Interferons as negative regulators of ILC2s in allergic lung inflammation and respiratory viral infections.

Group 2 innate lymphoid cells (ILC2s), characterized by a lack of antigen receptors, have been regarded as an important component of type 2 pulmonary immunity. Analogous to Th2 cells, ILC2s are capable of releasing type 2 cytokines and amphiregulin, thus playing an essential role in a variety of diseases, such as allergic diseases and virus-induced respiratory diseases. Interferons (IFNs), an important family of cytokines with potent antiviral effects, can be triggered by microbial products, microbial exposure, and pathogen infections. Interestingly, the past few years have witnessed encouraging progress in revealing the important role of IFNs and IFN-producing cells in modulating ILC2 responses in allergic lung inflammation and respiratory viral infections. This review underscores recent progress in understanding the role of IFNs and IFN-producing cells in shaping ILC2 responses and discusses disease phenotypes, mechanisms, and therapeutic targets in the context of allergic lung inflammation and infections with viruses, including influenza virus, rhinovirus (RV), respiratory syncytial virus (RSV), and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2).

Lacticaseibacillus casei CNRZ1874 supplementation promotes M1 alveolar macrophage activation and attenuates Mycoplasma pneumoniae pneumonia.

AIMS: To evaluate the protective effect of intestinal supplementation with Lacticaseibacillus casei CNRZ1874 on the inflammatory response induced by Mycoplasma pneumoniae in C57BL/6 J mice, and provide a potential strategy for alleviating M. pneumoniae pneumonia. METHODS AND RESULTS: C57BL/6 J mice were gavaged with L. casei CNRZ1874 or PBS for 7 consecutive days, and then infected with M. pneumoniae on day 8. Treatment with L. casei CNRZ1874 significantly reduced M. pneumoniae loads in the lungs and alleviated the lung inflammation on day 3 and 10 after pathogen infection. Importantly, oral administration with L. casei CNRZ1874 promoted M1 alveolar macrophages activation as evidenced by increased expression of iNOS, TNF-α, and CXCL1, while inhibited M2 alveolar macrophages activation as the expression of Arg1 and Chi3l3 were significantly decreased. In consistent with the M1 alveolar macrophages activation and enhanced mycoplasma clearance, the level of TNF-α was increased while the level of IL-4 was decreased in lung tissue from the L. casei CNRZ1874 group compared with the control group. However, oral administration with L. casei CNRZ1874 may not influence adaptive immunity induced by M. pneumoniae as evaluated by M. pneumoniae specific antibodies and T cells responses in spleen. CONCLUSIONS: Intestinal supplementation with L. casei CNRZ1874 can promote M1 alveolar macrophages activation, which contributes to the clearance of M. pneumoniae and attenuation of M.pneumoniae pneumonia.

Mycoplasma pneumoniae downregulates RECK to promote matrix metalloproteinase-9 secretion by bronchial epithelial cells.

Airway epithelial cells function as both a physical barrier against harmful substances and pathogenic microorganisms and as an important participant in the innate immune system. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in modulating inflammatory responses during respiratory infections. However, the signalling cascade that induces MMP-9 secretion from epithelial cells infected with Mycoplasma pneumoniae remains poorly understood. In this study, we investigated the mechanism of MMP-9 secretion in airway epithelial cells infected with M. pneumoniae. Our data clearly showed that M. pneumoniae induced the secretion of MMP-9 from bronchial epithelial cells and upregulated its enzymatic activity in a time- and dose-dependent manner. Using specific inhibitors and chromatin co-precipitation experiments, we confirmed that the expression of MMP-9 is reliant on the activation of the Toll-like receptor 2 (TLR2) and TLR6-dependent mitogen-activated protein kinase/nuclear factor- κB/activator protein-1 (MAPK/NF-κB/AP-1) pathways. Additionally, epigenetic modifications such as histone acetylation and the nuclear transcription factor Sp1 also regulate MMP-9 expression. M. pneumoniae infection also decreased the expression of the tumour suppressor reversion-inducing cysteine-rich protein with Kazal motifs (RECK) by inducing Sp1 phosphorylation. Overexpression of RECK significantly impaired the M. pneumoniae-triggered increase in MMP-9 enzymatic activity, although the level of MMP-9 protein remained constant. The study demonstrated that M. pneumoniae-triggered MMP-9 expression is modulated by TLR2 and 6, the MAPK/NF-κB/AP-1 signalling cascade, and histone acetylation, and M. pneumoniae downregulated the expression of RECK, thereby increasing MMP-9 activity to modulate the inflammatory response, which could play a role in airway remodelling.

