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Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that stimulates glucose-mediated insulin production by pancreatic beta cells. It is also associated with protective effects in multiple tissues. GLP-1 receptor is highly expressed in pulmonary tissue, hinting possible pulmonary delivery of GLP-1 drugs. However, little is known about the role of GLP-1 signaling in the lung, especially in mucus hypersecretory obstructive lung diseases. Here, we showed that treatment with exendin-4, a clinically available GLP-1 receptor agonist, up-regulates mucin expression in normal airway epithelial cells and in the lung of normal mice, indicating mucus stimulatory effect of GLP-1 under physiological condition. Exendin-4 also increased mucin expression in in vitro cellular and in vivo murine models of obstructive lung diseases via the activation of p38 MAP kinase. Notably, mucin induction in vivo exacerbated key pulmonary abnormalities including emphysematous phenotypes, implying that GLP-1 signaling in the lung is detrimental under pulmonary obstructive condition. Another GLP-1 receptor agonist liraglutide had similar induction of mucin. Together, our studies not only demonstrate novel physiological and pathological roles of GLP-1 in the lung but may also caution against the clinical use of inhaled GLP-1 receptor agonists in the patients with obstructive lung diseases.

Regulatory and effector T helper (Th) cells are abundant at mucosal surfaces, especially in the intestine, where they control the critical balance between tolerance and inflammation. However, the key factors that reciprocally dictate differentiation along these specific lineages remain incompletely understood. Here we report that the interleukin-1 (IL-1) family member IL-36γ signals through IL-36 receptor, myeloid differentiation primary response gene 88, and nuclear factor-κBp50 in CD4+ T cells to potently inhibit Foxp3-expressing induced regulatory T cell (Treg) development, while concomitantly promoting the differentiation of Th9 cells via a IL-2-STAT5- (signal transducer and activator of transcription factor 5) and IL-4-STAT6-dependent pathway. Consistent with these findings, mice deficient in IL-36γ were protected from Th cell-driven intestinal inflammation and exhibited increased colonic Treg cells and diminished Th9 cells. Our findings thus reveal a fundamental contribution for the IL-36/IL-36R axis in regulating the Treg-Th9 cell balance with broad implications for Th cell-mediated disorders, such as inflammatory bowel diseases and particularly ulcerative colitis.

Since the inner ear is an important but sensitive organ of hearing, bacteria-driven immune responses must be tightly controlled. In this issue of Cell Host & Microbe, Jung et al. (2019) demonstrate that cleaved cochlin plays a dual role to trap bacteria and recruit immune cells to improve ear function.

Cystic fibrosis transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel, and its dysfunction, due to CFTR gene mutations, causes the lethal inherited disorder cystic fibrosis (CF). To date, widespread dysregulation of certain coding genes in CF airway epithelial cells is well studied and considered as the driver of pulmonary abnormality. However, the involvement of non-coding genes, novel classes of functional RNAs with little or no protein-coding capacity, in the regulation of CF-associated gene dysregulation is poorly understood. Here, we utilized integrative analyses of human transcriptome array (HTA) and characterized 99 coding and 91 non-coding RNAs that are dysregulated in CFTR-defective CF bronchial epithelial cell line CFBE41o-. Among these genes, the expression level of linc-SUMF1-2, an intergenic non-coding RNA (lincRNA) whose function is unknown, was inversely correlated with that of WT-CFTR and consistently higher in primary human CF airway epithelial cells (DHBE-CF). Further integrative analyses under linc-SUMF1-knockdown condition determined MXRA5, SEMA5A, CXCL10, AK022877, CTGF, MYC, AREG and LAMB3 as both CFTR- and linc-SUMF1-2-dependent dysregulated gene sets in CF airway epithelial cells. Overall, our analyses reveal linc-SUMF1-2 as a dysregulated non-coding gene in CF as well as CFTR-linc-SUMF1-2 axis as a novel regulatory pathway involved in CF-associated gene dysregulation.

Airway mucus hyperproduction and fluid imbalance are important hallmarks of cystic fibrosis (CF), the most common life-shortening genetic disorder in Caucasians. Dysregulated expression and/or function of airway ion transporters, including cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), have been implicated as causes of CF-associated mucus hypersecretory phenotype. However, the contributory roles of other substances and transporters in the regulation of CF airway pathogenesis remain unelucidated. Here, we identified a novel connection between CFTR/ENaC expression and the intracellular Zn2 + concentration in the regulation of MUC5AC, a major secreted mucin that is highly expressed in CF airway. CFTR-defective and ENaC-hyperactive airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the zinc importer ZIP2/SLC39A2 (ΔC-ZIP2), which lacks the C-terminal domain. Importantly, ΔC-ZIP2 levels correlated inversely with wild-type ZIP2 and intracellular Zn2 + levels. Moreover, the splice switch to ΔC-ZIP2 as well as decreased expression of other ZIPs caused zinc deficiency, which is sufficient for induction of MUC5AC; while ΔC-ZIP2 expression per se induced ENaC expression and function. Thus, our findings demonstrate that the novel splicing switch contributes to CF lung pathology via the novel interplay of CFTR, ENaC, and ZIP2 transporters., Highlights • Zinc deficiency is a common feature in both CFTR-defective (CF) and ENaC-hyperactive (CF-like) airway epithelial cells. • A splice switch from WT-ZIP2 to ΔC-ZIP2 as well as other ZIPs down-regulation caused zinc deficiency in CF and CF-like cells. • Lower intracellular Zn2 + levels contributed to CF-associated MUC5AC hypersecretion in airway epithelial cells. The role of zinc in the pathogenesis of CF lung disease is not well understood. We utilized human CF patient-derived cell lines and primary cells as well as murine CF model, and identified zinc deficiency as a common characteristic in CF models. Down-regulation of several zinc importers (ZIPs) in CF cells caused zinc deficiency, which is sufficient for induction of MUC5AC, a major secreted mucin that exacerbates CF pathogenesis. Especially, strong contribution of ΔC-ZIP2, a novel ZIP2 splice isoform, in the regulation of CF-associated MUC5AC hypersecretion was clearly demonstrated. The study refined the importance of zinc in airway homeostasis.

