Autophagy induces eosinophil extracellular traps formation and allergic airway inflammation in a murine asthma model

Studies have shown autophagy participation in the immunopathology of inflammatory diseases. However, autophagy role in asthma and in eosinophil extracellular traps (EETs) release is poorly understood. Here, we attempted to investigate the autophagy involvement in EETs release and in lung inflammation in an experimental asthma model. Mice were sensitized with ovalbumin (OVA), followed by OVA challenge. Before the challenge with OVA, mice were treated with an autophagy inhibitor, 3-methyladenine (3-MA). We showed that 3-MA treatment decreases the number of eosinophils, eosinophil peroxidase (EPO) activity, goblet cells hyperplasia, proinflammatory cytokines, and nuclear factor kappa B (NFκB) p65 immunocontent in the lung. Moreover, 3-MA was able to improve oxidative stress, mitochondrial energy metabolism, and Na⁺, K⁺-ATPase activity. We demonstrated that treatment with autophagy inhibitor 3-MA reduced EETs formation in the airway. On the basis of our results, 3-MA treatment can be an interesting alternative for reducing lung inflammation, oxidative stress, mitochondrial damage, and EETs formation in asthma.     

Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits

The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.     

Severe vitamin E deficiency modulates airway allergic inflammatory responses in the murine asthma model

Allergic asthma is a complex immunologically mediated disease associated with increased oxidative stress and altered antioxidant defenses. It was hypothesized that alpha-tocopherol (alpha-T) decreases oxidative stress and therefore its absence may influence allergic inflammatory process, a pathobiology known to be accompanied by oxidative stress. Therefore, selected parameters of allergic asthma sensitization and inflammation were evaluated following ovalbumin sensitization and re-challenge of alpha-T transfer protein (TTP) knock-out mice (TTP(-/-)) that have greatly reduced lung alpha-T levels (e.g.<5%) compared to their litter mate controls (TTP(+/+)). Results showed that severe alpha-T deficiency result in a blunted lung expression of IL-5 mRNA and IL-5 protein and plasma IgE levels compared with TTP(+/+) mice following immune sensitization and rechallenge, although lung lavage eosinophil levels were comparable in both genomic strains. It is concluded that the initial stimulation of immune responses by the TTP(-/-) mice were generally blunted compared to the TTP(+/+) mice, thus diminishing some aspects of subsequent allergic inflammatory processes.     

Suppressive effects of CTLA4-Ig on nasal allergic reactions in presensitized murine model

Ag-specific T cell activation requires the engagement of T cell receptor (TCR) with antigen in the context of MHC, and the engagement of appropriate costimulatory molecules. It is well established that B7/CD28-CTLA4 costimulatory pathway plays an important role in the induction of T helper (Th) cells in T-cell dependent immune reactions. In this study, we evaluated the effects of blocking the costimulatory pathway by systemic administration of CTLA4-Ig during repeated nasal antigen challenges in systemically presensitized mouse. The antigen-induced early phase nasal symptoms, nasal hyperresponsiveness to histamine and nasal eosinophilia were significantly suppressed by CTLA4-Ig treatment. Elevation of serum level of antigen-specific IgE, but not IgG1 or IgG2a was inhibited by the treatment. In relation to cytokine levels in the tissue extracts of the nasal mucosa, an up-regulation of IL-4 was significantly inhibited, however, the levels of IL-5 and IFN-gamma were not affected by the treatment. These results suggest that B7/CD28-CTLA4 costimulatory pathway plays an important role in on-going Th2-related allergic reactions in the nose.     

Bromelain exerts anti-inflammatory effects in an ovalbumin-induced murine model of allergic airway disease

OBJECTIVE: Bromelain, a clinically used pineapple extract and natural product, has reported anti-inflammatory and immunomodulatory activities. The purpose of this study was to determine the effect of bromelain treatment in an ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). METHODS: To establish AAD, mice were sensitized with intraperitoneal (i.p.) OVA/alum and challenged with daily OVA aerosols. Mice were treated i.p. with either saline, 2 or 6 mg/kg bromelain, twice daily for four consecutive days. Bronchoalveolar lavage leukocytes and cytokines, lung histology, airway hyperresponsiveness, and lymphocyte populations via flow cytometry were compared between groups. RESULTS: Bromelain treatment of AAD mice resulted in reduced total BAL leukocytes, eosinophils, CD4+ and CD8+ T lymphocytes, CD4+/CD8+ T cell ratio, and IL-13. CONCLUSION: Bromelain attenuated development of AAD while altering CD4+ to CD8+ T lymphocyte populations. The reduction in AAD outcomes suggests that bromelain may have similar effects in the treatment of human asthma and hypersensitivity disorders.     

