Platelet glycoprotein VI promotes metastasis through interaction with cancer cell-derived galectin-3.

Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis, but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM) signaling and thereby regulates diverse functions, including platelet adhesion, aggregation, and procoagulant activity. GPVI has been proposed as a safe antithrombotic target, because its inhibition is protective in models of arterial thrombosis, with only minor effects on hemostasis. In this study, the genetic deficiency of platelet GPVI in mice decreased experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses supported that mouse, as well as human GPVI, had platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knockout approach, we identified galectin-3 as the major counterreceptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed galectin-3 uses ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab')2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study revealed a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.

Inhibition of platelet GPVI induces intratumor hemorrhage and increases efficacy of chemotherapy in mice.

Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively rendered tumor vessels highly permeable and caused massive intratumoral hemorrhage. While these results established platelets as potential targets for antitumor therapy, their depletion is not a treatment option due to their essential role in hemostasis. Thus, a detailed understanding of how platelets safeguard vascular integrity in tumors is urgently demanded. Here, we show for the first time that functional inhibition of glycoprotein VI (GPVI) on the platelet surface with an antibody (JAQ1) F(ab)2 fragment rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion while not inducing systemic bleeding complications. The intratumor bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells, confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, JAQ1 F(ab)2-mediated GPVI inhibition increased intratumoral accumulation of coadministered chemotherapeutic agents, such as Doxil and paclitaxel, thereby resulting in a profound antitumor effect. In summary, our findings identify platelet GPVI as a key regulator of vascular integrity specifically in growing tumors and could serve as a basis for the development of antitumor strategies based on the interference with platelet function.

Cutting Edge: Imbalanced Cation Homeostasis in MAGT1-Deficient B Cells Dysregulates B Cell Development and Signaling in Mice.

Cation homeostasis, in relation to various immune-suppressive diseases, is a novel field of investigation. Recently, patients with a loss-of-function mutation in magnesium transporter 1 (MAGT1) were reported to present a dysregulated Mg2+ homeostasis in T lymphocytes. Using Magt1-knockout mice (Magt1-/y ), we show that Mg2+ homeostasis was impaired in Magt1-/y B cells and Ca2+ influx was increased after BCR stimulation, whereas T and NK cell function was unaffected. Consequently, mutant B cells displayed an increased phosphorylation of BCR-related proteins differentially affecting protein kinase C activation. These in vitro findings translated into increased frequencies of CD19+ B cells and marginal zone B cells and decreased frequencies of plasma cells among CD45+ splenocytes in vivo. Altogether, our study demonstrates for the first time, to our knowledge, that abolished MAGT1 function causes imbalanced cation homeostasis and developmental responses in B cells. Therefore, this study might contribute to a further understanding of B cell-related pathologies.

T cell-derived IL-10 determines leishmaniasis disease outcome and is suppressed by a dendritic cell based vaccine.

In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.

Combined in vivo depletion of glycoprotein VI and C-type lectin-like receptor 2 severely compromises hemostasis and abrogates arterial thrombosis in mice.

OBJECTIVE: Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motif-bearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. APPROACH AND RESULTS: We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. CONCLUSIONS: These results demonstrate that GPVI and CLEC-2 can be simultaneously downregulated in platelets in vivo and reveal an unexpected functional redundancy of the 2 receptors in hemostasis and thrombosis. These findings may have important implications of the potential use of anti-GPVI and anti-CLEC-2-based agents in the prevention of thrombotic diseases.

Natural killer cells support the induction of protective immunity during dendritic cell-mediated vaccination against Leishmania major.

Dendritic cell (DC)-mediated vaccination against Leishmania major induces a parasite-specific T helper 1 (Th1) response and long-lasting protective immunity in susceptible mice. As the cytokine interleukin-12 required for induction of this Th1 response is not derived from the transferred DC, but has to be produced by the vaccinated host, we examined cross-presentation of transferred DC via resident DC of the host and cross-activation with natural killer (NK) cells as mechanisms supporting the induction of protective immunity after DC-mediated vaccination. Co-culture with DC that had been conditioned ex vivo by loading with L. major lysate and stimulation with CpG-containing oligodeoxynucleotides did not result in the activation of naive DC in vitro. Furthermore, L. major antigen from conditioned DC was not cross-presented to a significant extent in vivo. In contrast, co-culture of DC with NK cells led to cross-activation of both cell populations with induction of interferon-γ, which was dependent on the activation status of the conditioned DC. Transient depletion of NK cells during vaccination of L. major-susceptible mice with conditioned DC resulted in reduced protection. Our findings indicate that cross-presentation of conditioned DC after DC-based vaccination against L. major plays a minor role in the induction of protective immunity. However, we demonstrated for the first time that the capacity of DC to mediate protection against L. major is supported by cross-activation with NK cells of the host and NK-cell-derived interferon-γ.

