Viral infection of the central nervous system is complicated by the mostly irreplaceable nature of neurons, as the loss of neurons has the potential to result in permanent damage to brain function. However, whether neurons or other cells in the CNS sometimes survive infection and the effects of infection on neuronal function are largely unknown. To address this question, I used the rJHM strain (rJ) of mouse hepatitis virus, (MHV), a neurotropic coronavirus, which causes acute encephalitis in susceptible strains of mice. To determine whether neurons or other CNS cells survive acute infection with this virulent virus, I developed a recombinant JHMV that expresses Cre recombinase (rJ-Cre) and infected mice that universally expressed a silent (floxed) version of tdTomato. Infection of these mice with rJ-Cre resulted in expression of tdTomato in host cells. The results showed that some cells were able to survive the infection, as demonstrated by continued tdTomato expression after virus antigen could no longer be detected. Most notably, interneurons in the olfactory bulb, which are known to inhibit other neurons, represented a large fraction of the surviving cells. The results described in this thesis indicated that some neurons are resistant to virus-mediated cell death and provide a framework for studying the effects of prior coronavirus infection on neuron function. Another aspect of this thesis concerns the role of microglia in virus infections. Recent findings have highlighted roles for microglia in orchestrating normal development and refining neural network connectivity in the healthy central nervous system (CNS). Microglia are not only vital cells in maintaining CNS homeostasis, but also respond to injury, infection, and disease, by undergoing proliferation, and changes in transcription and morphology. Understanding the specific role of microglia in responding to viral infection is complicated by the presence of non-microglial myeloid cells with potentially overlapping function in the healthy brain, and by the rapid infiltration of hematopoietic myeloid cells into the brain in diseased states. Here, I used a CSF1R inhibitor that depletes microglia to examine the specific roles that microglia play in response to infection with a neurotropic coronavirus (MHV). My results show that microglia were required during the first days post infection to limit MHV replication, subsequent tissue damage and lethality. Additionally, microglia depletion resulted in ineffective T cell responses. These results reveal nonredundant, critical roles for microglia in the early innate and virus-specific T cell responses and for subsequent host protection from viral encephalitis. Overall these studies provide new insight into cells that survive MHV infection and into the role microglia play in the immune response to MHV. Specifically, many neurons, especially interneurons of the olfactory bulb survive MHV infection and microglia play a role in both the innate and adaptive immune response to viruses.

Sepsis occurs when infection enters the circulation resulting in harmful or lethal levels of pro- and anti-inflammatory cytokines that affects nearly 2 million people in the U.S. annually. Improved identification and treatment options have increased survival of patients shortly after sepsis. However, this management of sepsis subsequently revealed survivors have increased long-term mortality rates due to altered immune responses that increase susceptibility to secondary complications. My thesis further revealed how sepsis impacts CD8 T cell-mediated immunity that protects the host against sepsis unrelated complications that will be useful for improving the outcome of sepsis survivors. Using vaccinia virus (VacV) to generate circulating (TCIRCM) and skin resident (TRM) memory CD8 T cells, I showed sepsis preferentially diminishes the number and function of TCIRCM, while TRM in barrier tissues were unaffected in mice that received a low-severity model of sepsis. Despite optimal number and ‘sensing and alarming’ function of skin TRM, the capacity of these cells to promote a tissue-wide recall response was lost after sepsis that increased viral burden upon homologous skin infection. Decreased sensitivity of vascular endothelium to TRM-derived IFN-γ was the underlying mechanism that diminished localized recall responses after sepsis. Overall, my data stress the importance of understanding how sepsis-induced lesions in T cell-extrinsic factors contribute to diminished T cell-mediated immunity observed after sepsis. Sepsis influence on T cell-mediated immunity has previously been explored using model pathogens. However, sepsis survivors are susceptible to an array of secondary complications, including cancer, which often occurs in patients with no previous history of malignancy. This suggested the immunoparalysis phase of sepsis increases cancer incidence and/or progression that may contribute to the increased long-term mortality of this population. I showed that previously-septic mice had increased cancer-associated mortality weeks/months after sepsis recovery due to diminished frequency and function of tumor-infiltrating CD8 T cells (CD8 TILs) that mediate partial control of B16 melanoma. CD8 T cells with high expression of inhibitory receptors (denoted PD-1hi) that retain some effector functions in the tumor were preferentially eliminated in sepsis survivors. The rapid loss of PD-1hi CD8 TILs diminished the window of efficacy for checkpoint blockade therapy designed to improve the number and function of this T cell subset. Thus, the chronic immunoparalysis phase of sepsis is defined by diminished tumor-specific CD8 T cell responses that increases susceptibility to cancer mortality. Most sepsis patients have comorbidities at the time of sepsis onset but the capacity of sepsis to influence comorbidity burden remains unknown. Because cancer is the highest risk factor for sepsis, I examined the capacity of sepsis to influence the outcome of hosts bearing solid tumors. Sepsis was performed after B16 inoculation and recapitulated clinical data showing increased sepsis mortality in cancer patients. However, sepsis mortality only occurred in mice bearing large tumors, while hosts with less progressed tumors survived the septic event and had improved cancer prognosis. CD8 T cells were required for this phenomenon and the number of vasculature-excluded CD8 TILs was not altered after sepsis. Despite this, I showed CD8 TILs from mice that survived sepsis had increased activation and effector functions suggesting sepsis has the unexpected capacity to reinvigorate this subset. In total, sepsis impact on CD8 T cell responses alters host outcome to additional disease states that commonly affect sepsis patients, a notion that will be important for a growing number of sepsis survivors.

