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The most widely used influenza vaccines are prepared by chemical inactivation. However, chemical, especially formalin, treatment-induced modifications of the antigenic structure of the virus are frequently associated with adverse effects including low efficacy of protection, unexpected immune responses, or exacerbation of disease. Gamma-irradiation was suggested as an alternative influenza virus inactivation method due to its great features of completely inactivating virus while not damaging the structures of protein antigens, and cross-protective ability against heterologous strains. However, immunological features of gamma radiation-inactivated influenza vaccine have not been fully understood. In this study, we aimed to investigate the humoral and cellular immune responses of gamma radiation-inactivated influenza vaccine. The gamma irradiation-inactivated influenza vaccine (RADVAX(FluA)) showed complete viral inactivation but retained normal viral structure with functional activities of viral protein antigens. Intranasal immunization of RADVAX(FluA) provided better protection against influenza virus infection than formalin-inactivated influenza virus (FIV) in mice. RADVAX(FluA) greatly enhanced the production of virus-specific serum IgG and alveolar mucosal IgA, which effectively neutralized HA (hemagglutinin) and NA (neuraminidase) activities, and blocked viral binding to the cells, respectively. Further analysis of IgG subclasses showed RADVAX(FluA)-immunized sera had higher levels of IgG1 and IgG2a than those of FIV-immunized sera. In addition, analysis of cellular immunity found RADVAX(FluA) induced strong dendritic cells (DC) activation resulting in higher DC-mediated activation of CD8(+) T cells than FIV. The results support improved immunogenicity by RADVAX(FluA).

Background/aims Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-β1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-β1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. Methods We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. Results Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-β1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-β1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. Conclusion These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.

Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to β-lactams and for those who are pregnant.

Salmonella entericasubsp.entericaserovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Streptococcus pneumoniae is an important human pathogen responsible for more than 2 million deaths annually worldwide. The airway epithelium acts as the first-line of defense against pneumococcal infections by regulating acute inflammation against invading pneumococcus. Despite the intact adaptive immunity, failure in early defense due to loss of pattern recognition receptors (PRRs) and/or acute phase proteins (APPs) results in detrimental damage and death. C-reactive protein (CRP), the first found APP, is a member of the pentraxin family of proteins and an important soluble PRR for pneumococcus. CRP and another short pentraxin, serum amyloid P, are critical for acute defense against pneumococcal infection. However, the role of the long pentraxin PTX3 in regulating pneumococcal infections is unknown. In this study, PTX3 expression was upregulated by pneumococcus in epithelial cells and in lungs of mice. In addition, PTX3 potentiated pneumococcal inflammation; overexpression of PTX3 enhanced pneumococcus-induced cytokine expression, whereas knock-down of PTX3 with siPTX3 inhibited the cytokine expression. Furthermore, PTX3 deficiency indeed ameliorated acute inflammation and protected mice against death following pneumococcal infection. Pneumococcal toxin pneumolysin was responsible for PTX3 expression and upregulated PTX3 expression via JNK MAPK signaling. These data implicate PTX3 as a novel therapeutic target for the control of acute inflammation by pneumococcus.

Streptococcus pneumoniae is the most common respiratory bacterial pathogen among cases of community-acquired infection in young children, older adults, and individuals with underlying medical conditions. Although capsular polysaccharide-based pneumococcal vaccines have contributed to significant decrease in invasive pneumococcal infections, these vaccines have some limitations, including limited serotype coverage, lack of effective mucosal antibody responses, and high costs. In this study, we investigated the safety and immunogenicity of a live, whole-cell pneumococcal vaccine constructed by deleting the gene for prolipoprotein diacylglyceryl transferase (lgt) from the encapsulated pneumococcal strain TIGR4 (TIGR4Δlgt) for protection against heterologous pneumococcal strains. Pneumococcal strain TIGR4 was successfully attenuated by deletion of lgt, resulting in the loss of inflammatory activity and virulence. TIGR4Δlgt colonized the nasopharynx long enough to induce strong mucosal IgA and IgG2b-dominant systemic antibody responses that were cross-reactive to heterologous pneumococcal serotypes. Finally, intranasal immunization with TIGR4Δlgt provided serotype-independent protection against pneumococcal challenge in mice. Taken together, our results suggest that TIGR4Δlgt is an avirulent and attractive broad-spectrum pneumococcal vaccine candidate. More broadly, we assert that modulation of such “master” metabolic genes represents an emerging strategy for developing more effective vaccines against numerous infectious agents.

