Macrophage migration inhibitory factor promotes glucocorticoid resistance of neutrophilic inflammation in a murine model of severe asthma.

BACKGROUND: Severe neutrophilic asthma is resistant to treatment with glucocorticoids. The immunomodulatory protein macrophage migration inhibitory factor (MIF) promotes neutrophil recruitment to the lung and antagonises responses to glucocorticoids. We hypothesised that MIF promotes glucocorticoid resistance of neutrophilic inflammation in severe asthma. METHODS: We examined whether sputum MIF protein correlated with clinical and molecular characteristics of severe neutrophilic asthma in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. We also investigated whether MIF regulates neutrophilic inflammation and glucocorticoid responsiveness in a murine model of severe asthma in vivo. RESULTS: MIF protein levels positively correlated with the number of exacerbations in the previous year, sputum neutrophils and oral corticosteroid use across all U-BIOPRED subjects. Further analysis of MIF protein expression according to U-BIOPRED-defined transcriptomic-associated clusters (TACs) revealed increased MIF protein and a corresponding decrease in annexin-A1 protein in TAC2, which is most closely associated with airway neutrophilia and NLRP3 inflammasome activation. In a murine model of severe asthma, treatment with the MIF antagonist ISO-1 significantly inhibited neutrophilic inflammation and increased glucocorticoid responsiveness. Coimmunoprecipitation studies using lung tissue lysates demonstrated that MIF directly interacts with and cleaves annexin-A1, potentially reducing its biological activity. CONCLUSION: Our data suggest that MIF promotes glucocorticoid-resistance of neutrophilic inflammation by reducing the biological activity of annexin-A1, a potent glucocorticoid-regulated protein that inhibits neutrophil accumulation at sites of inflammation. This represents a previously unrecognised role for MIF in the regulation of inflammation and points to MIF as a potential therapeutic target for the management of severe neutrophilic asthma.

HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling.

Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling.

LL-37 and HMGB1 induce alveolar damage and reduce lung tissue regeneration via RAGE.

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, it is still unknown whether RAGE directly contributes to alveolar epithelial damage and abnormal repair responses. We hypothesize that RAGE activation not only induces lung tissue damage but also hampers alveolar epithelial repair responses. The effects of the RAGE ligands LL-37 and HMGB1 were examined on airway inflammation and alveolar tissue damage in wild-type and RAGE-deficient mice and on lung damage and repair responses using murine precision cut lung slices (PCLS) and organoids. In addition, their effects were studied on the repair response of human alveolar epithelial A549 cells, using siRNA knockdown of RAGE and treatment with the RAGE inhibitor FPS-ZM1. We observed that intranasal installation of LL-37 and HMGB1 induces RAGE-dependent inflammation and severe alveolar tissue damage in mice within 6 h, with stronger effects in a mouse strain susceptible for emphysema compared with a nonsusceptible strain. In PCLS, RAGE inhibition reduced the recovery from elastase-induced alveolar tissue damage. In organoids, RAGE ligands reduced the organoid-forming efficiency and epithelial differentiation into pneumocyte-organoids. Finally, in A549 cells, we confirmed the role of RAGE in impaired repair responses upon exposure to LL-37. Together, our data indicate that activation of RAGE by its ligands LL-37 and HMGB1 induces acute lung tissue damage and that this impedes alveolar epithelial repair, illustrating the therapeutic potential of RAGE inhibitors for lung tissue repair in emphysema.

RAGE and TLR4 differentially regulate airway hyperresponsiveness: Implications for COPD.

