Invasive fungal disease (IFD) can be a severe treatment complication in patients with myeloid malignancies, but current risk models do not incorporate disease-specific factors, such as somatic gene mutations. Germline GATA2 deficiency is associated with a susceptibility to IFD. To determine whether myeloid gene mutations were associated with IFD risk, we identified 2 complementary cohorts of patients with myeloid malignancy, based on (1) the diagnosis of invasive aspergillosis (IA), or (2) the presence of GATA2 mutations identified during standard clinical sequencing. We found somatic GATA2 mutations in 5 of 27 consecutive patients who had myeloid malignancy and developed IA. Among 51 consecutive patients with GATA2 mutations identified in the evaluation of myeloid malignancy, we found that IFD was diagnosed and treated in 21 (41%), all of whom had received chemotherapy or had undergone an allogeneic stem cell transplant. Pulmonary infections and disseminated candidiasis were most common. The 90-day mortality was 52% among patients with IFD. Our results indicate that patients with somatic GATA2 mutations are a vulnerable subgroup of patients with myeloid malignancy who have high risk for treatment-associated IFD and suggest that a focused approach to antifungal prophylaxis be considered.

Atherosclerotic plaque formation is driven by the continued expansion of cholesterol loaded foamy macrophages within the arterial intima. Foamy macrophages are primarily derived from newly recruited monocytes, but factors regulating monocyte specification toward foamy macrophage differentiation and prolonged survival in plaque remain poorly understood. We used trajectory analysis of integrated single cell RNA-seq data, along with a genome-wide CRISPR screening approach to identify Triggering Receptor Expressed on Myeloid Cells 2 (Trem2) as a candidate regulator for foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up additional oxidized low density lipoprotein (LDL) cholesterol in vitro. Competitive chimera experiments showed that Trem2-deficient macrophages were less competent to form foamy macrophages when competed against Trem2-sufficient macrophages in vivo. In addition, myeloid specific conditional deletion of Trem2 resulted in a dramatic attenuation of plaque progression, even when targeted in established atherosclerotic lesions. This was independent of changes in circulating inflammatory cytokines, monocyte recruitment, or serum cholesterol levels, but due to a reduction in plaque macrophage proliferation and enhanced cell death. Mechanistically, we link Trem2-deficient macrophages with an inability for cells to sense cholesterol loading and failure to upregulate efflux molecules. Accumulation of cholesterol in the endoplasmic reticulum enhanced activation of the ER-stress response that increased susceptibility for cholesterol-toxicity and cell death in foamy Trem2-deficient macrophages. Overall, this study identifies Trem2 as a regulator of foamy macrophage differentiation, atherosclerotic plaque growth, and as a putative therapeutic target for future intervention studies.

COVID-19 outcomes are linked to host immune responses and may be impacted by antiviral therapy. We investigated antibody and cytokine responses in ACTT-1 study participants enrolled at our center. We studied serum specimens from 19 hospitalized adults with COVID-19 randomized to treatment with remdesivir or placebo. We assessed SARS-CoV-2 antibody responses and identified cytokine signatures using hierarchical clustering. We identified no clear immunologic trends attributable to remdesivir treatment. Seven subjects were initially seronegative at study enrollment, and all four deaths occurred in this group with more recent symptom onset. We identified three dominant cytokine signatures, demonstrating different disease trajectories.

Atherosclerosis is driven by the accumulation of lipid in artery walls, leading to plaque formation. Plaque expansion is driven by monocyte infiltration where they differentiate into heterogeneous macrophage populations, including foamy macrophages. To understand genes that regulate monocyte differentiation toward foamy macrophages, we performed pseudotime analysis of murine scRNA-seq from atherosclerotic plaques. This analysis identified Trem2, a lipid sensor expressed on myeloid cells, as a potential regulator of foamy cell formation. These results were further supported by an in vitro genome-wide crispr screen performed on oxLDL-treated foamy macrophages, which identified Trem2 as a candidate regulator for oxLDL uptake. To test the role of macrophage Trem2 in atherosclerotic plaque formation, we generated Trem2 fl/fl CX3CR1 CreER LDLR -/- mice that delete Trem2 only in macrophages upon tamoxifen treatment (TAM). TAM enriched Western Diet (WD) feeding for 8 weeks lead to significantly decreased plaque formation in the aortic arch and aortic sinus of Trem-deficient mice compared to litermate control. Importantly, Trem2flox animals displayed comparable serum cholesterol levels to control mice following WD feeding. Finally, to determine whether Trem2 is required for foamy macrophage formation in vivo, we performed mixed bone marrow chimera of 50% Trem2 -/- marrow and 50% control marrow into Ldlr -/- recipients fed with western diet. Plaque associated Trem2-deficient macrophages failed to develop into foamy macrophages when in competition with WT macrophages, despite normal ratios of circulating monocytes. Overall, this data identifies Trem2 as a novel regulator of foamy macrophage formation and atherosclerotic plaque growth that could be targeted therapeutically to treat CVD.

OBJECTIVES/GOALS: Phagocytes, diverse cells that ingest material, are the primary cell type infected by Mycobacterium tuberculosis (Mtb) and the executors of protective mechanisms. T cells play a critical role by helping phagocytes control the infection. Understanding the precise T cell-dependent mechanisms by which phagocytic cell types contain Mtb is critical. METHODS/STUDY POPULATION: To determine the impact T cells have on different phagocyte cell populations’ host defense mechanisms, groups of wild–type and T cell deficient TCRa-/- mice were infected with an Mtb strain expressing fluorescent mScarlet protein. At four weeks post-infection, a time when T cell help contributes to control of Mtb, lungs were homogenized and cells sorted based on detection of mScarlet, indicating Mtb-infected cells. Cell suspensions from each mouse background were underwent single-cell RNA sequencing analysis to reveal the heterogenous cellular transcriptional response of different phagocyte populations. RESULTS/ANTICIPATED RESULTS: We found that Mtb-infected phagocytes from wild-type and TCRa-/- mouse lungs contain the same dominant cell phenotypic clusters, but these have different patterns of gene expression. Without T cells, phagocytes are prone to a more inflammatory phenotype. DISCUSSION/SIGNIFICANCE: This will translate fundamental biological data to test the hypothesis that Mtb encounters different environmental stresses exerted by different phagocytic cell types. This work could reveal host intracellular niches that enable bacterial persistence and elucidate new pathways that could be targeted for traditional antibiotic therapies for TB.