Dissertation Summaries

The Effect of Exogenous Leptin on Murine Dendritic Cell Morphology and Function

Christine Delgado, MS

University of Texas at El Paso, 2009

Leptin is a pleiotropic hormone primarily secreted by white adipose tissue. Although originally found to regulate food intake and energy expenditure, it is now known to also significantly affect the immune system. In this study we assessed the effect of leptin on bone marrow derived dendritic cell (BM-DC) cytoskeletal structure and functionality. Our preliminary data demonstrates that the addition of physiological concentrations of exogenous leptin to BM-DC causes cytoskeletal rearrangement specifically that of actin as evidenced by increased number of lamellipodia/dendrites and increased staining for actin within the lamellipodia/dendrite region. This suggests that leptin is a necessary component of DC–T cell interaction in that leptin may be responsible for enhanced migration of the DC to draining lymph nodes and potentiating the extent of the physical contact between these cells. We therefore hypothesized that leptin increases the number and length of dendrites in DCs by causing a rearrangement of the cytoskeleton, specifically actin, which in turn leads to an increased capacity to migrate and to activate T cells. To test this hypothesis we used BM-DCs from C57Bl/6 (B6) mice enriched by magnetic bead cell sorting. The effect of leptin on BM-DC cytoskeletal rearrangement was assessed by light and confocal microscopy; actin polymerization was determined by Western blot analysis for cell content of F-actin versus G-actin in fractioned cellular lysates of treated cells. The functional effects of leptin on BM-DC were evaluated in vitro by assessing DC migration and DC ability to active antigen-specific T cells. The differentially treated BM-DC was evaluated in transwell migration assays to assess their ability to migrate towards a DC-specific chemokine CCL19. Finally, treated BM-DC ability to activate T cells was assessed in vitro by measuring activation of an antigen-specific T cell hybridoma or of antigen-specific primary T cells (both specific to ovalbumin) as a function of IL-2 and INF-γ production or proliferation, respectively. We found that there is only a slight increase in actin content within the cell post treatment of leptin; however this increase is not significant. The addition of an exogenous physiological concentration of leptin to BM-DC enhanced polymerization of actin and therefore the formation of dendrites, both in number and length and thickness. This effect was particularly evident in the leptin and LPS duo treatment. Functionally the presence of leptin appears to enhance migration and promote T cell activation. This is likely due to the increased surface area, as well as dendrite number length and thickness which increase the likelihood of DC–T cell interactions, therefore potentiating the extent of the physical contact between these cells. This is further supported by enhanced T cell activation by leptin and LPS treated cells as measured by incorporation of tritiated thymidine, IL-2 and INF-γ production. The data obtained from this study suggest leptin plays an important role in immunological responses, and may specifically contribute to the pro-inflammatory state and increased susceptibility of autoimmune disorders found in obesity.

Unraveling the Mysteries of Sjogren’s Syndrome: A Closer Look at the Effects of Hormones and Genetics Over Time Using the NOD.B10.H2b Mouse Model

Vanessa M. Seamon, PhD

Florida Atlantic University, 2009

Sjögren’s Syndrome (SS) is characterized by lymphocytic infiltration, destruction and dysfunction of the lacrimal and salivary glands, and the presence of serum autoantibodies. Although, approximately 0.5% of the population suffers from SS, there is a female predominance of 9:1 compared with males. Most women with SS are postmenopausal; however, not all women who are postmenopausal develop SS. Therefore, we postulate that a decrease in the circulating levels of hormones creates an environment favorable to the development of SS in a predisposed genetic background.

In order to carry out our studies, we used the NOD.B10.H2 b mouse model of SS, and ovariectomized (OVX) them as a model for the postmenopausal condition. We removed the lacrimal glands and measured the gene expression and protein levels of several cytokines and chemokines known to be upregulated in patients with SS such as: IL-1β, IL-10, INF-γ, TNF-α, CCL9, and CXCL13. We also stained for markers of B cells (B220+) and T cells (CD4+ and CD8+), and counted positively stained cleaved caspase-3 cells as an indication of apoptosis. These experiments were done 3, 7, and 21 days post-OVX and compared to sham operated animals. In order to determine whether the changes observed with OVX were triggered mainly by a genetic predisposition, a nonprediposed OVX and sham operated mouse (C57BL/10) was used as control.

