TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells.

Rationale: TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function. Objective: To characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics. Methods and Results: Pressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for ≥1 minute and in some instances remained for ≥4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a controlled and spatially defined single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of Trpv4 compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of Lyve1 , Cd163 , Folr2 , Mrc1 , Ccl8 , Apoe , Cd209f , Cd209d , and Cd209g ; and at least half of these macrophages also expressed Timd4. This subset of macrophages also highly expressed Txa2s , which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction. Conclusions: Clinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema.

TREM2 deficiency reprograms intestinal macrophages and microbiota to enhance anti-PD-1 tumor immunotherapy.

The gut microbiota and tumor-associated macrophages (TAMs) affect tumor responses to anti-programmed cell death protein 1 (PD-1) immune checkpoint blockade. Reprogramming TAM by either blocking or deleting the macrophage receptor triggering receptor on myeloid cells 2 (TREM2) attenuates tumor growth, and lack of functional TREM2 enhances tumor elimination by anti-PD-1. Here, we found that anti-PD-1 treatment combined with TREM2 deficiency in mice induces proinflammatory programs in intestinal macrophages and a concomitant expansion of Ruminococcus gnavus in the gut microbiota. Gavage of wild-type mice with R. gnavus enhanced anti-PD-1-mediated tumor elimination, recapitulating the effect occurring in the absence of TREM2. A proinflammatory intestinal environment coincided with expansion, increased circulation, and migration of TNF-producing CD4+ T cells to the tumor bed. Thus, TREM2 remotely controls anti-PD-1 immune checkpoint blockade through modulation of the intestinal immune environment and microbiota, with R. gnavus emerging as a potential probiotic agent for increasing responsiveness to anti-PD-1.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC) that targets the α4ß7- mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) axis. To determine the mechanisms of action of VDZ, we examined five distinct cohorts of patients with UC. A decrease in naïve B and T cells in the intestines and gut-homing (ß7+) plasmablasts in circulation of VDZ-treated patients suggested that VDZ targets gut-associated lymphoid tissue (GALT). Anti-α4ß7 blockade in wild-type and photoconvertible (KikGR) mice confirmed a loss of GALT size and cellularity because of impaired cellular entry. In VDZ-treated patients with UC, treatment responders demonstrated reduced intestinal lymphoid aggregate size and follicle organization and a reduction of ß7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcγR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated mechanism of action of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC.

Distinct roles for LTalpha3 and LTalpha1beta2 produced by B cells contribute to their multi-faceted impact on ileitis.

B lymphocytes may facilitate chronic inflammation through antibody production or secretion of cytokines, including lymphotoxin (LT)-a1b2 associated with development of lymphoid tissue. Tertiary lymphoid structures (TLS) characterize human and murine ileitis by suppressing outflow from the ileum. Here, we show that B cell-derived secretory IgA protected against ileal inflammation, whereas B cell-derived LTa guarded against ileitis-associated loss of body mass. We initially hypothesized this protection resulted from formation of TLS that suppressed lymphatic outflow and thereby restrained systemic spread of inflammatory signals, but B cell-selective deletion of LTb did not exacerbate weight loss, despite eliminating TLS. Instead, weight loss driven by the cachectic cytokine TNF was exacerbated when LTa3, another ligand for TNF receptors, was selectively neutralized. Thus, B cells' multi-faceted impact on ileitis includes generating secretory IgA, expressing LTa1b2 to drive formation of TLS, and producing LTa3 for protecting against weight loss in the presence of TNF.

Resident dendritic cell density in the lymph node paracortex is preDC-estined.

Dendritic cells (DCs) are relatively short lived, yet DC frequencies in lymph nodes are stable. In this issue of Immunity, Ugur et al. reveal that type 1 conventional DCs (cDC1s) are maintained in the deep paracortex of the lymph node from a supply of preDCs that proliferate in nearby medullary vessels. Transition from preDC to cDC1 is regulated by Flt3L sensing.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Targeting the α4ß7-MAdCAM-1 axis with vedolizumab (VDZ) is a front-line therapeutic paradigm in ulcerative colitis (UC). However, mechanism(s) of action (MOA) of VDZ remain relatively undefined. Here, we examined three distinct cohorts of patients with UC (n=83, n=60, and n=21), to determine the effect of VDZ on the mucosal and peripheral immune system. Transcriptomic studies with protein level validation were used to study drug MOA using conventional and transgenic murine models. We found a significant decrease in colonic and ileal naïve B and T cells and circulating gut-homing plasmablasts (ß7+) in VDZ-treated patients, pointing to gut-associated lymphoid tissue (GALT) targeting by VDZ. Murine Peyer's patches (PP) demonstrated a significant loss cellularity associated with reduction in follicular B cells, including a unique population of epithelium-associated B cells, following anti-α4ß7 antibody (mAb) administration. Photoconvertible (KikGR) mice unequivocally demonstrated impaired cellular entry into PPs in anti-α4ß7 mAb treated mice. In VDZ-treated, but not anti-tumor necrosis factor-treated UC patients, lymphoid aggregate size was significantly reduced in treatment responders compared to non-responders, with an independent validation cohort further confirming these data. GALT targeting represents a novel MOA of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC, and for the development of new therapeutic strategies.

