| The definition of tumor is that the abnormal growth of one cell in the tissue that lost its ability to regulate the cell growth on the gene level, resulting from uncontrolled, progressive multiplication of cells and serving no physiological function. Six hallmarks of cancer have been summarized through current research:1. the potential of unlimited growth;2. the characteristic to escape programmed cell death (PCD);3. the ability to provide growth signals for themselves;4. the insensitivity to growth-inhibitory signals;5. the properties to promote angiogenesis;6. with characteristics of tissue invasion and metastasis. However, much more researches recently indicate that inflammation is the seventh hallmark of cancer, named as cancer-related inflammation (CRI).Cancer is not independent of inflammation, in fact, as early as the19th century, Rudolf Virchow discovered the presence of a large number of leukocytes within the tumor, thus the author puts forward the hypothesis:the possible association exists between inflammation and cancer, but researchers did not obtain conclusive evidence confirming that inflammation and inflammatory microenvironment plays an important role in the development of cancer until recent two or three decades. It has been reported that cancers caused by germline mutations only account for a small fraction of all numbers (about10%), however, cancers caused by the somatic mutations and environmental factors account for the majority of total cancers (approximately90%), wherein chronic inflammation was involved in the formation of inflammatory microenvironment that results in the initiation and development of cancer. The statistical data from the research conducted by Aggarwal et al. in2009have shown that up to20%of cancers were related to chronic infection, for instance, it is the common view that hepatitis B viruses (HBV) and hepatitis C viruses (HCV) infection increases the risk of hepatocellular carcinoma (HCC); persistent H. pylori infection is closely related to gastric cancer; Schistosomiasis or Bacteroides infection is closely associated with bladder cancer or colon cancer, respectively. Infection-induced inflammation belongs to the normal host immune defense, aiming to eliminate pathogens. However, the functional destruction of host immune system caused by pathogens leads to persistent chronic inflammation that promotes occurrence and development of tumors, but it is noteworthy that not all of the chronic inflammation increased risk of cancer, for example, researchers have discovered that in the initial prostate biopsies patients who have signs of inflammation may have a reduced risk of being diagnosed with prostate cancer in prostate biopsies in future; psoriasis may reduce cancer risk. In summary, the complex relationship between inflammation and cancer is like a double-edged sword, as inflammation can promote tumor progression, however, it also inhibits the growth of tumor, so far, the key factors to determine the two different processes are not fully elucidated, and the mechanism still needs to be explained by tumor immunity.In tumor microenvironment, in addition to cancer cells and stromal cells, the immune cells are also important components which include monocytes, macrophages, neutrophils, myeloid-derived suppressor cells (MDSC), mast cells, natural killer cells (NK cells), dendritic cells (DC), T lymphocytes, B lymphocytes, which communicate with each other through cell-cell interaction or in a cytokine-regulation manner. Innate and adaptive immune responses both play an important role in monitoring and clearing mutant tumor cells. Monocytes and macrophages are considered as important parts of innate immune system. It has been reported that macrophages in local tumor can account for50%of the proportion of tumors, suggesting the highly important role of macrophages in tumor immunity. Tumor associated macrophages (TAMs) have been propagated as M2type macrophage (also named as alternatively activated macrophages), promoting tumor growth, angiogenesis, invasion and metastasis. TAMs were also involved in the inhibition of anti tumor effect of other immune cells, and significant negative correlation exists between high numbers of TAM in local tumor and prognosis. M2macrophages were characterized with special membrane receptors and chemokine