Background/Aims: Nogo-B, a member of the reticulon family of proteins, is mainly located in the endoplasmic reticulum (ER). Here, we investigate the function and mechanism of Nogo-B in the regulation of TLR4-associated immune responses in the macrophage cell line of RAW264.7. Methods: Nogo-B was up- and down-regulated through the use of appropriate adenoviral vectors or siRNA, and the effects of Nogo-B on macrophages under liposaccharide (LPS) stimulation were evaluated via western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), flow cytometric analysis, and transwell assay. Results: Our data indicates that the protein of Nogo-B was down-regulated in a time- and dose-dependent manner following LPS administration in the macrophage. Nogo-B overexpression increased the production of inflammatory cytokines (MCP-1, TNF-α, IL-1β, and TGF-β), enhanced macrophage migration activities, activated major histocompatibility complex II (MHC II), and elevated the expression of macrophage scavenger receptor 1(MSR1), all of which suggest that Nogo-B is necessary for immune responses and plays an important role in regulating macrophage recruitment. Mechanistically, Nogo-B may enhance TLR4 expression in macrophage surfaces, activate mitogen-activated protein kinase (MAPK) pathways, and initiate inflammatory responses. Conclusion: These findings illustrate the key regulatory functions of Nogo-B in facilitating LPS-mediated immune responses through promoting the phosphorylation of MAP kinase.

Heart failure (HF) is characterized by cardiac dysfunction along with autonomic unbalance that is associated with increased renin-angiotensin system (RAS) activity and elevated levels of proinflammatory cytokines (PICs). Renal denervation (RD) has been shown to improve cardiac function in HF, but the protective mechanisms remain unclear. The present study tested the hypothesis that RD ameliorates isoproterenol- (ISO-) induced HF through regulation of brain RAS and PICs. Chronic ISO infusion resulted in remarked decrease in blood pressure (BP) and increase in heart rate and cardiac dysfunction, which was accompanied by increased BP variability and decreased baroreflex sensitivity and HR variability. Most of these adverse effects of ISO on cardiac and autonomic function were reversed by RD. Furthermore, ISO upregulated mRNA and protein expressions of several components of the RAS and PICs in the lamina terminalis and hypothalamic paraventricular nucleus, two forebrain nuclei involved in cardiovascular regulations. RD significantly inhibited the upregulation of these genes. Either intracerebroventricular AT1-R antagonist, irbesartan, or TNF-αinhibitor, etanercept, mimicked the beneficial actions of RD in the ISO-induced HF. The results suggest that the RD restores autonomic balance and ameliorates ISO-induced HF and that the downregulated RAS and PICs in the brain contribute to these beneficial effects of RD.

Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense; however, an overactive response is detrimental to the host. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in normal development and leukemogenesis. However, its role in regulating NF-κB–dependent inflammation remains unknown. In this article, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae- and TNF-α–induced NF-κB–dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA-binding activity. Moreover, expression of EVI1 itself is induced by nontypeable Haemophilus influenzae and TNF-α in an NF-κB–dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB–dependent inflammation. Thus, our study provides important insights into the novel role for EVI1 in negatively regulating NF-κB–dependent inflammation, and it may also shed light on the future development of novel anti-inflammatory strategies.

The phenotypic change of vascular smooth muscle cells (VSMCs), from a 'contractile' phenotype to a 'synthetic' phenotype, is crucial for pathogenic vascular remodeling in vascular diseases such as atherosclerosis and restenosis. Ca(2+)/calmodulin-stimulated phosphodiesterase 1 (PDE1) isozymes, including PDE1A and PDE1C, play integral roles in regulating the proliferation of synthetic VSMCs. However, the underlying molecular mechanism(s) remain unknown. In this study, we explore the role and mechanism of PDE1 isoforms in regulating β-catenin/T-cell factor (TCF) signaling in VSMCs, a pathway important for vascular remodeling through promoting VSMC growth and survival. We found that inhibition of PDE1 activity markedly attenuated β-catenin/TCF signaling by downregulating β-catenin protein. The effect of PDE1 inhibition on β-catenin protein reduction is exerted via promoting glycogen synthase kinase 3 (GSK3)β activation, β-catenin phosphorylation and subsequent β-catenin protein degradation. Moreover, PDE1 inhibition specifically upregulated phosphatase protein phosphatase 2A (PP2A) B56γ subunit gene expression, which is responsible for the effects of PDE1 inhibition on GSK3β and β-catenin/TCF signaling. Furthermore, the effect of PDE1 inhibition on β-catenin was specifically mediated by PDE1A but not PDE1C isozyme. Interestingly, in synthetic VSMCs, PP2A B56γ, phospho-GSK3β and phospho-β-catenin were all found in the nucleus, suggesting that PDE1A regulates nuclear β-catenin protein stability through the nuclear PP2A-GSK3β-β-catenin signaling axis. Taken together, these findings provide direct evidence for the first time that PP2A B56γ is a critical mediator for PDE1A in the regulation of β-catenin signaling in proliferating VSMCs.

PKCζ has emerged as a pathologic mediator of endothelial cell dysfunction, based on its essential role in tumor necrosis factor α (TNFα)–mediated inflammation. In contrast, extracellular signal–regulated kinase 5 (ERK5) function is required for endothelial cell homeostasis as shown by activation of Krüppel-like factor 2 (KLF2), increased endothelial nitric-oxide synthase (eNOS) expression, and inhibition of apoptosis. We hypothesized that protein kinase C ζ (PKCζ) activation by TNFα would inhibit the ERK5/KLF2/eNOS pathway. TNFα inhibited the steady laminar flow–induced eNOS expression, and this effect was reversed by the dominant-negative form of PKCζ (Ad.DN-PKCζ). In addition, ERK5 function was inhibited by either TNFα or the transfection of the catalytic domain of PKCζ. This inhibition was reversed by PKCζ small interfering RNA. PKCζ was found to bind to ERK5 under basal conditions with coimmunoprecipitation and the mammalian 2-hybrid assay. Furthermore, PKCζ phosphorylates ERK5, and mutation analysis showed that the preferred site is S486. Most importantly, we found that the predominant effect of TNFα stimulation of PKCζ was to decrease eNOS protein stability that was recapitulated by transfecting Ad.ERK5S486A mutant. Finally, aortic en face analysis of ERK5/PKCζ activity showed high PKCζ and ERK5 staining in the athero-prone region. Taken together our results show that PKCζ binds and phosphorylates ERK5, thereby decreasing eNOS protein stability and contributing to early events of atherosclerosis.