The Beneficial Role of Probiotic Lactobacillus in Respiratory Diseases.

Respiratory diseases cause a high incidence and mortality worldwide. As a natural immunobiotic, Lactobacillus has excellent immunomodulatory ability. Administration of some Lactobacillus species can alleviate the symptoms of respiratory diseases such as respiratory tract infections, asthma, lung cancer and cystic fibrosis in animal studies and clinical trials. The beneficial effect of Lactobacillus on the respiratory tract is strain dependent. Moreover, the efficacy of Lactobacillus may be affected by many factors, such as bacteria dose, timing and host background. Here, we summarized the beneficial effect of administered Lactobacillus on common respiratory diseases with a focus on the mechanism and safety of Lactobacillus in regulating respiratory immunity.

Biological functions of IL-17-producing cells in mycoplasma respiratory infection.

Mycoplasmas are the smallest and simplest bacteria that lack a cell wall but have the capability of self-replication. Among them, Mycoplasma pneumoniae is one of the most common causes of community-acquired pneumonia. The hallmark of mycoplasma respiratory diseases is the persistence of lung inflammation that involves both innate and adaptive immune responses. In recent years, a growing body of evidence demonstrates that IL-17 plays an important role in respiratory mycoplasma infection, and associates with the pathologic outcomes of infection, such as pneumonitis and asthma. Numerous studies have shown that a variety of cells, in particular Th17 cells, in the lung can secrete IL-17 during respiratory mycoplasma infection. In this article, we review the biological functions of distinct IL-17-producing cells in mycoplasma respiratory infection with a focus on the effect of IL-17 on the outcomes of infection.

T-B cell epitope peptides induce protective immunity against Mycoplasma pneumoniae respiratory tract infection in BALB/c mice.

Mycoplasma pneumoniae is the most common pathogen of community-acquired pneumonia in humans. Due to its high rates of antibiotic resistance, vaccination has become the best method to control the dissemination of M. pneumoniae. The recombinant carboxyl terminus of the P1 (P1C) protein is an immunodominant antigen, but it has negative effects such as poor stability and lower purity. In the current study, T-B epitopes of the P1C protein were predicted according to bioinformatics analysis and assessed for efficacy in peptide vaccination. BALB/c mice were subcutaneously inoculated with the T-B epitope peptides four times and then infected with M. pneumoniae through the respiratory tract. The results showed that the T-B epitope peptides of the P1C protein (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) induced strong antigen-specific serum antibody responses and cellular immune responses with high levels of serum IgG, IgA antibodies and Th1-biased (IFN-γ and IL-2) cytokines. Immunization with T-B epitope peptides significantly reduced the M. pneumoniae burden and the degree of inflammation in the challenged mice. Furthermore, the levels of IFN-γ and TNF-α in the supernatants of lung homogenates were observably reduced compared to those in the PBS group. Overall, our findings demonstrate that T-B epitopes (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) play significant roles in the P1C protein and can be used to induce powerful humoral and cellular immune responses to provide significant protection against M. pneumoniae pulmonary infection, which provides new insight into the design of potential multiepitope vaccines to prevent host infection by M. pneumoniae.

Mycoplasma pneumoniae Infections: Pathogenesis and Vaccine Development.

Mycoplasma pneumoniae is a major causative agent of community-acquired pneumonia which can lead to both acute upper and lower respiratory tract inflammation, and extrapulmonary syndromes. Refractory pneumonia caused by M. pneumonia can be life-threatening, especially in infants and the elderly. Here, based on a comprehensive review of the scientific literature related to the respective area, we summarize the virulence factors of M. pneumoniae and the major pathogenic mechanisms mediated by the pathogen: adhesion to host cells, direct cytotoxicity against host cells, inflammatory response-induced immune injury, and immune evasion. The increasing rate of macrolide-resistant strains and the harmful side effects of other sensitive antibiotics (e.g., respiratory quinolones and tetracyclines) in young children make it difficult to treat, and increase the health risk or re-infections. Hence, there is an urgent need for development of an effective vaccine to prevent M. pneumoniae infections in children. Various types of M. pneumoniae vaccines have been reported, including whole-cell vaccines (inactivated and live-attenuated vaccines), subunit vaccines (involving M. pneumoniae protein P1, protein P30, protein P116 and CARDS toxin) and DNA vaccines. This narrative review summarizes the key pathogenic mechanisms underlying M. pneumoniae infection and highlights the relevant vaccines that have been developed and their reported effectiveness.

