The macrophage receptors that mediate phagocytosis of Borrelia burgdorferi, the Lyme disease spirochete, are unknown despite this cell type’s importance in promoting pathogen clearance and inflammation-mediated tissue damage. We now demonstrate that the β2 integrin, Complement Receptor 3 (CR3), mediates the phagocytosis of opsonized and non-opsonized spirochetes by murine macrophages and human monocytes. Although, expression of the surface proteins, CspA and OspE, protects B. burgdorferi from complement-mediated phagocytosis, the versatility of CR3 counteracts the ability of B. burgdorferi to interfere with complement activation and complement-derived opsonins, thus minimizing the bacteria’s anti-complement strategy. Interaction of the spirochete with the integrin is not sufficient to internalize B. burgdorferi; however, phagocytosis occurs when the GPI-anchored protein, CD14, is coexpressed in CHO-CR3 cells. CR3-mediated phagocytosis occurs independently of MyD88-induced or inside-out signals but requires the translocation of the integrin to cholesterol rich microdomains. Interestingly, the absence of CR3 leads to marked increases in production of TNF in vitro and in vivo, in spite of reduced spirochetal uptake. Overall, our data establish CR3 as a MtD88–independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory out put of macrophages. Macrophages are critical cellular components of the immune response to infectious agents. During infection with B. burgdorferi, macrophages infiltrate the cardiac tissue and induce the activation of invariant NKT cells, leading to the production of the protective cytokine IFNγ. The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen activated protein kinases, such as p38 MAP kinase. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response with B. burgdorferi . The inhibition of p38 MAP kinase does not alter the ability of macrophages to phagocytose B. burgdorferi; however, inhibition of p38 during infection with B. burgdorferi results in increased carditis. Through the generation of transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, we demonstrate that this kinase regulates the production of the iNKT attracting chemokine, MCP-1 and the infiltration of these cells to the cardiac tissue during infection. Overall, the inhibition of p38 MAP kinase during infection with B. burgdorferi specifically in macrophages results in the deficient infiltration of iNKT cells and their diminished production of IFNγ, leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.

<p> COPD is characterized by an abnormal regulatory T cell (Treg) response with a shift towards a Th1 and Th17 cell responses. However, it is unclear if the function of Treg cells is impaired by smoking and in COPD. In addition, the miRNA profile of Treg cells in COPD is unknown and whether miRNA deregulation contributes to COPD immunopathogenesis. We set the objective to study Treg cell function isolated from peripheral blood of patients with COPD versus controls and to compare their miRNA profiles. We also were interested in exploring the function of some of the differentially expressed Treg cell miRNAs. We assessed the Treg cell function by observing their suppressive activity on autologous effector T cells and analyzed their miRNA expression initially by microarray analysis then conducted real time RT-PCR validation for selected miRNAs. In Silico target gene analysis for the validated miRNAs suggested that miR-199-5p is particularly relevant to Treg cell physiology so its function was investigated further using CCD-986Sk and MOLT-4 cells. We found no difference in Treg cell function between COPD and controls but we were able to identify 6 and 96 miRNAs that were differentially expressed in COPD versus control Treg cells. We confirmed that miR-199a-5p was repressed by approximately 4 fold in Treg cells of COPD patients compared to cells in healthy smokers. Importantly, miR-199a-5p had significant overrepresentation of its target genes in the Treg cell transcriptome, with many targets associated with the TGF-&beta; activation pathway. We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan&reg; arrays of the Human TGF-&beta; Pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favor of a Th1 and Th17 response.</p>

