Microbe-induced TLR4 trafficking is needed to promote innate immunity and an inflammatory reaction. We showed that increased TLR4 expression in immature enterocytes is associated with severe inflammation and the development of necrotizing enterocolitis (NEC). Thus, we need to determine the intracellular regulatory mechanisms for TLR4 trafficking in fetal enteric inflammation. Here, we show that both clathrin- and caveolin-dependent endocytosis are necessary for the prevention of the fetal colonic (FHC) IL-1β response to endotoxin (LPS, a TLR4 agonist). In response to LPS stimulation, the inhibitor of clathrin, chlorpromazine (CPZ), and caveolin 1, Methyl-β- cyclodextrin (MβCD), significantly increased TLR4 mRNA and surface protein expression by retaining TLR4 in the early endosome. An increased TLR4 resulted in increased proinflammatory cytokine IL-1β mRNA and protein induction in a time dependent manner. TICAM2, elicited by TLR4, mediates down-stream TLR4 signaling. MβCD did not increase TICAM2 mRNA expression indicating that CPZ and MβCD affect TLR4 signaling differently. In addition, both CPZ or MβCD alone and CPZ or MβCD plus LPS reduced IL-10 mRNA expression, which functions to reduce LPS IL-1β. Collectively, these data identify an additional role for intestinal expression of clathrin and caveolin in anti-inflammation via gram negative pathogen LPS through regulation of TLR4 trafficking and cytokine expression in immature enterocytes. Study of the endocytosis pathway is likely to lead to successful strategies for prevention, treatment and improved outcomes of immature inflammatory diseases such as NEC. / 2020-07-03T00:00:00Z

Activation induced Cytidine Deaminase (AID) contributes to the generation of antibody affinity by participating in two reactions, class switch recombination (CSR) and somatic hypermutation (SHM). Both reactions occur after VDJ recombination, subsequent to antigen exposure. During CSR, a deletion and recombination event occur to alter the effector function from IgM to either IgG, IgE, or IgA. SHM then occurs, which introduces point mutations at a high frequency into the variable regions of both the immunoglobulin heavy and light chains. These point mutations increase the antibody binding affinity for antigen, and antibodies with greatest affinity for antigen will be positively selected and further expanded during an immune response. The ability of AID to act as a mutator gene underscores the importance of understanding its regulation throughout the genome. Action of AID on genes outside of the Ig loci can lead to genomic instability. Hyperactivity of AID has been shown to cause chromosomal translocations and other oncogenic malignancies. Loss of AID can lead to immunodeficiencies. Therefore, it is imperative to understand how AID identifies and interacts with target sequences and mutates both strands of the DNA. Previous studies have identified DNA secondary structure such as R loops, transcription factors, miRNA, and phosphorylation as events important for determining AID's ability to access its substrate sequences. However, none of these studies demonstrated how AID mutates both strands of DNA, reminiscent to its in vivo mode of action.The focus of this thesis is to identify how AID mutates both strands of the DNA duplex, and how target genes are identified. To this end, we have discovered that AID functionally interacts with the cellular non-coding RNA degradation complex, RNA exosome. We observe that the RNA exosome stimulates AID activity on both strands of DNA in in vitro reconstituted reactions. The RNA exosome/AID complex binds to switch (S) sequences in a manner that is both transcription- and AID-dependent. Knockdown of exosome core component ExoSc3 results in defects in CSR. Additionally, this work focuses on the role of the neddylation (Nedd8) of AID in recruitment to its target sequences. Neddylation, a 10kDa modifier, is a small ubiquitin like modifier which functions in a variety of cellular processes. We have used a combination of proteomics, computational approaches, and candidate screening to identify and validate the role of E1, E2 and E3 in CSR. We have identified NEDD4 as the AID-specific E3 Neddylation ligase and demonstrated its requirement for CSR in mouse B cells. Using mass spectrometry, we have identified AID neddylation sites from in vitro neddylated AID proteins. We observe that mutation of these AID-neddylation sites affects AID/RNA exosome interaction and CSR efficiency in B cells. These observations point towards a role of NEDD4 in recruiting AID/RNA exosome complex to the immunoglobulin locus. Additionally, we confirm the role of NEDD4 as an E3 ubiquitin ligase of RNA polymerase. In both cell lines and primary cells, we observe an increase of germline transcripts and S region resident RNA polymerase in the absence of NEDD4. We propose NEDD4 ubiquitination can promote the degradation of stalled RNA polymerase complexes at the Ig S region, facilitating exosome access to germline transcripts and AID access to the template strand.

