Immunology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21687

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    IL-17A(+) CD4(+) T-Cells and Neutrophils Contribute to Lung Pathology during Murine Mycoplasma Infection
    (2017-03-14) Simecka, Jerry; Mize, Maximillion
    Background: Current research cannot fully explain how mycoplasma promote airway inflammation. It is no surprise that current vaccines promote lung damage. We showed that T-cells drive resistance and pathology in mice infected with Mycoplasma pulmonis. T-cells are important in vaccine-associated immunity. Our goal is to improve the effectiveness of current vaccines by understanding how T-cells contribute to disease outcome. IL-17A is secreted by T-cells and activates neutrophils during infection. However, overzealous IL-17A production activates immune responses that cause disease. We found elevated IL-17A mRNA and protein levels in mice infected with M. pulmonis. Lung lesions in cattle infected with Mycoplasma mycoides contain IL-17A. What IL-17A does during disease is still not known. I hypothesis that IL-17A contributes to disease pathology during murine mycoplasma infection. Methods: M. pulmonis is a natural pathogen of mice, infection mimics other mycoplasma diseases. Here, BALB/c mice were infected with M. pulmonis as previously described. Disease was monitored in mice receiving either PBS or antibodies against IL-17A and Ly6G. Flow cytometry and immunostaining were used to identify phagocytes and IL-17A+ lymphocytes in the lung. Results: T-cells produce IL-17A during infection with M. pulmonis. IL-17A+ T-cells exist in the lungs prior to infection and may contribute to the rapid recruitment of neutrophils into the respiratory tract immediately after inoculation. αβ CD4+ T-cells was the predominant T-cell population producing IL-17A throughout infection. By Day 14, αβ T-cells in the lungs and lower respiratory lymph nodes were able to secrete IL-17A alone, or in combination with IFN-γ. Expression of ROR-γt was not required for IL-17A production by αβ T-cells in the lung. Immunostaining revealed that IL-17A+ CD4+, and not IL-17A+ CD8+, were located within inflammatory lesions. Neutralizing IL-17A reduced host damage without impacting bacterial burden. Neutrophilic lesions were lower in response to IL-17A neutralization. Depletion of neutrophils was more effective at reducing pathology when compared to IL-17A neutralization. Combining IL-17A neutralization with neutrophil depletion failed to further reduce the disease pathogenesis. Conclusions: Neutrophils and IL-17A+ T-cells contribute to pathogenesis during murine mycoplasma infection. These cells may act independently to promote inflammation. IL-17A may exacerbate neutrophil-dependent pathology.
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    Expression of Cancer Stem Cell (CSC) Specific Transcription Factors and Cell Surface PCNA and Their Role in CSC Escape From NK Cell Effector Function
    (2017-03-14) Horton, Nathan; Mathew, Porunelloor A.; Malaer, Joseph
    Purpose: Natural Killer (NK) cells participate in the innate immune response against cancer and infection without prior sensitization. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of target cells. Cancer cells may evade NK-mediated killing by expressing or secreting ligands for NK cell inhibitory receptors. The Natural Cytotoxicity Receptor (NCR) family, comprised of NKp30, NKp44, and NKp46, is classically described as a group of activating receptors that induce NK cell activation and cytotoxicity. Notably, NKp44 functions as an activating or inhibitory receptor depending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with Human Leukocyte Antigen I (HLA I) and forms the inhibitory ligand for NKp44, resulting in the inhibition of NK function. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. Methods: Diffuse B cell lymphoma (DB) cells were labeled and sorted for cell surface PCNA expression via fluorescence activated cell sorting (FACS). Total RNA was isolated, converted to cDNA, and the transcription factors NANOG, SOX2, and Oct-4, which are associated with CSC phenotype, were analyzed by qRT-PCR from sorted PCNA+ and PCNA- cells. NK receptor-ligand interactions were blocked by incubating DB cells with anti-PCNA, anti-NKp44, or control antibodies and a chromium release killing assay was performed. Results: Cell sorting and qRT-PCR confirmed DB cells with cell surface PCNA have increased expression of transcription factors compared to PCNA- cells. Blocking the interaction of NKp44 and PCNA enhanced the killing of DB by NK cells. Conclusions: Cell surface PCNA is associated with increased CSC transcription factor expression. Additionally, cell surface PCNA on CSC may facilitate escape from NK cell killing by interacting with NKp44 and transmission of an inhibitory signal. Characterization of stem cell transcription factors and cell surface PCNA may provide novel immunotherapeutic targets to destroy CSC and thus prevent cancer metastasis.
