• Evolution of Antigen Presenting Cells in the Spleen

      Flowers, Emily Maya; Flajnik, M. F. (Martin F.); 0000-0003-4803-3824 (2021)
      XL cells are an MHC class IIhigh, cell surface-absorbed-Ig+ population of antigen presenting cells in the B cell follicle of the amphibian spleen. XL cells bear native antigen after immunization. Previous work proposed that XL cells are “double-duty” antigen presenting cell (APC), presenting antigen to both T and B cells. In mammals these functions are generally partitioned between conventional dendritic cells (cDC) presenting to T cells and follicular dendritic cells (FDC) presenting to B cells. In this PhD I have expanded on that premise, by comparing XL cells and peritoneal macrophages via RNA sequencing, functional assays, and in vivo kinetics of antigen handling. Like XL cells, Xenopus peritoneal macrophages are an MHC-IIhigh / Ig+ population. However, peritoneal macrophages are highly adherent, esterase-positive, and phagocytic, while my data demonstrate that XL cells are weakly adherent, esterase-low/negative, and weakly phagocytic. The peritoneal macrophage transcriptome is consistent with the broad myeloid lineage, and MAFB and CEBPA expression specifically within the macrophage lineage. XL cells express higher levels of genes associated with myeloid cDCs and cDC precursors, specifically ID2, BATF3, IRF4, and CD209. When formally testing the similarity between Xenopus and mammalian cell types, XL cells were enriched for cDC genes. In vivo, XL cells retain antigen through approximately day 20 after immunization – far shorter than the FDC antigen retention. Overall XL cells transcriptionally resemble mammalian cDC but share functional genes with FDC. We posit that XL cells are the myeloid precursor to FDC function, but also represent an evolutionary precursor to dedicated cDC lineages.
    • Nanoparticle-driven Immunoengineering: Characterization of the Effects of Polymer-based Nanoparticles in Regulating Macrophage-mediated Inflammatory Responses

      Lasola, Jackline Joy Martín; Kim, Anthony J.; 0000-0002-1287-6830 (2021)
      Nanoparticles (NPs) possess the exciting potential to engineer specific immune responses during different disease states. Although NPs are typically considered for their customizability to promote controlled delivery of a therapeutic drug or biologic, their intrinsic immunomodulatory activity remains poorly understood. Our lab had previously shown that cargo-less poly(lactic acid) (PLA)-based NPs can suppress LPS-induced proinflammatory cytokine release in bone marrow macrophages (BMMΦs). The work described in this dissertation sought to understand the mechanisms by which these particles elicit their protective effects in response to LPS stimulation. It was hypothesized that PLA-based NPs impart a multimodal, immune modifying protective effect upon BMMΦ and other innate immune cells that can be fine-tuned through alterations of the NP physicochemical properties. This activity can then be harnessed to abrogate states of severe inflammation, such as that seen during LPS-induced endotoxemia and sepsis. Using biochemical and immunological assays to assess cellular and molecular interactions between NPs and BMMΦs, we show that the surfactant characteristics of the PLA-based NPs drive the kinetics of BMMΦ uptake and act to disrupt pathogen-associated molecular pattern (PAMP) interactions at the BMMΦ cell surface. In combination with NP uptake, the polymer composition of the NP contributes to abrogate inflammatory cell signaling resulting in decreased cytokine secretions in response to LPS stimulation. Additional studies of the protein corona establish that these effects are partially driven by the constituents of the protein corona in a manner dependent on the physiological state of the biofluids in which NPs are delivered. These in vitro observations culminate in the modeling of severe inflammation via murine LPS-induced endotoxemia to establish the ability of PLA-based NPs to increase survival, decrease severity of the signs of systemic inflammation, and hasten recovery following onset of endotoxemia. The significance of this study is that it formally characterizes the intrinsic immunomodulatory activity of PLA- based NPs, a well-established nanomedicine platform. This illuminates fundamental insights into the multimodal effects of polymeric NP nano-bio interactions. This promises to aid in rational design of future polymeric NPs of increasing complexity for a wide variety of immune- mediated diseases and disorders.
    • The Role of STAT1 in SARS Coronavirus Pathogenesis

