controls (n = 32, HCs). We assessed the interictal gene expression levels of miR-155, IL-1β, TNF-α, and IL-10 in peripheral blood monocytes using rtPCR. The monocytic differentiation toward the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes was assessed in circulating monocytes with flow cytometry analysis and cell sorting.ResultsmiR-155 gene expression was higher in CM-MO group (2.68 ± 2.47 Relative Quantification - RQ) when compared to EM group (1.46 ± 0.85 RQ, p = 0.006) and HCs (0.44 ± 0.18 RQ, p = 0.001). In addition, miR-155 gene expression was higher in EM group when compared to HCs (p = 0.001). A multivariate analysis confirmed the difference between EM and CM-MO groups after correction for age, sex, smoking habit, preventive treatment, aura, presence of psychiatric or other pain conditions. We found higher gene expression of IL-1β, TNF-α, and lower gene expression of IL-10 in migraine participants when compared to HCs (p = 0.001 for all comparisons). TNF-α and IL-10 genes alterations were more prominent in CM-MO when compared to EM participants (p = 0.001). miR-155 positively correlated with IL-1β (p = 0.001) and TNF-α (p = 0.001) expression levels. Finally, in people with CM-MO, we described an up-regulated percentage of events in both M1 and M2 monocytic profiles.ConclusionsOur study shows for the first time a specific profile of activation of miR-155 gene expression levels in monocytes of selected migraine subpopulations, more pronounced in subjects with CM-MO. Interestingly, mir-155 expression correlated with markers of activation of the inflammatory and immune systems. The CM-MO subpopulation showed a peculiar increase of both pro-inflammatory and anti-inflammatory monocytes which worths further investigation.Trial registrationwww.clinicaltrials.gov. (NCT05891808).

The Effect of Total Glucosides of Peony on Lipopolysaccharide Induced Monocyte Immune Inflammation

This study aimed to explore the effect of total glucosides of peony (TGP) on the lipopolysaccharide induced secretion of IL-1, IL-6 and TNF-α in monocytes. Human peripheral blood was used to isolate and culture monocytes, with 25μg/mL Porphyromonas gingivalis lipopolysaccharide (Pg LPS) as a stimulating factor, then we observed the inhibit influence of 1, 5, 10, 20, 50μg/mL concentrations of TGP on the secretion of IL-1, IL-6, and TNF-α. Enzyme-linked immunosorbent assay (ELISA) was used on the concentration determination of IL-1, IL-6, and TNF-α. This study discovered that TGP had a significant inhibitory effect on Pg LPS induced monocyte secretion of IL-1 and IL-6, and the inhibitory effect on IL-1 was enhanced with the increase of TGP concentration. However, when the TGP reached a certain concentration, the inhibitory effect on IL-6 weakens. No effect was observed on the inhibitory effect of TNF-α. The results of the experiments showed that TGP have a significant inhibitory effect on the secretion of IL-1 and IL-6 by monocytes induced by Pg LPS within a certain concentration range, which might be one of its mechanisms in treating periodontitis.

Lupinus mutabilis Sweet (Tarwi) γ-conglutin Modulates TNF-α and IL-1α Transcriptional Expression in Human Monocytes Infected with Leishmania (V.) peruviana

