Inflammatory Status in Trained and Untrained Mice at Different Pollution Levels.

Atmospheric pollution can be defined as a set of changes that occur in the composition of the air, making it unsuitable and/or harmful and thereby generating adverse effects on human health. The regular practice of physical exercise (PE) is associated with the preservation and/or improvement of health; however, it can be influenced by neuroimmunoendocrine mechanisms and external factors such as air pollution, highlighting the need for studies involving the practice of PE in polluted environments. Herein, 24 male C57BL/6 mice were evaluated, distributed into four groups (exposed to a high concentration of pollutants/sedentary, exposed to a high concentration of pollutants/exercised, exposed to ambient air/sedentary, and exposed to ambient air/exercised). The exposure to pollutants occurred in the environmental particle concentrator (CPA) and the physical training was performed on a treadmill specially designed for use within the CPA. Pro- and anti-inflammatory markers in blood and bronchoalveolar lavage (BALF), BALF cellularity, and lung tissue were evaluated. Although the active group exposed to a high concentration of pollution showed a greater inflammatory response, both the correlation analysis and the ratio between pro- and anti-inflammatory cytokines demonstrated that the exercised group presented greater anti-inflammatory activity, suggesting a protective/adaptative effect of exercise when carried out in a polluted environment.

A single dose of angiotensin-(1-7) resolves eosinophilic inflammation and protects the lungs from a secondary inflammatory challenge.

OBJECTIVE: Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator. It is not known whether the pro-resolving effects of Ang-(1-7) are sustained and protect the lung from a subsequent inflammatory challenge. This study sought to investigate the impact of treatment in face of a second allergic or lipopolysaccharide (LPS) challenge. METHODS: Mice, sensitized and challenged with ovalbumin (OVA), received a single Ang-(1-7) dose at the peak of eosinophilic inflammation, 24 h after the final OVA challenge. Subsequently, mice were euthanized at 48, 72, 96, and 120 h following the OVA challenge, and cellular infiltrate, inflammatory mediators, lung histopathology, and macrophage-mediated efferocytic activity were evaluated. The secondary inflammatory stimulus (OVA or LPS) was administered 120 h after the last OVA challenge, and subsequent inflammatory analyses were performed. RESULTS: Treatment with Ang-(1-7) resulted in elevated levels of IL-10, CD4+Foxp3+, Mres in the lungs and enhanced macrophage-mediated efferocytic capacity. Moreover, in allergic mice treated with Ang-(1-7) and then subjected to a secondary OVA challenge, inflammation was also reduced. Similarly, in mice exposed to LPS, Ang-(1-7) effectively prevented the lung inflammation. CONCLUSION: A single dose of Ang-(1-7) resolves lung inflammation and protect the lung from a subsequent inflammatory challenge highlighting its potential therapeutic for individuals with asthma.

Protective effects of Surfacen® in allergen-induced asthma mice model.

Airway obstruction with increased airway resistance in asthma, commonly caused by smooth muscle constriction, mucosal edema and fluid secretion into the airway lumen, may partly be due to a poor function of pulmonary surfactant. Surfacen®, a clinical pulmonary surfactant, has anti-inflammatory action, but its effect on asthma has not been studied. This work aimed to evaluate the effect of Surfacen® in a murine allergen-induced acute asthma model, using house dust mite allergens. In a therapeutic experimental setting, mice were first sensitized by being administered with two doses (sc) of Dermatophagoides siboney allergen in aluminum hydroxide followed by one intranasal administration of the allergen. Then, sensitized mice were administered with aerosol of hypertonic 3% NaCl, Salbutamol 0.15 mg/kg, or Surfacen® 16 mg in a whole-body chamber on days 22, 23, and 24. Further, mice were subjected to aerosol allergen challenge on day 25. Surfacen® showed bronchial dilation and inhibition of Th2 inflammation (lower levels of IL-5 and IL-13 in broncoalveolar lavage) which increased IFN-γ and unchanged IL-10 in BAL. Moreover, Sufacen® administration was associated with a marked inhibition of the serum specific IgE burst upon allergen exposure, as well as, IgG2a antibody increase, suggesting potential anti-allergy effects with inclination towards Th1. These results support also the effectiveness of the aerosol administration method to deliver the drug into lungs. Surfacen® induced a favorable pharmacological effect, with a bronchodilator outcome comparable to Salbutamol, consistent with its action as a lung surfactant, and with an advantageous anti-inflammatory and anti-allergic immunomodulatory effect.

