<p>Foam cell formation from arterial intima macrophages and defective clearance of apoptotic foam cells drive the progression of the inflammatory disease atherosclerosis. The role of innate immune protein C1q in autoimmune disease and pathogen defense is well characterized, however its role in atherosclerosis remains largely uninvestigated. Prior studies have characterized the complement independent role of C1q in polarizing macrophages towards an anti-inflammatory phenotype during uptake of apoptotic cells and modified lipoproteins. To further understand the role of C1q in programming human monocyte-derived macrophages during foam cell formation, we used RNA-sequencing to elucidate pathways that are modulated by C1q during clearance of atherogenic lipoproteins. Expression of genes in JAK-STAT, PPAR, apoptotic, and TLR signaling pathways were modulated by C1q in this study. In addition, C1q suppressed STAT1 and PPAR transcriptional activity. This study identifies potential molecular mechanisms that support a beneficial role for C1q in early atherosclerosis. </p>

This dissertation was to study the sites of B cell development, the mechanisms of Ig diversification, the time when diversification occurs, the B cell repertoire during development and usage of Ig light chains in cattle. The expression of TdT, RAG-1 and RAG-2 was detected in spleen in young animals but not in old animals indicating that gene rearrangement of B cells takes place in spleen and is restricted to a short period of time during early development in cattle. To determine the mechanisms by which cattle create a diverse Ig repertoire, genomic rearrangement patterns and V$\lambda$ sequences of cDNA and germline genes were examined. Few rearrangements, potential donor sequences in germline pseudogenes and point mutations in the cDNAs were found. These data suggest that cattle primarily use gene conversion to create a diverse Ig repertoire and somatic hypermutation may be used to refine the Ig repertoire upon antigen challenge. To investigate when diversification occurs, V$\lambda$ IPP cDNA were compared between young and old animals. Many diverse nucleotides were found in young animals as that in old animals indicating that the diversification takes place in the very early stage of development. To determine the peripheral Ig repertoire during development, expression of peripheral light chains of an animal at different ages was examined. Diversity clustered in the CDRs was increased and different clones which were present in the periphery at a low level in neonatal animals were found to be expanded in older animals. These data indicate that the peripheral Ig repertoire is ligand-selected and clonally expanded by positive selection. To investigate the control of $\kappa$/$\lambda$ usage, expression and germline $\kappa$/$\lambda$, V$\kappa$ and C$\kappa$ repertoire were examined. The ratio of expressed $\kappa$/$\lambda$ was high in IPP and spleen, but it was low in the periphery. Additionally, similar numbers of $\kappa$/$\lambda$ genes were found in the germline and V$\lambda$ cDNA sequences were found to be as diverse as that of V$\lambda$. In contrast, C$\kappa$ possesses a low homology to other primarily $\kappa$ expressing mammals. These data suggest that a post-transcriptional control may govern the usage of $\kappa$/$\lambda$ in cattle.

Runx1 regulates the expression of several important target genes and plays critical roles in the process of hematopoiesis. Runx1 by itself is a poor regulator of transcription and instead nucleates transcription complexes through its C-terminus to transactivate or repress the expression of target genes. We generated a C-terminally deleted Runx1 construct (Runx1.d190), which lacks important co-factor sites, to further investigate the function of Runx1 in development. A potential role for Runx1 in regulating the expression of another potent transcriptional regulator, c-Myc, has been suggested by published studies, which show that Runx1 and c-Myc collaborate in oncogenesis. In these studies, we show that endogenous Runx1 binds to three Runx consensus sites upstream of the c-Myc transcriptional start site in Jurkat T cells and murine primary splenocytes. Retroviral transduction of Jurkat T cells with Runx1.d190 results in the increased transcription of c-Myc as determined by microarray analysis. In order to monitor c-Myc expression in response to early-acting and transient Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Treatment of murine primary splenocytes with TAT-Runx1.d190 protein results in a transient increase in the transcription of c-Myc and a corresponding increase in c-Myc protein levels. This effect is dependent on the ability of Runx1.d190 to bind to DNA. These data demonstrate that Runx1 directly regulates c-Myc expression in a C-terminally and DNA-binding dependent manner. In these studies, we also investigate the effects of the truncation of Runx1 C-terminus on hematopoietic stem cells (HSCs). We found that treatment of bone marrow cells enriched for HSCs with TAT-Runx1.d190 results in a 12.5 fold increase in hematopoietic precursors compared to untreated precursors in vitro as determined by Colony Forming Cell assays. We also show that hematopoietic precursors treated with TAT-Runx1.d190 are able to able to differentiate normally both in vitro and in vivo and thus represent functional hematopoietic precursors. Our findings show that we are able to transiently expand hematopoietic precursors ex vivo by treating the cells with a Runx1 construct that lacks the C-terminus. Collectively, this work demonstrates that Runx1 regulates the expression of c-Myc and the expansion of hematopoietic precursors in a C-terminally dependent manner.