Background: γ-Conglutin is a hemagglutinating lectin found in the seeds of the Andean legume Lupinus mutabilis. Leishmania (Viannia) peruviana is the etiological agent of cutaneous leishmaniasis, which is endemic in Peru. Leishmaniasis is a neglected disease that lacks an effective and non-toxic treatment. The aim of this study was to evaluate the effect of γ-conglutin from L. mutabilis Patón Grande ecotype (CoPG) on human monocyte cultures before infection with L. (V.) peruviana and to determine the production of molecules that stimulate cellular immunity. Methods: CoPG was isolated by chromatography from a hexane extract of L. mutabilis seeds. Monocytes were obtained by density gradient centrifugation. The effect of CoPG on their viability was determined by the MTT assay. To assess superoxide and nitric oxide production, monocyte cultures were pre-treated with CoPG and then infected with L. (V.) peruviana. Transcriptional expression of TNF-α and IL-1α in CoPG-treated and parasite-infected monocytes was determined by RT-PCR. Result: CoPG retained its hemagglutinating activity throughout the isolation process. The purified protein had a molecular weight of 47 KDa. Besides, CoPG at a 20 µg mL-1 induced the production of superoxide on the treated monocytes. This CoPG concentration also promoted the overexpression of IL-1α and TNF-α on the infected monocytes. The present study showed that CoPG has a novel in vitro immunomodulatory effect. The discovery of bioactive proteins from L. mutabilis may uncover a wide range of new applications for this Andean plant.

The bone-liver interaction modulates immune and hematopoietic function through Pinch-Cxcl12-Mbl2 pathway.

Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that bone marrow stromal cells (BMSCs) lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Prx1-Cre; Pinch1f/f; Pinch2-/- transgenic mice have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type BMSCs. Single cell sequencing analyses reveal dramatic alterations in subpopulations of the BMSCs in Pinch mutant mice. Pinch loss in Prx1+ cells blocks differentiation and maturation of hematopoietic cells in the bone marrow and increases production of pro-inflammatory cytokines TNF-α and IL-1β in monocytes. We find that Pinch is critical for expression of Cxcl12 in BMSCs; reduced production of Cxcl12 protein from Pinch-deficient BMSCs reduces expression of the Mbl2 complement in hepatocytes, thus impairing the innate immunity and thereby contributing to infection and death. Administration of recombinant Mbl2 protein restores the lethality induced by Pinch loss in mice. Collectively, we demonstrate that the novel Pinch-Cxcl12-Mbl2 signaling pathway promotes the interactions between bone and liver to modulate immunity and hematopoiesis and may provide a useful therapeutic target for immune and infectious diseases.

The Antinociceptive Effects of Combined Treatment With Atorvastatin and Vitamin C in the Chronic Constriction Injury Model of Rats.

Neuropathic pain (NP) is caused by damage to the somatosensory system. Nerve damage often results in chronic pain states, including hyperalgesia and allodynia. This study aims to evaluate the anti-nociceptive effects of atorvastatin, vitamin C, and their combination on various laboratory tests in an experimental model NP in rats. To assess the analgesic effects of atorvastatin (5 and 10 mg/kg), vitamin C (500 mg/kg), and their co-administration on chronic constriction injury (CCI) was induced in rats. Behavioral tests, motor nerve conduction velocity (MNCV), pro-inflammatory cytokines, and oxidative markers were measured. Furthermore, histopathological examination was performed. In the present study, it was found that the CCI model can significantly cause hyperalgesia and allodynia on the 21st postoperative day. It was found that the co-administration of vitamin C and atorvastatin has attenuating effects on allodynia and hyperalgesia. Co-administration of vitamin C and atorvastatin also improved MNCV. In the treatment groups, the inflammatory reactions and oxidative markers decreased. Moreover, the co-administration of atorvastatin and vitamin C decreased the perineural inflammation around the sciatic nerve. The results of this study showed that vitamin C potentiates the analgesic effects of atorvastatin in this model of experimental pain, and simultaneous consumption of these medications may be considered as effective therapeutics for NP. The protective properties of atorvastatin, and vitamin C, and their combination on the NP that were assessed can be regarded as a novelty for this study. The co-administration of atorvastatin and vitamin C significantly decreases inflammatory cytokines.The co-administration of atorvastatin and vitamin C significantly decreases stress oxidant markers.The co-administration of atorvastatin and vitamin C significantly attenuated nociceptive effects. Nerve damage causes the deposition of inflammatory factors and or oxidative stress at the site of injury, which in turn activates glial cells that are involved in increasing the inflammatory process by producing and releasing pro-inflammatory agents and oxidative stress. Among statins, atorvastatin is a drug to reduce inflammation, and its effectiveness has been recorded as an antioxidant effect. Vitamin C is known as a neuroprotective agent. Ascorbate inhibits the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in monocytes in high doses (20 mM) by inhibiting them. The rats were randomly divided into 7 groups of 10 animals as follows: 1: Sham-operated, 2: Chronic constriction injury (CCI), 3: CCI+vitamin C (500 mg/kg), 4: CCI+atorvastatin (5 mg/kg), 5: CCI+atorvastatin (10 mg/kg), 6: CCI+vitamin C (500 mg/kg)+atorvastatin (5 mg/kg), and 7: CCI+vitamin C (500 mg/kg)+atorvastatin (10 mg/kg). The results of the present study indicated that the anti-inflammatory, antioxidant, and neuroprotective properties of vitamin C and atorvastatin improved the effects of CCI in an empirical neuropathic in rats. Moreover, it was shown that the associated treatment with vitamin C and atorvastatin can reduce inflammatory factors, such as TNF-α and IL-6, and oxidative markers, such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and malonaldehyde (MDA), while the nerve conduction velocity enhanced and inflammation decreased in histology studies in CCI rats.

