Suppressor of cytokine signaling 1 (SOCS1) is a negative feedback inhibitor of cytoplasmic Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling. SOCS1 also contains a nuclear localization sequence (NLS), yet the in vivo importance of nuclear translocation is unknown. We generated transgenic mice containing mutated Socs1ΔNLS that fails to translocate in the cell nucleus (MGLtg mice). Whereas mice fully deficient for SOCS1 die within the first three weeks due to excessive interferon signaling and multiorgan inflammation, mice expressing only non-nuclear Socs1ΔNLS (Socs1-/-MGLtg mice) were rescued from early lethality. Canonical interferon gamma signaling was still functional in Socs1-/-MGLtg mice as shown by unaltered tyrosine phosphorylation of STAT1 and whole genome expression analysis. However, a subset of NFB inducible genes was dysregulated. Socs1-/-MGLtg mice spontaneously developed low-grade inflammation in the lung and had elevated Th2-type cytokines. Upon ovalbumin sensitization and challenge, airway eosinophilia was increased in Socs1-/-MGLtg mice. Decreased transepithelial electrical resistance (TER) in trachea epithelial cells from Socs1-/-MGLtg mice suggests disrupted epithelial cell barrier. The results indicate that nuclear SOCS1 is a regulator of local immunity in the lung and unravel a so far unrecognized function for SOCS1 in the cell nucleus.

Tumor-associated macrophages (TAM) are crucial participants in malignant progression. Acquisition of M2 (alternatively activated) phenotype by TAM during tumor progression enhances the immunosuppressive and tumor-supportive properties of TAM resulting in tumor invasion, metastasis and angiogenesis. Previously we have found that stabilin-1, a multifunctional scavenger/sorting receptor, is a marker of M2 macrophages that is expressed by TAM in several murine tumor models. However, its role in tumor progression was not defined. In order to identify the role of stabilin-1 in tumor progression, the model of mammary adenocarcinoma (TS/A) was established in BALB/c mice with stabilin-1 knockout. The growth of TS/A mammary adenocarcinoma in stabilin-1 knockout (ko) mice was suppressed by 36%. To identify the role of stabilin-1 in TAM biology and reveal functions of stabilin-1 that are important for tumor progression, isolation of high purity TAM from TS/A murine adenocarcinoma was established and optimized. Flow cytometry analysis revealed that adhesion/internalisation of extracellular SPARC was decreased in the isolated stabilin-1 ko TAM compared to wt TAM. Immunofluorescent/confocal microscopy showed that transport of SPARC into the endocytic pathway was significantly impaired in the stabilin-1 ko TAM. Since SPARC is known to inhibit the development of solid tumors including breast cancer we hypothesize that knockout of stabilin-1 in TAM induces accumulation of extracellular SPARC resulting in suppression of tumor growth. Analysis of two cohorts of female patients with breast carcinoma of different stages demonstrated that stabilin-1 is expressed on significant part of tumor associated macrophages. Three types of TAM were identified by co-staining with anti-stabilin-1 RS1 antibody and anti-CD68 antibody: CD68+stabilin-1-, CD68+stabilin-1+ and CD68-stabilin-1+. The highest levels of stabilin-1 expression and highest amount of stabilin-1+ TAM were found on stages I and IIa, suggesting that stabilin-1 is required for tumor growth at early stages of tumor progression. Our data indicate that stabilin-1 expression on TAM is necessary on the early stages of tumor growth in human cancer. Genetic knockout of stabilin-1 results in decrease in tumor growth in mouse adenocarcinoma model. In addition, tumor-associated macrophages deficient in stabilin-1 expression have significantly decreased ability for the endocytic clearance of SPARC. Citation Format: Vladimir Ryabov, Ilja Ovsij, Aida Avdic, Kai Schledzewski, Alexei Gratchev, Nan Wang, Bernd Arnold, Sergij Goerdt, Frederick Pfister, Alexander Marx, Limin Zheng, Julia Kzhyshkowska. Stabilin-1 is expressed on tumor-associated macrophages in breast cancer and supports tumor growth in animal model of breast adenocarcinoma by clearance of SPARC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1668. doi:10.1158/1538-7445.AM2014-1668

