Thesis (Ph. D.)--Ohio State University, 20043. / Title from first page of PDF file. Document formatted into pages; contains xiv, 115 p.; also includes graphics. Includes abstract and vita. Advisor: Clay B. Marsh, Dept.of Pathology. Includes bibliographical references (p. 102-115).

The fetal-maternal interface is an immunologically privileged site where the semi-allogeneic fetus is protected from the maternal immune system. Macrophage represents the second major type (20-30%) of the decidual leukocyte. It functions as important regulator of pregnancy processes such as fetal tolerance, placental development and onset of labor. Changes in macrophage number and activity have been associated with fetal loss and pregnancy complications, including intrauterine growth restriction and preeclampsia. Glycodelin-A (Gd-A) is an abundant glycoprotein with ubiquitous distribution in the first trimester deciduas. It is suggested to be involved in early placental development by its regulatory activities on various immune cells. In this study, it is hypothesized that Gd-A has regulatory role in monocyte/macrophage functions and differentiation. The first objective examined the role of Gd-A in the biological activities of monocytes and macrophages. Gd-A was found to bind to the monocytic cell lines THP-1 and U937, blood monocytes, granulocyte macrophage colony-stimulating factor (GM-CSF)-differentiated macrophages, phorbol 12-myristate 13-acetate-differentiated macrophage and blood macrophages. Gd-A did not affect the viability, proliferation, cell death and phagocytic activity of monocytes and macrophages. The second objective examined the effect of Gd-A on the cytokine secretion of monocytes and macrophages. Gd-A treatment increased the IL-6 production in monocytes and macrophages. IL-6 was associated with the development and growth of the fetal-placental unit. Gd-A also induced the extracellular signal regulated kinases (ERK) activation. The involvement of ERK in stimulating IL-6 production was confirmed by using pharmacological inhibitors. Monocytes in the blood stream are attracted and residue into the deciduas, which differentiated into macrophage under the influence of various decidual soluble factors. Therefore, the third objective studied the possible involvement of Gd-A on the differentiation of monocytes into macrophages. Co-treatment of Gd-A during the GM-CSF-induced differentiation of monocytes stimulated the indoleamine 2,3-dioxygenase (IDO-1) expression and activity in differentiated macrophages. These differentiated macrophages inhibited the proliferation of autologous peripheral blood mononuclear cell in the co-culture system by G0/G1 cell cycle arrest. IDO-1 is one of the reported decidual macrophage markers. It has been suggested to be involved in establishing the tolerogenic environment during pregnancy through L-tryptophan depletion. The increase in IDO-1 expression may be regulated by PKC signaling pathway. Taken together, this thesis reported a novel role of Gd-A on the monocyte differentiation. The present results enhance our knowledge on the regulation of early placentation in human and may shed light on understanding the pathology of complicated pregnancy due to macrophage dysfunction. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Macrophages have well-established roles in the primary response to pathogens and hold essential functions during innate and adaptive immunity. Under activation by different growth factors and cytokines, human monocytes have been shown to differentiate and polarize into two main types of macrophage, classically-activated macrophages (caMφ) and alternatively-activated macrophages (aaMφ), displaying distinct properties and phenotypes. For instance, caMφ secrete pro-inflammatory cytokines, whereas aaM secrete anti-inflammatory cytokines. Additionally, aaMφ displays stronger phagocytic ability and are equipped with different endosomal proteases. While it has been established that monocyte-derived macrophages can be infected by Influenza A virus, most studies utilized a macrophage population obtained by differentiation in the presence of autologous plasma. My research project aimed at systematically comparing susceptibility of the infection by Influenza A virus to the recently described caMφ and aaMφ. Here I show that monocytes cultured in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ or in presence of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-4 or IL-10 can be differentiated into distinct populations. According to immunophenotyping results, a distinct expression profile was observed for Cluster of Differentiation (CD) 36, CD86, Mannose Receptor (MR or CD206), and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209) among differentiated macrophages. Except for CD86 expression, my results were in accordance with previous reports and thus allowed me to classify all populations into caMφ (M1 macrophages), and aaMφ (M2a and M2c macrophages). I then assessed the susceptibility of the above mentioned macrophages to pandemic Influenza A/California/04/2009 H1N1 virus (CA04) infection. My results demonstrate a marked difference, caMφ showing low to moderate permissivity, whereas aaMφ – and in particular M2a macrophages – were consistently highly infected. In contrast, no difference was observed with Influenza A/WSN/1933 H1N1 virus (WSN/33) infection. Because sialic acids are regarded as the primary receptor for influenza virus, I investigated the cell surface distribution of sialic acids with α2-3 linkage (SAα-2,3) or α2-6 linkage (SAα-2,6) among the population of human macrophages. By