Microbiota regulates the turnover kinetics of gut macrophages in health and inflammation

The gut immune system has evolved to co-exist in a mutually beneficial symbiotic relationship with its microflora. Here, using a germ-free fate-mapping mouse model, we provide clear insight into how the enteric commensals determine the kinetics of macrophage turnover. The microbiome density along the gastrointestinal tract defines the persistence of ontogenically diverse macrophages, with the highest numbers of the long-lived F4/80hiTim4+ macrophage subset in the less densely colonized small intestine. Furthermore, the microbiome contributes to a tightly regulated monocyte-dependent replenishment of both long- and short-lived F4/80hi macrophages under homeostatic and inflammatory conditions. In the latter situation, the commensals regulate rapid replenishment of the depleted macrophage niche caused by the intestinal inflammation. The microbial ecosystem imprints a favorable cytokine microenvironment in the intestine to support macrophage survival and monocyte-dependent replenishment. Therefore, the host immune system-commensal cross-talk provides an efficient strategy to assure intestinal homeostasis.

Spatial deconvolution of HER2-positive breast cancer delineates tumor-associated cell type interactions

In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra- and interpatient heterogeneity; from which we discover shared gene signatures for immune and tumor processes. By integration with single cell data, we spatially map tumor-associated cell types to find tertiary lymphoid-like structures, and a type I interferon response overlapping with regions of T-cell and macrophage subset colocalization. We construct a predictive model to infer presence of tertiary lymphoid-like structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define a high resolution map of cellular interactions in tumors and provide tools generalizing across tissues and diseases.

Neutrophilic Asthma Is Associated With Smoking, High Numbers of IRF5+, and Low Numbers of IL10+ Macrophages

Asthma is a heterogenous disease with different inflammatory subgroups that differ in disease severity. This disease variation is hampering treatment and development of new treatment strategies. Macrophages may contribute to asthma phenotypes by their ability to activate in different ways, i.e., T helper cell 1 (Th1)-associated, Th2-associated, or anti-inflammatory activation. It is currently unknown if these different types of activation correspond with specific inflammatory subgroups of asthma. We hypothesized that eosinophilic asthma would be characterized by having Th2-associated macrophages, whereas neutrophilic asthma would have Th1-associated macrophages and both having few anti-inflammatory macrophages. We quantified macrophage subsets in bronchial biopsies of asthma patients using interferon regulatory factor 5 (IRF5)/CD68 for Th1-associated macrophages, CD206/CD68 for Th2-associated macrophages and interleukin 10 (IL10)/CD68 for anti-inflammatory macrophages. Macrophage subset percentages were investigated in subgroups of asthma as defined by unsupervised clustering using neutrophil/eosinophil counts in sputum and tissue and forced expiratory volume in 1 s (FEV1). Asthma patients clustered into four subgroups: mixed-eosinophilic/neutrophilic, paucigranulocytic, neutrophilic with normal FEV1, and neutrophilic with low FEV1, the latter group consisting mainly of smokers. No differences were found for CD206+ macrophages within asthma subgroups. In contrast, IRF5+ macrophages were significantly higher and IL10+ macrophages lower in neutrophilic asthmatics with low FEV1 as compared to those with neutrophilic asthma and normal FEV1 or mixed-eosinophilic asthma. This study shows that neutrophilic asthma with low FEV1 is associated with high numbers of IRF5+, and low numbers of IL10+ macrophages, which may be the result of combined effects of smoking and having asthma.

Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

Cell barrier function of resident peritoneal macrophages in post-operative adhesions

Post-operative adhesions are a leading cause of abdominal surgery-associated morbidity. Exposed fibrin clots on the damaged peritoneum, in which the mesothelial barrier is disrupted, readily adhere to surrounding tissues, resulting in adhesion formation. Here we show that resident F4/80HighCD206− peritoneal macrophages promptly accumulate on the lesion and form a ‘macrophage barrier’ to shield fibrin clots in place of the lost mesothelium in mice. Depletion of this macrophage subset or blockage of CD11b impairs the macrophage barrier and exacerbates adhesions. The macrophage barrier is usually insufficient to fully preclude the adhesion formation; however, it could be augmented by IL-4-based treatment or adoptive transfer of this macrophage subset, resulting in robust prevention of adhesions. By contrast, monocyte-derived recruited peritoneal macrophages are not involved in the macrophage barrier. These results highlight a previously unidentified cell barrier function of a specific macrophage subset, also proposing an innovative approach to prevent post-operative adhesions.

EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis

Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.