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter.

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

On Deep Landscape Exploration of COVID-19 Patients Cells and Severity Markers.

COVID-19 is a disease with a spectrum of clinical responses ranging from moderate to critical. To study and control its effects, a large number of researchers are focused on two substantial aims. On the one hand, the discovery of diverse biomarkers to classify and potentially anticipate the disease severity of patients. These biomarkers could serve as a medical criterion to prioritize attention to those patients with higher prone to severe responses. On the other hand, understanding how the immune system orchestrates its responses in this spectrum of disease severities is a fundamental issue required to design new and optimized therapeutic strategies. In this work, using single-cell RNAseq of bronchoalveolar lavage fluid of nine patients with COVID-19 and three healthy controls, we contribute to both aspects. First, we presented computational supervised machine-learning models with high accuracy in classifying the disease severity (moderate and severe) in patients with COVID-19 starting from single-cell data from bronchoalveolar lavage fluid. Second, we identified regulatory mechanisms from the heterogeneous cell populations in the lungs microenvironment that correlated with different clinical responses. Given the results, patients with moderate COVID-19 symptoms showed an activation/inactivation profile for their analyzed cells leading to a sequential and innocuous immune response. In comparison, severe patients might be promoting cytotoxic and pro-inflammatory responses in a systemic fashion involving epithelial and immune cells without the possibility to develop viral clearance and immune memory. Consequently, we present an in-depth landscape analysis of how transcriptional factors and pathways from these heterogeneous populations can regulate their expression to promote or restrain an effective immune response directly linked to the patients prognosis.

Relevance of angiotensin-(1-7) and its receptor Mas in pneumonia caused by influenza virus and post-influenza pneumococcal infection.

Resolution failure of exacerbated inflammation triggered by Influenza A virus (IAV) prevents return of pulmonary homeostasis and survival, especially when associated with secondary pneumococcal infection. Therapeutic strategies based on pro-resolving molecules have great potential against acute inflammatory diseases. Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator that acts on its Mas receptor (MasR) to promote resolution of inflammation. We investigated the effects of Ang-(1-7) and the role of MasR in the context of primary IAV infection and secondary pneumococcal infection and evaluated pulmonary inflammation, virus titers and bacteria counts, and pulmonary damage. Therapeutic treatment with Ang-(1-7) decreased neutrophil recruitment, lung injury, viral load and morbidity after a primary IAV infection. Ang-(1-7) induced apoptosis of neutrophils and efferocytosis of these cells by alveolar macrophages, but had no direct effect on IAV replication in vitro. MasR-deficient (MasR-/-) mice were highly susceptible to IAV infection, displaying uncontrolled inflammation, increased viral load and greater lethality rate, as compared to WT animals. Ang-(1-7) was not protective in MasR-/- mice. Interestingly, Ang-(1-7) given during a sublethal dose of IAV infection greatly reduced morbidity associated with a subsequent S. pneumoniae infection, as seen by decrease in the magnitude of neutrophil influx, number of bacteria in the blood leading to a lower lethality. Altogether, these results show that Ang-(1-7) is highly protective against severe primary IAV infection and protects against secondary bacterial infection of the lung. These effects are MasR-dependent. Mediators of resolution of inflammation, such as Ang-(1-7), should be considered for the treatment of pulmonary viral infections.

Dimethyl Fumarate Attenuates Lung Inflammation and Oxidative Stress Induced by Chronic Exposure to Diesel Exhaust Particles in Mice.