CD8 T cells have been demonstrated to be critical in the resolution of acute influenza A virus (IAV) infections. Previously our laboratory has demonstrated that the magnitude of the IAV-specific CD8 T cell response is inversely proportional to the initial IAV inoculum. The decrease in CD8 T cells observed during lethal dose IAV infections was shown tobe a result of apoptosis driven by FasL expressed on lymph node dendritic cells (LNDC) during lethal, but not sublethal, dose IAV infections. However the specific LNDC subset(s) responsible for FasL:Fas mediated apoptosis of IAV-specific CD8 T cells remains to be identified. The existence of multiple subsets of dendritic cells (DC) within the lymph node (LN) with distinct functions during viral infections suggest the possibility that a specific subset(s) may be responsible for this effect. Furthermore, the regulation of FasL expression on LNDC during lethal versus sublethal dose IAV infections was shown to be dependent on the levels of IL-12p40. However, the specific IL-12p40 containing cytokine, as well as which cells produce this cytokine within the LN remain unknown. Finally, whether or not the expression of FasL expression induced during infections is unique to IAV or if other pulmonary insults can mediate this effect is as of yet undetermined. Here we demonstrate that plasmacytoid DC (pDC), which accumulate in the lung draining LN, are the only LNDC subset eliminating IAV-specific CD8 T cells through FasL:Fas dependent mechanism both in vitro and in vivo during lethal dose IAV infections despite FasL expression by all LNDC subsets. Further we demonstrate that this disparity in LNDC FasL induced apoptosis is related to the individual DC subsets ability to present IAV antigen as pDC are the only LNDC subset incapable of viral antigen presentation via MHC class I during IAV infections. This dissertation further demonstrates that IL-12p40 homodimer (p402), which is produced by respiratory DC (rDC) and LNDC, and not IL-12p40 monomer controls FasL expression on LNDC during lethal dose IAV infections. Additionally I also demonstrate that IL-12Rβ1, which binds IL-12p40, is important for p402 mediated LNDC FasL expression. We further go on to demonstrate that rDC migration from the lungs to the LN is required for both LNDC p402 production and FasL expression. However, LNDC in isolation are unable maintain FasL expression suggesting their production of p402 may not me sufficient for FasL expression. Conversely, rDC are sufficient to induce FasL expression on LNDC from IL-12p40 deficient LN. Together these data suggest that rDC are a critical component of p402 mediated LNDC FasL expression. Finally, we demonstrate that the differential production of p402, and downstream LNDC FasL expression, observed during lethal versus sublethal dose IAV infections is not unique to IAV as intranasal stimulation with diverse TLR agonists also results in differential rDC p402 production and LNDC FasL expression. These data suggest that in addition to IAV, a multitude of pulmonary pathogens may regulate antigen-specific CD8 T cells through this pathway. Taken together the results present herein detail a mechanism of CD8 T cell regulation during IAV infections mediated through p402 induction of FasL expression on pDC within the LN. FasL expressing pDC then induce apoptosis of activated Fas+ IAV-specific CD8 T cells within the LN during lethal a IAV infections leading to a reduction in the number of IAV-specific CD8 T cells that reach the lung and as a result death of the host.