We found that gene expression of IL-1β, IL-10, and IFN-γ were upregulated in the lacrimal glands of the OVX NOD.B10.H2 b mice at 3 days post-OVX compared with sham operated animals. Gene expression of IL-1β, IL-10, IFN-γ, TNF-α, CCL9, and CXCL13 and protein levels of IL-1β, IL-10, and CCL9 were upregulated in the OVX NOD.B10.H2 b mice at 7 days post-OVX compared to sham operated animals. Also, at 7 days, an increase in B220+ B cells and an increase in cleaved caspase-3 were also observed in the OVX NOD.B10.H2 b mice lacrimal glands compared to sham operated animals. At 21 days, protein levels of IL-10 were also highly upregulated in the OVX NOD.B10.H2 b mice, together with an increase of B220+ B cells, a slight increase in the CD4/CD8 ratio and an increase on the number of caspase-3 positive cells. No changes were observed in any of the above parameters measured in the OVX C57BL/10 mice compared to the sham operated group, supporting our hypothesis that both, genetics and a decrease in the levels of hormones are necessary for SS to occur.

Alterations in Vitamin D Metabolism With Chronic Inflammation: Potential Implications in Inflammation-Induced Bone Loss

Yin Foong Lim, MS

Oklahoma State University, 2009

Inflammatory conditions are often associated with compromised serum 25-(OH)D 3 status and previous in vitro studies have shown that inflammatory cytokines may be involved. The objective of this study was to examine the effects of low dose LPS in vivo on inflammatory mediators and gene expression of VDR and hydroxylation enzymes involved in vitamin D metabolism. In experiment 1, 12-week-old male C57BL/6J mice (n = 12/group) were randomly assigned to LPS (0, 0.01, and 0.1 mg/kg bw/d) delivered by slow release pellets over 30 and 90 days. In experiment 2, 16-week-old male C57BL/6J mice (n = 10/group) were administered (i.p.) LPS (0, 0.1, 0.2 mg/kg bw/d) for 14 days. Body weight was not altered during either study. Low dose LPS increased hepatic TLR4, CD14, and IL-10 after 14 days. No alterations were observed in hepatic genes of interest after 30 days, but IL-10 and IL-1β were decreased after 90 days. Splenic IL-1β tended to increase (P = 0.05) after 14 days and IL-6 was increased in low dose LPS after 90 days. Hepatic CYP27A1 gene expression was decreased after 90 days, but not after 14 and 30 days. High dose LPS tended to decrease (P = 0.08) splenic VDR after 14 days, and decreased CYP27B1 after 90 days. Renal CYP27B1 was decreased and CYP24A1 was increased after 14 days. A significant increased in plasma 25-(OH)D3 was observed after 14 days, but not 30 days. Our data suggest that chronic low dose LPS has modest effects on VDR, decreases renal CYP27B1, and increases renal CYP24A1. Further studies should further explore how LPS alters renal gene expression of cytochrome P450 enzymes involved in vitamin D metabolism over time.

Analysis of Vaccine- and Infection-Induced Immune Responses Contributing to Suppression of Blood-Stage Malaria in the Plasmodium yoelii Rodent Model