Potential use of tuftsin in treatment of candida peritonitis in a murine model

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis caused by Candida albicans. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. Tuftsin was found to increase thioglycollate-elicited mouse peritoneal macrophage activity. 2x10(-7) M tuftsin enhanced two-fold cell association with radiolabelled candida, superoxide aniom production, and killing activity. Thus, a model consisting of mice undergoing peritoneal dialysis was developed in order to study the use of tuftsin as a therapeutic drug against peritoneal candidiasis. Administration of tuftsin (50 micrograms/mouse) before candidiasis induction with a lethal dose of candida (7x10(8) candida per mouse) improved mouse survival up to 70%, compared with 10% in the control group. The potential of tuftsin as a treatment for candidiasis was shown when the infection was induced with a sublethal dose of candida. Daily intraperitoneal injections of tuftsin (50 micrograms) to the sublethally infected mice caused a significant decrease in the number of candida recovered from the peritoneal cavity and from the blood (from 700 +/- 190 to 110 +/- 26 CFU/ml and from 100 +/- 26 CFU/ml to 17 +/- 8 CFU/ml, respectively). In addition, a larger number of peritoneal macrophages with greater phagocytic and killing activity were found in the tuftsin-treated mice. The effect of tuftsin may promote its potential use in the therapy of peritonitis in patients undergoing chronic peritoneal dialysis.     

Poly(ADP-ribose) polymerase-1 inhibition prevents eosinophil recruitment by modulating Th2 cytokines in a murine model of allergic airway inflammation: a potential specific effect on IL-5

We recently used a murine model of allergic airway inflammation to show that poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in the pathogenesis of asthma-related lung inflammation. In this study, we show that PARP-1 inhibition, by a novel inhibitor (TIQ-A) or by gene deletion, prevented eosinophilic infiltration into the airways of OVA-challenged mice. Such impairment of eosinophil recruitment appeared to take place after IgE production. OVA challenge of wild-type mice resulted in a significant increase in IL-4, IL-5, IL-10, IL-13, and GM-CSF secretions. Although IL-4 production was moderately affected in OVA-challenged PARP-1(-/-) mice, the production of IL-5, IL-10, IL-13, and GM-CSF was completely inhibited in ex vivo OVA-challenged lung cells derived from these animals. A single TIQ-A injection before OVA challenge in wild-type mice mimicked the latter effects. The marked effect PARP-1 inhibition exerted on mucus production corroborated the effects observed on the Th2 response. Although PARP-1 inhibition by gene knockout increased the production of the Th1 cytokines IL-2 and IL-12, the inhibition by TIQ-A exerted no effect on these two cytokines. The failure of lung cells derived from OVA-challenged PARP-1(-/-) mice to synthesize GM-CSF, a key cytokine in eosinophil recruitment, was reestablished by replenishment of IL-5. Furthermore, intranasal administration of IL-5 restored the impairment of eosinophil recruitment and mucus production in OVA-challenged PARP-1(-/-) mice. The replenishment of either IL-4 or IgE, however, did not result in such phenotype reversals. Altogether, these results suggest that PARP-1 plays a critical role in eosinophil recruitment by specifically regulating the cascade leading to IL-5 production.     

The role of sphingosine kinase in a murine model of allergic asthma

Asthma is an allergic disease characterized by chronic airway eosinophilia and pulmonary infiltration of lymphocytes, particularly of the Th2 subtype, macrophages and mast cells. Previous studies have shown a pivotal role for sphingosine kinase (SphK) on various proinflammatory cells, such as lymphocyte and eosinophil migration and mast cell degranulation. We therefore examined the roles of SphK in a murine model of allergic asthma. In mice previously sensitized to OVA, i.p. administration of N,N-dimethylsphingosine (DMS), a potent SphK inhibitor, significantly reduced the total inflammatory cell infiltrate and eosinophilia and the IL-4, IL-5, and eotaxin levels in bronchoalveolar lavage fluid in response to inhaled OVA challenge. In addition, DMS significantly suppressed OVA-induced inflammatory infiltrates and mucus production in the lungs, and airway hyperresponsiveness to methacholine in a dose-dependent manner. OVA-induced lymphocyte proliferation and IL-4 and IL-5 secretion were reduced in thoracic lymph node cultures from DMS-treated mice. Moreover, similar reduction in inflammatory infiltrates, bronchoalveolar lavage, IL-4, IL-5, eotaxin, and serum OVA-specific IgE levels was observed in mice with SphK1 knock-down via small interfering RNA approach. Together, these data demonstrate the therapeutic potential of SphK modulation in allergic airways disease.     