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    Culture with Dextrose Reduced Macrophage Viability in A Dose Dependent Manner: Implications for Prolotherapy
    (2017-03-14) Castillo, Rudy; Conway, Allison; Rabago, David; Nourani, Booby; Hodge, Lisa; Marvin, Hannah
    Purpose: Prolotherapy, coined from proliferant therapy, is an alternative injection-based therapy that has been used in clinical practice for over 80 years to treat various chronic musculoskeletal conditions. Modern hypotheses suggest prolotherapy promotes growth of normal cells and tissues to improve ligament mechanics, and decrease pain through inflammatory mechanisms. The most common injectant contains dextrose (D-glucose), a natural form of glucose found in the body. Recent literature indicates adults with symptomatic knee pain received the most relief from intra-articular injection of dextrose, compared to saline injections, or exercise. This study aims to begin to explain the mechanism of action of dextrose in the inflammatory response. Specifically, we hypothesized that dextrose would suppress the release of inflammatory mediators from LPS-activated macrophages. Methods: To test this hypothesis, murine RAW 264.7 macrophages were cultured in vitro with phosphate-buffered saline (PBS) or dextrose solution at 2.5%, 5%, and 10% of total volume per well, with or without 500ng lipopolysaccharide (LPS). Twenty-four hours after incubation at 37°C with 5% CO2, culture supernatants were stored and assayed for nitrite (NO2-) using Griess reagent. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide. Results: Dextrose did not significantly alter the production of NO2- in macrophages cultured without LPS. However, in LPS-activated macrophages, dextrose significantly (p2- compared to respective PBS controls. Specifically, 2.5% dextrose suppressed NO2-release by 78.31%, while 5% and 10% dextrose completely inhibited production of NO2- . Cell viability was also significantly (p Conclusions: Dextrose reduced viability and suppressed the production of NO2- by macrophages in vitro. Prolotherapy may protect against inflammation by reducing the inflammatory activities of macrophages. Future studies will examine the effect of dextrose on macrophage function in vivo using animal models.
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    The Role of ecSOD in Neutrophil Containment of Listeria Monocytogenes
    (2017-03-14) Swanta, Naomi; Berg, Rance E.; Okunnu, Busola
    Background: Extracellular superoxide dismutase (ecSOD) is an antioxidant that serves to minimize host tissue damage during reactive oxygen species (ROS) mediated immune responses. Listeria monocytogenes (LM) is an intracellular bacteria that is often used to study host pathogen interactions during intracellular bacterial infections. Previously, we showed that ecSOD activity is detrimental to the host during infection with LM. Furthermore, using depletion studies, we determined that neutrophils, a set of innate immune cells which are known for ROS generation, from mice that lack ecSOD (ecSOD KO) are more protective during LM infection. Materials and Methods: Using ecSOD congenic mice (expressing differing activities of ecSOD as indicated by their titles; ecSOD HI, ecSOD Wild Type, ecSOD Knockout), flow cytometry and a unique LM termed actA:LMGFP, we set out to determine how ecSOD activity modulates the protective capabilities of neutrophils during LM infection. Hypothesis: We hypothesized that ecSOD activity would hinder the ability of neutrophils to keep LM contained in the phagosome and therefore suppress their ability to ultimately kill the bacteria. Results: In vitro, a higher percentage of neutrophils from the liver, spleen, bone marrow, and peritoneal cavity ecSOD KO mice, allowed for phagosomal escape in comparison to the ecSOD expressing neutrophils. In vivo, at a high dose of infection, a similar trend was observed in the spleen, although, the opposite occurred in the liver. However, using MFI as an indicator of the relative number of bacteria per neutrophil, we observed that the ratio of cell associated to cytosolic bacteria was higher in ecSOD KO neutrophils in comparison to neutrophils with ecSOD activity. The next step was to determine if ecSOD modulates neutrophil protective mechanism downstream of phagosomal containment, mainly, autophagy. It was observed that phagosomal escape correlates with the initiation of autophagy in bone marrow neutrophils. However the effect on neutrophils from other organs is currently inconclusive. Conclusions: EcSOD activity does appear to modulate neutrophil association with LM. Lack of ecSOD activity increases association of the bacteria with neutrophils but simultaneously decreases the phagosomal escape of the bacteria into the cytosol. However, the presence of cytosolic bacteria induces the initiation of autophagy as a mechanistic means of keeping LM contained which should eventually lead to killing by the neutrophil.
