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.

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.

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.

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.

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.

Effects of high-fat diet on liver injury after small bowel resection.

BACKGROUND: The optimal regimen for enteral nutritional support in the management of children with short bowel syndrome (SBS) is not well characterized. A high fat, enteral diet is theoretically beneficial due to increased caloric density and enhanced structural adaptation. We therefore sought to determine the long-term effects of a high fat diet (HFD) on liver injury, a common complication of SBS, compared to a standard chow (SC) diet. METHODS: Using a parenteral nutrition-independent model of resection-associated liver injury, C57BL/6 mice underwent a sham operation or a 50% or 75% proximal small bowel resection (SBR). Mice in each group were then fed either a HFD (35% kcal fat) or SC (13% kcal fat). At post-operative week 15, markers of liver injury were quantified. RESULTS: Liver triglyceride levels were increased from 7- to 19-fold in mice on the HFD compared to mice fed SC in the sham, 50%, and 75% resection groups. Serum ALT (2.2-fold increase in 75% resected mice compared to sham controls) and AST (2.0- and 2.7-fold increases in 50% and 75% resected mice, respectively) levels as well as fibrotic liver staining were elevated only in resected mice fed a HFD. CONCLUSION: Long-term enteral feeding of HFD in our murine SBS model is associated with hepatic steatosis and liver injury. Our observation that liver steatosis and injury occur independent of parenteral nutrition suggests that enteral feeding composition and magnitude of intestinal loss may make a significant contribution to intestinal failure-associated liver disease.

Neutrophils promote VLA-4-dependent B cell antigen presentation and accumulation within the meninges during neuroinflammation.

The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibody-independent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4-dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS.

Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity.

Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood "macrophage disappearance reaction." Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage-induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity.

Lymph nodes go with the flow.

In this issue, Bovay et al. (https://doi.org/10.1084/jem.20180217) invoke a compelling model of interplay between the venous and lymphatic vasculature in regulating the developmental genesis and early expansion of LNs. This work supports an emerging model that lymph-venous crosstalk supports LN functionality at all stages.

GRPR antagonist protects from drug-induced liver injury by impairing neutrophil chemotaxis and motility.

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL-8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life-threatening tissue damage that ensues. A GRPR (gastrin-releasing peptide receptor) antagonist impairs IL-8-induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen-induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen-overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2-induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8-driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.

Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils.

Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.