No description available.

Interferon regulatory factor-8 (Irf-8), a hematopoietic transcriptional regulator, controls myeloid-cell proliferation and coordinates innate and adaptive host immune responses. Mice from the BXH-2 recombinant inbred strain carry an endogenous R294C mutation in Irf-8. This loss-of-function mutation induces clonal infiltration of undifferentiated Mac-1+/Gr-1 + granulocytic precursors in BXH-2 mice, extramedullary hematopoiesis, and splenomegaly similar to those seen in human chronic myeloid leukemia. It also renders the host permissible to the otherwise avirulent Mycobacterium bovis (BCG), and negatively affects survival or recovery of these mice to other infectious pathogens. Here, we generated a polyc1onal anti-Irf-8 antibody to better characterize the effects of the R294C mutation on Irf-8 protein expression, stability, and inducibility in hematopoietic and non-hematopoietic tissues. We found that mutant Irf-8C294-expressing tissues consistently displayed reduced Irf-8 abundance compared to their wild-type counterparts in both primary splenocytes and following transfection into heterologous cells, presumably due to decreased stability or increased rate of degradation of the mutant isoform. Results also indicate that native Irf-8 is also expressed in the heart, and to a lesser extent, in the kidneys. Since neither of these organs is well-known to be associated with hematopoietic or immune functions, this finding strengthens the possibility that Irf-8 may exert additional regulatory functions in other cellular contexts. Taken together, our study provides a better understanding about the molecular features of the mutant Irf-8 C294 protein and contributes to a growing body of evidence in support of Irf-8 expression in non-hematopoietic tissues.

No description available.

The Innate immune system is the first line of defense against invading microbial pathogens. It is a fast-acting and non-antigen-specific defense system, which employs germline encoded surveillance systems capable of responding to a broad-spectrum of pathogens. The innate immune system involves a variety of immune cells, which express different profiles of surveillance or detection receptors. Upon sensing pathogens, these receptors trigger cell signalling to turn on transcription of inflammatory cytokines, chemokines, anti-microbial peptides and type I Interferons. These effectors have direct effects on the control of pathogen load and also activate the adaptive immune system, which is ultimately required to clear infections. The type I interferons (IFNs) are the principal cytokines strongly induced during infection with viruses and are required for direct control of viral replication and modulation of cells of the adaptive immune response. The signalling pathways induced in order to activate type I IFNs are dependent on the interferon regulatory factors (IRFs). Striving for survival, microbes have evolved various strategies to subvert/impair these critical defense molecules. In this thesis work, I have used Human Metapneumoviruses (HMPVs), a relatively newly described family of paramyxoviruses as model viruses to explore the role of pattern recognition receptors (PRRs) and the IRF family of transcription factors in the innate immune response. These studies revealed that the recognition of HMPV viral pathogen-associated molecular patterns (PAMPs) by immune cells is different in different cell types. Retinoic acid-inducible gene-I (RIG-I), a cytosolic RNA helicases senses HMPV-A1 virus for triggering type I IFN activation by detecting its 5’- triphosphate viral RNA in most human cells, including cell lines and primary monocytes. An exception to these findings was plasmacytoid dendritic cells (PDCs), where Toll-like receptor (TLR)-7 is the primary sensor involved in detecting HMPV viruses. By comparing the innate immune response to two HMPV strains, we found that these two closely related strains had very different immune stimulatory capabilities. HMPV-1A strain triggered type I IFNs in monocytes, PDCs and cells of epithelial origin. In contrast, a related strain, HMPV-B1 failed to trigger IFN responses in most cell types. Our studies suggested that the phosphoprotein (P) of HMPV-B1 could prevent the viral RNA from being detected by RIG-I, thus inhibiting the induction of type I IFN production in most cell type examined. This finding adds to our understanding of the mechanisms by which viruses are sensed by surveillance receptors and also unveils new means of viral evasion of host immune responses. Although IRFs are extensively studied for their role in regulating type I IFN activation, especially in TLR and RIG-I like receptor (RLR) signalling pathways upon viral infection, a clear understanding of how this family of transcription factors contributes to anti-viral immunity was lacking. Studies conducted as part of this thesis revealed that in addition to IRF3 and IRF7, which play a central role in anti-viral immunity downstream of most PRRs (e.g. TLRs, RLRs, DNA sensors), the related factor IRF5 was also an important component of innate anti-viral defenses. Using IRF5-deficient mice we studied in detail the role of IRF5 in coordinating antiviral defenses by examining its involvement in signalling downstream of TLRs. These studies led us to examine the role of IRF5 in the regulation of type I IFNs as well as inflammatory cytokines in different cell types. While most TLRs that induced IFNβ showed normal responses in IRF5-deficient mice, CpG-B-induced IFNβ production in CD11c+CDCs isolated from mouse spleen but not those generated in vitro from bone marrow required IRF5. This was in contrast to responses with lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (polyIC), ligands for TLR4 and 3, respectively. Moreover, we found that in contrast to IRF3 and/or IRF7, IRF5 was important in coordinating the expression of inflammatory cytokines such as TNFα downstream of some TLRs. In addition to our studies to examine the requirement for IRF5 in TLR signaling, we also showed that muramyl peptide (MDP) from Mycobacterium tuberculosis (Mtb) could activate type I IFNs via IRF5. This was the first evidence linking IRF5 to a non-TLR-driven pathway. IRF5 activation in this case was downstream of a novel nucleotide-binding oligomerization domain containing (NOD)-2/receptor-interacting serine-threonine kinase (RIP)-2 signaling pathway. Collectively, the studies outlined in this thesis have assisted in providing a framework to understand the role of TLRs, RLRs and IRFs in the immune response to paramyxoviruses and have unveiled new mechanisms of activation of the IRFs as well as new mechanisms by which pathogens subvert or evade these important innate defense mechanisms.

Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders. Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease. Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.

CD8+ T cell differentiation is a complex process that requires integration of signals from the TCR, co-stimulatory molecules and cytokines. Ligation of the peptide-MHC complex with the cognate TCR initiates a downstream signaling cascade of which the IL-2 inducible T-cell kinase (ITK) is a key component. Loss of ITK results in a measured reduction in T cell activation. Consequently, Itk deficient mice have defects in thymic selection, CD8+ T cell expansion and differentiation in response to virus infections, and generate a unique population of innate-like CD8+ T cells. The mechanisms that translate TCR and ITK-derived signals into distinct gene transcription programs that regulate CD8+ T cell differentiation are not defined. Our microarray screen identified IRF4 as a potential transcription factor mediating the differentiation of innate-like T cells, and antiviral CD8+ T cell in response to acute and chronic LCMV infections. Innate-like CD8+ T cells are characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor Eomesodermin (Eomes). One component of this altered development is a non-CD8+ T cell-intrinsic role for IL-4. We show that IRF4 expression is induced upon TCR signaling and is dependent on ITK activity. In contrast to WT cells, activation of IRF4-deficient CD8+ T cells leads to rapid and robust expression of Eomes, which is further enhanced by IL-4 stimulation. These data indicate that ITK signaling promotes IRF4 up-regulation following CD8+ T cell activation and that this signaling xii pathway normally suppresses Eomes expression, thereby regulating the differentiation pathway of CD8+ T cells. ITK deficient mice also have reduced expansion of CD8+ T cells in response to acute LCMV infections. We show that IRF4 is transiently upregulated to differing levels in murine CD8+ T cells, based on the strength of TCR signaling. In turn, IRF4 controls the magnitude of the CD8+ T cell response to acute virus infection in a dose-dependent manner. Furthermore, the expression of key transcription factors such as T cell factor 1 and Eomesodermin are highly sensitive to graded levels of IRF4. In contrast, T-bet expression is less dependent on IRF4 levels and is influenced by the nature of the infection. These data indicate that IRF4 is a key component that translates the strength of TCR signaling into a graded response of virus-specific CD8+ T cells. The data from these studies indicated a pivotal role of IRF4 in regulating the expression of T-bet and Eomes. During persistent LCMV infections, CD8+ T cells differentiate into T-bethi and Eomeshi subsets, both of which are required for efficient viral control. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in favor of Eomes, leading to lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV Clone 13 infection. Altering this ratio in favor of T-bet xiii restores the differentiation of T-bet+ Eomes- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8+ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.

Diss. (sammanfattning) Uppsala : Univ., 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 4 uppsatser.