Bronchial Secretory Immunoglobulin A Deficiency Correlates With Airway Inflammation and Progression of Chronic Obstructive Pulmonary Disease

Rationale: Although airway inflammation can persist for years after smoking cessation in patients with chronic obstructive pulmonary disease (COPD), the mechanisms of persistent inflammation are largely unknown

BMP pathway regulation of and by macrophages.

Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC) treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle

NF-κB Activation Exacerbates, but Is not Required for Murine Bmpr2-Related Pulmonary Hypertension

Aim: The present study investigates the role of NF-κB in Bmpr2-related pulmonary hypertension (PH) using a murine model of PH with inducible overexpression of a cytoplasmic tail Bmpr2 mutation. Methods and Results: Electrophoretic mobility shift assay for nuclear extracts in Bmpr2R899X mouse lung and immunohistochemistry for NF-κB p65 in human PAH lung demonstrate that NF-κB is activated in end-stage disease. Acute inflammation or expression of a constitutively active NF-κB elicits a strong suppression of the BMP pathway in mice inversely correlating to activation of NF-κB targets. However, Bmpr2 mutation does not result in NF-κB activation in early disease development as assessed by luciferase reporter mice. Moreover, Bmpr2 mutant mice in which NF-κB activation is genetically blocked develop PH indistinguishable from that without the block. Finally, delivery of a virus causing NF-κB activation strongly exacerbates development of PH in Bmpr2 mutant mice, associated with increased remodeling. Conclusion: NF-κB activation exacerbates, but is not required for Bmpr2-related PH. Pulmonary vascular-specific activation of NF-κB may be a “second hit” that drives penetrance in heritable PH

Host A2B Adenosine Receptors Promote Carcinoma Growth1

Recent studies suggest that tumor-infiltrating immune cells can benefit the tumor by producing factors that promote angiogenesis and suppress immunity. Because the tumor microenvironment is characterized by high adenosine levels, we hypothesized that the low-affinity A2B adenosine receptor located on host immune cells may participate in these effects. In the current study, we tested this hypothesis in a Lewis lung carcinoma isograft model using A2B receptor knockout (A2BKO) mice. These mice exhibited significantly attenuated tumor growth and longer survival times after inoculation with Lewis lung carcinoma compared to wild type (WT) controls. Lewis lung carcinoma tumors in A2BKO mice contained significantly lower levels of vascular endothelial growth factor (VEGF) compared to tumors growing in WT animals. This difference was due to VEGF production by host cells, which comprised 30 ± 2% of total tumor cell population. Stimulation of adenosine receptors on WT tumor-infiltrating CD45+ immune cells increased VEGF production fivefold, an effect not seen in tumor-associated CD45+ immune cells lacking A2B receptors. In contrast, we found no significant difference in VEGF production between CD45- tumor cells isolated from WT and A2BKO mice. Thus, our data suggest that tumor cells promote their growth by exploiting A2B adenosine receptor-dependent regulation of VEGF in host immune cells

Neutrophil-Derived IL-1 beta Impairs the Efficacy of NF-kappa B Inhibitors against Lung Cancer

Although epithelial NF-kappa B signaling is important for lung carcinogenesis, NF-kappa B inhibitors are ineffective for cancer treatment. To explain this paradox, we studied mice with genetic deletion of IK kappa beta in myeloid cells and found enhanced tumorigenesis in Kras(G12D) and urethane models of lung cancer. Myeloid-specific inhibition of NF-kappa B augmented pro-IL-1 beta processing by cathepsin G in neutrophils, leading to increased IL-1 beta and enhanced epithelial cell proliferation. Combined treatment with bortezomib, a proteasome inhibitor that blocks NF-kappa B activation, and IL-1 receptor antagonist reduced tumor formation and growth in vivo. In lung cancer patients, plasma IL-1 beta levels correlated with poor prognosis, and IL-1 beta increased following bortezomib treatment. Together, our studies elucidate an important role for neutrophils and IL-1 beta in lung carcinogenesis and resistance to NF-kappa B inhibitors

IκB Kinase Activity Drives Fetal Lung Macrophage Maturation along a Non-M1/M2 Paradigm

In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11b(low)F4/80(high) cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms

Interaction of soluble macrophage derived factors and smooth muscle growth.

<p>(<b>A</b>) Schematic representation of the BMP receptor complex and the location of BMPR2 mutations and the site of action of the BMPR1-SMAD signaling inhibitor DM3189. (<b>B</b>) Confluent cultures of A7r5 VSMC or A7r5 cells stably expressing human BMPR2 alleles that interfere with ligand binding (F14), truncate the protein in the kinase domain (KD) or truncate the cytoplasmic tail (CD) were wounded by drawing a pipette across the center of the culture. Companion wells were similarly wounded and co-cultured with macrophages on insert and the area of the exposed plastic quantitated. Data presented as the percent of closure at 48 hr in the absence (white bars) or presence of macrophages (shaded bars) for each cell line. (<b>C</b>) Quantification of effect of macrophage co-culture on A7r5 cells expressing listed BMPR2 alleles. Cells defective in ligand binding or truncated in the kinase domain fail to respond to macrophage co-culture (F14 and KD) cells with intact kinase domain remain macrophage responsive and respond similarly even in the presence of a cytoplasmic tail truncation.(CD, WT and A7r5 VSMC). Both presence of macrophages and constructs were significant by two-way ANOVA at p<.01; *indicates p<.01 by post-hoc t-test.</p