      Page, Carly A.; Frieman, Matthew B. (2012)
      Infection with Severe acute respiratory syndrome Coronavirus (SARS-CoV) often caused severe end stage lung disease with characteristics of acute lung injury, especially in elderly populations. The virus-host interactions that govern this severe response are not well understood. We have previously shown that STAT1 signaling plays an important role in control of SARS-CoV pathogenesis and this control is independent of the role of STAT1 in interferon signaling. STAT1-/- mice have greater weight loss, worsened lung pathology and an altered immune response consisting of elevated Alternatively activated macrophages (AAMs) following infection with SARS-CoV. We hypothesized that STAT1 is playing a role in the polarization of the immune response, specifically in macrophages, resulting in a worsened outcome and long-term lung disease. To test this hypothesis, we first created bone marrow chimeras to determine the lineage of cells that contributes to the enhanced pathology when STAT1 is deleted. Upon histological examination, mice lacking STAT1 in cells of the hematopoietic lineage display more severe lung pathology following infection with a mouse adapted SARS-CoV (rMA15). In order to examine the contribution of the macrophage population to SARS-CoV pathogenesis, we developed a conditional knockout strain of mice utilizing the LysM promoter to delete STAT1 in monocytes and macrophages. Following infection with SARS-CoV, LysM/STAT1 mice have a delay in viral clearance and enhanced lung pathology characterized by enhanced fibroblast proliferation as well as numerous lymphoid aggregates at 9 days post-infection. This pathology is suggestive of a pre-fibrotic state, which has been observed in some human patients following SARS-CoV infection. Induction of AAMs was blocked through concurrent deletion of STAT6 with STAT1, which successfully ameliorated the enhanced disease phenotype. These double deleted mice displayed very limited pathology following infection. STAT1-/- mice were also studied at 21 days post-infection to evaluate long-term effects of SARS-CoV infection. STAT1-/- mice maintained more severe lung pathology at this late time point, displayed continued presence of AAM-associated proteins and increased levels of activated collagen. We propose that STAT1 is important in controlling macrophage activation and polarization during SARS-CoV infection in order to help limit immune mediated pathologies and long-term progression to fibrotic disease.
    • The Role of Type-2 IL-4 Receptor and its Downstream Genes in Nematode Infection

      Sun, Rex; Shea-Donohue, Terez (2015)
      Over one third of the world's population is infected with nematodes with the highest prevalence in third world countries. It is well established that nematode infection induces a highly polarized Th2 immune response in the host that contributes to nematode expulsion. IL-13, one of the major cytokines for Th2 response, shares a common receptor with IL-4 and has overlapping physiological effects. Studies with various knockouts showed important roles of IL-4 and IL-13 in both lung and gut pathologies, but has been insufficient to distinguish the effects of these two cytokines. The central hypothesis of this project is that IL-13 acting through the type 2 IL-4R leads to activation of STAT6-dependent transcription of genes that participate in the epithelial host defense against enteric pathogens. To investigate this hypothesis, we will infect mice deficient in IL-13R?1 to examine the development and maintenance of Th2 immunity. Neutrophils have been shown to play an indirect role in modulating Th2 responses in nematode infection by eliminating bacteria associated with the invading nematodes to prevent a localized Th1 up-regulation. Neutrophil elastase, one of the IL-13/STAT6 dependent genes, is a critical component in the anti-microbial activity of neutrophils and the major protease contained in neutrophil-secreted granules and neutrophil extracellular traps (NETs). The second aim of the project will examine the role of neutrophils elastase in models of enteric nematode infection and its contribution to host immunity.
    • Tumor necrosis factor-alpha posttranscriptional gene expression regulation and messenger ribonucleic acid poly(A) tail metabolism in macrophages

      Crawford, Eric Keith; Hasday, Jeffrey D. (1996)
      Tumor necrosis factor-alpha (TNF-alpha)is both essential for host survival and potentially fatal if inappropriately expressed. The extreme and divergent actions of TNF-alpha underscore the importance of understanding the mechanisms that regulate its expression. TNF-alpha expression in macrophages is regulated at the levels of transcript initiation and elongation, but transcriptional control appears to have a lesser role in TNF-alpha regulation than posttranscriptional processes. Despite ongoing basal TNF-alpha transcription, TNF-alpha messenger ribonucleic acid (mRNA) is not translated in resting macrophages, but following cell activation, translational efficiency increases several hundred fold. Most eukaryotic transcripts are 3' polyadenylated during intranuclear mRNA processing. The poly(A) tail generally stabilizes mRNA and enhances ribosome recruitment and translational efficiency. In oocytes and embryos, rapid cytoplasmic poly(A) tail shortening and elongation are used to orchestrate expression of some gene transcripts. In this work we show that in resting macrophages, cytoplasmic TNF-alpha mRNA has a markedly attenuated or absent poly(A) tail and is not associated with ribosomes. Following stimulation with lipopolysaccharide (LPS), fully adenylated and polysome associated TNF-alpha mRNA appears coincident with onset of TNF-alpha protein synthesis. Macrophages contains no detectable stores of TNF-alpha protein, yet in cells pretreated with actinomycin D to block new transcription, LPS induces a gradual increase in length of the cytoplasmic pool of hypoadenylated TNF-alpha mRNA as well as secretion of TNF-alpha protein. These data suggest that removal of the poly(A) tail blocks initiation of translation in unstimulated macrophages and LPS inactivates this process allowing synthesis of translatable polyadenylated TNF-alpha mRNA. We propose that the increase in size of cytoplasmic TNF-alpha mRNA and subsequent TNF-alpha protein secretion, in the presence of transcriptional inhibition, is due to cytoplasmic readenylation and translation of the readenylated transcript. Readenylation of preformed hypoadenylated TNF-alpha mRNA may provide the macrophage with an alternative pathway for producing small amounts of TNF-alpha.