In vivo intranasal anti-CD23 treatment inhibits allergic responses in a murine model of allergic rhinitis

Although CD23-dependent transcytosis of IgE and IgE-derived immune complexes across respiratory epithelial cells is likely to play a pivotal role in the initiation and development of airway allergic inflammation, there is currently a lack of physiological support for this phenomena to suggest that the targeting of CD23 could be used as a means of therapeutic intervention. The present study was designed to detect the CD23 expression in the nasal mucosa of allergic rhinitis (AR) murine model by immunohistochemistry and western blotting, and to investigate whether intranasal anti-CD23 treatment could inhibit allergen-induced upper airway inflammation in the AR model. This is the first report to show that CD23 was constitutively expressed in murine nasal epithelial cells, and its expression was significantly up-regulated in the AR murine model. In vivo, the up-regulation of CD23 expression was correlated with increased serum IL-4 levels. Following intranasal anti-CD23 treatment, nasal symptoms were alleviated and histopathologic examination showed a significant decrease in eosinophilic infiltration. Meanwhile, ELISA analysis showed levels of serum leukotriene C4 (LTC4), eosinophil cation protein (ECP), ovalbumin (OVA)-specific IgE and IL-4 also significantly decreased, as were LTC4 and OVA-specific IgE in the nasal lavage fluid. Furthermore, Western blotting analysis showed that ECP expression in the nasal mucosa was down-regulated. Finally, flow cytometric analysis revealed anti-CD23 treatment inhibited Th2 cell responses. These results indicate that intranasal anti-CD23 treatment can reduce allergic responses in a murine model of allergic rhinitis.     

Tumor necrosis factor release from lipopolysaccharide-stimulated human monocytes: Lipopolysaccharide tolerance in vitro

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.     

Polyopes affinis alleviates airway inflammation in a murine model of allergic asthma

Marine algae have been utilized in food as well as medicine products for a variety of purposes. The purpose of this study was to determine whether an ethanol extract of Polyopes affinis (P.affinis) can inhibit the pathogenesis of T helper 2 (Th2)-mediated allergen-induced airway inflammation in a murine model of asthma. Mice that were sensitized and challenged with ovalbumin (OVA) evidenced typical asthmatic reactions such as the following: an increase in the number of eosinophils in the bronchoalveolar lavage (BAL) fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways as well as the narrowing of the airway luminal; the development of airway hyperresponsiveness (AHR); the presence of pulmonary Th2 cytokines; and the presence of allergenspecific immunoglobulin E (IgE) in the serum. The successive intraperitoneal administration of P. affinis ethanolic extracts before the last airway OVA-challenge resulted in a significant inhibition of all asthmatic reactions. These data suggest that P. affinis ethanolic extracts possess therapeutic potential for the treatment of pulmonary allergic disorders such as allergic asthma.     

Tumor necrosis factors alpha and beta differ in their capacities to generate interleukin 1 release from human endothelial cells

The capacity of the tumor necrosis factors, TNF-alpha and TNF-beta, products of activated macrophages and lymphocytes, respectively, to stimulate interleukin 1 (IL-1) release from endothelial cells derived from human umbilical veins was examined in vitro. Recombinant TNF-alpha caused IL-1 release by 4 hr with maximal levels of 17 U/ml by 24 hr; half-maximal stimulation occurred at approximately 80 pM. In contrast, recombinant TNF-beta was a relatively poor stimulus for IL-1 release. Even at concentrations as high as 600 pM, only 3 U of IL-1/ml were recovered; maximal IL-1 release (10 to 12 U/ml) required up to 5 nM TNF-beta. Natural, glycosated human TNF-beta was comparable in activity to recombinant TNF-beta. TNF-beta did not directly inhibit the IL-1 comitogenesis assay, nor was there evidence that TNF-beta induced the release of an IL-1 inhibitor, in that supernatants generated in the presence of TNF-beta did not inhibit thymocyte proliferation to a recombinant IL-1 standard. Binding of the recombinant TNF to endothelial monolayers was assessed by using [125I]TNF-alpha in competition studies with cold TNF-alpha and TNF-beta. Binding of TNF-alpha was half-maximal at 80 pM with an average of 664 receptors/cell and Kd = 0.043 nM. Although TNF-beta was capable of fully competing for [125I]TNF-alpha binding, half-maximal binding occurred at 800 pM TNF-beta. These data suggest that the TNF receptors on human endothelial cells may reflect the structural differences between these two homologous cytokines.     