Fibroblast growth factor-inducible 14 (FN14) is a highly inducible cytokine receptor linked to a number of intracellular signaling pathways, including the nuclear factor-kappaB (NFkB) and mitogen-activated protein kinase (MAPK) pathways, as well as to physiological processes, such as angiogenesis, tissue repair, and regeneration. Although FN14 is expressed at low levels in normal tissue, it is highly expressed after tissue injury and appears to mediate activities crucial to wound repair and regeneration. In vitro, FN14 expression is induced by numerous growth factors, as well as by its own ligand TNF-like weak inducer of apoptosis (TWEAK). Given these results, it is thought that FN14 plays an important physiological role in the response to acute injury, and that FN14 activity is primarily regulated at the level of ligand and receptor expression. Various pathological states, however, also exhibit increased expression and activity of FN14. Tumor progression, chronic autoimmune disease, and neuroinflammation are just a few of the conditions in which increased expression or function of FN14 have been implicated. In addition, it has been shown, in vitro, that overexpression of FN14 can result in ligand-independent signaling by the receptor. This signaling in the absence of TWEAK has been associated with increased proliferation and invasiveness of several types of cancer cells. Thus, although FN14 plays an important physiological role after acute injury, it appears that dysregulation of FN14 expression may contribute to various chronic pathological states. Since FN14 protein expression is so highly inducible, and TWEAK-FN14 signaling can further amplify receptor levels, this begs the question whether unregulated receptor levels can influence the pathological switch through altered signaling and resultant cell proliferation, migration, or chemokine expression. It seems likely that there are cellular mechanisms in place to prevent excess accumulation of FN14. To date, however, the stability of FN14 or mechanisms of FN14 downregulation, factors which would presumably have dramatic effects on the sustained activity of the receptor, have not been explored. The main focus of this project was to explore the cellular mechanisms involved in downregulation of FN14 levels or signaling capacity which are likely important for preventing inappropriate and uncontrolled signaling. One mechanism through which receptor signaling can be attenuated is by ligand-induced downregulation. In some cases, as with the receptor Notch, ligand-independent turnover can occur as well. During the course of this project, both of these turnover mechanisms were evaluated for FN14. It was determined that the receptor undergoes both rapid ligand-dependent and ligand-independent turnover, and that these two processes are distinct and synergistic. A described endocytic motif in the FN14 cytoplasmic tail does not seem to be required for either method of turnover, which means that the endocytic mechanism remains to be elucidated. We focused on dissecting the ligand-independent turnover of FN14 due to the novel nature of this regulatory mechanism. It appears that FN14 is constitutively expressed, trafficked to the cell surface, endocytosed, and degraded in lysosomes. This constitutive trafficking to sites of degradation seems to require only the extracellular domain of the receptor, suggesting that FN14 may interact with a trafficking partner using this domain. Although FN14 seems to be constitutively degraded in the steady-state, growth factor treatment stabilizes protein levels, in a manner independent of transcriptional upregulation. This finding supports the notion that FN14 levels are regulated not only at the level of expression, but also at the level of protein stability. Ultimately, this work describes previously unrecognized aspects of FN14 biology, which may enhance our understanding of how FN14 activity is dysregulated in chronic pathological states. Using this information, it might be possible to design peptides to alter FN14 trafficking patterns in a therapeutically applicable manner.

Dendritic cells (DC) are cells of the immune system that are specialized in sensing and responding to pathogens. They coordinate innate and adaptive immune responses to different types of pathogens through the release of inflammatory cytokines, secretion of interferon, and presentation of antigen to naïve T cells. Several different subsets of dendritic cells have been distinguished in the spleen and intestine based on their ability to perform these different functions. However, the specific signaling pathways and transcription factors involved in DC subset differentiation and the precise functions of these subsets are not well understood. Plasmacytoid dendritic cells (PDCs) are a subset of dendritic cells that are specialized at the secretion of type I interferon. Through conditional targeting of E2-2, a transcription factor essential for the specification of the PDC lineage, we have demonstrated a vital role for PDCs in the control of acute and chronic viral infections. In addition, deletion of E2-2 was employed to characterize novel E2-2-dependent, alternative CD8 DC fates. Furthermore, we have identified a common pathway for the differentiation of the key T cell-priming populations of CD11b DCs in the spleen and intestine. Specific targeting of Notch2 revealed that splenic CD11b DCs are comprised of at least two functionally and developmentally distinct populations that can be distinguished by their expression of the surface molecule Esam. Deletion of Notch2 in dendritic cells also led to the specific loss of CD103CD11b DCs from the lamina propria of the intestine and a corresponding reduction in IL-17-producing T cells. Throughout these studies, the identification of specific developmental pathways of DC subsets has provided key insights into their different functions.

