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Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. The BMP receptor activin receptor-like kinase 1 (ALK1) is well known for its role in blood vessel formation; however, its role within the immune system has never been revealed to our knowledge. Here, we found that BMP9/BMP10/ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen, and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages showed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6ChiCX3CR1lo monocytes/macrophages to pro-resolving Ly6CloCX3CR1hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2−/− neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair., Neutrophils and macrophages are both involved in the initiation of inflammation, but whether and how they may participate in inflammation resolution is unclear. Here the authors show that neutrophils may mediate the conversion of macrophage into a pro-resolution phenotype via reactive oxygen species production to promote liver repair.

Background & Aims Production of neutralizing antibodies against hepatitis B surface antigen (HBsAg) is dysregulated in patients with persistent hepatitis B virus (HBV) infection. We investigated mechanisms by which this immune response to the virus is disrupted and whether it can be restored to promote clearance of HBV. Methods Immune-competent C57BL/6N and C57BL/6J, as well as mice deficient in follicular helper T cells (Tfh-cell-deficient), B cells, or Foxp3+ T-regulatory cells (Treg cell deficient), were given hydrodynamic injections of pAAV/HBV1.2 plasmids. Some mice were given injections of sorted Tfh cells, pan-B cells, Treg cells, or a blocking antibody against CTLA4. Production of antibodies against HBsAg and clearance of HBV were assessed by flow cytometry, enzyme-linked immunosorbent assay, polymerase chain reaction, and immunohistochemical analyses. We obtained blood samples from patients with HBV infection and isolated Treg cells. We measured the ability of Treg cells to suppress production of interleukin 21 (IL21) in CD4+ T cells. Results Immune-competent C57BL/6N and C57BL/6J mice transfected with the plasmid encoding HBV had features of viral clearance and viral persistence observed in humans. A Tfh-cell response to HBsAg was required for clearance of HBV and was suppressed by Treg cells in mice with persistent HBV infection. Depletion of Treg cells or inhibition of Treg-cell function (with blocking antibody against CTLA4) restored the Tfh-cell response against HBsAg and clearance of HBV in mice. Impaired Tfh-cell response to HBsAg was observed in blood from patients with chronic HBV infection, responsiveness was restored by depletion of Treg cells or blocking antibody against CTLA4. Conclusions In studies of HBV-infected mice and blood from patients with chronic HBV infection, we found a Tfh-cell response to HBsAg of to be required for HBV clearance, and that this response was blocked by Treg cells. Inhibiting Treg-cell activity using neutralizing antibody against CTLA4 restored the ability of Tfh cells to clear HBV infection; this approach might be developed for treatment of patients with chronic HBV infection.

Kupffer cells (KCs) are the resident macrophages of the liver, and they respond to and counteract metabolic stresses, such as those imposed by high-fat diet feeding in mouse models. However, little is known regarding the role of these cells in maintaining metabolic homeostasis under metabolically normal conditions. In this study, we found that depletion of KCs in vivo led to enhanced lipolysis in adipose tissue by increasing the expression of FGF21, a metabolic regulator, in hepatocytes. IL-1β secreted from KCs contributed to the suppression of FGF21 expression in hepatocytes. FGF21 overexpression led to a lean phenotype and enhanced lipolysis in mice. KC depletion resulted in a lack of IL-1β signaling in the liver, leading to elevated expression of FGF21 in hepatocytes. FGF21 promoted lipolysis in adipose tissue and led to hyperlipidemia and decreased body weight. The secretion of IL-1β in KCs was mediated by bacterial products. Antibiotic treatment also led to enhanced lipolysis. Therefore, the current study identified a physiological role of KCs in the regulation of adipose lipolysis.

Macrophages have been suggested to contribute to constructing a cancer stem cell (CSC) niche. However, whether and how macrophages regulate the activity of CSCs through juxtacrine signaling are poorly understood. Here we report LSECtin, a transmembrane protein highly expressed on tumor-associated macrophages (TAMs), enhances stemness of breast cancer cells (BCCs). We identified BTN3A3, a B7 family member with previously unknown functions as the receptor for LSECtin on BCCs responsible for stemness-promoting effect of LSECtin. In mice bearing human tumor xenografts, either macrophage-specific ablation of LSECtin or silencing of BTN3A3 in BCCs decreased CSC frequency and tumor growth. Admixture of LSECtin-positive macrophages increased the tumorigenic activity of BCCs dependent on BTN3A3. Disruption of the LSECtin-BTN3A3 axis with BTN3A3-Fc or anti-BTN3A3 mAb has a therapeutic effect on breast cancer. These findings define a juxtacrine signaling mechanism by which TAMs promote cancer stemness. Targeting this axis in the CSC niche may provide potential therapies to breast cancer.

