Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries characterized by leukocyte accumulation in the vessel wall. Both innate and adaptive immune responses contribute to atherogenesis, but the identity of atherosclerosis-relevant antigens and the role of antigen presentation in this disease remain poorly characterized. We developed live-cell imaging of explanted aortas to compare the behavior and role of APCs in normal and atherosclerotic mice. We found that CD4 + T cells were capable of interacting with fluorescently labeled (CD11c-YFP + ) APCs in the aortic wall in the presence, but not the absence, of cognate antigen. In atherosclerosis-prone Apoe -/-CD11c-YFP + mice, APCs extensively interacted with CD4 + T cells in the aorta, leading to cell activation and proliferation as well as secretion of IFN-γ and TNF-α. These cytokines enhanced uptake of oxidized and minimally modified LDL by macrophages. We conclude that antigen presentation by APCs to CD4 + T cells in the arterial wall causes local T cell activation and production of proinflammatory cytokines, which promote atherosclerosis by maintaining chronic inflammation and inducing foam cell formation.
The power of a single engineered organism is limited by its capacity for genetic modification. To circumvent the constraints of any singular microbe, a new frontier in synthetic biology is emerging: synthetic ecology, or the engineering of microbial consortia. Here we develop communication systems for such consortia in an effort to allow for complex social behavior across different members of a community. We posit that such communities will outpace monocultures in their ability to perform complicated tasks if communication among and between members of the community is well regulated. Quorum sensing was identified as the most promising candidate for precise control of engineered microbial ecosystems, due to its large diversity and established utility in synthetic biology. Through promoter and protein modification, we engineered two quorum sensing systems (rpa and tra) to add to the extensively used lux and las systems. By testing the cross-talk between all systems, we thoroughly characterized many new inducible systems for versatile control of engineered communities. Furthermore, we’ve identified several system pairs that exhibit useful types of orthogonality. Most notably, the tra and rpa systems were shown to have neither signal crosstalk nor promoter crosstalk for each other, making them completely orthogonal in operation. Overall, by characterizing the interactions between all four systems and their components, these circuits should lend themselves to higher-level genetic circuitry for use in microbial consortia.
Microbial ecologists are increasingly turning to small, synthesized ecosystems1–5 as a reductionist tool to probe the complexity of native microbiomes6,7. Concurrently, synthetic biologists have gone from single-cell gene circuits8–11 to controlling whole populations using intercellular signaling12–16. The intersection of these fields is giving rise to new approaches in waste recycling,17 industrial fermentation18, bioremediation19, and human health16,20. These applications share a common challenge7 well known in classical ecology21,22; stability of an ecosystem cannot arise without mechanisms that prohibit the faster growing species from eliminating the slower. Here, we combine orthogonal quorum sensing systems and a population control circuit with diverse self-limiting growth dynamics in order to engineer two ‘ortholysis’ circuits capable of maintaining a stable co-culture of metabolically competitive strains in microfluidic devices. While no successful co-cultures are observed in a two-strain ecology without synthetic population control, the ‘ortholysis’ design dramatically increases the co-culture rate from 0% to approximately 80%. Agent-based and deterministic modeling reveal that our system can be adjusted to yield different dynamics, including phase-shifted, anti-phase or synchronized oscillations as well as stable steady-state population densities. The ‘ortholysis’ approach establishes a paradigm for constructing synthetic ecologies by developing stable communities of competitive microbes without the need for engineered codependency.
Atherosclerosis is an immune-mediated disease associated with lipid accumulation and formation of atherosclerotic plaques in the major arteries of the body. Vascular dendritic cells (DC) are present throughout the adventitia of the normal mouse aorta and expand under atherosclerotic conditions, but their function remains incompletely understood. To test whether DC can mediate productive antigen presentation directly in the aortic wall, we used two-photon microscopy. CD11cYFP tagged DC were motile in mouse aortas and interacted with T cells in an antigen dependent manner. In atherosclerotic Apoe-/- CD11cYFP mice, DC interacted with transferred T cells from Apoe-/-, but not C57BL/6 mice. Interaction with DC decreased T cell migration velocity and induced secretion of IFN-γ, TNF-α and IL-17, which enhanced oxLDL uptake by primary macrophages. Using flow cytometry of digested whole aorta from C57BL/6 and Apoe-/- mice fed either Western diet or regular chow diet, we found CD11b+CD11c+ and CD11b-CD11c+ DC in the aortic wall of C57BL/6 mice, and this population expanded in Apoe-/- mice on western diet. Under these conditions, CD8a+ and CD103 DC appear among the CD11c+ DC population. Taken together, our data show the emergence of distinct subsets of DC in the mouse aorta during the progression of atherosclerosis and demonstrate that the aortic wall can be a site of active antigen presentation leading to the induction of a pro-atherogenic immune response.
For risk assessment purposes, the 780 km 2 Savannah River Site (SRS) has been partitioned spatially into Integrator Operable Units (IOUs) that correspond to the watersheds of the Savannah River tributaries that drain the SRS. The streams within each watershed are "integrators" because they receive contaminants transported by surface or subsurface flow from all sources within their watersheds. Ecological receptors within the streams are exposed to these contaminants, and their health reflects the severity of contamination within the watershed. The Upper Three Runs (UTR) drainage is one of the six IOUs on the SRS. The UTR IOU includes five IOU subunits that correspond to the upper (UTR-upper), middle (UTR-middle), and lower (UTR-lower) reaches of UTR; Tinker Creek; and Tims Branch. Also included in the UTR IOU are three relatively small tributary streams: Mill Creek, McQueen Branch, and Crouch Branch. Tinker Creek, Mill Creek, and UTR-upper are largely undisturbed by SRS operations. UTR-middle, UTRlower, Tims Branch, Crouch Branch, and McQueen Branch are potentially impacted by SRS waste sites or industrial operations.Assessment of the ecological effects of contaminants in the SRS IOUs involves 1) the measurement of contaminant levels in sediment, fish, crayfish, and surface water; 2) the use of contaminant exposure models that estimate potential contaminant doses to ecological receptors; and 3) field bioassessments of the fish and invertebrate assemblages inhabiting SRS streams. The data generated by these studies provide a broad basis for a weight-of-evidence characterization of the extent and severity of contaminant related ecological impacts on SRS aquatic ecosystems.There was little evidence that metal concentrations were unusually elevated in UTR-middle and UTR-lower, both located downstream from SRS waste sites and industrial areas. Concentrations of metals in sediments and fish in these subunits were comparable to those in reference subunits that were unaffected by SRS operations. Contaminant exposure models showed that most metals in UTR-middle and UTR-lower were not present at levels high enough to harm mammals and birds, with the possible exceptions of Hg and Al in UTR-lower. However, these two metals exceeded toxicity benchmarks in reference subunits as well as UTR lower. Elevated levels of Hg in UTRlower probably resulted from the aerial deposition of Hg from non-SRS sources (as shown for other streams in the Savannah River basin) coupled with the presence of wetlands in the vicinity of UTR-lower that facilitated Hg methylation. Al exceedances may have been related to naturally high Al levels in SRS soils. Bioassessments based on fish and macroinvertebrate assemblage structure confirmed the conclusion that UTR was unimpaired by metals released by SRS operations. Two sampling methods -a multiple habitat sampling protocol and Hester-Dendy artificial substrates -showed that macroinvertebrate community structure in potentially impacted reaches of UTR was comparable or superior to community structu...
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