The IPP had previously been implicated as an important site for Ig diversification in sheep and cattle but the early site for Ig diversification in sheep remained in question. Recently, fetal spleen has been shown to be a potential site of B cell development in cattle which is phylogenetically close to sheep (Lucier et al., 1998). In this study, in order to solve the problem of when, where, and how the primary immunoglobulin repertoire of sheep is generated and diversified before the onset of diversification in IPP, various tissues of fetuses at the first trimester were examined for the expression of λ light chain genes and the degree of Vλ diversity. Thus, this study has provided significant evidence for the following conclusions on early Ig Vλ diversification in sheep: (1) Two germline Vλ genes, 5.1 and 5.3 were identified as predominant participants in Ig λ light chain gene rearrangement. (2) A new Jλ gene was found and shown to be utilized in Ig λ light chain gene rearrangement. (3) At 63 days of gestation, there is little diversity seen in the Ig λ light chain repertoire outside the spleen. However, even at this early stage, there is significant diversity of λ light chain within spleen. (4) Fetal spleen is already a reservoir of extensive Ig diversity by the end of the first trimester. (5) Spleen is an earlier site of Vλ diversity than IPP, a bursa-equivalent GALT in sheep. (6) Fetal spleen may provide a partially diversified B cell stock from which a small number of precursor B cells emigrate into the IPP and undergo subsequent clonal expansion and additional diversification within the IPP follicles in sheep. (7) This study shows that multiple sites are involved in the diversification of the Vλ repertoire in sheep.

It is widely accepted that activating mutations of genes encoding the Notch family of transmembrane receptors, specifically Notch1, are associated with oncogenic transformation. Previous data from our lab has shown that an active form of Notch1 (Nic) provides a protective effect against apoptosis in D011.10 T cells, and that this effect may be attributed to Nic binding the pro-apoptotic protein Nur77. Nur77 is an immediate early gene that is upregulated during negative selection of thymocytes and activation-induced apoptosis in D011.10 T cells. Nur77 upregulation is tightly regulated and requires MEF2D, NFAT, and the co-activator, p300, to effectively respond to apoptotic stimuli. In this report we show that Nic has the ability to interfere with the induction of transcription of Nur77, and that this interference is directly related to the inability of p300 to bind the Nur77 promoter in the presence of Nic. We also show that blocking Notch activation through gamma secretase-inhibitors or siRNA directed against Notch1 in T cell acute lymphoblastic leukemia (T-ALL) cell lines restores Nur77 upregulation in response stimuli. These observations support a model in which during thymocytes negative selection activating mutations of Notch1 inhibit the upregulation of a crucial proapoptotic molecule. Studies to determine the mechanism by which Nur77 induces apoptosis have indentified a unique translocation of Nur77 from the nucleus to the cytosol. It has been determined that once in the cytosol Nur77 interacts with members of the Bcl-2 family of proteins at the mitochondrial membrane. This interaction induces a conformational change of Bcl-2 so that is becomes pro-apoptotic instead of protective. Of similar interest is the role that Nur77 itself plays during the induction of activation-induced apoptosis which may be independent of Bcl-2 conformational change. In an effort to describe possible functions of Nur77, DO11.10 cells that have Nur77 under a tet-inducible promoter were observed for changes IP3R. Initial results from our lab suggest that Nur77 alone has the ability to induce cell death in DO11.10 cells using this tet-inducible system. Interestingly we have been able to identify distinct changes in IP3R isoforms during stimulation induced apoptosis and Nur77-dependent apoptosis. Current experiments are focused on a mechanism beyond the known function of the Nur77/Bcl-2 interaction; that Nur77 may also be acting as a physical barrier between the known anti-apoptotic interaction of IP3R and Bcl-2, leading to sustained calcium flux.

Biology is the study of life and life systems. Until recently, biologists have concentrated in examining how cells proliferate, differentiate, and survive in biological systems. Although such studies reveal extremely interesting insights into the complexity of living organisms, there is much more to this story. Just as cells live, cells must also eventually die. The study of how and why cells die has become the focus much scientific research over the past decade. Because the regulation of the number and specificity of cells in the immune system is critical to the life of a mammalian organism, researchers began to investigate how this strict control was accomplished. It was found that large quantities of immature T cells die in the course of their development by a specific type of cell death process coined apoptosis. This Ph.D. dissertation was directed towards examining the mechanisms involved in the regulation of thymocyte apoptosis in a murine model system. The first portion of the project involved isolating differentially regulated genes using either a plus/minus or subtractive hybridization screening strategy The second component of this dissertation investigated possible roles molecular oxygen and/or free radicals play during thymocyte apoptosis. Results from these studies both identified numerous putative death transcripts as well as revealed the requirement for oxygen during cell death in thymocytes induced by specific stimuli.

