Chronic ethanol exposure decreases H3K27me3 in the Il6 promoter region of macrophages and generates persistent dysfunction on neutrophils during fungal infection.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the effects of ethanol exposure on epigenetic markers in bone marrow (BM) and their impact on inflammatory response during Aspergillus fumigatus infection. RESULTS: Chronic ethanol exposure decreased H3K27me3 enrichment in the Il6 promoter region while increased H3K4me3 enrichment in Tnf. Chimeric mice were generated by transplanting BM from mice exposed to ethanol or water. Infection of ethanol-chimeric mice culminated in higher clinical scores, although there was no effect on mortality. However, previous chronic exposure to ethanol affects persistently the inflammatory response in lung tissue, demonstrated by increased lung damage, neutrophil accumulation and IL-6, TNF and CXCL2 production in ethanol-chimeric mice, resulting in a decreased neutrophil infiltration into the alveolar space. Neutrophil killing and phagocytosis were also significantly lower. Moreover, BM derived macrophages (BMDM) from ethanol-chimeric mice stimulated with A. fumigatus conidia exhibited higher levels of TNF, CXCL2 and IL-6 release and a higher killing activity. The Il6 promoter of BMDM from ethanol-chimeric mice exhibited a reduction in H3K27me3 enrichment, a finding also observed in BM donors exposed to ethanol. CONCLUSIONS: These evidences demonstrate that prior chronic alcohol exposure of bone-marrow modify immune effector cells functions impairing the inflammatory response during A. fumigatus infection. These findings highlight the persistent impact of chronic ethanol exposure on infectious disease outcomes.

Inhibition of CXCR2 plays a pivotal role in re-sensitizing ovarian cancer to cisplatin treatment.

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients' high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.

Anti-inflammatory and antioxidant potential, in vivo toxicity, and polyphenolic composition of Eugenia selloi B.D.Jacks. (pitangatuba), a Brazilian native fruit.

Brazilian native fruits are a rich source of polyphenolic compounds that can act as anti-inflammatory and antioxidant agents. Here, we determined the polyphenolic composition, anti-inflammatory mechanism of action, antioxidant activity and systemic toxicity in Galleria mellonella larvae of Eugenia selloi B.D.Jacks. (synonym Eugenia neonitida Sobral) extract (Ese) and its polyphenol-rich fraction (F3) obtained through bioassay-guided fractionation. Phenolic compounds present in Ese and F3 were identified by LC-ESI-QTOF-MS. The anti-inflammatory activity of Ese and F3 was tested in vitro and in vivo through NF-κB activation, cytokine release and neutrophil migration assays. The samples were tested for their effects against reactive species (ROO•, O2•-, HOCl and NO•) and for their toxicity in Galleria mellonella larvae model. The presence of hydroxybenzoic acid, ellagitannins and flavonoids was identified. Ese and F3 reduced NF-κB activation, cytokine release and neutrophil migration, with F3 being three-fold more potent. Overall, F3 exhibited strong antioxidant effects against biologically relevant radicals, and neither Ese nor F3 were toxic to G. mellonella larvae. In conclusion, Ese and F3 revealed the presence of phenolic compounds that decreased the inflammatory parameters evaluated and inhibited reactive oxygen/nitrogen species. E. selloi is a novel source of bioactive compounds that may provide benefits for human health.

Acute Lung Injury in Response to Intratracheal Instillation of Lipopolysaccharide in an Animal Model of Emphysema Induced by Elastase.