Short-chain fatty acids inhibit the activation of T lymphocytes and myeloid cells and induce innate immune tolerance.

The intestinal microbiota contributes to gut immune homeostasis, where short-chain fatty acids (SCFAs) function as the major mediators. We aimed to elucidate the immunomodulatory effects of acetate, propionate, and butyrate. With that in mind, we sought to characterise the expression of SCFA receptors and transporters as well as SCFAs' impact on the activation of different immune cells. Whereas all three SCFAs decreased tumour necrosis factor (TNF)-α production in activated T cells, only butyrate and propionate inhibited interferon (IFN)-γ, interleukin (IL)-17, IL-13, and IL-10 production. Butyrate and propionate inhibited the expression of the chemokine receptors CCR9 and CCR10 in activated T- and B-cells, respectively. Similarly, butyrate and propionate were effective inhibitors of IL-1β, IL-6, TNF-α, and IL-10 production in myeloid cells upon lipopolysaccharide and R848 stimulation. Acetate was less efficient at inhibiting cytokine production except for IFN-α. Moreover, SCFAs inhibited the production of IL-6 and TNF-α in monocytes, myeloid dendritic cells (mDC), and plasmacytoid dendritic cells (pDC), whereas acetate effects were relatively more prominent in pDCs. In monocytes and mDCs, acetate was a less efficient inhibitor, but it was equally effective in inhibiting pDCs activation. We also studied the ability of SCFAs to induce trained immunity or tolerance. Butyrate and propionate- but not acetate- prevented Toll-like receptor-mediated activation in SCFA-trained cells, as demonstrated by a reduced production of IL-6 and TNF-α. Our findings indicate that butyrate and propionate are equally efficient in inhibiting the adaptive and innate immune response and did not induce trained immunity. The findings may be explained by differential SCFA receptor and transporter expression profiles of the immune cells.

CD73 Dysregulates Monocyte Anti-Tumor Activity in Multiple Myeloma.