Air pollution is mainly caused by burning of fossil fuels, such as diesel, and is associated with increased morbidity and mortality due to adverse health effects induced by inflammation and oxidative stress. Dimethyl fumarate (DMF) is a fumaric acid ester and acts as an antioxidant and anti-inflammatory agent. We investigated the potential therapeutic effects of DMF on pulmonary damage caused by chronic exposure to diesel exhaust particles (DEPs). Mice were challenged with DEPs (30 µg per mice) by intranasal instillation for 60 consecutive days. After the first 30 days, the animals were treated daily with 30 mg/kg of DMF by gavage for the remainder of the experimental period. We demonstrated a reduction in total inflammatory cell number in the bronchoalveolar lavage (BAL) of mice subjected to DEP + DMF as compared to those exposed to DEPs alone. Importantly, DMF treatment was able to reduce lung injury caused by DEP exposure. Intracellular total reactive oxygen species (ROS), peroxynitrite (OONO), and nitric oxide (NO) levels were significantly lower in the DEP + DMF than in the DEP group. In addition, DMF treatment reduced the protein expression of kelch-like ECH-associated protein 1 (Keap-1) in lung lysates from DEP-exposed mice, whereas total nuclear factor κB (NF-κB) p65 expression was decreased below baseline in the DEP + DMF group compared to both the control and DEP groups. Lastly, DMF markedly reduced DEP-induced expression of nitrotyrosine, glutathione peroxidase-1/2 (Gpx-1/2), and catalase in mouse lungs. In summary, DMF treatment effectively reduced lung injury, inflammation, and oxidative and nitrosative stress induced by chronic DEP exposure. Consequently, it may lead to new therapies to diminish lung injury caused by air pollutants.

4-Carvomenthenol ameliorates the murine combined allergic rhinitis and asthma syndrome by inhibiting IL-13 and mucus production via p38MAPK/NF-κB signaling pathway axis.

The aim of this study was to analyze the 4-carvomenthenol (carvo) oral treatment on the experimental model of the combined allergic rhinitis and asthma syndrome (CARAS). BALB/c mice were OVA-sensitized on day zero and 7th (50 µg/mL OVA in 10 mg/mL Al (OH)3) and OVA-challenged (5 mg/mL, 20 µL/animal) for three weeks. In the last week, the animals were dally challenged with aerosol of OVA and the carvo treatment (12.5, 25 or 50 mg/kg) occurred one hour before each OVA-challenge. Data were analyzed and p < 0.05 was considered significant. Carvo (12.5-50 mg/kg) decreased significantly the eosinophil migration into the nasal (NALF) and bronchoalveolar (BALF) cavities as well as on the nasal and lung tissues of sick animals. The treatment also decreased mucus production on both tissue sections stained with PAS (periodic acid-Schiff satin). In addition, the histological analyzes demonstrated that sick mice presented hyperplasia and hypertrophy of the lung smooth muscle layer followed by increasing of extracellular matrix and carvo (50 mg/kg) inhibited these asthmatic parameters. We analyzed the allergic rhinitis signals as nasal frictions and sneezing and observed that carvo decreased these two signals as well as serum OVA-specific IgE titer, type 2 cytokine synthesis, mainly IL-13, with increasing of IL-10 production. Decreasing of IL-13 production corroborated with decreasing of mucus production and these effects were dependent on p38MAPK/NF-κB(p65) signaling pathway inhibition. Therefore, these data demonstrated that a monoterpene of essential oils presents anti-allergic property on an experimental model of CARAS suggesting a new drug prototype to treat this allergic syndrome.

Alveolar Macrophages Are Key Players in the Modulation of the Respiratory Antiviral Immunity Induced by Orally Administered Lacticaseibacillus rhamnosus CRL1505.

The oral administration of Lacticaseibacillus rhamnosus CRL1505 differentially modulates the respiratory innate antiviral immune response triggered by Toll-like receptor 3 (TLR3) activation in infant mice, improving the resistance to Respiratory Syncytial Virus (RSV) infection. In this work, by using macrophages depletion experiments and a detailed study of their production of cytokines and antiviral factors we clearly demonstrated the key role of this immune cell population in the improvement of both viral elimination and the protection against lung tissue damage induced by the CRL1505 strain. Orally administered L. rhamnosus CRL1505 activated alveolar macrophages and enhanced their ability to produce type I interferons (IFNs) and IFN-γ in response to RSV infection. Moreover, an increased expression of IFNAR1, Mx2, OAS1, OAS2, RNAseL, and IFITM3 was observed in alveolar macrophages after the oral treatment with L. rhamnosus CRL1505, which was consistent with the enhanced RSV clearance. The depletion of alveolar macrophages by the time of L. rhamnosus CRL1505 administration abolished the ability of infant mice to produce increased levels of IL-10 in response to RSV infection. However, no improvement in IL-10 production was observed when primary cultures of alveolar macrophages obtained from CRL1505-treated mice were analyzed. Of note, alveolar macrophages from the CRL1505 group had an increased production of IL-6 and IL-27 suggesting that these cells may play an important role in limiting inflammation and protecting lung function during RSV infection, by increasing the maturation and activation of Treg cells and their subsequent production of IL-10. In addition, we provided evidence of the important role of CD4+ cells and IFN-γ in the activation of alveolar macrophages highlighting a putative pathway through which the intestinal and respiratory mucosa are communicated under the influence of L. rhamnosus CRL1505.