It has been well established that HMG-CoA reductase inhibitors (statins) cause adverse side effects in skeletal muscle ranging from mild to fatal myotoxicity upon dose, drug interaction, and exercise. However, the underlying mechanisms by which statins induce myotoxicity have not been fully addressed. Recent reports showed that statins induce endoplasmic reticulum (ER) stress and cell death in immune cells and myoblasts in vitro. Therefore, the goal of study is to investigate the molecular mechanism by which statins induce skeletal muscle cell death and myopathy via the regulation of ER stress. Biochemical data showed that TUDCA, an ER stress inhibitor, inhibited atorvastatin- and simvastatin-induced protein cleavages of PARP-1 and caspase-3, respectively. Actually, statin treatment activated marker proteins of unfolded protein responses (UPR) including ATF6, CHOP, and spliced XBP1 and these responses were inhibited by TUDCA. In addition, statin treatment induced mRNA levels of UPR marker genes, suggesting that statins activate ER stress in a transcriptional regulation. The physiological relevance of ER stress in statin-induced myopathy was demonstrated in a mouse model of myopathy, in which instillation of simvastatin and atorvastatin led to myopathy. Notably, the reduction of muscular endurance in response to statin instillation was significantly improved in TUDCA treating group compared to vehicle control group. Moreover, CHOP deficiency mice showed restoration of statin-induced reduction of muscular endurance, suggesting that statin induces myopathy via ER stress and in a CHOP-dependent manner. Taken together, these findings indicate that statins specifically induce myopathy in an ER stress-dependent manner, suggesting the therapeutic potential of ER stress regulation in preventing adverse effects of statin.

Recent studies have indicated that protease-activated receptor-1 (PAR-1) is involved in cytoprotective and anti-inflammatory responses in endothelial cells (ECs). However, the role of PAR-1 in laminar flow-mediated atheroprotective responses remains unknown. Herein, we investigated whether PAR-1 regulates laminar flow-mediated mechanotransduction in ECs. Confocal analysis showed that PAR-1 was internalized into early endosomes in response to laminar flow. In addition, flow cytometry analysis showed that cell surface expression of PAR-1 was reduced by laminar flow, suggesting that PAR-1 was activated in response to laminar flow. Depletion of PAR-1 using human PAR-1 siRNA inhibited unidirectional laminar flow-mediated actin stress fiber formation and cellular alignment as well as atheroprotective gene expressions in HUVECs. Moreover, PAR-1 knockdown inhibited laminar flow-stimulated eNOS phosphorylation, and inhibited the phosphorylations of Src, AMPK, ERK5 and HDAC5. Furthermore, PAR-1 depletion inhibited laminar flow-mediated anti-inflammatory responses as demonstrated by reduced TNFα-induced VCAM-1 expression and by monocyte adhesion to HUVECs, and prevented laminar flow-mediated anti-apoptotic response. An investigation of the role of PAR-1 in vasomotor modulation using mouse aortic rings revealed that acetylcholine-induced vasorelaxation was diminished in PAR-1 deficient mice compared to littermate controls. Taken together, these findings suggest that PAR-1 be viewed as a novel pharmacologic target for the treatment of vascular diseases, including atherosclerosis.

