Annexin-1-deficient mice exhibit spontaneous airway hyperresponsiveness and exacerbated allergen-specific antibody responses in a mouse model of asthma.

BACKGROUND: Glucocorticoids are the mainstream drugs used in the treatment and control of inflammatory diseases such as asthma. Annexin-1 (ANXA1) is an anti-inflammatory protein which has been described as an endogenous protein responsible for some anti-inflammatory glucocorticoid effects. Previous studies have identified its importance in other immune diseases such as rheumatoid arthritis and cystic fibrosis. ANXA1-deficient ((-/-)) mice are Th2 biased, and ANXA1 N-terminus peptide exhibits anti-inflammatory activity in a rat model of pulmonary inflammation. OBJECTIVE: ANXA1 protein is found in bronchoalveolar lavage fluid from asthmatics. However, the function of ANXA1 in the pathological development of allergy or asthma is unclear. Thus, in this study we intended to examine the effect of ANXA1 deficiency on allergen-specific antibody responses and airway responses to methacholine (Mch). METHODS: ANXA1(-/-) mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA. Airway resistance, lung compliance and enhanced pause (PenH) were measured in naïve, sensitized and saline or allergen-challenged wild-type (WT) and ANXA1(-/-) mice. Total and allergen-specific antibodies were measured in the serum. RESULTS: We show that allergen-specific and total IgE, IgG2a and IgG2b levels were significantly higher in ANXA1(-/-) mice. Furthermore, naïve ANXA1(-/-) mice displayed higher airway hypersensitivity to inhaled Mch, and significant differences were also observed in allergen-sensitized and allergen-challenged ANXA1(-/-) mice compared with WT mice. CONCLUSIONS: In conclusion, ANXA1(-/-) mice possess multiple features characteristic to allergic asthma, such as airway hyperresponsiveness and enhanced antibody responses, suggesting that ANXA1 plays a critical regulatory role in the development of asthma. CLINICAL RELEVANCE: We postulate that ANXA1 is an important regulatory factor in the development of allergic disease and dysregulation of its expression can lead to pathological changes which may affect disease progression.

Honeybee venom secretory phospholipase A2 induces leukotriene production but not histamine release from human basophils.

The role of basophils in an anaphylactic response is well recognized but is usually masked by mast cells, which contain similar mediators for the induction of generalized vasodilatation and laryngeal constriction. The rapid onset of systemic anaphylactic symptoms, particularly in insect stings and ingested food, suggest that basophils, a circulating pool of cells containing histamine and other potent mediators such as leukotrienes, may be more involved in systemic anaphylaxis than originally thought. We wished to examine if secretory phospholipase A2, a systemic allergen found in honey bee venom (HBV-sPLA2) may activate basophils directly leading to rapid systemic mediator release. Basophils were isolated from human blood and stimulated with increasing concentrations of HBV-sPLA2. We found that physiological concentrations of HBV-sPLA2 induce rapid leukotriene C4 production from purified human basophils within 5 min, while interleukin (IL)-4 expression and production was induced at later time-points. Histamine release was not induced, signifying that HBV-sPLA2 did not induce generalized degranulation. Surface expression of CD63, CD69 and CD11b were up-regulated following HBV-sPLA2 treatment. Stimulation of basophils with anti-immunoglobulin E (IgE) following treatment with HBV-sPLA2 did not induce more leukotriene release. To investigate the mechanism of leukotriene production, 9-12 octadecadiynioc acid, a cyclooxygenase-1 (COX-1) and 15-lipoxygenase inhibitor, was used and this abrogated leukotriene production. These results indicate that HBV-sPLA2 can directly activate human basophils in vitro to induce leukotriene production.

Influenza A virus enhances its propagation through the modulation of Annexin-A1 dependent endosomal trafficking and apoptosis.

The influenza virus infects millions of people each year and can result in severe complications. Understanding virus recognition and host responses to influenza infection will enable future development of more effective anti-viral therapies. Previous research has revealed diverse yet important roles for the annexin family of proteins in modulating the course of influenza A virus (IAV) infection. However, the role of Annexin-A1 (ANXA1) in IAV infection has not been addressed. Here, we show that ANXA1 deficient mice exhibit a survival advantage, and lower viral titers after infection. This was accompanied with enhanced inflammatory cell infiltration during IAV infection. ANXA1 expression is increased during influenza infection clinically, in vivo and in vitro. The presence of ANXA1 enhances viral replication, influences virus binding, and enhances endosomal trafficking of the virus to the nucleus. ANXA1 colocalizes with early and late endosomes near the nucleus, and enhances nuclear accumulation of viral nucleoprotein. In addition, ANXA1 enhances IAV-mediated apoptosis. Overall, our study demonstrates that ANXA1 plays an important role in influenza virus replication and propagation through various mechanisms and that we predict that the regulation of ANXA1 expression during IAV infection may be a viral strategy to enhance its infectivity.

Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis.

The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKß. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

trans-Resveratrol, an extract of red wine, inhibits human eosinophil activation and degranulation.

BACKGROUND AND PURPOSE: trans-Resveratrol, a non-flavonoid polyphenol found abundantly in red wine possesses antiproliferative and anti-inflammatory activity in various inflammatory disease conditions. However, the effect of trans-resveratrol on eosinophil activation in relation to allergy has not been investigated. EXPERIMENTAL APPROACH: Human eosinophils were isolated and purified from whole blood and incubated for 16 h with trans-resveratrol. Eosinophil chemotaxis, activation and degranulation, and apoptosis were investigated. The effect of trans-resveratrol on the inhibition of p38 and ERK1/2 activation was examined. KEY RESULTS: Treatment of human eosinophils with trans-resveratrol at concentrations <100 microM for 16 h did not induce eosinophil apoptosis. Similar results were seen after 24 h and 48 h incubations. trans-Resveratrol (<100 microM) significantly inhibited eosinophil peroxidase release after activation with IL-5 (IC(50)=2.9+/-0.9 microM) or C5a (IC(50)=3.9+/-0.5 microM) after 5 min priming with cytochalasin B (CB). Similarly, the production of leukotriene C4 after stimulation with calcium ionophore, and eosinophil chemotaxis in response to eotaxin, as well as CD11b upregulation and CD62 L shedding was also significantly reduced by trans-resveratrol, at concentrations above 5 microM. All the activators induced p38 and ERK1/2 phosphorylation maximal at 2 min of activation. trans-Resveratrol potently inhibited p38 and ERK1/2 activation after calcium ionophore and CB and C5a activation. CONCLUSIONS AND IMPLICATIONS: trans-Resveratrol is effective at inhibiting human eosinophil activation and degranulation at concentrations <100 microM, while not inducing apoptosis. This potent anti-inflammatory activity of trans-resveratrol and possibly its metabolites on eosinophils may be worth investigating for the treatment of eosinophil-related allergic diseases.