Multiple myeloma (MM) is characterized by immune cell dysfunction in the tumor microenvironment (TME). We aimed at evaluating the effect of CD73, an overexpressed factor in some tumors, on anti-tumor immune function in the TME of MM. We analyzed the expression of CD73 in T-, B-, and natural killer (NK)-lymphocytes and monocytes in bone marrow (BM), peripheral blood (PB) from MM patients and healthy controls, and residual CD138+ cells using flow cytometry. The anti-tumor activity of these monocytes was confirmed by co-culture with RPMI-8226 cells treated with a CD73 inhibitor. We determined the interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ levels using a cytometric bead array. Monocyte phagocytosis in cell culture sediment was then observed and measured. CD73 was highly expressed in T-, B-, and NK-lymphocytes and monocytes from the BM and PB isolated from patients with MM. Compared with healthy controls, MM samples exhibited significantly higher CD73 expression and TNF-α, IFN-γ, IL-10 levels in monocytes. Inhibiting CD73 in BM immune cells from MM samples significantly increased the secretion of IL-2, TNF-α, and IFN-γ, as well as the killing ability of immune cells. However, monocyte phagocytosis was seldom observed. Inhibiting CD73 in MM monocytes significantly increased the secretion of IL-2, TNF-α, and IFN-γ in monocytes and improved monocyte killing and phagocytosis. Monocytes from MM exhibited weakened anti-tumor effects, and CD73 was involved in forming an immunosuppressive microenvironment. Inhibiting CD73 partly restored the anti-tumor activity of monocytes, a potential strategy for the treatment of MM.

611: HOST IMMUNE ENDOTYPING IN PEDIATRIC ACUTE RESPIRATORY FAILURE

Introduction: Acute respiratory failure (ARF) is a common reason for admission to the pediatric intensive care unit (PICU). The ability to immune endotype critically ill children with ARF to understand if adaptive or innate defects belie severity of illness, and then modulate these defects by evaluating therapies in an ex vivo fashion may be paradigm shifting. We have previously shown that T-cell IFN-γ (adaptive) and monocyte TNF-α (innate) production, as quantitated by the Enzyme-Linked Immune Absorbent Spot (ELISpot) assay, reflect immune competence in COVID-19 pneumonia, and illness severity is associated with decreased circulating lymphocytes with innate and adaptive immune suppression, evidenced by decreased IFN-ɣ and TNF-α production. This promising approach may have significant benefits in critically ill children, as it is unknown if specific measures of innate and adaptive immune function correlate with illness severity. We hypothesize that serial quantitative analysis of IFN-γ and TNF-α production by ELISpot will accurately reflect host immune status in critically ill children with ARF. Methods: In this IRB approved prospective cohort study, children admitted to the PICU with ARF had serial blood analysis using ELISpot for IFN-γ and TNF-α production after positive stimulus with CD3/28 and LPS respectively. ELISpot reporting for cytokine production is done as number of cells secreting the relevant cytokine; data was compared to relevant patient metadata and clinical outcomes. Results: We are currently enrolling patients, and have analyzed two patients with ARF secondary to pneumonia. Patient 1 had 19 cells secreting IFN-ɣ on day 1 and 69 cells on day 5; and 582 cells secreting TNF-α on day 1 and 1217 cells on day 5. Patient 2 had 18 cells secreting IFN-ɣ on day 1 and 144 cells on day 5; and 189 cells secreting TNF-α on day 1 and 466 cells on day 5. Conclusions: Preliminary analysis of IFN-γ and TNF-α production by ELISpot shows surprisingly more IFN-γ reduction early in disease course, which may be a driver of viral replication and severity of illness. Over 5 days, both patients had appropriately hyper inflammatory innate responses. With improved power and additional time points, there is potential to introduce adaptive stimulants early in disease trajectory that may alter clinical outcomes.

Inflammation parameters, monocyte subgroups and toll-like receptor expression before and after dialysis in patients with chronic kidney disease

