Macrophages (MΦ) as specialized immune cells are involved in rubella virus (RuV) pathogenesis and enable the study of its interaction with the innate immune system. A similar replication kinetics of RuV in the two human MΦ types, the pro-inflammatory M1-like (or GM-MΦ) and anti-inflammatory M2-like (M-MΦ), was especially in M-MΦ accompanied by a reduction in the expression of the innate immune receptor CD14. Similar to RuV infection, exogenous interferon (IFN) β induced a loss of glycolytic reserve in M-MΦ, but in contrast to RuV no noticeable influence on CD14 expression was detected. We next tested the contribution of CD14 to the generation of cytokines/chemokines during RuV infection of M-MΦ through the application of anti-CD14 blocking antibodies. Blockage of CD14 prior to RuV infection enhanced generation of virus progeny. In agreement with this observation, the expression of IFNs was significantly reduced in comparison to the isotype control. Additionally, the expression of TNF-α was slightly reduced, whereas the chemokine CXCL10 was not altered. In conclusion, the observed downmodulation of CD14 during RuV infection of M-MΦ appears to contribute to virus-host-adaptation through a reduction of the IFN response.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23783号 / 医博第4829号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 上野 英樹, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Opioid receptor-mediated regulation of chemokine receptors is vital for the host immune response, development, and neurological function. Previous studies have demonstrated that the kappa opioid receptor (KOR) activation results in decreased infectivity of human immunodeficiency virus 1 (HIV-1) in human peripheral blood mononuclear cells (PBMCs). We have found this effect is due to down-regulation of the major HIV-1 co-receptors, CCR5 and CXCR4. Using molecular techniques, CCR5 and CXCR4 mRNA levels drop dramatically following KOR activation. To dissect the mechanism involved, we used transcription factor binding arrays and compared control cell extracts to KOR activated cell extracts. We determined that the interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) could be involved in the KOR-mediated repression of CCR5 and CXCR4 transcription and protein expression. Using chemical inhibitors and small interfering RNA (siRNA) molecules, we determined that JAK2, STAT3, and IRF2 are critical members of this signal transduction pathway. The understanding of these particular mechanisms should prove to be beneficial for the development of potential pharmacological agents targeted at HIV-1 binding and infection since virus infection requires expression of the co-receptors CXCR4 and CCR5. Understanding the molecular basis for KOR-induced inhibition of co-receptor expression may provide a basis for the development of KOR agonist-based therapeutics to treat individuals infected with HIV. / Molecular Biology and Genetics

Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.

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