Susanne Roser-Page

Alterations in the immuno-skeletal interface drive bone destruction in HIV-1 transgenic rats.

Osteoporosis and bone fractures are increasingly recognized complications of HIV-1 infection. Although antiretroviral therapy itself has complex effects on bone turnover, it is now evident that the majority of HIV-infected individuals already exhibit reduced bone mineral density before therapy. The mechanisms responsible are likely multifactorial and have been difficult to delineate in humans. The HIV-1 transgenic rat recapitulates many key features of human AIDS. We now demonstrate that, like their human counterparts, HIV-1 transgenic rats undergo severe osteoclastic bone resorption, a consequence of an imbalance in the ratio of receptor activator of NF-κB ligand, the key osteoclastogenic cytokine, to that of its physiological decoy receptor osteoprotegerin. This imbalance stemmed from a switch in production of osteoprotegerin to that of receptor activator of NF-κB ligand by B cells, and was further compounded by a significantly elevated number of osteoclast precursors. With the advancing age of individuals living with HIV/AIDS, low bone mineral density associated with HIV infection is likely to collide with the pathophysiology of skeletal aging, leading to increased fracture risk. Understanding the mechanisms driving bone loss in HIV-infected individuals will be critical to developing effective therapeutic strategies.

Nicotine and fibronectin expression in lung fibroblasts: implications for tobacco-related lung tissue remodeling

Tobacco‐related lung diseases are associated with alterations in tissue remodeling and are characterized by increased matrix deposition. Among the matrix molecules found to be highly expressed in tobacco‐related lung diseases is fibronectin, a cell adhesive glycoprotein implicated in tissue injury and repair. We hypothesize that nicotine, a component of tobacco, stimulates the expression of fibronectin in lung fibroblasts via the activation of intracellular signals that lead to increased fibronectin gene transcription. In support of this, we found that nicotine stimulated the expression of fibronectin in lung fibroblasts and that its stimulatory effect was associated with activation of protein kinase C and mitogen‐activated protein kinases, increased levels of intracellular cAMP, and phosphorylation and DNA binding of the transcription factor CREB. Increased transcription of the gene was dependent on cAMP‐response elements (CREs) present on the 5′ end of its gene promoter. The stimulatory effect of nicotine on fibronectin expression was abolished by α‐bungarotoxin, an inhibitor of α7 nicotinic acetylcholine receptors (α7 AChRs). Of note, nicotine increased the expression of α7 nAChRs on fibroblasts. Our data suggest that nicotine induces lung fibroblasts to produce fibronectin by stimulating α7 nAChR‐ dependent signals that regulate the transcription of the fibronectin gene.

Predisposition for disrepair in the aged lung.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury. Methods: Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-&bgr;1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations. Results: The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-&bgr; receptor 1 and TGF-&bgr;1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation. Conclusion: Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-&bgr;1 expression and signaling and are populated by Thy-1–negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.

Fibronectin stimulates human lung carcinoma cell growth by inducing cyclooxygenase-2 (COX-2) expression.