Abstract Objectives The data of the monocyte subgroups and expressed toll like receptors (TLR) in the innate immune system response, which develop against chronic inflammation in patients with predialysis chronic kidney disease (CKD) and in patients who undergo dialysis treatment, are limited. We aimed to investigate the effect of the dialysis procedure on the current chronic inflammatory condition and which role of monocyte subgroups ratios, the expressions of TLR2/4 and serum Tumor necrosis factor alpha (TNF-α) levels involved in the innate immune response process. Methods We investigated monocyte subgroups, TLR2/TLR4 expressions and serum TNF-α levels in 30 predialysis CKD patients, 90 CKD patients undergoing dialysis and 30 healthy control subjects. Monocyte subgroup percentages and TLR2/TLR4 expressions were determined using the flow cytometry, serum TNF-α levels were investigated using the enzyme-linked immunosorbent assay (ELISA). Results In the dialysis patients, the percentages of classical (p=0.0001) and non-classical (p=0.078) monocytes were found to be higher when compared with the predialysis CKD patients. The percentages of TLR4 expression on non-classical monocytes was higher in dialysis and predialysis patients compared with the healthy controls (p<0.0001, p=0.796). Serum TNF-α level was significantly higher in dialysis and predialysis patients compared with the healthy controls (p=0.013, p=0.022) and a positive correlation between the classical monocyte subgroup and TNF-α was observed (r=0.285, p=0.006). Conclusions Increased percentages of non-classical monocytes, TLR4 expressions and serum TNF-α levels observed in the predialysis CKD patients and dialysis patients might be related to inflammation.

Antioxidant, Immunomodulatory and Potential Anticancer Capacity of Polysaccharides (Glucans) from Euglena gracilis G.A. Klebs

The present study was carried out to determine the bioactivity of polysaccharides extracted from Euglena gracilis (EgPs). These were characterized by FT-IR and GC-MS. Cytotoxicity analyses (MTT) were performed on healthy human gingival fibroblast cell lines (HGF-1), obtaining an IC50 of 228.66 µg mL-1, and cell lines with anticancer activity for colon cancer (HCT-116), breast cancer (MCF-7), human leukemia (U-937, HL-60) and lung cancer (NCl-H460), showing that EgPs have anticancer activity, mainly in HTC-116 cells (IC50 = 26.1 µg mL-1). The immunological assay determined the immunomodulatory capacity of polysaccharides for the production of proinflammatory cytokines IL-6 and TNF-α in murine macrophages (RAW 264.7) and TNF-α in human monocytes (THP-1). It was observed that the EgPs had a stimulating capacity in the synthesis of these interleukins. The antioxidant capacity of polysaccharides and their biomass were analyzed using the ABTS method (18.30 ± 0.14% and (5.40 ± 0.56%, respectively, and the DPPH method for biomass (17.79 ± 0.57%). We quantitatively profiled HGF-1 proteins by liquid chromatography-tandem mass spectrometry analysis, coupled with 2-plex tandem mass tag labelling, in normal cells. In total, 1346 proteins were identified and quantified with high confidence, of which five were considered to be overexpressed. The data is available through ProteomeXchange, under identifier PXD029076.

Immunomodulatory Properties of Packed Red Blood Cells during Storage

Introduction: Red blood cell (RBC) transfusion may affect the recipient immune system. During RBC storage in an unphysiological environment, RBC quality and function are impaired, the cells bleb extracellular vesicles (EVs), and other bioactive substances accumulate in the storage medium. EVs can carry reactive biomolecules and mediate cell-cell interactions. Thus, EVs could explain RBC transfusion related immunomodulation, particularly after prolonged storage. Methods: We exposed peripheral blood mononuclear cells (PBMCs) to allogeneic RBC supernatant (SN) and EVs from fresh and longer-stored RBC units, diluted plasma, and storage solution SAGM, and studied activation and proliferation of T-cells by flow cytometry, and cytokine secretion of LPS-stimulated PBMCs by enzyme-linked immunosorbent assay (ELISA). Results: Both fresh and longer-stored RBC SN but not EVs induced immunomodulation in recipient cells. RBC SN and diluted plasma augmented the proliferation of particularly CD8⁺ T-cells in a 4-day proliferation assay. T-cell activation by SN was evident already after 5 h as shown by upregulation of CD69. SN suppressed monocyte TNF-α and increased IL-10 secretion while diluted plasma increased secretion of both cytokines. Conclusion: This in vitro study demonstrates that stored RBC SN will have mixed immunomodulatory effects depending on responder cells and conditions, independent of RBC storage age. Fresh RBCs containing relatively few EVs can induce immune responses. Residual plasma in the products may contribute to these effects.