using lectin staining with Maackia amurensis lectin (MAL) II and Sambucus nigra lectin (SNA), which bind sialic acids with α2-3 linkage (SAα-2,3) and α2-6 linkage (SAα-2,6) respectively, I found all the monocyte-derived macrophages exhibited a comparable expression of SAα-2,3 and SAα-2,6, which unlikely explain the differential susceptibility to infection by CA04. In addition to sialic acids, C-type lectins were also proposed to mediate entry of influenza viruses into macrophages. All macrophages expressed CD206 but only M2a expressed CD209. However assay aiming at interfering with CD209 binding (MAb blocking assay or EGTA treatment) did not inhibit pdmH1N1 infection. Surprisingly, infection in presence of EGTA, which is believed to reduce the functional ability of C-type lectins, exacerbated susceptibility of the macrophages. Altogether my results show that susceptibility to Influenza A virus infection of in vitro differentiated primary human macrophages is unlikely to rely on the sialic acid expression profile and is dependent on viral strain. Further studies are needed to understand what difference from caMφ and aaMφ – either phenotypic and/or biochemical – confer them distinct susceptibilities to some viral subtype/strain of Influenza A. / published_or_final_version / Pathology / Master / Master of Philosophy

The seasonal Influenza A viruses are respiratory pathogens causing epidemics annually with mild illnesses, while sporadically, novel influenza viruses emerge and trigger pandemics associated with more widespread and sometimes severe disease. The biological basis for severity of influenza disease remains unclear though it is recognized that the interplay between the influenza viruses and the host immune responses both contribute to viral pathogenesis. As macrophages are key sentinels of the innate immune response and play a crucial role in being the “first responders” as well as contributing to shaping the subsequent (pathogen‐specific) adaptive immune response, the objective of this research was to bring insights on the outcomes of the interactions of influenza viruses with the macrophages. The occurrence of Antibody‐Dependent Enhancement (ADE) of Influenza infection in macrophages was investigated. ADE occurs when non‐neutralized virus‐antibody complexes find alternative entry routes into host cells, mainly through the Fc‐receptor pathway and has been demonstrated predominantly in macrophages. Addition of human serum from some individuals to influenza A virus (either H5 pseudoparticles or pandemic (H1N1) virus) led to enhanced infection of murine macrophage‐like cells as illustrated by a two to five fold increase in detection of influenza M‐gene copies. Immunofluorescence microscopy indicated that serum‐mediated pandemic (H1N1) infection led to an increase in the number of infected cells than in controls. As the fold change in viral gene copies paralleled the fold increase of infected cells I concluded that ADE infection provide pandemic (H1N1) virus with increased opportunity to infect cells rather than simply increase the viral load per cell. In order to strengthen our results, and make them more physiologically relevant, experiments were then performed with human primary cells with clinical sera. However, ADE was not demonstrated in primary human macrophages, suggesting that ADE may be cell type or host specific. The second research question investigated was whether the different state of human primary macrophage differentiation or activation in vitro determined the susceptibility to influenza infection. Recently, work by others has shown a diverse range of macrophage phenotypes that arise by differences in macrophage differentiation and activation. In addition to the classical activation pathway (caMΦ), new mechanisms of activation, designated as alternative activation (aaMΦ), have been reported. Classically and alternatively activated macrophages display different phenotypes and properties, such as molecule expression patterns, cytokine secretion, and gene signatures. This study constitutes the first systematic comparison of Influenza A virus infection of these different subsets of human primary monocyte‐derived macrophages. When assessed for their permissiveness to different influenza A viruses, aaMΦΦshowed greater susceptibility to influenza A infection than caMΦ. This work also documents the receptor patterns and the gene expression profile of these macrophages in response to influenza virus infection in vitro. The results point to differences in susceptibility of the classically and alternatively activated human macrophages to pandemic H1N1 and other influenza A viruses and reveal intrinsic differences between these macrophage subtypes. Further investigations are needed to define the cellular and molecular determinants that define susceptibility of different macrophage subsets to influenza A infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Background and Aim Hepatocellular Carcinoma (HCC) is the fifth most frequent malignancy worldwide with high mortality and recurrence rate. Chronic inflammation is a dominant risk factor for the malignancy and the roles of tumor associated immunological infiltrates during the disease development and progression remain unclear. The significance of alternative activated macrophages (M2) with pro-tumor phenotypes has been demonstrated in many cancers except HCC. M2 macrophages are associated with tumor angiogenesis, invasion and growth which represent a potential target to be investigated. In this study, we intend to investigate the role of M2 macrophage on HCC. Materials