Background: Increasing evidence has suggested that differentiation of adventitial fibroblasts (AFs) to myofibroblasts plays an important role in arterial remodeling. The molecular mechanisms by which myofibroblast formation is regulated still remain largely unknown. This study aimed to evaluate the role of cyclic nucleotide phosphodiesterase 1A (PDE1A) in the formation of adventitial myofibroblasts induced by transforming growth factor (TGF)-β1. Methods and Results: AFs were cultured by the explant method. Western blot and immunocytochemistry were applied for α-smooth muscle actin (SMA) or protein kinase C (PKC) α protein analysis. Results showed that TGF-β1 upregulated PDE1A protein expression in rat aortic AFs and pharmacological inhibition of PDE1A blocked TGF-β1-induced α-SMA expression, a marker of myofibroblast formation, suggesting that the upregulation of PDE1A may mediate TGF-β1-induced AF transformation. Moreover, calphostin C (a PKC inhibitor) inhibited TGF-β1-induced α-SMA expression, whereas phorbol-12-myristate-13-acetate (a PKC activator) induced it. Finally, the upregulation of PKCα expression by TGF-β1 was also inhibited by PDE1A inhibition. Conclusions: Taken together, our data suggest that TGFβ1 induces α-SMA expression and myofibroblast formation via a PDE1A-PKCα-dependent mechanism. Our study thus unveils a novel signaling mechanism underlying TGF-β1-induced adventitial myofibroblast formation.

The activation of transcription factor NF-kappaB is controlled by two main pathways: the classical canonical (RelA/p65-p50)- and the alternative noncanonical (RelB/p52)-NF-kappaB pathways. RelB has been shown to play a protective role in RelA/p65-mediated proinflammatory cytokine release in immune-inflammatory lymphoid cells. Increased infiltration of macrophages and lymphoid cells occurs in lungs of patients with chronic obstructive pulmonary disease, leading to abnormal inflammation. We hypothesized that RelB, and its signaling pathway, is differentially regulated in macrophages and B cells and in lung cells, leading to differential regulation of proinflammatory cytokines in response to cigarette smoke (CS). CS exposure increased the levels of RelB and NF-kappaB-inducing kinase associated with recruitment of RelB on promoters of the IL-6 and macrophage inflammatory protein-2 genes in mouse lung. Treatment of macrophage cell line, MonoMac6, with CS extract showed activation of RelB. In contrast, RelB was degraded by a proteasome-dependent mechanism in B lymphocytes (human Ramos, mouse WEHI-231, and primary mouse spleen B cells), suggesting that RelB is differentially regulated in lung inflammatory and lymphoid cells in response to CS exposure. Transient transfection of dominant negative IkappaB-kinase-alpha and double mutants of NF-kappaB-inducing kinase partially attenuated the CS extract-mediated loss of RelB in B cells and normalized the increased RelB level in macrophages. Taken together, these data suggest that RelB is differentially regulated in response to CS exposure in macrophages, B cells, and in lung cells by IkappaB-kinase-alpha-dependent mechanism. Rapid degradation of RelB signals for RelA/p65 activation and loss of its protective ability to suppress the proinflammatory cytokine release in lymphoid B cells.

Glucocorticoids are highly effective in the control of many inflammatory and immune diseases. Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, the molecular basis for the inhibitory effect of glucocorticoids on mucin overproduction, a hallmark of chronic respiratory diseases, still remains unclear. Here we show that glucocorticoids markedly inhibit up-regulation of MUC5AC induced by NTHi, a major human bacterial pathogen causing chronic obstructive pulmonary disease and otitis media. Inhibition of NTHi-induced MUC5AC expression by dexamethasone occurs at the level of p38 MAPK via glucocorticoid receptor. Moreover, glucocorticoids up-regulate MKP-1 expression, which in turn leads to p38 dephosphorylation and the subsequent inhibition of NTHi-induced MUC5AC expression. These studies provide new insight into the molecular mechanism underlying glucocorticoid therapy and may lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with NTHi infections.

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.

Gram-positive bacterium Streptococcus pneumoniae is an important human pathogen that colonizes the upper respiratory tract and is also the major cause of morbidity and mortality worldwide. S. pneumoniae causes invasive diseases such as pneumonia, meningitis, and otitis media. Despite the importance of pneumococcal diseases, little is known about the molecular mechanisms by which S. pneumoniae-induced inflammation is regulated, especially the negative regulatory mechanisms. Here we show that S. pneumoniae activates nuclear factor of activated T cells (NFAT) signaling pathway and the subsequent up-regulation of inflammatory mediators via a key pneumococcal virulence factor, pneumolysin. We also demonstrate that S. pneumoniae activates NFAT transcription factor independently of Toll-like receptors 2 and 4. Moreover, S. pneumoniae induces NFAT activation via both Ca2+-calcineurin and transforming growth factor-β-activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase (MKK) 3/6-p38α/β-dependent signaling pathways. Interestingly, we found for the first time that tumor suppressor cylindromatosis (CYLD) acts as a negative regulator for S. pneumoniae-induced NFAT signaling pathway via a deubiquitination-dependent mechanism. Finally, we showed that CYLD interacts with and deubiquitinates TAK1 to negatively regulate the activation of the downstream MKK3/6-p38α/β pathway. Our studies thus bring new insights into the molecular pathogenesis of S. pneumoniae infections through the NFAT-dependent mechanism and further identify CYLD as a negative regulator for NFAT signaling, thereby opening up new therapeutic targets for these diseases.

Mucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium P. aeruginosa, an important human respiratory pathogen causing cystic fibrosis, utilizes reactive oxygen species (ROS) to up-regulate MUC5AC mucin expression. Pseudomonas aeruginosa lipopolysaccharide (PA-LPS) induces production of ROS through protein kinase C (PKC)-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases transforming growth factor-alpha (TGF-alpha), which in turn, leads to up-regulate MUC5AC expression. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with P. aeruginosa infections.

The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10–1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis–dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

In review of the past studies on NF-kappaB regulation, most of them have focused on investigating how NF-kappaB is activated by a single inducer at a time. Given the fact that, in mixed bacterial infections in vivo, multiple inflammation inducers, including both nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae, are present simultaneously, a key issue that has yet to be addressed is whether NTHi and S. pneumoniae simultaneously activate NF-kappaB and the subsequent inflammatory response in a synergistic manner. Here, we show that NTHi and S. pneumoniae synergistically induce NF-kappaB-dependent inflammatory response via activation of multiple signaling pathways in vitro and in vivo. The classical IKKbeta-IkappaBalpha and p38 MAPK pathways are involved in synergistic activation of NF-kappaB via two distinct mechanisms, p65 nuclear translocation-dependent and -independent mechanisms. Moreover, casein kinase 2 (CK2) is involved in synergistic induction of NF-kappaB via a mechanism dependent on phosphorylation of p65 at both Ser536 and Ser276 sites. These studies bring new insights into the molecular mechanisms underlying the NF-kappaB-dependent inflammatory response in polymicrobial infections and may lead to development of novel therapeutic strategies for modulating inflammation in mixed infections for patients with otitis media and chronic obstructive pulmonary diseases.

The transforming growth factor beta (TGF-beta) pathway represents an important signaling pathway involved in the regulation of diverse biological processes, including cell proliferation, differentiation, and apoptosis. Despite the known role of TGF-betaR-mediated signaling in suppressing immune response, its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here, we show for the first time that TGF-betaR-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacterium nontypeable Hemophilus influenzae (NTHi) in vitro and in vivo. The positive regulation of TLR2 induction by TGF-betaR is mediated via a dual mechanism involving distinct signaling pathways. One mechanism involves functional cooperation between the TGF-betaR-Smad3/4 pathway and NF-kappaB pathway. Another involves MAP kinase phosphatase 1 (MKP-1)-dependent inhibition of p38 MAPK, a known negative regulator for TLR2 induction. Moreover, we showed that TbetaR-mediated signaling is probably activated by NTHi-derived TGF-beta mimicry molecule via an autocrine-independent mechanism. Thus, our study provides new insights into the role of TGF-beta signaling in positively regulating host defense response by tightly controlling the expression level of TLR2 during bacterial infections and may lead to new therapeutic strategies for modulating host defense and inflammatory response.

Toll-like receptor 2 (TLR2) plays an important role in host defense against bacterial pathogens. Activation of TLR2 signaling not only induces the activation of innate immunity and instructs the development of the acquired immunity but also leads to the detrimental inflammatory responses in inflammatory and infectious diseases. To avoid detrimental inflammatory responses, TLR2 signaling must be tightly regulated. In contrast to the relative known positive regulation of TLR2 signaling, its negative regulation, however, is largely unknown. In addition the distal signaling components that link TLR2 to its downstream signaling pathways have yet to be further defined. In the present study we have provided direct evidence for the negative regulation of TLR2 signaling by the tumor suppressor cylindromatosis (CYLD). We showed that activation of TLR2 signaling by TLR2 ligands including peptidoglycan (PGN), MALP-2, and Pam3CSK4 induces activation of IKKs-IkappaBalpha and MKK3/6-p38 pathways not only by TRAF6 but also by TRAF7, a recently identified TRAF family member. The activation of both pathways leads to the transcription of TNF-alpha, IL-1beta, and IL-8 as well as CYLD. CYLD in turn leads to the inhibition of TRAF6 and TRAF7 likely via a deubiquitination-dependent mechanism. The present studies thus unveil a novel autoregulatory feedback mechanism that negatively controls TLR2-IKKs-IkappaBalpha/MKK3/6-p38-NF-kappaB-dependent induction of immune and inflammatory responses via negatively cross-talking with both TRAF6 and TRAF7. These findings provide novel insights into autoregulation and negative regulation of TLR signaling.

Epidermal growth factor receptor (EGFR) has been shown to play important roles in regulating diverse biological processes, including cell growth, differentiation, apoptosis, adhesion, and migration. Its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here we show for the first time that EGFR acts as a negative regulator for TLR2 induction by the bacterium nontypeable Haemophilus influenzae (NTHi) in vitro and in vivo. The negative regulation of TLR2 induction by EGFR is mediated via an Src-MKK3/6-p38 alpha/beta MAP kinase-dependent mechanism. Moreover, direct activation of EGFR signaling by the bacterium NTHi-derived EGF-like factor appears to be responsible for triggering the downstream Src-MKK3/6-p38 MAPK signaling, which in turn leads to the negative regulation of TLR2 induction. Finally, exogenous EGF increases NTHi invasion of host epithelial cells, thereby demonstrating the biological significance of TLR2 regulation by EGFR signaling. The evidence we provided in the present study may suggest a novel strategy utilized by bacteria to attenuate host defensive and immune response by negatively regulating the expression of host defense receptor TLR2. These studies may bring new insight for fully understanding the important role of EGFR signaling in regulating host defense and immune response by tightly controlling TLR2 induction during bacterial infections.