A role for mast cells and mast cell tryptase in driving neutrophil recruitment in LPS-induced lung inflammation via protease-activated receptor 2 in mice.

OBJECTIVE: This study aims to investigate the role of protease-activated receptor (PAR) 2 and mast cell (MC) tryptase in LPS-induced lung inflammation and neutrophil recruitment in the lungs of C57BL/6 mice. METHODS: C57BL/6 mice were pretreated with the PAR2 antagonist ENMD-1068, compound 48/80 or aprotinin prior to intranasal instillation of MC tryptase or LPS. Blood leukocytes, C-X-C motif chemokine ligand (CXCL) 1 production leukocytes recovered from bronchoalveolar lavage fluid (BALF), and histopathological analysis of the lung were evaluated 4 h later. Furthermore, we performed experiments to determine intracellular calcium signaling in RAW 264.7 cells stimulated with LPS in the presence or absence of a protease inhibitor cocktail or ENMD-1068 and evaluated PAR2 expression in the lungs of LPS-treated mice. RESULTS: Pharmacological blockade of PAR2 or inhibition of proteases reduced neutrophils recovered in BALF and LPS-induced calcium signaling. PAR2 blockade impaired LPS-induced lung inflammation, PAR2 expression in the lung and CXCL1 release in BALF, and increased circulating blood neutrophils. Intranasal instillation of MC tryptase increased the number of neutrophils recovered in BALF, and MC depletion with compound 48/80 impaired LPS-induced neutrophil migration. CONCLUSION: Our study provides, for the first time, evidence of a pivotal role for MCs and MC tryptase in neutrophil migration, lung inflammation and macrophage activation triggered by LPS, by a mechanism dependent on PAR2 activation.

Effects of VAChT reduction and α7nAChR stimulation by PNU-282987 in lung inflammation in a model of chronic allergic airway inflammation.

The cholinergic anti-inflammatory pathway has been shown to regulate lung inflammation and cytokine release in acute models of inflammation, mainly via α7 nicotinic receptor (α7nAChR). We aimed to evaluate the role of endogenous acetylcholine in chronic allergic airway inflammation in mice and the effects of therapeutic nAChR stimulation in this model. We first evaluated lung inflammation and remodeling on knock-down mice with 65% of vesicular acetylcholine transport (VAChT) gene reduction (KDVAChT) and wild-type(WT) controls that were subcutaneously sensitized and then inhaled with ovalbumin(OVA). We then evaluated the effects of PNU-282987(0.5-to-2mg/kg),(α7nAChR agonist) treatment in BALB/c male mice intraperitoneal sensitized and then inhaled with OVA. Another OVA-sensitized-group was treated with PNU-282987 plus Methyllycaconitine (MLA,1 mg/kg, α7nAChR antagonist) to confirm that the effects observed by PNU were due to α7nAChR. We showed that KDVAChT-OVA mice exhibit exacerbated airway inflammation when compared to WT-OVA mice. In BALB/c, PNU-282987 treatment reduced the number of eosinophils in the blood, BAL fluid, and around airways, and also decreased pulmonary levels of IL-4,IL-13,IL-17, and IgE in the serum of OVA-exposed mice. MLA pre-treatment abolished all the effects of PNU-282987. Additionally, we showed that PNU-282987 inhibited STAT3-phosphorylation and reduced SOCS3 expression in the lung. These data indicate that endogenous cholinergic tone is important to control allergic airway inflammation in a murine model. Moreover, α7nAChR is involved in the control of eosinophilic inflammation and airway remodeling, possibly via inhibition of STAT3/SOCS3 pathways. Together these data suggest that cholinergic anti-inflammatory system mainly α7nAChR should be further considered as a therapeutic target in asthma.