Mast cells are tissue resident, innate immune cells that provide protection against parasitic and bacterial infections and venom poisoning. Mast cells also play a pathogenic role in atopy and allergic diseases. Atopy and allergic diseases are increasing in the developed world and are predicted to continue to increase at an alarming rate. Current treatment options include corticosteroids, anti-histamines, anti-IgE and avoidance of allergen. These interventions have limitations: some patients are steroid resistant; anti-histamines have low efficacy since they need to be administered early during allergen exposure; and anti-IgE is costly. Thus there is a clinical need for new treatment options. An efficient approach is to re-purpose FDA-approved drugs. Selective serotonin reuptake inhibitors (SSRIs) are a class of anti-depressants used to treat depression and other psychiatric disorders. SSRIs have been shown to possess anti-inflammatory properties, but the mechanism of action is unclear. The possibility to treat allergic diseases with SSRIs has not been studied. Using primary mouse bone marrow derived, ex vivo cultured mouse peritoneal, and primary skin derived human mast cells, we show that the SSRI fluoxetine suppresses IgE-mediated degranulation, cytokine production, and inflammatory lipid secretion. Several other SSRIs showed similar effects on mouse mast cells. Cytokine suppression occurs at a transcriptional level, as evidenced by decreased signaling downstream of the IgE receptor and reduced cytokine mRNA induction. We found that fluoxetine-mediated suppression requires the purinergic receptor, P2X3. Furthermore, we show that IgE stimulation elicits rapid ATP release from mast cells, and that ATP and purinergic signaling is a positive feedback regulator of mast cell activation. Fluoxetine can also suppress ATP-mediated cytokine production, degranulation, and lipid production most likely via NFkb suppression and diminished purinergic receptor expression. Importantly, fluoxetine effects are consistent in an in vivo passive systemic anaphylaxis (PSA) model and in a house dust mite (HDM) airway hyperresponsiveness and lung inflammation model of asthma. In the PSA model, fluoxetine reduced hypothermia and cytokine production. In the asthma model, the drug suppressed bronchoresponsiveness as well as pulmonary mast cell hyperplasia and eosinophilia, and the recruitment of Th2 cells, neutrophils, eosinophils, and lymphocytes to the bronchoalveolar space, as well as cytokine levels in the bronchoalveolar fluid in sensitized mice. Overall, we show that fluoxetine broadly suppresses mast cell activation in vitro and in vivo, most likely by impeding an ATP-P2X3 positive feedback loop.

CD4 and CD8 T cells are constantly exposed to inflammatory signals that influence diverse functional outcomes during infections and certain autoimmune disorders. One of the signals controlling CD4 and CD8 T cell functions is the inflammatory cytokine IL-12. Previous studies have focused on how IL-12 regulates CD4 and CD8 T cell functions when present during or after the activation of the T cell receptor (TCR). However, based on murine studies, we have only recently begun to appreciate that exposure to inflammatory signals, driven in part by IL-12, could alter how CD4 and CD8 T cells respond to TCR stimulation. Although intriguing, these studies have left several questions unanswered. Does IL-12 similarly regulate the function of human T cells? If so, what is the exact molecular mechanism by which IL-12 mediates these effects? To address these critical questions, we examined how IL-12 pretreatment altered human CD4 and CD8 T cell responses to subsequent TCR stimulation. In CHAPTER III, we examined how prior exposure to IL-12 alters the responses of human CD4 T cells to subsequent TCR stimulation. Some of our key findings were that IL-12 pretreatment increased the production of IFN-γ, TNF-α, IL-13, IL-4 and IL-10 after TCR stimulation, suggesting that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. Based on the intracellular staining and mRNA expression data, we concluded that the IL-12-mediated increased production of a range of cytokines was a consequence of at least two separate mechanisms, increased mRNA expression for IFN-γ and increased release of TNF-α, IL-13, IL-4 and IL-10. In CHAPTER IV, we explored the mechanisms by which IL-12 pretreatment altered human CD4 T cell responses to TCR stimulation. We observed that IL-12 pretreatment increased the phosphorylation of AKT, P38 and LCK following TCR stimulation without altering other TCR signaling molecules, suggesting that this potentially mediates the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that were not transcriptionally regulated was partially driven by increased oxidative metabolism. Collectively our results uncovered a novel function of IL-12 in regulating human CD4 T cell responses; specifically, it enhanced the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism. Then, in CHAPTER V, we examined the effects of IL-12 pretreatment in altering the responses of human CD8 T cells to subsequent TCR stimulation. Our key finding was that pretreatment of human CD8 T cells with IL-12 resulted in increased IFN-γ and TNF-α cytokine mRNA and protein production following subsequent TCR challenge. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules. Together our results suggest that prior exposure to IL-12 potentiates human CD8 T cell responses to TCR stimulation possibly by altering the activation of TCR signaling pathways. In the end, our results increase our understanding of the physiologic properties of human CD4 and CD8 T cell and provide mechanistic insight into novel functions for IL-12. Our results also provide insights into potential avenues to improve the current uses of IL-12 in therapeutics.