A cytotoxic effect of human lactoferrin fusion with Fc domain of IgG.

Lactoferrin (LTF) is a natural iron-binding protein with a potential for clinical utility in many human immune disorders, including cancer. A fusion of LTF with the Fc domain of IgG2 (FcLTF) was designed with inherent properties of an extended the half-life in circulation. Furthermore, the effects of LTF and FcLTF were assessed for influence on the activity of natural killer (NK) cells isolated from human peripheral blood, on the NK-92 cell line, and on human monocytes. The NK cytotoxic activity induced by LTF and FcLTF was determined against the human leukemia K562 cell line, and also for monocytes, by measuring TNFα and granzyme B production, and in an assay for Jurkat cell viability. Selected gene expression in NK-92 cells and monocytes, induced by LTF and FcLTF, was performed by Real Time PCR. No significant difference was observed in NK-92 cytotoxicity stimulated by LTF and FcLTF. The effects on NK cells isolated from the human peripheral blood were varied, possibly due to the immunoregulatory nature of LTF sensing the immune status of donors. Furthermore, only the FcLTF group strongly stimulated production of TNFα and granzyme B in isolated monocytes. In addition, only supernatants from the monocyte cultures treated with FcLTF decreased the viability of Jurkat cells. The ability of FcLTF to induce TNFα in monocytes was strongly inhibited by anti-CD32 and moderately inhibited by anti-CD14 antibody. Lastly, it was demonstrated that FcLTF, strongly induced expression of PI3K, with subsequent activation of AKT/mTOR signaling pathway. Overall, it was demonstrated that this novel fusion molecule may be a perferred choice for clinical utility than the wild type LTF.

DIFFERENTIAL CYTOKINE RESPONSE OF IMMUNOCOMPETENT CELLS TO THE INDUCTION OF RESISTANT, REVERTANT AND SENSITIVE ESCHERICHIA COLI

The work is devoted to the study of the influence of the resistance phenotype of E. coli subcultures on the functionality of evolutionarily conserved pathogen-associated antigens (PAMP) and a special class of pathogen-associated molecular patterns indicating the viability of microbes (vitaPAMP) on immunocompetent human peripheral blood cells. In this work, we used the resistant E. coli strain ATCC BAA-2523, the sensitive E. coli strain ATCC 8739, and the revertant subculture of Escherichia coli obtained under experimental conditions from the E. coli strain ATCC BAA-196. Using enzyme immunoassay, we performed a comparative analysis of mononuclear (PBMC), monocytic (MON), and lymphocytic (LIM) cytokine responses to the induction of resistant, revertant, and susceptible E. coli subcultures by living and fixed cells. It turned out that the PAMP structures of subcultures of fixed E. coli cells are predominantly recognized by human PBMCs. While the pathogen-associated molecular patterns of living E. coli cells are recognized by different target cells, therefore, PBMCs primarily respond to the structures of the vitaPAMP-resistant subculture, while the structures of the revertant E. coli – human MON react to the structures of the vitaPAMP, which is a sensitive strain and PBMC – and MON respond to vitaPAMP. It has also been shown that, in response to fixed cultures of E. coli S and E. coli R, there is a decrease in the threshold level of production of IL-1β, IL-6 and TNF-α in both PBMCs and monocytes, compared with the level of cytokine production induced by live bacteria. Whereas fixed cells of revertant E. coli caused a higher production of pro-inflammatory cytokines PBMC than living cells of this culture.

