Medical Biology

Interleukin-6 (IL-6) family cytokines, including IL-6, oncostatin M (OSM), interleukin- 11 (IL-11) and Leukemia Inhibitory Factor (LIF), are pivotal modulators of numerous cellular processes such as acute phase and immune responses, cellular activation, differentiation, proliferation and survival. Binding of these cytokines to their receptors and the common gp130 subunit activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Suppressor of cytokine signalling-3 (SOCS3) is a major regulator of the JAK/STAT pathway. This thesis examines the impact of the IL-6 cytokine family (gp130 cytokines) on cartilage and the potential regulatory role for SOCS3 in chondrocytes. For this purpose, we generated a transgenic mouse line expressing Cre recombinase under the control of the mouse type II collagen promoter (Col2a1). Tissue-specific recombination of genes using the loxP-Col2a1-Cre mouse provides a means to investigate the deletion of a gene of interest in chondrocytes. This thesis begins by characterising the expression of Cre recombinase in organs and tissues of the SOCS3 conditional knockout mouse (Socs3Δ/Δcol2). Col2a1-Cre expression was found in both articular chondrocytes and cartilage explants, resulting in the excision of Socs3. However, some Col2a1-Cre expression was also observed in the eye, kidney, liver, heart and a small population of neutrophils. Because previous studies have also demonstrated Col2a1-Cre expression in synovial fibroblasts using a similar model, Col2a1-Cre expression and potential SOCS3 deletion was evaluated in these cells. I demonstrated that synovial fibroblasts from Socs3Δ/Δcol2 mice stimulated with gp130 cytokines failed to show any reduction in Socs3 mRNA expression, or differences in cytokine production. The consequences of Cre recombinase expression and Socs3 deletion in a proportion of synovial fibroblasts therefore appear to be minimal. Chondrocytes are not only responsible for maintaining cartilage homeostasis, but also play a critical role in skeletal development. Having characterised the specificity of SOCS3 deletion in Socs3Δ/Δcol2 mice, I proceeded to investigate the consequences of SOCS3-deficiency in chondrocytes during skeletal development. In an ageing cohort study, I found consistently reduced total body weight in Socs3Δ/Δcol2 mice compared to controls. Further examination showed retardation of longitudinal bone growth in Socs3Δ/Δcol2 mice. Immunohistochemistry showed reduced proliferation of SOCS3-deficient growth plate chondrocytes. These results suggest a key regulatory role for SOCS3 in growth plate chondrocytes during bone growth. I next examined the specific contribution of chondrocytes to joint inflammation and cartilage degradation in response to gp130 cytokines. Stimulation of primary chondrocytes with gp130 cytokines activated the JAK/STAT signalling cascade. SOCS3 deletion in chondrocytes prolonged STAT1, STAT3 and STAT5 signalling. In response to gp130 cytokines, isolated chondrocytes and cartilage explant cultures from Socs3Δ/Δcol2 mice showed enhanced aggrecanase expression, cartilage degradation, and increased production of multiple inflammatory mediators. These findings highlight the potential roles of chondrocytes during joint inflammation. In particular, OSM was found to be a potent mediator of catabolic and inflammatory responses in chondrocytes. Intra-articular injection of gp130 cytokines induced, to varying degrees, joint inflammation and cartilage degradation in wild-type mice. Prolonged JAK/STAT signalling in Socs3Δ/Δcol2 mice exacerbated these responses. Using the methylated bovine serum albumin (mBSA)/interleukin-1 (IL-1) model of acute inflammatory monoarthritis, I showed that chondrocytes play a significant role in driving joint inflammation and cartilage and bone erosion, at least comparable to the hematopoietic compartment. These results were reproduced in the K/BxN serum transfer model, a well-established model of inflammatory arthritis. To investigate the divergent responses of chondrocytes to gp130 cytokines, transcriptional profiling was performed on isolated wild-type and SOCS3-deficient chondrocytes stimulated with each of the gp130 cytokines. A wide range of biological processes was altered in chondrocytes in response to gp130 cytokine stimulation, including apoptosis, cytokine and chemokine activity and inflammatory signalling cascades. OSM had the most potent influence on genes associated with “Positive regulation of inflammatory responses” and these were amplified by the absence of SOCS3. The major findings of this thesis that contribute to a greater understanding of the role of chondrocyte in skeletal development and inflammatory arthritis are: 1. SOCS3 plays a key role in regulating chondrocyte function in the growth plate during bone growth 2. gp130 cytokines, to varying degrees, can induce catabolic and inflammatory responses in chondrocytes in vitro 3. OSM appears to be at least as important as IL-6 in mediating inflammatory responses in chondrocytes and may be a novel target for therapeutic blockade in inflammatory arthritis 4. SOCS3 plays a critical role in limiting chondrocyte responses to gp130 cytokines 5. Prolonged JAK/STAT signalling in SOCS3-deficient chondrocytes exacerbates joint inflammation, cartilage degradation and bone erosion in vivo.

Malaria remains a major cause of morbidity and mortality throughout the tropical world. The emergence of parasite strains resistant to chloroquine and other commonly used anti-malarial drugs provides an urgent call for vaccine development and novel drug discovery. The clinical manifestations of a P. falciparum infection can range from a mild febrile illness to life-threatening severe malaria, however the determinants of disease severity are not well understood. Although the early, innate immune response to malaria serves to limit parasite growth and reduce clinical manifestations, endogenous mediators produced in response to bioactive parasite products may contribute to pathology associated with malaria. The P. falciparum glycosylphosphatidylinositol (GPI) anchor has been described as a malarial toxin, capable of stimulating pro-inflammatory cytokine responses in vitro and in vivo. Vaccination with a synthetic form of the GPI glycan protects mice from experimental cerebral malaria and death. However attempts to investigate the bioactivity of GPI have been hindered by a lack of suitable reagents for its study. Monoclonal antibodies were raised to P. falciparum GPI and used as probes to investigate the role of GPI in parasite cell biology. The expression of free GPI in late schizonts, dynamic localization of GPI during merozoite invasion, and growth inhibition in the presence of an anti-GPI monoclonal antibody suggest that GPI may have a role in invasion of erythrocytes. Furthermore, the pan-species and multiple life stage reactivity of anti-GPI antibodies have important implications for vaccine development. The second part of this thesis describes a study of cellular cytokine and chemokine responses in a severe malaria case control study in Papua New Guinean children. Cytokines and chemokines produced by convalescent peripheral blood mononuclear cells (PBMC) in response to stimulation with parasitized red blood cells or Toll-like receptor 2 (TLR2) and TLR4 agonists were significantly associated with severe malaria. A more comprehensive understanding of the role of these endogenous mediators in malaria pathogenesis will aid in the development of effective vaccines and may provide the opportunity for novel interventions.

A general study of factors stimulating formation of colonies of granulocytic and monocytic cells from mouse bone marrow cells grown in agar cultures has been made. Factors from mouse serum, urine and tissue extracts, human serum and urine and mouse embryo cell “conditioned medium” were examined. (For full abstract open document)

Medical Biology - Theses

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