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    Pro-inflammatory condition-induced tumor dormancy in pre-metastatic reservoir thymus
    (2017-03-14) Su, Dong-Ming; Sizova, Olga
    Hypothesis: Cancer patients suffering from metastatic relapse is the major cause of death. This is attributed to some undetectable minimal number of tumor cells, which are able to resist radio-chemotherapy, being in dormant state at some organs of the body. The largest T-lymphoid organ, thymus, has just recently been suggested as this kind of organ to be a pre-metastatic reservoir for tumor cell dormancy, and eventually relapse. We hypothesize that infection-, drug- and aging-resulted thymic atrophy induces inflammatory conditions to create this tumor pre-metastatic reservoir. Materials and Methods: To test our hypothesis we utilized various cancer cell lines (both human- and mouse- derived) and inoculated them into different groups of mice to see how specific tumor microenvironments (young thymus/naturally and induced involuted thymus/T-and B-cell lacking) have an effect on cancer cells retention and survival during chemotherapy. Results: Our observations in tumor-inoculated mouse model show that various cancer cells could be retained in the thymus. This retention ratio (in the thymus vs. lymph nodes) was particularly high in the naturally-aging- or drug-caused atrophied thymus. We found that genotoxic chemotherapy (Doxorubicin) can lead to changes in thymic microenvironment, characterized by activation of p53 in thymic epithelial cells (TECs), induction of TEC senescence, and an increase of pro-inflammatory factors. These thymic conditions were able to confer tumor cell capacity to anti-apoptosis. We also found that dormant tumor cells in the thymus have a capacity to induce tumor recurrence in the distant organs. Conclusions: Our pilot experiments suggest that activation of p53 in TECs induced by DNA-damage upon Doxorubicin treatment promotes senescence, in which SASP (senescence-associated secretory phenotype) is activated, thereby leading to tumor cell dormancy/chemo-resistance. This work is clinically relevant because our findings may help to determine a new target to prevent tumor relapse after chemotherapy.