Expression of SARS-CoV-2 receptor ACE2 and coincident host response signature varies by asthma inflammatory phenotype.

BACKGROUND: More than 300 million people carry a diagnosis of asthma, with data to suggest that they are at a higher risk for infection or adverse outcomes from severe acute respiratory syndrome coronavirus 2. Asthma is remarkably heterogeneous, and it is currently unclear how patient-intrinsic factors may relate to coronavirus disease 2019. OBJECTIVE: We sought to identify and characterize subsets of patients with asthma at increased risk for severe acute respiratory syndrome coronavirus 2 infection. METHODS: Participants from 2 large asthma cohorts were stratified using clinically relevant parameters to identify factors related to angiotensin-converting enzyme-2 (ACE2) expression within bronchial epithelium. ACE-2-correlated gene signatures were used to interrogate publicly available databases to identify upstream signaling events and novel therapeutic targets. RESULTS: Stratifying by type 2 inflammatory biomarkers, we identified subjects who demonstrated low peripheral blood eosinophils accompanied by increased expression of the severe acute respiratory syndrome coronavirus 2 receptor ACE2 in bronchial epithelium. Genes highly correlated with ACE2 overlapped with type 1 and 2 IFN signatures, normally induced by viral infections. T-cell recruitment and activation within bronchoalveolar lavage cells of ACE2-high subjects was reciprocally increased. These patients demonstrated characteristics corresponding to risk factors for severe coronavirus disease 2019, including male sex, history of hypertension, low peripheral blood, and elevated bronchoalveolar lavage lymphocytes. CONCLUSIONS: ACE2 expression is linked to upregulation of viral response genes in a subset of type 2-low patients with asthma with characteristics resembling known risk factors for severe coronavirus disease 2019. Therapies targeting the IFN family and T-cell-activating factors may therefore be of benefit in a subset of patients.

Warifteine and methylwarifteine inhibited the type 2 immune response on combined allergic rhinitis and asthma syndrome (CARAS) experimental model through NF-кB pathway.

CARAS is an airway inflammation of allergic individuals, with a type 2 immune response. The pharmacotherapy is based on drugs with relevant side effects. Thus, the goal of this study evaluated the alkaloids warifteine (War) and methylwarifteine (Mwar) from Cissampelos sympodialis in CARAS experimental model. Therefore, BALB/c mice were ovalbumin (OVA) sensitized and challenged and treated with both alkaloids. Treated animals showed a decrease (p < 0.05) of allergic signs as sneezing and nasal rubbings, histamine nasal hyperreactivity, and inflammatory cell migration into the nasal (NALF) and the bronchoalveolar (BALF) fluids, main eosinophils. In the systemic context, only Mwar reduced eosinophilia, however, both alkaloids reduced the serum levels of OVA-specific IgE. Histological analysis revealed that the alkaloids decreased the inflammatory cells into the subepithelial and perivascular regions of nasal tissue and the peribronchiolar and perivascular regions of lung tissue. Hyperplasia/hypertrophy of nasal and lung goblet cells were reduced in alkaloid treated animals; however, the treatment did not change the number of mast cells. The lung hyperactivity was attenuated by reducing hyperplasia of fibroblast and collagen fiber deposition and hypertrophy of the lung smooth muscle layer. The immunomodulatory effect was by decreasing of type 2 and 3 cytokines (IL-4/IL-13/IL-5 and IL-17A) dependent by the increasing of type 1 cytokine (IFN-γ) into the BALF of treated sick animals. Indeed, both alkaloids reduced the NF-кB (p65) activation on granulocytes and lymphocytes, indicating that the alkaloids shut down the intracellular transduction signals underlie the transcription of TH2 cytokine gens.

Insulin Modulates the Immune Cell Phenotype in Pulmonary Allergic Inflammation and Increases Pulmonary Resistance in Diabetic Mice.

