Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKβ/NF-κB signaling

Background

Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages.

Methodology/Principal Findings

Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages.

Conclusions/Significance

These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.     

Evidence for involvement of Th17 type responses in post kala azar dermal leishmaniasis (PKDL)

Background

Post kala-azar dermal leishmaniasis (PKDL), a dermal sequel of visceral leishmaniasis, caused by Leishmania donovani, constitutes an important reservoir for the parasite. Parallel functioning of counter acting immune responses (Th1/Th2) reflects a complex immunological scenario, suggesting the involvement of additional regulatory molecules in the disease pathogenesis.

Methodology/Principal Findings

In the present study, human cytokine/chemokine/receptor specific cDNA array technique was employed to identify modulations in gene expression of host immuno-determinants during PKDL, followed by evaluation of Th17 type responses by analyzing mRNA and protein expression of Th17 markers (IL-23, IL-17, RORγt) and performing functional assays using Leishmania antigen (TSLA) or recombinant (rec)IL-17. Array analysis identified key immuno-regulatory molecules including cytokines (TNF-α, IFN-γ, IL-10, IL-17), chemokines (MCP-1, MIP-1α), apoptotic molecules (FasL, TRAIL, IRF-1) and receptors (CD40, Fas). Up regulation in lesional expression of Th17 markers was observed during PKDL compared to control (IL-17 and IL-23, P = 0.0008; RORγt, P = 0.02). In follow-up samples, chemotherapy significantly down regulated expression of all markers. In addition, lesional expression of IL-17 was confirmed at protein level by Immuno-histochemistry. Further, systemic presence of Th17 responses (IL-17 and IL-23) was observed in plasma samples from PKDL patients. In functional assays, TSLA stimulated the secretion of IL-17 and IL-23 from PBMCs of PKDL patients, while recIL-17 enhanced the production of TNF-α as well as nitric oxide (NO) in PKDL compared to control (TNF-α, P = 0.0002; NO, P = 0.0013). Further, a positive correlation was evident between lesional mRNA expression of IL-17 and TNF-α during PKDL.

Conclusion/Significance

The results highlight key immune modulators in PKDL and provide evidence for the involvement of Th17 type responses in the disease pathogenesis.     

Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background

The mechanisms by which humans regulate pro- and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum.

Methodology/Principal Findings

We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-α receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-α, IL-10/interferon (IFN)-γ, IL-10/IL-6 and sTNFRII/TNF-α ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-α receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species.

Conclusions

Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.     

Effect of pregnancy on serum cytokines in SLE patients

Introduction

The aim of this study was to evaluate an extensive panel of cytokines involved in immune regulation during pregnancy in patients with systemic lupus erythematosus (SLE) and in healthy women.

Methods

A total of 47 consecutive successful pregnancies in 46 SLE patients and 56 pregnancies in 56 matched healthy subjects, as controls, were prospectively studied. Serum interleukin (IL)-1-α, IL-1-β, IL-2, IL-6, IL-8, IL-10, IL-12p70, interferon (INF)-γ and tumor necrosis factor (TNF)-α were detected in sera obtained at the first and third trimester of pregnancy by a highly sensitive, multiplexed sandwich ELISA.

Results

Medians (pg/ml) of serum levels of most helper T (Th)1-type cytokines were significantly lower in the third trimester compared with those observed in the first trimester of pregnancy in healthy women: INF-γ 2.0 vs 3.4, TNF-α 10.2 vs 11.5, IL-1-α 0.9 vs 1.1, IL-1-β 0.6 vs 1.0, IL-2 3.0 vs 3.5, and IL-12p70 4.9 vs 5.6 (P-values < 0.02 for all). By contrast, only the IL-1-α serum levels were lower in the third trimester compared with the first trimester in SLE patients (P = 0.006). IFN-γ/IL-6 and IFN-γ/IL-10 ratios were higher in controls than in SLE (P = 0.002, and P = 0.001, respectively); moreover, they were significantly reduced in the third compared to the first trimester of pregnancy in healthy women, but not in SLE.

