Thesis (Ph. D.)--University of Chicago, Committee on Immunology, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-116). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-116). Also available in print.

Interleukin-17A (IL-17A)is an important pro-inflammatory cytokine that plays a critical role in host defenses against diverse pathogens. Studies have shown that IL-17Aplays protective role against sub-lethal H1 and H3 subtypes influenza infections, but it is unclear about the role of IL-17A in the highly pathogenic H5N1 and lethal H1N1 influenza virus infection. B cell is an important effector cell type in anti-influenza immunity. Although roles of B cell in influenza infection have been extensively investigated, it is unclear whether and how IL-17AregulatesB cell response during influenza infection. I examined the role of IL-17A against influenza infection by challengingIL-17A knockout (KO) and wild-type (WT) mice with highly pathogenic H5N1 and lethal H1N1 influenza viruses. Following challenge, IL-17AKO mice exhibited significantly lower survival rate, profoundly reduced body weight, more severe tissue damage and higher viral burden in the lung tissues. These evidences suggest that IL-17Aplays a protective role in lethal influenza infection. To study whether IL-17Amodulates B cell response against influenza, I found that both B-1a and B-2 cells were detected in the lung tissue and pulmonary draining lymph node, Mediastinal lymph node (MedLN),as early as 2days post-infection. Meanwhile, B-1a cells predominantly contributed to the early virus-specific IgM in the respiratory tract. However, virus-specific IgM markedly reduced in IL-17A KO mice when compared with WT controls. Adoptive transfer of B-1a cells or B-1a cell-derived antibodies conferred protection in IL-17A KO mice. These results demonstrate that IL-17A plays a critical role in modulating early antibody production of B-1a cells against lethal influenza infection. To further elucidate how IL-17A regulates B-1a cell response, I observed that B-1a cells migrated into MedLN and lung tissues during infection and underwent plasmacytic differentiation with increased antibody production in airways. IL-17A deficiency impaired these processes of B-1a cells, while intra-nasally instillation of IL-17A restored B-1a cell response by promoting both B-1a cell migration and plasmacytic differentiation. By inducing blimp-1 expression in B-1a cells in an NF-κB dependent pathway, IL-17A directly promoted plasmacytic differentiation of B-1a cells both in vivo and in vitro. Furthermore, chromatin immuno-precipitation analysis confirmed that NF-κB directly bound to the promoter of blimp-1 gene and promoted blimp-1 expression in B-1a cells following IL-17A stimulation. To determine the functional significance of IL-17A signaling in modulating B cell response against influenza infection, I first uncovered markedly reduced B cell response, predominantly B-1a cell response in IL-17A KO mice, showing reduced local migration and impaired plasmacytic differentiation in the early stage of infection. Next, intra-nasal administration of IL-17A into IL-17A KO mice significantly restored this B-1a cell response. Moreover, I detected expression of IL-17A receptor in B-1a cells. IL-17A treatment could promote antibody production from B-1a cells by inducing blimp-1 expression in an NF-κB dependent pathway. Taken together, these findings identify a novel role of IL-17A in actingas an immune modulator of B cell response against influenza infection, which will contribute to a fuller understanding of B cell biology and anti-viral response in host defense. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Much of the research that has been done on the fish immune system has focussed on innate immunity. Very little is known about the adaptive immune system of fish and how it is regulated. This study has identified and characterised key cytokines that had not been found in any salmonids species to date, and that are potentially involved in T helper 1 (Th1) and Th17 type responses. The cloning of trout and salmon IL-1β3, and identification of a salmon IL-1β4 pseudogene revealed two types of IL-1β genes exist in teleost fish. Trout IL-1β3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1β3 also has a role in inflammation and host defence. The IL-12 cytokine family are heterodimeric proteins that consist of an α-chain (p19, p28 or p35) and β-chain (p40 or EBI3). Due to the teleost wide whole genome duplication and/or salmonid whole genome duplication events, fish, especially salmonids, may have many paralogues of each subunit. Indeed, two distantly related p40 subunits termed p40A and p40B, as well as an EBI3 gene and a p35 gene have been previously cloned in rainbow trout. The cloning and sequence analysis of a p19 subunit gene was described in rainbow trout for the first time The expression and modulation of the (now known) five subunits (p19, p35, p40b, p40c and EBI3) of the trout IL-12 family members was comparatively examined in vivo in healthy fish and in fish after viral or parasite infection, and in vitro after stimulation with PAMPs, immune stimulants, suppressants, and recombinant trout cytokines. Bioactivity testing of two recombinant proteins of rainbow trout IL-12 paralogues was next studied. The recombinant IL-12A (p40c/p35a) and IL-12B (p40b/p35a) proteins were added to head kidney cultures and immune gene changes examined by real time PCR. This experiment 7 showed both up and down regulation of a number of the genes analysed, and revealed that the recombinant IL-12A and IL-12B proteins possess some shared bioactivities, but that some differences in function were also apparent. Fish in the Salmonidae family are characterized by having a relatively recent tetraploid ancestry, where a common ancestor of salmon and trout experienced whole genome duplication. Hence modern day species may be considered pseudo-tetraploid, as they are in the process of reverting to a stable diploid state. To gain further insight into the number of IL-12 family member paralogues present in salmonids, attention was turned to Atlantic salmon due to the availability of the initial genome sequencing contigs. Nine IL-12 cytokine family subunits (p19a, p19b, p35a1, p35a2, p35b1, p40c, p40b1, p40b2, and EBI3) were identified in Atlantic salmon and then comparatively examined in vivo in healthy fish and in fish after Poly I:C immune stimulation and in vitro after stimulation of head kidney cells with PAMPs, immune stimulants and suppressants, and recombinant trout cytokines. Having these genes available for study, along with what is currently known about the teleost immune system, will allow investigations into the adaptive immune responses of fish to a level not previously possible. Understanding these types of responses in fish and how they are regulated will help in the development of essential therapeutic strategies in fish.