receptors, such as mannose receptor (CD206), CD163, CXCR1, CXCR2, CCR2and also with secretion of several cytokines and chemokines, including IL-10, IL-1receptor antagonist (IL-lra), CCL17, CCL18, and CCL22.Dendritic cells (DCs) participate in the initiation, maintenance and regulation of the immune response, and play the key role in immune tolerance or immune activation. DCs are considered as only antigen-presenting cells (APC) those could activate naive T cells. DC could induce naive T cells into T helper cells1(Thl cells) or Th2cells, such as Thl cells with secretion of IL-2, IL-12p70, IFN-y, and Th2cells with the secretion of IL-4, IL-10, TGF-P, which are involved in inflammatory response and cell-mediated immune response. Given the important role of DC in anti-tumor immunity, DC-based immunotherapy (also known as DC vaccine) has become the hot topic in tumor immunotherapy and researchers has obtained good results from experiments in vitro, but there exists many problems in clinical applications, including weak anti-tumor immune response, great difference among individuals and so on. In fact, DCs in local tumor are tailored into tolerogenic DC with reduced antigen-presenting ability and secretion of Th2-type cytokines which can not effectively kill tumor cells. Significant differences on anti-tumor therapeutic effect of DC vaccines in vitro and in vivo remind the researchers that tumor microenvironment in vivo may modulate and even dominate the nature and function of DC. The main characteristics of the tumor microenvironment contains low pH values, hypoxia, and nutrient deficiencies, in addition, modification on DC function by pathogens in the tumor microenvironment also play a crucial and unignored role in inhibiting DC function.During the chronic infection process, to clarify how did pathogens and their specific virulence factors regulate the immune system and their mechanisms will provide direct insight into molecular mechanisms of inflammation or cancer biotherapy. Moraxella catarrhalis was originally considered as normal flora of the respiratory tract, but a growing number of studies indicate that Moraxella catarrhalis is one kind of pathogenic bacteria that can induce otitis media in children, and it is also responsible for the exacerbation with chronic obstructive pulmonary disease (COPD) through inducing apoptosis of alveolar epithelial cells. More than90%of M. catarrhalis produce β-lactamase that is resistant to ampicillin, so the research and development of M. catarrhali vaccine can solve the problem that is about increasingly serious antibiotic resistance, but the progress of M. catarrhalis vaccine research is slow, and one of the main factors is the lack of animal models for research. Mouse pulmonary clearance model is the widely used model, which is designed to measure clearance of bacteria from the lungs following bacterial challenge directly into the airways, but there is difference between mouse and human, as M. catarrhalis was cleared in mouse within24hours, but with the long-term existence in human respiratory tract after infection, so this model can not simulate the human respiratory tract infection. In view of this, it is necessary to seek better animal model to simulate human respiratory system infection and to conduct the research on M. catarrhalis vaccine in vitro is particularly necessary.Researchers have discovered that a lot of monocytes and dendritic cells were recruited to inflammation sites by chemokines after M. catarrhalis infection. Monocytes and DCs are highly heterogeneous populations, and monocytes can be polarized to into pro-inflammatory M1macrophages or anti-inflammatory M2macrophages by pathogen and its virulence factor, also, DC maturation was affected by pathogen and other factors in the micro-environment, which promote or inhibit DC maturation. Monocytes/macrophages are regarded as the sentinel cells of innate immune response, while DCs play the key role in regulating innate and adaptive immune response, thus regulation of M. catarrhalis on two cell populations may be directly or indirectly affects, even determines results of the immune response in lung after infection. However, the regulation on monocyte differentiation and DC maturation induced by M. catarrhalis has not been fully elucidated. Although there is no proof to