Introduction: Reports have shown that the onset of diabetes mellitus (DM) in patients previously diagnosed with asthma decreases asthmatic symptoms, whereas insulin aggravates asthma. The present study evaluated the modulatory effect of insulin on the development of allergic airway inflammation in diabetic mice. Materials and Methods: To evaluate the effects of relative insulin deficiency, an experimental model of diabetes was induced by a single dose of alloxan (50 mg/kg, i.v.). After 10 days, the mice were sensitized with ovalbumin [OVA, 20 µg and 2 mg of Al(OH)3, i.p.]. A booster immunization was performed 6 days after the first sensitization [20 µg of OVA and 2 mg of Al(OH)3, i.p.]. The OVA challenge (1 mg/mL) was performed by daily nebulization for 7 days. Diabetic animals were treated with multiple doses of neutral protamine Hagedorn (NPH) before each challenge with OVA. The following parameters were measured 24 h after the last challenge: (a) the levels of p38 MAP kinase, ERK 1/2 MAP kinases, JNK, STAT 3, and STAT 6 in lung homogenates; (b) the serum profiles of immunoglobulins IgE and IgG1; (c) the concentrations of cytokines (IL-4, IL-5, IL-10, IL-13, TNF-α, VEGF, TGF-ß, and IFN-γ) in lung homogenates; (d) cells recovered from the bronchoalveolar lavage fluid (BALF); (e) the profiles of immune cells in the bone marrow, lung, thymus, and spleen; and (f) pulmonary mechanics using invasive (FlexiVent) and non-invasive (BUXCO) methods. Results: Compared to non-diabetic OVA-challenged mice, OVA-challenged diabetic animals showed decreases in ERK 1 (2-fold), ERK 2 (7-fold), JNK (phosphor-54) (3-fold), JNK/SAPK (9-fold), STAT3 (4-fold), the levels of immunoglobulins, including IgE (1-fold) and IgG1 (3-fold), cytokines, including Th2 profile cytokines such as IL-4 (2-fold), IL-5 (2-fold), IL-13 (4-fold), TNF-α (2-fold), VEGF (2-fold), and TGF-ß (2-fold), inflammatory infiltrates (14-fold), T cells, NK cells, B cells and eosinophils in the bone marrow, lung, thymus and spleen, and airway hyperreactivity. STAT6 was absent, and no eosinophilia was observed in BALF. Insulin treatment restored all parameters. Conclusion: The data suggested that insulin modulates immune cell phenotypes and bronchial hyperresponsiveness in the development of allergic airway inflammation in diabetic mice.

Copaiba oil suppresses inflammation in asthmatic lungs of BALB/c mice induced with ovalbumin.

Asthma is a chronic inflammatory disease that represents high hospitalizations and deaths in world. Copaiba oil (CO) is popularly used for relieving asthma symptoms and has already been shown to be effective in many inflammation models. This study aimed to investigate the immunomodulatory relationship of CO in ovalbumin (OVA)-induced allergic asthma. The composition of CO sample analyzed by GC and GC-MS and the toxicity test was performed in mice at doses of 50 or 100 mg/kg (by gavage). After, the experimental model of allergic asthma was induced with OVA and mice were orally treated with CO in two pre-established doses. The inflammatory infiltrate was evaluated in bronchoalveolar lavage fluid (BALF), while cytokines (IL-4, IL-5, IL-17, IFN-γ, TNF-α), IgE antibody and nitric oxide (NO) production was evaluated in BALF and lung homogenate (LH) of mice, together with the histology and histomorphometry of the lung tissue. CO significantly attenuated the number of inflammatory cells in BALF, suppressing NO production and reducing the response mediated by TH2 and TH17 (T helper) cells in both BALF and LH. Histopathological and histomorphometric analysis confirmed that CO significantly reduced the numbers of inflammatory infiltrate in the lung tissue, including in the parenchyma area. Our results indicate that CO has an effective in vivo antiasthmatic effect.

Chronic exposure to diesel particles worsened emphysema and increased M2-like phenotype macrophages in a PPE-induced model.