The response of lungs with emphysema to an acute lung injury (ALI) remains unclear. This study compared the lung response to intratracheal instillation of lipopolysaccharide (LPS) in rats with and without emphysema. Twenty-four Wistar rats were randomized to four groups: control group (C-G), ALI group (ALI-G), emphysema group (E-G), emphysema and ALI group (E-ALI-G). Euthanasia and the following analysis were performed 24 h after ALI induction: lung histology, bronchoalveolar lavage (BAL), mRNA expression of inflammatory mediators, and blood gas measures. The histological analysis showed that animals of ALI-G (0.55 ± 0.15) and E-ALI-G (0.69 ± 0.08) had a higher ALI score compared to C-G (0.12 ± 0.04) and E-G (0.16 ± 0.04) (p < 0.05). The analysis of each component of the score demonstrated that ALI-G and E-ALI-G had greater alveolar and interstitial neutrophil infiltration, as well as greater amount of alveolar proteinaceous debris. Comparing the two groups that received LPS, there was a trend of higher ALI in the E-ALI-G, specially due to a higher neutrophil infiltration in the alveolar spaces and a higher septal thickening. Total cell count (E-G = 3.09 ± 0.83; ALI-G = 4.45 ± 1.9; E-ALI-G = 5.9 ± 2.1; C-G = 0.73 ± 0.37 × 105) and neutrophil count (E-G = 0.69 ± 0.35; ALI-G = 2.53 ± 1.09; E-ALI-G = 3.86 ± 1.4; C-G = 0.09 ± 0.07 × 105) in the BAL were higher in the groups E-G, ALI-G, and E-ALI-G when compared to C-G (p < 0.05). The IL-6, TNF-α, and CXCL2 mRNA expressions were higher in the animals that received LPS (ALI-G and E-ALI-G) compared to the C-G and E-G (p < 0.05). No statistically significant difference was observed in the BAL cellularity and in the expression of inflammatory mediators between the ALI-G and the E-ALI-G. The severity of ALI in response to intratracheal instillation of LPS did not show difference in rats with and without intratracheal-induced emphysema.

Gestational Hypothyroxinemia Imprints a Switch in the Capacity of Astrocytes and Microglial Cells of the Offspring to React in Inflammation.

Hypothyroxinemia (Hpx) is a highly frequent condition characterized by low thyroxine (T4) and normal 3,3',5'-triiodothyronine (T3) and thyroid stimulating hormone (TSH) levels in the blood. Gestational Hpx is closely related to cognitive impairment in the human offspring. In animal models gestational Hpx causes impairment at glutamatergic synapsis, spatial learning, and the susceptibility to suffer strong autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying these phenotypes are unknown. On the other hand, it has been shown that astrocytes and microglia affect the outcome of EAE. In fact, the activation of astrocytes and microglia in the central nervous system (CNS) contributes to EAE progression. Thus, in this work, the reactivity of astrocytes and microglia from rats gestated in Hpx was evaluated aiming to understand whether these cells are targets of gestational Hpx. Interestingly, microglia derived from the offspring gestated in Hpx were less reactive compared to microglia derived from offspring gestated in euthyroidism. Instead, astrocytes derived from the offspring gestated in Hpx were significantly more reactive than the astrocytes from the offspring gestated in euthyroidism. This work contributes with novel information regarding the effects of gestational Hpx over astrocytes and microglia in the offspring. It suggests that astrocyte could react strongly to an inflammatory insult inducing neuronal death in the CNS.

Emphysema induced by elastase enhances acute inflammatory pulmonary response to intraperitoneal LPS in rats.

Abnormalities in lungs caused by emphysema might alter their response to sepsis and the occurrence of acute lung injury (ALI). This study compared the extension of ALI in response to intraperitoneal lipopolysaccharide (LPS) injection in Wistar rats with and without emphysema induced by elastase. Adult male Wistar rats were randomized into four groups: control, emphysema without sepsis, normal lung with sepsis and emphysema with sepsis. Sepsis was induced, and 24 h later the rats were euthanised. The following analysis was performed: blood gas measurements, bronchoalveolar lavage (BAL), lung permeability and histology. Animals that received LPS showed significant increase in a lung injury scoring system, inflammatory cells in bronchoalveolar lavage (BAL) and IL-6, TNF-α and CXCL2 mRNA expression in lung tissue. Animals with emphysema and sepsis showed increased alveolocapillary membrane permeability, demonstrated by higher BAL/serum albumin ratio. In conclusion, the presence of emphysema induced by elastase increases the inflammatory response in the lungs to a systemic stimulus, represented in this model by the intraperitoneal injection of LPS.

