The effect of IκK-16 on lipopolysaccharide-induced impaired monocytes

This study focuses on impaired monocyte function, which occurs in some patients after trauma, major elective surgery, or sepsis. This monocyte impairment increases the risk of secondary infection and death. We aimed to determine the influence IκK-16 had on monocytes using an ex-vivo model of human monocyte impairment. We included the effects of the well-studied comparators interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on impaired monocytes. Primary human monocytes were stimulated with 10 ng/mL of lipopolysaccharide (LPS) for 16 h and then challenged with 100 ng/mL LPS to assess the monocyte inflammatory response. Treatment regimens, consisting of either IκK-16, IFN-γ, or GM-CSF, were administered to impaired monocytes near the time of initial LPS stimulation. Stimulation with 10 ng/mL LPS initially promoted a pro-inflammatory response but subsequently impaired production of both tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) and decreased HLA-DR expression. IκK-16 treatment attenuated TNF-α production and programmed death-ligand 1 (PD-L1) expression and increased IL-10 and CD14 expression. IFN-γ treatment increased TNF-α production as well as PD-L1 and HLA-DR expression. In conclusion, limiting early inflammation with IκK-16 suppresses TNF-α production and PD-L1 expression but enhances IL-10 production and preserves CD14 expression for potential future exposure to infective stimuli.     

Protective effect of Ulinastatin against murine models of sepsis: Inhibition of TNF-α and IL-6 and augmentation of IL-10 and IL-13

AimExcessive production of inflammatory mediators during invasive infection plays a key role in the pathogenesis of sepsis. In an attempt to improve survival of patients with this lethal syndrome, agents were developed to selectively inhibit mediators in this inflammatory response. Ulinastatin (UTI), a human protease inhibitor, inhibits the enhanced production of pro-inflammatory molecules. However, it is unknown if Ulinastatin treatment could result in protective effects for sepsis. The aim of this study was to investigate the role of Ulinastatin on septic rats.MethodsSixty male Wistar rats were divided into six groups, 10 of each: sham-operation plus PBS (5 ml), cecal ligation and puncture (CLP) plus PBS (5 ml), CLP plus UTI (5000 U/kg), CLP plus UTI (10,000 U/kg), CLP plus UTI (20,000 U/kg) and sham-operation plus UTI (10,000 U/kg). Rats in the UTI groups after CLP operation were treated with Ulinastatin by intraperitoneal injection at different doses and then compared with untreated sepsis control animals.ResultsThe intestinal concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) were significantly higher in septic rats than those in normal rats. Ulinastatin administration effectively suppressed the levels of TNF-α and IL-6, whereas it markedly enhanced the levels of IL-10 and IL-13.ConclusionUlinastatin may possess a protective role in the septic process by inhibiting TNF-α and IL-6, and augmenting IL-10 and IL-13 concentrations in intestine of septic rats.     

Urban air pollution produces up-regulation of myocardial inflammatory genes and dark chocolate provides cardioprotection

Air pollution is a serious environmental problem. Elderly subjects show increased cardiac morbidity and mortality associated with air pollution exposure. Mexico City (MC) residents are chronically exposed to high concentrations of fine particulate matter (PM_2.5) and PM-associated lipopolysaccharides (PM-LPS). To test the hypothesis that chronic exposure to urban pollution produces myocardial inflammation, female Balb-c mice age 4 weeks were exposed for 16 months to two distinctly different polluted areas within MC: southwest (SW) and northwest (NW). SW mice were given either no treatment or chocolate 2 g/9.5 mg polyphenols/3 times per week. Results were compared to mice kept in clean air. Key inflammatory mediator genes: cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and the LPS receptor CD14 (cluster of differentiation antigen 14) were measured by real-time polymerase chain reaction. Also explored were target NFκB (nuclear factor κB), oxidative stress and antioxidant defense genes.TNF-α, IL-6, and COX-2 were significantly increased in both NW and SWMC mice (p = 0.0001). CD14 was up-regulated in SW mice in keeping with the high exposures to particulate matter associated endotoxin. Chocolate administration resulted in a significant down-regulation of TNF-α (p < 0.0001), IL-6 (p = 0.01), and IL-1β (p = 0.02). The up-regulation of antioxidant enzymes and the down-regulation of potent oxidases, toll-like receptors, and pro-apoptotic signaling genes completed the protective profile.Exposure to air pollution produces up-regulation of inflammatory myocardial genes and endotoxin plays a key role in the inflammatory response. Regular consumption of dark chocolate may reduce myocardial inflammation and have cardioprotective properties in the setting of air pollution exposures.     