Chronic exposure to ambient levels of air pollution induces respiratory illness exacerbation by increasing inflammatory responses and apoptotic cells in pulmonary tissues. The ineffective phagocytosis of these apoptotic cells (efferocytosis) by macrophages has been considered an important factor in these pathological mechanisms. Depending on microenvironmental stimuli, macrophages can assume different phenotypes with different functional actions. M1 macrophages are recognized by their proinflammatory activity, whereas M2 macrophages play pivotal roles in responding to microorganisms and in efferocytosis to avoid the progression of inflammatory conditions. To verify how exposure to air pollutants interferes with macrophage polarization in emphysema development, we evaluated the different macrophage phenotypes in a PPE- induced model with the exposure to diesel exhaust particles. C57BL/6 mice received intranasal instillation of porcine pancreatic elastase (PPE) to induce emphysema, and the control groups received saline. Both groups were exposed to diesel exhaust particles or filtered air for 60 days according to the groups. We observed that both the diesel and PPE groups had an increase in alveolar enlargement, collagen and elastic fibers in the parenchyma and the number of macrophages, lymphocytes and epithelial cells in BAL, and these responses were exacerbated in animals that received PPE instillation prior to exposure to diesel exhaust particles. The same response pattern was found inCaspase-3 positive cell analysis, attesting to an increase in cell apoptosis, which is in agreement with the increase in M2 phenotype markers, measured by RT-PCR and flow cytometry analysis. We did not verify differences among the groups for the M1 phenotype. In conclusion, our results showed that both chronic exposure to diesel exhaust particles and PPE instillation induced inflammatory conditions, cell apoptosis and emphysema development, as well as an increase in M2 phenotype macrophages, and the combination of these two factors exacerbated these responses. The predominance of the M2-like phenotype likely occurred due to the increased demand for efferocytosis. However, M2 macrophage activity was ineffective, resulting in emphysema development and worsening of symptoms.

Airway exposure to Staphylococcal enterotoxin type B (SEB) enhances the number and activity of bone marrow neutrophils via the release of multiple cytokines.

BACKGROUND: The lung infections by Staphylococcus aureus are strongly associated with its ability to produce enterotoxins. However, little is known about the mechanisms underlying trafficking of bone marrow (BM) neutrophils during airway inflammation induced by Staphylococcal enterotoxin B (SEB). We therefore aimed to investigate the effects of mouse airways SEB exposure on BM neutrophil counts and its adhesive properties as well as on the release of cytokines/chemokines that orchestrate BM neutrophils trafficking to lung tissue. METHODS: Male BALB/c mice were intranasally exposed to SEB (1 µg), and at 4, 16 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. BM neutrophils adhesion, MAC-1 and LFA1-α expressions (by flow cytometry) as well as measurement of cytokine and/or chemokines levels were assayed after SEB-airway exposure. RESULTS: Prior exposure to SEB promoted a marked influx of neutrophils to BAL and lung tissue, which was accompanied by increased counts of BM immature neutrophils and blood neutrophilia. BM neutrophil expressions of LFA1-α and MAC-1 were unchanged by SEB exposure whereas a significant enhancement of adhesion properties to VCAM-1 was observed. The early phase of airway SEB exposure was accompanied by high levels of GM-CSF, G-CSF, IFN-γ, TNF-α and KC/CXCL1, while the latter phase by the equilibrated actions of SDF1-α and MIP-2. CONCLUSION: Mouse airways exposure to SEB induces BM cytokines/chemokines release and their integrated actions enhance the adhesion of BM neutrophils leading to acute lung injury.

MHTP, a synthetic tetratetrahydroisoquinoline alkaloid, attenuates lipopolysaccharide-induced acute lung injury via p38MAPK/p65NF-κB signaling pathway-TLR4 dependent.

INTRODUCTION: This study investigated the mechanism of action of a synthetic tetrahydroisoquinoline alkaloid, MHTP, in an experimental model of acute lung injury (ALI) in two distinct moments: 72 h and 10 days. METHODOLOGY: To realize this study, 2.5 mg/kg of lipopolysaccharide (LPS) was intranasally administered in BALB/c mice, and nasal instillation of MHTP (1.25; 2.5; 5.0; 10 or 20 mg/kg) was administrated at 1, 24, and 48 h after LPS challenge. The data were statistically analyzed and p < 0.05 was considered statistically significant. RESULTS: MHTP treatment (2.5, 5.0, 10 or 20 mg/kg) significantly decreased neutrophil migration into the bronchoalveolar lavage fluid (BALF), tissue inflammatory cell infiltration, edema, and hemorrhage as well as collagen fiber deposition on the perialveolar regions at both moments. TNF-α and IL-6 levels were significantly diminished in the MHTP-treated animals at 72 h and maintained them, at a basal level, at 10-day observation. These effects of MHTP are due to downregulating p38MAPkinese/p65NFκB signaling pathway-TLR4 dependent. Also, the MHTP treatment promoted a survival rate at 100% and improved their body weights during the 10-day observation. Unlike, the LPS group (non-treated LPS challenged animals) presented less than 50% of surviving rate at 72 h and the animals that survived did not improve their physiological state at 10-day observation. CONCLUSIONS: These data showed for the first time the beneficial and effective activity of a nasal treatment with a synthetic tetrahydroisoquinoline alkaloid on an experimental model of ALI and pointed out the molecular mechanism related to it.

