Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial-mesenchymal Transition in Aggressive Variant Prostate Cancers.

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.

Role of IL-27 in HSV-1-Induced Herpetic Stromal Keratitis.

Herpetic stromal keratitis (HSK) is a painful and vision-impairing disease caused by recurrent HSV-1 infection of the cornea. The virus replication in the corneal epithelium and associated inflammation play a dominant role in HSK progression. Current HSK treatments targeting inflammation or virus replication are partially effective and promote HSV-1 latency, and long-term use can cause side effects. Thus, understanding molecular and cellular events that control HSV-1 replication and inflammation is crucial for developing novel HSK therapies. In this study, we report that ocular HSV-1 infection induces the expression of IL-27, a pleiotropic immunoregulatory cytokine. Our data indicate that HSV-1 infection stimulates IL-27 production by macrophages. Using a primary corneal HSV-1 infection mouse model and IL-27 receptor knockout mice, we show that IL-27 plays a critical role in controlling HSV-1 shedding from the cornea, the optimum induction of effector CD4+ T cell responses, and limiting HSK progression. Using in vitro bone marrow-derived macrophages, we show that IL-27 plays an antiviral role by regulating macrophage-mediated HSV-1 killing, IFN-ß production, and IFN-stimulated gene expression after HSV-1 infection. Furthermore, we report that IL-27 is critical for macrophage survival, Ag uptake, and the expression of costimulatory molecules involved in the optimum induction of effector T cell responses. Our results indicate that IL-27 promotes endogenous antiviral and anti-inflammatory responses and represents a promising target for suppressing HSK progression.

Targeting Wnt/ß-catenin signaling using XAV939 nanoparticles in tumor microenvironment-conditioned macrophages promote immunogenicity.

The aberrant activation of Wnt/ß-catenin signaling in tumor cells and immune cells in the tumor microenvironment (TME) promotes malignant transformation, metastasis, immune evasion, and resistance to cancer treatments. The increased Wnt ligand expression in TME activates ß-catenin signaling in antigen (Ag)-presenting cells (APCs) and regulates anti-tumor immunity. Previously, we showed that activation of Wnt/ß-catenin signaling in dendritic cells (DCs) promotes induction of regulatory T cell responses over anti-tumor CD4+ and CD8+ effector T cell responses and promotes tumor progression. In addition to DCs, tumor-associated macrophages (TAMs) also serve as APCs and regulate anti-tumor immunity. However, the role of ß-catenin activation and its effect on TAM immunogenicity in TME is largely undefined. In this study, we investigated whether inhibiting ß-catenin in TME-conditioned macrophages promotes immunogenicity. Using nanoparticle formulation of XAV939 (XAV-Np), a tankyrase inhibitor that promotes ß-catenin degradation, we performed in vitro macrophage co-culture assays with melanoma cells (MC) or melanoma cell supernatants (MCS) to investigate the effect on macrophage immunogenicity. We show that XAV-Np-treatment of macrophages conditioned with MC or MCS significantly upregulates the cell surface expression of CD80 and CD86 and suppresses the expression of PD-L1 and CD206 compared to MC or MCS-conditioned macrophages treated with control nanoparticle (Con-Np). Further, XAV-Np-treated macrophages conditioned with MC or MCS significantly increased IL-6 and TNF-α production, with reduced IL-10 production compared to Con-Np-treated macrophages. Moreover, the co-culture of MC and XAV-Np-treated macrophages with T cells resulted in increased CD8+ T cell proliferation compared to Con-Np-treated macrophages. These data suggest that targeted ß-catenin inhibition in TAMs represents a promising therapeutic approach to promote anti-tumor immunity.

Targeting ß-catenin using XAV939 nanoparticle promotes immunogenic cell death and suppresses conjunctival melanoma progression.

Many tumors dysregulate Wnt/ß-catenin pathway to promote stem-cell-like phenotype, tumorigenesis, immunosuppression, and resistance to targeted cancer immunotherapies. Therefore, targeting this pathway is a promising therapeutic approach to suppress tumor progression and elicit robust anti-tumor immunity. In this study, using a nanoparticle formulation for XAV939 (XAV-Np), a tankyrase inhibitor that promotes ß-catenin degradation, we investigated the effect of ß-catenin inhibition on melanoma cell viability, migration, and tumor progression using a mouse model of conjunctival melanoma. XAV-Nps were uniform and displayed near-spherical morphology with size stability for upto 5 days. We show that XAV-Np treatment of mouse melanoma cells significantly suppresses cell viability, tumor cell migration, and tumor spheroid formation compared to control nanoparticle (Con-Np) or free XAV939-treated groups. Further, we demonstrate that XAV-Np promotes immunogenic cell death (ICD) of tumor cells with a significant extracellular release or expression of ICD molecules, including high mobility group box 1 protein (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP). Finally, we show that local intra-tumoral delivery of XAV-Nps during conjunctival melanoma progression significantly suppresses tumor size and conjunctival melanoma progression compared to Con-Nps-treated animals. Collectively, our data suggest that selective inhibition of ß-catenin in tumor cells using nanoparticle-based targeted delivery represents a novel approach to suppress tumor progression through increased tumor cell ICD.