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hemopoietic cell line, B6SUtA. These cells were first used to identify the mIL-3 surface receptor as a monomeric 67 kDa protein with a pI of approximately 6.2. Further studies suggested the presence of an additional mIL-3 binding protein with an apparent molecular mass of 140 kDa. Then, in an attempt to gain some insights into the mechanism of action of mIL-3, molecules other than mIL-3 were tested to determine their effects on cell proliferation. Murine granulocyte -macrophage colony-stimulating factor (mGM-CSF) was found to be as potent as mIL-3 in stimulating B6SUtA cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, both stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA cells exposed for brief periods to mIL-3, mGM-CSF or TPA were analyzed for changes in phosphorylation patterns using metabolic [superscript]32p-labeling. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68 kDa cytosolic protein, while all three agents stimulated the serine-specific phosphorylation of a 67 kDa membrane protein. Furthermore, using antibodies to phosphotyrosine, it appeared that mIL-3 stimulated tyrosine phosphorylation of 67 kDa and 140 kDa membrane proteins, as well as of 40, 55 and 90 kDa cytosolic proteins. The 90 kDa protein was also tyrosine phosphorylated in response to mGM-CSF, suggesting that this phosphorylation results from a common step in mIL-3 and mGM-CSF-stimulated signaling pathways. These phosphotyrosine containing proteins were not detected in TPA-treated cells. Moreover, evidence from a variety of studies is presented that the 140 kDa but not the 67 kDa mIL-3 receptor becomes phosphorylated on tyrosine residues when B6SUtA cells bind mIL-3. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated, in part, by pro-inflammatory cytokines such a sI L- I P, TNFa andI L-6. Many factors may contribute to cytokine imbalances in this disease, for example, biochemical modulation of PBMCsa ndt heir membranes A. key membrane proteini s the Na/KATPase( sodium pump) responsible for ionic homeostasis Sodiump ump activity on rheumatoid PBMCsw as found to be markedly depressed when compared with healthy control cells possibly through an oxidative mechanism. Inhibition of the sodium pump by a cardiac glycoside inhibitor, ouabain, transiently upregulated[N a'ji levels and rapidly induced IL-10 and TNFa mRNA and protein in human PBMCs. In contrast, IL-6 production was significantly depressed. The sodium ionophore, monensin, caused a similar Na-dependent cytokine response to that of ouabain. This cytokine profile however, was reversed when studying rheumatoids ynovial fibroblasts where ouabain induced I L-6; IL- I and TNFa, on the other hand, were not expressed. An elevation in intracellulars odiumc an causea secondary rise in intracellular calcium levels through the action of a Na/Ca2+ exchanger. In studies using the calcium ionophore, A23187, it was observed that an elevation in [Ca 2+]i brought aboutt he induction of IL- IP and TNF(xi n PBMCs with a corresponding repression of IL-6 production. The data obtained in this study suggest that impaired N a/K-ATPase activity in rheumatoid cells, through elevations in intracellular cation levels, might help promote over-production of IL- IP and TNF(x by monocytes and IL-6 by synovial fibroblasts. This pattern of cytokine production conforms to that observed in rheumatoid synovial tissue in situ, thus supporting a role for this biochemical defect in contributing to the perpetuation of the chronic inflammatory state.

The WHO predicts that by the year 2035 the world will be facing a “cancer tidal wave”. This has spurred on the development of many cancer immunotherapies. The adoptive transfer of ex vivo expanded NK cells is one such therapy that could have high efficacy and target specificity. However, the adoptive transfer of NK cells has some negative side effects. Fortunately, these are not due to the direct effects of the NK cells. Instead, toxicity arises from the systemic administration of IL-2 which supports NK cell function. To sidestep the need for IL-2 injections our project investigated the effect of stimulating NK cells with interleukin 12, 15, and 18 in vitro. Our hope is that one-day pre-stimulation of NK cells with these cytokines in vitro before their adoptive transfer will maintain NK cell activation and survival in vivo. Our research has revealed that ex vivo expanded NK cells stimulated with IL-12 and IL-18 +/- IL-15 significantly upregulates the expression of IFN-γ, TNF-α, CXCL-8, CCL3L1, and LTA. Furthermore, production of these cytokines can continue up to 72 hours post stimulation in vitro. If the production of these cytokines continues after adoptive transfer of NK cells into cancer patients it could drastically alter the anti-inflammatory milieu of the cancer patient. Our attention was then turned to elucidating the factors responsible for the long term activation of the NK cells in the IL-12 and IL-18 +/- IL-15 conditions. We have determined that the increase in production of proinflammatory cytokines is due to direct increases in IFN-γ transcription. The results of these trials will direct the future use of NK cells in clinical trials. Specifically, there is great potential for this research to be used to predict potential negative side effects of using ex vivo expanded and stimulated NK cells as a cancer immunotherapy. / Thesis / Master of Science (MSc)

No description available.