Patricia M. Petritus, PhD

Drexel University College of Medicine, Pennsylvania, 2009

Immunization with Plasmodium yoelii merozoite surface protein-8 (Py MSP-8) protects mice from lethal malaria but does not prevent infection. Using this MSP-based vaccine model, vaccine- and infection-induced immune responses that contribute to protection were investigated. Analysis of prechallenge sera from rPy MSP-8 immunized C57BL/6 and BALB/c mice revealed high and comparable levels of antigen-specific IgG but differences in isotype profile and specificity for conformational epitopes were noted. As both strains of mice were similarly protected against P. yoelii, vaccine-induced responses did not correlate with protection. However, passive immunization studies suggested that protection resulted from differing immune responses. Studies with cytokine-deficient mice showed that protection was induced by immunization of C57BL/6 mice only when IL-4 and IFN-γ were both present. In BALB/c mice, the absence of either IL-4 or IFN-γ led to predictable shifts in the IgG isotype profile but did not reduce the magnitude of the antibody response induced by rPy MSP-8 immunization. Immunized IL-4^−/− BALB/c mice were protected against P. yoelii. Surprisingly, immunized IFN-γ^−/− BALB/c mice initially controlled parasite growth but eventually succumbed to infection. Analysis of cytokine production revealed that P. yoelii infection induced 2 distinct peaks of IFN-γ in rPy MSP-8 immunized BALB/c mice. Further evaluation determined that it was the early production of IFN-γ that was necessary for protection in immunized and challenged mice. Maximal parasite growth in rPy MSP-8 immunized mice occurred during a period of sustained TGF-β production. TGF-β neutralization did not lead to an uncontrolled, pathologic proinflammatory response but resulted in modestly improved protection in rPy MSP-8 immunized mice. There were no significant differences in antibody or cytokine responses between isotype- and anti-TGF-β-treated, rPy MSP-8 immunized mice. Interestingly, rPy MSP-8 immunized IFN-γ^−/− BALB/c mice, which normally succumb to lethal P. yoelii malaria, were protected when TGF-β was neutralized. These data suggest that in rPy MSP-8 immunized mice, TGF-β is not necessary for down-modulating inflammatory responses and is detrimental to disease outcome in the absence of IFN-γ. Combined, the data indicate that suppression of lethal P. yoelii in rPy MSP-8 immunized mice requires high titers of Py MSP-8 antibody and infection-induced IFN-γ, which is associated with development of the protective response to additional parasite antigens.

Characterization of Cytochrome P450 4F Subfamily: Response in Traumatic Brain Injury and Gene Regulation

Ying Wang, PhD

University of Texas Graduate School of Biomedical Sciences at Houston, 2009

CYP4F enzymes metabolize endogenous molecules including arachidonic acid, leukotrienes, and prostaglandins. The involvement of these eisosanoids in inflammation has led to the hypothesis that CYP4Fs may modulate inflammatory conditions after traumatic brain injury (TBI). In rat, TBI elicited changes in mRNA expression of CYP4Fs as a function of time in the cerebrum region. These changes in CYP4F mRNA levels inversely correlated with the cerebral leukotriene B4 (LTB4) level following injury at the same time points. TBI also resulted in changes in CYP4F protein expression and localization around the injury site, where CYP4F1 and CYP4F6 immunoreactivity increased in surrounding astrocytes and CYP4F4 immunoreactivity shifted from endothelia of cerebral vessels to astrocytes. The study with rat primary astrocytes indicated that pro-inflammatory cytokines TNF-α and IL-1β could affect the transcription of CYP4Fs to a certain degree, whereas the changing pattern in the primary astrocytes appeared to be different from that in the in vivo TBI model.

In addition, the regulation of CYP4F genes has been an unsolved issue although factors including cytokines and fatty acids appear to affect CYP4Fs expression in multiple models. In this project, HaCaT cells were used as an in vitro cellular model to define signaling pathways involved in the regulation of human CYP4F genes. Retinoic acids inhibited CYP4F11 expression, whereas cytokines TNF-α and IL-1β induced transcription of CYP4F11 in HaCaT cells. The induction of CYP4F11 by both cytokines could be blocked by a JNK specific inhibitor, indicating the involvement of the JNK pathway in the up-regulation of CYP4F11. Retinoic acids are known to function in gene regulation through nuclear receptors RARs and RXRs. The RXR agonist LG268 greatly induced transcription of CYP4F11, whereas RAR agonist TTNPB obviously inhibited CYP4F11 transcription, indicating that the down-regulation of CYP4F11 by retinoic acid was mediated by RARs, and that inhibition of CYP4F11 by retinoic acid may also be related to the competition for RXR receptors. Thus, the CYP4F11 gene is regulated by signaling pathways including the RXR pathway and the JNK pathway. In contrast, the regulation mechanism of other CYP4Fs by retinoic acids appears to be different from that of CYP4F11.