Fragments of antigen-loaded dendritic cells (DC) and DC-derived exosomes induce protective immunity against Leishmania major.

Upon loading with parasite antigen and adoptive transfer, dendritic cells (DC) are able to confer protection against the protozoan parasite Leishmania major. In the present study, we investigated whether viable DC are required for inducing protection. We provide evidence that L. major antigen-loaded DC that had been fixed with paraformaldehyde or exposed to UV irradiation, and even disrupted cells, are able to serve as an effective vaccine. Furthermore, we demonstrate the potential of DC-derived exosomes to mediate protective immunity against cutaneous leishmaniasis. The route of antigen presentation to recipient T cells involves uptake of intravenously injected DC fragments into late endosomal compartments of splenic DC in the recipient. In vitro studies showed that DC fragments induce T-cell proliferation and interleukin 12 secretion by splenocytes. Together, these findings suggest that the development of a cell-free vaccine for immunoprophylaxis against leishmaniasis and other infectious diseases is feasible.

Phospholipase PlaB is a new virulence factor of Legionella pneumophila.

We previously identified Legionella pneumophila PlaB as the major cell-associated phospholipase A/lysophospholipase A with contact-dependent hemolytic activity. In this study, we further characterized this protein and found it to be involved in the virulence of L. pneumophila. PlaB was mainly expressed and active during exponential growth. Active PlaB was outer membrane-associated and at least in parts surface-exposed. Transport to the outer membrane was not dependent on the type I (T1SS), II (T2SS), IVB (T4BSS) or Tat secretion pathways. Furthermore, PlaB activity was not dependent on the presence of the macrophage infectivity potentiator (Mip) or the major secreted zinc metalloproteinase A (MspA). Despite the fact that PlaB is not essential for replication in protozoa or macrophage cell lines, we found that plaB mutants were impaired for replication in the lungs and dissemination to the spleen in the guinea pig infection model. Histological sections monitored less inflammation and destruction of the lung tissue after infection with the plaB mutants compared to L. pneumophila wild type. Taken together, PlaB is the first phospholipase A/lysophospholipase A with a confirmed role in the establishment of Legionnaires' disease.

Split immune response after oral vaccination of mice with recombinant Escherichia coli Nissle 1917 expressing fimbrial adhesin K88.

Enterotoxigenic Escherichia coli (ETEC) are a leading cause of diarrhoea in piglets and newborn calves. Massive efforts have therefore been made to develop a vaccine for the induction of protective mucosal immunity against ETEC. Since it has been shown that the probiotic strain E. coli Nissle 1917 (EcN) can serve as a safe carrier for targeted delivery of recombinant molecules to the intestinal mucosa, we constructed the recombinant strain EcN pMut2-kanK88 (EcN-K88) stably expressing the determinant for the K88 fimbrial adhesin of ETEC on the bacterial surface. After oral application of EcN-K88 to mice for one week, EcN-K88 as well as wild-type EcN and EcN mock-transformed with the plasmid vector only could be detected in faecal samples for a minimum of 7 days after the last feeding, indicating that EcN can transiently colonise the murine intestine. Oral application of EcN-K88 resulted in significant IgG serum titres against K88 as early as 7 days after the initial feeding with EcN-K88, but no significant IgA titres. In contrast, we failed to detect any specific T cell responses towards the K88 antigen both in spleen and mesenteric lymph nodes. Although dendritic cells readily upregulated maturation and activation markers in response to K88 stimulation, accompanied by secretion of interleukin (IL)-12, IL-6, IL-10, and tumour necrosis factor, restimulation of T cells from mice having received EcN-K88 with K88-loaded dendritic cells did not result in detectable T cell proliferation and IL-2 secretion, but rather induced an IL-10 bias. While the serum antibody responses clearly demonstrate that K88 is recognized by the humoral immune system, our findings indicate that oral application of probiotic EcN expressing the K88 fimbrial adhesin does not induce a selective T cell response towards the antigen.

Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice.

DC-based vaccination against Leishmania major induces a parasite-specific Th1 response and long-lasting protective immunity in susceptible mice. Since distinct DC subsets have been proposed to direct the predominant development of either Th1 or Th2 cells, we analyzed the capability of plasmacytoid DC (pDC) to induce protection and elicit a Th1 response against L. major. Pulsing with L. major lysate induced the activation and maturation of semi-mature murine pDC that had been isolated from the spleen, as indicated by up-regulation of the co-stimulatory molecules CD86 and CD80, but did not enhance the level of IFN-alpha secretion by pDC. Vaccination of susceptible mice with L. major lysate-pulsed pDC induced highly effective T cell-mediated immunity against subsequent infection with L. major parasites. Surprisingly, the protection was not accompanied by a polarized Th1 cytokine profile. Co-activation of pDC with CpG-containing oligodeoxynucleotides, which has been shown to be critical for activating the protective potential of myeloid DC, was not required for the protective effect of L. major antigen-pulsed pDC. These findings demonstrate that antigen-loaded pDC are able to induce T cell-mediated protection against a parasite disease and that experimental leishmaniasis is a suitable model to elucidate the mechanisms underlying DC-based vaccination against infections.