Development of OX40 agonists for canine cancer immunotherapy.

Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

ADAMTS7 Attenuates House Dust Mite-Induced Airway Inflammation and Th2 Immune Responses.

PURPOSE: ADAMTS7 is a secreted metalloproteinase enzyme and proteoglycan associated with the early progression of coronary artery disease. However, there is limited information regarding the role of ADAMTS7 in lung adaptive immunity and inflammation. Thus, we sought to assess whether ADAMTS7 expression in the lung modulates house dust mite (HDM)-induced airway inflammation and Th2 immune response. METHODS: The role of ADAMTS7 in HDM-induced airway disease was assessed in ADAMTS7-deficient (ADAMTS7-/-) mice and compared with the wild-type control mice by flow cytometry, ELISA, and histopathology. Furthermore, the antigen priming capability of dendritic cells (DC) was determined ex vivo by employing coculture with CD4+ OT-II cells. RESULTS: ADAMTS7-/- mice develop an augmented eosinophilic airway inflammation, mucous cell metaplasia, and increased Th2 immune response to inhaled HDM. In addition, allergen uptake by lung DC and migration to draining mediastinal lymph node were significantly increased in ADAMTS7-/- mice, which shows an enhanced capacity to mount allergen-specific T-cell proliferation and effector Th2 cytokine productions. We propose that the mechanism by which ADAMTS7 negatively regulates DC function involves attenuated antigen uptake and presentation capabilities, which reduces allergic sensitization and Th2 immune responses in the lung. CONCLUSION: In aggregate, we provide compelling evidence that ADAMTS7 plays a pivotal role in allergic airway disease and Th2 immunity and would be an attractive target for asthma.

Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) level determines steroid-resistant airway inflammation in aging.

Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly, which correlates with the changes seen in adults with asthma. However, whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here, we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism, we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR), mixed granulomatous airway inflammation comprising eosinophils and neutrophils, and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate, lung, and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes, which progressively declined with age and further by HDM-induced inflammation. Moreover, we found increased glycolytic reprogramming, maturation/activation of DCs, the proliferation of OT-II cells, and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity.

Irg1/itaconate metabolic pathway is a crucial determinant of dendritic cells immune-priming function and contributes to resolute allergen-induced airway inflammation.

Itaconate is produced from the mitochondrial TCA cycle enzyme aconitase decarboxylase (encoded by immune responsive gene1; Irg1) that exerts immunomodulatory function in myeloid cells. However, the role of the Irg1/itaconate pathway in dendritic cells (DC)-mediated airway inflammation and adaptive immunity to inhaled allergens, which are the primary antigen-presenting cells in allergic asthma, remains largely unknown. House dust mite (HDM)-challenged Irg1-/- mice displayed increases in eosinophilic airway inflammation, mucous cell metaplasia, and Th2 cytokine production with a mechanism involving impaired mite antigen presentations by DC. Adoptive transfer of HDM-pulsed DC from Irg1-deficient mice into naïve WT mice induced a similar phenotype of elevated type 2 airway inflammation and allergic sensitization. Untargeted metabolite analysis of HDM-pulsed DC revealed itaconate as one of the most abundant polar metabolites that potentially suppress mitochondrial oxidative damage. Furthermore, the immunomodulatory effect of itaconate was translated in vivo, where intranasal administration of 4-octyl itaconate 4-OI following antigen priming attenuated the manifestations of HDM-induced airway disease and Th2 immune response. Taken together, these data demonstrated for the first time a direct regulatory role of the Irg1/itaconate pathway in DC for the development of type 2 airway inflammation and suggest a possible therapeutic target in modulating allergic asthma.

Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients.

Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients.

IFN-λ Regulates Neutrophil Biology to Suppress Inflammation in Herpes Simplex Virus-1-Induced Corneal Immunopathology.

