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Gilliland, H. N.

Publications and source records attributed to Gilliland, H. N..

2 recordsLinked to original sources

Differential activation of NF-κB and HIF-1α between alveolar-like macrophages and myeloid-derived macrophages drive inflammatory differences following Mycobacterium abscessus infection.

Pulmonary infections caused by Mycobacterium abscessus (Mab), a rapidly growing nontuberculous mycobacterium (NTM), are on the rise in patients with chronic or acquired lung disease. In contrast to immunocompetent individuals, these patient cohorts exhibit abnormal pulmonary function that result from chronic inflammation and mucus build-up. Treatment regimens rely on multi-drug cocktails yet Mabs natural recalcitrance to common antibiotics extends treatment timelines and increases the frequency of treatment failures. Thus, it is important to understand the mechanisms by which immunocompetent individuals clear Mab with relative ease while susceptible individuals do not. In the lungs, macrophages are the first immune cell Mab encounters following infection, with both resident alveolar macrophages and recruited myeloid derived macrophages playing important roles during infection control. However, the specific role of these distinct macrophage populations in regulating control and inflammatory responses during Mab remains limited due to a lack of ex vivo models that recapitulate the functions of different macrophage subsets. Here, we leverage a fetal-liver derived alveolar macrophage (FLAM) model to define early inflammatory responses occurring at the Mab-macrophage interface compared to bone-marrow derived macrophages (BMDMs). Even though both FLAMs and BMDMs similarly control intracellular Mab, the inflammatory response between these macrophage populations is significantly different. BMDMs robustly activated NF-{kappa}B transcriptional targets that include important chemokines and inflammatory cytokines like TNF, FLAMs transiently induced these genes following Mab infection. While activation of FLAMs or BMDMs with IFN{gamma} prior to Mab infection did not alter Mab intracellular dynamics, it did drive FLAMs to be more inflammatory, yet important differences remained compared to BMDMs, including the lower expression of the inducible nitric oxide synthase. This was reversed with chemical activation of HIF1. We conclude that FLAMs and BMDMs differentially respond to Mab infection due to differences in signaling networks activated following innate immune sensing, with FLAMs being more hypoinflammatory than BMDMs. More broadly our results highlight a need to better understand the initial interactions with Mab and distinct macrophage populations to define pathways that contribute to pulmonary protection or disease.

immunology↗

A genome-wide screen in macrophages defines host genes regulating the uptake of Mycobacterium abscessus.

The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies. Mycobacterium abscessus (Mab) is a highly antibiotic resistant, rapidly growing non-tuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3 and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but not Escherichia coli or latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease. IMPORTANCEPathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given that M. abscessus is broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required for M. abscessus uptake. We identified new macrophage uptake regulators during M. abscessus infection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions during M. abscessus infection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.

microbiology↗