Mechanosensitive mTORC1 signaling maintains lymphatic valves.

Homeostatic maintenance and repair of lymphatic vessels are essential for health. We investigated the dynamics and the molecular mechanisms of lymphatic endothelial cell (LEC) renewal in adult mesenteric quiescent lymphatic vasculature using label-retention, lineage tracing, and cell ablation strategies. Unlike during development, adult LEC turnover and proliferation was confined to the valve regions of collecting vessels, with valve cells displaying the shortest lifespan. Proliferating valve sinus LECs were the main source for maintenance and repair of lymphatic valves. We identified mechanistic target of rapamycin complex 1 (mTORC1) as a mechanoresponsive pathway activated by fluid shear stress in LECs. Depending on the shear stress level, mTORC1 activity drives division of valve cells or dictates their mechanic resilience through increased protein synthesis. Overactivation of lymphatic mTORC1 in vivo promoted supernumerary valve formation. Our work provides insights into the molecular mechanisms of maintenance of healthy lymphatic vascular system.

Lessons of Vascular Specialization From Secondary Lymphoid Organ Lymphatic Endothelial Cells.

Secondary lymphoid organs, such as lymph nodes, harbor highly specialized and compartmentalized niches. These niches are optimized to facilitate the encounter of naive lymphocytes with antigens and antigen-presenting cells, enabling optimal generation of adaptive immune responses. Lymphatic vessels of lymphoid organs are uniquely specialized to perform a staggering variety of tasks. These include antigen presentation, directing the trafficking of immune cells but also modulating immune cell activation and providing factors for their survival. Recent studies have provided insights into the molecular basis of such specialization, opening avenues for better understanding the mechanisms of immune-vascular interactions and their applications. Such knowledge is essential for designing better treatments for human diseases given the central role of the immune system in infection, aging, tissue regeneration and repair. In addition, principles established in studies of lymphoid organ lymphatic vessel functions and organization may be applied to guide our understanding of specialization of vascular beds in other organs.

Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis.

The vasculature is a key regulator of leukocyte trafficking into the central nervous system (CNS) during inflammatory diseases including multiple sclerosis (MS). However, the impact of endothelial-derived factors on CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream of Cholesterol-25-hydroxylase (Ch25h), promote neuroinflammation but their functions in the CNS are not well-understood. Using floxed-reporter Ch25h knock-in mice, we trace Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrate that Ch25h ablation specifically from ECs attenuates experimental autoimmune encephalomyelitis (EAE). Mechanistically, inflamed Ch25h-deficient CNS ECs display altered lipid metabolism favoring polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) expansion, which suppresses encephalitogenic T lymphocyte proliferation. Additionally, endothelial Ch25h-deficiency combined with immature neutrophil mobilization into the blood circulation nearly completely protects mice from EAE. Our findings reveal a central role for CNS endothelial Ch25h in promoting neuroinflammation by inhibiting the expansion of immunosuppressive myeloid cell populations.

c-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling. Maf inactivation perturbed the villus zonation program by increasing carbohydrate-related transcripts while suppressing transcripts linked to amino-acid and lipid absorption. The formation of cytoplasmic lipid droplets, shuttling dietary fat to chylomicrons, was impaired upon Maf loss indicating its role in dietary lipid handling. Maf inactivation under homeostatic conditions expanded tuft cells and led to compensatory gut lengthening, preventing weight loss. However, delayed Maf-/- enterocyte maturation impaired weight recovery after acute intestinal injury, resulting in reduced survival. Our results identify c-MAF as a regulator of the intestinal villus zonation program, while highlighting the importance of coordination between stem/progenitor and differentiation programs for intestinal regeneration.

In and out: Leishmania metastasis by hijacking lymphatic system and migrating immune cells.

The lymphatic system plays a crucial role in mounting immune response against intracellular pathogens, and recent studies have documented its role in facilitating tumor dissemination linked largely with cancer cells. However, in mucocutaneous leishmaniasis (MCL) caused by Leishmania Viannia subgenus showing infectious metastasis and resulting in severe distant secondary lesions, the route of escape of these parasites to secondary sites has not yet been investigated in detail. Our results demonstrated that when infection was associated with inflammation and additionally exacerbated by the presence of dsRNA viral endosymbiont (LRV1), lymphatic vessels could serve as efficient routes for infected cells to egress from the primary site and colonize distant organs. We challenged this hypothesis by using the intracellular Leishmania protozoan parasites Leishmania guyanensis (Lgy) associated with or without a dsRNA viral endosymbiont, exacerbating the infection and responsible for a strong inflammatory response, and favoring metastasis of the infection. We analyzed possible cargo cells and the routes of dissemination through flow cytometry, histological analysis, and in vivo imaging in our metastatic model to show that parasites disseminated not only intracellularly but also as free extracellular parasites using migrating immune cells, lymph nodes (LNs), and lymph vessels, and followed intricate connections of draining and non-draining lymph node to finally end up in the blood and in distant skin, causing new lesions.

ADAMTS18+ villus tip telocytes maintain a polarized VEGFA signaling domain and fenestrations in nutrient-absorbing intestinal blood vessels.

