Short-term roxithromycin treatment attenuates airway inflammation via MAPK/NF-κB activation in a mouse model of allergic asthma

Objective

We investigated whether roxithromycin reduces ovalbumin-specific allergic asthma symptoms in mice, and we further investigated the inhibitory mechanism of roxithromycin in ovalbumin-specific allergic asthma.

Methods

Mice were divided into five groups (n?=?10 for each): control group, roxithromycin-treated groups (5, 20 and 40?mg/kg) and ovalbumin-challenged group. We measured the recruitment of inflammatory cells into the bronchoalveolar lavage fluid (BALF) or the lung tissues by Kwik-Diff and hematoxylin and eosin (H&;E) staining, goblet cell hyperplasia by alcian blue–periodic acid–Schiff (AB-PAS) staining, airway hyperresponsiveness (AHR) by whole-body plethysmograph chamber, cytokine and immunoglobulin E (IgE) levels by ELISA, and the activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB) in the lung tissues by Western blotting.

Results

Treatment with roxithromycin resulted in fewer inflammatory cells in the BALF and peribronchial areas, and decreased AHR, goblet cell hyperplasia, IgE levels and inflammatory cytokines, as well as MAP kinases and NF-κB activation, which are increased in lung tissues of mice with ovalbumin-induced allergic asthma.

Conclusions

Our data suggest that oral administration of roxithromycin suppresses ovalbumin-induced airway inflammation and AHR by regulating the inflammatory cytokines via MAP kinases/NF-κB pathway in inflammatory cells. Based on these results, we suggest that roxithromycin may be used as a therapeutic agent for allergy-induced asthma.     

Inclusion body myositis: laser microdissection reveals differential up-regulation of IFN-γ signaling cascade in attacked versus nonattacked myofibers

Sporadic inclusion body myositis (IBM) is a muscle disease with two separate pathogenic components, degeneration and inflammation. Typically, nonnecrotic myofibers are focally surrounded and invaded by CD8(+) T cells and macrophages. Both attacked and nonattacked myofibers express high levels of human leukocyte antigen class I (HLA-I) molecules, a prerequisite for antigen presentation to CD8(+) T cells. However, only a subgroup of HLA-I(+) myofibers is attacked by immune cells. By using IHC, we classified myofibers from five patients with sporadic IBM as attacked (A(IBM)) or nonattacked (N(IBM)) and isolated the intracellular contents of myofibers separately by laser microdissection. For comparison, we isolated myofibers from control persons (H(CTRL)). The samples were analyzed by microarray hybridization and quantitative PCR. HLA-I up-regulation was observed in A(IBM) and N(IBM), whereas H(CTRL) were negative for HLA-I. In contrast, the inducible chain of the interferon (IFN) γ receptor (IFNGR2) and several IFN-γ-induced genes were up-regulated in A(IBM) compared with N(IBM) and H(CTRL) fibers. Confocal microscopy confirmed segmental IFNGR2 up-regulation on the membranes of A(IBM), which positively correlated with the number of adjacent CD8(+) T cells. Thus, the differential up-regulation of the IFN-γ signaling cascade observed in the attacked fibers is related to local inflammation, whereas the ubiquitous HLA-I expression on IBM muscle fibers does not require IFNGR expression.     

Influence of β2-adrenergic receptor polymorphisms on asthma exacerbation in children with severe asthma regularly receiving salmeterol

BackgroundNew evidence suggests that different β₂-adrenergic receptor (β2AR) polymorphisms may influence asthma control in patients receiving long-acting β₂-agonists (LABAs) as regular therapy.ObjectivesTo determine the influence of β2AR polymorphisms on asthma exacerbations in children with severe asthma from Argentina receiving inhaled corticosteroid (ICS) and LABAs regularly.MethodsNinety-seven children with severe asthma were genotyped for polymorphisms of β2AR at codons 16 and 27. The number of severe exacerbations, the time of first asthma exacerbation, and the number of hospitalizations during 12 months were assessed. Changes on pulmonary function from the beginning to the end of the study were also evaluated.ResultsThe number of overall asthma exacerbations and the proportion of children with these events were similar among β2AR genotypes at position 16 (Arg/Arg, Arg/Gly, and Gly/Gly) and at position 27 (Gln/Gln, Gln/Glu, and Glu/Glu). The time to first asthma exacerbation was similar among individuals carrying different β2AR polymorphisms. No β2AR genotype association was found in relation to the number of hospitalizations. Longitudinal analysis of forced expiratory volume in 1 second from baseline to the end of the study also showed no differences among β2AR genotypes at position 16 or 27. No association was observed among the 3 most common haplotypes (Arg/Arg-Gln/Gln, Gly/Gly-Gln/Gln, and Gly/Gly-Glu/Glu) and the number of participants with asthmatic crisis or with the overall number of exacerbations.Conclusionβ2AR polymorphisms were not associated with an increased risk of having asthma exacerbations or lung function decline in a population of Argentinian children with severe asthma receiving ICS and LABAs regularly.     