HSV-1 infection of the cornea causes a severe immunoinflammatory and vision-impairing condition called herpetic stromal keratitis (SK). The virus replication in corneal epithelium followed by neutrophil- and CD4+ T cell-mediated inflammation plays a dominant role in SK. Although previous studies demonstrate critical functions of type I IFNs (IFN-α/ß) in HSV-1 infection, the role of recently discovered IFN-λ (type III IFN), specifically at the corneal mucosa, is poorly defined. Our study using a mouse model of SK pathogenesis shows that HSV-1 infection induces a robust IFN-λ response compared with type I IFN production at the corneal mucosal surface. However, the normal progression of SK indicates that the endogenous IFN responses are insufficient to suppress HSV-1-induced corneal pathology. Therefore, we examined the therapeutic efficacy of exogenous rIFN-λ during SK progression. Our results show that rIFN-λ therapy suppressed inflammatory cell infiltration in the cornea and significantly reduced the SK pathologic condition. Early rIFN-λ treatment significantly reduced neutrophil and macrophage infiltration, and IL-6, IL-1ß, and CXCL-1 production in the cornea. Notably, the virucidal capacity of neutrophils and macrophages measured by reactive oxygen species generation was not affected. Similarly, ex vivo rIFN-λ treatment of HSV-1-stimulated bone marrow-derived neutrophils significantly promoted IFN-stimulated genes without affecting reactive oxygen species production. Collectively, our data demonstrate that exogenous topical rIFN-λ treatment during the development and progression of SK could represent a novel therapeutic approach to control HSV-1-induced inflammation and associated vision impairment.

Pyruvate kinase M2 in lung APCs regulates Alternaria-induced airway inflammation.

Sensitivity to allergenic fungi (Alternaria alternata) is associated with acute, severe asthma attacks. Antigen presenting cells (APCs) in the lung sense environmental perturbations that induce cellular stress and metabolic changes and are critical for allergic airway inflammation. However, the mechanisms underlying such environmental sensing by APCs in the lung remains unclear. Here we show that acute Alternaria challenge rapidly induces neutrophil accumulation in airways, and alter expressions of Pyruvate Kinase (PKM2) and hypoxia-inducible factor -1α (Hif-1α) that correlates with proinflammatory mediator release. Blockade of IL33 signaling in vivo led to reduce oxidative stress and glycolysis in lung APCs. Lung-specific ablation of CD11c+ cells abrogates Alternaria-induced neutrophil accumulation and inflammation. Furthermore, administration of Alternaria into the airways stimulated APCs and elevate the expression of Glut-1. Mechanistically, we establish that PKM2 is a critical modulator of lung APC activation in Alternaria-induced acute inflammation. Allosteric activation of PKM2 by a small molecule ML265 or siRNA-mediated knock down correlated negatively with glycolysis and activation of APCs. These results collectively demonstrates that PKM2-mediated glycolytic reprogramming by fungal allergen Alternaria influences lung APC activation, thereby promotes acute airway inflammation. Our data support a model in which Alternaria sensitization in airways induce a circuitry of glycolysis and PKM2 regulation that confers an acute activation of APCs in the lung, whose targeting might represent a strategy for asthma treatment.

Dendritic Cell-Restricted Progenitors Contribute to Obesity-Associated Airway Inflammation via Adam17-p38 MAPK-Dependent Pathway.

Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely, how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK), which is blocked with a selective p38α/ß MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically, we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether, these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation.

Dimethyl fumarate abrogates dust mite-induced allergic asthma by altering dendritic cell function.

INTRODUCTION: Allergic asthma is the most common inflammatory disease of upper airways. Airway dendritic cells (DCs) are key antigen presenting cells that regulate T helper 2 (Th2)-dependent allergic inflammation. Recent studies have shown critical role of airway DCs in the induction of Th2-mediated allergic inflammation and are attractive therapeutic targets in asthma. However, molecular signaling mechanism that regulate DCs function to Th2 immune responses are poorly understood. Here we aim to evaluate the immunomodulatory effect of dimethyl fumarate (DMF), an FDA approved small molecule drug, in the house dust mite (HDM)-induced experimental model of allergic asthma. METHODS: DMF was administered intranasally in the challenge period of HDM-induced murine model of experimental asthma. Airway inflammation, airway hyperreactivity, Th2/Th1 cytokine were assessed. The effect of DMF on DC function was further evaluated by adoptive transfer of HDM-pulsed DMF treated DCs to wild-type naïve mice. RESULTS: DMF treatment significantly reduced HDM-induced airway inflammation, mucous cell metaplasia, and airway hyperactivity to inhaled methacholine. Mechanistically, DMF interferes with the migration of lung DCs to draining mediastinal lymph nodes, thereby attenuates the induction of allergic sensitization and Th2 immune response. Notably, adoptive transfer of DMF treated DCs to naïve mice with HDM challenge similarly reduces the features of allergic asthma. CONCLUSION: This identifies a novel function of DMF on DC-mediated adaptive immune responses in the setting of HDM-induced airway inflammation. Taken together, our results offer a mechanistic rationale for DMF use to target DCs in local lung environment as antiasthmatic therapy.

Low-density lipoprotein receptor-related protein 1 attenuates house dust mite-induced eosinophilic airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. OBJECTIVE: We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. METHODS: LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. RESULTS: Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. CONCLUSION: This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

Metabolic Plasticity in Dendritic Cell Responses: Implications in Allergic Asthma.