Conclusions

In SLE patients, Th1/Th2 cytokine serum level ratio does not decrease during pregnancy progression as much as in healthy pregnant women. This could account for the observation of a low frequency of disease flares in the third trimester of gestation.     

TNF-α involvement in insulin resistance induced by experimental scorpion envenomation

Background

Scorpion venom induces systemic inflammation characterized by an increase in cytokine release and chemokine production. There have been few experimental studies assessing the effects of scorpion venom on adipose tissue function in vivo.

Methodology/Principal Findings

To study the adipose tissue inflammation (ATI) induced by Androctonus australis hector (Aah) venom and to assess possible mechanisms of ATI, mice (n = 6, aged 1 month) were injected with Aah (0.45 mg/kg), toxic fraction of Aah (FTox-G50; 0.2 mg/kg) or saline solution (control). Inflammatory responses were evaluated by ELISA and cell sorting analyses in adipose tissue 45 minutes and 24 hours after injection. Quantitative real-time PCR was used to assess the regulation of genes implicated in glucose uptake. The titers of selected inflammatory cytokines (IL-1β, IL-6 and TNF-α) were also determined in sera and in insulin target tissues. The serum concentration of IL-1β rose 45 minutes after envenomation and returned to basal level after 24 hours. The pathophysiological effects of the venom after 24 hours mainly involved M1-proinflammatory macrophage infiltration in adipose tissue combined with high titers of IL-1β, IL-6 and TNF-α. Indeed, TNF-α was strongly induced in both adipose tissue and skeletal muscle. We studied the effects of Aah venom on genes implicated in insulin-stimulated glucose uptake. Insulin induced a significant increase in the expression of the mRNAs for hexokinase 2 and phosphatidylinositol 3-kinase in both skeletal muscle and adipose tissue in control mice; this upregulation was completely abolished after 24 hours in mice envenomed with Aah or FTox-G50.

Conclusions/Significance

Our findings suggest that Aah venom induces insulin resistance by mechanisms involving TNF-α-dependent Map4k4 kinase activation in the adipose tissue.     

Elevated serum uric acid is associated with high circulating inflammatory cytokines in the population-based Colaus study

Background

The relation of serum uric acid (SUA) with systemic inflammation has been little explored in humans and results have been inconsistent. We analyzed the association between SUA and circulating levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor- α (TNF-α) and C-reactive protein (CRP).

Methods and Findings

This cross-sectional population-based study conducted in Lausanne, Switzerland, included 6085 participants aged 35 to 75 years. SUA was measured using uricase-PAP method. Plasma TNF-α, IL-1β and IL-6 were measured by a multiplexed particle-based flow cytometric assay and hs-CRP by an immunometric assay. The median levels of SUA, IL-6, TNF-α, CRP and IL-1β were 355 µmol/L, 1.46 pg/mL, 3.04 pg/mL, 1.2 mg/L and 0.34 pg/mL in men and 262 µmol/L, 1.21 pg/mL, 2.74 pg/mL, 1.3 mg/L and 0.45 pg/mL in women, respectively. SUA correlated positively with IL-6, TNF-α and CRP and negatively with IL-1β (Spearman r: 0.04, 0.07, 0.20 and 0.05 in men, and 0.09, 0.13, 0.30 and 0.07 in women, respectively, P<0.05). In multivariable analyses, SUA was associated positively with CRP (β coefficient ± SE = 0.35±0.02, P<0.001), TNF-α (0.08±0.02, P<0.001) and IL-6 (0.10±0.03, P<0.001), and negatively with IL-1β (−0.07±0.03, P = 0.027). Upon further adjustment for body mass index, these associations were substantially attenuated.