Malaria is still a significant public health problem in the Tropics, with an estimated 200 million cases a year and more than 1 million deaths, mostly in young children in sub-Saharan Africa. Plasmodium falciparum is the parasite responsible for the majority of the morbidity and mortality due to malaria. We know from the historical use of malaria to treat neurosyphilis that there were several different strains of P. falciparum, some of which were more pathogenic and had higher multiplication rates than others. High multiplication rates of P. falciparum isolates have been associated with severe disease in Thailand, but not in Kenya or Mali. In determining what differences exist between fast- and slow-growing malaria parasites, and understanding their relationship with clinical outcome, we may discover a way of targeting those parasites that cause most disease. This thesis describes a genetic analysis of the determinants of growth and pathogenicity in the rodent malaria parasite, Plasmodium chabaudi. The use of rodent malaria parasite strains for genetic analysis has several experimental, ethical and financial advantages over the use of human malaria parasites. In addition, rodent malaria parasite strains also vary significantly in their growth and pathogenicity, making them excellent candidates for a genetic analysis of these characteristics. The first section of this thesis is concerned with the characterisation of the growth, pathogenicity and transmissibility of two strains, DS and DK, of the rodent malaria parasite P. c. adami. The DS strain is fast-growing, pathogenic, non-selective in its invasion of red blood cells and a poor transmitter to mosquitoes. The DK strain is slow-growing, non-pathogenic, selective in its invasion of red blood cells and a good transmitter to mosquitoes. In the second section of this thesis is a detailed study of the growth characteristics of DS and DK in mixed infections, relative to their growth in single infections. Both sections provide information relevant for the main objective of this thesis, but also contribute to the body of work on pathogenicity and transmissibility, and pathogenicity and strain behaviour in mixed infections, which has been carried out in rodent malaria parasites to-date. The third section of the thesis contains the results of a genetic analysis of the difference in growth between P. c. adami strains DS and DK, using the Linkage Group Selection (LGS) technique. On several occasions, DS and DK were crossed in the mosquito vector and, following selection for fast growth in mice, the cross progeny were initially screened with genome-wide, quantitative AFLP markers. Markers specific to the slow-growing parent DK which were greatly reduced in intensity after selection were found on P. chabaudi chromosomes 6, 7 and 9. This result suggests that the difference in growth between the two strains is determined by multiple genetic loci. The selection on chromosomes 7 and 9 was then looked at in greater detail, using SNP-based markers quantified by Pyrosequencing™. It was found, consistently, that a region at one end of DS chromosome 9 was inherited as a single, non-recombining unit in cross progeny selected for fast growth. As this was the region most strongly selected against, it suggests that a gene (or genes) in this region has a major role in the determination of growth characteristics, and therefore pathogenicity, in P. c. adami. Narrowing down this region further, in order to identify the candidate gene(s), remains a key future objective.