Dual Impact of Parental Disease-Associated Mutations on Offspring Behavior in Mice

Bojana Zupan, PhD

Weill Medical College of Cornell University, New York, 2009

The genetics of many heritable disorders are not well understood due to the complex relationship between multiple genes and the environment. Psychiatric disorders are particularly interesting in this respect as they are modulated by multiple genetic factors while remaining susceptible to environmental stimuli. The environment of prenatal and/or early postnatal developmental periods is one such stimulus, and it is dependent on the maternal genotype. If genetic variability alters maternal physiology and behavior during this time, then we postulate that the overall genetic impact on the offspring is measured by the effect produced by the transmitted genes as well as by the effect produced by the maternal environment induced by those genes. This is the basic premise of our Dual Impact Model, which applies this supposition to offspring disease susceptibility.

Using 2 mouse lines, one a model of a human disorder, the Fragile X Syndrome (FXS), and the other a mouse lacking the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α), we generated both littermate and nonlittermate wild-type and knockout mice, allowing us to discern between genetic and environmental influences on behavior. We found that parental-genotype dependent modifications of the environment contribute to life-long behavioral changes in offspring. The convergence of the offspring and maternal genotype dependent effects likely leads to the full phenotype associated with a genetic abnormality/variability. This has potential implications in the treatment of genetic disorders as targeting the mechanisms of offspring genotype-dependent phenotypes may not be sufficient to relieve all symptoms. In these cases, a dual target approach is necessary.

Strikingly, some disease-associated behaviors were frequently observed in genetically unaffected offspring, indicating that these parental genotype-dependent environmental effects can increase disease susceptibility in genetically unaffected offspring. The importance of the maternal genotype on modulating environmental stimuli is further supported by cross-fostering experiments showing that pre and/or postnatal maternal genotype effects (depending on the strain) are sufficient to modify adult behavior of genetically normal animals. These findings bring to attention the need to better understand the epigenetic mechanisms by which certain environmental stimuli exert their effects so that inadvertent programming of offspring behavioral processes can be avoided.

Interleukin-11 Regulates Autoimmune Demyelination

Blake T. Gurfein, PhD

Mount Sinai School of Medicine of New York University, 2009

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Selective destruction of myelin is the foremost pathological hallmark of the early stages of MS disease progression. Later in the disease course progressive axonal transection is also observed and is associated with permanent neurologic disability. Current therapies for MS target inflammation but do not directly address neuroprotection or lesion repair. Cytokines of the gp130 family regulate the survival and differentiation of both neural and immune cells, and we recently identified expression of the family member interleukin-11 (IL-11) in active MS plaques. Here, we show that IL-11 regulates the clinical course and neuropathology of experimental autoimmune encephalomyelitis (EAE), a demyelinating model that mimics many of the clinical and pathologic features of MS. Importantly, the effects of IL-11 are achieved via a combination of immunoregulation and direct neuroprotection. IL-11 receptor-alpha null (IL-11Ra^−/−) mice displayed a significant increase in clinical severity and neuropathology of EAE compared with wild-type littermates. Inflammation, demyelination, and oligodendrocyte loss were all exacerbated in IL-11Ra^−/− animals. Conversely, wild-type mice treated with IL-11 displayed milder clinical signs and neuropathology than vehicle-treated controls. In cocultures of murine MOG 35-55-specific CD4+ T lymphocytes and CD11c+ antigen-presenting cells (APCs), IL-11 treatment resulted in a significant decrease in T cell-derived effector cytokine production. This unexpected effect was generated by modulation of CD11c+ APC-mediated lymphocyte activation, and was associated with a decrease in the size of the CD11c+ cell population. Conversely, IL-11 strongly reduced apoptosis and potentiated mitosis in primary cultures of mouse oligodendrocyte progenitors (OPCs). Collectively, these data reveal that IL-11 regulates inflammatory demyelination via a unique combination of immunoregulation and neuroprotection. IL-11 signaling may represent a therapeutic avenue to restrict CNS inflammation and potentiate oligodendrocyte survival in autoimmune demyelinating disease.