The effect of sex, menstrual cycle phase and oral contraceptive use on intestinal permeability and ex-vivo monocyte TNFα release following treatment with lipopolysaccharide and hyperthermia

PurposeInvestigate the impact of sex, menstrual cycle phase and oral contraceptive use on intestinal permeability and ex-vivo tumour necrosis factor alpha (TNFα) release following treatment with lipopolysaccharide (LPS) and hyperthermia. MethodsTwenty-seven participants (9 men, 9 eumenorrheic women (MC) and 9 women taking an oral contraceptive pill (OC)) completed three trials. Men were tested on 3 occasions over 6 weeks; MC during early-follicular, ovulation, and mid-luteal phases; OC during the pill and pill-free phase. Intestinal permeability was assessed following a 4-hour dual sugar absorption test (lactulose: rhamnose). Venous blood was collected each trial and stimulated with 100 μg·mL−1 LPS before incubation at 37 °C and 40 °C and analysed for TNFα via ELISA. ResultsL:R ratio was higher in OC than MC (+0.003, p = 0.061) and men (+0.005, p = 0.007). Men had higher TNFα responses than both MC (+53 %, p = 0.004) and OC (+61 %, p = 0.003). TNFα release was greater at 40 °C than 37 °C (+23 %, p < 0.001). ConclusionsMen present with lower resting intestinal barrier permeability relative to women regardless of OC use and displayed greater monocyte TNFα release following whole blood treatment with LPS and hyperthermia. Oral contraceptive users had highest intestinal permeability however, neither permeability or TNFα release were impacted by the pill cycle. Although no statistical effect was seen in the menstrual cycle, intestinal permeability and TNFα release were more variable across the phases.

Reduced proinflammatory activity of outer membrane vesicles of Tannerella forsythia treated with quorum sensing inhibitors.

Outer membrane vesicles (OMVs) of bacteria harbor physiologically active molecules, and quorum sensing inhibitors (QSIs) are expected to regulate bacterial virulence. In this study, we analyzed the proinflammatory activity of OMVs of the periodontal pathogen Tannerella forsythia treated with d-arabinose and d-galactose as QSIs, which inhibit the biofilm formation of periodontal pathogens and autoinducer 2 activity. Compared to OMVs of nontreated T. forsythia (TF OMVs), OMVs released from QSI-treated T. forsythia, designated TF ara-OMVs and TF gal-OMVs, showed reduced production of TNF-α, IL-1β, IL-6, and IL-8 in THP-1 monocytes through decreased activation of NF-κB/MAPKs. Using a human NF-κB reporter cell line and bone marrow-derived macrophages from TLR2-/- mice, TF ara-OMVs and TF gal-OMVs showed less activation of TLR2 than TF OMVs. These results demonstrated that QSIs provide a dual advantage against bacterial infection by inhibiting bacterial biofilm formation and generating OMVs with reduced proinflammatory activity.

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&amp;lt;0.05) and oxLDL (56% and 30%, p&amp;lt;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&amp;lt;0.05) and IL1-β (41% and 44%, p&amp;lt;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.

Ablation of uncoupling protein 3 affects interrelated factors leading to lipolysis and insulin resistance in visceral white adipose tissue.

The physiological role played by uncoupling protein 3 (UCP3) in white adipose tissue (WAT) has not been elucidated so far. In the present study, we evaluated the impact of the absence of the whole body UCP3 on WAT physiology in terms of ability to store triglycerides, oxidative capacity, response to insulin, inflammation, and adipokine production. Wild type (WT) and UCP3Knockout (KO) mice housed at thermoneutrality (30°C) have been used as the animal model. Visceral gonadic WAT (gWAT) from KO mice showed an impaired capacity to store triglycerides (TG) as indicated by its lowered weight, reduced adipocyte diameter, and higher glycerol release (index of lipolysis). The absence of UCP3 reduces the maximal oxidative capacity of gWAT, increases mitochondrial free radicals, and activates ER stress. These processes are associated with increased levels of monocyte chemoattractant protein-1 and TNF-α. The response of gWAT to in vivo insulin administration, revealed by (ser473)-AKT phosphorylation, was blunted in KO mice, with a putative role played by eif2a, JNK, and inflammation. Variations in adipokine levels in the absence of UCP3 were observed, including reduced adiponectin levels both in gWAT and serum. As a whole, these data indicate an important role of UCP3 in regulating the metabolic functionality of gWAT, with its absence leading to metabolic derangement. The obtained results help to clarify some aspects of the association between metabolic disorders and low UCP3levels.