Conclusions

SUA was associated positively with IL-6, CRP and TNF-α and negatively with IL-1β, particularly in women. These results suggest that uric acid contributes to systemic inflammation in humans and are in line with experimental data showing that uric acid triggers sterile inflammation.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

Multidimensional single cell based STAT phosphorylation profiling identifies a novel biosignature for evaluation of systemic lupus erythematosus activity

Introduction

Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE), a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE.

Methods

Phospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA) patients and 13 healthy donors (HDs). A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10) were also monitored.

Results

We observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF), and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells.

Conclusions

The basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.     

Cumulative inflammatory load is associated with short leukocyte telomere length in the Health, Aging and Body Composition Study

Background

Leukocyte telomere length (LTL) is an emerging marker of biological age. Chronic inflammatory activity is commonly proposed as a promoter of biological aging in general, and of leukocyte telomere shortening in particular. In addition, senescent cells with critically short telomeres produce pro-inflammatory factors. However, in spite of the proposed causal links between inflammatory activity and LTL, there is little clinical evidence in support of their covariation and interaction.

Methodology/Principal Findings

To address this issue, we examined if individuals with high levels of the systemic inflammatory markers interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) had increased odds for short LTL. Our sample included 1,962 high-functioning adults who participated in the Health, Aging and Body Composition Study (age range: 70–79 years). Logistic regression analyses indicated that individuals with high levels of either IL-6 or TNF-α had significantly higher odds for short LTL. Furthermore, individuals with high levels of both IL-6 and TNF-α had significantly higher odds for short LTL compared with those who had neither high (OR = 0.52, CI = 0.37–0.72), only IL-6 high (OR = 0.57, CI = 0.39–0.83) or only TNF-α high (OR = 0.67, CI = 0.46–0.99), adjusting for a wide variety of established risk factors and potential confounds. In contrast, CRP was not associated with LTL.

Conclusions/Significance

Results suggest that cumulative inflammatory load, as indexed by the combination of high levels of IL-6 and TNF-α, is associated with increased odds for short LTL. In contrast, high levels of CRP were not accompanied by short LTL in this cohort of older adults. These data provide the first large-scale demonstration of links between inflammatory markers and LTL in an older population.     

Akt regulates IL-10 mediated suppression of TNFα-induced cardiomyocyte apoptosis by upregulating Stat3 phosphorylation

Background

We have already reported that TNF-α increases cardiomyocyte apoptosis and IL-10 treatment prevented these effects of TNF-α. Present study investigates the role of Akt and Jak/Stat pathway in the IL-10 modulation of TNF-α induced cardiomyocyte apoptosis.

Methodology/Principal findings

Cardiomyocytes isolated from adult Sprague Dawley rats were exposed to TNF-α (10 ng/ml), IL-10 (10 ng/ml) and TNF-α+IL-10 (ratio 1) for 4 h. Exposure to TNF-α resulted in an increase in cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay. IL-10 by itself had no effect, but it prevented TNF-α induced apoptosis. IL-10 treatment increased Akt levels within cardiomyocytes and this change was associated with an increase in Jak1 and Stat3 phosphorylation. Pre-exposure of cells to Akt inhibitor prevented IL-10 induced Stat3 phosphorylation. Furthermore, in the presence of Akt or Stat3 inhibitor, IL-10 treatment was unable to block TNF-α induced cardiomyocyte apoptosis.

Conclusion

It is suggested that IL-10 modulation of TNF-α induced cardiomyocyte apoptosis is mediated by Akt via Stat3 activation.     

A novel peptide derived from human pancreatitis-associated protein inhibits inflammation in vivo and in vitro and blocks NF-kappa B signaling pathway

Background

Pancreatitis-associated protein (PAP) is a pancreatic secretory protein belongs to the group VII of C-type lectin family. Emerging evidence suggests that PAP plays a protective effect in inflammatory diseases. In the present study, we newly identified a 16-amino-acid peptide (named PAPep) derived from C-type lectin-like domain (CTLD) of human PAP with potent anti-inflammatory activity using both in vivo and in vitro assays.