Human monocytoid cells as a model to study Toll-like receptor-mediated activation.

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors.

Nitric oxide and metalloproteinases in canine articular ligaments: a comparison between the cranial cruciate, the medial genual collateral and the femoral head ligament.

Osteoarthritis due to cranial cruciate ligament (CCL) rupture or hip dysplasia is one of the most important causes of chronic lameness in dogs. This study aimed at comparing nitric oxide (NO) production by the CCL with that of the femoral head ligament (FHL) and the medial collateral ligament (MCL), and investigating the pathway of NO production and the concomitant metalloproteinase (MMP) activity in the presence or absence of an inflammatory stimulus. Ligaments of normal dogs were subjected to different stimuli, and NO and MMP activity from explant culture supernatants were compared. The results showed that in explant cultures of the canine CCL more NO was produced than in those of the other two ligaments. A higher level of NO was produced when CCLs were exposed to the inducible nitric oxide synthase (iNOS)-inducing cocktail TNF/IL-1/LPS, and NO synthesis could be inhibited by both l-NMMA, a general nitric oxide synthase (NOS) inhibitor and l-NIL, a specific iNOS inhibitor. However, a correlation between NO synthesis and iNOS expression levels as determined by immunohistochemistry was not observed. In contrast to CCL, no evidence for iNOS-dependent NO synthesis was observed for MCL and FHL. The CCL produced less MMP than MCL and FHL, and no correlation between MMP and NO could be demonstrated. MMP activity in the CCL increased significantly after 48 h of incubation with the inflammatory stimulus. The results suggest that in canine osteoarthritis NO synthesized by canine CCL plays a more important role in the pathogenesis of osteoarthritis of the stifle than that synthesized by FHL and MCL.

Toll-like receptor-4 is involved in eliciting an LPS-induced oxidative burst in neutrophils.

The lipopolysaccharide (LPS) receptor complex of mononuclear phagocytes is composed of Toll-like receptor-4 (TLR4), MD-2 and CD14. Other phagocyte populations may express similar LPS receptors. The transmembrane glycoprotein TLR4 was shown to induce or upregulate a variety of gene products, which collectively are the mediators of an LPS effect. In this study, an involvement of TLR4 in mediation of an oxidative burst was determined using murine peritoneal exsudate neutrophils and lucigenin-enhanced chemiluminescence (CL). The CL response was dependent on the LPS dose and the presence of serum, putatively a source of lipopolysaccharide-binding protein (LBP). In the absence of serum, a CL signal was elicited by 4 microg/ml LPS in peritoneal exsudate cells (PEC) from TLR4-sufficient (C3H/HeN) but not TLR4 deficient (C3H/HeJ) mice. The signal obtained in PEC from TLR4-sufficient mice was completely abrogated by superoxide dismutase (SOD), which indicated that the response depended on the formation of superoxide anion, and was also seen in purified neutrophils but not purified macrophages (Mphi). In the presence of serum, lower LPS concentrations (e.g. 40 ng/ml) elicited a strong CL response in PEC from TLR4-sufficient, and a weak signal in cells from TLR-4-deficient mice. This suggests that TLR4 engagement is involved in promoting an oxidative burst in murine neutrophils.

[The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes]. / Zur Rolle von Stickoxyd bei der Listerienenzephalitis der Wiederkäuer und bei intrazisternal infizierten Ratten.

Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts. In ruminants, infection with L. monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy. Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis. iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat. iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats. This is indirect evidence for differences in the ability to produce NO in the three species. Presence of iNOS and NT were inversely correlated with the numbers of bacteria. While microabscesses of goats contained high amounts of L. monocytogenes they occurred only rarely in cattle. To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed. Eleven day old infant rats were infected intracisternally with a low dose of L. monocytogenes. This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality. Listeria proliferated strongly in the inflammatory lesions during the first days of infection, reached a peak at day 4 and were eliminated until day 7. The presence of bacteria was closely accompanied by high numbers of iNOS-expressing M phi and the formation of NT. Administration of the iNOS inhibitor L-NIL or the radical scavenger PBN resulted in rapid death of the treated animals. However, the increase in bacterial numbers was one order of magnitude higher for animals treated with PBN compared with L-NIL administration. This shows that NO plays an important role in the control of a brain infection with Listeria, but suggests that reactive oxidants other than NO are also involved. In conclusion, our findings point to a possible involvement of the differences in the ability to express iNOS and subsequent NO production in the different clinical outcome of listeria encephalitis in cattle and small ruminants.