Stronger Toll-like receptor 1/2, 4, and 7/8 but less 9 responses in peripheral blood mononuclear cells in non-infectious exacerbated asthmatic children

Toll-like receptors (TLR) initiate innate and often affect adaptive immune response. This study aimed to determine if TLR response and T regulatory cell (Treg) function in peripheral blood mononuclear cells (PBMC) correlate with clinical severity in non-infectious asthma. TLR1–9 expression and representative response cytokine TNF-α, IL-6, and IFN-β secretions were analyzed after stimulation by TLR1–9 ligands from 17 non-infectious asthmatic children. TNF-α production was higher in TLR1/2 (median 385.4 vs. 250.3 pg/ml in 1 μg/ml Pam3CSK4, p = 0.0078), TLR4 (2392.4 vs. 1355.9 in 1 μg/ml LPS; p = 0.0005), and TLR7/8 (10,776.2 vs. 4237.0 pg/ml in 1 μg/ml R848, p = 0.0079) of patients in exacerbation than those in convalescence and healthy controls despite equal TLR expression. TNF-α production stimulated by TLR9 agonist was significantly lower in exacerbation (17.7 vs. 34.9 pg/ml in 1 μg/ml ODN2216, p = 0.0175), while IL-6 production had similar patterns but was significantly lower in TLR3 signaling (119.7 vs. 245.0 pg/ml in 0.1 μg/ml poly(I:C), p = 0.0033). IFN-β production by TLR3 agonist also decreased in exacerbation but not statistically significant. Six older children showed decreased FOXP3 percentage in CD4 + CD25^high and decreased suppression capability in exacerbation but restored in stabilization (82.8% vs. 90.0%, p = 0.0061 and 60.9% vs. 81.7%, p = 0.0071; respectively). In conclusion, normalizing imbalanced TLR signaling and enhancing Treg cell capability may guide possible therapeutic strategies for non-infectious asthma in exacerbation.     

A double blind randomized experimental study on the use of IgM-enriched polyclonal immunoglobulins in an animal model of pneumonia developing shock

BackgroundPatients with severe pneumonia often develop septic shock. IgM-enriched immunoglobulins have been proposed as a potential adjuvant therapy for septic shock. While in vitro data are available on the possible mechanisms of action of IgM-enriched immunoglobulins, the results of the in vivo experimental studies are non-univocal and, overall, unconvincing. We designed this double blinded randomized controlled study to test whether IgM-enriched immunoglobulins administered as rescue treatment in a pneumonia model developing shock, could either limit lung damage and/or contain systemic inflammatory response.MethodsThirty-eight Sprague Dawley rats were ventilated with injurious ventilation for 30 min to prime the lung. The rats were subsequently randomized to received intratracheal instillation of either lipopolysaccharide (LPS) (12 mg/kg) or placebo followed by 3.5 h of protective mechanical ventilation. IgM-enriched immunoglobulins at 25 mg/h (0.5 mL/h) or saline were intravenously administered in the last hour of mechanical ventilation. During the experiment, gas exchange and hemodynamic measurements were recorded. Thereafter, the animals were sacrificed, and blood and organs were stored for cytokines measurements.ResultsDespite similar lung and hemodynamic findings, the administration of IgM-enriched immunoglobulins compared to placebo significantly modulates the inflammatory response by increasing IL-10 levels in the bloodstream and by decreasing TNF-α in bronchoalveolar lavage (BAL) fluid. Furthermore, in vitro data suggest that IgM-enriched immunoglobulins induce monocytes production of IL-10 after LPS stimulation.ConclusionsIn an in vivo model of pneumonia developing shock, IgM-enriched immunoglobulins administered as rescue treatment enhance the anti-inflammatory response by increasing blood levels of IL-10 and reducing TNF-α in BAL fluid.     