The molecular mechanisms responsible for TNF-alpha-mediated MUC2 intestinal mucin up-regulation in HM3 colon adenocarcinoma cells were analyzed using promoter-reporter assays of the 5'-flanking region of the MUC2 gene. Chemical inhibitors, mutant reporter constructs, and EMSA confirmed I-kappaB/NF-kappaB pathway involvement. Wortmannin, LY294002 and dominant negative Akt, as well as dominant negative NF-kappaB-inducing kinase (NIK) inhibited MUC2 reporter transcription, indicating that both phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathway and NIK pathways mediate the effects of TNF-alpha. Wortmannin inhibited NF-kappaB binding and transcriptional activity without inhibiting NF-kappaB translocation to the nucleus, indicating that PI3K/Akt signaling activates NF-kappaB transcriptional activity directly. Our results demonstrate that TNF-alpha up-regulates MUC2 in human colon epithelial cells via several signaling pathways, involving both NIK and PI3K/Akt, which converge at the common IKK/I-kappaB/NF-kappaB pathway. TNF-alpha activated JNK, but JNK inhibitor SP600125 and dominant negative cJun consistently activated transcription, revealing a negative role for this signaling pathway. Thus TNF-alpha causes a net up-regulation of MUC2 gene expression in cultured colon cancer cells because NF-kappaB transcriptional activation of this gene is able to counter-balance the suppressive effects of the JNK pathway. However, the existence of this inhibitory JNK pathways suggests a mechanism whereby--in the absence of NF-kappaB activation--TNF-alpha production during inflammation in vivo could actually inhibit MUC2 production, giving rise to the defective mucosal protection which characterizes inflammatory bowel disease.

Mucin overproduction is a hallmark of nontypeable Haemophilus influenzae (NTHi) infections. The molecular mechanisms underlying up-regulation of mucin in NTHi infections especially during the initial phase remain unknown. Here we show that P6, a 16-kDa outer membrane lipoprotein well conserved in NTHi, up-regulates MUC5AC mucin gene transcription in vitro and in vivo. Moreover, P6 induces MUC5AC transcription via TLR2-MyD88-IRAK1-TRAF6-TAK1-dependent p38 MAPK-AP1 and IKKbeta-IkappaBalpha-NF-kappaB signaling pathways. This study may bring new insights into the molecular pathogenesis of NTHi-induced infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with NTHi infections.

The transcription factor NF-kappaB regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-kappaB are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-kappaB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-kappaB essential modulator, also known as IkappaB kinase gamma). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-kappaB by TNF-alpha and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-kappaB signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-kappaB-dependent induction of CYLD by TNF-alpha and NTHi. These findings provide novel insights into the autoregulation of NF-kappaB activation.

Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen causing otitis media in children and exacerbation of chronic obstructive pulmonary disease in adults. Like most other bacterial infections, NTHi infections are also characterized by inflammation, which is mainly mediated by cytokines and chemokines such as tumor necrosis factor α (TNF-α). Among a variety of transcription regulators, NF-κB has been shown to play a critical role in regulating the expression of large numbers of genes encoding inflammatory mediators. In review of the current studies on NF-κB regulation, most of them have focused on investigating how NF-κB is activated by a single inducer at a time. However, in bacteria-induced inflammation in vivo , multiple inducers including both exogenous and endogenous mediators are present simultaneously. A key issue that has yet to be addressed is whether the exogenous inducers such as NTHi and the endogenous factors such as TNF-α activate NF-κB in a synergistic manner. We show that NTHi and TNF-α, when present together, synergistically induce NF-κB activation via two distinct signaling pathways: NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves NF-κB-inducing kinase-IκB kinase β/γ-dependent phosphorylation and degradation of IκBα, whereas the NF-κB translocation-independent pathway involves mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 1-dependent activation of MAPK kinase 3/6-p38 MAPK pathway. In addition, the same signaling pathways are also involved in synergistic induction of TNF-α, IL-1β, and IL-8. These studies should deepen our understanding of the molecular mechanisms underlying the combinatorial regulation of inflammation and lead to development of therapeutic strategies for NTHi-induced infections.

The human β-defensin 2 (HBD 2) is a potent anti-bacterial peptide with a wide spectrum of activity. MEF (myeloid elf-1-like factor) or Elf4, a member of the ETS transcription factor family, has been shown to up-regulate the basal expression of the HBD 2 gene in epithelial cells. The mammalian cell nucleus is organized into distinct nuclear domains, one of which is the PML (promyelocytic leukemia) nuclear body involved in the regulation of transcription. Here, we show that PML stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous HBD 2 expression. Keywords:: β-defensin 2, myeloid elf-1-like factor, promyelocytic leukemia nuclear body

Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. We have demonstrated recently that glucocorticoids synergistically enhance nontypeable Haemophilus influenzae (NTHi)-induced expression of Toll-like receptor 2 (TLR2), an important TLR family member that has been shown to play a critical role in host immune and defense response. However, the molecular mechanisms underlying the glucocorticoid-mediated enhancement of TLR2 induction still remain unknown. Here we show that glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAPK phosphatase-1 (MKP-1) that, in turn, leads to dephosphorylation and inactivation of p38 MAPK, the negative regulator for TLR2 expression. Moreover, increased expression of TLR2 in epithelial cells greatly enhances the NTHi-induced expression of several key cytokines, including tumor necrosis factor-alpha and interleukins 1beta and 8, thereby contributing significantly to host immune and defense response. These studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPKs.

Transforming growth factor-beta (TGF-beta) and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response. The involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin, a primary innate defensive response for mammalian airways, however, still remains unknown. Here, we report that the bacterium nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, utilizes the TGF-beta-Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKKbeta/gamma-IkappaBalpha pathway to mediate NF-kappaB-dependent MUC2 mucin transcription. The NTHi-induced TGF-beta receptor Type II phosphorylation occurred at as early as 5 min. Pretreatment of NTHi with TGF-beta neutralization antibody reduced up-regulation of MUC2 transcription. Moreover, functional cooperation of NF-kappaB p65/p50 with Smad3/4 appears to positively mediate NF-kappaB-dependent MUC2 transcription. These data are the first to demonstrate the involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF-beta signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.

MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Src tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 up-regulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer.

beta-defensin 2 is produced by a variety of epithelial cell types in the body and exhibits potent antimicrobial activity against a variety of pathogens, including the bacteria that are most commonly associated with otitis media (OM). The human beta-defensin 2 (hBD-2) gene is an NF-kappa B regulated gene and a variety of proinflammatory stimuli can induce its expression. Although the presence of molecules of innate immunity such as lysozyme and lactoferrin has been demonstrated in the middle ear, to date there have been no reports on the expression of beta-defensin 2. In the present study, we demonstrate that beta-defensin 2 is expressed in the middle ear mucosa of humans and rats. We also show that it is expressed in a human middle ear epithelial cell line and that its expression is induced by proinflammatory stimuli such as interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS). Moreover, we demonstrate that the transcriptional activation of hBD-2 gene by IL-1 alpha is mediated through an Src-dependent Raf-MEK1/2-ERK signaling pathway.

Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that causes chronic otitis media with effusion (COME) in children and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms underlying mucin overproduction in NTHi infections still remain unclear. Here, we show that NTHi strongly up-regulates MUC5AC mucin transcription only after bacterial cell disruption. Maximal up-regulation is induced by heat-stable bacterial cytoplasmic proteins, whereas NTHi surface membrane proteins induce only moderate MUC5AC transcription. These results demonstrate an important role for cytoplasmic molecules from lysed bacteria in the pathogenesis of NTHi infections, and may well explain why many patients still have persistent symptoms such as middle ear effusion in COME after intensive antibiotic treatment. Furthermore, our results indicate that activation of p38 mitogen-activated protein kinase is required for NTHi-induced MUC5AC transcription, whereas activation of phosphoinositide 3-kinase-Akt pathway leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 mitogen-activated protein kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi infections and lead to novel therapeutic intervention for COME and COPD.

Nontypeable Haemophilus influenzae (NTHi), a Gram-negative bacterium, is the primary cause of otitis media in children and the exacerbation of chronic obstructive pulmonary disease in adults. A hallmark of both diseases is an overactive inflammatory response, including the upregulation of chemokines, such as interleukin-8 (IL-8). An appropriate inflammatory response is essential for eradicating pathogens. However, excessive inflammation can cause host tissue damage. Therefore, expression of IL-8 must be tightly regulated. We previously reported that NTHi induces IL-8 expression in an ERK-dependent manner. We also have shown that the deubiquitinase cylindromatosis (CYLD) suppresses NTHi-induced inflammation. However, the underlying molecular mechanism of how CYLD negatively regulates ERK-mediated IL-8 production is largely unknown. Here, we examine both human lung epithelial A549 cells and lung of Cyld −/− mice to show that CYLD specifically targets the activation of ERK. Interestingly, CYLD enhances NTHi-induced upregulation of another negative regulator, MAP Kinase Phosphatase-1 (MKP-1), which, in turn, leads to reduced ERK activation and subsequent suppression of IL-8. Taken together, the CYLD suppression of ERK-dependent IL-8 via MKP-1 may bring novel insights into the tight regulation of inflammatory responses and also lead to innovative therapeutic strategies for controlling these responses by targeting key negative regulators of inflammation.

Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-κB, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-κB in human epithelial cells via two distinct signaling pathways, NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves activation of NF-κB inducing kinase (NIK)–IKKα/β complex leading to IκBα phosphorylation and degradation, whereas the NF-κB translocation-independent pathway involves activation of MKK3/6–p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase pathways may occur at transforming growth factor-β activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-κB activation. In addition, several key inflammatory mediators including IL-1β, IL-8, and tumor necrosis factor-α are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-κB via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-κB via TLR2–TAK1-dependent NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.

Atherosclerosis, the major cause of myocardial infarction and stroke, is a chronic arterial disease characterized by lipid deposition and inflammation in the vessel wall. Cholesterol, in low-density lipoprotein (LDL), plays a critical role in the pathogenesis of atherosclerosis. Vinpocetine, a derivative of the alkaloid vincamine, has long been used as a cerebral blood flow enhancer for treating cognitive impairment. Recent study indicated that vinpocetine is a potent anti-inflammatory agent. However, its role in the pathogenesis of atherosclerosis remains unexplored. In the present study, we show that vinpocetine significantly reduced atherosclerotic lesion formation in ApoE knockout mice fed with a high-fat diet. In cultured murine macrophage RAW264.7 cells, vinpocetine markedly attenuated oxidized LDL (ox-LDL) uptake and foam cell formation. Moreover, vinpocetine greatly blocked the induction of ox-LDL receptor 1 (LOX-1) in cultured macrophages as well as in the LOX-1 level in atherosclerotic lesions. Taken together, our data reveal a novel role of vinpocetine in reduction of pathogenesis of atherosclerosis, at least partially through suppressing LOX-1 signaling pathway. Given the excellent safety profile of vinpocetine, this study suggests vinpocetine may be a therapeutic candidate for treating atherosclerosis.

Lysozyme is expressed in serous, but not mucous, cells of the tracheobronchial glands and thereby constitutes a marker of the serous cell lineage in these glands. To identify DNA regulatory elements and transcription factors mediating the commitment of progenitor cells to the serous cell lineage, we have characterized the regulatory activity and DNA-protein interactions of the 5'-flanking region of the bovine lysozyme gene lys 5a. Results obtained from these studies indicate that although approximately 94 bp of 5'flanking DNA are necessary for high level expression in transient transfection assays, an evolutionarily conserved promoter within 66 bp of the transcription start site is sufficient to confer serous cell-specific expression. Farther upstream, within 6.1 kb of the 5' flanking region, are 4 silencers. Analysis of the serous cell-specific lysozyme promoter by electrophoretic mobility shift assay (EMSA) revealed the presence of binding sites for 3 serous cell nuclear proteins, designated LSF1, LSF2 and LSF3. Binding of LSF2 and LSF3 was localized to a 20-mer subdomain (-50/-30) of the cell-specific promoter using binding competition assays. More accurate identification of the protein binding site(s) was achieved through the use of mutagenesis, which implicated the motif 5' AAGGAAT 3' (-46/-40) in both protein binding and serous cell-specific transcriptional activity. This motif has previously been identified as a binding site for ets protein transcription factors, suggesting that serous cell-specific regulation of lys 5a transcription is partly controlled by the binding of ets-like protein(s) to the motif 5'AGGAAGT3'.