Lipid absorption and overall intestinal lymphatic transport are impaired following partial small bowel resection in mice.

Short bowel syndrome (SBS) is associated with diminished levels of serum fats caused by unknown mechanisms. We have shown that mesenteric lymphatics remodel to a more primitive state one week after small bowel resection (SBR); therefore, this study focuses on the effect of chronic lymphatic remodeling and magnitude of resection on intestinal lipid uptake and transport. C57BL6 and Prox1 creER-Rosa26LSLTdTomato (lymphatic reporter) mice underwent 50% or 75% proximal SBR or sham operations. Functional transport of lipids and fecal fat content was measured and lymphatic vasculature was compared via imaging. There was a significant reduction in functional transport of cholesterol and triglyceride after SBR with increasing loss of bowel, mirrored by a progressive increase in fecal fat content. We also describe significant morphological changes in the lymphatic vasculature in both the lamina propria and mesentery. Intestinal lymphatic drainage assay in vivo demonstrated a marked reduction of systemic absorption after resection. Intestinal lymphatic vessels significantly remodel in the setting of chronic SBS. This remodeling may account at least in part for impaired intestinal uptake and transport of fat via the compromised lymphatic architecture. We believe that these changes may contribute to the development of intestinal failure associated liver disease (IFALD), a major morbidity in patients with SBS.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

LYVE1+ macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth.

Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1hi MHC IIlo-hi CX3CR1gfplo/- and LYVE1lo/- MHC IIhi CX3CR1gfphi subsets) in the mesenteric and parietal mesothelial linings of the peritoneum. These macrophages resembled LYVE1+ macrophages within surface membranes of numerous organs. Fate-mapping approaches and analysis of newborn mice showed that LYVE1hi macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1+ stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1hi macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment contains at least four resident macrophage populations and that LYVE1hi mesothelial macrophages drive tumor growth independently of the omentum.

Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Enterically derived high-density lipoprotein restrains liver injury through the portal vein.

The biogenesis of high-density lipoprotein (HDL) requires apoA1 and the cholesterol transporter ABCA1. Although the liver generates most of the HDL in the blood, HDL synthesis also occurs in the small intestine. Here, we show that intestine-derived HDL traverses the portal vein in the HDL3 subspecies form, in complex with lipopolysaccharide (LPS)-binding protein (LBP). HDL3, but not HDL2 or low-density lipoprotein, prevented LPS binding to and inflammatory activation of liver macrophages and instead supported extracellular inactivation of LPS. In mouse models involving surgical, dietary, or alcoholic intestinal insult, loss of intestine-derived HDL worsened liver injury, whereas outcomes were improved by therapeutics that elevated and depended upon raising intestinal HDL. Thus, protection of the liver from injury in response to gut-derived LPS is a major function of intestinally synthesized HDL.

Visceral obesity and insulin resistance associate with CD36 deletion in lymphatic endothelial cells.

Disruption of lymphatic lipid transport is linked to obesity and type 2 diabetes (T2D), but regulation of lymphatic vessel function and its link to disease remain unclear. Here we show that intestinal lymphatic endothelial cells (LECs) have an increasing CD36 expression from lymphatic capillaries (lacteals) to collecting vessels, and that LEC CD36 regulates lymphatic integrity and optimizes lipid transport. Inducible deletion of CD36 in LECs in adult mice (Cd36ΔLEC) increases discontinuity of LEC VE-cadherin junctions in lacteals and collecting vessels. Cd36ΔLEC mice display slower transport of absorbed lipid, more permeable mesenteric lymphatics, accumulation of inflamed visceral fat and impaired glucose disposal. CD36 silencing in cultured LECs suppresses cell respiration, reduces VEGF-C-mediated VEGFR2/AKT phosphorylation and destabilizes VE-cadherin junctions. Thus, LEC CD36 optimizes lymphatic junctions and integrity of lymphatic lipid transport, and its loss in mice causes lymph leakage, visceral adiposity and glucose intolerance, phenotypes that increase risk of T2D.

Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders.

The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS.

Neurotensin is an anti-thermogenic peptide produced by lymphatic endothelial cells.

The lymphatic vasculature plays important roles in the physiology of the organs in which it resides, though a clear mechanistic understanding of how this crosstalk is mediated is lacking. Here, we performed single-cell transcriptional profiling of human and mouse adipose tissue and found that lymphatic endothelial cells highly express neurotensin (NTS/Nts). Nts expression is reduced by cold and norepinephrine in an α-adrenergic-dependent manner, suggesting a role in adipose thermogenesis. Indeed, NTS treatment of brown adipose tissue explants reduced expression of thermogenic genes. Furthermore, adenoviral-mediated overexpression and knockdown or knockout of NTS in vivo reduced and enhanced cold tolerance, respectively, an effect that is mediated by NTSR2 and ERK signaling. Inhibition of NTSR2 promoted energy expenditure and improved metabolic function in obese mice. These data establish a link between adipose tissue lymphatics and adipocytes with potential therapeutic implications.