support the existence of the positive association between M. catarrhalis and lung cancer, yet it is worth noting that the Gram-negative bacteria were isolated in68%patients who suffer from lung cancer, of which the most important are Haemophilus influenzae and M. catarrhalis. This fact raises a new question:whether M. catarrhalis that exist in the lung of patients who also suffer from lung cancer regulate or modulate tumor immunity?Monocytes and DCs may encounter multiple stimulants presented on the bacterial surface, such as outer membrane protein UspAl (ubiquitous surface protein A1) of M. catarrhalis and lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria including M. catarrhali. Monocytes can be induced to M1macrophage with LPS stimulation and DC maturation are also enhanced by LPS. It has been reported that pathogen and its virulence factor play an important role in M2polarization and the inhibition of DC maturation. For example, monocytes were polarized to M2macrophages by virulence factor LcrV of Yersinia sp.; hepatic DC maturation was inhibited by intestinal commensal bacteria products through the activation of IL-6/STAT3pathway, thus the question is that whether M. catarrhalis UspAl also induce M2polarization and the inhibition of DC maturation as other virulence factors as mentioned above? M. catarrhalis UspAl binds with N terminal domain of carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) and it has been proved that T cell activation and function were inhibited by CEACAM1signal through ITIM activation. Engagement of UspAl with CEACAM1has been reported to be involved in the regulation not only of epithelial function but also of T cell function upon CEACAM1cross-linking. However, it is not clear that whether M. catarrhalis UspAl (and in particular the recombinant UspAl fragment rD-7that binds to CEACAM1) affects monocyte differentiation and DC maturation through UspAl-CEACAM1signal. To reveal this question, the two classical cell populations of innate and adaptive immune system (monocyte/macrophages and dendritic cells) were stimulated with recombinant protein rD-7based on outer membrane protein UspAl of Moraxella catarrhalis and the effects on monocyte differentiation and DC maturation were determined respectively in vitro. Our study may provide the insight for the lung cancer patients with M. catarrhalis infections who were also treated with DC vaccine and the development of M. catarrhali vaccine.Part One The study on monocytes differentiation modulated by Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7and mechanism of rD-7-driven differentiationObjective:To investigate the effect of Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7on human monocyte differentiation and the mechanism of rD-7-driven differentiation.Methods:1. Production and identification of recombinant rD-7, rD-7/D and r6-8proteins. Recombinant protein rD-7and control protein (rD-7/D and r6-8) were produced using the pQE30expression system. Recombinant proteins carrying a His tag were purified by affinity chromatography using Ni-NTA agarose. Purified proteins were detected using an anti-His-tag antibody and in the case of rD-7, also a soluble CEACAMl-Fc construct by western blotting. The samples were treated with Polymyxin B to remove endotoxin and then sterilized with0.2μm filter. The endotoxin levels in all purified proteins were less than1.0EU/μg as determined by Limulus Amoebocyte Lysate method.2. Digestions of rD-7and rD-7/D with Proteinase K. Samples of the purified recombinant molecules were digested using200μg/ml of Proteinase K (PK) at37℃for3h and the digested samples were heated at95℃for1h to inactive the enzyme. The absence of intact proteins in the relevant samples was confirmed using SDS-PAGE.3. Isolation of CD14+/CD15+cells from healthy human peripheral blood. Peripheral bloods from healthy volunteers under inform consent was collected in the sterile collection bag, and PBMCs were prepared with density gradient centrifugation using Histopaque-1077. CD14+monocytes and CD15+cells were positively selected from PBMCs by using CD14and CD15immunomagnetic beads respectively. 