Methodology/Principal Findings

We assessed the anti-inflammatory effect of PAPep on endotoxin-induced uveitis (EIU) in rats and demonstrated that intravitreal pretreatment of PAPep concentration-dependently attenuated clinical manifestation of EIU rats, reduced protein leakage and cell infiltration into the aqueous humor (AqH), suppressed tumor necrosis factor (TNF)-α, interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein (MCP)-1 production in ocular tissues, and improved histopathologic manifestation of EIU. Furthermore, PAPep suppressed the LPS-induced mRNA expression of TNF-α and IL-6 in RAW 264.7 cells, inhibited protein expression of ICAM-1 in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) as well as U937 cells adhesion to HUVECs. Western blot analysis in ocular tissues and different cell lines revealed that the possible mechanism for this anti-inflammatory effect of PAPep may depend on its ability to inhibit the activation of NF-kB signaling pathway.

Conclusions/Significance

Our studies provide the first evidence that the sequence of PAPep is within the critically active region for the anti-inflammatory function of PAP and the peptide may be a promising candidate for the management of ocular inflammatory diseases.     

Comprehensive Analysis of Inflammatory Immune Mediators of the Intraocular Fluid Aspirated from the Foldable Capsular Vitreous Body Filled-Eyes

Purpose

To analyze the level of human inflammatory immune mediators in the intraocular fluid aspirated from foldable capsular vitreous body (FCVB) filled-eyes during FCVB removal surgery, 3 months after implantation.

Methods

8 samples of intra-FCVB fluids (n = 8) were collected from 8 FCVB filled patients in our previous FCVB exploratory clinical trial. The intra-FCVB fluids were aspirated from the FCVB filled-eyes during the FCVB removal surgeries at the third month. For the control groups, the vitreous fluids were collected from patients with idiopathic macular hole (n = 9) or rhegmatogenous retinal detachment (n = 6) during pars plana vitrectomy. A multiplex immunoassay was used to determine levels of 9 cytokines (IL-1β, IL-2, IL-6, IL-8, IL-10, IL-17, TNF-α, IFN-γ and VEGF) in these samples. The VEGF level of some intra-FCVB fluids (n = 6) were re-tested using enzyme-linked immunosorbent assay (ELISA).

Results

In the intra-FCVB fluids, 9 cytokines concentrations of most samples (n = 5) measured by Multiplex immunoassay showed low values, except for Patient 02, 06, and 09. The VEGF concentrations of some intra-FCVB fluids (n = 6) tested by ELISA were in accordance with Multiplex immunoassay results. For all eight patients (n = 8), the concentrations of IL-1β, IL-2, IL-6, TNF-α, IFN-γ and VEGF were slightly higher as compared to the idiopathic macular hole control group. While, the concentrations of IL-8, IL-10, and IL-17 were not statistically significant different compared with the idiopathic macular hole control samples. Most cytokines concentrations (IL-2, IL-6, IL-8, IL-10, IL-17, TNF-α, IFN-γ, VEGF) were not statistically significant different compared to the rhegmatogenous retinal detachment control group except IL-1β.

Conclusions

The FCVB had sufficient porosity to allow cytokines to pass through. This study first discovered that the FCVB possesses favorable permeability of proteins in the human eye.     

Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced Inflammatory Response through Multiple Mechanisms in Macrophages

Background

Apigenin is a non-toxic natural flavonoid that is abundantly present in common fruits and vegetables. It has been reported that apigenin has various beneficial health effects such as anti-inflammation and chemoprevention. Multiple studies have shown that inflammation is an important risk factor for atherosclerosis, diabetes, sepsis, various liver diseases, and other metabolic diseases. Although it has been long realized that apigenin has anti-inflammatory activities, the underlying functional mechanisms are still not fully understood.