Up-Conversion Luminescent Nanoparticles for Molecular Imaging, Cancer Diagnosis and Treatment.

In the past few years, we have witnessed great development and application potential of various up-conversion luminescent nanoparticles (UCNPs) in the nanomedicine field. Based on the unique luminescent mechanism of UCNPs and the distinguishable features of cancer biomarkers and the microenvironment, an increasing number of smart UCNPs nanoprobes have been designed and widely applied to molecular imaging, cancer diagnosis, and treatment. Considerable technological success has been achieved, but the main obstacles to oncology nanomedicine is becoming an incomplete understanding of nano-bio interactions, the challenges regarding chemistry manufacturing and controls required for clinical translation and so on. This review highlights the progress of the design principles, synthesis and surface functionalization preparation, underlying applications and challenges of UCNPs-based probes for cancer bioimaging, diagnosis and treatment that capitalize on our growing understanding of tumor biology and smart nano-devices for accelerating the commercialization of UCNPs.

Proteome array of antibody responses to Chlamydia trachomatis infection in nonhuman primates.

AIMS: Nonhuman primates have been used to investigate pathogenic mechanisms and evaluate immune responses following Chlamydia trachomatis inoculation. This study aimed to systemically profile antibody responses to C. trachomatis infection in nonhuman primates. MATERIALS AND METHODS: Sera were obtained from 4 pig-tailed and 8 long-tailed macaques which were intravaginally or ocularly infected with live C. trachomatis organisms, and analyzed by C. trachomatis proteome array of antigens. KEY FINDINGS: The sera from 12 macaques recognized total 172 C. trachomatis antigens. While 84 antigens were recognized by pig-tailed macaques intravaginally infected with serovar D strain, 125 antigens were recognized by long-tailed macaques ocularly infected with serovar A, and 37 antigens were recognized by both. Ocular inoculation with virulent A2497 strain induced antibodies to more antigens. Among the antigens uniquely recognized by A2497 strain infected macaques, outer membrane complex B antigen (OmcB) induced robust antibody response. Although macaques infected by less virulent A/HAR-13 strain failed to develop antibodies to OmcB, reinfection by A2497 strain induced high levels of antibodies to OmcB. SIGNIFICANCE: Proteome array has revealed a correlation of chlamydial infection invasiveness with chlamydial antigen immunogenicity, and identified antibody responses to OmcB potentially as biomarkers for invasive infection with C. trachomatis.

Exogenous Hydrogen Sulfide Regulates Mycoplasma pneumoniae Lipid-Associated Membrane Proteins to Induce Expression of Heme Oxygenase-1 and Proinflammatory Cytokines.

This study was designed to investigate the effect of exogenous hydrogen sulfide (H2S) on the secretion of Heme oxygenase (HO-1) and proinflammatory cytokines in human mononuclear cell line THP-1 stimulated by lipid-associated membrane proteins (LAMPs) prepared from Mycoplasma pneumoniae (M. pneumoniae) and explore its regulatory mechanism. Cultured cells were stimulated with M. pneumoniae LAMPs after pretreatment with H2S to analyze the production of proinflammatory cytokines and HO-1 by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that THP-1 cells, which were stimulated by LAMPs after pretreatment with H2S, had decreased production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by inhibiting the mitogen-activated protein kinases (MAPKs)/nuclear factor-kappa B (NF-κB) signaling pathway and increased expression of HO-1 by activating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Our results indicate that H2S may play an important role in attenuating inflammation induced by M. pneumoniae LAMPs due to its ability to decrease the production of IL-6 and IL-8 and increase the expression of the HO-1. These findings support further studies for possible clinical applications.

Three polypeptides screened from phage display random peptide library may be the receptor polypeptide of Mycoplasma genitalium adhesion protein.

