Immunology
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Item Divergent Roles of Extracellular Superoxide Dismutase During Intracellular Bacterial Infection(2016-03-23) Break, Timothy; Okunnu, Busola; Swanta, Naomi; Berg, Rance E.; Witter, AlexandraExtracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using congenic mice with varying levels of ecSOD activity (ecSOD HI, WT and KO), we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) from degradation and promotes egress of immature neutrophils out of the bone marrow and into the liver where they are unable to provide protection against LM. Since ecSOD can be produced by cells from the hematopoietic lineage as well as somatic cells, the potential contribution of ecSOD produced by cells from each lineage required further investigation. In order to determine the relative contributions of ecSOD produced from either hematopoietic-derived cells or somatic cells, we generated bone marrow chimera mice using ecSOD KO mice and C57Bl/6 mice. Briefly, host mice were irradiated to eliminate hematopoietic lineage cells and reconstituted with bone marrow cells isolated from donor mice. Control groups consisted of ecSOD KO mice reconstituted with bone marrow from ecSOD KO donors (KO -> KO) or C57Bl/6 mice with bone marrow from C57Bl/6 mice (WT -> WT). Experimental groups consisted of ecSOD KO mice reconstituted with bone marrow from C57Bl/6 mice (WT -> KO) or C57Bl/6 mice with bone marrow from ecSOD KO mice (KO -> WT). All mice were then infected with LM and evaluated for neutrophil recruitment and bacterial burden. We observed that ecSOD produced by hematopoietic cells leads to increased bacterial burden during LM infection, while ecSOD produced from somatic cells is essential for increased neutrophil recruitment. Collectively, our data suggest that ecSOD produced by both hematopoietic cells and somatic cells is involved in our observed phenomena; however, the contribution of ecSOD from each cell lineage is skewed towards either increased neutrophil recruitment or increased susceptibility to LM infection, but not both. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item The Role of ecSOD in Phagosomal Containment of Listeria Monocytogenes(2016-03-23) Witter, Alexandra; Swanta, Naomi; Berg, Rance E.; Okunnu, BusolaExtracellular superoxide dismutase (ecSOD) is a secreted enzyme associated with the extracellular matrix that plays a protective role during reactive oxygen species (ROS) mediated inflammatory responses. Listeria monocytogenes is an intracellular gram positive bacteria that causes listeriosis in infected individuals and animals. EcSOD has been shown to modulate the innate immune response to Listeria infection. With the use of ecSOD congenic mice (ecSOD HI, ecSOD WT, ecSOD KO), we have established that high ecSOD activity leads to decreased bacterial clearance despite an increase in neutrophil recruitment. Whereas, no ecSOD activity results in reduced neutrophil recruitment but effective bacterial clearance. Importantly, neutrophils in the livers of ecSOD KO mice show superior protective capabilities in comparison to neutrophils in ecSOD HI mice. To deduce the mechanism by which ecSOD contributes to neutrophil effector function during Listeria infection, we made use of different strains of GFP expressing Listeria monocytogenes. Neutrophils isolated from both the bone marrow and the liver of the ecSOD congenic mice were able to effectively phagocytose the bacteria. However, we observed that there was more bacterial escape from the phagosome of neutrophils isolated from the bone marrow of ecSOD KO mice in comparison to the ecSOD HI and WT mice. In contrast, there was no observable difference in the ability of neutrophils isolated from the livers of the ecSOD congenic mice to prevent Listeria escape from the phagosome. These data are contradictory to the previously reported protective nature of the ecSOD KO neutrophils and their importance for bacterial clearance from the livers of infected mice. The neutrophils used for the current in vitro studies were isolated from the tissues in the absence of infection. Neutrophils are generally not present in large numbers in tissues in the absence of infection, therefore, we inferred that the isolated neutrophils were not functionally active. In conclusion, our results suggest that our in vitro observations are not supportive of the previous in vivo data regarding the ability of ecSOD to modulate neutrophil function.Item P53-dependent stromal senescence-induced tumor dormancy in the pre-metastatic reservoir thymus(2016-03-23) Su, Dong-Ming PhD; Sizova, OlgaCancer patient survival rate has significantly improved with popularization of early diagnostics and advancement in the therapy. However, after surgical removal of primary tumor and subsequent radio-chemotherapy, some cancer patients can still suffer from recurrence of the same tumor in secondary sites of the body several years later. Metastatic tumor is the major cause of death among cancer patients. This metastatic recurrence/relapse is attributed to some minimal number of tumor cells, which are able to resist radio-chemotherapy, being in sleeping/dormant state at some organs of the body. It is known that cancer cells can go to distant places of the body via vasculature and lymphatics, however, the largest lymph organ – thymus has just recently been suggested as a potential reservoir for tumor cell dormancy, and eventually relapse. However, the mechanisms responsible for tumor dormancy in the thymus are unknown. We believe that cancer cells can migrate to thymus and become dormant due