The small intestinal villus tip is the first point of contact for lumen-derived substances including nutrients and microbial products. Electron microscopy studies from the early 1970s uncovered unusual spatial organization of small intestinal villus tip blood vessels: their exterior, epithelial-facing side is fenestrated, while the side facing the villus stroma is non-fenestrated, covered by pericytes and harbors endothelial nuclei. Such organization optimizes the absorption process, however the molecular mechanisms maintaining this highly specialized structure remain unclear. Here we report that perivascular LGR5+ villus tip telocytes (VTTs) are necessary for maintenance of villus tip endothelial cell polarization and fenestration by sequestering VEGFA signaling. Mechanistically, unique VTT expression of the protease ADAMTS18 is necessary for VEGFA signaling sequestration through limiting fibronectin accumulation. Therefore, we propose a model in which LGR5+ ADAMTS18+ telocytes are necessary to maintain a "just-right" level and location of VEGFA signaling in intestinal villus blood vasculature to ensure on one hand the presence of sufficient endothelial fenestrae, while avoiding excessive leakiness of the vessels and destabilization of villus tip epithelial structures.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

IFN-γ-dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis.

Tumor-associated lymphatic vessels promote metastasis and regulate antitumor immune responses. Here, we assessed the impact of cytotoxic T cells on the local lymphatic vasculature and concomitant tumor dissemination during an antitumor response. Interferon-γ (IFN-γ) released by effector T cells enhanced the expression of immunosuppressive markers by tumor-associated lymphatic endothelial cells (LECs). However, at higher effector T cell densities within the tumor, T cell-based immunotherapies induced LEC apoptosis and decreased tumor lymphatic vessel density. As a consequence, lymphatic flow was impaired, and lymph node metastasis was reduced. Mechanistically, T cell-mediated tumor cell death induced the release of tumor antigens and cross-presentation by tumor LECs, resulting in antigen-specific LEC killing by T cells. When LECs lacked the IFN-γ receptor expression, LEC killing was abrogated, indicating that IFN-γ is indispensable for reducing tumor-associated lymphatic vessel density and drainage. This study provides insight into how cytotoxic T cells modulate tumor lymphatic vessels and may help to improve immunotherapeutic protocols.

Apelin-driven endothelial cell migration sustains intestinal progenitor cells and tumor growth.

Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.

The C5a-C5aR1 complement axis is essential for neutrophil recruitment to draining lymph nodes via high endothelial venules in cutaneous leishmaniasis.

Neutrophils are specialized innate immune cells known for their ability to fight pathogens. However, the mechanisms of neutrophil trafficking to lymph nodes are not fully clear. Using a murine model of dermal infection with Leishmania parasites, we observe a transient neutrophil influx in draining lymph nodes despite sustained recruitment to the infection site. Cell-tracking experiments, together with intravital two-photon microscopy, indicate that neutrophil recruitment to draining lymph nodes occurs minimally through lymphatics from the infected dermis, but mostly through blood vessels via high endothelial venules. Mechanistically, neutrophils do not respond to IL-1ß or macrophage-derived molecules. Instead, they are guided by the C5a-C5aR1 axis, using L-selectin and integrins, to extravasate into the draining lymph node parenchyma. We also report that C5, the C5a precursor, is locally produced in the draining lymph node by lymphatic endothelial cells. Our data establish and detail organ-specific mechanisms of neutrophil trafficking.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

Mechanosensitive ACKR4 scavenges CCR7 chemokines to facilitate T cell de-adhesion and passive transport by flow in inflamed afferent lymphatics.

T cell migration via afferent lymphatics to draining lymph nodes (dLNs) depends on expression of CCR7 in T cells and CCL21 in the lymphatic vasculature. Once T cells have entered lymphatic capillaries, they slowly migrate into contracting collecting vessels. Here, lymph flow picks up, inducing T cell detachment and rapid transport to the dLNs. We find that the atypical chemokine receptor 4 (ACKR4), which binds and internalizes CCL19 and CCL21, is induced by lymph flow in endothelial cells lining lymphatic collectors, enabling them to scavenge these chemokines. In the absence of ACKR4, migration of T cells to dLNs in TPA-induced inflammation is significantly reduced. While entry into capillaries is not impaired, T cells accumulate in the ACKR4-deficient dermal collecting vessel segments. Overall, our findings identify an ACKR4-mediated mechanism by which lymphatic collectors facilitate the detachment of lymph-borne T cells in inflammation and their transition from crawling to free-flow toward the dLNs.

Stem-cell-like T cells have a specific entry gate to the tumor.

Tumor-infiltrated T cells with stem-cell-like properties are important for determining the immunotherapy response. In this issue of Cancer Cell, Asrir and colleagues show that their entry requires specialized tumor-associated endothelial cells that resemble immature and inflamed lymph node vessels and that immunotherapy enhances the recruitment capacity of these endothelial cells.

The double life of TLR2.

TLR2 located in the endothelium promotes angiogenesis and inflammation.

Lef1 restricts ectopic crypt formation and tumor cell growth in intestinal adenomas.