Dendritic cells (DCs) are highly specialized in antigen presentation and play a pivotal role in the initiation, progression, and perpetuation of adaptive immune responses. Emerging immune pathways are being recognized increasingly for DCs and their subsets that differentially regulate T lymphocyte function based on the type and interactions with the antigen. However, these interactions not only alter the signaling process and DC function but also render metabolic plasticity. The current review focuses on the metabolic cues of DCs that coordinate DC activation and differentiation and discuss whether targeting these fundamental cellular processes have implications to control airway inflammation and adaptive immunity. Therefore, strategies using metabolism-based therapeutic manipulation of DC functions could be developed into novel treatments for airway inflammation and asthma.

A Critical Role for P2X7 Receptor-Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury.

Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.

Platelet-derived Wnt antagonist Dickkopf-1 is implicated in ICAM-1/VCAM-1-mediated neutrophilic acute lung inflammation.

Neutrophil infiltration represents the early acute inflammatory response in acute lung injury. The recruitment of neutrophils from the peripheral blood across the endothelial-epithelial barrier into the alveolar airspace is highly regulated by the adhesion molecules on alveolar epithelial cells (AECs). Wnt/ß-catenin signaling is involved in the progression of inflammatory lung diseases including asthma, emphysema, and pulmonary fibrosis. However, the function of Wnt/ß-catenin signaling in acute lung inflammation is unknown. Here, we identified platelet-derived Dickkopf-1 (Dkk1) as the major Wnt antagonist contributing to the suppression of Wnt/ß-catenin signaling in AECs during acute lung inflammation. Intratracheal administration of Wnt3a or an antibody capable of neutralizing Dkk1 inhibited neutrophil influx into the alveolar airspace of injured lungs. Activation of Wnt/ß-catenin signaling in AECs attenuated intercellular adhesion molecule 1 (ICAM-1)/vascular cell adhesion molecule 1 (VCAM-1)-mediated adhesion of both macrophages and neutrophils to AECs. Our results suggest a role for Wnt/ß-catenin signaling in modulating the inflammatory response, and a functional communication between platelets and AECs during acute lung inflammation. Targeting Wnt/ß-catenin signaling and the communication between platelets and AECs therefore represents potential therapeutic strategies to limit the damage of acute pulmonary inflammation.

Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase.

DNA-dependent protein kinase (DNA-PK) mediates double-stranded DNA break repair, V(D)J recombination and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

MicroRNA-206 regulates surfactant secretion by targeting VAMP-2.

Lung surfactant secretion is a highly regulated process. Our previous studies have shown that VAMP-2 is essential for surfactant secretion. In the present study we investigated the role of miR-206 in surfactant secretion through VAMP-2. VAMP-2 was confirmed to be a target of miR-206 by 3'-untranslational region (3'-UTR) luciferase assay. Mutations in the predicated miR-206 binding sites reduced the binding of miR-206 to the 3'-UTR of VAMP-2. miR-206 decreased the expression of VAMP-2 protein and decreased the lung surfactant secretion in alveolar type II cells. In conclusion, miR-206 regulates lung surfactant secretion by limiting the availability of VAMP-2 protein.

The very low density lipoprotein receptor attenuates house dust mite-induced airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

The very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands and plays a key role in brain development. Although the VLDLR mediates pleiotropic biological processes, only a limited amount of information is available regarding its role in adaptive immunity. In this study, we identify an important role for the VLDLR in attenuating house dust mite (HDM)-induced airway inflammation in experimental murine asthma. We show that HDM-challenged Vldlr(-/-) mice have augmented eosinophilic and lymphocytic airway inflammation with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia. A genome-wide analysis of the lung transcriptome identified that mRNA levels of CD209e (DC-SIGNR4), a murine homolog of DC-SIGN, were increased in the lungs of HDM-challenged Vldlr(-/-) mice, which suggested that the VLDLR might modify dendritic cell (DC) function. Consistent with this, VLDLR expression by human monocyte-derived DCs was increased by HDM stimulation. In addition, 55% of peripheral blood CD11c(+) DCs from individuals with allergy expressed VLDLR under basal conditions. Lastly, the adoptive transfer of HDM-pulsed, CD11c(+) bone marrow-derived DCs (BMDCs) from Vldlr(-/-) mice to the airways of wild type recipient mice induced augmented eosinophilic and lymphocytic airway inflammation upon HDM challenge with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia, as compared with the adoptive transfer of HDM-pulsed, CD11c(+) BMDCs from wild type mice. Collectively, these results identify a novel role for the VLDLR as a negative regulator of DC-mediated adaptive immune responses in HDM-induced allergic airway inflammation.