DGKα DNA vaccine relieves airway allergic inflammation in asthma model possibly via induction of T cell anergy

Induction of T cell immune tolerance is thought to be a good method for treatment of asthma. Diacylglycerol kinases alpha (DGKα), enzymes that catalyze phosphorylation of diacylglycerol to produce phosphatidic acid, could inhibit diacylglycerol (DAG)-mediated signaling following T-cell receptor engagement and prevent T cell hyperactivation, thus playing important roles in the induction of T cell anergy. In the present study, we aimed to investigate the effects of DNA vaccine encoding DGKα gene administration on allergen-induced airway allergic inflammation in the murine model of asthma. Animal models were created and plasmid containing DGKα were constructed. Cytokine production was detected after the administration of DGKα gene plasmid. Immunization of mice with alum-adsorbed ovalbumin (OVA) followed by challenged with inhalation of aerosolized OVA resulted in the development of airway allergic inflammation. Administration of DGKα gene before the aerosolized OVA challenge significantly decreased the allergic airway inflammation and eosinophil infiltration in bronchoalveolar lavage fluid (BALF). Immunization with DGKα DNA vaccine decreased OVA-specific IgE and interleukin 13 (IL-13) levels in sera, and increased the IFN-γ level in BALF. The results of the present study provide evidence for the potential utility of the administration of DGKα DNA vaccine as an approach to gene therapy for asthma.     

Nuclear factor-κB mediates the phenotype switching of airway smooth muscle cells in a murine asthma model

Airway smooth muscle cells (ASMCs) phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to airway proliferative diseases such as allergic asthma. Nuclear Factor-κB (NF-κB) has been reported as a key regulator for the occurrence and development of asthma. However, little is known regarding its role in ASM cell phenotypic modulation. To elucidate the role of NF-κB in regulating ASM cells phenotypic modulation, we investigated the effects of NF-κB on ASM cells contractile marker protein expression, and its impact on proliferation and apoptosis. We found that chronic asthma increased the activation of NF-κB in the primary murine ASM cells with a concomitant marked decrease in the expression of contractile phenotypic marker protein including smooth muscle alpha-actin (α-SMA). Additionally, we used the normal ASM cells under different processing to build the phenotype switching when we found the activation of NF-κB. Meanwhile, the expression of α-SMA in asthma was significantly increased by the NF-κB blocker. NF-κB blocker also suppressed asthma mouse ASM cell proliferation and promoted apoptosis. These findings highlight a novel role for the NF-κB in murine ASM cell phenotypic modulation and provide a potential target for therapeutic intervention for asthma.     

Synergistic effect of IFN-γ gene on LIGHT-induced apoptosis in HepG2 cells via down regulation of Bcl-2

To detect the expression of anti-apoptotic factor Bcl-2 and Survivin in transferred HepG2 cells and evaluate the synergistic effect of IFN-γ gene on LIGHT-induced apoptosis signal transduction pathways, the full-length ORF of LIGHT and IFN-γ gene were cloned into pcDNA4 and verified by DNA sequencing. After being optimized by EGFP, recombinant LIGHT and IFN-γ were transferred into the HepG2 cells mediated by a cationic liposome in vitro. The expression of LIGHT and IFN-γ was identified in the supernatants by ELISA. The HepG2 cells were divided into three groups: the control, LIGHT gene transfection alone, and simultaneous transfection of LIGHT and IFN-γ genes. The cell apoptosis and expression of Bcl-2 and Survivin in cell lysate were detected through FCM. After transfection, the apoptosis rate of HepG2 cells was increased with the prolonged time, and the apoptosis rate of LIGHT group was higher than the control group, while the LIGHT/IFN-γ group was higher than the LIGHT group P < 0.01). The expression of Bcl-2 and Survivin in LIGHT group and LIGHT/IFN-γ group decreased dramatically compared with the control group. LIGHT gene alone can result in significant inhibition of HepG2 cells proliferation. INF-γ can synergistically precede LIGHT-induced apoptotic processes through down-regulation of Bcl-2 expression, but not survivin expression.     

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer

Oncogenic activation accompanied by escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.     

