Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases. MUC5AC mucin is a major component of airway mucus, and its expression is modulated by a TNF-α-converting enzyme (TACE)–EGF receptor pathway that can be activated by reactive oxygen species (ROS). Dual oxidase 1 (Duox1), a homologue of glycoprotein p91^phox, is expressed in airway epithelium and generates ROS. We hypothesize that Duox1 activates TACE, cleaving pro-TGF-α into soluble TGF-α, resulting in mucin expression. To examine this hypothesis, we stimulated both normal human bronchial epithelial cells and NCI-H292 airway epithelial cells with phorbol 12-myristate 13-acetate and with human neutrophil elastase. These stimuli induced TACE activation, TGF-α release, and mucin expression, effects that were inhibited by ROS scavengers, implicating ROS in TACE activation. Inhibition of epithelial NADPH oxidase or knockdown of Duox1 expression with small interfering RNA prevented ROS generation, TGF-α release, and mucin expression by these stimuli, implicating Duox1 in TACE activation and mucin expression. Furthermore, the PKCδ/PKCθ inhibitor rottlerin prevented the effects induced by phorbol 12-myristate 13-acetate and human neutrophil elastase, suggesting that PKCδ and PKCθ are involved in Duox1 activation. From these results, we conclude that Duox1 plays a critical role in mucin expression by airway epithelial cells through PKCδ/PKCθ-Duox1-ROS-TACE-pro-ligand-EGF receptor cascade.     

Generation of Th17 cells in response to intranasal infection requires TGF-β1 from dendritic cells and IL-6 from CD301b+ dendritic cells

Intranasal (i.n.) infections preferentially generate Th17 cells. We explored the basis for this anatomic preference by tracking polyclonal CD4⁺ T cells specific for an MHC class II-bound peptide from the mucosal pathogen Streptococcus pyogenes. S. pyogenes MHC class II-bound peptide-specific CD4⁺ T cells were first activated in the cervical lymph nodes following i.n. inoculation and then differentiated into Th17 cells. S. pyogenes-induced Th17 formation depended on TGF-β1 from dendritic cells and IL-6 from a CD301b⁺ dendritic cell subset located in the cervical lymph nodes but not the spleen. Thus, the tendency of i.n. infection to induce Th17 cells is related to cytokine production by specialized dendritic cells that drain this site.Th17 cells are a subset of CD4⁺ helper T cells that orchestrate protective immunity to extracellular bacterial and fungal pathogens, predominantly at epithelial surfaces (1). T-cell antigen receptor (TCR) recognition of an MHC class II-bound peptide (p:MHCII) on an antigen-presenting cell causes Th17 cells to secrete the signature cytokine IL-17A, which acts primarily by increasing chemokine production in epithelial tissues to enable the recruitment, activation, and migration of neutrophils and monocytes (1).In vitro studies have shown that naive CD4⁺ T cells differentiate into Th17 cells when stimulated by p:MHCII ligands in the presence of transforming growth factor-β1 (TGF-β1) and IL-6 (2-4). Similarly, TGF-β1 is required for Th17 differentiation during the induction of experimental autoimmune encephalomyelitis (EAE) (5). Th17 cells are also generated during intranasal (i.n.) infection by Streptococcus pyogenes, and TGF-β1 receptor signaling and IL-6 are involved (6, 7). However, Th17 cell formation in the small intestine does not depend on TGF-β1 (8) and requires IL-1β but not IL-6 (9). Additionally, Kuchroo and colleagues reported that although Th17 differentiation was normally dependent on IL-6, it could occur without it through an IL-21–dependent pathway if regulatory T cells were absent (10).Infection via the i.n. route may induce Th17 cells because nasal-associated lymphoid tissue (NALT) contains specialized antigen-presenting cells that preferentially produce IL-1β or IL-6 and TGF-β1. Here, we tested this idea by studying the primary immune response to i.n. infection with S. pyogenes bacteria (11). We found that S. pyogenes p:MHCII-specific Th17 cell formation depended on TGF-β1 from dendritic cells and IL-6 from a CD301b⁺ dendritic cell subset located in the cervical lymph nodes (CLNs) but not the spleen.     

