MADISON, SHARON LYNN. The effects of PM2.5 on allergic inflammation in mast cell deficient mice. (Under the direction of Bruce Hammerberg.) Animal models of asthma have confirmed epidemiological findings that exposure to fine particulate matter (PM2.5) can enhance asthmatic symptoms, including eosinophilic inflammation and airway hyperresponsiveness. Critics have dismissed the possibility that these studies utilizing artificial exposure scenarios, like intratracheal instillation (i.t.), can be legitimately extrapolated to human risk largely due to the fact that the doses required for this type of model exceed the normal ambient concentrations of PM2.5. In order to improve the credibility of the findings from previous animal studies utilizing the i.t. method for delivery of aqueous particle suspensions to the lung, and to determine the biological mechanisms responsible for the observed enhancement of allergic inflammation following PM2.5 exposure, large-scale air samplers have been developed making it possible to directly expose wild type (WT) and genetically altered mice to fine, concentrated ambient particles (CAPs). In this study allergic asthma was modeled in both WT and mast cell deficient (MCD) mice by local (L) or systemic (S) sensitization to ovalbumin (OVA). Two weeks later mice were challenged with OVA (day 0) and then exposed to CAPs (day 0 & 1) with numerous endpoints collected (day 0-2). Overall, there was a temporal difference in the bronchoalveolar lavage cell profile between L and S sensitized mice, and the contribution of mast cells (MC) to this differential response was best observed for neutrophils at day 0 and day 1. Compared to air exposed mice, CAPs depressed total inflammatory cell infiltrates in the bronchoalveolar lavage fluid at day 0 and day 1 after OVA challenge for all groups. This overwhelming difference of limited cellular infiltration of monocytes and neutrophils in the bronchoalveolar lavage fluid following CAPs exposure, and the significant difference between the L and S sensitization protocols, confound interpretation for all of the factors examined. However, the specific finding that CAPs can enhance eosinophil recruitment by day 2 after OVA challenge indicates that the results from previous animal studies utilizing i.t. PM2.5 exposures do in fact support the epidemiological associations linking PM2.5 exposures with the enhancement of allergic inflammation indicative of the asthmatic phenotype. Given the strict regulation of immunological tolerance at mucosal surfaces like the lung, the genetic variability of different mouse strains, and the daily changes in ambient PM2.5 composition, the findings of this study prompt many unique questions. However, the bottom line is that this study demonstrates that ambient PM2.5 does alter Th2-like responses in mice by enhancing pulmonary BAL eosinophils in the late phase response (day 2), and that mast cells are critical to their recruitment.

CD4+CD25+ T regulatory cells represent an important subset of lymphocytes whose function is to suppress autoimmune disease and control normal immune responses. There is much research indicating a direct role for TGF-beta expressed on the surface of Tregs in the suppressor function of these cells. TGF-beta, whether bound to the cell surface or secreted, is produced as part of a complex that renders the cytokine inactive. Thrombospondin is the primary activator of biologically latent TGF-beta. This research demonstrates that thrombospondin is expressed on the surface of T lymphocytes isolated from the blood and lymph nodes of normal and FIV positive felines. Thrombospondin is expressed at significantly higher levels on CD4+CD25+ T lymphocytes, but can be induced in culture by activating T helper cells in the presence of LPS and IL2. We also observed that the CD4+CD25- T helper cells isolated from FIV negative control lymph nodes were able to markedly upregulate surface thrombospondin expression compared with similarly stimulated CD4+CD25- T helper cells from FIV positive sources. These findings are initial steps in working to understand the mechanism behind latent TGF-beta activation on CD4+CD25+ T regulatory cells and the role this cell type plays in FIV/HIV pathogenesis.

Developing effective vaccines against the porcine reproductive and respiratory syndrome virus (PRRSV) has proved difficult, highlighting the need for basic information on the nature of the immune response against this virus and the mechanisms of resistance that the virus employs. In this investigation our goal was to characterize the immune response against the major outer membrane protein of the virus, GP5, in pigs experimentally infected with a North Carolina isolate of PRRSV known as the NC Powell strain. In addition, we compared this response with the immune response seen after vaccination with purified recombinant GP5 (rGP5) protein. Humoral immune responses were monitored by western blot and immunofluorescence while T cell responses were monitored using proliferation assays and flow cytometry. Our results show strong humoral recognition of rGP5 protein during both natural and vaccine induced Ab responses. In addition, epitope mapping via western blot revealed that Ab responses were directed largely against the C-terminal endodomain of rGP5 protein in both experimentally infected and vaccinated pigs. We also investigated T cell responses to rGP5 protein. Our experiments revealed that T cells from vaccinated animals also responded to both rGP5 protein and inactivated NC Powell strain of PRRSV suggesting that T cells may play an important role in vaccine-induced resistance. Interestingly, we found that the inactivated NC Powell strain of PRRSV caused a strong proliferative response in naïve T cells from control animals, perhaps indicating the presence of a superantigen as a component of this highly virulent strain of PRRSV.