Liver injury after small bowel resection is prevented in obesity-resistant 129S1/SvImJ mice.

Intestinal failure-associated liver disease is a major morbidity associated with short bowel syndrome. We sought to determine if the obesity-resistant mouse strain (129S1/SvImJ) conferred protection from liver injury after small bowel resection (SBR). Using a parenteral nutrition-independent model of resection-associated liver injury, C57BL/6J and 129S1/SvImJ mice underwent a 50% proximal SBR or sham operation. At postoperative week 10, hepatic steatosis, fibrosis, and cholestasis were assessed. Hepatic and systemic inflammatory pathways were evaluated using oxidative markers and abundance of tissue macrophages. Potential mechanisms of endotoxin resistance were also explored. Serum lipid levels were elevated in all mouse lines. Hepatic triglyceride levels were no different between mouse strains, but there was an increased accumulation of free fatty acids in the C57BL/6J mice. Histological and serum markers of hepatic fibrosis, steatosis, and cholestasis were significantly elevated in resected C57BL/6J SBR mice as well as oxidative stress markers and macrophage recruitment in both the liver and visceral white fat in C57BL/6J mice compared with sham controls and the 129S1/SvImJ mouse line. Serum endotoxin levels were significantly elevated in C57BL/6J mice with significant elevation of hepatic TLR4 and reduction in PPARα expression levels. Despite high levels of serum lipids, 129S1/SvImJ mice did not develop liver inflammation, fibrosis, or cholestasis after SBR, unlike C57BL/6J mice. These data suggest that the accumulation of hepatic free fatty acids as well as increased endotoxin-driven inflammatory pathways through PPARα and TLR4 contribute to the liver injury seen in C57BL/6J mice with short bowel syndrome.NEW & NOTEWORTHY Unlike C57BL/6 mice, the 129S1/SvImJ strain is resistant to liver inflammation and injury after small bowel resection. These disparate outcomes are likely due to the accumulation of hepatic free fatty acids as well as increased endotoxin-driven inflammatory pathways through PPARα and TLR4 in C57BL/6 mice with short bowel syndrome.

Sensory Nerves Regulate Transcriptional Dynamics of Lymph Node Cells.

The nervous system plays important roles in homeostasis and inflammatory responses in tissues. However, the regulation of lymph nodes (LN) by nerves remains largely unknown. Huang et al. demonstrate that LNs are innervated by unique peptidergic nociceptors that signal to various endothelial, stromal, and immune cell types in LNs.

TNF-mediated alveolar macrophage necroptosis drives disease pathogenesis during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infants under 2 years old. Necroptosis has been implicated in the outcomes of respiratory virus infections. We report that RSV infection triggers necroptosis in primary mouse macrophages and human monocytes in a RIPK1-, RIPK3- and MLKL-dependent manner. Moreover, necroptosis pathways are harmful to RSV clearance from alveolar macrophages. Additionally, Ripk3-/- mice were protected from RSV-induced weight loss and presented with reduced viral loads in the lungs.Alveolar macrophage depletion also protected mice from weight loss and decreased lung RSV virus load. Importantly, alveolar macrophage depletion abolished the upregulation of Ripk3 and Mlkl gene expression induced by RSV infection in the lung tissue.Autocrine tumor necrosis factor (TNF)-mediated RSV-triggered macrophage necroptosis and necroptosis pathways were also involved in TNF secretion even when macrophages were committed to cell death, which can worsen lung injury during RSV infection. In line, Tnfr1-/- mice had a marked decrease in Ripk3 and Mlkl gene expression and a sharp reduction in the numbers of necrotic alveolar macrophages in the lungs. Finally, we provide evidence that elevated nasal levels of TNF are associated with disease severity in infants with RSV bronchiolitis.We propose that targeting TNF and/or the necroptotic machinery may be valuable therapeutic approaches to reduce the respiratory morbidity caused by RSV infection in young children.

Lymphatic and Blood Network Analysis During Obesity.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Peripheral nerve resident macrophages share tissue-specific programming and features of activated microglia.

Whereas microglia are recognized as fundamental players in central nervous system (CNS) development and function, much less is known about macrophages of the peripheral nervous system (PNS). Here, by comparing gene expression across neural and conventional tissue-resident macrophages, we identified transcripts that were shared among neural resident macrophages as well as selectively enriched in PNS macrophages. Remarkably, PNS macrophages constitutively expressed genes previously identified to be upregulated by activated microglia during aging, neurodegeneration, or loss of Sall1. Several microglial activation-associated and PNS macrophage-enriched genes were also expressed in spinal cord microglia at steady state. We further show that PNS macrophages rely on IL-34 for maintenance and arise from both embryonic and hematopoietic precursors, while their expression of activation-associated genes did not differ by ontogeny. Collectively, these data uncover shared and unique features between neural resident macrophages and emphasize the role of nerve environment for shaping PNS macrophage identity.