Tissue homeostasis and remodeling are processes that involve high turnover of biological macromolecules. Many of the waste molecules that are by-products or degradation intermediates of biological macromolecule turnover enter the circulation and are subsequently cleared by liver sinusoidal endothelial cells (LSEC). Besides the mannose receptor, stabilin-1 and stabilin-2 are the major scavenger receptors expressed by LSEC. To more clearly elucidate the functions of stabilin-1 and -2, we have generated mice lacking stabilin-1, stabilin-2, or both stabilin-1 and -2 (Stab1–/– Stab2–/– mice). Mice lacking either stabilin-1 or stabilin-2 were phenotypically normal; however, Stab1–/– Stab2–/– mice exhibited premature mortality and developed severe glomerular fibrosis, while the liver showed only mild perisinusoidal fibrosis without dysfunction. Upon kidney transplantation into WT mice, progression of glomerular fibrosis was halted, indicating the presence of profibrotic factors in the circulation of Stab1–/– Stab2–/– mice. While plasma levels of known profibrotic cytokines were unaltered, clearance of the TGF-β family member growth differentiation factor 15 (GDF-15) was markedly impaired in Stab1–/– Stab2–/– mice but not in either Stab1–/– or Stab2–/– mice, indicating that it is a common ligand of both stabilin-1 and stabilin-2. These data lead us to conclude that stabilin-1 and -2 together guarantee proper hepatic clearance of potentially noxious agents in the blood and maintain tissue homeostasis not only in the liver but also distant organs.

IFN-gamma regulates multiple processes in the immune system. Although its antimicrobial effector functions are well described, less is known about the mechanisms by which IFN-gamma regulates CD8(+) T cell homeostasis. With the help of adoptive T cell transfers, we show in this study that IFN-gammaR signaling in CD8(+) T cells is dispensable for expansion, contraction, and memory differentiation in response to peptide vaccination. In contrast, host IFN-gammaR signaling counterregulates CD8(+) T cell responses and the generation of effector memory T cell processes, which are partially regulated by CD11b(+) cells. Similar to vaccination-induced proliferation, host IFN-gammaR signaling limits the expansion of naive CD8(+) T cells and their differentiation into effector memory-like T cells in lymphopenic mice. In contrast to peptide vaccination, IFN-gammaR signaling in CD8(+) T cells contributes to memory fate decision in response to lymphopenia, an effect that is fully reversed by high-affinity TCR ligands. In conclusion, we show that host IFN-gammaR signaling controls the magnitude of CD8(+) T cell responses and subsequent memory differentiation under lymphopenic and nonlymphopenic conditions. In contrast, IFN-gammaR signaling in CD8(+) T cells does not affect cell numbers under either condition, but it directs memory fate decision in response to weak TCR ligands.

Deficiency of the mitochondrial enzyme 2‐methyl‐3‐hydroxybutyryl‐CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease‐causing gene is HSD17B10 and encodes 17β‐hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss‐of‐function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10−/− mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine‐restricted diet are required.

IL-7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL-7 production. To study Il7 gene regulation in vivo, we generated a novel IL-7-reporter mouse, which allows the non-invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL-7-producing cells. With these IL-7-reporter mice, we identify thymus, skin and intestine as major sources of IL-7 in vivo. Importantly, we show that IFN-gamma and the commensal microflora promote steady-state IL-7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN-gamma signaling in intestinal epithelial cells strongly reduces their IFN-gamma-driven IL-7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN-gamma production by lymphocytes, which in turn promotes epithelial cell IL-7 production and the survival of IL-7-dependent lymphocytes.