HIV-1 infection of the central nervous system (CNS) results in neurological impairments in subpopulation of HIV-infected individuals which range from from mild cognitive/motor disorder (MCMD) to HIV-associated dementia (HAD). HIV-1 associated neurological diseases are still a big problem even with the introduction of combined antiretroviral therapy. However the mechanisms of CNS infection and pathogenesis that lead to HAD are still not completely clear. HIV-1 CNS infection occurs soon after peripheral infection. Subsequent to infection, the CNS may act as a protected anatomical reservoir for lentiviruses and may also give rise to the development of or sequestration of unique quasispecies. The choroid plexus (ChP) has been demonstrated to be an important site for lentivirus infection and contains a mixture of viral quasispecies including both systemic and brain derived isolates. Since the appearance of viral RNA is particularly prominent in the cerebrospinal fluid (CSF), the ChP-CSF pathway may contribute to infection and viral diversity in the CNS. In the present study, we investigated lentiviral infection and evolution within the CNS by directly infusing virus into the CSF using an FIV animal model. Cell-free NCSU1 FIV or cell-associated FIV (FIV infected ChP macrophages) was directly injected into the right lateral ventricle of the brain. Negative controls were sham inoculated with uninfected ChP macrophages or cell-free culture supernatant and positive controls were infected systemically with cell-free FIV by intraperitoneal (i.p.) injection. Intracerebroventricular (i.c.v.) inoculation with cell-free FIV resulted in high levels of plasma FIV RNA detected as early as 1-2 weeks post inoculation in all 6 i.c.v. cats, and the plasma viremia preceded the detection of CSF viral RNA. Compared to i.p. cats, i.c.v. cats exhibited much higher levels of CSF FIV RNA and FIV DNA in the brain, increased ratios of CSF to plasma viral load (>1 at several time points), and a unique rebound CSF viral peak (5 of 6 cats). Infusion of FIV-infected ChP-Mac induced an acute inflammatory response and a slight suppression of the CD4+:CD8+ ratio, but failed to produce a detectable infection. After cell-free inoculation, FIV env variants, amplified by the poymerase chain reaction (PCR) and isolated using the heteroduplex tracking assay (HTA), exhibited clear compartmentalization between the CNS and periphery. Unique or enriched variants rapidly appeared in the CSF. Similar variation was seen in FIV proviral DNA isolated from cortical and subcortical brain regions. Compared to the initial viral peak in CSF, the second CSF viral peak displayed considerable change from both the first CSF peak and matched samples of plasma. FIV env diversity was highest in the CNS tissue and was unrelated to matched PBMCs collected at the same time indicating that the sequences were not due to PBMC trafficking. In addition, three unique variants were found to be selectively enriched in the CNS. Taken together, these results demonstrated that 1) CSF provides an efficient pathway for the transfer of infectious virus to the periphery, 2) virus trafficking through the CSF promotes infection of the CNS and viral diversification, 3) CSF virus may derive from both the local productive infection and blood, and 4)virus within the CNS experienced a relatively rapid and independent evolution relative to virus in the periphery.