to DNA damage caused by chemotherapy. Under the certain condition, those cancer cells are able to disseminate into other organs of the body leading to tumor relapse. It is necessary to study tumor micro-environmental elements in the thymus, so they could be targeted by the anti-tumor therapy. Our preliminary observation shows that administration of a chemotherapeutic drug (Doxorubicin) into young wild-type mice leads to activation of p-53 protein in thymus epithelial cells and induces thymus shrinkage. To link activation of p-53 in thymic epithelial cells and tumor dormancy we injected human breast cancer cells in young and aged mice with subsequent Doxorubicin administration. We found that tumor cells were harder to be killed in the aged involuted thymus with increased p53 in thymic epithelial cells. Our preliminary data suggest that activation of p53 in thymic epithelial cells induced by DNA-damage upon Doxorubicin promotes cell senescence of thymic epithelial cells, which leads to tumor cell dormancy/chemo-resistance. This work is clinically relevant because our findings might be able to find new strategies to prevent tumor relapse after chemotherapy.Item The role of HMGB1 during Listeria Infection.(2016-03-23) Witter, Alexandra; Okunnu, Olubusola; Berg, Rance E.; Swanta, NaomiAbstract 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 infections. LM stimulates the immune system via pathogen-associated molecular patterns (PAMPs) such as lipoproteins which interact with pattern recognition receptors (PRRs) including toll-like receptors (TLR2). These interactions induce activation of immune cells resulting in the production of cytokines such as TNFa, IL-1 and IL-6. PRRs also induce immune activity in response to damage-associated molecular patterns (DAMPs) which are released from host cells in response to cellular damage. High Mobility Group Box 1 (HMGB1) protein is a DAMP that has been shown to be actively secreted from macrophages during sterile inflammation resulting in the production of TNFa by binding to toll-like receptor 4 (TLR4). Even though HMGB1 has been shown to be an active player during sterile inflammation, nothing has been published about its function during immune responses to any pathogenic infection. b. Purpose The purpose of our study is to understand the effect LM infection has on transcription, translation and post translational modification of HMGB1. We are also studying the effect of HMGB1 on inflammatory cytokines such as TNFa, IL-1 and IL-6, neutrophil recruitment to infected organs, and bacterial burden during LM infection. c. 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. The aforementioned cells were also incubated overnight with 10ng/mL of HMGB1, and supernatants were harvested to measure TNFa by ELISA. d. 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 TNFa production in spleen and bone marrow cells. e. Conclusion Our results indicate that HMGB1 production is not influenced by LM infection. Furthermore, our data suggest that HMGB1 may not influence immune responses against LM in vitro. Further studies are required to elucidate the in vivo functions of HMGB1 during LM infection. b. Purpose The purpose of our study is to understand the effect LM infection has on transcription, translation and post translational modification of HMGB1. We are also studying the effect of HMGB1 on inflammatory cytokines such as TNFa, IL-1 and IL-6, neutrophil recruitment to infected organs, and bacterial burden during LM infection. c. 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. The aforementioned cells were also incubated overnight with 10ng/mL of HMGB1, and supernatants were harvested to measure TNFa by ELISA. d. 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 TNFa production in spleen and bone marrow cells. e. Conclusion Our results indicate that HMGB1 production is not influenced by LM infection. Furthermore, our data suggest that HMGB1 may not influence immune responses against LM in vitro. Further studies are required to elucidate the in vivo functions of HMGB1 during LM infection.Item Differential Effects of Thoracic Duct Lymph on Pulmonary Macrophages(2016-03-23) Conway, Allison; Hodge, Lisa; Castillo, Rudy A.Purpose: The gut-lung axis remains a poorly defined mechanism that could impact etiology and treatment of gut and respiratory diseases. Early literature first described this gut-lung crosstalk in inflammatory bowel disease patients with chronic bronchopulmonary disease. Recent literature describes the role of intestinal lymph during acute respiratory distress syndrome (ARDS). In these studies, intestinal ischemia and reperfusion injury in animal models induces lung injury suggesting factors, such as cytokines and lipids, released from the gut during gastrointestinal shock can contribute to ARDS. The gut lymphatics provide a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Our lab has previously demonstrated lymph-enhancing techniques enhanced the flux of cytokines, chemokines and reactive oxygen and nitrogen species in thoracic and mesenteric lymph. We propose factors released from the gut travel through the lymphatics to the lung and suppress the immune response in the lung. Methods: To test this hypothesis, alveolar macrophages from bronchoalveolar lavage fluid (BALF), lung tissue macrophages (after BALF collected) and intraperitoneal (IP) macrophages from IP lavage were isolated from healthy F344 rats. In addition, a rat alveolar macrophage cell line (NR8383) was used for in vitro studies. Macrophages were cultured for 12 hours and canine thoracic duct lymph (TDL) at 10% total volume