Key Factors in T Follicular Helper Cell Development

Amanda Catherine Poholek, PhD

Yale University, Connecticut, 2009

The effector subset of CD4 T cells that is responsible for mediating T-dependent antibody responses has been classified as follicular helper T cells (T FH cells). TFH cells, defined by expression of the surface markers CXCR5, PD-1, and ICOS are located in germinal centers where they provide help for B cell maturation through the secretion of IL-21 and expression of CD4OL; however the transcription factor that is required for their development is unknown. The transcriptional repressor Bcl6 is upregulated in T FH cells, yet the role that it plays in T FH cells has not been described. We have found downregulation of P-selectin glycoprotein ligand-1 (PSGL1) as a new surface marker for T FH cells, and show that Bcl6 is required for the development of T FH cells that are PSGL1 lo. Activated antigen-specific CD4 PSGL1 lo T cells are enriched for expression of CXCR5 and PD-1, and highly upregulate IL-21 and Bcl6. In addition, we find that Bcl6 is required in CD4 T cells for development of germinal centers, presumably because T FH cells are required for their formation or development. Over-expression of Bcl6 leads to an increase in PSGL1 lo T FH cells; however, it cannot rescue T FH cell development in the absence of ICOS. Consistent with others, we find B cells are required for full development of T FH cells, but not for downregulation of PSGL1, suggesting there are multiple steps in development of the subset. These data demonstrate that Bcl6, independent of ICOS signaling, is critical for the development of this lineage. We also show that T FH cells are not limited to secreting IL-21, as activated CD4 T cells that express the T FH cell surface markers CXCR5, PD-1 and are PSGL1 lo can also make IFN-γ or IL-4. In addition, subsets of TFH cells located in the germinal center are positive for IFN-γ or IL-4. This challenges the model that T FH cells are incapable of secreting cytokines associated with other CD4 T cell subsets, and suggests an alternative model in which some plasticity exists. This work has significantly contributed to our understanding of T FH cell development, and has identified a new marker that may have functional relevance to the T FH cell population.

Modulation of Macrophage Responses to Borrelia Burgdorferi in Acute Murine Lyme Carditis

Chris M. Olson, Jr., PhD

University of Massachusetts–Amherst, 2009

The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant natural killer T (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. Here, we report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 (B6) mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFN-γ, which we demonstrate controls the severity of murine Lyme carditis by at least 2 mechanisms. First, IFN-γ greatly enhances the recognition of Borrelia burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Second, IFN-γ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate acute murine Lyme carditis through the action of IFN-γ, which appears to self-renew through a positive feedback loop during infection.

Inflammation during infection with B. burgdorferi is dependent on the ability of the spirochete to evade local mechanisms of clearance. Even though macrophages are the main infiltrating cell during Lyme carditis, the identification of a receptor capable of mediating phagocytosis of B. burgdorferi has been elusive. Here, we demonstrate that the integrin CR3 is able to mediate binding to the spirochete and facilitate phagocytosis in a complement-dependent and independent manner. Expression of CR3, but not CR4, in CHO cells markedly enhanced their capacity to interact with B. burgdorferi, in the absence and presence of complement opsonization. Furthermore, the interaction between CR3 and B. burgdorferi is dependent on the metal-ion-dependent adhesion site (MIDAS) and could be blocked with EDTA. Inhibition of CR3 with blocking antibody was able to completely abrogate phagocytosis of B. burgdorferi by the macrophage-like RAW264.7 cells and partially block uptake by bone marrow-derived macrophages (BMMs), a finding that was recapitulated with CD11b-deficient BMMs. We further show that activation with recombinant IFN-γ increases the transcription of CD11b and CD18, which correlates with increased surface expression of CR3, and that the effect of IFN-γ on the phagocytosis of B. burgdorferi is circumscribed to CR3 activity, because inhibition of CR3 is able to completely diminish the effect of IFN-γ on the phagocytosis of the B. burgdorferi. Last, our results demonstrate that CR3 is a negative regulator of proinflammatory cytokine induction in macrophages responding to B. burgdorferi. Overall, our data demonstrate roles for CR3 in the binding, phagocytosis and proinflammatory cytokine elicited by B. burgdorferi and shed light on the role of IFN-γ in mediating the clearance of the spirochete during Lyme disease.