Cytokine-induced transient monocyte IL-7Ra expression and the serum milieu in tuberculosis.

Bacterial components and cytokines induce IL-7 receptor (IL-7Rα) expression in monocytes. Aberrant low IL-7Rα expression of monocytes has been identified as a feature of tuberculosis immunopathology. Here, we investigated the mechanisms underlying IL-7Rα regulation of monocytes and tuberculosis serum effects on IL-7Rα expression. Serum samples from tuberculosis patients and healthy controls, cytokine candidates, and mycobacterial components were analyzed for in vitro effects on IL-7Rα expression of primary monocytes, monocyte-derived macrophages (MDM), and monocyte cell lines. IL-7Rα regulation during culture and the role of FoxO1 were characterized. In vitro activation-induced IL-7Rα expression in human monocytes and serum samples from tuberculosis patients boosted IL-7Rα expression. Although pathognomonic tuberculosis cytokines were not associated with serum effects, we identified cytokines (i.e., GM-CSF, IL-1β, TNF-α, IFN-γ) that induced IL-7Rα expression in monocytes and/or MDM comparable to mycobacterial components. Blocking of cytokine subsets (i.e., IL-1β/TNF-α in monocytes, GM-CSF in MDM) largely diminished IL-7Rα expression induced by mycobacterial components. Finally, we showed that in vitro-induced IL-7Rα expression was transient and dependent on constitutive FoxO1 expression in primary monocytes and monocyte cell lines. This study demonstrated the crucial roles of cytokines and constitutive FoxO1 expression for transient IL-7Rα expression in monocytes.

Metabolic and Inflammatory Profiles of Post-Bariatric Patients with Weight Recidivism.

Background Bariatric surgery promotes expressive weight loss, improving the metabolic and inflammatory profiles. The behavior of these indicators in bariatric patients with weight recidivism is unknown. We aimed to investigate both profiles in bariatric patients with high ratio of weight regain (RWR), comparing them with nonsurgical patients with obesity.MethodsForty patients with obesity subjected to Roux-en-Y gastric bypass (RYGB) with high RWR composed the bariatric group, and 40 controls matched for BMI, age, and gender were recruited as nonsurgical group. Between-group comparisons were performed for clinical history, physical examination, biochemical, metabolic, and inflammatory profiles.ResultsBariatric group was composed of a group with an excess weight loss of 85.9 ± 16.8%, a RWR of 56.5 ± 19.7%, and a time since surgery of 10.7 ± 4.3 years. We noticed a lower proportion of patients with type 2 diabetes mellitus and dyslipidemia (P ≤ 0.05) and lower neck and waist circumferences (P ≤ 0.05) in this group. No differences between groups were observed concerning hip circumference, blood pressure, heart rate, total cholesterol, LDL-c, acid uric, creatinine, ALT, ASP, interferon-γ (INF-γ), interferon gamma–induced protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), TNF-α, interleukin-1β (IL-1 β), interleukin-17 (IL-17), and interleukin-10 (IL-10). Of note, fasting glucose; HbA1c; triglycerides; and, surprisingly, IL-6 levels were lower (P ≤ 0.05) in the bariatric group than nonsurgical one while HDL-c level was higher (P < 0.001).ConclusionExpressive post-bariatric weight loss, even in patients with high RWR, suggests a possible metabolic benefit/protection in the long term. Probably decreased circulating levels of IL-6 are involved in it.Trial RegistrationNCT04193397.