Role of Splenic Macrophages in the Initiation of Tolerance to Apoptotic Cell Associate Antigens
AbstractSystemic Autoimmune disease occurs due to the breakdown of tolerance to self-antigens caused in part by impaired clearance of apoptotic cells. The spleen is a primary site for generation of the tolerogenic response to selfantigens in the periphery. The marginal zone (MZ), which contains the specialized macrophage (MΦs) populations: Marginal Zone Macrophages (MZMs) and Metallophilic Marginal zone Macrophages (MMMs), as well as B cells and dendritic cells (DCs) play a requisite role in capture of apoptotic cells and the initiation of tolerance to associated self antigens. Moreover, defective MZ cellular architecture may lead to increased auto-reactivity that exacerbates autoimmune like disease progression. MZMs are specialized to recognize and capture apoptotic cells and promote peripheral tolerance to apoptotic cells and associated antigens. However, the mechanism by which MZMs enforce this tolerance is not known. Thus, the overall goal of our project is to fill the lapse in the scientific knowledge and understanding of the mechanism(s) by which the splenic stromal MΦs drive immune tolerance to apoptotic cell associated antigens.
AffiliationDepartment of Biochemistry and Molecular Biology
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p65fl/fl/LysMCre Transgenic Mouse Model Shows Altered Nf-Kb Signaling In MacrophagesHoward, Shelby; Talkad, Aditi; Oza, Eesha; Department of Biological Sciences (2016-03)We have produced and begun characterizing a transgenic mouse model, p65fl/fl/LysMCre, that lacks canonical nuclear factor-kappaB (NF-kB) signaling (p65) in cells of the myeloid lineage, which includes macrophages. NF-kB pathway activity is very important in normal immune function, synaptic plasticity, and memory, and aberrant NF-kB activity is associated with autoimmune disease, and importantly, cancer. Macrophages can be present in very large numbers in a variety of cancers, and can lead to tumor progression through promotion of tumor inflammation, angiogenesis, invasion, and metastasis. This animal model will allow our group to pursue experiments involved in better understanding how stromal macrophages communicate with cancer cells through the NF-kB pathway, and how loss of canonical NF-kB signaling in cells of the myeloid lineage might weaken the tumor and make it more susceptible to standard treatments. Characterization of the model thus far reveals that p65 protein is indeed absent in macrophages derived from bone marrow monocytes, and that NF-kB signaling is altered when stimulated with lipopolysaccharide. We have just begun co-culture experiments with p65 deleted macrophages and glioma cells, and anticipate altered communication when compared to culture with control macrophages. Funding Source: Cancer Center Collaboration Grant
Effects of Chronic Alcohol and Glucose Exposure on Viability of Alveolar MacrophagesKeller, Elizabeth; Department of Biological Sciences (Augusta University, 2019-05)The general population widely acknowledges the potential adverse health risks associated with the lifestyle factors of chronic alcohol abuse and obesity. These factors are manifested in the form of routine elevated blood ethanol and glucose levels, respectively. There is evidence which shows that the lungs are secondary organs affected by such physiological conditions. Healthy lungs are protected against infection and harmful airborne particles by macrophages—one of the working entities of the immune system. However, when these immune-responsive cells are compromised and unable to adequately perform normal functions, lung health may deteriorate. Therefore, a healthy pulmonary alveolar macrophage population is vital for the preservation of adequate lung function and for the prevention of respiratory infections and related complications. Chronic alcohol exposure and elevated glucose concentrations have been shown to suppress alveolar macrophage function, in turn possibly lowering the lungs’ first line of defense against foreign particles and infection. The objective of this study is to determine the effects of exogenous ethanol and increased glucose concentration on macrophage size and viability in relation to their compromised functionality within the human system through an in vitro study. This study tested alveolar macrophage response to conditions of elevated glucose levels, exogenous ethanol, and a joint treatment in a laboratory setting. Elevated glucose levels were representative of hyperglycemic conditions which often occur in states of obesity, while ethanol treatment simulated chronic alcohol exposure. NR8383 rat alveolar macrophages were grown in vitro in 25 cm3 flasks with each treatment. In addition to treatments, a control was maintained to ensure integrity of the experiment. Each conditional treatment was subjected to weekly viability testing and imaging for size analysis. The data was compared among the different treatment groups to gain a better understanding of how alveolar macrophages were affected by the specified conditions. The hypothesized theory anticipates an increased cell count in the glucose treated cells, but a decreased cell count in the ethanol and combination treatments. All three treatments were expected to yield smaller cell size, thus impairing macrophage function. Future studies plan to expose each treatment group to the bacterial agent lipopolysaccharide, or LPS, to test the initiated response of the macrophages and determine levels of functionality. LPS is commonly found on the outer membrane of gram-negative bacteria and is a reliable stimulant of the mammalian immune system. Differential responses of the treatment groups to LPS will indicate the overall health and functionality of the macrophages, helping to determine the effects that chronic alcohol and glucose exposure manifest on alveolar macrophages. Results from the current experiment will be analyzed to hypothesize clinically relevant responses.
CHARACTERIZATION OF NF-¿B DEFICIENT BONE-MARROW MACROPHAGESPepper, Anthony; Fischer, Jeffrey; Department of Biological Sciences; Bradford, Jennifer; Department of Biological Sciences; Augusta University (2018-02-12)The aim of this study was tocharacterize bone-marrow derived macrophages (BMDMs) that lack canonical nuclear factor-kappaB (NF-?B) signaling. The macrophages for the study were obtained by harvesting the bone-marrow fromp65LysMCre (KO) mice and LysMCrecontrolmice.To determine NF-?B deletion efficiency, p65 (a transcription factor in the canonical pathway) protein levels were evaluated by fluorescent microscopyin bothKOand control BMDMs that had been stimulated withlipopolysaccharide(LPS).The induction ofiNOSwasmonitoredin KO and control BMDMswhenactivated by NF-?B stimulatorsIFN-?andLPS.The regulation of iNOSwas assessedby comparing macrophages that had been treated withLPS, IFN-?, or both to a control treatment under fluorescent microscopy. In addition to staining, a nitric oxide assay was employed to help determine the extent of iNOS activity.The macrophages were also visualized under light microcopyby comparing macrophagesthat were stimulated with LPS andIFN-?tounstimulated cellsusing fluorescence microscopy.Currently,a caspase assay is in progress to help further evaluate the effects of p65 losswithin macrophages.