Neuroprotective and Neuroregenerative Strategies After Contusion Spinal Cord Injury

Harra Rebecca Sandrow-Feinberg, PhD

Drexel University College of Medicine, Pennsylvania, 2009

Spinal cord injury (SCI) results in a devastating and permanent injury to the central nervous system. Damage to the spinal cord interrupts transmission of ascending and descending information, which creates sensory and motor deficits. The degree of impairment is dependent upon the level and severity of the injury. Cervical (C) contusion injuries are most common resulting in a bruising or compression of the cord. Due to the prevalence of this form of SCI the rodent model chosen was a severe unilateral cervical contusion SCI. A mid cervical injury was created with the Infinite Horizon impactor using a force of 200 Kdyne. The first study using a C4 injury investigated function after aspiration of the lesion site and apposition of one end of a predegenerated peripheral nerve graft during an acute (7 days) and chronic (28 days) situation. A change in injury level was requires for subsequent studies because of the remarkable spontaneous recovery after the 2 initial studies with a C4 injury. Results indicated that function atter the C4 injury was not further impaired with removal of the lesion site debris of either time point. Remaining studies used the C5 injury model to investigate the neuroprotective and/or neuroregenerative consequences of acute treatment with a modified anti-inflammatory agent, pegylated interferon-β (PEG IFN-β). This model also was used for development and for further study of a forced exercise rehabilitation strategy. Short term studies evaluated biochemical changes in spinal cord tissue in response to SCI, exercise or PEG IFN-β alone. Exercise resulted in significantly increased levels of brain-derived neurotrophic factor and reduced inflammatory cell infiltration caudal to the injury. PEG IFN-β treatment resulted in a decrease of some pro-inflammatory cytokines and an increased in some anti-inflammatory cytokines. Long term behavioral assessments revealed accelerated early recovery that plateaued by 5e weeks post SCI and exercise. Each therapeutic approach led to beneficial effects but in combination they did not act synergistically. These results suggest several possible strategies for neuroprotection and partial recovery after SCI though further studies are needed to achieve full recovery.

The Role of Interferon-tau (IFNT) in Luteal Gene Expression, Steroidogenesis, and Luteal Lifespan in the Ewe

Rebecca Clark Bott, PhD

Colorado State University, 2009

Interferon-tau (IFNT) was evaluated for endocrine actions on the corpus luteum (CL). The hypothesis was that infusion of IFNT would increase luteal expression of interferon-stimulated gene (ISG)-15, and the length of time for ewes to return to estrus. Osmotic pumps containing 200 mg IFNT or BSA (n = 12 each) were connected to the uterine vein of nonpregnant ewes 10 days postestrus. Messenger RNA encoding ISG15 was elevated in CL from pregnant and IFNT-infused ewes (P < 0.05) compared to nonpregnant and BSA-treated ewes, respectively. Luteal mRNA encoding ISG15 from ewes treated with IFNT was greater than in ewes treated with BSA (P < 0.05). Serum concentrations of progesterone were not different in ewes that received infusions of BSA or IFNT. Progesterone decreased by 6 h (P < 0.05) in ewes that received BSA+PGF or IFNT+PGF, but did not differ in ewes that received infusions of IFNT^+/− PGF at 8, 10, or 12 h after PGF. There were no differences in prostaglandin E synthase (PGES) or prostaglandin F synthase (PGFS), or in prostaglandin dehydrogenase (PGDH), steroidogenic acute regulatory protein (StAR), peripheral type benzodiazepine receptor (PBR), cytochrome P450 side chain cleavage enzyme (CYP-11A), or 3b-hydroxysteroid dehydrogenase (3b-HSD). Seven day infusion of IFNT during the time frame of maternal recognition of pregnancy resulted in 20% of IFNT-treated ewes returning to estrus by d19 compared to 100% of BSA-treated ewes (P < 0.01). In conclusion IFNT acts systemically, alters gene expression in the corpus luteum, and decreases the number of ewes returning to estrus by d19.