Induced elastin regeneration by chronically activated smooth muscle cells for targeted aneurysm repair

Elastin breakdown in vascular aneurysms is mediated by cytokines such as tumor necrosis factor α (TNF-α, which induces vascular smooth muscle cell (SMC) activation and regulates their deposition of matrix. We previously demonstrated that exogenous supplementation with TGF-β1 (1 ng ml^?1) and hyaluronan oligomers (0.786 kDa, 0.2 μg ml^?1) cues the upregulation of elastin matrix synthesis by healthy cultured SMCs. Here, we determine whether these cues likewise enhance elastin matrix synthesis and assembly by TNF-α-stimulated SMCs, while restoring their healthy phenotype. Adult rat aortic SMCs were treated with TNF-α alone or together with TGF-β1/hyaluronan oligomeric cues and the release of inflammatory markers were monitored during over a 21 day culture. Biochemical analysis was used to quantify cell proliferation, matrix protein synthesis and cross-linking efficiency, while immunofluorescence and electron microscopy were used to analyze the elastin matrix quality. It was observed that SMC activation with TNF-α (10 ng ml^?1) induced matrix calcification and promoted production of elastolytic MMP-2 and MMP-9. However, these effects were attenuated by the addition of TGF-β1 and HA oligomer cues to TNF-α-stimulated cultures, which also enhanced tropoelastin and collagen production, improved elastin matrix yield and cross-linking, promoted elastin fiber formation and suppressed elastase activity, although the release of MMP-2 and MMP-9 was not affected. Overall, the results suggest that TGF-β1 and HA oligomers are potentially useful in suppressing SMC activation and inducing regenerative elastin repair within aneurysms.     

Alteration of embryonic AT2-R and inflammatory cytokines gene expression induced by prenatal exposure to lipopolysaccharide affects renal development

Prenatal exposure to LPS(lipopolysaccharide) results in renal damage in offspring rats, but the mechanism is unknown. The present study was to explore the role of angiotensin II and inflammation in the development of renal damage induced by prenatal exposure to LPS. The pregnant rats were randomly divided into two groups, i.e., control group, LPS group. The rats in the two groups were administered intraperitoneally with vehicle or 0.79 mg/kg LPS on 8th, 10th and 12th day during gestation. The mRNA expression of angiotensinogen, renin, AT₁-R, AT₂-R, TNF-α and IL-6 in embryos were assessed. Renal Ang II-positive cells, monocytes/macrophages, lymphocytes, collagen I and TUNEL-positive cells were identified by immunohistochemical staining in newborn and 7-week-old offspring rats. The number of glomeruli and creatinine clearance rate were determined in offspring at 7 weeks of age. The results showed that prenatal LPS decreased AT₂-R mRNA expression but increased TNF-α and IL-6 mRNA expression in embryos. Prenatal LPS decreased renal angiotensin II-positive cells in newborn offspring rats, while these increased in 7-week-old offspring rats. Prenatal LPS decreased glomerular number and creatinine clearance rate but increased renal infiltrating monocytes/macrophages and lymphocytes at 7 weeks of age. Prenatal LPS also increased TUNEL-positive cells and collagen I expressions in newborn rats and 7-week-old offspring rats.ConclusionAlteration of embryonic AT₂-R and inflammatory cytokines gene expression induced by prenatal exposure to lipopolysaccharide affects renal development.     

Two inhibitors of pro-inflammatory cytokine release,interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes

The biological activity of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α depends on the level of TNF-α itself, the expression of the p55 and p75 cell surface receptors for TNF on target cells and the concentrations of the natural inhibitors of TNF-α, the soluble p55 and p75 TNF receptors (TNF-R). Interleukin (IL)-10 and IL-4 are known to inhibit TNF-α production by monocytes. We, therefore, investigated the effects of IL-10 and IL-4 on the cell surface expression and release of TNF-R by human monocytes to determine whether these cytokines also indirectly modulated the biological activity of TNF-α. Exposure to IL-10 (1-10 U/ml) for 24 or 48 h increased soluble p75 TNF-R expression and concomitantly reduced surface expression of p75 TNF-R. Further, IL-l α-stimulated production of TNF-α was diminished by IL-10 and only a small proportion of this TNF-α was bioactive, consistent with increased production of inhibitory soluble TNF-R. IL-10 also induced down-regulation of surface p55 TNF-R on monocytes, and increased release of soluble p55 TNF-R. However, the expression of soluble p55 TNF-R was much lower than soluble p75 TNF-R, indicating that it contributed less importantly to neutralization of TNF-α under these conditions. Like IL-10, IL-4 supressed the release of TNF-α by monocytes. In contrast to IL-10, however, IL-4 (0.1-10 ng/ml) supressed the release of soluble p75 TNF-R from monocytes in a dose-dependent manner. Release of soluble p55 TNF-R was also supressed by IL-4. IL-10, therefore, reduces the pro-inflammatory potential of TNF in three ways: by down-regulating surface TNF-R expression whilst increasing production of soluble TNF-R and inhibiting the release of TNF-α itself. This suggests that IL-10 may be useful in the treatment of diseases where overexpression of TNF-α occurs.     