Tobacco use is the most important risk factor for the development of lung carcinoma. One characteristic shared by tobacco‐related lung diseases is altered lung connective tissue content and composition. In particular, tobacco results in increased expression of fibronectin (FN), a matrix glycoprotein implicated in lung development, injury and repair and in tumor cell invasion. We hypothesized that excessive deposition of FN in lung might promote lung carcinoma cell proliferation. Consistent with this hypothesis, we found that FN stimulated human lung carcinoma cell proliferation and diminished apoptosis in vitro, and that this effect was mediated through the integrin α5β1 and associated with upregulation of cyclooxygenase‐2 (COX‐2) mRNA and protein expression, and increased prostaglandin E2 (PGE2) biosynthesis. The stimulatory effect of FN on COX‐2 was blocked by the specific COX‐2 inhibitor NS‐398 and by inhibitors of protein kinase C (PKC), Calphostin C, and extracellular signal‐regulated kinases (Erks), PD98095. Electrophoretic mobility shift assays revealed that FN increased the nuclear binding activity of cyclic AMP response element binding protein (CREB) and CCAAT/enhancer‐binding protein (C/EBP), 2 proteins known to play important roles in the regulation of COX‐2 promoter activity. Transient transfection assays with wild‐type and mutated constructs of the human COX‐2 gene promoter revealed that the stimulatory effect of FN was prevented when either the CRE or the NF‐IL6 (C/EBP) sites were mutated. Taken together, the results indicate that FN stimulates human lung carcinoma cell proliferation and diminishes apoptosis by inducing COX‐2 gene expression and PGE2 biosynthesis. Activation of PKC and Erk and DNA‐protein interactions at CRE and NF‐IL6 (C/EBP) sites in the COX‐2 gene promoter appear to play key roles in this process. This work demonstrates that signaling through specific matrix‐binding β1 integrins (i.e., α5β1) resulting from exaggerated deposition in lung of the matrix glycoprotein fibronectin might promote lung carcinoma cell growth.

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.

Extracellular Matrix Influences Alveolar Epithelial Claudin Expression and Barrier Function

The lung is dynamically remodeled in response to injury, which alters extracellular matrix composition, and can lead to either healthy or impaired lung regeneration. To determine how changes in extracellular matrix can influence alveolar epithelial barrier function, we examined the expression and function of tight junction proteins by rat alveolar epithelial type II cells cultured on one of three different matrix components: type I collagen or fibronectin, matrix glycoproteins which are highly expressed in injured lungs, or laminin, a basement membrane matrix component. Of note, alveolar epithelial cells cultured for 2 days on fibronectin formed high-resistance barriers and showed continuous claudin-3 and claudin-18 localization to the plasma membrane, as opposed to cells cultured on either type I collagen or laminin, which had low resistance monolayers and had areas of cell-cell contact that were claudin deficient. The barrier formed by cells cultured on fibronectin also had preferential permeability to chloride as compared with sodium. Regardless of the initial matrix composition, alveolar epithelial cells cultured for 5 days formed high-resistance barriers, which correlated with increased claudin-18 localization to the plasma membrane and an increase in zonula occludens-1. Day 5 cells on laminin had significantly higher resistance than cells on either fibronectin or type I collagen. Thus, although alveolar epithelial cells on fibronectin formed rapid barriers, it was at the expense of producing an optimized barrier.

Polyketal microparticles for therapeutic delivery to the lung

Inflammation in the setting of interstitial lung disease (ILD) occurs in the distal alveolar spaces of the lung, which presents significant challenges for therapeutic delivery. The development of aerosolizable microparticles from non-immunogenic polymers is needed to enable the clinical translation of numerous experimental therapeutics that require localization to the deep lung and repeated delivery for optimal efficacy. Polyketals (PK), a family of polymers, have several unique properties that make them ideal for lung delivery, specifically their hydrolysis into non-acidic, membrane-permeable compounds and their capacity to form microparticles with the aerodynamic properties needed for aerosolization. In this study, we tested the lung biocompatibility of microparticles created from a polyketal polymer, termed PK3, following intratracheal instillation in comparison to commonly used PLGA microparticles. We furthermore tested the initial efficacy of PK3 microparticles to encapsulate and effectively deliver active superoxide dismutase (SOD), a free radical scavenging enzyme, in a model of lung fibrosis. Our findings indicate that PK3 microparticles display no detectable level of alveolar or airway inflammation, whereas PLGA induced a small inflammatory response. Furthermore, SOD-loaded into PK3 microparticles maintained its activity upon release and, when delivered via PK3 microparticles, inhibited the extent of lung fibrosis.