This work is an investigative look behind the mechanisms of neutrophil migration. Each of three chapters involves exploration into a different signaling pathway important for migration downstream of chemoattractant stimulation through inhibition of a kinase or disruption of the function of an effector and examination of the effects on migration, adhesion, and actin reorganization in primary human or equine neutrophils. Chapter II examines the requirement for the signaling molecule p38 Mitogen Activated Kinase (MAPK) in equine neutrophil chemotaxis through use of the p38 specific inhibitor SB203580. SB203580 reduced LTB4- and PAF-induced migration and disrupted the ability of cells to polarize, but did not affect b2 integrin-dependent adhesion or surface b2 integrin expression. Chapter III is a comprehensive inquiry into the regulation of the phosphorylation of serine 157 of the cytoskeletal protein Vasodilator-stimulated Phosphoprotein (VASP). The rapid and transient phosphorylation of VASP serine 157 corresponded with F-actin levels in chemoattractant-stimulated human neutrophils. fMLF-induced serine 157 phosphorylation was abolished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced serine 157 phosphorylation was unaffected by PKC inhibitors, PKG inhibitors, and the CamKII inhibitor KN-62. Inhibition of adhesion did not alter fMLF-induced VASP phosphorylation or dephosphorylation. This study demonstrated that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent and adhesion-independent VASP serine 157 phosphorylation. Chapter IV probed into the function of the actin binding protein and PKC substrate Myristoylated Alanine-Rich C-kinase Substrate (MARCKS) through utilization of a cell permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with 50 μM MANS, but not a scrambled control peptide, significantly inhibited their migration and adhesion in response to fMLF, IL8, or LTB4. MANS significantly reduced F-actin content in neutrophils 30s after fMLF-induced polymerization, but did not alter the ability of cells to polarize, spread, or upregulate surface b2 integrin expression. These data provided evidence that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Tregs). Tregs have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Tregs become infected and activated during the acute stage of FIV infection leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU-1 and blood and lymph node biopsies were collected at 1 week intervals following inoculation. Real-Time PCR was used to determine plasma viremia and relative number of FIV copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of activation markers. Real-time RT-PCR was also used to assess relative increases in FoxP3 and TGF- mRNA levels over time. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays and inhibition of cellular proliferation was assessed by incorporation of tritiated thymidine and CFSE. Our results show that peak viremia levels correlate with maximal infectivity in lymph node CD4+CD25+ populations. FIV-gag-mRNA levels are higher in CD4+CD25+ T cells than CD4+CD25- lymph node T cells. Activation of FoxP3 and increased expression of TGF1 in CD4+CD25+ cells correlates with peak plasma viremia and FIV-gag-mRNA levels in CD4+CD25+ T cells. Regulatory function can be detected in CD4+CD25+ T cells during the acute phase of FIV infection. Our findings support the hypothesis that early activation of immunosuppressor function in Treg cells may limit an effective anti-FIV response contributing to the establishment of the chronic infection and the immunodeficiency caused by this virus.

Feline Immunodeficiency Virus (FIV) and Human Immunodeficiency Virus (HIV) are lentiviruses that are remarkable similar in their genomic organization, receptor usage and pathogenesis. Based on this FIV has evolved into a well-established small animal model for studying AIDS. FIV and HIV cause a progressive depletion of T cells via a still unknown mechanism though numerous studies support a role of membrane expressed HIV env glycoprotein in apoptotic killing of CD4+ T cells. HIV env glycoprotein is a heterodimer of surface expressed gp120 that binds to CD4 and a chemokine receptor and transmembrane gp41 that mediates fusion and syncytia formation. The role of the fusion process in HIV env-mediated apoptosis remains controversial even though evidence suggests that cytopathic effect of HIV is related to the fusogenic potential of env glycoprotein. Blocking HIV env receptor interactions either at the level of gp120 or gp41 blocks both syncytia formation and apoptosis. This suggests a crucial role for HIV gp41 in fusion, as well as apoptosis. The hydrophobic pre-transmembrane (pre-TM) region of HIV gp41 is important for membrane fusion and sequence analysis reveals a similar region in FIV gp41. The current study was undertaken to determine the role of different regions of FIV env in mediating fusion and apoptosis in bystander cells and to determine whether the two phenomena are related. FIV env interactions with target cells were blocked at the level of gp120 or gp41 and the effect of these on fusion and apoptosis studied. The role of FIV gp41 pre-TM region in fusion and apoptosis was also determined. Our findings support a role of FIV env in apoptotic loss of T cells and this phenomenon correlates with env-mediated fusion.

Epidemiological studies have noted an increase in adverse health effects with increasing levels of air pollution. One major area of concern is the incidence of respiratory infections, specifically influenza. An air pollutant that has raised concern in recent years is diesel exhaust (DE) due to an increase the amount of diesel engines in use. Previous laboratory studies have reported that DE exposure prior to an influenza infection increases viral titers but the mechanism of how this occurs is still unknown. Herein, studies were designed to investigate three main areas associated with DE enhanced influenza infection. 1) Assess whether DE affects host defense responses against pathogens, 2) Determine if pre-exposure to DE increases susceptibility to influenza in vivo, 3) Investigate whether exposure to DE increases the severity of an ongoing established influenza infection. DE exposure alone increased proinflammatory cytokines, adhesion molecules, decreased expression and production of surfactant proteins (SP)-A and D as well as clara cell secretory protein (CCSP). The molecules downregulated by DE are important for binding viral and bacterial pathogens therefore making the lung more susceptible to infection. This was confirmed when mice were exposed to DE and then subsequently infected with influenza A. One day post infection mice pre-exposed to DE had a significant increases in influenza induced inflammation and viral titers that were associated with a decrease in SP-A and SP-D. Mice exposed to DE during an established influenza infection also had a significant increase in viral titers and pulmonary inflammation throughout the course of infection. This DE-enhanced influenza infection was associated with an upregulation of the Th2 cytokine IL-4 which has previously been shown to delay clearance. However with antioxidant treatment to combat the oxidative stress induced by DE, pulmonary inflammation and IL-4 expression returned to baseline levels. Taken together these data indicate that exposure to DE either before or during influenza infection has immunomodulatory effects that can be detrimental to the host with increased viral proliferation and morbidity associated with the disease.