BACKGROUND: The receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) is implicated in COPD. Although these receptors share common ligands and signalling pathways, it is not known whether they act in concert to drive pathological processes in COPD. We examined the impact of RAGE and/or TLR4 gene deficiency in a mouse model of COPD and also determined whether expression of these receptors correlates with airway neutrophilia and airway hyperresponsiveness (AHR) in COPD patients. METHODS: We measured airway inflammation and AHR in wild-type, RAGE-/- , TLR4-/- and TLR4-/- RAGE-/- mice following acute exposure to cigarette smoke (CS). We also examined the impact of smoking status on AGER (encodes RAGE) and TLR4 bronchial gene expression in patients with and without COPD. Finally, we determined whether expression of these receptors correlates with airway neutrophilia and AHR in COPD patients. RESULTS: RAGE-/- mice were protected against CS-induced neutrophilia and AHR. In contrast, TLR4-/- mice were not protected against CS-induced neutrophilia and had more severe CS-induced AHR. TLR4-/- RAGE-/- mice were not protected against CS-induced neutrophilia but were partially protected against CS-induced mediator release and AHR. Current smoking was associated with significantly lower AGER and TLR4 expression irrespective of COPD status, possibly reflecting negative feedback regulation. However, consistent with preclinical findings, AGER expression correlated with higher sputum neutrophil counts and more severe AHR in COPD patients. TLR4 expression did not correlate with neutrophilic inflammation or AHR. CONCLUSIONS: Inhibition of RAGE but not TLR4 signalling may protect against airway neutrophilia and AHR in COPD.

Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis.

The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.

Development of a Soluplus budesonide freeze-dried powder for nasal drug delivery.

OBJECTIVE: The aim of this work was to develop an amorphous solid dispersions/solutions (ASD) of a poorly soluble drug, budesonide (BUD) with a novel polymer Soluplus® (BASF, Germany) using a freeze-drying technique, in order to improve dissolution and absorption through the nasal route. SIGNIFICANCE: The small volume of fluid present in the nasal cavity limits the absorption of a poorly soluble drug. Budesonide is a corticosteroid, practically insoluble and normally administered as a suspension-based nasal spray. METHODS: The formulation was prepared through freeze-drying of polymer-drug solution. The formulation was assessed for its physicochemical (specific surface area, calorimetric analysis and X-ray powder diffraction), release properties and aerodynamic properties as well as transport in vitro using RPMI 2650 nasal cells, in order to elucidate the efficacy of the Soluplus-BUD formulation. RESULTS: The freeze-dried Soluplus-BUD formulation (LYO) showed a porous structure with a specific surface area of 1.4334 ± 0.0178 m2/g. The calorimetric analysis confirmed an interaction between BUD and Soluplus and X-ray powder diffraction the amorphous status of the drug. The freeze-dried formulation (LYO) showed faster release compared to both water-based suspension and dry powder commercial products. Furthermore, a LYO formulation, bulked with calcium carbonate (LYO-Ca), showed suitable aerodynamic characteristics for nasal drug delivery. The permeation across RPMI 2650 nasal cell model was higher compared to a commercial water-based BUD suspension. CONCLUSIONS: Soluplus has been shown to be a promising polymer for the formulation of BUD amorphous solid suspension/solution. This opens up opportunities to develop new formulations of poorly soluble drug for nasal delivery.

Allergen-induced IL-6 trans-signaling activates γδ T cells to promote type 2 and type 17 airway inflammation.

BACKGROUND: A variant in the IL-6 receptor (IL-6R) gene increases asthma risk and is predicted to decrease IL-6 classic signaling and increase IL-6 trans-signaling. This suggests that inhibition of IL-6 trans-signaling, but not classic signaling, might suppress allergic airway inflammation. OBJECTIVES: We sought to determine whether IL-6 signaling contributes to (1) acute experimental asthma induced by clinically relevant allergens and (2) variation in asthma clinical phenotypes in asthmatic patients. METHODS: Mice were sensitized to house dust mite (HDM) or cockroach at day 0, treated with IL-6R inhibitors at day 13, and challenged with the same allergen at days 14 to 17. End points were measured 3 hours after the final challenge. IL-6 and soluble IL-6 receptor (sIL-6R) expression in induced sputum of asthmatic patients was correlated with asthma clinical phenotypes. RESULTS: Both HDM and cockroach induced a type 2/type 17 cytokine profile and mixed granulocytic inflammation in the airways. Both allergens increased IL-6 expression in the airways, but only cockroach induced sIL-6R expression. Therefore HDM challenge promoted IL-6 classic signaling but not trans-signaling; in this model treatment with anti-IL-6R did not suppress airway inflammation. In contrast, cockroach-induced inflammation involved activation of IL-6 trans-signaling and production of IL-17A by γδ T cells. Anti-IL-6R, selective blockade of sIL-6R, or γδ T-cell deficiency significantly attenuated cockroach-induced inflammation. Asthmatic patients with high airway IL-6 and sIL-6R levels were enriched for the neutrophilic and mixed granulocytic subtypes. CONCLUSION: Experimental asthma associated with both high IL-6 and high sIL-6R levels in the airways is attenuated by treatment with IL-6R inhibitors.

Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation.

BACKGROUND: The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization. OBJECTIVES: To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization. METHODS: TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively. RESULTS: The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1α, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation. CONCLUSION: The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention.

Development of an adaptation framework to implement a new professional pharmacy service (PPS) to a new environment.

The effective provision of professional pharmacy services is critical to support the delivery of primary health care. Structured frameworks and theoretical strategies are required to facilitate successful service implementation processes, outcomes and sustainability. This commentary discusses the considerations of what framework (adoption versus adaptation) would be suitable when implementing a new professional pharmacy service to a new environment. Utilizing Minor Ailments Services (MASs) as an exemplar as a professional pharmacy service case study, the research that underpinned these considerations enabled the development of a sequential, phased framework. There is the potential to utilize this framework for future evolving professional pharmacy services in the new setting.

RAGE and its ligand amyloid beta promote retinal ganglion cell loss following ischemia-reperfusion injury.

Introduction: Glaucoma is a progressive neurodegenerative disease associated with age. Accumulation of amyloid-beta (Aß) proteins in the ganglion cell layer (GCL) and subsequent retinal ganglion cell (RGC) loss is an established pathological hallmark of the disease. The mechanism through which Aß provokes RGC loss remains unclear. The receptor for the advanced glycation end product (RAGE), and its ligand Aß, have been shown to mediate neuronal loss via internalizing Aß within the neurons. In this study, we investigated whether the RAGE-Aß axis plays a role in RGC loss in experimental glaucoma. Methods: Retinal ischemia was induced by an acute elevation of intraocular pressure in RAGE-/- and wild-type (WT) control mice. In a subset of animals, oligomeric Aß was injected directly into the vitreous of both strains. RGC loss was assessed using histology and biochemical assays. Baseline and terminal positive scotopic threshold (pSTR) were also recorded. Results: Retinal ischemia resulted in 1.9-fold higher RGC loss in WT mice compared to RAGE-/- mice (36 ± 3% p < 0.0001 vs. 19 ± 2%, p = 0.004). Intravitreal injection of oligomeric Aß resulted in 2.3-fold greater RGC loss in WT mice compared to RAGE-/- mice, 7-days post-injection (55 ± 4% p = 0.008 vs. 24 ± 2%, p = 0.02). We also found a significant decline in the positive scotopic threshold response (pSTR) amplitude of WT mice compared to RAGE-/- (36 ± 3% vs. 16 ± 6%). Discussion: RAGE-/- mice are protected against RGC loss following retinal ischemia. Intravitreal injection of oligomeric Aß accelerated RGC loss in WT mice but not RAGE-/-. A co-localization of RAGE and Aß, suggests that RAGE-Aß binding may contribute to RGC loss.

TLR2 ligand engagement upregulates airway smooth muscle TNFα-induced cytokine production.

Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 µg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.

TGF-ß regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells.

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-ß (TGF-ß), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-ß on ASMC inflammatory responses is the induction of IL-6 release. TGF-ß also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-ß on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-ß in ASMCs is unclear. In this study, we show that TGF-ß induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-ß. Moreover, these findings suggest a potential role for NAC in preventing TGF-ß-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-ß.