Plasma cell neoplasms (PCN), including multiple myeloma, are tumors of terminally differentiated B cells. Despite a significant research effort, and numerous advances in therapy, most tumors of this B cell lineage remain incurable. To this end, understanding factors which are critical for the development of PCN may lead to new avenues for therapy. Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine which supports the growth, proliferation, and survival of myeloma cells. We found that inflammation, and in particular, IL-6 is critical for the development of PCN. In order to determine if tumor microenvironment (TME) or B cell-derived IL-6 was more important in PCN development, we utilized an adoptive transfer system of tumor formation. By adoptively transferring premalignant B cells into recipients, and then providing the B cells with an inflammatory microenvironment through the use of pristane, we were able to generate donor tumors in recipient mice. Utilizing this method, a series of adoptive transfers were performed to determine the primary source of IL-6 in murine PCN development. We discovered that TME-derived IL-6, and not B cell-derived IL-6, is most critical for PCN development. Furthermore, in studying the lesions in B cell development which lead to tumor formation, we discovered that IL-6 collaborates with the proto-oncogene c-Myc in spontaneous germinal center (GC) formation. The spontaneous GCs were accompanied by a robust follicular T helper cell response. In characterizing the genetic lesions which lead to the GC formation, we discovered that Myc-transgenic mice develop a significant increase in the population of B1a B cells. Furthermore, these B1a B cells infiltrate the spontaneous GCs of double transgenic Myc/IL-6 mice. Lastly, utilizing our adoptive transfer method, we determined that the germinal center response is necessary for the development of PCN in mice. Lastly, we focused our efforts on another oncogene which collaborates with IL-6, BCL-2. Double transgenic BCL-2/IL-6 mice develop PCN and spontaneous GCs. Of interest however, the adoptive transfer of BCL-2/IL-6 B cells results in tumor formation without the use of pristane. Furthermore, the adoptive transfer recipients develop bone lesions, hind limb paralysis, and a monoclonal gammopathy. This model closely recapitulates many of the pathophysiological features seen in human PCN. This new model promises to be important for future studies into PCN development and treatment.

No description available.

Due to shared routes of transmission, co-infection with Hepatitis C (HCV) and Human Immuno-deficiency Virus (HIV) has become prevalent worldwide, with approximately one-third of all HIV-infected individuals and about 10% of all HCV infected individuals in North America being co-infected with the other virus (1). Recent advances in HIV treatment have increased the life expectancy of HIV-infected patients, resulting in HCV-associated disease to develop into a major cause of morbidity and mortality among the co-infected population. HIV is consistently shown to have a negative effect on different aspects of HCV disease, from increased HCV RNA levels (2) to aggravated liver fibrosis and more rapid progression to cirrhosis and end-stage liver disease (3). The host immune responses play a major role in not only controlling HCV infection, but also in causing hepatic inflammation and damage. Despite major advances in the understanding of the pathogenesis of these two infections, the mechanisms underlying the role of immune responses in more rapid progression of HCV disease in HCV/HIV co-infection is not quite clear. This thesis is generated based on an investigation to understand why HIV infection worsens HCV pathogenesis. This question is addressed throughout this thesis from different immunological aspects, with a focus on the function of T-cells. I have demonstrated that in HCV/HIV co-infection, functional HIV-specific T-cells accumulate in the liver and produce an array of cytokines, including INF- and TNF-, which may represent a bystander role for HIV in the aggravation of HCV-induced liver damage. My data also demonstrate that co-expression of two defined T-cell exhaustion markers, Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, in the setting of HIV co-infection. Both total and HCV-specific peripheral T-cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared to HCV mono-infection. In co-infection, HCV-specific CD8+ T-cells showed greater frequencies of Tim-3/PD-1 dual-expression than those being HIV-specific, indicating a greater degree of exhaustion in the former. Additionally, I demonstrated that some HIV mono-infected individuals may contain CD8+ T-cells that cross-recognize two defined HLA-A2-restricted epitopes within the HIV and HCV proteome, the HIV-Gag: SLYNTVATL and HCV-NS5b: ALYDVVSKL. This T-cell cross-reactivity was further elaborated in the context of HCV/HIV co-infection, demonstrating that degeneracy of HIV-specific T-cells may play a role in the immuno-pathology of co-infection. Altogether, these data could be integrated into the foundation of potential mechanisms involved in the immunopathogenesis of HCV/HIV co-infection, and be applied to further investigation in basic science and clinical studies in this field.