Otitis media (OM) is the most common childhood bacterial infection and the major cause of conductive hearing loss in children. Mucus overproduction is a hallmark of OM. Streptococcus pneumoniae is the most common Gram-positive bacterial pathogen causing OM. Among many mucin genes, MUC5AC has been found to be greatly up-regulated in the middle ear mucosa of human patients with OM. We previously reported that S. pneumoniae up-regulates MUC5AC expression in a MAPK ERK-dependent manner. We also found that MAPK phosphatase-1 (MKP-1) negatively regulates S. pneumoniae-induced ERK-dependent MUC5AC up-regulation. Therapeutic strategies for up-regulating the expression of negative regulators such as MKP-1 may have significant therapeutic potential for treating mucus overproduction in OM. However, the underlying molecular mechanism by which MKP-1 expression is negatively regulated during S. pneumoniae infection is unknown. In this study we show that phosphodiesterase 4B (PDE4B) mediates S. pneumoniae-induced MUC5AC up-regulation by inhibiting the expression of a negative regulator MKP-1, which in turn leads to enhanced MAPK ERK activation and subsequent up-regulation of MUC5AC. PDE4B inhibits MKP-1 expression in a cAMP-PKA-dependent manner. PDE4-specific inhibitor rolipram inhibits S. pneumoniae-induced MUC5AC up-regulation both in vitro and in vivo. Moreover, we show that PDE4B plays a critical role in MUC5AC induction. Finally, topical and post-infection administration of rolipram into the middle ear potently inhibited S. pneumoniae-induced MUC5AC up-regulation. Collectively, these data demonstrate that PDE4B mediates ERK-dependent up-regulation of mucin MUC5AC by S. pneumoniae by inhibiting cAMP-PKA-dependent MKP-1 pathway. This study may lead to novel therapeutic strategy for inhibiting mucus overproduction. Background: Mucus overproduction is a hallmark of otitis media (OM) induced by Streptococcus pneumoniae. Results: PDE4B mediates S. pneumoniae-induced MUC5AC up-regulation by inhibiting the expression of a negative regulator MKP-1. Conclusion: PDE4-specific inhibitor rolipram inhibits S. pneumoniae-induced MUC5AC up-regulation. Significance: Identifying PDE4B as a molecular target for inhibiting MUC5AC by up-regulating MKP-1 may have significant therapeutic potential for treating OM.

Respiratory systems are constantly being challenged by pathogens. Lung epithelial cells serve as a first line of defense against microbial pathogens by detecting pathogen-associated molecular patterns (PAMPs) and activating downstream signaling pathways, leading to a plethora of biological responses required for shaping both the innate and adaptive arms of the immune response. Acute-phase proteins (APPs), such as type 1 plasminogen activator inhibitor (PAI-1), play important roles in immune/inflammatory responses. PAI-1, a key regulator for fibrinolysis and coagulation, acts as an APP during acute phase response (APR) such as acute lung injury (ALI), inflammation, and sepsis. However, the role of PAI-1 in the pathogenesis of these diseases still remains unclear, especially in bacterial pneumonia. In this study, we showed that PAI-1 expression is upregulated following nontypeable Haemophilus influenzae (NTHi) infection. PAI-1 knockout (KO) mice failed to generate early immune responses against NTHi. Failure of generating early immune responses in PAI-1 KO mice resulted in reduced bacterial clearance and prolonged disease process, which in turn led to enhanced inflammation at late stage of infection. Moreover, we also found that NTHi induces PAI-1 via activation of TLR2–MyD88–MKK3–p38 MAPK signaling pathway. These data suggest that PAI-1 plays critical role in earl host defense response against NTHi infection. Our study thus reveals a novel role of PAI-1 in infection caused by NTHi, one of the most common gram-negative bacterial pathogens in respiratory systems.

In the host immune system, the leukocytes are often exposed to multiple pathogens including bacteria and viruses. The principal challenge for the host is to efficiently detect the invading pathogen and mount a rapid defensive response. Leukocytes recognize invading pathogens by directly interacting with pathogen-associated molecular patterns via Toll-like receptors (TLRs) expressed on the leukocyte surfaces. In this study, we provide direct evidence that bacterial LPS enhances the host antiviral response by up-regulating TLR3 expression in human peripheral blood monocytes and monocytic cell lines, THP1 cells. Moreover, LPS induces TLR3 expression via a TLR4-MyD88-IRAK-TRAF6-NF-κB-dependent signaling pathway. Interestingly, CYLD, an important deubiquitinase, acts as a negative regulator of TLR3 induction by LPS. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response; furthermore, it identifies CYLD for the first time as a critical negative regulator of bacterial LPS-induced response.

In the host immune system, leukocytes are often exposed to multiple inflammation inducers. NF-κB is of considerable importance in leukocyte function because of its ability to activate the transcription of many proinflammatory immediate-early genes. Tremendous efforts have been made toward understanding how NF-κB is activated by various inducers. However, most research on NF-κB regulation has been focused on understanding how NF-κB is activated by a single inducer. This is unlike the situation in the human immune system where multiple inflammation inducers, including both exogenous and endogenous mediators, are present concurrently. We now present evidence that the formylated peptide f-Met-Leu-Phe (fMLP), a bacterial chemoattractant, synergizes with TNFα to induce NF-κB activation and the resultant inflammatory response in vitro and in vivo. The mechanism of synergistic activation of NF-κB by bacterial fMLP and TNFα may be involved in the induction of RelA acetylation, which is regulated by p38 MAPK. Thus, this study provides direct evidence for the synergistic induction of NF-κB-dependent inflammatory responses by both exogenous and endogenous inducers. The ability of fMLP to synergize with TNFα and activate NF-κB represents a novel and potentially important mechanism through which bacterial fMLP not only attracts leukocytes but also directly contributes to inflammation by synergizing with the endogenous mediator TNFα.