Establishing consensus for the education, training and assessment requirements of community pharmacy staff to deliver minor ailments services: A modified Delphi study.

INTRODUCTION: Minor ailments services (MASs) are pharmacy-based and support individuals to manage minor conditions. MASs are delivered by community pharmacists and non-pharmacist staff. Limited information exists regarding education, training, assessment requirements, and suitability of existing processes to support MAS delivery. The purpose of this study was to determine consensus amongst multiple stakeholder participants regarding these processes. METHODS: A modified Delphi process was utilized. Phase 1 consisted of stakeholder participants completing two rounds of an online questionnaire responding to Likert-items (Round 1 [R1] [n = 46]; Round 2 [R2] [n = 34]). Phase 2 consisted of three teleconference rounds discussing items that did not achieve consensus in the previous two rounds. Consensus was defined as ≥80% panel agreement. RESULTS: Forty MASs stakeholders participated in the study. MASs stakeholder participants included community pharmacists (n = 7), pharmacy student graduate pharmacist (n = 4), non-pharmacist staff (n = 13), faculty staff/academics (n = 5), general practitioners (n = 5), and individuals affiliated with pharmacy professional organizations (n = 6). Consensus was achieved on 22 of 46 statements in R1, 8 of 34 statements in R2, and 21 of 27 statements in Phase 2. CONCLUSIONS: It may be useful for MAS education and training to consider the clinical and non-clinical elements of service delivery. Training should be available to all community pharmacy staff. The results of this study may be useful to policymakers and professional organizations to enhance existing curricula or inform training guidelines for MAS delivery.

Exploration of health professional stakeholders' views and experiences regarding minor ailments services' education, training and assessment.

Background Minor ailments services are structured pharmacy-based primary health care services that manage minor conditions. Limited training, education and assessment exists to promote the delivery of minor ailments services by pharmacy staff and it is unclear if the existing training and education processes meet professional requirements. Objective To explore the views and experiences of health professional stakeholders such as community pharmacists, intern pharmacists, medicines counter assistants and general medical practitioners with regards to minor ailments services education, training and assessment practices and preferences. Setting This study explored the views and experiences of health professional stakeholders in Australia. Method Semi-structured interviews were conducted, audio recorded, transcribed verbatim and then coded thematically using QSR Nvivo12. Main outcome measure Stakeholders' views and experiences regarding minor ailments services education, training and assessment practices and preferences. Results Twenty-eight interviews were conducted (community pharmacists n = 12; medicines counter assistants n = 4; intern pharmacists n = 9; general medical practitioners n = 3). Thematic analysis generated three themes: (1) pharmacy staff who require minor ailment service training; (2) acceptability and willingness to complete additional training; (3) learning preferences and approaches. Stakeholders reported considerations for the diverse roles in service delivery and fit for purpose tailored training. Conclusion Detailed practice guidelines may facilitate clarity of an individual staff member's role. Education and training in both clinical and non-clinical aspects of the service may be beneficial and may improve minor ailments service uptake and outcomes.

Investigating MIF in Mouse Models of Severe Corticosteroid-Resistant Neutrophilic Asthma.

Experimental mouse models of asthma are widely used to investigate the underlying mechanisms of this complex and heterogeneous disease. Using mouse models of ovalbumin-induced asthma, previous investigators have established a crucial role for MIF in the development of type 2-mediated eosinophilic asthma. Surprisingly, however, the role of MIF in other phenotypes of asthma has received little attention. MIF is an important mediator of neutrophilic inflammation, and also acts to antagonize the actions of corticosteroids. Thus, MIF may play a role in the development of severe forms of asthma in which airway neutrophilia and corticosteroid insensitivity are major features. In this chapter, we provide an experimental protocol that may be used to investigate the role of MIF in a mouse model of severe corticosteroid-resistant neutrophilic asthma.

Educational needs of community pharmacy staff in minor ailment service delivery: A systematic scoping review.