Epithelial barrier disruption is a major cause of inflammatory bowel disease (IBD); however, the cellular and molecular regulation of intestinal epithelial homeostasis remains largely undefined. Here, we show that the C-type lectin receptor LSECtin (Clec4g) on macrophages is required for protection against dextran sulfate sodium-induced colitis. Mechanistically, LSECtin promotes apoptotic cell clearance by macrophages and induces the production of antiinflammatory/tissue repair factors in an engulfment-dependent manner, which in turn stimulates epithelial cell proliferation. Deletion of LSECtin results in defective engulfment by colon macrophages, leading to aberrant proresolving factor production and impaired intestinal epithelium repair. Collectively, our findings suggest that LSECtin-dependent corpse clearance by macrophages can direct intestinal regeneration and maintenance of the mucosal barrier after injury.

Tumor-associated macrophages (TAMs) are major components of the tumor microenvironment. Although a role for TAMs in promoting tumor progression has been revealed, the differentiation mechanisms and intrinsic signals of TAMs regulated by the tumor microenvironment remain unclear. Here we constructed an in vitro TAMs cell model, TES-TAMs, which is from tumor-extract-stimulated bone-marrow-derived macrophages. We performed a comparative proteomics analysis of bone-marrow-derived macrophages and TES-TAMs, which indicated that TES-TAMs possessed characteristic molecular expression of TAMs. Intriguingly, the signal pathways enriched in up-regulated differentially expressed proteins of TAMs demonstrated that glycolysis metabolism reprogramming may play an important role in TAM differentiation. We found that hexokinase-2, a key mediator of aerobic glycolysis, and the downstream proteins PFKL and ENO1 were remarkably increased in both TES-TAMs and primary TAMs from our MMTV-PyMT mice model. This phenomenon was then verified in human THP-1 cell lines stimulated by tumor extract solution from breast cancer patient. Taken together, our study provides insight into the induction of TAM differentiation by the tumor microenvironment through metabolic reprogramming.

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response., Author Summary Ebola virus (EBOV), a highly virulent pathogen, causes a severe hemorrhagic fever syndrome. The fatal infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is thought to contribute to disease pathogenesis, as high amounts of shed GP from virus-infected cells are detected in patients, and activate macrophages and dendritic cells (DCs) to produce proinflammatory cytokines. Here, we show that LSECtin plays an important role in GP-mediated inflammatory responses in human DCs. LSECtin is a DAP12-coupled receptor able to initiate specific signaling events in human DCs. LSECtin interacts with Ebola GP and results in DAP12 phosphorylation. LSECtin knockdown impairs the production of proinflammatory cytokines induced by Ebola GP. Thus, this study suggests that LSECtin may contribute to Ebola GP-mediated pathogenicity.

Liver sinusoidal endothelial cell lectin (LSECtin) was recently reported to suppress intrahepatic T cell immunity and to limit immune-mediated liver injury. However, its role in the outcome and pathogenesis of viral infection has not yet been elucidated. Using a mouse model infected with a hepatotropic adenovirus, we found that the absence of LSECtin led to a higher frequency of intrahepatic effector CTLs. These cells produced higher levels of antiviral cytokines and cytotoxic factors and exhibited an increased expression of the transcription factors T-bet and Runx3. This phenotype observed in the LSECtin-knockout cells mediated a more efficient virus-specific cytotoxity compared with that of wild-type cells. As a consequence, LSECtin deficiency significantly accelerated liver adenovirus clearance. In contrast, LSECtin upregulation in the liver delayed viral clearance; this delayed clearance was accompanied by the downregulation of the antiviral activity of CTLs. We further constructed an immunocompetent mouse model of acute hepatitis B viral infection to demonstrate that LSECtin significantly delayed the clearance of hepatitis B virus from blood and infected hepatocytes by limiting the frequency of hepatitis B virus–specific IFN-γ–producing cells. Consistent with this function, LSECtin was upregulated in the liver of mouse models of viral hepatitis. Taken together, our results suggest that LSECtin may facilitate the reduction of liver inflammation at the cost of delaying virus clearance and that this effect might be hijacked by the virus as an escape mechanism.