4. Monocytes activation. Freshly isolated monocytes were stimulated with2.5μg/ml of the recombinant proteins rD-7, r6-8or rD-7/D, or with LPS (2μg/ml) respectively. After24h stimulation, cells were harvested and the surface molecules expression was analyzed by flow cytometry and culture supernatants were collected and stored at-80℃for cytokine detection. To block the binding of rD-7to CEACAM1in some experiments, monocytes were treated with polyclonal anti CEACAM antibody A0115or IgG control (50μg/ml) for1h prior to the addition of the recombinant molecules as above. After additional23h, cells were collected and the surface molecules expressions were detected by using flow cytometry.5. The detection of molecular expression on monocytes by using flow cytometry. The CEACAM1expression on freshly isolated monocytes was determined using flow cytometry. In monocyte activation experiment, monocytes stimulated with rD-7, r6-8, rD-7/D or LPS were collected and CD80, CD86and CD206expressions on cells were detected by using flow cytometry.6. The study of the binding of rD-7on CD14+or CD15+cells and the blocking effect of A0115on rD-7binding on cells. For detection of surface-bound rD-7or rD-7/D and their inhibition by anti-CEACAM antibody, freshly isolated monocytes or CD15+cells were first incubated in the presence of A0115(50μg/ml) or its IgG control (50μg/ml) for30min on ice and then with the recombinant bacterial molecules (2.5μg/ml) for1h on ice. After treatment, the surface binding of rD-7on CD14+or CD15+cells were analyzed by using flow cytometry.7. Detection of the levels of IL-6, IL-10, IL-12p70and TNF-a from monocytes stimulated by rD-7with Luminex assay. Freshly isolated monocytes were stimulated by rD-7or rD-7/D for24h, and then the supernatants were collected. The levels of IL-6, IL-10, IL-12p70and TNF-a were detected using MILLIPLEX (?) MAP Human Cytokine/Chemokine Magnetic Bead Panel I and results were analyzed using MAGPIX system.8. Analysis of chemokines/cytokine profiles from monocytes stimulated by rD-7or rD-7/D with proteome profiler antibody arrays. Freshly isolated monocytes were stimulated by rD-7or rD-7/D for24h, the supernatants were collected and the chemokines/cytokine profiles were detected using Proteome Profiler human cytokine array Panel A kit.Results:1. Modulation of monocyte differentiation by M. catarrhalis UspAl-derived recombinant fragment rD-7. Compared to the unstimulated group, CD206expression on CD14+cells was significantly increased by the recombinant protein rD-7(P<0.001), however, CD80expression was significantly decreased (P<0.05) and CD86expression was not significantly changed; CD80(P<0.05) and CD206(P<0.001) expression on CD14+cells were significantly enhanced by LPS but CD86expression was reduced (P<0.05); the expressions of CD80, CD206, CD86on CD14+cells were not significantly affected by r6-8.2. The increased MFI of CD14+cells expressing CD206modulated by rD-7was dependent on the intact protein. CD206expression on CD14+cells was significantly increased by rD-7(P<0.001) compared with unstimulated cells, whilst digested rD-7lost the ability to increase CD206expression on monocytes (P<0.001) compared with monocytes stimulated with rD-7, suggesting the increased MFI of CD14+cells expressing CD206modulated by rD-7was dependent on the intact protein instead of endotoxin.3. CD14+monocytes express CEACAM1, the receptor of rD-7. The expression of CEACAM1on CD14+monocytes was detected by flow cytometry using the polyclonal antibody A0115as well as an anti-CEACAM1mAb (R and D Systems MAb2244). Low numbers of monocytes expressing CEACAM1were detected by both antibodies.4. rD-7-induced CD206expression on monocytes is independent of the ability to bind CEACAM1. Compared to the unstimulated control, CD206expression on CD14+monocytes was significantly increased by rD-7(P<0.001) or rD-7/D (P< 0.001) at a similar level. Digested rD-7and digested rD-7/D lost the ability to increase CD206expression on CD14+cells. CD206expression on CD14+cells was significantly increased by LPS (P<0.001); however, CD206expression was still enhanced by LPS treated with Proteinase K (P<0.001). Flow cytometry analysis revealed the rD-7binding on CD15+cells (P<0.001) instead of rD-7/D. Compared with IgG control, A0115blocked the rD-7binding on CD15+cells (P<0.05).5. Confirmation of CEACAM-independent binding and stimulation of CD206expression on CD14+cells by rD-7and rD-7/D by antibody inhibition assays. Both rD-7(P<0.05) and rD-7/D (P<0.05) bound to the