Methodology and Principal Findings

In the present study, we examined the effect of apigenin on LPS-induced inflammatory response and further elucidated the potential underlying mechanisms in human THP-1-induced macrophages and mouse J774A.1 macrophages. By using the PrimePCR array, we were able to identify the major target genes regulated by apigenin in LPS-mediated immune response. The results indicated that apigenin significantly inhibited LPS-induced production of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α through modulating multiple intracellular signaling pathways in macrophages. Apigenin inhibited LPS-induced IL-1β production by inhibiting caspase-1 activation through the disruption of the NLRP3 inflammasome assembly. Apigenin also prevented LPS-induced IL-6 and IL-1β production by reducing the mRNA stability via inhibiting ERK1/2 activation. In addition, apigenin significantly inhibited TNF-α and IL-1β-induced activation of NF-κB.

Conclusion and Significance

Apigenin Inhibits LPS-induced Inflammatory Response through multiple mechanisms in macrophages. These results provided important scientific evidences for the potential application of apigenin as a therapeutic agent for inflammatory diseases.     

Unlike for human monocytes after LPS activation, release of TNF-α by THP-1 cells is produced by a TACE catalytically different from constitutive TACE

Background

Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α.

Methodology/Principal Findings

Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation.

Conclusions/Significance

On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.     

Hydrogen sulfide attenuated tumor necrosis factor-α-induced inflammatory signaling and dysfunction in vascular endothelial cells

Background

Hydrogen sulfide (H₂S), the third physiologically relevant gaseous molecule, is recognized increasingly as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the effects and mechanisms of sodium hydrosulfide (NaHS, a H₂S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.

Methodology and Principal Findings

Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.

Significance

H₂S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.     

Circulating and PBMC Lp-PLA2 associate differently with oxidative stress and subclinical inflammation in nonobese women (menopausal status)

Background

This study aimed to determine the association of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) activity in circulation and peripheral blood mononuclear cells (PBMCs) with inflammatory and oxidative stress markers in nonobese women and according to menopausal status. Lp-PLA₂ activity, a marker for cardiovascular risk is associated with inflammation and oxidative stress.

Methodology/Principal Findings

Eighty postmenopausal women (53.0±4.05 yr) and 96 premenopausal women (39.7±9.25 yr) participated in this study. Lp-PLA₂ activities, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β in plasma as well as in PBMCs were measured. Plasma ox-LDL was also measured. Postmenopausal women demonstrated higher circulating levels of ox-LDL and IL-6, as well as IL-6, TNF-α, and IL-1β in PBMCs, than premenopausal women. In both groups, plasma Lp-PLA₂ activity positively correlated with Lp-PLA₂ activity in PBMCs and plasma ox-LDL. In premenopausal women, Lp-PLA₂ activities in plasma and PBMCs positively correlated with IL-6, TNF-α, and IL-1β in PBMCs. In postmenopausal women, plasma ox-LDL positively correlated with PBMC cytokine production. In subgroup analysis of postmenopausal women according to plasma ox-LDL level (median level: 48.715 U/L), a significant increase in Lp-PLA₂ activity in the plasma but not the PBMCs was found in the high ox-LDL subgroup. Plasma Lp-PLA₂ activity positively correlated with unstimulated PBMC Lp-PLA₂ activity in the low ox-LDL subgroup (r = 0.627, P<0.001), whereas in the high ox-LDL circulating Lp-PLA₂ activity positively correlated with plasma ox-LDL (r = 0.390, P = 0.014) but not with Lp-PLA₂ activity in PBMCs.

Conclusions/Significance

The lack of relation between circulating Lp-PLA₂ activity and Lp-PLA₂ activity in PBMCs was found in postmenopausal women with high ox-LDL. This may indicate other sources of circulating Lp-PLA₂ activity except PBMC in postmenopausal women with high ox-LDL. We also demonstrated that circulating Lp-PLA₂ and PBMC secreted Lp-PLA₂ associate differently with markers of oxidative stress and sub clinical inflammation in nonobese women, particularly according to the menopausal states.     