Activin A,a product of fetal Leydig cells,is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion

Formation of tubular structures relies upon complex interactions between adjacent epithelium and mesenchyme. In the embryonic testes, dramatic compartmentalization leads to the formation of testis cords (epithelium) and the surrounding interstitium (mesenchyme). Sertoli cells, the epithelial cell type within testis cords, produce signaling molecules to orchestrate testis cord formation. The interstitial fetal Leydig cells, however, are thought only to masculinize the embryo and are not known to be involved in testis cord morphogenesis. Contrary to this notion, we have identified activin A, a member of the TGF-β protein superfamily, as a product of the murine fetal Leydig cells that acts directly upon Sertoli cells to promote their proliferation during late embryogenesis. Genetic disruption of activin βA, the gene encoding activin A, specifically in fetal Leydig cells resulted in a failure of fetal testis cord elongation and expansion due to decreased Sertoli cell proliferation. Conditional inactivation of Smad4, the central component of TGF-β signaling, in Sertoli cells led to testis cord dysgenesis and proliferative defects similar to those of Leydig cell-specific activin βA knockout testes. These results indicate that activin A is the major TGF-β protein that acts directly on Sertoli cells. Testicular dysgenesis in activin βA and Smad4 conditional knockout embryos persists into adulthood, leading to low sperm production and abnormal testicular histology. Our findings challenge the paradigm that fetal testis development is solely under the control of Sertoli cells, by uncovering an active and essential role of fetal Leydig cells during testis cord morphogenesis.     

EGF receptor-independent action of TGF-alpha protects Naked2 from AO7-mediated ubiquitylation and proteasomal degradation

Naked family members (Drosophila Naked Cuticle and mammalian Naked1 and Naked2) have been identified as inducible antagonists of canonical Wnt signaling. We recently reported that Naked2, but not Naked1, interacts with the cytoplasmic tail of TGF-α, thereby coating TGF-α-containing exocytic vesicles and directing these vesicles to the basolateral corner of polarized epithelial cells. Here, we show that Naked2 is a short-lived protein with a half-life of 60 min caused by its rapid ubiquitin-mediated proteasomal degradation. Overexpression of TGF-α stabilizes Naked2 protein in an EGF receptor (EGFR)-independent manner; a physical interaction between the cytoplasmic tail of TGF-α and Naked2 is necessary and sufficient for this protection. We have identified a RING finger protein, AO7/RNF25, as a ubiquitin ligase for Naked2, and we have shown that overexpression of TGF-α reduces binding of AO7 to Naked2. These results identify an EGFR-independent action of TGF-α, in which it protects Naked2 from proteasomal degradation, thus ensuring its delivery to the basolateral surface of polarized epithelial cells.     

Consistent loss of functional transforming growth factor β receptor expression in murine plasmacytomas

Murine plasmacytomas are tumors of Ig-secreting plasma cells that can be induced in genetically susceptible BALB/c mice. The deregulation of the c-myc protooncogene is a critical oncogenic event in the development of plasmacytomas (PCTs) although it is not sufficient for their malignant transformation. We have demonstrated that PCTs produce active transforming growth factor β (TGF-β) in vitro. Because TGF-β is a potent negative regulator of the proliferation and differentiation of B lymphocytes, we examined its role in plasmacytomagenesis by comparing responsiveness to TGF-β of nonneoplastic plasma cells and PCTs. The nontransformed plasma cells that accumulate in interleukin 6 transgenic mice undergo accelerated apoptosis upon treatment with TGF-β, but the 15 PCTs studied, including primary and transplanted tumors as well as established cell lines, were refractory to TGF-β-mediated growth inhibition and apoptosis. Although PCTs lack functional TGF-β receptors as demonstrated by chemical crosslinking to radiolabeled TGF-β1, they nonetheless contain mRNA and protein for both type I and II TGF-β receptors, suggesting a potential defect in receptor trafficking or processing. The results clearly show the consistent inactivation of TGF-β receptors in plasmacytoma cells, demonstrating for the first time that interruption of a tumor suppressor pathway contributes to plasmacytomagenesis.     

Role of TGF-beta in proliferative vitreoretinal diseases and ROCK as a therapeutic target

Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-β is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-β is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-β's importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-β2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-β blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted α-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-β in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-β and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.     