Studies were undertaken to examine immunoglobulin repertoire diversification in cattle. Diversification was examined in a number of organs from late first trimester bovine fetuses and from the ileal Peyer’s patch (IPP) follicles of young calves. To investigate the diversification in IPP follicles, individual IPP follicles were isolated by microdissection and diversification of the lambda variable region was examined by RT-PCR and subsequent cloning and sequencing. When intrafollicular sequences from a 4 week old calf were determined and compared, two major groups could be delineated. An examination of these groups revealed clear genealogical relationships that implicated both gene conversion and untemplated somatic hypermutation as the mechanisms responsible for diversification of VX within the IPP follicles. Diversification of Vλ was also examined in early (95–110 gestational day) fetal organs. The organs examined included fetal spleen, blood, liver, thymus, ileum and bone marrow. Sequences obtained from the various organs revealed that while Vλ sequences were highly diversified in spleen, very little VλX diversification was seen in the blood, liver, ileum or bone marrow. The sequences obtained from spleen indicated that both gene conversion and untemplated somatic hypermutation could be taking place in fetal spleen. Evidence for diversification in fetal spleen was also obtained by examining expression of recombination activating genes (RAG). An examination of fetal tissues for the expression of RAG-1 found that RAG-1 transcripts were present only in fetal thymus, bone marrow and spleen. The presence of both RAG-1 transcripts and a highly diversified population of Vλ sequences implicates the fetal spleen as an organ where both Vλ rearrangement and diversification might take place in cattle.

Cattle represent an economically important species whose immune system seems to depart from the standard human and murine models. Little has been documented about bovine lymphocyte activation in vitro, such work is needed for comparative immunology and for us to understand bovine immunology. In general, lymphocyte activation is accompanied by many molecular changes at the mRNA level. The unique characteristics of lymphocyte activation imply that a unique set of genes is associated with this biological process. However, there is very little known (in any species) about lymphocyte-specific genes that are differentially up-regulated after activation. In this thesis, bovine lymphocyte activation was first stimulated in vitro. Secondly, representational difference analysis (RDA) method was used to clone mRNAs that are exclusively present in activated bovine lymphocytes. Subsequently, the cDNAs were analyzed by DNA sequencing and homology search against the Genbank database. Clone E8 was identified as a potential G-protein-coupled receptor. E8 is up-regulated in activated bovine lymphocytes at 2 hours post stimulation. When up-regulated, E8 mRNA level remains constant from 2 hours to at least 72 hours post stimulation. Similar kinetic expression of E8 is observed following either LPS or Con A stimulations. Expression of E8 was also detected in murine lymphocytes upon activation with LPS or Con A and with similar kinetic expression. E8 showed increased level of expression when human T and B lymphocytes were activated by cross-linking of antigen receptors along with costimulatory molecules. E8 expression was found not to be associated with resting non-lymphoid tissues, activated non-lymphoid cell lines, nor activated macrophages and neutrophils. Therefore, E8 represents an early gene specific to lymphocyte activation. The size of the full-length transcript of clone E8 was estimated at about 2.2 kb. A full-length cDNA was obtained by the RACE procedure. Sequence alignment revealed that E8 is homologous to EB11, a human gene induced by EBV; CXCR1, as well as human CCR4 and CCR5 genes. Potential biological functions of this gene are discussed.