Notch1 specifically upregulates expression of the cytokine interferon-γ in peripheral T cells through activation of NF-κB. However, how Notch mediates NF-κB activation remains unclear. NF-κB activation occurs within minutes of TCR engagement and this activation is sustained for at least 48 hours following TCR signaling. We used either γ-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members or siRNA against Notch1 to reduce endogenous expression of Notch1 specifically. We demonstrate that GSI blocked the later, sustained NF-κB activation, but did not affect the initial activation of NF-κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF-κB and competes with IκBα, leading to retention of NF-κB in the nucleus, and that N1IC can directly regulate IFN-γ expression through complexes formed on the IFN-γ promoter. Additionally, within the immunological synapse, cytosolic Notch1 associates with CARMA1 and BCL10, mediating a direct interaction among them. Upon TCR and CD28 stimulation, Notch1 directly interacts with PKC&thetas; and the IKK complex, leading to IKK-mediated activation of NF-κB. In the absence of Notch1, there is no formation of CARMA1/BCL10/Malt1 complex, which is required for IKK activation. Taken together, these data suggest that Notch1 plays two roles during T cell activation: as an activation scaffold in the cytoplasm and as a transcriptional activator in the nucleus.

Notch signaling is critical for the regulation of differentiation, proliferation and apoptosis in many cell types. Notch receptors and various Notch ligands have been shown to have a regulatory effect on cell cycle progression during the processes of development and differentiation. Cyclins are proteins that regulate cell cycle check-points, thereby controlling cell cycle progression. The D-cyclins, specifically, are required for overcoming the G1/S checkpoint. It has been shown previously (Ronchini et al) that NotchIC regulates the expression of Cyclin D1, one member of the Cyclin-D family. It has also been reported (Sicinska et al.) that Cyclin D3-/- mice display impaired thymocyte development and do not develop Notch1-induced leukemia. Based on these observations, we hypothesized that cyclin D3 may be a downstream target of Notch signaling in T cells. We observed that T cell receptor signaling increases Cyclin D3, cdk4 and cdk6 expression in peripheral T cells and inhibiting Notch activity reduces Cyclin D3, cdk4 and cdk6 expression in activated T cells. Using reporter assays, as well as chromatin immunoprecipitation, we show that the transactivation domain of Notch1 is critical for regulating the Cyclin D3 promoter. We demonstrate that cyclin D3-cdk4 and cdk6 is also responsible for cell cycle progression in Notch-dependent human T-ALL cell lines. We show that Cyclin D3 and cdk4/6 are important targets in constitutively active Notch signaling in leukemic T cells, as they can partially override the G1 arrest observed with GSI treatment. Together, our data indicate that Notch signaling controls peripheral and leukemic T cell proliferation. Furthermore, we have begun to outline a possible mechanism for the regulated expression of Cyclin D3 and cdk4/6 in leukemic T cells, and through our future experiments we hope to reveal the oncogenic potential of Cyclin D3 as a target of dysregulated Notch1 signaling.

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.

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.