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    The effect of ecSOD on HMGB1 and it's role during Listeria infection
    (2017-03-14) Witter, Alexandra; Okunnu, Olubusola; Berg, Rance E.; Swanta, Naomi
    Background: Listeria monocytogenes (LM) is a gram-positive, intracellular foodborne pathogen which can cause severe disease in immunocompromised individuals and is a leading cause of death from foodborne infection. During LM infection, reactive oxygen species (ROS) represent a first line of defense, which not only destroys the pathogen but can also cause collateral tissue damage. Extracellular superoxide dismutase (ecSOD) is a potent antioxidant that protects host tissue by regulating ROS concentrations. Using congenic mice that express varying levels of ecSOD, our lab has previously demonstrated that ecSOD activity decreases resistance to LM infection and pro-inflammatory cytokine production. In contrast, ecSOD enhances neutrophil recruitment to the liver during LM infection. High-mobility group box 1 protein (HMGB1) is a ROS modulated protein, which has been shown to promote neutrophil recruitment when critical cysteines are reduced but promotes pro-inflammatory responses when these residues are oxidized. We hypothesize that ecSOD activity reduces cysteine residues in HMGB1, thus enhancing neutrophil recruitment while decreasing pro-inflammatory responses during LM infection. Purpose: The purpose of our study is to understand the effect ecSOD has on transcription, translation and post-translational modification of HMGB1. We are also studying the effect of HMGB1 on inflammatory cytokine production such as TNFa, IL-1 and IL-6 and neutrophil recruitment to infected organs during LM infection in ecSOD congenic mice. Methods: C57BL/6 mice were infected with LM and at 3 days post infection immune cells were isolated from the spleen and bone marrow. The cells were used for rt-PCR to study HMGB1 transcription, and western blotting to study the translation of HMGB1. Furthermore, the aforementioned cells from ecSOD congenic mice were also incubated overnight with 10ng/mL of HMGB1 and supernatants were harvested to measure TNFa by ELISA. Results: Our results indicate that LM does not have a significant effect on HMGB1 transcription and translation in the spleen and bone marrow. We also show that at 10ng/mL, HMGB1 does not enhance or induce TNFa production in spleen and bone marrow cells from ecSOD congenic mice. Conclusions: Our results indicate that HMGB1 production is not influenced by LM infection in C57BL/6 mice. Furthermore, our data suggest that HMGB1 may not influence immune responses against LM in ecSOD congenic mice in vitro. Further studies are required to elucidate the in vivo functions of HMGB1 during LM infection.
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    Thoracic Duct Lymph Suppresses the Inflammatory Response of Macrophages in vitro
    (2017-03-14) Hodge, Lisa; Castillo, Rudy A.
    Purpose: The gastrointestinal lymphatic vessels redistribute a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Recent literature suggests that during gastrointestinal injury, soluble factors released from the mesentery redistribute duct to the lung via the thoracic duct where they initiate inflammation and contribute to multiple organ dysfunction syndrome (MODS). Alternatively, normal mesenteric lymph has been shown to suppress inflammation in vivo and in vitro. Specifically, under inflammatory conditions, normal mesenteric lymph reduced the expression of cell adhesion molecules and myeloperoxidase in pulmonary tissue and reduced expression of cell adhesion molecules on pulmonary endothelial cells. However, the role of normal lymph on phagocyte function remains unknown. Importantly, macrophages have been to shown to contribute to MODS. The aim of this study was to investigate the effect of normal lymph on macrophage function. Specifically, we hypothesized that normal thoracic duct lymph (TDL) would suppress the release of inflammatory mediators by LPS-activated macrophages. Methods: To test this hypothesis, under anesthesia the thoracic ducts of eight mongrel dogs were cannulated and lymph was collected. The TDL was centrifuged to remove cells and the TDL supernatant was frozen and stored at -80oC. Murine RAW 264.7 macrophages were cultured in vitro with TDL at 0.5, 1, 2, 5 or 10% total volume per well or phosphate-buffered saline at 5 or 10% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C with 5% CO2. After incubation, cell cultures were centrifuged to remove cells and the supernatants were assayed for nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a) production. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide to distinguish live cells from apoptotic cells. Results: TDL did not augment the production of NO2-, TNF-a or alter cell viability by macrophages cultured in media alone. However, when macrophages were activated with LPS, TDL suppressed the release of NO and TNF-a. Specifically, the addition of TDL at 5% total volume per well suppressed NO2- production (15±0.6 uM) and TNF-a production (5016±425 pg/mL) compared to LPS. Culture with LPS and/or TDL did not alter cell viability. Conclusions: Our data suggests that during stimulation with LPS, a biological factor in lymph suppressed the release of inflammatory mediators by macrophages. Furthermore, cell viability was unaltered, suggesting that that TDL altered macrophage function. Future studies will focus on the ability of lymph to suppress the inflammatory response in disease models.