Dendritic cells (DC) are uniquely well-equipped, professional antigen-presenting cells (APC), with the ability to initiate and regulate immune responses. In transplantation, DC of both donor and host origin contribute to graft rejection by inducing T cell activation and proliferation, via the direct and indirect pathways of allorecognition, respectively. Evidence has also accumulated, however, that DC, particularly in an immature state, can promote tolerance induction and prolong organ allograft survival. Rapamycin is a potent immunosuppressant pro-drug that is well-recognized for its inhibitory effects on T cell proliferation. Despite extensive research on rapamycins impact on lymphocytes, little is known to date regarding its effects on DC. The central hypothesis in these studies was that, rapamycin interferes with the DC maturation and enhances their tolerogenic potential. We first analyzed the influence of rapamycin, in pharmacologically-relevant concentrations, on the maturation, functional activation and T cell stimulatory potential of murine myeloid DC. Herein we show that rapamycin targets DC antigen (Ag)-uptake and IL-4-mediated maturation both in vitro (in bone marrow-derived DC), and in vivo (in freshly-isolated DC, following in vivo administration of rapamycin). Exposure to rapamycin impairs inflammatory cytokine production and effective T cell stimulation by DC. Furthermore, rapamycin-treated DC induce Ag-specific T cell anergy. Next, we determined that presentation of alloAgs to T cells by rapamycin-pretreated DC of host origin, under in vivo (pre-transplant) steady-state conditions, could induce hyporesponsiveness to subsequent challenge and prolong organ (heart) graft survival. A single infusion of these cells, seven days prior to transplant, led to a significant improvement in transplant outcome in an Ag-specific manner. Furthermore, repeated infusion resulted in marked prolongation of graft survival. These studies demonstrate, for the first time, that the immunosuppressive action of rapamycin can be ascribed, in part, to its inhibitory effects on DC and that rapamycin can potentiate the tolerogenic properties of DC. They also reveal the potential of rapamycin-treated DC as therapeutic vectors of transplant tolerance.

A major challenge to combating ocular herpes simplex virus type 1 (HSV-1) infection lies in controlling the protective and immunopathological potential of the immune system. Numerous studies have shown that the immune system, especially CD8 T cells, plays an integral role in the establishment and maintenance of viral latency. In contrast, CD4 T cells mediate an immunoinflammatory response in the infected cornea termed herpes stromal keratitis (HSK) that leads to scarring and blindness. Here we address several hypotheses related to the role of CD4 T cells following ocular HSV-1 infection. First, we asked if stimulation through the costimulatory pair CD134/OX40L is required for the activation of CD4 T cells within the infected cornea and subsequent inflammation. Our findings established that both CD134 and OX40L are expressed in the cornea during HSK. However, blocking CD134/OX40L interactions did not alleviate HSK. Our second hypothesis was that HSV-specific CD4 T cells are required to initiate HSK, but bystander activation becomes important during the later, chronic stages of disease. To address this hypothesis we isolated and cloned CD4 T cells from infected corneas, demonstrated their reactivity to viral antigens, and confirmed their capacity to mediate HSK in nude mice. These cells will be used in future studies to investigate the relative role of antigen-specific and bystander activated CD4 T cells in HSK. Next, we tested the hypothesis that while CD4 T cells are the primary mediators of inflammation in the cornea, CD8 T cells can also mediate inflammation in their absence. Our findings established that CD8 T cells can mediate HSK, but CD8 T cell-mediated HSK is transient and only induced when high doses of virus are used to infect the cornea. We also tested our hypothesis that CD4 T cell help is required for the proper generation and maintenance of memory CD8 T cells at the site of viral latency. Our findings demonstrate that CD4 T cells control the contraction phase of the effector CD8 T cell response within the TG, the generation of CD8 memory precursors, and directly or indirectly influence viral genome burden within sensory neurons following ocular infection.