Influenza viral neuraminidase primes bacterial coinfection through TGF-β–mediated expression of host cell receptors

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.Secondary bacterial pneumonia or coinfection is the leading cause of viral-associated mortality during influenza A virus (IAV) pandemics (1, 2). The synergistic lethality of IAV and bacterial coinfection has been observed in animal models (3), suggesting a causative relationship between IAV infection and secondary bacterial pneumonia. Increased bacterial adherence post-IAV has been well recognized (4); however, the underlying mechanisms remain elusive. It has been demonstrated that IAV neuraminidase (NA) promotes the adherence of Streptococcus pneumoniae to lung epithelial cells, and viral NA activity has been associated with the levels of bacterial adherence and mortality in coinfected mice (5). In addition, inhibitors of NA, such as oseltamivir, reversed the effects of NA on bacterial adherence (6). These findings suggest that IAV NA contributes substantially to coinfection.ECM proteins, such as fibronectin (Fn), collagen, and laminin, interact with integrins, which transduce signals to regulate cell growth, differentiation, migration, and other cellular activities. ECM proteins and integrins are receptors that bind to microbial surface components recognizing adhesive matrix molecules (MSCRAMM) for bacterial adherence and invasion (4, 7). The expression of these cellular adhesion molecules can be up-regulated through TGF-β (8). This cytokine is secreted as an inactive or latent protein that subsequently is activated through various mechanisms (9). Schultz-Cherry and Hinshaw (10) reported that latent TGF-β is activated through IAV NA, and recently these authors demonstrated that viral NA triggers TGF-β activation through the removal of sialic acid motifs from latent TGF-β (11). These findings suggest that TGF-β might play a role in IAV-enhanced bacterial adherence.Adherence to host tissue is a critical initial step to establish infection. The most frequently observed bacteria in coinfections are S. pneumoniae, group A Streptococcus pyogenes (GAS), Staphylococcus aureus, and Haemophilus influenza (1, 12, 13). These bacteria require ECM components or integrins as receptors for adherence (14–17). We previously demonstrated that the invasion of host cells by GAS is promoted through the TGF-β–enhanced expression of integrin and Fn (8). These observations suggest that the activation of TGF-β through IAV NA might promote the expression of cellular receptors, facilitating bacterial adherence and leading to increased host susceptibility to coinfection.The goal of the present study was to define the mechanisms underlying the increased bacterial adherence post-IAV infection. We showed that expression of α5 integrin/Fn was up-regulated in response to IAV infection or viral NA treatment and reversed through the inhibition of TGF-β signaling, indicating that IAV increased the expression of host receptors through NA-activated TGF-β. In addition, IAV-mediated bacterial adherence required the Fn-binding protein of GAS, and the adherence of coinfective pathogens to IAV-infected cells was impeded by TGF-β inhibitors, suggesting that the bacteria commonly observed in coinfection likely share a similar mechanism for initiating an infection. Interventions targeting these mechanisms might reduce the incidence and severity of postinfluenza bacterial pneumonia.     

Polarizing the T helper 17 response in Citrobacter rodentium infection via expression of resistin-like molecule α

Citrobacter rodentium infection is a murine model of pathogenic Escherichia coli infection that allows investigation of the cellular and molecular mechanisms involved in host-protective immunity and bacterial-induced intestinal inflammation. We recently demonstrated that following C. rodentium infection, the absence of Resistin-Like Molecule (RELM) α resulted in attenuated Th17 cell responses and reduced intestinal inflammation with minimal effects on bacterial clearance. In this addendum, we investigated the cytokine modulatory effects of RELMα and RELMα expression in the intestinal mucosa following C. rodentium infection. We show that in addition to promoting Th17 cytokine responses, RELMα inhibits Th2 cytokine expression and Th2-cytokine effector macrophage responses in the C. rodentium-infected colons. Second, utilizing reporter C. rodentium, we examined RELMα expression and macrophage recruitment at the host pathogen interface. We observed infection-induced macrophage infiltration and RELMα expression by intestinal epithelial cells. The influence of infection-induced RELMα on macrophage recruitment in the intestine is discussed.     

Targeted deletion of Smad4 shows it is required for transforming growth factor β and activin signaling in colorectal cancer cells

Smad4 (DPC4) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) β and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-β, as well as from the TGF-β family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-β unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis.     

Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon

Crohn''s disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARγ functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of β-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Pparγ mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARγ-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn''s colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARγ-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.     