Mycoplasma genitalium adhesion protein (MgPa) is a major adhesin of M. genitalium, a human pathogen associated with a series of genitourinary tract diseases. MgPa plays a very important role in M. genitalium adhering to the host cells. However, the exact receptor peptides or proteins of MgPa are still poorly understood so far. Three polypeptides (V-H-W-D-F-R-Q-W-W-Q-P-S), (D-W-S-S-W-V -Y-R-D-P-Q-T) and (H-Y-I-D-F-R-W) were previously screened from a phage display random peptide library using recombinant MgPa (rMgPa) as a target molecule. In this study, three polypeptides were artificially synthesized and investigated as to whether they are potential receptors of MgPa. We found that rMgPa specifically bound to three synthesized polypeptides as determined via an indirect enzyme-linked immunosorbent assay (ELISA). Moreover, three polypeptides were further identified by indirect immunofluorescence microscopy (IFM). We confirmed that rMgPa and M. genitalium can adhere to SV-HUC-1 cells in vitro and that anti-rMgPa antibody and three synthesized polypeptides can partially inhibit the adherence of rMgPa and M. genitalium to SV-HUC-1 cells. In summary, these three polypeptides may be the essential receptor peptides of MgPa, and may aid in enhancing the understanding of biological function of MgPa and the possible pathogenic mechanism of M. genitalium.

Cyclophilin A is the potential receptor of the Mycoplasma genitalium adhesion protein.

The Mycoplasma genitalium adhesion protein (MgPa), the most important outer membrane protein of M. genitalium, plays a vital role in the adhesion to and invasion of host cells by M. genitalium. Identification of MgPa receptors will help elucidate the pathogenic mechanism of M. genitalium. However, the receptor protein of MgPa has not been reported to date. In this study, an MgPa-binding protein with a molecular weight of approximately 17 kDa was screened from SV-HUC-1 cell membrane proteins by a modified virus overlay protein binding assay (VOPBA). Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the protein components of the 17-kDa protein. The results demonstrated that the MgPa-binding protein was most likely Cyclophilin A (CyPA). The binding activity and distribution of CyPA in SV-HUC-1 cells were detected using indirect ELISA, western blotting, far-western blotting and indirect immunofluorescence. We found that recombinant MgPa (rMgPa) could bind with CyPA from SV-HUC-1 cell membrane proteins and to recombinant CyPA, which indicated that CyPA was predominant component of the 17-kDa protein band and can interact with rMgPa. In addition, an indirect immunofluorescence assay showed that CyPA was partially distributed on the membrane surfaces of SV-HUC-1 cells and could partially inhibit the adhesion of rMgPa and M. genitalium to SV-HUC-1 cells. Co-localization assays further indicated that rMgPa and M. genitalium can interact with CyPA. These results suggested that the CyPA located on SV-HUC-1 cell membranes may be the potential receptor of MgPa, which could provide an experimental basis for elucidating the function of MgPa and the possible pathogenic mechanism of M. genitalium.

Oral Chlamydia vaccination induces transmucosal protection in the airway.

Although Chlamydia has been frequently detected in the gastrointestinal tracts of both humans and animals, it is not associated with any gastrointestinal pathology. We have recently shown that gastrointestinal Chlamydiamuridarum is not only non-pathogenic but also induces protective immunity in the genital tract. We now report that the transmucosal immunity induced by a single oral immunization with C.muridarum protected the mouse airway from a subsequent challenge infection. The oral immunization significantly reduced chlamydial burden in the airway as early as day 3 after intranasal challenge. As a result, the airway chlamydial spreading to extra-airway tissues was completely prevented on day 3 and significantly reduced on day 9. The immunized mice were protected from any significant systemic toxicity caused by the intranasal challenge since there was no significant bodyweight drop in the immunized mice. This robust protection correlated well with Chlamydia-specific antibodies that recognize chlamydial organism surface antigens and T cell responses that are dominated with a Th1 phenotype. The immunized mice developed high ratios of IgG2b/c over IgG1 levels and IFNγ-producing over IL-5- or IL-13-producing lymphocytes. Thus, we have demonstrated that oral vaccination with C. muridarum can induce Th1-dominant transmucosal immunity in the airway. Together with previous studies, we propose that non-pathogenic colonization of Chlamydia in the gastrointestinal tract be explored as an oral delivery system for inducing protection against infections and pathologies in extra-gastrointestinal tissues.

Nonpathogenic Colonization with Chlamydia in the Gastrointestinal Tract as Oral Vaccination for Inducing Transmucosal Protection.