Higher TNFα responses in young males compared to females are associated with attenuation of monocyte adenylyl cyclase expression

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation.Males and females, age groups 20–30 years and 50–70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6 h, p = 0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p = 0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases.     

Maturation of human iDCs by IL-18 plus PGE2, but not by each stimulus alone,induced migration toward CCL21 and the secretion of IL-12 and IFN-γ

Dendritic cells (DCs) are potent antigen-presenting cells that initiate the primary immune response and whose functional properties in vivo depend on the maturation stimulus. We describe the functional properties of human monocyte-derived DCs after the maturation of immature DCs (iDCs) for 2 days with LPS (100 ng/ml), PGE2 (1 μg/ml), CD40L (1 μg/ml) or IL-18 (200 ng/ml) and with CD40L + PGE2 and IL-18 + PGE2 mixtures at the same concentrations as above. Neither IL-18 nor PGE2 alone stimulated IL-12 or IFN-γ secretion. When administered simultaneously to 1 × 10⁶ iDCs/ml, IL-18 + PGE2 induced the secretion of 131.4 ± 6.7 pg IL-12/ml and 355 ± 87 pg IFN-γ/ml but there was no detectable IL-10 secretion. However, PGE2 alone stimulated the secretion of 208 ± 89 pg IL-10/ml whereas IL-18 alone did not stimulate the secretion of IL-10, IL-12, TNF-α or INF-γ. When the mixture of CD40L + PGE2 was used, only migration toward CCL19 and CCL21 was induced. CD40L did not stimulate the secretion of IL-10, IL-12, TNF-α or IFN-γ and did not stimulate migration toward CCL19 or CCL21. The extent of stimulation of T cell proliferation was essentially the same for all stimuli at the concentrations given above. New properties such as IL-12 and INF-γ secretion and migration toward CCL21 emerged when a mixture of IL-18 + PGE2 was employed. These data show that when the pairs of stimuli reported here were used simultaneously their effect was not additive. This system can be used to prepare mDCs with properties useful for cell therapy and also as a model to investigate the mechanisms of cytokine secretion and cell migration.     

Life-long endurance exercise in humans: Circulating levels of inflammatory markers and leg muscle size

Human aging is associated with a loss of skeletal muscle and an increase in circulating inflammatory markers. It is unknown whether endurance training (Tr) can prevent these changes. Therefore we studied 15 old trained (O-Tr) healthy males and, for comparison, 12 old untrained (O-Un), 10 Young-Tr (Y-Tr) and 12 Young-Un (Y-Un). Quadriceps size, VO₂ peak, CRP, IL-6, TNF-α and its receptors, suPAR, lipid profile, leucocytes and glucose homeostasis were measured. Tr was associated with an improved insulin profile (p < 0.05), and lower leucocyte (p < 0.05) and triglyceride levels (p < 0.05), independent of age. Aging was associated with poorer glucose control (p < 0.05), independent of training. The age-related changes in waist circumference, VO₂ peak, cholesterol, LDL, leg muscle size, CRP and IL-6 were counteracted by physical activity (p < 0.05). A significant increase in suPAR with age was observed (p < 0.05). Most importantly, life-long endurance exercise was associated with a lower level of the inflammatory markers CRP and IL-6 (p < 0.05), and with a greater thigh muscle area (p < 0.05), compared to age-matched untrained counterparts. These findings in a limited group of individuals suggest that regular physical endurance activity may play a role in reducing some markers of systemic inflammation, even within the normal range, and in maintaining muscle mass with aging.     