surface of CD14+monocytes, and A0115did not block the binding as observed compared with IgG control. Freshly isolated monocytes were blocked with A0115before stimulation with rD-7or rD-7/D for24h. Both rD-7(P<0.01) and rD-7/D (P<0.01) still significantly enhanced CD206expression on CD14+cells and this remained unaffected in the presence of A0115.6. IL-6, TNF-a, IL-10and IL-12p70secretion from monocytes stimulated by rD-7and rD-7/D. IL-10, IL-12p70, IL-6and TNF-a in supernatants from monocytes stimulated with2.5μg/ml rD-7or rD-7/D were quantified by Luminex assays, confirming that rD-7and rD-7/D did not increase the secretion of IL-10or IL-12p70, whilst neither rD-7nor rD-7/D induced proinflammatory cytokines IL-6and TNF-a secretion compared with the unstimulated group from the four donors.7. Cytokines and chemokine expression profiles from monocytes modulated by rD-7or rD-7/D. A significant increase in IL-1ra secretion produced by monocytes stimulated with rD-7(P<0.001) or rD-7/D (P<0.001) for24h was observed, and the quantity of IL-lra released following treatment with rD-7or rD-7/D was similar to that from the LPS-stimulated group. In addition, IL-8secretion was also increased by either rD-7(P<0.05) or rD-7/D (P<0.01) when compared to the unstimulated group. IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-17, IL-32a, MCP-1, MIP-1β, CXCL10and CXCL12were undetectable in rD-7-or rD-7/D-stimulated groups. There was no significant change for C5a, CD40L, G-CSF, GM-CSF, CXCL1, CCL1, sICAM-1, IFN-γ, IL-1α, IL-1β, IL-6, IL-13, IL-16, IL-17E, IL-23, IL-27, CXCL11, MIF, CCL3, Serpin E1, CCL5, TNF-a, sTREM-1secretion between the rD-7or rD-7/D group and the unstimulated group.Conclusions:Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7modulated monocyte differentiation to CD14+CD206+phenotype; the modulation was dependent on intact rD-7protein instead of endotoxin; CD80expression on CD14+cells were decreased by rD-7; CEACAM1, the receptor of rD-7was expressed on monocyte, however, monocytes differentiation modulated by rD-7was independent of CE AC AMI; the secretion of the anti-inflammatory cytokine IL-1ra was significantly induced by rD-7. Monocyte differentiation was significantly modified by Moraxella catarrhalis UspAl-derived recombinant fragment rD-7, revealing the regulation of Moraxella catarrhalis virulence factor UspAl on innate immune response, thus our study will provide the insight for the lung cancer patients with M. catarrhalis infections who were also treated with DC vaccine and the development of M. catarrhali vaccine. Part Two The study on dendritic cells maturation modulated by Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7and mechanism of rD-7-driven DC maturationObjective:To investigate the effect of Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7on human DC maturation and the mechanism of rD-7-driven maturation. Methods:1. Production and identification of recombinant rD-7and rD-7/D proteins. The procedure of production of recombinant proteins has been described in part one. The endotoxin levels in all purified proteins were less than1.0EU/μg as determined by Limulus Amoebocyte Lysate method.2. Isolation of CD14+monocytes from healthy human peripheral blood. Peripheral bloods from healthy volunteers under inform consent was collected in the sterile collection bag, and PBMCs were prepared with density gradient centrifugation using Histopaque-1077. CD14+monocytes were positively selected from PBMCs by using CD14immunomagnetic beads.3. Induction and identification of DC. DC medium containing1000U/ml rh GM-CSF,500U/ml rh IL-4,10%human AB serum was prepared, and freshly isolated CD14+monocytes were resuspended by DC medium to adjust the cell concentration of5x105/ml.2ml cell suspension were grown in24-well plate and plated in the humidified incubator at37℃with5%CO2.200μl fresh DC medium were changed on day3and day5. Immature DCs were yielded on day6, and imDCs were induced to mDCs by LPS (2μg/ml) for additional24h.4. DC activation. Freshly isolated CD14+monocytes was induced to imDCs on Day6with rh GM-CSF and rh IL-4, and imDCs were stimulated with rD-7(2.5μg/ml), rD-7/D (2.5μg/ml) or LPS (2μg/ml) for24h, and the expression of CD80, CD83, CD86, HLA-DR on DCs were detected by flow cytometry. In blocking CEACAM experiment, imDCs were blocked with