Idiopathic pulmonary fibrosis is a progressive and chronic lung disease of unknown cause. Pathologically, the interstitium of the lungs becomes thick and stiff, which eventually cause the symptom of breathlessness. It has been established that the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway plays a critical role in the pathogenesis of lung fibrosis. TGF-β1–mediated activation of the mitogen-activated protein kinase family affects Smad signaling. Extracellular signal–regulated kinase (ERK) 5, an atypical member of mitogen-activated protein kinase, promotes cardiac hypertrophy characterized with increased expression of fibrotic and extracellular matrix genes. However, the role of ERK5 in pulmonary fibrosis remains unknown. Herein, we investigated whether ERK5 regulates the pathogenesis of pulmonary fibrosis in both in vitro and in vivo systems. Pharmacological inhibition of mitogen activated protein kinase kinase 5/ERK5 with BIX02189 and depletion of ERK5 with siRNA-ERK5 inhibited TGF-β1–induced extracellular matrix production in lung epithelial cells and fibroblasts. Inhibition of ERK5 also blocked the TGF-β1 signal to Smad3 transcriptional activity. However, TGF-β1–induced Smad3 phosphorylation and nuclear translocation were not affected by inhibition of ERK5. Notably, ERK5 regulates TGF-β1–induced fibrogenic signaling via Smad3 acetylation. Furthermore, ERK5 inhibitor, BIX02189, inhibited lung fibrosis and improved survival rate in the bleomycin-induced lung fibrosis model. Our findings indicate that ERK5 plays a critical role in TGF-β1–induced pulmonary fibrosis via enhancing Smad3 acetylation. This study may lead to a novel therapeutic strategy for treating lung fibrosis.

Laminar flow protects from atherosclerosis in endothelium.Laminar flow induces Nrf2 activation dependent on ERK5 activation, leading to up-regulation of downstream genes of Nrf2.ERK5 requires Nrf2 activation to exert cytoprotective effect on HUVEC. ERK5 inhibitor BIX02189 regulates Nrf2 activation in vivo.Identifying ERK5 as a molecular target for regulating flow-mediating Nrf2-dependent gene expression may have significant therapeutic potential for treating atherosclerosis. Atherosclerosis is often observed in areas where disturbed flow is formed, whereas atheroprotective region is found in areas where steady laminar flow is developed. It has been reported that some genes activated by blood flow play important roles in vascular function and pathogenesis of atherosclerosis. Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction and disease under laminar flow. Krüppel-like factor 2 (KLF2) and NF-E2-related factor 2 (Nrf2) are major transcriptional factors that contribute to anti-atherogenic responses under laminar flow. Implication of ERK5 in laminar flow-mediated regulation of KLF2-dependent gene has been established, whereas the role of ERK5 in laminar flow-mediated activation of Nrf2 pathway has not been addressed yet. In this study, we found that the blockage of ERK5 either by genetic depletion with siRNA or by biochemical inactivation with a specific chemical compound inhibited laminar flow-induced up-regulation of Nrf2-dependent gene expressions, whereas activation of ERK5 increased transcriptional activity and nuclear translocation of Nrf2, which suggests that ERK5 mediates laminar flow-induced up-regulation of Nrf2-dependent gene expression. Further functional studies showed that ERK5 provides protection against oxidative stress-induced cytotoxicity dependent on Nrf2. Molecular interaction between ERK5 and Nrf2 was further induced by laminar flow. Finally, flow-dependent nuclear localization of Nrf2 was inhibited by BIX02189, a specific inhibitor of MEK5, in aorta of mice in vivo. Collectively, these data demonstrate that laminar flow-induced activation of ERK5-Nrf2 signal pathway plays a critical role for anti-inflammatory and anti-apoptotic mechanism in endothelial cells.

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.

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.

Significance Despite the critical role for myeloid differentiation factor 88 (MyD88) in mediating pathogen-induced innate and adaptive immune responses, how functional activity of MyD88 is tightly controlled remains unknown. Among various types of posttranslational modification, ubiquitination and deubiquitination of host signaling molecules play an important role in tightly regulating immune response. However, inducible ubiquitination, in particular proteolysis-independent polyubiquitination as well as deubiquitination of MyD88, remains largely unclear. Here, we demonstrate that deubiquitinase CYLD negatively regulates MyD88-mediated inflammation by directly interacting with MyD88 and deubiquitinating nontypeable Haemophilus influenzae -induced lysine 63-linked polyubiquitination of MyD88. Thus, our studies may not only bring insights into the negative regulation of Toll-like receptor–MyD88-dependent signaling but may also lead to the development of a previously unidentified therapeutic strategy for uncontrolled inflammation.

Epithelial cells represent the first line of host innate defense against invading microbes by elaborating a range of molecules involved in pathogen clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. How mucin is induced in upper respiratory Streptococcus pneumoniae infections is unknown. In this study, we show that pneumolysin is required for up-regulation of MUC5AC mucin via TLR4-dependent activation of ERK in human epithelial cells in vitro and in mice in vivo. Interestingly, a “second wave” of ERK activation appears to be important in mediating MUC5AC induction. Moreover, IκB kinase (IKK) α and IKKβ are distinctly involved in MUC5AC induction via an ERK1-dependent, but IκBα-p65- and p100-p52-independent, mechanism, thereby revealing novel roles for IKKs in mediating up-regulation of MUC5AC mucin by S. pneumoniae.