Expression of a dominant-negative type II transforming growth factor β (TGF-β) receptor in the epidermis of transgenic mice blocks TGF-β-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor β (TGF-β) is required to mediate the growth inhibitory effect of TGF-β on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-β receptor (ΔβRII) in the epidermis. The ΔβRII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10–14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F₁ mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from ΔβRII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-β-induced growth inhibition. These data document the role of the type II TGF-β receptor in mediating TGF-β-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Mutant surfactant A2 proteins associated with familial pulmonary fibrosis and lung cancer induce TGF-β1 secretion

Mutations in the genes encoding the lung surfactant proteins are found in patients with interstitial lung disease and lung cancer, but their pathologic mechanism is poorly understood. Here we show that bronchoalveolar lavage fluid from humans heterozygous for a missense mutation in the gene encoding surfactant protein (SP)-A2 (SFTPA2) contains more TGF-β1 than control samples. Expression of mutant SP-A2 in lung epithelial cells leads to secretion of latent TGF-β1, which is capable of autocrine and paracrine signaling. TGF-β1 secretion is not observed in lung epithelial cells expressing the common SP-A2 variants or other misfolded proteins capable of increasing cellular endoplasmic reticulum stress. Activation of the unfolded protein response is necessary for maximal TGF-β1 secretion because gene silencing of the unfolded protein response transducers leads to an ∼50% decrease in mutant SP-A2–mediated TGF-β1 secretion. Expression of the mutant SP-A2 proteins leads to the coordinated increase in gene expression of TGF-β1 and two TGF-β1–binding proteins, LTBP-1 and LTBP-4; expression of the latter is necessary for secretion of this cytokine. Inhibition of the TGF-β autocrine positive feedback loop by a pan–TGF-β–neutralizing antibody, a TGF-β receptor antagonist, or LTBP gene silencing results in the reversal of TGF-β–mediated epithelial-to-mesenchymal transition and cell death. Because secretion of latent TGF-β1 is induced specifically by mutant SP-A2 proteins, therapeutics targeted to block this pathway may be especially beneficial for this molecularly defined subgroup of patients.     

Perturbation of transforming growth factor (TGF)-ß1 association with latent TGF-β binding protein yields inflammation and tumors

Transforming growth factor-β (TGF-β) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-β is often released as part of an inactive tripartite complex consisting of TGF-β, the TGF-β propeptide, and a molecule of latent TGF-β binding protein (LTBP). The interaction of TGF-β and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-β from this state is crucial for signaling. To examine the contribution of LTBP to TGF-β function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1^C33S/C33S mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-β1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1^−/− animals. Tgfb1^C33S/C33S mice exhibited decreased levels of active TGF-β1, decreased TGF-β signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-β complex is important for proper TGF-β1 function and that Tgfb1^C33S/C33S mice are hypomorphs for active TGF-β1. Moreover, although mechanisms exist to activate latent TGF-β1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.     

Specific glycosphingolipids mediate epithelial-to-mesenchymal transition of human and mouse epithelial cell lines

Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with d-threo-1-(3′,4′-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-β, which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 or TGF-β is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines. (ii) TGF-β treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-β-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition).     

Compromised peroxisomes in idiopathic pulmonary fibrosis,a vicious cycle inducing a higher fibrotic response via TGF-β signaling

Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-β) receptor II knockout mice indicating a role for TGF-β signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-β1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-β signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-β) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-β–induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.Idiopathic pulmonary fibrosis (IPF) is a chronic, devastating, and lethal fibrotic disorder in human lung. IPF is characterized by a worsening of pulmonary function and persistent alterations of the lung parenchyma as a result of fibrotic foci formation by activated fibroblasts/myofibroblasts and excessive production and deposition of extracellular matrix components (ECM) (1–4). It is well accepted that transforming growth factor beta (TGF-β) signaling plays a critical role in IPF development. Inhibition of TGF-β signaling by blocking its downstream Smad3 gene expression protects against bleomycin-induced fibrosis in animal models (5, 6). In addition, there is increasing evidence that tumor necrosis factor alpha (TNF-α) also plays an important role in initiation and perpetuation of the fibrotic processes, possibly by activating TGF-β signaling pathway (7). However, the mechanisms by which TGF-β and TNF-α promote the fibrotic response in IPF are incompletely known.Peroxisomes are single membrane bounded ubiquitous organelles, present in all types of cells. Particularly, type II alveolar epithelial cells and club cells (Clara) in the lung have highly abundant peroxisomes (8). These organelles are involved in a variety of metabolic pathways, including degradation of reactive oxygen species (ROS) and bioactive lipid mediators (prostaglandins and leukotriens) and synthesis of antioxidant lipids (polyunsaturated fatty acids, plasmalogens, etc.) (9). Absence or dysfunction of peroxisomes results in increased cellular oxidative stress, leading to severe pathological consequences in many organ systems (10, 11). Lung is one of the organs with highest exposure to various forms of reactive oxygen and nitrogen species (ROS and RNS) due to oxygen and different environmental oxidants in the inspired air, causing oxidation of cellular DNA, proteins and lipids, consequently a direct lung injury (12). Studies have shown that the most severe phenotype of a peroxisome biogenesis disorder (e.g., Zellweger syndrome) is associated with progressive liver fibrosis or cirrhosis, leading to early death of the patients during childhood (11). Moreover, mice with peroxisome dysfunction caused by PEX11β knockout died during their first days of life and exhibit morphological alterations of the lungs (13). In contrast, treatment of rats with an agonist specific for peroxisome proliferator-activated receptor alpha (PPAR-α) significantly ameliorated tubulointerstitial renal fibrosis (14). Despite the fact that peroxisomal metabolism might play an important role in other tissue fibrosis, the role of peroxisomes in lung fibrosis onset and progression seen in IPF patients has never been reported (1, 15).Herein, using human IPF and control fibroblast cultures as well as a bleomycin-induced mouse lung fibrosis model, we demonstrate that peroxisomal biogenesis and metabolism is compromised in the lung and in fibroblasts of IPF patients, in which a down-regulation of peroxisomal proteins leads to activation and release of profibrotic factors such as TGF-β1 and collagen. In contrast, peroxisome proliferation by treatment with PPAR-α agonist (ciprofibrate, WY14643) significantly reduces the TGF-β1–induced myofibroblast differentiation in IPF fibroblast cultures.     

TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury

TGF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which—together with patch-clamp and flow cytometry studies—identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys⁴³. Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β–dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.The acute respiratory distress syndrome (ARDS) is a devastating syndrome characterized by alveolar flooding (edema), which impairs gas exchange, leading to respiratory failure (1). The high mortality rate of 35–45% observed in patients with ARDS and the lack of any pharmacological therapy (1) underscores the need to better understand the pathomechanisms of this lethal disease, in the hope of facilitating improved clinical management of affected patients.Alveolar edema occurs as a consequence of increased fluid influx into the alveolar airspaces from the vasculature, across the thin alveolo-capillary barrier (2), as well as a failure of transepithelial Na⁺ and Cl⁻ ion transport, which drives fluid clearance from the alveolar airspaces. Transepithelial sodium transport is undertaken by the concerted action of several ion transporters, namely the Na⁺/K⁺-ATPase (3) and the epithelial sodium channel (ENaC) (4, 5), which actively transport Na⁺ out of the fluid lining the alveolar airspaces (epithelial lining fluid, ELF). This process generates an osmotic gradient that clears water from the alveolar airspaces (6). This fluid clearance process is defective in ARDS patients with compromised alveolo-capillary barrier function, and it is widely believed that edema fluid must be cleared for patients with ARDS to survive (7, 8).TGF-β is a key mediator of acute lung injury (ALI), where TGF-β is activated locally by integrin α_vβ₆ (9) in cooperation with protease-activated receptor-1 (10), to increase epithelial and endothelial permeability and promote alveolar flooding. In further support of a role for TGF-β in ALI, two studies have demonstrated increased TGF-β levels in lung fluids from patients with ALI/ARDS (11, 12), and in these patients lower TGF-β levels correlate with more ventilator-free and intensive care unit-free days (11). Some evidence has also implicated TGF-β in transepithelial ion transport in vitro, where TGF-β down-regulated gene expression of one of three ENaC subunits (13), temporally modulated gene expression of the Na⁺/K⁺-ATPase (14), and impacted Cl⁻ transport (15). In animal models of ALI/ARDS, administration of a soluble type II TGF-β receptor, which sequesters free TGF-β, attenuated the degree of pulmonary edema (9), confirming a role for TGF-β in disturbed lung fluid dynamics associated with experimental ALI/ARDS, however, a role for TGF-β in regulating alveolar fluid reabsorption has not been established.In this study, a unique TGF-β signaling pathway is described, whereby TGF-β—acting through the Tgfbr1/Smad2/3 axis (16)—recruits phosphoinositide-metabolizing enzymes and an NADPH oxidase to generate reactive oxygen species (ROS), which drive αβγENaC complex internalization from the lung epithelial cell surface and, hence, block the sodium-transporting capacity of alveolar epithelial cells. Using animal and isolated organ models of edema resolution, we demonstrate that TGF-β, applied at clinically relevant doses, rapidly blocked the transepithelial ion fluxes necessary for alveolar fluid reabsorption, and indeed alveolar fluid reabsorption itself. Given the rapid onset and progression of ARDS and the critical role played by ENaC-mediated alveolar fluid clearance in the survival of ARDS patients, the pathway described here has important implications for the understanding of the pathological mechanisms that promote formation or persistence of alveolar edema in ARDS patients. This idea is highlighted by the findings reported here that identify TGF-β, exclusively, as the factor in the lung fluids of ARDS patients responsible for promoting loss of ENaC from the lung epithelial cell surface. In addition to revealing an entirely unique TGF-β signaling pathway that regulates ion channel trafficking, these data point to a pathway that may be amenable to pharmacological manipulation in patients with ARDS, a devastating and lethal syndrome for which no pharmacological therapy currently exists.     