Antiretroviral therapy induces a rapid increase in bone resorption that is positively associated with the magnitude of immune reconstitution in HIV infection

Objective:Antiretroviral therapy (ART) paradoxically intensifies bone loss in the setting of HIV infection. Although the extent of bone loss varies, it occurs with virtually all ART types, suggesting a common pathway that may be aligned with HIV disease reversal. Using an animal model of immunodeficiency we recently demonstrated that immune activation associated with CD4+ T-cell reconstitution induces increased production of the osteoclastogenic cytokines RANKL and TNF&agr; by immune cells, driving enhanced bone resorption and loss in bone mineral density. Design:To confirm these findings in humans, we investigated the early kinetics of CD4+ T-cell recovery in relation to biomarkers of bone turnover and osteoclastogenic regulators in a prospective 24-week cohort study. Methods:Clinical data and blood sampling for HIV-RNA PCR, CD4+ T-cell counts, bone turnover biomarkers, and osteoclastogenic regulators were obtained from ART-naïve HIV-infected study participants initiating standard doses of lopinavir/ritonavir plus tenofovir disoproxil fumarate/emtricitabine at baseline and at weeks 2, 8, 12, and 24 post ART. Results:C-terminal telopeptide of collagen (CTx) a sensitive biomarker of bone resorption rose by 200% above baseline at week 12, remaining elevated through week 24 (&agr;<0.01), and was associated with significant increases in plasma levels of osteoclastogenic regulators [receptor activator of NF-kB ligand (RANKL), tumor necrosis factor alpha, (TNF&agr;)]. Importantly, the magnitude of CD4+ T-cell recovery correlated significantly with CTx (rs = 0.387, &agr;=0.01). Conclusion:Our data suggest that ART-induced bone loss occurs early, is aligned with early events of immune reconstitution, and these immune changes provide a unifying mechanism to explain in part the skeletal decline common to all ART.

Role of T-cell reconstitution in HIV-1 antiretroviral therapy-induced bone loss

HIV infection causes bone loss. We previously reported that immunosuppression-mediated B-cell production of receptor activator of NF-κB ligand (RANKL) coupled with decline in osteoprotegerin correlate with decreased bone mineral density (BMD) in untreated HIV-infection. Paradoxically, antiretroviral therapy (ART) worsens bone loss although existing data suggest that such loss is largely independent of specific antiretroviral regimen. This led us to hypothesize that skeletal deterioration following HIV disease reversal with ART may be related to T-cell repopulation and/or immune-reconstitution. Here we transplant T cells into immunocompromised mice to mimic ART-induced T-cell expansion. T-cell reconstitution elicits RANKL and TNFα production by B-cells and/or T-cells, accompanied by enhanced bone resorption and BMD loss. Reconstitution of TNFα- or RANKL-null T-cells and pharmacological TNFα antagonist all protect cortical, but not trabecular bone, revealing complex effects of T-cell-reconstitution on bone turnover. These findings suggest T-cell repopulation and/or immune-reconstitution as putative mechanisms for bone loss following ART initiation.

In Utero Nicotine Exposure Promotes M2 Activation in Neonatal Mice Alveolar Macrophages

Background:Maternal smoking in utero has been associated with adverse health outcomes including lower respiratory tract infections in infants and children, but the mechanisms underlying these associations continue to be investigated. We hypothesized that nicotine plays a significant role in mediating the effects of maternal tobacco smoke on the function of the neonatal alveolar macrophage (AM), the resident immune cell in the neonatal lung.Methods:Primary AMs were isolated at postnatal day 7 from a murine model of in utero nicotine exposure. The murine AM cell line MH-S was used for additional in vitro studies.Results:In utero nicotine increased interleukin-13 and transforming growth factor–β1 (TGFβ1) in the neonatal lung. Nicotine-exposed AMs demonstrated increased TGFβ1 and increased markers of alternative activation with diminished phagocytic function. However, AMs from mice deficient in the α7 nicotinic acetylcholine receptor (α7 nAChR) had less TGFβ1, reduced alternative activation, and improved phagocytic functioning despite similar in utero nicotine exposure.Conclusion:In utero nicotine exposure, mediated in part via the α7 nAChR, may increase the risk of lower respiratory tract infections in neonates by changing the resting state of AM toward alternative activation. These findings have important implications for immune responses in the nicotine-exposed neonatal lung.