Inflammation is a multifactorial response towards noxious stimuli, however appropriate regulation and resolution of inflammation is crucial for the prevention of chronic inflammatory diseases such as atherosclerosis. The endocannabinoid (eCB) system is an endogenous immunomodulatory system which consists of a series of lipophilic ligands that signal via two G-protein-coupled receptors. Cannabinoid receptor 1 (CB1R) is mainly expressed in the central nervous system and its activation has psychoactive effects. Cannabinoid receptor 2 (CB2R) is mainly expressed on leukocytes and receptor activation has anti-inflammatory actions in mouse models of atherosclerosis and chronic inflammatory pain. It is considered that CB2R activation is involved in modulation of the recruitment of inflammatory cells, especially monocytes/macrophages; however the exact mechanism of action has not been fully elucidated. We hypothesised that activation of CB2R modulates monocyte/ macrophage recruitment and signalling, thus providing a homeostatic mechanism to limit macrophage activation in inflammatory responses. The high lipophilicity of cannabinoid ligands and their lack of selectivity for CB2R over CB1R limits CB2R drug development. In collaboration with Dr Angela Russell, we used virtual screening and a CB2R cAMP assay that we validated to discover a novel CB2R agonist, 3-((2’-Cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-b]indole, (DIAS2). In collaboration with Dr Russell’s group who did chemical synthesis, we extended this novel scaffold to include over 80 compounds. Using the same hCB2R cAMP screening assay we demonstrated that 16 compounds with the same scaffold are at active at CB2R in the nanomolar range. At least 3 compounds, including DIAS2, were found to be ≥ 300-fold selective for CB2R over CB1R in cAMP assays and radioligand binding studies. In the inflammatory model of zymosan-induced peritonitis, DIAS2 dose-dependently inhibited inflammatory monocyte recruitment by 50% at highest dose of 5 mg/kg with no effect on neutrophils. In further zymosan-induced peritonitis experiments 5 mg/kg of DIAS2 and a structurally-similar CB2R agonist from the same family of triazino-indoles inhibited monocyte recruitment while a different CB2R agonist (JWH-133) at 5 mg/kg did not inhibit monocyte recruitment. Analysis of peritoneal exudates showed that the inhibition of monocyte recruitment was not associated with changes in the levels of JE, MIP-1α and nitric oxide but was associated with increased levels of the chemokine KC. Using in vitro cell biology approaches, we demonstrated that 10μΜ dose of both DIAS2 and JWH-133 reduced forskolin-induced cAMP production in primary murine macrophages. Also 2.5 to 10 μΜ οf JWH-133 and HU-308 dose-dependently induced primary murine macrophage chemotaxis which could be blocked a CB2R antagonist (SR 144528, 1 μΜ) while DIAS2 at doses up to 10 μΜ was not a chemoattractant. Accordingly HU-308 and JWH-133 were at least 3-fold more efficacious than DIAS2 at recruiting β-arrestin to the murine CB2R. Moreover in studies with primary murine macrophages 10 μΜ dose of JWH-133 and HU-308 induced ERK1/2 and Akt phosphorylation within 30 minutes, while 2-AG (an endogenous eCB ligand) and DIAS2 at 10 μΜ had no such effect. In summary, we have discovered a novel family CB2R agonists and demonstrated that some devoid of chemotactic active CB2R agonists can reduce monocyte recruitment in vivo while other chemoattractant CB2R agonists have no in vivo anti-inflammatory effect. We propose that non-chemotactic CB2R agonists represent a new class of anti-inflammatory drugs with a novel mode of action.

Intestinal homeostasis is a delicate balance between suppression of immune responses against innocuous antigens and stimulation of immune responses against pathogens. Type I interferon (IFN-1) cytokines have both immunostimulatory and immunomodulatory effects. Colon mononuclear phagocytes (MP) constitutively produced IFN-1 in a TRIFdependent manner. We explored the function of endogenous IFN-1 in the colon using the T cell adoptive transfer model of colitis. Transfer of CD4⁺CD45RB^hi naïve T cells from wild type (WT) or IFNAR subunit 1 knockout (IFNAR1^-/-) mice into RAG^-/- hosts resulted in similar onset and severity of colitis. In contrast, RAG^-/- x IFNAR1^-/- double knockout (DKO) mice developed accelerated severe colitis compared to RAG^-/- hosts when transferred WT CD4⁺CD45RB^hi T cells. Although WT or IFNAR1^-/- regulatory T (Treg) cells equally prevented disease caused by CD45RB^hi naïve T cells, WT Treg cells co-transferred with naïve CD4⁺ T cells into DKO recipients failed to expand or maintain Foxp3 expression and gained effector functions in the colon. IFNAR signaling on host hematopoietic cells inhibited T cell-mediated colitis, but not innate colitis. MPs isolated from the colon lamina propria (cLP) required IFNAR signaling for the production of the anti-inflammatory cytokines, IL-10, IL-27, and IL-1RA, but not for the production of classic pro-inflammatory cytokines. IFN-1-dependent secretion of IL-1RA was particularly important in inhibiting the migration of inflammatory DCs with potent T cell proliferative capacity from the cLP to the mesenteric lymph nodes. Finally, preliminary results suggested that IFN-1 may shape the commensal microbiota, but is not essential for controlling specific colitis-inducing bacteria.