TGF-ß Signaling Prevents MHC Class II-Expressing Lymphatic Endothelial Cells from Reactivating Human Allogenic Memory CD4+ T Cells.

Lymphatic endothelial cells (LECs) express MHC class II (MHC-II) upon IFN-γ stimulation, yet recent evidence suggests that LECs cannot activate naive or memory CD4+ T cells. In this article, we show that IFN-γ-activated human dermal LECs can robustly reactivate allogeneic human memory CD4+ T cells (hCD4+ TMs), but only when TGF-ß signaling is inhibited. We found that in addition to upregulating MHC-II, IFN-γ also induces LECs to upregulate glycoprotein A repetitions predominant, which anchors latent TGF-ß to the membrane and potentially inhibits T cell activation. Indeed, hCD4+ TM proliferation was substantially increased when LEC-CD4+ TM cultures were treated with a TGF-ß receptor type 1 inhibitor or when glycoprotein A repetitions predominant expression was silenced in LECs. Reactivated hCD4+ TMs were characterized by their proliferation, CD25 expression, and cytokine secretion. CD4+ TM reactivation was dependent on LEC expression of MHC-II, confirming direct TCR engagement. Although CD80 and CD86 were not detected on LECs, the costimulatory molecules OX40L and ICOSL were upregulated upon cytokine stimulation; however, blocking these did not affect CD4+ TM reactivation by LECs. Finally, we found that human dermal LECs also supported the maintenance of Foxp3-expressing hCD4+ TMs independently of IFN-γ-induced MHC-II. Together, these results demonstrate a role for LECs in directly modulating CD4+ TM reactivation under inflammatory conditions and point to LEC-expressed TGF-ß as a negative regulator of this activation.

Cross-reactivity of T cell-specific antibodies in the bank vole (Myodes glareolus).

The bank vole is a common Cricetidae rodent that is a reservoir of several zoonotic pathogens and an emerging model in eco-immunology. Here, we add to a developing immunological toolkit for this species by testing the cross-reactivity of commercially available monoclonal antibodies (mAbs) to the bank vole lymphocyte differentiation molecules and a transcription factor. We show that a combination of mAbs against CD4, CD3, and Foxp3 allows flow cytometric distinction of the main subsets of T cells: putative helper CD4+, cytotoxic CD8+ (as CD3+CD4-) and regulatory CD4+Foxp3+. We also provide a comparative analysis of amino acid sequences of CD4, CD8αß, CD3εγδ and Foxp3 molecules for a number of commonly studied Cricetidae rodents and discuss mAb cross-reactivity patterns reported so far in this rodent family. We found that in case of mAbs targeting the extracellular portions of commonly used T cell markers, sequence similarity is a poor prognostic of cross-reactivity. Use of more conserved, intracellular molecules or molecule fragments is a more reliable approach in non-model species, but the necessity of cell fixation limit its application in, e.g. functional studies.

BacSp222 bacteriocin as a novel ligand for TLR2/TLR6 heterodimer.

OBJECTIVE AND DESIGN: BacSp222 bacteriocin is a bactericidal and proinflammatory peptide stimulating immune cells to produce selected cytokines and NO in NF-ĸB dependent manner. This study aims to identify the receptor which mediates this activity. METHODS: We applied fluorescently labeled BacSp222 and a confocal microscopy imaging to analyze the direct interaction of the bacteriocin with the cells. Reporter HEK-Blue cells overexpressing human toll-like receptors (TLR2, TLR4, TLR5 or TLR2/TLR1 and TLR2/TLR6 heterodimers) were stimulated with BacSp222, and then the activity of NF-ĸB-dependent secreted embryonic alkaline phosphatase (SEAP) was measured. In turn, formylated peptide receptor (FPR) or TLR2 antagonists were used to verify bacteriocin-stimulated TNF production by murine monocyte-macrophage cell lines. RESULTS: BacSp222 undergoes internalization into cells without disturbing the cell membrane. FPR antagonists do not affect TNF produced by BacSp222-stimulated murine macrophage-like cells. In contrast, BacSp222 stimulates NF-ĸB activation in HEK-Blue overexpressing TLR2 or TLR2/TLR6 heterodimer, but not TLR2/TLR1, TLR4 or TLR5 receptors. Moreover, TLR2-specific antagonists inhibit NF-ĸB signaling in BacSp222-stimulated HEK-Blue TLR2/TLR6 cells and reduce TNF release by BacSp222-treated RAW 264.7 and P388.D1. CONCLUSIONS: BacSp222 is a novel ligand for TLR2/TLR6 heterodimer. By binding TLR complex the bacteriocin undergoes internalization, inducing proinflammatory signaling that employs MyD88 and NF-ĸB pathways.

Bacteriocin BacSp222 and Its Succinylated Forms Exhibit Proinflammatory Activities Toward Innate Immune Cells .