Influence of a mutation in IFN-γ receptor 2 (IFNGR2) in human cells on the generation of Th17 cells in memory T cells

The T cell subsets involved in inflammatory reactions are mainly the IFN-γ secreting Th1 cells and IL17-producing Th17 cells. Although Th17 cells are primed in the thymus, there is evidence that Th17 cells can be generated from effector memory CD4⁺ T cells. Cytokines as IL-6, TGF-β, IL-21 and IL-23 involved in development of Th17 cells are well described. Here we analyzed the impact of a mutation in the IFN-γ receptor 2 (IFN-γR2) on the induction of Th17 cells. By isolation of T cells and monocytes of a patient with this mutation we could demonstrate an inhibitory role of IFN-γ signaling as IFN-γR2-deficient monocytes induce a higher percentage of IL-17⁺ cells from both healthy and IFN-γR2-deficient CD4⁺ T cells. This data confirm the interference of these two T helper subsets and points to a balance of Th1 and Th17 cells obtained by their own cytokine production and their interplay with APCs.     

The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.     

An airway epithelial iNOS–DUOX2–thyroid peroxidase metabolome drives Th1/Th2 nitrative stress in human severe asthma

Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H₂O₂. Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H₂O₂, but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H₂O₂ formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H₂O₂ to nitrogen dioxide radical (NO₂^•) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS–DUOX2–TPO–NO₂^• metabolome drives nitrative stress in HAEC and likely in SA.     

M2 polarization of tumor-associated macrophages is dependent on integrin β3 via peroxisome proliferator-activated receptor-γ up-regulation in breast cancer

Macrophages are particularly abundant and play an important role throughout the tumor progression process, namely, tumor-associated macrophages (TAM) in the tumor microenvironment. TAM can be polarized to disparate functional phenotypes, the M1 and M2 macrophages. M1-like type macrophages are defined as pro-inflammatory cells involved in killing cancer cells, while M2-like type cells can specially promote tumor growth and metastasis, tissue remodeling and immunosuppression. In this study, we first found that integrin β3 was highly expressed on the surface of TAM, both in vivo and in vitro, that displayed the M2-like characteristics. Under intervention of CYC or triptolide, the integrin β3 inhibitors, the M2 polarization of TAM could be inhibited. Moreover, in the cell model of M2 polarization, either blockade or knockout/knockdown of integrin β3 could also suppress macrophage M2 polarization, which suggested that the M2 polarization was dependent on integrin β3. Using knockdown of peroxisome proliferator-activated receptor-γ (PPARγ), an M2 regulator, we found that expression and activation of PPARγ participated in M2 polarization that was mediated by integrin β3. Finally, to verify the activity of integrin β3 inhibitors on TAM in vivo, 4T1 tumor-bearing mice were treated with CYC or triptolide; in response, the M1/M2 ratio of TAM was up-regulated, while the infiltration of total lymphocytes into tumor tissue was not altered. In general, our study found a connection between integrin β3 and macrophage polarization, which provides a strategy for facilitating M2 to M1 repolarization and reconstructing the tumor immune microenvironment.     

Protective effects of hyperbaric oxygen treatment against spinal cord injury in rats via toll-like receptor 2/nuclear factor-κB signaling

Spinal cord injury (SCI) is a serious medical problem with high mortality and disability rates. Hyperbaric oxygen (HBO) treatment is beneficial for neurological recovery after SCI, but the underlying mechanisms await characterization. This study examined whether HBO treatment following SCI in rats exerts a neuroprotective effect through activation of the toll-like receptor (TLR) 2/nuclear factor (NF)-κB signaling pathway. The SC of rats was injured via T10 laminectomy. Experimental animals (n = 144) were divided into four groups: sham-operated (SH), SH + HBO, SCI, and SCI + HBO. Each group was subdivided into six subgroups (n = 6 per group) that were examined at 12 h, and 1, 2, 3, 7, and 14 days post-injury. Functional recovery in the hind limb was evaluated using the Basso, Beattie, and Bresnahan (BBB) scoring system. The expression of TLR2 and NF-кB was assessed by real-time polymerase chain reaction and Western blotting, while interleukin-1 (IL)-1β and tumor necrosis factor (TNF)-α levels were measured by enzyme-linked immunosorbent assay. TLR2 and NF-кB levels and histological scores were higher in the SCI than in the SH and SH + HBO groups at various time points. HBO treatment decreased TLR2 and NF-кB expression and histological scores as well as IL-1β and TNF-α levels compared to the SCI group at early post-injury stages. In addition, BBB scores were improved in the SCI + HBO relative to the SCI group at 7 and 14 days. HBO treatment may mitigate secondary injury to the SC by inhibiting inflammatory responses induced by TLR2/NF-кB signaling, thereby promoting functional recovery and improving neurological outcome.     