Main pathways of action of Brazilian red propolis on the modulation of neutrophils migration in the inflammatory process.

BACKGROUND: Brazilian propolis is popularly used as treatment for different diseases including the ones with inflammatory origin. Brazilian red propolis chemical profile and its anti-inflammatory properties were recently described however, its mechanism of action has not been investigated yet. AIM: Elucidate Brazilian red propolis major pathways of action on the modulation of neutrophil migration during the inflammatory process. METHODS: The ethanolic extract of propolis (EEP) activity was investigated for neutrophil migration into the peritoneal cavity, intravital microscopy (rolling and adhesion of leukocytes), quantification of cytokines TNF-α, IL-1ß and chemokines CXCL1/KC, CXCL2/MIP-2, neutrophil chemotaxis induced by CXCL2/MIP-2, calcium influx and CXCR2 expression on neutrophils. RESULTS: EEP at 10mg/kg prevented neutrophil migration into peritoneal cavity (p < 0.05), reduced leukocyte rolling and adhesion on the mesenteric microcirculation (p < 0.05) and inhibited the release TNF-α, IL-1ß, CXCL1/KC and CXCL2/MIP-2 (p < 0.05). EEP at 0.01, 0.1 and 1µg/ml reduced the CXCL2/MIP-2-induced neutrophils chemotaxis (p < 0.05) without affect cell viability (p > 0.05).EEP at 1µg/ml decreased the calcium influx induced by CXCL2/MIP-2 (p<0.05). On the other hand, none of EEP concentrations tested altered CXCR2 expression by neutrophils (p>0.05). CONCLUSION: Brazilian red propolis appears as a promising anti-inflammatory natural product which mechanism seems to be by reducing leukocyte rolling and adhesion; TNF-α, IL-1ß, CXCL1/KC and CXCL2/MIP-2 release; CXCL2/MIP-2-induced chemotaxis and calcium influx.

Protective effect of quercetin on acute lung injury in rats with sepsis and its influence on ICAM-1 and MIP-2 expression.

This study aimed to explore the protective effect of quercetin on acute lung injury (ALI) in rats with sepsis and the related mechanism. Rats were administered different doses of quercetin intraperitoneally, and blood samples and lung tissue were collected at 24 h after treatment. Arterial blood gases, lung water content, protein content, and cell counts in bronchoalveolar lavage fluid (BALF) were measured. Morphological changes in lung tissue pathology were observed under a light microscope. Serum intercellular adhesion molecule (ICAM)-1 and macrophage inflammatory protein 2 (MIP-2) levels were detected and ICAM-1 and MIP-2 mRNA expression in lung tissue was determined. Compared with that in the control model group, arterial blood gases, lung water content, protein content, and cell counts in BALF improved in the high- and low-dose quercetin groups (P < 0.05), with maximal improvement observed for the high-dose quercetin (P < 0.05). Lesions on the lungs improved in the high- and low-dose quercetin groups than those in the control model group, and the high-dose quercetin group showed better improvement than the low-dose group (P < 0.05). Compared with that in the sham-operated group, both serum and lung tissue ICAM-1 and MIP-2 expression increased significantly in the model group (P < 0.05). The quercetin groups presented lower ICAM-1 and MIP-2 expression than the control model group, with the lowest expression observed in the high-dose group (P < 0.05). Quercetin may protect against ALI in rats with sepsis by inhibiting ICAM-1 and MIP-2 expression.

Pulmonary and hepatic injury after sub-chronic exposure to sublethal doses of microcystin-LR.