NK cells are innate lymphocytes that are crucial for host immunity during infections. Discrimination between healthy and infected cells is facilitated through a sum of inhibitory and stimulatory signals. Host cells can modulate the expression of NK ligands in response to infection, transformation, and stress, while viruses can exploit these mechanisms to prevent the killing of the infected cells. This thesis focuses on the interaction between the NK receptor, NKR-P1B, and its ligand, Clr-b. First, we used a reporter cell-based protocol to establish hybridomas producing monoclonal antibodies against a putative viral immunoevasin, RCTL. We express CD3ζ-fusion proteins on the reporter cells, which we then use to immunize and screen hybridoma specificities. Our results demonstrate a rapid, efficient, and high-throughput method of monoclonal antibody screening, and provide the framework for our work on cytomegalovirus evasion of the NKR-P1B:Clr-b system. Next, we show that RCMV infection results in a notable downregulation of rClr-b at the protein and transcript levels. Conversely, RCTL is upregulated during infection, and binds to the same NK-inhibitory receptor as Clr-b, NKR-P1B. In the absence of RCTL, RCMV-mediated Clr-b loss leads to increased NK-killing of infected targets and an NK-dependent reduction of viral titers in vivo. Notably, NKR-P1B is highly polymorphic, and certain rat NKR-P1B alleles have lost binding to the viral RCTL but not to the host Clr-b molecule, suggesting co-evolution between the host and the virus. In the next chapter, we address some of the mechanisms responsible for CMV-mediated Clr-b downregulation, and show that Clr-b downregulation also occurs to mice in response to MCMV infection. Moreover, early gene expression (host or viral) appears to be required for Clr-b downregulation. Interestingly, engagement of any one conventional pattern recognition receptor is insufficient to mimic MCMV-mediated Clr-b downregulation. Similarly, fibroblasts lacking various intermediates for the interferon or inflammasome pathways still downregulate mClr-b, with the exception of the DNA sensor, Zbp-1. Lastly, a recently identified autocatalytic motif conserved in the rat and mouse Clr-b transcripts, the hammerhead ribozyme, appears to be involved in Clr-b regulation. Taken together, our results explore a novel and important role for NKR-P1B:Clr-b interactions in viral immunity.

Due to shared routes of transmission, co-infection with Hepatitis C (HCV) and Human Immuno-deficiency Virus (HIV) has become prevalent worldwide, with approximately one-third of all HIV-infected individuals and about 10% of all HCV infected individuals in North America being co-infected with the other virus (1). Recent advances in HIV treatment have increased the life expectancy of HIV-infected patients, resulting in HCV-associated disease to develop into a major cause of morbidity and mortality among the co-infected population. HIV is consistently shown to have a negative effect on different aspects of HCV disease, from increased HCV RNA levels (2) to aggravated liver fibrosis and more rapid progression to cirrhosis and end-stage liver disease (3). The host immune responses play a major role in not only controlling HCV infection, but also in causing hepatic inflammation and damage. Despite major advances in the understanding of the pathogenesis of these two infections, the mechanisms underlying the role of immune responses in more rapid progression of HCV disease in HCV/HIV co-infection is not quite clear. This thesis is generated based on an investigation to understand why HIV infection worsens HCV pathogenesis. This question is addressed throughout this thesis from different immunological aspects, with a focus on the function of T-cells. I have demonstrated that in HCV/HIV co-infection, functional HIV-specific T-cells accumulate in the liver and produce an array of cytokines, including INF- and TNF-, which may represent a bystander role for HIV in the aggravation of HCV-induced liver damage. My data also demonstrate that co-expression of two defined T-cell exhaustion markers, Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, in the setting of HIV co-infection. Both total and HCV-specific peripheral T-cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared to HCV mono-infection. In co-infection, HCV-specific CD8+ T-cells showed greater frequencies of Tim-3/PD-1 dual-expression than those being HIV-specific, indicating a greater degree of exhaustion in the former. Additionally, I demonstrated that some HIV mono-infected individuals may contain CD8+ T-cells that cross-recognize two defined HLA-A2-restricted epitopes within the HIV and HCV proteome, the HIV-Gag: SLYNTVATL and HCV-NS5b: ALYDVVSKL. This T-cell cross-reactivity was further elaborated in the context of HCV/HIV co-infection, demonstrating that degeneracy of HIV-specific T-cells may play a role in the immuno-pathology of co-infection. Altogether, these data could be integrated into the foundation of potential mechanisms involved in the immunopathogenesis of HCV/HIV co-infection, and be applied to further investigation in basic science and clinical studies in this field.