Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item THE ENDOGENOUS REGULATION OF THE HUMAN MACROPHAGE ACTIVATION RESPONSE(2020) Hamidzadeh, Kajal; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are innate immune cells that participate in host defense to invading pathogens. They are powerful producers of cytokines and inflammatory mediators due to their efficient recognition of pathogen associated molecular patterns (PAMPs) via toll like receptors (TLRs). We and others have shown that the activation response to PAMPs is transient. In the present work, we demonstrate that stimulated macrophages produce adenosine and prostaglandin E2, which function as regulators of the macrophage activation response. Macrophages also upregulate receptors for these regulators to terminate inflammation and promote wound healing. We performed high throughput RNA sequencing to characterize the transcriptomes of human monocyte-derived macrophages in response to stimulation with LPS + Adenosine or LPS + PGE2. These cells exhibited a decrease in inflammatory transcripts and an increase in transcripts associated with cell growth and repair when compared to cells stimulated in the absence of these regulators. Macrophages can be generated from precursor cells in response to two different growth factors; M-CSF (macrophage colony stimulating factor) and GM-CSF (granulocyte-macrophage colony stimulating factor). M-CSF is expressed constitutively in a variety of tissues, while GM-CSF is expressed primarily in the lung, but can be induced in other tissues under inflammatory conditions. We demonstrate that human macrophages differentiated in M-CSF readily adopt an anti-inflammatory, growth promoting phenotype in response to LPS + Adenosine or LPS + PGE2, while macrophages differentiated in GM-CSF do not. This observation suggests that M-CSF derived human macrophages may be better able to alter their activation state in response to surrounding signals in order to maintain homeostasis. GM-CSF derived macrophages, in contrast, may undergo a more prominent activation response that is associated with inflammation and tissue destruction due to their inability to efficiently respond to resolving molecules.Item Transcriptomic profiling of Leishmania parasites and host macrophages during an infection(2015) Dillon, Laura Anne Liefer; El-Sayed, Najib M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leishmania parasites cause leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The parasite's life cycle is divided between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must avoid being killed by the innate and adaptive immune responses. We performed transcriptomic profiling using RNA-seq to simultaneously identify global changes in gene expression in Leishmania parasites across multiple lifecycle stages and in infected macrophages from both murine and human hosts. Using a novel approach based on a dual statistical test to identify genes that were differentially expressed relative to both uninfected macrophages and macrophages that had ingested inert particles, we were able to filter out genes that were differentially regulated as part of a general phagocytic response and thereby select genes specific to Leishmania infection. The most substantial and dynamic Leishmania-specific differential expression responses were observed during early infection, while changes observed later were common to phagocytosis more generally. An evaluation of RNA processing events within the parasite revealed precise UTR boundaries for a majority of genes and widespread alternative trans-splicing and polyadenylation. Collection of data from multiple biological replicates, the use of matched host control samples, careful statistical analysis of variation, and removal of batch effects enabled the detection of biological differences between samples and timepoints with high confidence and sensitivity. Pathway and gene ontology analyses provided insights into the higher level processes activated across parasite developmental stages and during intracellular infection to reveal signatures of Leishmania differentiation and infection.Item THE IDENTIFICATION AND CHARACTERIZATION OF AN INTRINSIC CD39/A2R-BASED REGULATORY MECHANISM THAT GOVERNS MACROPHAGE ACTIVATION RESPONSES(2014) Cohen, Heather Bloom; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are acutely sensitive to changes within their environment and can readily develop into a variety of activation states important for both the progression and resolution of inflammation. In response to immunological threats, macrophages must be able to effectively clear infections without sacrificing the integrity of the affected tissue. Thus, these cells must successfully balance their activation responses in order to preserve tissue function and the overall health of the host. The failure to properly regulate macrophage activation responses often manifests in the clinic in a variety of disease scenarios including sepsis, chronic inflammatory disorders, and cutaneous Leishmaniasis. While many factors that drive the initiation of macrophage activation are known, it remains unclear what governs the transition to an immunosuppressive state. This study reveals that macrophages can control their own activation status through the coordination between the ecto-ATPase, CD39, and the adenosine 2a and 2b receptors (A2Rs). The first part of this work shows that soon after toll-like receptor (TLR) stimulation, macrophages secrete and convert ATP into immunosuppressive adenosine via CD39. Moreover, we show that CD39 on macrophages is necessary to induce regulatory macrophage development and prevent severe immunopathology in a mouse model of septic shock. The next sets of data demonstrate that TLR activation also enhances A2bR expression, thus completing the CD39-initiated autoregulatory circuit to limit inflammatory macrophage responses. The second part of this work demonstrates that the chronic inflammatory disease-asociated cytokine, IFN-gamma, prevents TLR-induced A2bR expression and consequently promotes the hyper-production of inflammatory cytokines by macrophages thereby revealing a novel mechanism by which IFN-gamma; maintains overactive macrophages. The final chapter illustrates that while the A2bR is the dominant adenosine receptor mediating the inhibition of inflammatory cytokine production, A2aR signaling inhibits nitric oxide generation and that its expression may be hijacked by intracellular parasites to evade innate host defense mechanisms. Thus, this study demonstrates that inflammatory macrophage activation is inherently transient and that macrophages can reprogram themselves. These results culminate in the discovery of a novel immunomodulatory mechanism reliant on macrophage purinergic signaling and offer new targets and strategies to more effectively treat myriad inflammatory and infectious diseases.