Objective Adhesion molecules play an important role in tumour metastasis. The liver is a frequent target for the metastasis of several tumour types. However, virtually no liver-specific adhesion molecules have been described in terms of organ-specific metastasis. This study aimed to determine the role of liver sinusoidal endothelial cell lectin (LSECtin) in colon carcinoma metastasis to the liver. Design The role of LSECtin in colon carcinoma metastasis to the liver was determined by LSECtin knockout nude mice and anti-LSECtin antibody. LSECtin promoting the migration of LS174T and LoVo cells was determined by transwell experiment. The serum levels of soluble LSECtin in patients were elevated by ELISA. Results LSECtin was found to adhere to LS174T and LoVo colon cancer cells in vitro and in vivo. Deficiency or blocking of LSECtin significantly decreased hepatic metastases of LS174T and LoVo cells. Primary colon cancer cells from patients also exhibited remarkably low rates of hepatic metastasis in LSECtin knockout mice. LSECtin promoted the migration of LS174T and LoVo cells and increased the expression of c-Met in these cells. Serum soluble LSECtin was detected at significantly higher levels in colon cancer patients with or without hepatic metastases compared with healthy controls and was also increased in colon cancer patients with metastases compared with those without metastases. Conclusion The results indicate that LSECtin plays an important role in colorectal carcinoma liver metastasis and may be a promising new target for intervention in metastasis formation.

Recently, LSECtin was identified as a co-inhibitory molecule involved in the control of T cell-mediated acute liver injury. However, it is not known whether LSECtin also suppresses the function of different CD4(+) T-cell subsets. In this study, we demonstrate that LSECtin-mediated signaling potently inhibits the proliferation and IL-2 production of total, naive and pre-activated CD4(+) T cells. However, exogenous IL-2 does not overcome the inhibitory effect on the proliferation of naive CD4(+) T cells. Based on the LSECtin-mediated inhibition of pre-activated CD4(+) T cells, we further analyzed the role of LSECtin in different effector CD4(+) T-cell subsets including Th1, Th2 and Th17. We observed that LSECtin significantly suppressed the production of their signature cytokines suggesting that LSECtin may also play an inhibitory role in the effector phase. LSECtin did not inhibit the CD4(+) T-cell proliferation induced by PMA (phorbol myristate acetate) and ionomycin suggesting that the LSECtin-mediated inhibition reduces the activation of the proximal TCR signalsome. The inhibition of CD4(+) T-cell activation mediated by LSECtin is associated with the up-regulation of Cbl-b and has no effect on GRAIL, Itch and SHP-2 phosphatase. Furthermore, LSECtin-initiated inhibition is compromised in Cbl-b mutant CD4(+) T cells. Taken together, these data demonstrate that LSECtin-mediated signaling up-regulates the threshold of CD4(+) T cell activation via tuning the expression of Cbl-b.

Background & Aims The liver is an organ with paradoxic immunologic properties and is known for its tolerant microenvironment, which holds important implications for hepatic diseases. The molecular basis for this local immune suppression, however, is poorly understood. In this study, we aimed to determine the role of liver sinusoidal endothelial cell lectin (LSECtin), a recently identified member of the dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) family, in the regulation of hepatic T-cell immune response. Methods The regulation of T-cell effector function by LSECtin was determined by co-stimulated T cells with anti-CD3/CD28 monoclonal antibody and LSECtin protein, or co-culture of T-cell receptor transgenic T cells with mouse LSECs in vitro. We generated LSECtin knockout mice and prepared recombinant LSECtin protein and complementary DNA plasmids to analyze the role of LSECtin in hepatic T-cell immune regulation in vivo. Results We showed that LSECtin specifically recognized activated T cells and negatively regulated their immune responses. In mice with T-cell–mediated acute liver injury, the lack of LSECtin accelerated the disease owing to an increased T-cell immune response, whereas the exogenous administration of recombinant LSECtin protein or plasmid ameliorated the disease via down-regulation of T-cell immunity. Conclusions Our results reveal that LSECtin is a novel regulator of T cells and expose a crucial mechanism for hepatic T-cell immune suppression, perhaps opening up a new approach for treatment of inflammatory diseases in the liver.