Chlamydia has been detected in the gastrointestinal tracts of humans and animals. We now report that gastrointestinal Chlamydia muridarum is able to induce robust transmucosal protection in mice. C. muridarum colonization in the gastrointestinal tract correlated with both a shortened course of C. muridarum genital tract infection and stronger protection against subsequent genital tract challenge infection. Mice preinoculated intragastrically with C. muridarum became highly resistant to subsequent C. muridarum infection in the genital tract, resulting in prevention of pathology in the upper genital tract. The transmucosal protection in the genital tract was rapidly induced, durable, and dependent on major histocompatibility complex (MHC) class II antigen presentation but not MHC class I antigen presentation. Although a deficiency in CD4+ T cells only partially reduced the transmucosal protection, depletion of CD4+ T cells from B cell-deficient mice completely abolished the protection, suggesting a synergistic role of both CD4+ T and B cells in the gastrointestinal C. muridarum-induced transmucosal immunity. However, the same protective immunity did not significantly affect C. muridarum colonization in the gastrointestinal tract. The long-lasting colonization with C. muridarum was restricted to the gastrointestinal tract and was nonpathogenic to either gastrointestinal or extragastrointestinal tissues. Furthermore, gastrointestinal C. muridarum did not alter the gut microbiota or the development of gut mucosal resident memory T cell responses to a nonchlamydial infection. Thus, Chlamydia may be developed into a safe and orally deliverable replicating vaccine for inducing transmucosal protection.

Nrf2 regulates the inflammatory response, including heme oxygenase-1 induction, by mycoplasma pneumoniae lipid-associated membrane proteins in THP-1 cells.

A series of inflammatory responses caused by Mycoplasma pneumoniae largely depend on the lipid-associated membrane proteins (LAMPs). Nuclear factor E2-related factor 2 (Nrf2), a transcription factor, is considered to be a critical modulator of inflammatory responses and cellular redox homeostasis. Monocytes play an important role in the invasion and immunity to resist pathogens. Here, we investigated the role of Nrf2 in the anti-inflammatory response stimulated by LAMPs using the human monocyte cell line THP-1. LAMPs were shown to affect the localization of Nrf2, and the levels of reactive oxygen species and inflammatory reactants, including nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines (IL-6, IL-8), were highly elevated in LAMP-stimulated Nrf2-silenced THP-1 cells. Moreover, LAMPs induced the levels of mRNA and the expression of heme oxygenase-1 (HO-1). In summary, our results demonstrated that LAMPs cause nuclear translocation of Nrf2, which further suppresses the expression of inflammatory reactants in THP-1 cells.

Intravenous Inoculation with Chlamydia muridarum Leads to a Long-Lasting Infection Restricted to the Gastrointestinal Tract.

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, it remains unclear whether the chlamydial organisms can be introduced into the gastrointestinal tract via pathways independent of the oral and anal routes. We have recently shown that Chlamydia muridarum spreads from the genital tract to the gastrointestinal tract potentially via the circulatory system. To test whether hematogenous C. muridarum can spread to and establish a long-lasting colonization in the mouse gastrointestinal tract, we inoculated mice intravenously with a luciferase-expressing C. muridarum strain and monitored its distribution. After tail vein inoculation, most luciferase-generated bioluminescence signals were detected in the mouse abdominal area throughout the experiment. The ex vivo imaging revealed that the abdominal signals came from the gastrointestinal tract tissues. Simultaneous monitoring of chlamydial organisms in individual organs or tissues revealed an initial stage of systemic spreading followed by a long-lasting infection in the gastrointestinal tract. A retro-orbital vein inoculation of the C. muridarum organisms at a lower dose in a different mouse strain also led to colonization of the gastrointestinal tract. We have demonstrated that intravenous C. muridarum inoculation can result in colonization of the gastrointestinal tract, suggesting that the chlamydial organisms may use the sexual behavior-independent circulation pathway to infect the gastrointestinal tract.

The Chlamydia muridarum Organisms Fail to Auto-Inoculate the Mouse Genital Tract after Colonization in the Gastrointestinal Tract for 70 days.

Chlamydia muridarum is known to colonize in the gastrointestinal tract for long periods of time, which has been hypothesized to serve as a reservoir for spreading to the genital tract. To test this hypothesis, a luciferase-expressing C. muridarum was used to establish a long-lasting infection in the mouse gastrointestinal tract following either intragastric or intrarectal inoculations. In vivo imaging revealed significant bioluminescent signals mainly in the mouse abdominal area throughout the experiments. Ex vivo imaging localized the signals to the mouse gastrointestinal tract, which was confirmed by monitoring the C. muridarum organisms in the mouse organs/tissues. Despite the long-lasting colonization in the gastrointestinal tract and active shedding of infectious organisms in the rectal swabs, the organisms did not cause any significant infection or pathology in the genital tract throughout the experiments, which was reproduced in multiple strains of mice and with an increased inoculation dose to the gastrointestinal tract. The above observations have demonstrated that the long-lasting C. muridarum organisms from the gastrointestinal tract are inefficient in auto-inoculating the genital tract, suggesting that the gastrointestinal tract Chlamydia may utilize an indirect mechanism to affect its pathogenicity in the genital tract.