We had previously shown that microcystin-LR (MCLR) could induce lung and liver inflammation after acute exposure. The biological outcomes following prolonged exposure to MCLR, although more frequent, are still poorly understood. Thus, we aimed to verify whether repeated doses of MCLR could damage lung and liver and evaluate the dose-dependence of the results. Male Swiss mice received 10 intraperitoneal injections (i.p.) of distilled water (60 µL, CTRL) or different doses of MCLR (5 µg/kg, TOX5), 10 µg/kg (TOX10), 15 µg/kg (TOX15) and 20 µg/kg (TOX20) every other day. On the tenth injection respiratory mechanics (lung resistive and viscoelastic/inhomogeneous pressures, static elastance, and viscoelastic component of elastance) was measured. Lungs and liver were prepared for histology (morphometry and cellularity) and inflammatory mediators (KC and MIP-2) determination. All mechanical parameters and alveolar collapse were significantly higher in TOX5, 10, 15 and 20 than CTRL, but did not differ among them. Lung inflammatory cell content increased dose-dependently in all TOX groups in relation to CTRL, being TOX20 the largest. The production of KC was increased in lung and liver homogenates. MIP-2 increased in the liver of all TOX groups, but in lung homogenates it was significantly higher only in TOX20 group. All TOX mice livers showed steatosis, necrosis, inflammatory foci and a high degree of binucleated hepatocytes. In conclusion, sub-chronic exposure to MCLR damaged lung and liver in all doses, with a more important lung inflammation in TOX20 group.

Inflammatory Cytokines Contribute to Asbestos-Induced Injury of Mesothelial Cells.

BACKGROUND: Several diseases have been related to asbestos exposure, including the pleural tumor mesothelioma. The mechanism of pleural injury by asbestos fibers is not yet fully understood. The inflammatory response with release of mediators leading to a dysregulation of apoptosis may play a pivotal role in the pathophysiology of asbestos-induced pleural disease. OBJECTIVE: To determine whether pro-inflammatory cytokines produced by asbestos-exposed pleural mesothelial cells modify the injury induced by the asbestos. METHODS: Mouse pleural mesothelial cells (PMC) were exposed to crocidolite or chrysotile asbestos fibers (3.0 µg/cm(2)) for 4, 24, or 48 h and assessed for viability, necrosis and apoptosis, and the production of cytokines IL-1ß, IL-6 and macrophage inflammatory protein-2 (MIP-2). Cells exposed to fibers were also treated with antibodies anti-IL-1ß, anti-IL-6, anti- IL-1ß+anti-IL-6 or anti-MIP-2 or their irrelevant isotypes, and assessed for apoptosis and necrosis. Non-exposed cells and cells treated with wollastonite, an inert particle, were used as controls. RESULTS: Mesothelial cells exposed to either crocidolite or chrysotile underwent both apoptosis and necrosis and released cytokines IL-1ß, IL-6 and MIP-2. In the crocidolite group, apoptosis and the levels of all cytokines were higher than in the chrysotile group, at comparable concentrations. Neutralization of IL-1ß andIL-6, but not MIP-2, inhibited apoptosis and necrosis, especially in the cells exposed to crocidolite fibers. CONCLUSIONS: Both crocidolite and chrysotile asbestos fibers induced apoptosis and produced an acute inflammatory response characterized by elevated levels of IL-1ß, IL-6 and MIP-2 in cultured mouse PMC. IL-1ß and IL-6, but not MIP-2, were shown to contribute to asbestos-induced injury, especially in the crocidolite group.

The effect of composition, size, and solubility on acute pulmonary injury in rats following exposure to Mexico city ambient particulate matter samples.