Purpose: The zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 - an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules. Methods: The study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin - nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides. Results: BacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide. Conclusion: All naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.

In vivo Studies on Pharmacokinetics, Toxicity and Immunogenicity of Polyelectrolyte Nanocapsules Functionalized with Two Different Polymers: Poly-L-Glutamic Acid or PEG.

BACKGROUND: The functionalization of a nanoparticle surface with PEG (polyethylene glycol) is an approach most often used for extending nanomaterial circulation time, enhancing its delivery and retention in the target tissues, and decreasing systemic toxicity of nanocarriers and their cargos. However, because PEGylated nanomedicines were reported to induce immune response including production of anti-PEG antibodies, activation of the complement system as well as hypersensitivity reactions, hydrophilic polymers other than PEG are gaining interest as its replacement in nanomaterial functionalization. Here, we present the results of in vivo evaluation of polyelectrolyte nanocapsules with biodegradable, polyelectrolyte multilayer shells consisting of poly-l-lysine (PLL) and poly-l-glutamic (PGA) acid as a potential drug delivery system. We compared the effects of nanocapsules functionalized with two different "stealth" polymers as the external layer of tested nanocapsules was composed of PGA (PGA-terminated nanocapsules, NC-PGA) or the copolymer of poly-l-lysine and polyethylene glycol (PEG-terminated nanocapsules, NC-PEG). METHODS: Nanocapsules pharmacokinetics, biodistribution and routes of eliminations were analysed postmortem by fluorescence intensity measurement. Toxicity of intravenously injected nanocapsules was evaluated with analyses of blood morphology and biochemistry and by histological tissue analysis. DNA integrity was determined by comet assay, cytokine profiling was performed using flow cytometer and detection of antibodies specific to PEG was performed by ELISA assay. RESULTS: We found that NC-PGA and NC-PEG had similar pharmacokinetic and biodistribution profiles and both were eliminated by hepatobiliary and renal clearance. Biochemical and histopathological evaluation of long-term toxicity performed after a single as well as repeated intravenous injections of nanomaterials demonstrated that neither NC-PGA nor NC-PEG had any acute or chronic hemato-, hepato- or nephrotoxic effects. In contrast to NC-PGA, repeated administration of NC-PEG resulted in prolonged increased serum levels of a number of cytokines. CONCLUSION: Our results indicate that NC-PEG may cause undesirable activation of the immune system. Therefore, PGA compares favorably with PEG in equipping nanomaterials with stealth properties. Our research points to the importance of a thorough assessment of the potential influence of nanomaterials on the immune system.

Analysis of toxicity and anticancer activity of micelles of sodium alginate-curcumin.

BACKGROUND: Curcumin is a natural polyphenol with anti-inflammatory, chemopreventive and anticancer activity. However, its high hydrophobicity and poor bioavailability limit its medical application. The development of nanocarriers for curcumin delivery is an attractive approach to overcome its low bioavailability and fast metabolism in the liver. We synthesized a blood compatible alginate-curcumin conjugate, AA-Cur, which formed colloidally stable micelles of approximately 200 nm and, as previously shown, exerted strong cytotoxicity against mouse cancer cell lines. Here we analyze in vivo toxicity and antitumor activity of AA-Cur in two different mouse tumor models. METHOD: Potential toxicity of intravenously injected AA-Cur was evaluated by: i) analyses of blood parameters (morphology and biochemistry), ii) histology, iii) DNA integrity (comet assay), and iv) cytokine profiling (flow cytometry). Antitumor activity of AA-Cur was evaluated by measuring the growth of subcutaneously inoculated colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in control mice vs mice treated with AA-Cur. RESULTS: Injections of four doses of AA-Cur did not reveal any toxicity of the conjugate, thus indicating the safety of its use. AA-Cur elicited moderate anti-tumor activity toward colon MC38-CEA or breast 4T1 carcinomas. CONCLUSION: The tested conjugate of alginate and curcumin, AA-Cur, is non-toxic and safe, but exhibits limited anticancer activity.

ADAM17 Promotes Motility, Invasion, and Sprouting of Lymphatic Endothelial Cells.

Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFß2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

Antibody-based antiangiogenic and antilymphangiogenic therapies to prevent tumor growth and progression.

Blood and lymphatic vessel formation is an indispensable factor for cancer progression and metastasis. Therefore, various strategies designed to block angiogenesis and lymphangiogenesis are being investigated in the hope to arrest and reverse tumor development. Monoclonal antibodies, owing to their unequalled diversity and specificity, might be applied to selectively inhibit the pathways that cancer cells utilize to build up a network of blood vessels and lymphatics. Among the possible targets of antibody-based therapies are proangiogenic and prolymphangiogenic growth factors from the VEGF family and the receptors to which they bind (VEGFRs). Here, we present molecular mechanisms of angiogenesis and lymphangiogenesis exploited by tumors to progress and metastasise, with examples of antibody-based therapeutic agents directed at interfering with these processes. The expanding knowledge of vascular biology helps to explain some of the problems encountered in such therapies, that arise due to the redundancy in signaling networks controlling the formation of blood and lymphatic vessels, and lead to tumor drug resistance. Nonetheless, combined treatments and treatments focused on newly discovered proangiogenic and prolymphangiogenic factors give hope that more prominent therapeutic effects might be achieved in the future.