Impact of TNF-α and LTα gene polymorphisms on genetic susceptibility in Indian SLE patients

The promoter polymorphisms of tumour necrosis factor-α (TNF-α) and intronic Lymphotoxin-α (LTα) have been implicated as genetic risk factors for systemic lupus erythematosus (SLE) in various ethnic groups. The aim of this study was to investigate an impact of TNF-α (?308G/A; 238G/A) and LTα (+252A/G) gene polymorphisms in disease susceptibility among Indian 200 SLE patients along with 201 healthy controls. The gene polymorphisms were studied by using direct DNA sequencing and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Serum levels were measured by multiplex assay. Allelic frequencies of TNF-α ?308A (OR = 2.3, p = 0.0001, Pc = 0.0003) and LTα +252G (OR = 2.1, p < 0.0001, Pc < 0.001) were significantly higher in SLE patients. Frequency of haplotype-AGG was found to be higher in patients than controls (OR = 12.2, p = 0.0050). Serum levels of TNF-α and LTα also were found to be significantly higher in patients showing variant alleles. TNF-α ?308G/A + A/A genotypes (p < 0.01) and LTα +252 A/G + G/G genotypes (p < 0.02) were significantly associated with renal disorders and haematological manifestations. SLE patients with ?308G/A + A/A genotypes showed higher prevalence of anti-dsDNA antibodies (OR = 3.9, p = 0.0014, Pc = 0.0098) and anti-Sm antibodies (OR = 4.1, p = 0.0002, Pc = 0.0014). The present study suggests TNF-α ?308A and LTα +252G as risk alleles for disease susceptibility associated with higher serum levels of TNF-α and LTα and concomitant discrete clinical features among Indian SLE patients.     

A similar pro/anti-inflammatory cytokine balance is present in the airways of competitive athletes and non-exercising asthmatics

Purpose

Intensive exercise modifies airway inflammation and infection susceptibility. We aimed to determine the effect of exercise on pro- and anti-inflammatory cytokine (TNF-α, IL-1ra, IL-10) and innate immunity protein (HSPA1, sCD14) levels in exhaled breath condensate (EBC) and nasal secretions of competitive athletes, non-exercising asthmatics and healthy controls (HC).

Association of cytokine gene polymorphisms with the complications of allogeneic haematopoietic stem cell transplantation

The purpose of our study was to confirm the prevalence of the association between single nucleotide polymorphisms present in genes encoding cytokines and the complications occurring after haematopoietic stem cell transplantation (HSCT). 108 recipients and 81 donors were typed for TNF-α ( ?308), TGF-β1 (codon 10, 25), IL-10 (?1082, ?819, ?592), IL-6 (?174) and INF-γ (+874). Our studies have shown a tendency toward association between the occurrence of acute form of graft versus host disease (aGVHD) and IL-6 genotype. Homozygote C/C was less likely to develop aGVHD (p = 0,09). Genotype GCC/ATA in IL-10 recipient gene alone had protective effect against the occurrence of aGVHD (p = 0,01). Furthermore, GCC/ATA protected the host against developing the disease in the clinically relevant grades (II-IV) (p = 0,03). In addition, the recipient’s T/T G/G genotype (TGF-β1) predisposed to the development of both acute (p = 0,06 – trend) and chronic (p = 0,04) GVHD and also severe aGVHD (p = 0,004). We also observed a statistically significant association between the genotype of recipient and the risk of infection – the protective function of the G/C IL-6 in the bloodstream infections (p = 0,001). Our results suggest that IL-6, IL-10 and TGF-β1 genotypes of recipient are the most associated with the risk of complications after HSCT.     

Growth and differentiation factor-5 (GDF-5) in the human intervertebral annulus cells and its modulation by IL-1ß and TNF-α in vitro

Growth and differentiation factor-5 (GDF-5) is a member of the TGF-ß superfamily which regulates cell division and differentiation. GDF-5 attracted high interest because of its role in skeletal development, especially in cartilaginous sites. Little is known, however, about the role of GFD-5 in disc cell biology. The present work demonstrated the immunohistologic presence of GDF-5 in human outer and inner annulus tissue. Microarray analysis of annulus cells showed significant upregulation of GDF-5 expression in herniated vs. non-herniated lumbar discs (2.14-fold change, p = 0.021). In vitro three-dimensional culture studies challenged human annulus cells with IL-1ß and TNF-α, two proinflammatory cytokines known to be elevated in the human degenerating disc. Exposure resulted in significant downregulation of GDF-5 during both TNF-α exposure (5.83-fold change, p = 0.044) and IL-1ß exposure (3.38-fold change, p = 0.015). In vitro findings suggest that the degenerating disc milieu, with high proinflammatory cytokine levels, may limit expression of GDF-5, resulting in limited regenerative capacity of the intact disc.