Particulate matter (PM)-associated metals can contribute to adverse cardiopulmonary effects following exposure to air pollution. The aim of this study was to investigate how variation in the composition and size of ambient PM collected from two distinct regions in Mexico City relates to toxicity differences. Male Wistar Kyoto rats (14 wk) were intratracheally instilled with chemically characterized PM10 and PM2.5 from the north and PM10 from the south of Mexico City (3 mg/kg). Both water-soluble and acid-leachable fractions contained several metals, with levels generally higher in PM10 South. The insoluble and total, but not soluble, fractions of all PM induced pulmonary damage that was indicated by significant increases in neutrophilic inflammation, and several lung injury biomarkers including total protein, albumin, lactate dehydrogenase activity, and γ-glutamyl transferase activity 24 and 72 h postexposure. PM10 North and PM2.5 North also significantly decreased levels of the antioxidant ascorbic acid. Elevation in lung mRNA biomarkers of inflammation (tumor necrosis factor [TNF]-α and macrophage inflammatory protein [MIP]-2), oxidative stress (heme oxygenase [HO]-1, lectin-like oxidized low-density lipoprotein receptor [LOX]-1, and inducibile nitric oxide synthase [iNOS]), and thrombosis (tissue factor [TF] and plasminogen activator inhibitor [PAI]-1), as well as reduced levels of fibrinolytic protein tissue plasminogen activator (tPA), further indicated pulmonary injury following PM exposure. These responses were more pronounced with PM10 South (PM10 South > PM10 North > PM2.5 North), which contained higher levels of redox-active transition metals that may have contributed to specific differences in selected lung gene markers. These findings provide evidence that surface chemistry of the PM core and not the water-soluble fraction played an important role in regulating in vivo pulmonary toxicity responses to Mexico City PM.

Low-level laser therapy associated to N-acetylcysteine lowers macrophage inflammatory protein-2 (MIP-2) mRNA expression and generation of intracellular reactive oxygen species in alveolar macrophages.

OBJECTIVE: The aim of this work was to investigate the low-level laser therapy (LLLT) effect on alveolar macrophages (AM) activated by oxidative stress and lipopolysaccharide (LPS). BACKGROUND DATA: LLLT has been reported to actuate positively relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased MIP-2 mRNA expression and intracellular reactive oxygen species (ROS) generation observed in acute lung inflammation (ALI) can be influenced by LLLT. MATERIALS AND METHODS: Rat AM cell line (AMJ2-C11) was cultured with LPS or H(2)O(2) and laser irradiated. MIP-2 mRNA and ROS production in the AM were evaluated by Real Time-PCR and the 2',7'-dichlorofluorescin diacetate (DCFH-DA) respectively. The NF-κB protein in the AM was measured by the enzyme linked immunoassay method. To investigate the antioxidant effect of laser, the AM were prebathed with N-acetylcysteine (NAC) and then irradiated with laser. LLLT was also studied in the presence of an inhibitor of NF-κB (BMS 205820). In addition, the effect of LLLT on NF-κB protein was investigated. RESULTS: LLLT attenuated the MIP-2 mRNA expression and intracellular ROS generation after LPS or H(2)O(2). When the AM were pretreated with NAC, the laser effect was potentiated. BMS 205820 suppresses the effect of LLLT on MIP-2 mRNA expression and ROS generation, stimulated by LPS or H(2)O(2). On NF-κB transcription factor, both the LLLT and NAC reduced this protein in the AM exposed to LPS or H(2)O(2). The synergistic effect between LLLT and NAC on the reduction the NF-κB was also evidenced. CONCLUSION: Results indicate that there is a synergistic action of LLLT with NAC on MIP-2 mRNA expression from LPS- or H(2)O(2)-stimulated AM, and that both ROS intracellular generation and NF-kB signaling seem to be involved.

Low intensity laser therapy (LILT) in vivo acts on the neutrophils recruitment and chemokines/cytokines levels in a model of acute pulmonary inflammation induced by aerosol of lipopolysaccharide from Escherichia coli in rat.