High-mobility group box 1 (HMGB-1) has been reported as a "late" proinflammatory mediator in sepsis. In vitro data have shown that HMGB-1 can induce activation of intracellular signaling pathways via interaction with at least three pattern recognition receptors: Toll-like receptor (TLR) 2, TLR-4, and the receptor for advanced glycation end products (RAGE). The objective of this study was to investigate the role of these receptors in the in vivo response to HMGB-1. Therefore, we first performed a time-series experiment with wild-type (Wt) mice. High-mobility group box 1 induced time-dependent elevations of TNF-alpha, IL-6, monocyte chemoattractant protein 1, and thrombin-antithrombin complex levels in peritoneal lavage fluid and plasma. This inflammatory reaction was accompanied by a prominent and sustained rise in neutrophil counts in the peritoneal cavity. We next administered HMGB-1 to Wt, TLR-2, TLR-4, and RAGE mice. All genotypes showed similar plasma levels of TNF-alpha, IL-6, IL-10, and thrombin-antithrombin complex at 2 h after intraperitoneal injection of HMGB-1. Compared with Wt mice, both TLR-4 and RAGE mice displayed lower TNF-alpha and IL-6 concentrations and lower neutrophil numbers in their peritoneal lavage fluid. In contrast, TLR-2 mice showed increased levels of TNF-alpha and IL-6 in their peritoneal cavity relative to Wt mice. These data indicate that HMGB-1 induces release of cytokines, activation of coagulation, and neutrophil recruitment in vivo via a mechanism that at least in part depends on TLR-4 and RAGE.

We have shown previously that mast cells o(MC) and MC-derived mediators are crucial for earlyb stages of zymosan-induced peritonitis in Swiss and Balb/c mice. Present results revealed the same phenomena in males of C57BI/6 mice as in the MC-depleted animals (by application of a compound 48/80) the intraperitoneal accumulation of exudatory fluid and cells, measured at 0.5, 6 and 24 hours after zymosan injection, was significantly diminished. In particular, an early vascular permeability and PGE(2) content at 30 min of peritonitis, and exudatory leukocyte numbers and MCP-1 content at 6 hours of peritonitis were significantly lower in MC-deprived mice then in those with normal MC population. The peritoneal cavity of intact C57BI/6 males contains less then 1% of mast cells defined as cells filled with metachromatically stained granules (counted in a haemocytometer) and those with MC-specific c-kit receptors (determined both by quantitative PCR at mRNA level and by flow cytometry on cell surface). After zymosan injection numbers of cells with metcahromatically-stained granules significantly decrease reaching minimum at 6 hours of peritonitis with a parallel significant increase of c-kit positive MCs. These contradictory results might be explained by zymosan-induced degranulation of mast cells and concomitant influx of newly arriving mast cells through the omental milky spots and/or in situ MC proliferation.