Active Nanofibrous Membrane Effects on Gingival Cell Inflammatory Response

Alpha-melanocyte stimulating hormone (α-MSH) is involved in normal skin wound healing and also has anti-inflammatory properties. The association of α-MSH to polyelectrolyte layers with various supports has been shown to improve these anti-inflammatory properties. This study aimed to evaluate the effects of nanofibrous membrane functionalized with α-MSH linked to polyelectrolyte layers on gingival cell inflammatory response. Human oral epithelial cells (EC) and fibroblasts (FB) were cultured on plastic or electrospun Poly-ε-caprolactone (PCL) membranes with α-MSH covalently coupled to Poly-L-glutamic acid (PGA-α-MSH), for 6 to 24 h. Cells were incubated with or without Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). Cell proliferation and migration were determined using AlamarBlue test and scratch assay. Expression of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and transforming growth factor-beta (TGF-β) was evaluated using RT-qPCR method. Cell cultures on plastic showed that PGA-α-MSH reduced EC and FB migration and decreased IL-6 and TGF-β expression in Pg-LPS stimulated EC. PGA-α-MSH functionalized PCL membranes reduced proliferation of Pg-LPS stimulated EC and FB. A significant decrease of IL-6, TNF-α, and TGF-β expression was also observed in Pg-LPS stimulated EC and FB. This study showed that the functionalization of nanofibrous PCL membranes efficiently amplified the anti-inflammatory effect of PGA-α-MSH on gingival cells.     

Maternal imbalance between pro-angiogenic and anti-angiogenic factors in HIV-infected women with pre-eclampsia

Abstract

Angiogenic imbalance contributes to the development of preeclampsia. We evaluated the protein expression of the proangiogenic placental growth factor (PlGF) and transforming growth factor beta 1 (TGF-β₁) compared with the anti-angiogenic soluble fms-like tyrosine kinase receptor (sFlt1) and soluble endoglin (sEng) in HIV-infected normotensive and pre-eclamptic pregnancies.Blood was obtained from 110 pregnant women, enrolled in four groups, namely, HIV-negative normotensives (27); HIV-positive normotensives (31); HIV-negative pre-eclamptics (27) and HIV-positive pre-eclamptics (25), and was used to measure PlGF, TGF-β1, sFlt1 and sEng levels.Increased sFlt1 and sEng levels were associated with the pre-eclamptics (HIV negative and positive) compared with their counterparts. Decreased PlGF levels were observed between the HIV-negative pre-eclamptics versus HIV-negative normotensives, but levels differed significantly (p = 0.02) among the normotensives (HIV negative and positive). TGF-β₁ remained unchanged across all groups. Higher sEng/TGF-β₁ ratios were associated with the pre-eclamptics (HIV negative and positive) compared with their counterparts. This study demonstrated increased sFlt1 and sEng levels in pre-eclamptic compared with normotensive pregnancies, irrespective of the HIV status.