It has been suggested that low intensity laser therapy (LILT) acts on pulmonary inflammation. Thus, we investigate in this work if LILT (650nm, 2.5mW, 31.2mW/cm(2), 1.3J/cm(2), laser spot size of 0.08cm(2) and irradiation time of 42s) can attenuate edema, neutrophil recruitment and inflammatory mediators in acute lung inflammation. Thirty-five male Wistar rats (n=7 per group) were distributed in the following experimental groups: control, laser, LPS, LPS+laser and dexamethasone+LPS. Airway inflammation was measured 4h post-LPS challenge. Pulmonary microvascular leakage was used for measuring pulmonary edema. Bronchoalveolar lavage fluid (BALF) cellularity and myeloperoxidase (MPO) were used for measuring neutrophil recruitment and activation. RT-PCR was performed in lung tissue to assess mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin (IL-10), cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage inflammatory protein-2 (MIP-2) and intercellular adhesion molecule-1 (ICAM-1). Protein levels in both BALF and lung were determined by ELISA. LILT inhibited pulmonary edema and endothelial cytoskeleton damage, as well as neutrophil influx and activation. Similarly, the LILT reduced the TNF-α and IL-1ß, in lung and BALF. LILT prevented lung ICAM-1 up-regulation. The rise of CINC-1 and MIP-2 protein levels in both lung and BALF, and the lung mRNA expressions for IL-10, were unaffected. Data suggest that the LILT effect is due to the inhibition of ICAM-1 via the inhibition of TNF-α and IL-1ß.

Mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of IL-1beta, MIP-2 and LTB(4).

OBJECTIVE: In the present study, the role of macrophages and mast cells in mineral trioxide aggregate (MTA)-induced release of neutrophil chemotactic factor was investigated. STUDY DESIGN: MTA suspension (50 mg/mL) was plated over inserts on macrophages or mast cells for 90 minutes. Untreated cells served as controls. Cells were washed and cultured for 90 minutes in RPMI without the stimuli. Macrophages and mast cell supernatants were injected intraperitoneally (0.5 mL/cavity), and neutrophil migration was assessed 6 hours later. In some experiments, cells were incubated for 30 minutes with dexamethasone (DEX, 10 muM/well), BWA4C (BW, 100 muM/well) or U75302 (U75, 10 muM/well). The concentration of Leukotriene B(4) (LTB(4)) in the cell-free supernatant from mast cells and macrophage culture was measured by ELISA. RESULTS: Supernatants from MTA-stimulated macrophages and mast cells caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages and mast cells was significantly inhibited by DEX, BW, or U75. Macrophages and mast cells expressed mRNA for interleukin-1 (IL-1)beta and macrophage inflammatory protein-2 (MIP-2) and the pretreatment of macrophages and mast cells with DEX, BW, or U75 significantly altered IL-1beta and MIP-2 mRNA expression. LTB(4) was detected in the MTA-stimulated macrophage supernatant but not mast cells. CONCLUSIONS: MTA-induces the release of neutrophil chemotactic factor substances from macrophages and mast cells with participation of IL-1beta, MIP-2, and LTB(4).

Mucosal innate response stimulation induced by lipopolysaccharide protects against Bordetella pertussis colonization.

Non-specific enhancement of the airways innate response has been shown to impair lung infections in several models of infection such diverse as influenza A, Streptococcus pneumoniae, and Aspergillus niger. Our aim was to evaluate whether a similar event could operate in the context of Bordetella pertussis respiratory infection, not only to enrich the knowledge of host-bacteria interaction but also to establish immunological basis for the development of new control strategies against the pathogen. Using a B. pertussis intranasal infection model and coadministration of different TLR agonists at the moment of the infection, we observed that the enhancement of innate response activation, in a TLR4-dependent way, could efficiently impair B. pertussis colonization (P < 0.001). While LPS from different microbial sources were equally effective in promoting this effect, flagellin and poly I:C coadministration, in spite of inducing expression of innate response markers TNFalpha, CXCL2, CXCL10 and IL6, was not effective to prevent B. pertussis colonization. Our results indicate that during the early stage of infection, specific anti-microbial mechanisms triggered by TLR4 stimulation are able to impair B. pertussis colonization. These findings could complement our current view of the role of TLR4-dependent processes that contribute to anti-pertussis immunity.