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.

We have identified a novel signaling pathway that leads to expression of matrix metalloproteinase-9 (MMP-9) in murine macrophages in response to the bacterial endotoxin, LPS. We showed that p38 kinase was essential for this induction and observed that LPS-induced MMP-9 expression was sensitive to rottlerin, a putative protein kinase Cδ (PKCδ) inhibitor. However neither infection with a retrovirus expressing a dominant negative mutant of PKCδ nor down-regulation of PKCδ by prolonged PMA treatment affected MMP-9 expression, thus excluding involvement of PKCδ. Interestingly, LPS-induced MMP-9 expression and p38 kinase phosphorylation were shown to be suppressed by the antioxidant N-acetylcysteine and the flavoenzyme inhibitor diphenyleneiodonium chloride, but not by pyrrolidine dithiocarbamate, an NF-κB inhibitor. In addition, LPS was found to induce the production of mitochondrial reactive oxygen species (ROS) and this effect was rottlerin-sensitive, suggesting an inhibitory effect of rottlerin on mitochondrial ROS. LPS-induced MMP-9 expression and p38 kinase phosphorylation were also inhibited by rotenone, a specific inhibitor of mitochondrial complex I, supporting the role of mitochondrial ROS in LPS signaling to MMP-9. Finally, we showed that the ROS-p38 kinase cascade targets the transcription factor AP-1. Taken together, our findings identify a ROS-p38 kinase-AP-1 cascade as a novel pathway mediating LPS signaling to MMP-9 expression in macrophages.

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.

Epidemiological studies indicate that methylglyoxal (MGO) plasma levels are closely linked to diabetes and the exacerbation of diabetic cardiovascular complications. Recently, it was established that endoplasmic reticulum (ER) stress importantly contributes to the pathogenesis of diabetes and its cardiovascular complications. The objective of this study was to explore the mechanism by which diabetes instigates cardiomyocyte apoptosis and cardiac dysfunction via MGO-mediated myocyte apoptosis. Intriguingly, the MGO activated unfolded protein response pathway accompanying apoptotic events, such as cleavages of PARP-1 and caspase-3. In addition, Western blot analysis revealed that MGO-induced myocyte apoptosis was inhibited by depletion of CHOP with siRNA against Ddit3, the gene name for rat CHOP. To investigate the physiologic roles of CHOP in vivo, glucose tolerance and cardiac dysfunction were assessed in CHOP-deficient mice. No significant difference was observed between CHOP KO and littermate naive controls in terms of the MGO-induced impairment of glucose tolerance. In contrast, myocyte apoptosis, inflammation, and cardiac dysfunction were significantly diminished in CHOP KO compared with littermate naive controls. These results showed that CHOP is the key signal for myocyte apoptosis and cardiac dysfunction induced by MGO. These findings suggest a therapeutic potential of CHOP inhibition in the management of diabetic cardiovascular complications including diabetic cardiomyopathy.

Aims bFGF is a potent mitogen of cells associated with fibrosis. Although ERK5 has been reported to play roles in the development of fibrosis, its roles in regulating bFGF-induced fibrotic responses are not understood, especially in lung fibroblasts. The authors investigated the role of ERK5 in bFGF induction of cell proliferation and in induction of PAI-1, a critical regulator of the pathological features of fibrosis, in lung fibroblasts. Main methods The role played by ERK5 in bFGF-induced PAI-1 expression was elucidated by perturbing the ERK5 signaling pathway using a specific chemical inhibitor and siRNA of ERK5. The effects of ERK5 signal perturbation on PAI-1 expression were measured at multiple levels by Q-PCR, immunoblotting, ELISA, and reporter gene analysis. The role of MEF2 in bFGF-induced activation of PAI-1 promoter activity via ERK5 was measured using a biotin-labeled DNA pull-down assay, and the effects of ERK5 on the mitogenic effects of bFGF were assessed using a MTT assay. Key findings In both primary human lung fibroblast and lung fibroblast cell lines, inhibition of ERK5 blocked bFGF-induced PAI-1 expression at both mRNA and protein levels and inhibited bFGF-induced PAI-1 promoter activity induction by bFGF. Upon stimulation with bFGF, MEF2 directly bound to the consensus sequence of the MEF2 binding site in the PAI-1 promoter. In addition, bFGF-induced PAI-1 up-regulation was inhibited by MEF2 siRNA, and bFGF-induced fibroblast proliferation was blocked by inhibiting ERK5. Significance This study reveals a novel role for the ERK5–MEF2 cascade, linking bFGF-induced PAI-1 expression and subsequent mitogenic processes in lung fibroblasts.

Nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) plays an important role in the development of invasive diseases, and is also critically involved in setting up respiratory bacterial and viral infections. We previously reported that pneumococcus, one of the commonly carried bacteria in the nasopharynx, regulates non-typeable Haemophilus influenzae-induced inflammation by upregulating the expression of Toll-like receptor 2 (TLR2). However, the underlying molecular mechanisms by which TLR2 expression is regulated during pneumococcal infections have not yet been well characterized. TBX21 is an important transcription factor of adaptive immunity, but there is an increasing body of evidence pointing to a role in regulating innate immunity. The expression of TBX21 was reported in epithelial cells, but the expression and role of TBX21 in respiratory epithelium, especially for regulating TLR2, has not yet been studied. In this study, we found that pneumococcus upregulates TBX21 expression in the respiratory epithelium. The effect of pneumococcus on TBX21 expression was dependent on its cytoplasmic toxin, pneumolysin. In addition, epithelial TBX21 expression was not regulated by the gram-negative bacterium non-typeable Haemophilus influenzae, peptidoglycan or endotoxin. Deficiency of TBX21 in mice or knocking down TBX21 in epithelial cells suppressed pneumococcus-induced TLR2 expression, but not that of TLR4 or TLR9. These results indicate that the adaptive immune regulator TBX21 participates in regulating innate immune responses, through regulation of TLR2 expression during pneumococcal infections.

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.

Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis., Lung injury initiates a series of wound-healing responses, which if unregulated, can lead to fibrosis. Li et al. show that the deubquitinase CYLD has a key role in the prevention of fibrosis by inhibiting transforming growth factor β-signalling through the direct deubiquitination of the protein kinase Akt.

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.

Background Inflammation is a hallmark of many serious human diseases. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are characterized by inflammation, which is mainly mediated by nuclear transcription factor-kappa B (NF-κB)-dependent production of proinflammatory mediators. 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 NF-κB activation and inflammation, however, remains largely unknown. Methodology/Principal Findings In the present study, we demonstrate that EGFR plays a vital role in NTHi-induced NF-κB activation and the subsequent induction of proinflammatory mediators in human middle ear epithelial cells and other cell types. Importantly, we found that AG1478, a specific tyrosine kinase inhibitor of EGFR potently inhibited NTHi-induced inflammatory responses in the middle ears and lungs of mice in vivo. Moreover, we found that MKK3/6-p38 and PI3K/Akt signaling pathways are required for mediating EGFR-dependent NF-κB activation and inflammatory responses by NTHi. Conclusions/Significance Here, we provide direct evidence that EGFR plays a critical role in mediating NTHi-induced NF-κB activation and inflammation in vitro and in vivo. Given that EGFR inhibitors have been approved in clinical use for the treatment of cancers, current studies will not only provide novel insights into the molecular mechanisms underlying the regulation of inflammation, but may also lead to the development of novel therapeutic strategies for the treatment of respiratory inflammatory diseases and other inflammatory diseases.

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α–induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α- or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-κB–dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca 2+ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.

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.