Mucosal epithelial cells in the respiratory tract act as the first line of host innate defense against inhaled microbes by producing a range of molecules for clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping the inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in defective mucosal defenses. Thus, tight regulation of mucin production is critical for maintaining an appropriate balance between beneficial and detrimental outcomes. Among various mechanisms, negative regulation plays an important role in tightly regulating mucin production. Here we show that the PAK4-JNK signaling pathway acted as a negative regulator for Streptococcus pneumoniae pneumolysin-induced MUC5AC mucin transcription. Moreover pneumolysin also selectively induced expression of MKP1 via a TLR4-dependent MyD88-TRAF6-ERK signaling pathway, which inhibited the PAK4-JNK signaling pathway, thereby leading to up-regulation of MUC5AC mucin production to maintain effective mucosal protection against S. pneumoniae infection. These studies provide novel insights into the molecular mechanisms underlying the tight regulation of mucin overproduction in the pathogenesis of airway infectious diseases and may lead to development of new therapeutic strategies.

The TGF-beta (transforming growth factor-beta) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-beta in inflammatory responses, its role in regulating NF-kappaB (nuclear factor-kappaB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-beta1 synergizes with proinflammatory cytokine TNF-alpha (tumour necrosis factor-alpha) to induce NF-kappaB activation and the resultant inflammatory response in vitro and in vivo. TGF-beta1 synergistically enhances TNF-alpha-induced NF-kappaB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-alpha-induced RelA acetylation and DNA-binding activity by TGF-beta1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-beta in inflammatory responses and provides new insight into the regulation of NF-kappaB by TGF-beta signalling.

Toll-like receptors (TLRs) are important sensors of microbial pathogens and mediators of innate immune responses. Although the signal transduction of TLRs is well elucidated, their basal regulation is largely unexplored. Here we show that the tumor suppressor p53 positively regulates the transcription of TLR3, a receptor for viral double-stranded RNA and poly(I-C), by binding to the p53 site in the TLR3 promoter. TLR3 expression was lower in HCT116 p53(-/-) cells than in HCT116 p53(+/+) cells. Activation of p53 by 5-fluorouracil increased the TLR3 mRNA in epithelial cell lines with wild-type p53 but not in cell lines harboring mutant p53. Knockdown of p53 by small interfering RNA decreased the TLR3 expression. TLR3 mRNA was also lower in liver and intestine of p53(-/-) mice than in p53(+/+) mice. Furthermore, the poly(I-C)-induced phosphorylation of IkappaB-alpha, nuclear translocation of NF-kappaB, and phosphorylation of interferon regulatory transcription factor 3, were drastically reduced in HCT116 p53(-/-) cells, indicating a dysregulation of the two signaling pathways governed by TLR3. Consequently, induction of interleukin-8 and beta interferon after poly(I-C) stimulation was impaired in HCT116 p53(-/-) cells. These results suggest that p53 influences TLR3 expression and function and highlight a role of p53 in innate immune response in epithelial cells.

Shear stress–induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H 2 O 2 and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow–induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H 2 O 2 and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H 2 O 2 and AGE–mediated reduction of shear stress–mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H 2 O 2 in shear stress–induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction.

Mucin overproduction is a hallmark of chronic respiratory diseases (CRD) such as chronic obstructive pulmonary disease and asthma, and otitis media. Despite the fact that nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae are co-existing under these disease conditions, little is known about how NTHi and S. pneumoniae induce mucin overproduction. Here we show that NTHi and S. pneumoniae, when present together, synergistically induce MUC5AC mucin transcription. TLR2/4-MyD88-TAK1 signaling cascade transmits signal to regulate the synergistic induction of MUC5AC. The activation of MKK3/6-p38 and ERK MAPK pathways are required for the synergistic induction of MUC5AC. Moreover, S. pneumoniae synergizes with NTHi to induce MUC5AC expression via AP-1-dependent mechanism. Thus, our studies provide direct evidence for the synergistic induction of MUC5AC in mixed infections and bring novel insights into our understanding of molecular mechanisms underlying polymicrobial infections in CRD and OM.

The incidence of mixed viral/bacterial infections has increased recently because of the dramatic increase in antibiotic-resistant strains, the emergence of new pathogens, and the resurgence of old ones. Despite the relatively well-known role of viruses in enhancing bacterial infections, the impact of bacterial infections on viral infections remains unknown. In this study, we provide direct evidence that nontypeable Haemophilus influenzae (NTHi), a major respiratory bacterial pathogen, augments the host antiviral response by up-regulating epithelial Toll-like receptor 7 (TLR7) expression in vitro and in vivo. Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAK-TRAF6-IKK-NF-kappaB-dependent signaling pathway. Interestingly, CYLD, a novel deubiquitinase, acts as a negative regulator of TLR7 induction by NTHi. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response and further identifies CYLD for the first time as a critical negative regulator of host antiviral response.

Mucin, a major component of mucus, plays a critical role in host mucosal defense response by participating in mucociliary clearance. However, if overproduced, overproduced mucus leads to airway mucus obstruction and conductive hearing loss. Despite extensive studies that focus on investigating how MUC5AC mucin is regulated by one inducer at a time, how MUC5AC is synergistically regulated by multiple factors remains unknown. Here we provide direct evidence for the first time that bacterial pathogen NTHi and human growth factor EGF synergize with each other to potently up-regulate MUC5AC mucin transcription. Moreover, activation of both p38 and ERK is required for synergistic induction of MUC5AC by NTHi and EGF. Finally, PAK2 and PAK4 are differentially involved in this synergistic induction of MUC5AC by acting upstream of p38 and ERK. Our studies bring novel insights into our understanding of synergistic regulation of MUC5AC mucin by both pathological and physiological inducers.

Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348-517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian two-hybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.

NontypeableHaemophilus influenzae(NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of ≤3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.