The purpose of the research has been to characterize the response of naïve or virus-specific swine T lymphocytes from different lymphoid compartments to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Peripheral blood mononuclear cells (PBMCs), tracheobronchial lymph node (TLN) and lateral retropharyngeal lymph node (LLN) cells were labeled with PKH67 green fluorescence dye to measure cell proliferation and surface phenotypes were examined using anti-CD4, anti-CD8 or anti-CD25 monoclonal antibodies. Stimulation with Concanavalin A was also included as a positive control. Our results show that potential virus-specific T lymphocytes were found in the peripheral blood, although no cell proliferation was found in cultures of lymph node cells. We did find that the percentages of CD8+ T cells in cultures of lymph node cells from the virus-infected pig increased after in vitro stimulation with the Powell virus compared to the lymph node cells cultured in media only, suggesting that CD8+ T lymphocytes may play a role in the virus clearance and immune memory to PRRSV.

Naturally occurring CD4+CD25+ T regulatory cells (Treg) play important roles in maintaining immunologic self-tolerance in addition to controlling the magnitude of anti-microbial immune responses. However, the capacity of these CD4+CD25+ Treg cells to control immune responses both in vivo and in vitro is not well established. CD4+CD25+ Treg cell-mediated suppression can control autoimmune diseases; transplantation tolerance and graft verses host disease and, in contrast hinder tumor immunity and immunity to infectious agents. As Treg cells have been reported to be involved in several diseases, this study focused on molecular characteristics that enables them to maintain anergy and also resistance to programmed cell death along with the effect of FIV-infection on regulation of the above phenotypic characteristics. Our results show that feline CD4+CD25+ Treg cells are phenotypically and functionally anergic as indicated by elevated levels of the cyclin dependent kinase inhibitors, CdkI¡¦s, (p21cip1,p16ink4, and p27kip1) , and resistance to mitogen-induced proliferation compared to their counter parts CD4+CD25- T cells. Importantly, CdkI¡¦s are constitutively over-expressed only in FIV-infected cats. As expected Treg cells from FIV-infected cats that over-expressed CdkI¡¦s expressed low levels of the cyclins (mainly cyclins D) and phosphorylated retinoblastoma protein (pRb) that are responsible for cell cycle progression. We investigated the role of TGF?Ò signaling and found that TGF?Ò1 plus ConA stimulation was able to convert CD4+CD25- T cells to CD4+CD25+ T cells with functional and phenotypic characteristics including upregulation of CdkI¡¦s and bcl-2. The differential expression of CdkI¡¦s and bcl-2 between the two CD4+ T cell subsets may be linked to TGF?Ò-Smad pathway. Consistent with upregulation of CdkI¡¦s and bcl-2, we found that although natural and TGF?Ò1 converted CD4+CD25+ Treg cells are anergic, they are more resistant to activation induced cell death compared to CD4+CD25- T cells functionally which correlated with increased bcl-2 to bax ratio in Treg cells. Thus, the molecular characterization of this unique population of Treg cells may be essential for understanding their role and function for developing effective therapeutics and vaccination especially against chronic infections such as Acquired Immune Deficiency Syndrome (AIDS).

The thymus is an organ derived from embryonic endoderm and mesoderm differentiation. It is located above the heart and is made up of two compartments, the thymic epithelial space and the perivascular space. The thymic epithelial space consists of the cortex and the medulla, which is where T cell development, maturation and induction of self tolerance occur in a process known as thymopoiesis. The thymus is susceptible to chronic and acute stressors that result in thymic involution. A consequence of thymic involution is reduced thymopoiesis, which affects the generation of a diverse T cell repertoire and establishment of central T cell tolerance. Many thymosuppressive and thymostimulatory cytokines are involved in thymopoiesis and thymic involution. Keratinocyte growth factor and IL-7 are two cytokines that function in driving early thymic progenitor proliferation and T cell development, respectively. We hypothesized that IL-7 and Keratinocyte growth factor, delivered via recombinant adenovirus, can improve thymopoiesis and T cell reconstitution in mice in an endotoxin model of acute thymic atrophy. Analysis of thymus weight, cellularity, phenotype and TCR gene rearrangement showed moderate increases in thymic function with delivery of IL-7 or Keratinocyte growth factor versus control. Taken together, these data suggested that IL-7 and Keratinocyte growth factor, delivered via recombinant adenoviruses, have thymostimulatory effects on the thymus in normal thymus or settings of acute thymic atrophy and maybe beneficial for future development as therapeutics.