Bacteraemic pneumonia is a common cause of sepsis in critically ill patients today and is characterized by dysregulation of inflammation. The genetic factors predisposing to bacteraemic pneumonia are not yet fully understood. Innate immunity is pivotal for host defence against invading bacteria, and nuclear factor-kappa B (NF-kappaB) is central to bacteria-induced inflammation and immune responses. The deubiquitinating enzyme CYLD has been identified as a key negative regulator for NF-kappaB. In the present study, we investigated the role of CYLD in innate immune response in Escherichia coli pneumonia. Upon E. coli inoculation, Cyld(-/-) mice were hypersusceptible to E. coli pneumonia with higher mortality. Innate immune response to E. coli was enhanced in Cyld(-/-) cells and mice. Cyld(-/-) cells exhibited enhanced NF-kappaB activation upon E. coli inoculation, and the enhanced NF-kappaB activation by E. coli was abolished by perturbing IkappaB kinase (IKK) signalling. Furthermore, IKK inhibitor rescued Cyld(-/-) mice from lethal infection during E. coli pneumonia along with reduced inflammation. Taken together, these data showed that CYLD acts as a crucial negative regulator for E. coli pneumonia by negatively regulating NF-kappaB. These findings provide novel insight into the regulation of bacteraemic pneumonia and related diseases and may help develop novel therapeutic strategies for these diseases.

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.

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.

Summary Streptococcus pneumoniae ( S. pneumoniae ) causes high early mortality in pneumococcal pneumonia, which is characterized by acute lung injury (ALI). The molecular mechanisms underlying ALI and the high early mortality remain unknown. Despite recent studies that identify deubiquitinating enzyme cylindromatosis (CYLD) as a key regulator for T cell development, tumor cell proliferation, and NF-κB transcription factor signaling, its role in regulating bacteria-induced lethality, however, is unknown. Here, we showed that CYLD deficiency protected mice from S. pneumoniae pneumolysin (PLY)-induced ALI and lethality. CYLD was highly induced by PLY, and it inhibited MKK3-p38 kinase-dependent expression of plasminogen activator inhibitor-1 (PAI-1) in lung, thereby potentiating ALI and mortality. Thus, CYLD is detrimental for host survival, thereby indicating a mechanism underlying the high early mortality of pneumococcal pneumonia.

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 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.

CARD-containing MAGUK protein 1 (CARMA1) plays a crucial role in regulating adaptive immune responses upon T-cell receptor (TCR) activation in T cells. Its role in regulating host mucosal innate immune response such as upregulation of mucin remains unknown. Here we show that CARMA1 acts as a key signaling mediator for synergistic upregulation of MUC5AC mucin by bacterium nontypeable Haemophilus influenzae (NTHi) and phorbol ester PMA in respiratory epithelial cells. NTHi-induced TLR-dependent TRAF6-MKK3-p38 MAPK signaling pathway synergizes with PKCθ-MEK-ERK signaling pathway. CARMA1 plays a crucial role in mediating this synergistic effect via TRAF6, thereby resulting in synergistic upregulation of MUC5AC mucin. Thus our study unveils a novel role for CARMA1 in mediating host mucosal innate immune response.

BackgroundNontypeableHaemophilus influenzae(NTHi) is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) via activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B in pulmonary alveolar epithelial cells.MethodsHuman alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA), and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The roles of Toll-like receptor (TLR) 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4in vitroand in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out micein vivo. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors.ResultsNTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cellin vitroand in the lungs of micein vivo. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B. Moreover, the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-kappa B. However, NTHi-induced DNA binding activity of NF-kappa B was not affected by the inhibition of p38 MAPK.ConclusionNTHi induces COX-2 and PGE2 expression in a p38 MAPK and NF-kappa B-dependent manner through TLR2 in lung epithelial cellsin vitroand lung tissuesin vivo. The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.

Background Toll-like receptor 2 (TLR2) plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. Streptococcus pneumoniae (S. pneumoniae) and nontypeable Haemophilus influenzae (NTHi) were previously reported to synergize with each other to induce inflammatory responses. Despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface TLR2. Results Here we provide direct evidence that S. pneumoniae synergizes with NTHi to upregulate TLR2 expression in lung and middle ear of the mice. Pneumolysin (PLY) appears to be the major virulence factor involved in this synergism. Moreover, S. pneumoniae PLY induces TLR2 expression via a TLR4-MyD88-NF-κB-dependent signaling pathway. Interestingly, tumor suppressor CYLD acts as a negative regulator of S. pneumoniae-induced TLR2 up-regulation via negative-crosstalk with NF-κB signaling. Conclusion Our study thus provides novel insights into the regulation of TLR2 expression in mixed bacterial infections.