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
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.
THE ROLE OF CXCL10 AND NF-KB SIGNALING IN MACROPHAGE INFLUENCE ON BREAST CANCER INVASIONMikulsky, Emilee; Department of Biological Sciences; Bradford, Jennifer; Augusta University (2019-02-13)Breast cancer is the second most deadly cancer with more than 260,000 people being diagnosed, and over 40,900 dying from it annually in the United States. This project focuses on triple negative breast cancer (TNBC), which is very aggressive due to the lack of hormone receptors. TNBC is characterized by an expansive stromal compartment that contains a large percentage of immune system macrophages, which correlates with poor patient prognosis. The Bradford lab has identified that the chemokine CXCL10 was found to be decreased at the mRNA and protein levels in TNBC cells when co-cultured with macrophages. The loss of CXCL10 might result in less destruction of tumors. To better understand the decrease in CXCL10, a cell invasion assay investigating invasion ability of the MDA-MB231 TNBC cell line was completed using macrophage conditioned media, with and without recombinant CXCL10, or a CXCL10 neutralizing antibody. We also investigated whether the NF-kappaB signaling pathway was involved by using primary bone marrow-derived macrophages from a NF-kappaB knockout mouse. The cell invasion assay showed that altering CXCL10 and NF-kappaB signaling in macrophages and/or the MDA-MB231 cells leads to differences in invasion ability.