Somatic mutations in APC or CTNNB1 genes lead to aberrant Wnt signaling and colorectal cancer (CRC) initiation and progression via-catenin­T cell factor/lymphoid enhancer binding factor TCF/LEF transcription factors. We found that Lef1 was expressed exclusively in Apc-mutant, Wnt ligand­independent tumors, but not in ligand-dependent, serrated tumors. To analyze Lef1 function in tumor development, we conditionally deleted Lef1 in intestinal stem cells of Apcfl/fl mice or broadly from the entire intestinal epithelium of Apcfl/fl or ApcMin/+ mice. Loss of Lef1 markedly increased tumor initiation and tumor cell proliferation, reduced the expression of several Wnt antagonists, and increased Myc proto-oncogene expression and formation of ectopic crypts in Apc-mutant adenomas. Our results uncover a previously unknown negative feedback mechanism in CRC, in which ectopic Lef1 expression suppresses intestinal tumorigenesis by restricting adenoma cell dedifferentiation to a crypt-progenitor phenotype and by reducing the formation of cancer stem cell niches.

Palmdelphin Regulates Nuclear Resilience to Mechanical Stress in the Endothelium.

BACKGROUND: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. METHODS: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. RESULTS: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. CONCLUSIONS: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology.

Soluble trivalent engagers redirect cytolytic T cell activity toward tumor endothelial marker 1.

Tumor endothelial marker 1 (TEM1) is an emerging cancer target with a unique dual expression profile. First, TEM1 is expressed in the stroma and neo-vasculature of many human carcinomas but is largely absent from healthy adult tissues. Second, TEM1 is expressed by tumor cells of mesenchymal origin, notably sarcoma. Here, we present two fully human anti-TEM1 single-chain variable fragment (scFv) reagents, namely, 1C1m and 7G22, that recognize distinct regions of the extracellular domain and possess substantially different affinities. In contrast to other, well-described anti-TEM1 binders, these fragments confer cytolytic activity when expressed as 2nd generation chimeric antigen receptors (CARs). Moreover, both molecules selectively redirect human T cell effector functions toward TEM1+ tumor cells when incorporated into experimental soluble bispecific trivalent engagers that we term TriloBiTEs (tBs). Furthermore, systemic delivery of 1C1m-tB prevents the establishment of Ewing sarcoma tumors in a xenograft model. Our observations confirm TEM1 as a promising target for cancer immunotherapy and illustrate the prospective translational potential of certain scFv-based reagents.

FOXC2 controls adult lymphatic endothelial specialization, function, and gut lymphatic barrier preventing multiorgan failure.

The mechanisms maintaining adult lymphatic vascular specialization throughout life and their role in coordinating inter-organ communication to sustain homeostasis remain elusive. We report that inactivation of the mechanosensitive transcription factor Foxc2 in adult lymphatic endothelium leads to a stepwise intestine-to-lung systemic failure. Foxc2 loss compromised the gut epithelial barrier, promoted dysbiosis and bacterial translocation to peripheral lymph nodes, and increased circulating levels of purine metabolites and angiopoietin-2. Commensal microbiota depletion dampened systemic pro-inflammatory cytokine levels, corrected intestinal lymphatic dysfunction, and improved survival. Foxc2 loss skewed the specialization of lymphatic endothelial subsets, leading to populations with mixed, pro-fibrotic identities and to emergence of lymph node-like endothelial cells. Our study uncovers a cross-talk between lymphatic vascular function and commensal microbiota, provides single-cell atlas of lymphatic endothelial subtypes, and reveals organ-specific and systemic effects of dysfunctional lymphatics. These effects potentially contribute to the pathogenesis of diseases, such as inflammatory bowel disease, cancer, or lymphedema.

Macrophage depletion induces edema through release of matrix-degrading proteases and proteoglycan deposition.

Colony-stimulating factor 1 receptor (CSF1R) blockade abates tumor-associated macrophage (TAM) infiltrates and provides marked clinical benefits in diffuse-type tenosynovial giant cell tumors. However, facial edema is a common adverse event associated with TAM elimination in patients. In this study, we examined molecular and cellular events associated with edema formation in mice and human patients with cancer treated with a CSF1R blocking antibody. Extended antibody treatment of mice caused marked body weight gain, an indicator of enhanced body fluid retention. This was associated with an increase of extracellular matrix-remodeling metalloproteinases (MMPs), namely MMP2 and MMP3, and enhanced deposition of hyaluronan (HA) and proteoglycans, leading to skin thickening. Discontinuation of anti-CSF1R treatment or blockade of MMP activity restored unaltered body weight and normal skin morphology in the mice. In patients, edema developed at doses well below the established optimal biological dose for emactuzumab, a CSF1R dimerization inhibitor. Patients who developed edema in response to emactuzumab had elevated HA in peripheral blood. Our findings indicate that an early increase of peripheral HA can serve as a pharmacodynamic marker for edema development and suggest potential interventions based on MMP inhibition for relieving periorbital edema in patients treated with CSF1R inhibitors.

Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels.

The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow-responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial-specific Tie2-Cre or the tamoxifen-inducible LEC-specific Prox1-CreERT2 line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow-induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel-type-specific endothelial cell identities.