β2 integrins are adhesion molecules on the surface of neutrophils. Avidity activation of β2 integrins includes transportation of pre-formed integrins to the cell surface and a conformational change in the integrin to a high-binding state. Upon binding ligand, β2 integrins initiate a signaling cascade that results in activation of the neutrophil to a pro-inflammatory state, and the inhibition of this signal can prevent further activation of the neutrophil. cAMP and it effector protein kinase A (PKA) exert a generally inhibitory effect upon neutrophil activation. PKA has been shown to inactivate myosin light chain kinase (MLCK). Myosin light chain (MLC) phosphorylation is crucial for actin-myosin complex formation, which is required for stability and contraction of the actin cytoskeleton in neutrophils as well as β2 integrin-dependent adhesion. We hypothesize that the inhibitory effect of PKA upon neutrophils is due to inhibition of β2 integrin avidity activation resulting in the subsequent inhibition of neutrophil activation. Furthermore we hypothesize that the effect of PKA upon β2 integrin avidity activation is mediated through PKA?s effect upon MLCK. We demonstrate that inhibition of PKA induces β2 integrin-dependent adhesion and that augmentation of cAMP prevented β2 integrin-dependent adhesion and subsequent respiratory burst activity. Further, we demonstrate via flow cytometric detection of antibodies directed against β2 integrins that pharmacologic inhibition of PKA activity results in overall increased β2 integrin expression on the neutrophil surface, as well as increased expression of the activated form of the integrin. This upregulation and activation of β2 integrins due to inhibition of PKA is abolished by pharmacologic MLCK inhibition. Inhibition of MLCK also blocked β2 integrin-dependent neutrophil adhesion achieved by inhibition of PKA, as well as neutrophil migration along towards a PKA inhibitor. These findings demonstrate that PKA regulation of β2 integrin affinity activation and subsequent neutrophil activation is via an MLCK-dependent pathway.

Canine babesiosis is an emerging infectious disease in the United States (US). An epidemic of Babesia gibsoni infections in the US was identified. An association between dog breed and B. gibsoni infections was detected. Babesia gibsoni-infected dogs were more likely to be American pit bull terriers and B. canis vogeli infected dogs were more likely to be greyhounds. An association between a recent dog bite and B. gibsoni infection was detected, implicating direct dog-to-dog transmission as a route of infection in the US. Several genes from canine Babesia spp. were characterized, including 18S ribosomal RNA (rRNA), internal transcribed spacer regions (ITS), cytochrome B (cytB), and rhoptry-associated protein-1 (RAP-1). These genetic data were used to develop a sensitive and specific diagnostic semi-nested polymerase chain reaction (PCR) test for canine babesiosis. Using this assay, a novel large Babesia organism was identified in a blood sample obtained from a clinical patient. Molecular and phylogenetic characterization of this large Babesia spp. determined that it was most closely related to B. bigemina. Lastly, an atovaquone and azithromycin drug combination was shown to be the first treatment to clear canine B. gibsoni infections.

Although numerous theories exist proposing mechanisms whereby autoimmune diseases may be initiated, as of yet none of these have been definitively shown to be responsible for the induction of any naturally-occurring disease. One of these theories, known as autoantigen complementarity, states that the initiator of an autoimmune response may not be the target autoantigen itself or an exogenous mimic, but instead is a peptide or protein that is âantisenseâ or âcomplementaryâ in shape and/or charge to the autoantigen. The first immune response is therefore production of an antibody specific for this complementary protein, followed by an anti-antibody (i.e. anti-idiotypic antibody) that reacts with the paratope of the first antibody and also recognizes the âsenseâ or self-protein due to surface contour, charge, and/or hydropathy complementarity. Our laboratory group has published evidence for autoantigen complementarity in one autoimmune glomerular disease, proteinase-3 specific antineutrophil cytoplasmic autoantibody glomerulonephritis. The overall objective of the work described here was to provide further evidence for complementary proteins as inciting antigens in autoimmune glomerulonephritis using anti-GBM disease as the classic antibody-mediated autoimmune glomerulopathy; our central hypothesis was that anti-GBM disease is caused by a protein or peptide complementary to the anti-GBM autoantigen. The design of synthetic peptides complementary in sequence to portions of the human and rat α3(IV)NC1 collagen domains, and the design and production of a recombinant complementary α3(IV)NC1 protein more likely to possess appropriate tertiary structure to be complementary in sequence and in structure to the full-length anti-GBM epitope are described. These antigens were used to demonstrate that a subset of patients with anti-GBM disease have anti-idiotypic antibodies specific for α3(IV)NC1-complementary peptides and proteins, that these antibodies are distinct from their pathogenic idiotypic partners, and that the anti-GBM antibodies bind to these anti-complementary protein antibodies as expected by idiotypic:anti-idiotypic partners. Finally, we describe the immunologic and clinicopathologic consequences of immunization of two rodent models with anti-GBM-complementary peptides, thus providing provisional evidence for autoantigen complementarity-induced anti-GBM disease.