per well and/or LPS (500ng per well) added for 24 hours. Supernatants were stored and used to measure nitric oxide (NO) and tumor necrosis factor alpha (TNFa) using Griess assay and ELISA. Results: AM were the most sensitive to LPS activation compared to LM and IP macrophages. TDL suppressed LPS-induced NO (52% decrease) and TNFa (25% decrease) production. However, TDL did not significantly suppress LPS activation in LM and IP macrophages. In NR8383, TDL suppressed LPS-induced NO (86% decrease) and TNFa (66% decrease) production. TDL alone did not activate NR8383, AM, LM or IP macrophages and did not affect cell viability after culture. Conclusions: Our results suggest a biological factor in lymph selectively suppresses LPS activation in alveolar macrophages. The mobilization of healthy lymph may protect the lung from chronic inflammation caused by pathogens and pulmonary disease. Future studies will focus on identifying factors in lymph that may modulate the immune response that may improve disease outcome.Item Analysis of expression of immune receptors in pediatric acute lymphoblastic leukemia (ALL)(2016-03-23) Mathew, Stephen O.; Mathew, Porunelloor A.; Bowman, Paul; Powers, SheilaAcute Lymphoblastic leukemia (ALL) is the most common type of cancer in children. It is characterized by overgrowth of the lymphocyte precursor (either B cell or T cell) that is nonfunctioning, and crowds out other immune cells. Current treatment options have a success rate of 80-90%. However, those who relapse have a survival rate of only around 25-40%. Also, side effects of current chemotherapy and radiation treatments have been shown to impact the normal growth and development of children. Research has shown that ALL of the B cell lineage is particularly resistant to killing by Natural Killer (NK) cells. NK cells are part of the innate immune system and specialize in killing tumor and virally infected cells. NK cell activation is dependent on a balance of inhibitory and activating receptors and their ligands expressed on the target cell. Our laboratory has previously cloned three immune receptors, 2B4, CS1 and LLT1, which have been shown to play a role in cancer, however their significance and role in childhood ALL have not been evaluated. In this study, we evaluated the expression of immune receptors 2B4, CS1, LLT1, NKp30 and NKp46 in pediatric ALL subjects both male and female in the age range of 3 – 18 yrs. ALL subjects and healthy subjects were enrolled at Cook Children’s Hospital and UNT Health Science Center, Fort Worth, TX with informed consent/assent according to IRB approval (UNTHSC IRB# 2008-094 & CCMC IRB# 2008-57). The blood samples were collected and peripheral blood mononuclear cells (PBMC) were isolated and analyzed by flow cytometry and real-time qPCR for expression of immune receptors. ALL subjects showed altered expression of immune receptors in the PBMC as compared to healthy subjects. There was an overall decrease in the expression of 2B4, CS1, LLT1 and NKp46 in the B lymphoblasts of ALL subjects as compared to healthy subjects. Expression and functional analysis of these receptors in a larger sample size will provide valuable insights to conduct future mechanistic studies to investigate the role of these immune receptors in ALL resistance and relapse. Funded by Cancer Research Foundation of North Texas (CRFNT).Item Impact of exogenous hyaluronan on dendritic cell activation and trafficking from skin(2016-03-23) Mummert, Mark; Hersh, JessicaIntroduction: Hyaluronan (HA) is a polysaccharide used as dermal filler in cosmetic dermatology. A minority of patients ( Purpose: This study is to determine whether HA at different molecular weights (M.W.) can be degraded into biologically active fragments inducing migration of APCs to draining lymph nodes causing contact hypersensitivity in mice. Materials and Methods: Mice, divided into 3 groups of 4, were subcutaneously injected in their ears with Lifecore Biomedical HA of graded Molecular Weights (M.W.) (Group 1: 851 kDa-1.19 MDa, Group 2: 66kDa-90 kDa, Group 3:kDa). The positive control of HA tetrasaccharide (Group 4) and negative control of PBS (Group 5) were from Sigma-Aldrich. All antibodies (Ab) were from BD Pharmingin. Female BALB/c mice were injected with 200 µg HA/ear or the contralateral ear was injected with PBS (40 µL/ear). Mice were sacrificed 24 hours post-injection, auricular lymph nodes were collected, and HA and PBS draining lymph nodes were pooled separately. Ears were harvested after lymph node collection, and stored in PBS wetted gauze at -20ºC. Pooled lymph nodes were homogenized; cells were counted using a hemocytometer. Cells were incubated for 30 minutes on ice with Fc blocking Ab, stained with a phycoerythrin labeled MHC II specific Ab, or phycoerythrin labeled isotype control IgG Ab for 30 minutes on ice. DCs in the auricular lymph nodes were assessed using flow cytometry. Epidermal sheets were prepared and stained with anti-MHC II specific Ab. The number and morphology of Langerhans cells were evaluated microscopically. Results: The control Ab showed insignificant binding while anti-MHC II Ab showed significant binding to a subpopulation of APCs stained for MHC II. HA with M.W. [greater than] 5000 Da did not cause MHC II positive cells to migrate out of the ears. None of the HA injections resulted in significant morphological changes or differences in Langerhans cell densities Conclusion: HA [greater than] 5,000 Da did not cause migration of APCs to draining lymph nodes. None of the injected HA preparations cause morphological changes or emigration of Langerhans cells.