Sentinel lymph node biopsy for several cancers has shown that metastatic tumour cells are preferentially arrested in the lymph node sinuses. To study the molecular components of this sinusoidal trap, gene profiling of lymph node (sinuses) versus tonsil (no sinuses) was performed. Among other groups of molecules, an intriguing gene signature of scavenger and lectin-like receptors was identified. Nine of the 13 genes were preferentially expressed in sinusoidal cells by immunohistochemistry. Using stabilin-2 and monoclonal antibody 3A5 as exclusive endothelial cell (EC) and macrophage (M phi) markers, respectively, lymph node sinusoidal ECs (stabilin-2+, LYVE-1+, DC-SIGNR+, MARCO+, stabilin-1+, MMR+) and sinusoidal M phi (MMR+, DC-SIGN+, sialoadhesin+, CD163+, stabilin1+) showed distinct, but overlapping expression patterns of the signature molecules by double labelling immunofluorescence. The number of stabilin-1+ sinusoidal M phi, however, varied considerably between samples, indicating turnover/differentiation dynamics in this sinusoidal cell population. In the hepatic sinuses, LYVE-1 and CD36 were strongly upregulated on both sinusoidal ECs and M phi, while DC-SIGNR and DC-SIGN were strongly down-regulated; in contrast to lymph node sinusoidal ECs, MARCO was confined to M phi (Kupffer cells) in the liver sinuses. As M phi are not present in the wall and lumen of splenic sinuses, splenic sinuses expressed a considerably reduced repertoire of scavenger/lectin receptors lacking sialoadhesin, CD36, CD163, and MARCO; in addition, DC-SIGNR was absent from splenic sinusoidal ECs, while DC-SIGN and thrombomodulin were strongly expressed. Interestingly, most of the signature molecules are known to mediate tumour cell adhesion in addition to their functions as scavenger or pattern recognition receptors. This study establishes a gene and tissue database platform to test the hypothesis that additive expression of the lymph node sinus signature genes in sinusoidal ECs and M phi may contribute to selective tumour cell metastasis in lymph nodes and liver including organspecific mechanisms, such as intraluminal retention or transmigration, while sparing the spleen. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased vascular permeability leading to vascular leakage is a central feature of all inflammatory reactions and is critical for the formation of an inflammatory exudate. The leakage occurs because of gap formation between endothelial cells and breakdown of the basement membrane barriers. The present study aimed to investigate the role of gelatinase B [matrix metalloproteinase 9 (MMP-9)], known to be involved in neutrophil exudation, in changes of vascular permeability at the early stages of acute zymosan peritonitis. We show that although MMP-9 is being released already within the first minutes of peritonitis, its lack, induced pharmacologically or genetically, does not decrease but rather increases vasopermeability. In mice treated with an inhibitor of gelatinases (A and B), a tendency to increased vasopermeability existed, and in MMP-9-/- mice [knockout (KO)], the difference was statistically significant in comparison with their controls. Moreover, in intact KO mice, significantly augmented production of prostaglandin E(2) (PGE(2)) of cyclooxygenase 1 (COX-1) origin was detected, and depletion of peritoneal macrophages, but not mast cells, decreased vasopermeability in KO mice. Thus, the increase of vasopermeability observed on KO mice is a result of the increased production of COX-1-derived PGE(2) by peritoneal macrophages. We conclude that genetic deficiency in gelatinase B might lead to the development of a compensatory mechanism involving the COX pathway.

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b(+), F4/80(+) tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1(+) macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVE-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1(+), stabilin-1(+) double-positive, while 9.9% were LYVE-1(+), stabilin-1(-) and 33.5% were LYVE-1(-), stabilin-1(+). Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1(+), stabilin-1(+) non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1(+) lymphatics and LYVE-1(+) tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.

In a mouse model of hepatocellular carcinogenesis, highly vascularized tumors develop through two distinct morphologic phases of neovascularization. We show that increased vascular caliber occurs first, followed by extensive vessel sprouting in late-stage carcinomas. To define molecular pathways in tumor neovascularization, endothelial cells were directly purified from normal liver and advanced tumors. Gene expression profiling experiments were then designed to identify genes enriched in the vascular compartment. We report that Cathepsin S is the major protease specifically overexpressed during vessel sprouting. We also show that the CC chemokines CCL2 and CCL3 are secreted by neovessels and stimulate proliferation through their cognate receptors in an autocrine fashion. This suggests that chemokine signaling represents the most prominent signaling pathway in tumor-associated endothelial cells and directly regulates vessel remodeling. Furthermore, high angiogenic activity is associated with attenuated lymphocyte extravasation and correlates with expression of the immunomodulatory cytokine interleukin 10. This is the first comprehensive study addressing liver-specific vascular changes in a murine autochthonous tumor model. These novel insights into liver angiogenesis infer an environmental control of neovascularization and have important implications for the design of antiangiogenic therapies.

