Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells.

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

Steatosis drives monocyte-derived macrophage accumulation in human metabolic dysfunction-associated fatty liver disease.

Background & Aims: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common complication of obesity with a hallmark feature of hepatic steatosis. Recent data from animal models of MAFLD have demonstrated substantial changes in macrophage composition in the fatty liver. In humans, the relationship between liver macrophage heterogeneity and liver steatosis is less clear. Methods: Liver tissue from 21 participants was collected at time of bariatric surgery and analysed using flow cytometry, immunofluorescence, and H&E microscopy. Single-cell RNA sequencing was also conducted on a subset of samples (n = 3). Intrahepatic triglyceride content was assessed via MRI and tissue histology. Mouse models of hepatic steatosis were used to investigate observations made from human liver tissue. Results: We observed variable degrees of liver steatosis with minimal fibrosis in our participants. Single-cell RNA sequencing revealed four macrophage clusters that exist in the human fatty liver encompassing Kupffer cells and monocyte-derived macrophages (MdMs). The genes expressed in these macrophage subsets were similar to those observed in mouse models of MAFLD. Hepatic CD14+ monocyte/macrophage number correlated with the degree of steatosis. Using mouse models of early liver steatosis, we demonstrate that recruitment of MdMs precedes Kupffer cell loss and liver damage. Electron microscopy of isolated macrophages revealed increased lipid accumulation in MdMs, and ex vivo lipid transfer experiments suggested that MdMs may serve a distinct role in lipid uptake during MAFLD. Conclusions: The human liver in MAFLD contains macrophage subsets that align well with those that appear in mouse models of fatty liver disease. Recruited myeloid cells correlate well with the degree of liver steatosis in humans. MdMs appear to participate in lipid uptake during early stages of MALFD. Impact and implications: Metabolic dysfunction associated fatty liver disease (MAFLD) is extremely common; however, the early inflammatory responses that occur in human disease are not well understood. In this study, we investigated macrophage heterogeneity in human livers during early MAFLD and demonstrated that similar shifts in macrophage subsets occur in human disease that are similar to those seen in preclinical models. These findings are important as they establish a translational link between mouse and human models of disease, which is important for the development and testing of new therapeutic approaches for MAFLD.

Accessory fimbrial subunits and PPAD are necessary for TLR2 activation by Porphyromonas gingivalis.

Porphyromonas gingivalis is an oral pathogen that promotes dysbiosis by quenching the bactericidal activity of the host immune system while maintaining chronic inflammation, leading to periodontitis. This involves the secretion of virulence factors such as P. gingivalis peptidyl arginine deiminase (PPAD), which converts the C-terminal Arg residues of bacterial and host-derived proteins and peptides into citrulline. We have previously shown that PPAD activity and major fimbriae (containing FimA) are necessary for P. gingivalis to activate Toll-like receptor 2 (TLR2). TLR2 is an important component of the innate immune system and plays a predominant role in the recognition of P. gingivalis by host cells. Here, we extend those findings to show that P. gingivalis strains deficient for PPAD and fimbriae induced almost identical transcriptional profiles in infected primary human gingival fibroblasts (PHGFs), but these differed substantially from the transcriptome elicited by the wild-type ATCC 33277 strain. Apparently, PPAD-modified fimbriae trigger the host cell response to P. gingivalis, as confirmed by showing that the proinflammatory host cell response mediated by TLR2 is dependent on PPAD activity and the presence of fimbriae, with type I fimbriae as the most potent TLR2 activators. We also found that PPAD-modified accessory fimbrial subunits (FimC, FimD, and FimE) alone or in combination are TLR2 ligands in a reporter cell line. Although FimA polymerization to form the fimbrial shaft was not required for TLR2 activation, the secretion and proteolytic maturation of FimA were necessary for signaling by accessory Fim proteins. This was supported by showing that the proinflammatory activation of PHGFs is dependent on PPAD and accessory fimbrial subunits. We conclude that accessory fimbrial subunits are modified by PPAD and stimulate the response to P. gingivalis infection in a TLR2-dependent manner.

Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Transcriptomic atlas and interaction networks of brain cells in mouse CNS demyelination and remyelination.

Demyelination is a hallmark of multiple sclerosis, leukoencephalopathies, cerebral vasculopathies, and several neurodegenerative diseases. The cuprizone mouse model is widely used to simulate demyelination and remyelination occurring in these diseases. Here, we present a high-resolution single-nucleus RNA sequencing (snRNA-seq) analysis of gene expression changes across all brain cells in this model. We define demyelination-associated oligodendrocytes (DOLs) and remyelination-associated MAFBhi microglia, as well as astrocytes and vascular cells with signatures of altered metabolism, oxidative stress, and interferon response. Furthermore, snRNA-seq provides insights into how brain cell types connect and interact, defining complex circuitries that impact demyelination and remyelination. As an explicative example, perturbation of microglia caused by TREM2 deficiency indirectly impairs the induction of DOLs. Altogether, this study provides a rich resource for future studies investigating mechanisms underlying demyelinating diseases.

Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction.

Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 improves CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Summary: Chronic stimulation of CARs containing the 41BB costimulatory domain leads to a novel state of T cell dysfunction that is distinct from T cell exhaustion.