Anti-inflammatory activity of SintMed65, an N-acylhydrazone derivative, in a mouse model of allergic airway inflammation.

Asthma is a chronic, complex and heterogeneous inflammatory illness, characterized by obstruction of the lower airways. About 334 million people worldwide suffer from asthma, and these estimates, as well as the severity of the disease, have increased in the last decades. Glucocorticoids are currently the most widely used drugs in the treatment and control of asthma symptoms, but their prolonged use can cause serious adverse effects. N-acylhydrazone derivatives have been tested in pre-clinical studies in models of inflammatory diseases. Here we tested SintMed65 (N'-[(1E)-3-(4-nitrophenylhydrazono)]-(2E)-propan-2-ylidene-3,5-dinitrobenzohydrazide), a compound belonging to a novel class of immunosuppressive drugs, in a mouse model of allergic airway inflammation. BALB/c mice were sensitized previously and challenged with ovalbumin for five consecutive days and SintMed65 treatment was performed orally 1 h prior to challenge with ovalbumin. Administration of SintMed65, as well as the reference drug dexamethasone, reduced cellularity and the number of eosinophils in the bronchoalveolar fluid (BALF). SintMed65 also reduced the production of Th2 cytokines IL-4, IL-5 and IL-13 in the BALF, and IL-4, IL-10 and CCL8 gene expression in lung, compared to vehicle-treated mice. Importantly, a reduction in the number of leukocytes and in the mucus production in lungs of SintMed65-treated mice was found, compared to the vehicle-treated group. In contrast, IgE production was not significantly altered after treatment with SintMed65. Our results demonstrate that compound SintMed65 possesses anti-inflammatory characteristics, suggesting its therapeutic potential for the treatment of allergic diseases.

Urban airborne particle exposure impairs human lung and blood Mycobacterium tuberculosis immunity.

RATIONALE: Associations between urban (outdoor) airborne particulate matter (PM) exposure and TB and potential biological mechanisms are poorly explored. OBJECTIVES: To examine whether in vivo exposure to urban outdoor PM in Mexico City and in vitro exposure to urban outdoor PM2.5 (< 2.5 µm median aerodynamic diameter) alters human host immune cell responses to Mycobacterium tuberculosis. METHODS: Cellular toxicity (flow cytometry, proliferation assay (MTS assay)), M. tuberculosis and PM2.5 phagocytosis (microscopy), cytokine-producing cells (Enzyme-linked immune absorbent spot (ELISPOT)), and signalling pathway markers (western blot) were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from healthy, non-smoking, residents of Mexico City (n=35; 13 female, 22 male). In vivo-acquired PM burden in alveolar macrophages (AM) was measured by digital image analysis. MEASUREMENTS AND MAIN RESULTS: In vitro exposure of AM to PM2.5 did not affect M. tuberculosis phagocytosis. High in vivo-acquired AM PM burden reduced constitutive, M. tuberculosis and PM-induced interleukin-1ß production in freshly isolated BAC but not in autologous PBMC while it reduced constitutive production of tumour necrosis factor-alpha in both BAC and PBMC. Further, PM burden was positively correlated with constitutive, PM, M. tuberculosis and purified protein derivative (PPD)-induced interferon gamma (IFN-γ) in BAC, and negatively correlated with PPD-induced IFN-γ in PBMC. CONCLUSIONS: Inhalation exposure to urban air pollution PM impairs important components of the protective human lung and systemic immune response against M. tuberculosis. PM load in AM is correlated with altered M. tuberculosis-induced cytokine production in the lung and systemic compartments. Chronic PM exposure with high constitutive expression of proinflammatory cytokines results in relative cellular unresponsiveness.