IFN-gamma has a dual function in the regulation of T cell homeostasis. It promotes the expansion of effector T cells and simultaneously programs their contraction. The cellular mechanisms leading to this functional dichotomy of IFN-gamma have not been identified to date. In this study we show: 1) that expansion of wild-type CD8(+) T cells is defective in IFN-gamma-deficient mice but increased in IFN-gamma R-deficient mice; and 2) that contraction of the effector CD8(+) T cell pool is impaired in both mouse strains. Furthermore, we show that CD11b(+) cells responding to IFN-gamma are sufficient to limit CD8(+) T cell expansion and promote contraction. The data presented here reveal that IFN-gamma directly promotes CD8(+) T cell expansion and simultaneously induces suppressive functions in CD11b(+) cells that counter regulate CD8(+) T cell expansion, promote contraction, and limit memory formation. Thus, innate immune cells contribute to the IFN-gamma-dependent regulation of Ag-specific CD8(+) T cell homeostasis.

IFN-gamma has a dual function in the regulation of T cell homeostasis. It promotes the expansion of effector T cells and simultaneously programs their contraction. The cellular mechanisms leading to this functional dichotomy of IFN-gamma have not been identified to date. In this study we show: 1) that expansion of wild-type CD8+ T cells is defective in IFN-gamma-deficient mice but increased in IFN-gammaR-deficient mice; and 2) that contraction of the effector CD8+ T cell pool is impaired in both mouse strains. Furthermore, we show that CD11b+ cells responding to IFN-gamma are sufficient to limit CD8+ T cell expansion and promote contraction. The data presented here reveal that IFN-gamma directly promotes CD8+ T cell expansion and simultaneously induces suppressive functions in CD11b+ cells that counter-regulate CD8+ T cell expansion, promote contraction, and limit memory formation. Thus, innate immune cells contribute to the IFN-gamma-dependent regulation of Ag-specific CD8+ T cell homeostasis.

Murine zymosan-induced peritonitis represents a well-defined model of acute inflammation. However, the molecular mechanisms by which leukocytes degrade basement membranes during extravasation into the peritoneum are not clear. Gelatinase B (MMP-9) is thought to participate in cellular migration, yet its role in leukocyte transmigration through endothelia during inflammation remains controversial. The aim of the present study was to evaluate the role of MMP-9 in the cell influx during zymosan-induced experimental peritonitis. In zymosan-treated Balb/c mice MMP-9 and its natural inhibitor (tissue inhibitor of metalloproteinase 1 – TIMP-1) were present in the peritoneal fluid and plasma at the time of peritoneal neutrophil (polymorphonuclear leukocyte – PMN) infiltration and persisted there until the time of monocytes/macrophages influx. To probe the function of gelatinases, gelatinase B-deficient mice (MMP-9−/−) were used as well as Balb/c mice treated with cyclic CTTHWGFTLC (INH), a specific peptide inhibitor of gelatinases. The studies revealed that in either group of mice deprived of MMP-9 activity, PMN infiltration was impaired at the time of their maximal extravasation (6h) while tumor necrosis factor α (TNF-α), cytokine-induced neutrophil chemoattractant (KC) and interleukin 10 (IL-10) levels were not changed. At later stages (24h post-zymosan) a significant increase in PMNs was observed in MMP-9−/− mice, but not in the inhibitor-treated mice, in comparison to their respective controls. Moreover, intraperitoneal (i.p.) injection of recombinant mouse pro-MMP-9 induced leukocyte accumulation in peritoneum. Collectively, the findings indicate that gelatinase B participates in leukocyte transmigration; however, its function can be compensated by other mechanisms.

Neonatal life is a unique developmental stage, in which the peripheral T-lymphocyte pool is evolving in a lymphopenic environment and the freshly generated T lymphocytes are extensively migrating through non-lymphoid tissues. Here, we emphasize the consequences of neonatal lymphopenia and tissue accessibility for T-lymphocyte tolerance induction. We propose that self-destructive T-lymphocyte responses are prevented in the neonate by limiting homeostatic T-lymphocyte expansion and thereby the activation of potentially autodestructive memory T lymphocytes. Furthermore, the extensive migration through tissues might lead to a dominant form of tolerance in the neonatal phase, which is maintained throughout adult life, during which the tissues are no longer readily accessible to T lymphocytes. Thus, it is not only dendritic cells that are crucial for self-tolerance: neonatal mechanisms are also required.