Beneficial lactobacilli: effects on the vaginal tract in a murine experimental model

Vaginal probiotics containing lactic acid bacteria with activity towards pathogenic microorganisms that cause urogenital tract infections have been proposed as a valid strategy for their prophylaxis and therapy. A murine experimental model was set up to evaluate the colonization capability of beneficial human lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells. Five Lactobacillus strains were intravaginally inoculated into previously estrogenized BALB/c mice. The significance of the effects observed in the vaginal tract was determined by analysis of variance using the general linear model. The numbers of viable vaginal lactobacilli were significantly higher at proestrous–estrous than those at the metaestrous–diestrous phase and decreased markedly on the days after inoculation. Lactobacilli inoculation did not cause cytological or histological modifications of the murine vaginal tract. Moreover, the intravaginal administration of Lactobacillus salivarius CRL (Centro de Referencia para Lactobacilos culture collection) 1328 and Lactobacillus gasseri CRL 1263 did not affect the amounts of granulocytes and macrophages present in vaginal washings. In conclusion, the results demonstrate that vaginal lactobacilli did not produce adverse effects on the murine vaginal tract. Therefore, they could be proposed as safe probiotic candidates to promote a balanced microbiota in the urogenital tract.     

A novel anti-inflammatory role of simvastatin in a murine model of allergic asthma

Statins, the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, are effective serum cholesterol-lowering agents in clinical practice, and they may also have anti-inflammatory properties. Asthma is characterized by chronic eosinophilic inflammation in the airways, which is thought to be regulated by the activity of T lymphocytes. We therefore examined the anti-inflammatory activity of simvastatin in a murine model of allergic asthma. In mice previously sensitized to OVA, simvastatin treatment, either orally or i.p., reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid in response to inhaled OVA challenge. Simvastatin therapy i.p. was also associated with a reduction in IL-4 and IL-5 levels in bronchoalveolar lavage fluid and, at higher doses, a histological reduction in inflammatory infiltrates in the lungs. OVA-induced IL-4, IL-5, IL-6, and IFN-gamma secretion was reduced in thoracic lymph node cultures from simvastatin-treated mice. Simvastatin treatment did not alter serum total IgE or OVA-specific IgG1 and IgG2a levels. These data demonstrate the therapeutic potential of statin-sensitive pathways in allergic airways disease.     

Regulatory role of B cells in a murine model of allergic airway disease

Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3(+) T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-beta induced conversion of CD4(+)CD25(-) T effector cells into functionally suppressive CD4(+)CD25(+)Foxp3(+) T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.     

Budesonide effects on Clara cell under normal and allergic inflammatory condition

Clara cells are nonciliated secretory cells implicated in lung homeostasis by the synthesis of immunomodulatory and host defense products, being one of the most important the CC16 protein. In this study, we compared the effects of budesonide (BUD), an inhaled corticoid, on Clara cell biology and its ability to reverse morphofunctional changes induced in an allergic airway hyper-responsiveness mouse model. In normal mice, exposure to BUD induced morphological changes compatible with a state of maximal differentiation on CC16 positive cells which developed a prominent cupola filled up with numerous mitochondria rich in CYP2E1, a member of the cytochrome P450 family. Consequently, CYP2E1 expression raised significantly. Exposure to OVA provoked hypertrophy of Clara cells and an increment in their number per millimeter of basal membrane. These cells acquired a mucous cell phenotype characterized by a notorious expansion of the secretory granular content. Synthesis of CC16 was greatly up-regulated concurrent to the finding of MUC5AC expression and the increment of epidermal growth factor receptor (EGFR). Mitochondrial content decreased significantly with a consequent reduction in CYP2E1 expression. After BUD treatment of OVA-challenged animals, the majority of Clara cells regained their normal morphology and functional characteristics; CYP2E1 levels raised when compared to the OVA exposed group. The BUD potential to differentiate Clara cells appeared to be important for the regression of the profound changes generated by the allergic injury. These results demonstrated the wide range of stimuli that can modify different aspects of Clara cell biology, and highlighted the effects of budesonide as a modulator of P450 enzymes, which probably contributes to a complementary antiinflamatory activity.     

Soluble recombinant human thrombomodulin suppresses inflammation-induced gastrointestinal tumor growth in a murine peritonitis model

Molecular and Cellular Biochemistry - In the original article, Figs.&nbsp;3b, 4a, c and 5d were published incorrectly. The correct version of the figures are provided in this correction.