Leptospiral Hemolysins Induce Proinflammatory Cytokines through Toll-Like Receptor 2-and 4-Mediated JNK and NF-κB Signaling Pathways

Background

Infection with pathogenic Leptospira species causes serious systemic inflammation in patients. Although a few leptospiral proinflammatory molecules have been identified, Leptospira likely encodes other unidentified strong inflammation stimulators. The pathogenic L. interrogans genome encodes numerous putative hemolysin genes. Since hemolysins from other bacteria can cause inflammatory reactions, we hypothesized that leptospiral hemolysins may function as proinflammatory stimulators that contribute to the strong inflammation associated with Leptospira infection.

Methodology/Principal Findings

We first used cytokine protein microarrays for systematic analysis of serum cytokine profiles in leptospirosis patients and leptospire-infected mice. We found that IL-1β, IL-6 and TNF-α were the main proinflammatory cytokines in the sera of both the patients and the mice. We then analyzed eight putative hemolysins in L. interrogans strain Lai. The results showed that five of them, Sph1, Sph2, Sph3, HlpA and TlyA were secreted and had hemolytic activity. More importantly, these five hemolysins induced the strong production of IL-1β, IL-6 and TNF-α in human and mouse macrophages (although a bit lower in the latter). Furthermore, blockade of TLR2 or TLR4 with either antibodies or inhibitors of the NF-κB or JNK signaling pathways significantly reduced the production of hemolysin-induced IL-1β, IL-6 and TNF-α. Macrophages isolated from TLR2-, TLR4-or double TLR2-and 4-deficient mice also confirmed that the leptospiral hemolysins that induce proinflammatory cytokines are both TLR2-and TLR4-dependent.

Conclusions/Significance

Our findings demonstrate that L. interrogans secretes many hemolysins that function as powerful inducers of proinflammatory cytokines through both TLR2-and TLR4-dependent JNK and NF-κB pathways.     

Long-term alterations of cytokines and growth factors expression in irradiated tissues and relation with histological severity scoring

Purpose

Beside its efficacy in cancer treatment, radiotherapy induces degeneration of healthy tissues within the irradiated area. The aim of this study was to analyze the variations of proinflammatory (IL-1α, IL-2, IL-6, TNF-α, IFN-γ), profibrotic (TGF-β1), proangiogneic (VEGF) and stem cell mobilizing (GM-CSF) cytokines and growth factors in an animal model of radiation-induced tissue degeneration.

Materials and Methods

24 rats were irradiated unilaterally on the hindlimb at a monodose of 30 Gy. Six weeks (n = 8), 6 months (n = 8) and 1 year (n = 8) after irradiation the mediators expression in skin and muscle were analyzed using Western blot and the Bio-Plex® protein array (BPA) technology. Additional histological severity for fibrosis, inflammation, vascularity and cellularity alterations scoring was defined from histology and immnunohistochemistry analyses.

Results

A significant increase of histological severity scoring was found in irradiated tissue. Skin tissues were more radio-sensitive than muscle. A high level of TGF-β1 expression was found throughout the study and a significant relation was evidenced between TGF-β1 expression and fibrosis scoring. Irradiated tissue showed a chronic inflammation (IL-2 and TNF-α significantly increased). Moreover a persistent expression of GM-CSF and VEGF was found in all irradiated tissues. The vascular score was related to TGF-β1 expression and the cellular alterations score was significantly related with the level of IL-2, VEGF and GM-CSF.

Conclusion

The results achieved in the present study underline the complexity and multiplicity of radio-induced alterations of cytokine network. It offers many perspectives of development, for the comprehension of the mechanisms of late injuries or for the histological and molecular evaluation of the mode of action and the efficacy of rehabilitation techniques.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.