and Methods M2 macrophages in 100 clinical specimens collected from HCC patients were detected by immunohistochemical (IHC) staining and quantitative PCR using common macrophage markers CD14 and CD68, and M2 specific markers CD163, Scavenger receptor class A (SA) and Mannose receptor (MR). The protein and transcript expression levels were further correlated with clinical pathological parameters. Phenotypic and functional characteristics of M1 and M2 macrophages derived from THP-1 cell line were validated and their roles in promoting HCC growth and invasion were studied in vitro co-culture system and in vivo orthotopic mice model. Secretory profiles of M2 macrophages after cocultivated with HCC cells were analyzed by cytokines antibody array screening. C-C motif chemokine 22 (CCL22) was identified to be upregulated in M2 macrophages in response to HCC cells. The functional roles of the chemokine in HCC was further studied by chemotaxis and migration assay. Underlying molecular mechanisms induced by CCL22 in HCC cells were determined. Results In clinical analysis, we first discovered that macrophages were widely expressed in liver tumor comprising 10-30% of total cell population. Noteworthy, the density of a M2 macrophage marker CD163 was found to had a significant prognostic impact as an independent factor associated disease free survival (HR: 3.79; 95% CI 1.3-10.4; p<0.01). Strong expression of the CD163+ population was also correlated with poor relapse free survival, multiple tumor nodules and increased venous infiltration. In vivo studies revealed that the tumor volume injected with M2 macrophages increased 3.26-fold (1.27cm3±0.36) compared to the control group (0.39cm3±0.05) (P=0.032). In contrast, mice injected with M1 macrophages had a significant reduction of tumor volume by 2.79-fold (0.14cm3±0.02) (P=0.044). Increasing rate of lung metastases (57%) was also observed in M2 treated group compared to the control (25%) (P<0.05). In vitro, M2 macrophages increased the number of HCC cells (MHCC97L) and migration events by 1.3-fold and 3.2-fold after cocultivation compared to negative control (P<0.05). In cytokine antibody array study, M2 macrophages derived CCL22 was discovered to be strongly induced by MHCC97L. Chemotaxis analysis confirmed that CCL22 increased MHCC97L migration capacities and excess level led to increased venous infiltration in HCC patients (p<0.05). Upon addition of CCL22, the epithelial mesenchymal transition through SNAIL activation in MHCC97L was observed. Conclusion Clinical analysis and both the in vitro and in vivo studies presented here collectively illustrated the significances of M2 macrophages in promoting tumor growth and migration through CCL22-CCR4 mechanism in HCC. Targeting these M2 macrophages and associated products in situ represents a novel approach for treating the disease. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Chronic obstructive pulmonary disease (COPD) is a progressive disease and characterized by persistent airflow limitation. Pathophysiologically it involves many components, including oxidative stress and inflammation of the airways and lungs. Although the primary cause of COPD is smoking, acute exacerbation due to infections can accelerate disease progression, which is a significant cause of morbidity, mortality and burden on healthcare costs. One-half of all acute exacerbations of COPD are associated with bacterial infection, with non-typeable Hemophilus influenzae being the most common pathogen. Staphylococcus pneumonia, one of the most common Gram-positive bacterial pathogens, is also involved in airway infections, either primary or subsequent to viral diseases. To COPD patients the common respiratory pathogens include Gram-negative and Gram-positive bacterial species. Lipopolysaccharide (LPS), a major component of the outer membrane of all Gram-negative bacteria, which is the predominant inducer of inflammatory responses, has been widely studied. On the other hand, less is known about Gram-positive bacteria, which do not contain LPS but express lipoteichoic acid (LTA) as an important proinflammatory constituent in their cell wall. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that plays an important role in regulating pulmonary function and pathogenesis of inflammation. In this study, we hypothesize that the serotoninergic system may be involved in LPS- and LTA-induced inflammation in COPD. Since monocyte recruitment to lung is a key step in COPD, this study aims to investigate the effects of LPS or LTA alone and in combination of 5-HT pretreatment on the release of pro-inflammatory cytokines in undifferentiated (i.e. monocytes) and differentiated THP-1 cells (i.e. macrophages). LPS, LTA or 5-HT alone induced the release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in both monocytes and macrophages. Our findings also showed that 5-HT pretreatment suppressed the LPS-induced IL-8 and MCP-1 release, suggesting that 5-HT might act as an anti-inflammatory mediator. On the other hand, 5-HT pretreatment enhanced the LTA-induced IL-8 and MCP-1 release, indicating that 5-HT might also act as a pro-inflammatory mediator. These results demonstrate that 5-HT may be involved in the differential modulation of inflammatory processes during Gram-negative or Gram-positive infections in COPD. / published_or_final_version / Medicine / Master / Master of Medical Sciences

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published_or_final_version / abstract / toc / Pharmacology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Chemistry / Master / Master of Philosophy