A0115or IgG isotype control (50μg/ml) for1h, and cells were stimulated with rD-7or rD-7/D for additional23h.5. Detection the levels of TNF-a, IL-10and IL-12p70. imDCs were stimulated with rD-7(2.5μg/ml), rD-7/D (2.5μg/ml) or LPS (2μg/ml) for24h, and then supernatants were collected and the levels of TNF-a, IL-10and IL-12p70were determined by ELISA. In blocking CEACAM experiment, imDCs were blocked with A0115or IgG isotype control (50μg/ml) for1h, and cells were stimulated with rD-7or rD-7/D for additional23h.Results:1. Modulation of DC maturation by M. catarrhalis UspAl-derived recombinant fragment rD-7. imDCs on day6were incubated with the CEACAM-binding recombinant molecule rD-7or LPS. The mean fluorescence intensities (MFIs) of cells expressing CD86(P<0.001) and HLA-DR (P<0.01) were significantly increased by rD-7compared with untreated imDCs. rD-7also increased MFI of cells expressing CD83(P<0.05) but to a lesser extent than LPS (P<0.001). Additionally, while LPS increased CD80expression (P<0.001), rD-7decreased its expression on DCs (P<0.01).2. CEACAM1, the receptor of rD-7, was expressed on human DCs. Myeloid-derived monocytes were induced by rh GM-CSF and rh IL-4for6days to yield immature DC or mature DC with additional24h LPS stimulation. The percentages of cells expressing CD11c were almost100%. CEACAM1was detected on imDCs and mDCs with monoclonal anti CEACAM1antibody MAb2244whilst the percentage of CEACAM1+DCs was low by using flow cytometry.3. Decreased CD80expression on DC modulated by rD-7was independent of CEACAM1signal. After imDCs were stimulated with either rD-7or rD-7/D for24h, CD80expression on DCs was significantly decreased to similar levels by rD-7(P<0.01) or rD-7/D (P<0.05), but positive control stimulation with LPS alone significantly increased CD80expression as observed (P<0.001). However, the decrease of CD80expression induced by rD-7was not blocked by using anti CEACAM antibody A0115.4. Increased CD83expression on DC modulated by rD-7was independent of CEACAM1signal. CD83expression on DCs was increased by rD-7/D (P<0.01) to similar levels in each case with using rD-7(P<0.05). Furthermore, control LPS also significantly increased CD83expression (P<0.001). Also, the increase of CD83expression induced by rD-7was not blocked by A0115 compared with IgG control.5. Increased CD86expression on DC modulated by rD-7was independent of CEACAM1signal. CD86expression on DCs was also increased by rD-7/D (P<0.001) to similar levels in each case with using rD-7(P<0.001). Furthermore, control LPS also significantly increased CD86expression (P<0.001). Compared with LPS, the increased CD86expression induced by rD-7(P<0.05) and rD-7/D (P<0.01) were more significant. Also, the increase of CD86expression induced by rD-7was not blocked by A0115compared with IgG control.6. Increased HLA-DR expression on DC modulated by rD-7was independent of CEACAM1signal. HLA-DR expression on DCs was all increased by rD-7(P<0.01), rD-7/D (P<0.01), LPS (P<0.01) to similar levels in each case and A0115could not block the increase of HLA-DR expression on DCs induced by rD-7compared with IgG control.7. TNF-a, IL-10and IL-12p70secretions from DCs stimulated by rD-7. TNF-a secretions was significantly increased by positive control LPS (P<0.05). TNF-a produced by DCs was slightly increased by rD-7and rD-7D to a similar level. Furthermore, IL-10and IL-12p70were not detectable using ELISA after rD-7or rD-7/D stimulation.Conclusions:Moraxella catarrhalis adhesin UspAl-derived recombinant fragment rD-7modulated DC activation and maturation. CD83, CD86and HLA-DR expressions were increased by rD-7. Unlike LPS, CD80expression on DC was decreased by rD-7. CEACAM1, the receptor of rD-7was expressed on DC; however, DC maturation modulated by rD-7was independent of CEACAM1. Unlike LPS, TNF-a, IL-10and IL-12p70secretion was not significantly affected by rD-7. DC maturation was significantly modified by Moraxella catarrhalis UspAl-derived recombinant fragment rD-7, revealing the regulation of Moraxella catarrhalis virulence factor UspAl on adaptive immune response, thus our study will provide the insight for the lung cancer patients with M. catarrhalis infections who were also treated with DC vaccine and the development of M. catarrhali vaccine. |
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