BACKGROUND: Minor ailment services (MASs) are structured, protocol driven pharmacy services established locally or nationally. Community pharmacy staff may benefit from education and training to deliver MASs. Our objective was to examine the evidence regarding training, education, and assessment requirements associated with the delivery of MASs by community pharmacists and other community pharmacy staff. METHODS: Two independent literature search strategies were conducted to examine the grey literature and scientific literature. Inclusion criteria consisted of English written literature related to the training of pharmacists, medicine counter assistants (MCAs), pharmacy technicians, and pharmacy students in the context of MASs. RESULTS: Sixty-six grey literature records (n = 57) and scientific articles (n = 9) met inclusion criteria. Most trainings targeted community pharmacists and focused on clinical care aspects that did not include guidance on service parameters and MAS delivery. Training lacked uniformity and varied in terms of time commitment, cost, curricula, and assessment processes. Limited training was identified for community pharmacy staff, particularly MCAs. IMPLICATIONS: MAS training is primarily provided for community pharmacists, with scant MAS training for community pharmacy support staff. Furthermore, existing training for any stakeholder group did not include guidance pertaining to service delivery. A structured training approach for the entire community pharmacy team is recommended to promote MAS outcomes and deliver a robust, high quality service. Detailed protocols and guidelines may be needed to ensure skilled MAS providers can deliver quality patient care.

Glucocorticoid suppression of CX3CL1 (fractalkine) by reduced gene promoter recruitment of NF-kappaB.

Glucocorticoids are an important anti-inflammatory treatment of many inflammatory diseases including asthma. However, the mechanisms by which they mediate their suppressive effects are not fully understood. Respiratory epithelial cells are a source of CX(3)CL1 (fractalkine), which mediates cell adhesion and acts as a chemoattractant for monocytes, T cells, and mast cells. We show, in lung A549 epithelial cells, that the tumor necrosis factor-alpha (TNF-alpha) and IFNgamma synergistically induced protein release and mRNA expression of CX(3)CL1 is inhibited by dexamethasone, without interfering with cytokine-induced nuclear translocation of NF-kappaB, and by an inhibitor of IkappaB kinase 2, AS602868. DNA binding assays confirmed the ability of NF-kappaB to bind to the proximal CX(3)CL1 promoter. Chromatin immunoprecipitation assays showed a 5-fold increase in the recruitment of NF-kappaB to the CX(3)CL1 gene promoter in response to IFNgamma/TNF-alpha; this too was reversed by dexamethasone. In contrast, dexamethasone did not displace NF-kappaB from the granulocyte-macrophage colony-stimulating factor gene promoter. We conclude that CX(3)CL1 expression is regulated through the NF-kappaB pathway and that dexamethasone inhibits CX(3)CL1 expression through a glucocorticoid receptor-dependent (RU486 sensitive) mechanism. This study also provides support for the action of glucocorticoids mediating their suppressive effects on expression by interfering with the binding of transcriptional activators at native gene promoters.

Transcriptional regulation of cytokine function in airway smooth muscle cells.

The immuno-modulatory properties of airway smooth muscle have become of increasing importance in our understanding of the mechanisms underlying chronic inflammation and structural remodeling of the airway wall in asthma and chronic obstructive pulmonary disease (COPD). ASM cells respond to many cytokines, growth factors and lipid mediators to produce a wide array of immuno-modulatory molecules which may in turn orchestrate and perpetuate the disease process in asthma and COPD. Despite numerous studies of the cellular effects of cytokines on cultured ASM, few have identified intracellular signaling pathways by which cytokines modulate or induce these cellular responses. In this review we provide an overview of the transcriptional mechanisms as well as intracellular signaling pathways regulating cytokine functions in ASM cells. The recent discovery of toll-like receptors in ASM cells represents a significant development in our understanding of the immuno-modulatory capabilities of ASM cells. Thus, we also review emerging evidence of the inflammatory response to toll-like receptor activation in ASM cells.