[Mycoplasma genitalium-derived lipid-associated membrane proteins negatively regulate cytokine secretion by inducing HO-1 expression in placental trophoblast cells].

OBJECTIVE: To observe the expression of heme oxygenase-1 (HO-1) in regulation of cytokines response induced by Mycoplasma genitalium-derived lipid-associated membrane proteins (LAMPs) in placental trophoblast cells. METHODS: Placental trophoblast cells were cultured in vitro and stimulated by 0.5-5 µg/mL LAMPs for 4 to 12 hours. Expression of HO-1 mRNA and protein, and nuclear translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) were detected by real-time quantitative PCR and Western blotting, respectively. The intracellular formation of reactive oxygen species (ROS) was detected by the fluorescent probe H2DCFDA. N-acetyl-cysteine (NAC) and nuclear factor erythroid-2 related factor 2 (Nrf2) siRNA were respectively used to analyze the roles of ROS and Nrf2 in mediating HO-1 expression. Finally, placental trophoblast cells were transfected with HO-1 siRNA, or preincubated by the HO-1 agonist cobalt protoporphyrin (CoPP) or its inhibitor zinc protoporphyrin (ZnPP), and LAMPs-induced secretion of TNF-α and IL-1ß were detected by ELISA. RESULTS: M. genitalium LAMPs induced the expression of HO-1 mRNA and protein, the accumulation of ROS and the nuclear translocation of Nrf2 in placental trophoblast cells. NAC treatment inhibited LAMPs-induced HO-1 expression and Nrf2 nuclear translocation, and the transfection of Nrf2 siRNA significantly abrogated HO-1 expression. Furthermore, HO-1 siRNA and ZnPP treatment increased LAMPs-induced TNF-α and IL-1ß secretion, while the HO-1 agonist CoPP treatment further decreased their production. CONCLUSION: M. genitalium LAMPs could induce placental trophoblast cells to express HO-1 through ROS/Nrf2 pathways. Up-regulation of HO-1 negatively regulates excessive production of cytokines.

[Mycoplasma genitalium lipid-associated membrane proteins induce endocervical epithelial cells to express ß-defensin-2].

OBJECTIVE: To observe the expression of human ß-defensin-2 (hBD-2) induced by Mycoplasma genitalium-derived lipid-associated membrane proteins (LAMPs) and its potential mechanism. METHODS: Human endocervical epithelial End1/E6E7 cells were cultured in vitro and stimulated by different concentrations of LAMPs for 48 hours, and the expressions of hBD-2 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Toll-like receptor 2 (TLR2) and TLR6 neutralizing antibodies for End1/E6E7 cell cultivation, and dominant negative plasmids for cell transfection were used to analyze the roles of TLR2, TLR6 and MyD88 in mediating hBD-2 expression. Nuclear translocation of the nuclear factor κB (NF-κB) p65 subunit, and its DNA-binding activity were detected by Western blotting and electrophoretic mobility shift assay (EMSA), respectively. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, was used to investigate the effect of NF-κB on hBD-2 expression. RESULTS: Mycoplasma genitalium-derived LAMPs induced the expressions of hBD-2 mRNA and protein in End1/E6E7 cells. The expressions could be abrogated by TLR2 and TLR6 neutralizing antibodies, or their dominant negative plasmids. In addition, dominant negative plasmids of MyD88 significantly decreased LAMPs-induced hBD-2 expression. Western blotting showed that p65 was translocated to the nucleus, and the DNA binding activity of NF-κB was enhanced after LAMPs treatment. Furthermore, PDTC treatment decreased LAMPs- induced hBD-2 expression. CONCLUSION: Mycoplasma genitalium-derived LAMPs can induce End1/E6E7 cells to express hBD-2, which may be involved in the TLR2, TLR6/Myd88/NF-κB pathways.

Macrophage-activating lipopeptide-2 requires Mal and PI3K for efficient induction of heme oxygenase-1.

AIMS: This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes. METHODS: Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay. RESULTS: MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002. CONCLUSIONS: These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.