ADAM17 silencing in mouse colon carcinoma cells: the effect on tumoricidal cytokines and angiogenesis.

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.

Exogenous nitric oxide inhibits shedding of ADAM17 substrates.

Both ADAM17, the secretase responsible for the shedding of ectodomains of numerous membrane proteins including TNF and its receptors, as well as nitric oxide synthesized by inducible nitric oxide synthase play regulatory roles in inflammation and tumor progression. We analyzed the effect of endogenous and exogenous nitric oxide on the expression and activity of ADAM17 in murine endothelial cells and a monocyte/macrophage cell line. We found that endogenous nitric oxide influenced neither ADAM17 mRNA level nor the shedding of two ADAM17 substrates, TNF and TNFR1. Exogenous NO significantly diminished the release of TNF and TNFR1 without affecting the ADAM17 transcript level. Our data seem contrary to a previous report that showed the activation of ADAM17 by nitric oxide (Zhang et al., 2000, J Biol Chem 275: 15839-15844). We discuss potential mechanisms of NO-mediated inhibition of ectodomain shedding and possible reasons of discrepancy between our results and the previous report.

Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts.

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala(76):Val(77) within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.

Effects of elastase and cathepsin G on the levels of membrane and soluble TNFalpha.

Neutrophil elastase (NE) and cathepsin G (CG), the proteolytic enzymes localized in azurophil granules of neutrophils (PMN), are involved in PMN responses to various stimuli. When released at sites of inflammation, they participate in the degradation of numerous proteins involved in the regulation of the immune response. In this study, we employed ADAM17(-/-) fibroblasts stably transfected with cDNA of human pro-tumor necrosis factor alpha (proTNFalpha) (ADAM17(-/-)TNF(+)) to investigate the effects of NE and CG on shedding and degradation of TNFalpha. Both NE and CG were found to diminish the level of membrane TNFalpha (mTNFalpha) as measured by flow cytometry. This process was accompanied by the accumulation of biologically active soluble TNFalpha (sTNFalpha) in the culture medium, as determined by an increase in both the cytotoxic activity of TNFalpha and its ability to serve as a co-stimulator in the induction of inducible nitric oxide synthase (iNOS). However, in contrast to CG, NE at high concentrations was able to degrade sTNFalpha released from the cell surface. Using soluble recombinant human TNFalpha, we identified Val(93)-Ala(94) and Val(117)-Glu(118) as the NE cleavage sites within the sTNFalpha molecule. Taken together, the ability of NE and CG to modulate levels of membrane and soluble forms of TNFalpha may contribute to the proinflammatory activity of neutrophils.

Inactivation of membrane tumor necrosis factor alpha by gingipains from Porphyromonas gingivalis.

Gingipains are cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. They consist of arginine-specific (HRgpA and RgpB) and lysine-specific (Kgp) proteinases. Gingipains strongly affect the host defense system by degrading some cytokines, components of the complement system, and several immune cell receptors. In an in vitro model, gingipains were shown to degrade soluble tumor necrosis factor alpha (TNF-alpha). However, since membrane TNF-alpha shows strong biological activity, especially in local inflammatory lesions, it was worth investigating whether gingipains might also destroy membrane TNF-alpha and limit its biological activities. To avoid a possible influence of gingipains on ADAM17, the secretase of TNF-alpha, the majority of experiments were performed using ADAM17-/- fibroblasts stably transfected with cDNA of human pro-TNF-alpha (ADAM17-/- TNF+). Arginine-specific gingipains (Rgp's) strongly diminished the level of TNF-alpha on the cell surface as measured by flow cytometry, and this process was not accompanied by an increased concentration of soluble TNF-alpha in the culture medium. Degradation of membrane TNF-alpha by Rgp's correlated with a strong decrease in TNF-alpha-mediated biological activities of ADAM17-/- TNF+ cells. First, the activation state of transcription factor NF-kappaB was suppressed; second, the cells were no longer able to induce apoptosis in HL-60 cells. Kgp was also able to cleave membrane TNF-alpha, but its effect was much weaker than that of Rgp's. Gingipains also limited the binding of native TNF-alpha to the target cells. Thus, gingipains are able not only to cleave soluble TNF-alpha but also to destroy the membrane form of the cytokine, which may additionally dysregulate the cytokine network.