Targeted inhibition of GATA-6 attenuates airway inflammation and remodeling by regulating caveolin-1 through TLR2/MyD88/NF-κB in murine model of asthma

The purpose of this study was to evaluate the effects of GATA-6 on airway inflammation and remodeling and the underlying mechanisms in a murine model of chronic asthma. Female BALB/c mice were randomly divided into four groups: phosphate-buffered saline control (PBS), ovalbumin (OVA)-induced asthma group (OVA), OVA+ siNC and OVA+ siGATA-6. In this mice model, GATA-6 expression level was significantly elevated and the expression in Caveolin-1 (Cav-1) inversely correlated with the abundance of GATA-6 in OVA-induced asthma of mice. Silencing of GATA-6 gene expression upregulated Cav-1 expression. Additionally, downregulation of GATA-6 dramatically decreased OVA-challenged inflammation, infiltration, and mucus production. Moreover, silencing of GATA-6 resulted in decreased levels of immunoglobulin E (IgE) and inflammatory mediators and reduced inflammatory cell accumulation, as well as inhibiting the expression of important mediators including matrix metalloproteinase (MMP)-2 and MMP-9, TGF-β1, and a disintegrin and metalloproteinase 8 (ADAM8) and ADAM33, which is related to airway remodeling. Further analysis confirmed that silencing of GATA-6 attenuated OVA-induced airway inflammation and remodeling through the TLR2/MyD88 and NF-κB pathway. In conclusion, these findings indicated that the downregulation of GATA-6 effectively inhibited airway inflammation and reversed airway remodeling via Cav-1, at least in part through downregulation of TLR2/MyD88/NF-κB, which suggests that GATA-6 represents a promising therapeutic strategy for human allergic asthma.     

Administration of a selective β2 adrenergic receptor antagonist exacerbates neuropathology and cognitive deficits in a mouse model of Alzheimer's disease

Currently, there are no available approaches to cure or slow down the progression of Alzheimer's disease (AD), which is characterized by the accumulation of extracellular amyloid-β (Aβ) deposits and intraneuronal tangles that comprised hyperphosphorylated tau. The β2 adrenergic receptors (β2ARs) are expressed throughout the cortex and hippocampus and play a key role in cognitive functions. Alterations in the function of these receptors have been linked to AD; however, these data remain controversial as apparent contradicting reports have been published. Given the current demographics of growing elderly population and the high likelihood of concurrent β-blocker use for other chronic conditions, more studies into the role of this receptor in AD animal models are needed. Here, we show that administration of ICI 118,551 (ICI), a selective β2AR antagonist, exacerbates cognitive deficits in a mouse model of AD, the 3xTg-AD mice. Neuropathologically, ICI increased Aβ levels and Aβ plaque burden. Concomitantly, ICI-treated 3xTg-AD mice showed an increase in tau phosphorylation and accumulation. Mechanistically, these changes were linked to an increase in amyloidogenic amyloid precursor protein processing. These results suggest that under the conditions used here, selective pharmacologic inhibition of β2ARs has detrimental effects on AD-like pathology in mice. Overall, these studies strengthen the notion that the link between β2ARs and AD is likely highly complex and suggest caution in generalizing the beneficial effects of β blockers on AD.     

Association of the IFN-γ (+874A/T) Genetic Polymorphism with Paranoid Schizophrenia in Tunisian Population

Since growing evidence suggests a significant role of chronic low-grade inflammation in the physiopathology of schizophrenia, we have hypothesized that functional genetic variant of the IFN gamma (IFN-γ; +874A/T; rs2430561) gene may be involved in the predisposition to schizophrenia. This research is based on a case–control study which aims to identify whether polymorphism of the IFN-γ gene is a risk factor for the development of schizophrenia. The RFLP-PCR genotyping of the IFN-γ gene was conducted on a Tunisian population composed of 218 patients and 162 controls. The IFN-γ (+874A/T) polymorphism analysis showed higher frequencies of minor homozygous genotype (TT) and allele (T) in all patients compared with controls (11.5 vs. 4.9%; p = 0.03, OR = 2.64 and 30.7 vs. 24.1%, p = 0.04, OR = 1.4, respectively). This correlation was confirmed for male but not for female patients. Also, the T allele was significantly more common among patients with paranoid schizophrenia when compared with controls (25.8 vs. 4.9%, p = 0.0001; OR = 6.7). Using the binary regression analysis to eliminate confounding factors as age and sex, only this last association remained significant (p = 0.03; OR = 1.76, CI = 1.05–2.93). In conclusion, our results showed a significant association between +874A/T polymorphism of IFN-γ and paranoid schizophrenia, suggesting that this single nucleotide polymorphism (SNP) or another at proximity could predispose to paranoid schizophrenia. Since the minor allele of this polymorphism was correlated with an increased expression of their product, our study validates the hypothesis of excessive pro-inflammatory cytokine in the physiopathology of paranoid schizophrenia.