Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Autoimmune Valvular Carditis Requires Endothelial Cell TNFR1 Expression.

BACKGROUND: Inflammation is a key driver of cardiovascular pathology, and many systemic autoimmune/rheumatic diseases are accompanied by increased cardiac risk. In the K/B.g7 mouse model of coexisting systemic autoantibody-mediated arthritis and valvular carditis, valve inflammation depends on macrophage production of TNF (tumor necrosis factor) and IL-6 (interleukin-6). Here, we sought to determine if other canonical inflammatory pathways participate and to determine whether TNF signaling through TNFR1 (tumor necrosis factor receptor 1) on endothelial cells is required for valvular carditis. METHODS: We first asked if type 1, 2, or 3 inflammatory cytokine systems (typified by IFNγ, IL-4, and IL-17, respectively) were critical for valvular carditis in K/B.g7 mice, using a combination of in vivo monoclonal antibody blockade and targeted genetic ablation studies. To define the key cellular targets of TNF, we conditionally deleted its main proinflammatory receptor, TNFR1, in endothelial cells. We analyzed how the absence of endothelial cell TNFR1 affected valve inflammation, lymphangiogenesis, and the expression of proinflammatory genes and molecules. RESULTS: We found that typical type 1, 2, and 3 inflammatory cytokine systems were not required for valvular carditis, apart from a known initial requirement of IL-4 for autoantibody production. Despite expression of TNFR1 on a wide variety of cell types in the cardiac valve, deleting TNFR1 specifically on endothelial cells protected K/B.g7 mice from valvular carditis. This protection was accompanied by reduced expression of VCAM-1 (vascular cell adhesion molecule), fewer valve-infiltrating macrophages, reduced pathogenic lymphangiogenesis, and diminished proinflammatory gene expression. CONCLUSIONS: TNF and IL-6 are the main cytokines driving valvular carditis in K/B.g7 mice. The interaction of TNF with TNFR1 specifically on endothelial cells promotes cardiovascular pathology in the setting of systemic autoimmune/rheumatic disease, suggesting that therapeutic targeting of the TNF:TNFR1 interaction could be beneficial in this clinical context.

Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) orchestrates a suppressive tumor microenvironment that fosters immunotherapy resistance. Tumor-associated macrophages (TAMs) are the principal immune cell infiltrating PDA and are heterogeneous. Here, by employing macrophage fate-mapping approaches and single-cell RNA sequencing, we show that monocytes give rise to most macrophage subsets in PDA. Tumor-specific CD4, but not CD8, T cells promote monocyte differentiation into MHCIIhi anti-tumor macrophages. By conditional major histocompatibility complex (MHC) class II deletion on monocyte-derived macrophages, we show that tumor antigen presentation is required for instructing monocyte differentiation into anti-tumor macrophages, promoting Th1 cells, abrogating Treg cells, and mitigating CD8 T cell exhaustion. Non-redundant IFNγ and CD40 promote MHCIIhi anti-tumor macrophages. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from that of tissue-resident macrophages following loss of macrophage MHC class II or tumor-specific CD4 T cells. Thus, tumor antigen presentation by macrophages to CD4 T cells dictates TAM fate and is a major determinant of macrophage heterogeneity in cancer.

Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis.

Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

Despite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Brown adipose tissue monocytes support tissue expansion.

Monocytes are part of the mononuclear phagocytic system. Monocytes play a central role during inflammatory conditions and a better understanding of their dynamics might open therapeutic opportunities. In the present study, we focused on the characterization and impact of monocytes on brown adipose tissue (BAT) functions during tissue remodeling. Single-cell RNA sequencing analysis of BAT immune cells uncovered a large diversity in monocyte and macrophage populations. Fate-mapping experiments demonstrated that the BAT macrophage pool requires constant replenishment from monocytes. Using a genetic model of BAT expansion, we found that brown fat monocyte numbers were selectively increased in this scenario. This observation was confirmed using a CCR2-binding radiotracer and positron emission tomography. Importantly, in line with their tissue recruitment, blood monocyte counts were decreased while bone marrow hematopoiesis was not affected. Monocyte depletion prevented brown adipose tissue expansion and altered its architecture. Podoplanin engagement is strictly required for BAT expansion. Together, these data redefine the diversity of immune cells in the BAT and emphasize the role of monocyte recruitment for tissue remodeling.