Chronic bacterial infections are caused by pathogens that persist within their hosts and avoid clearance by the immune system. Treatment and/or detection of such pathogens is difficult, and the resulting pathologies are often deleterious or fatal. There is an urgent need to develop protective vaccines and host-directed therapies that synergize with antibiotics to prevent pathogen persistence and infection-associated pathologies. However, many persistent pathogens, such as Mycobacterium tuberculosis, actively target the very host pathways activated by vaccination. These immune evasion tactics blunt the effectiveness of immunization strategies and are impeding progress to control these infections throughout the world. Therefore, it is essential that M. tuberculosis immune evasion-related pathogen virulence strategies are considered to maximize the effectiveness of potential new treatments. In this review, we focus on how Mycobacterium tuberculosis infects antigen-presenting cells and evades effective immune clearance by the adaptive response through (i) manipulating antigen presentation, (ii) repressing T cell-activating costimulatory molecules, and (iii) inducing ligands that drive T cell exhaustion. In this context, we will examine the challenges that bacterial virulence strategies pose to developing new vaccines. We will then discuss new approaches that will help dissect M. tuberculosis immune evasion mechanisms and devise strategies to bypass them to promote long-term protection and prevent disease progression.
There is an obvious need in the architecture, engineering, and construction (AEC) industry for improved project team integration through project delivery to ensure improved project outcomes. The literature reports that, among other methods, project partnering, when followed successfully, provides a great opportunity to improve project performance via improved collaboration among key project stakeholders (e.g., owner, designer, contractor) and reduce claims as a result while letting all project members stay in their traditional roles and work under any contractual framework, including design‐bid‐build. Despite its potential and history in the United States since the late 1980s and being classified as one of the best practices by the Construction Industry Institute in 1996, partnering continues to be underutilized. Existing research on partnering is mostly limited to public projects such as mega roadway and bridge projects. Guided by the literature, the aim of this research is to understand and report barriers to project partnering in the United States from both vertical/horizontal and public/private construction sectors. Via a comprehensive literature review, followed by a Delphi survey of partnering experts, this study systematically classified barriers to project partnering. In study results, implementation barriers to partnering during project delivery are more frequently pronounced than the barriers to its adoption. Of the top reported barriers to project partnering, the majority are cultural; project team related barriers show the greatest area of potential for improvement; and contrary to the literature, none is legislative. The study contributes to the body of knowledge by drawing attention to project delivery and management practices in the AEC industry to improve team collaboration and chances of successful implementation and adoption of integrative practices.
Alveolar macrophages (AMs) are tissue-resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. Although the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics, as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. In this study, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGF-b. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.
Alveolar macrophages (AMs) are a critical element of the innate immune response to inhaled agents, yet functional and genetic studies of this unique macrophage population are lacking. Current strategies to obtain large quantities of AMs are cumbersome and inefficient. This is due largely to both the high cost of time and resources involved in the extraction of AMs and the inability to effectively culture AMs ex vivo for extended periods of time. While bone marrow derived macrophages (BMDMs) are modeled in numerous immortalized cell lines, AMs currently lack an acceptable model that can be used in vitro. Recently, self-replicating cells derived from the fetal mouse liver, termed “MPI” cells, have been shown to possess AM-like characteristics. Here, we show that early after isolation, these cells are SiglecFhi, Cd11chi, and Cd14low, while also expressing high levels of Pparg, Marco, Itgax, and Car4, akin to AMs. Additionally, like AMs, MPI cells effectively efferocytose dead cell debris and phagocytose silica particles. While these cells lose their “AM-likeness” over time, addition of the cytokine TGF-β dramatically delays this shift away from the AM-like phenotype. Gene expression analysis shows that in contrast to cells treated with TGF-β, untreated MPI cells cease expressing Tgfbr1, the receptor for TGF-β, concurrent with the shift away from the AM-phenotype. Further, these cells are amenable to viral transduction, and we have successfully employed CRISPR/Cas9 targeted genetic editing in MPI cells. These findings further our understanding of MPI cells as an accessible and genetically tractable model for AMs that allow for long-term and large-scale studies that are not possible with AMs isolated ex vivo.
Alveolar macrophages (AMs) are a tissue resident macrophage population within the lung that are critical to maintain lung homeostasis and respond to inhaled antigens. Mechanistic investigations of AM function employing genetic approaches like CRISPR-Cas9 are hindered by the inability to obtain large numbers or maintain the AM-likeness of these cells ex vivo. We recently developed fetal liver-derived alveolar-like macrophages (FLAMs), a model that maintains the expression profile and immune functions of AMs long-term ex vivo. Importantly, FLAMs are genetically tractable using CRISPR-Cas9, enabling previously impossible interrogation of AM functions. Leveraging this innovative tool, we generated a genome-wide knockout FLAM library and are now completing forward genetic screens to dissect the regulation of AM maintenance and function. One such screen identified genes that regulate the surface expression of the AM-specific protein, Siglec-F. Among the numerous AM-associated pathways we identified as positive regulators of Siglec-F, the most robust signature was for genes related to the peroxisome. Thus, we hypothesize that the peroxisome is an essential signaling platform for AM functions. To test this prediction further we are using chemical and genetic approaches to dissect peroxisome functions in AMs. We are examining how peroxisomes modulate gene expression and metabolic flux in AMs and determining how this alters AM responses to stimuli. Together, these results highlight the utility of our novel genetic screening platform in FLAMs to unlock our understanding of AM functional mechanisms like never before. This work was supported by the Rackham Research Endowment Award from Michigan State University.
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