Nele Berghmans

CXCR4 and CCR5 ligands cooperate in monocyte and lymphocyte migration and in inhibition of dual-tropic (R5/X4) HIV-1 infection

One of the most important functions of chemokines and their receptors is the regulation of directional migration of leukocytes within tissues. In specific tissue compartments, cells are exposed to multiple chemokines presented in complex dimensional and temporal patterns. Therefore, a leukocyte requires the mechanisms to integrate the various directional signals it receives from different chemoattractants. In this study, we report that CCL3, CCL5, and CCL8, three potent mononuclear cell chemoattractants, are able to synergize with the homeostatic chemokine CXCL12 in the migration of CD14+ monocytes, CD3+ T‐lymphocytes, or PHA‐activated lymphoblasts. In addition, CCL5 augmented the CXCR4 ligand‐driven ERK phosphorylation in mononuclear cells. Furthermore, the synergistic effect between CCL5 and CXCL12 in monocyte chemotaxis is inhibited in the presence of specific CCR1 antibody and AMD3100, but not by maraviroc. In HIV‐1 infection assays, a combination of CXCL12 and CCL5 cooperated to inhibit the replication of the dual‐tropic (R5/X4) HIV‐1 HE strain. Finally, although the dual‐tropic HIV‐1 strain was barely suppressed by AMD3100 or maraviroc alone, HIV‐1 infection was completely blocked by the combination of these two receptor antagonists. Our data demonstrate the cooperation between CCL5 and CXCL12, which has implications in migration of monocytes/lymphocytes during inflammation and in HIV‐1 infection.

Isotypic neutralizing antibodies against mouse GCP-2/CXCL6 inhibit melanoma growth and metastasis

The chemokine granulocyte chemotactic protein (GCP)-2/CXCL6 promotes tumor growth as angiogenesis inducer and neutrophil chemoattractant. The neutralizing capacity and specificity of monoclonal mouse anti-murine (mu)GCP-2/CXCL6 antibodies were evidenced by granulocyte chemotaxis and signaling assays. The half-life of the non-antigenic antibody in the blood circulation was approximately 15 days. The titers remained constant upon weekly injection. Tumor growth and lymphogenic metastases of human melanoma over-expressing muGCP-2 were reduced in mice treated with anti-muGCP-2. Moreover, the drop in muGCP-2 antibody titer correlated with the melanoma tumor size. Taken together, we show that functional blocking of GCP-2 inhibits tumor growth and metastases.

Increase in Sialylation and Branching in the Mouse Serum N-glycome Correlates with Inflammation and Ovarian Tumour Progression

Ovarian cancer is the most lethal gynaecological cancer and is often diagnosed in late stage, often as the result of the unavailability of sufficiently sensitive biomarkers for early detection, tumour progression and tumour-associated inflammation. Glycosylation is the most common posttranslational modification of proteins; it is altered in cancer and therefore is a potential source of biomarkers. We investigated the quantitative and qualitative effects of anti-inflammatory (acetylsalicylic acid) and pro-inflammatory (thioglycolate and chlorite-oxidized oxyamylose) drugs on glycosylation in mouse cancer serum. A significant increase in sialylation and branching of glycans in mice treated with an inflammation-inducing compound was observed. Moreover, the increases in sialylation correlated with increased tumour sizes. Increases in sialylation and branching were consistent with increased expression of sialyltransferases and the branching enzyme MGAT5. Because the sialyltransferases are highly conserved among species, the described changes in the ovarian cancer mouse model are relevant to humans and serum N-glycome analysis for monitoring disease treatment and progression might be a useful biomarker.

Interferon-γ orchestrates the number and function of Th17 cells in experimental autoimmune encephalomyelitis

Th17 cells are suggested to be pathogenic in mediating experimental autoimmune encephalomyelitis (EAE), but their relation to interferon (IFN)-γ-producing Th1 cells in vivo is not well understood. We studied the numbers and functions of Th17 cells in CREAE in Biozzi ABH mice, both in peripheral lymphoid organs and in the central nervous system. Th1 and Th17 cells alternated in secondary lymphoid organs and infiltrated into the central nervous system during chronic relapsing experimental autoimmune encephalomyelitis (CREAE). In the absence of IFN-γ the numbers and secretion of Th17 cells was enhanced, whereas exogenous administration of IFN-γ decreased the Th17 cells. In mice with intact IFN-γ genes, in vivo neutralization of interleukin (IL)-17 protected against EAE development by enhancing the number of IFN-γ-producing cells. IFN-γ knockout mice were partially protected by anti-IL-17 antibodies by decreasing cell numbers and production of IL-17. Our findings suggest that, whereas IFN-γ as such is not necessary for EAE development in the mouse, the lack of suppression of Th17 cells by IFN-γ enhances the susceptibility to develop EAE. IFN-γ thus orchestrates the number and function of Th17 cells.

Intradermal air pouch leukocytosis as an in vivo test for nanoparticles

The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that – after first-line in vitro assays – characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.

Serum amyloid A1α induces paracrine IL‐8/CXCL8 via TLR2 and directly synergizes with this chemokine via CXCR2 and formyl peptide receptor 2 to recruit neutrophils

Cell migration depends on the ability of leukocytes to sense an external gradient of chemotactic proteins produced during inflammation. These proteins include chemokines, complement factors, and some acute phase proteins, such as serum amyloid A. Serum amyloid A chemoattracts neutrophils, monocytes, and T lymphocytes via its G protein‐coupled receptor formyl peptide receptor 2. We demonstrate that serum amyloid A1α more potently chemoattracts neutrophils in vivo than in vitro. In contrast to CD14+ monocytes, no rapid (within 2 h) induction of interleukin‐8/CXC chemokine ligand 8 or macrophage‐inflammatory protein‐1α/CC chemokine ligand 3 was observed in purified human neutrophils after stimulation of the cells with serum amyloid A1α or lipopolysaccharide. Moreover, interleukin‐8/CXC chemokine ligand 8 induction in monocytes by serum amyloid A1α was mediated by toll‐like receptor 2 and was inhibited by association of serum amyloid A1α with high density lipoprotein. This indicates that the potent chemotactic response of neutrophils toward intraperitoneally injected serum amyloid A1α is indirectly enhanced by rapid induction of chemokines in peritoneal cells, synergizing in a paracrine manner with serum amyloid A1α. We observed direct synergy between IL‐8/CXC chemokine ligand 8 and serum amyloid A1α, but not lipopolysaccharide, in chemotaxis and shape change assays with neutrophils. Furthermore, the selective CXC chemokine receptor 2 and formyl peptide receptor 2 antagonists, SB225002 and WRW4, respectively, blocked the synergy between IL‐8/CXC chemokine ligand 8 and serum amyloid A1α in neutrophil chemotaxis in vitro, indicating that for synergy their corresponding G protein‐coupled receptors are required. Additionally, SB225002 significantly inhibited serum amyloid A1α‐mediated peritoneal neutrophil influx. Taken together, endogenous (e.g., IL‐1β) and exogenous (e.g., lipopolysaccharide) inflammatory mediators induce primary chemoattractants such as serum amyloid A that synergize in an autocrine (monocyte) or a paracrine (neutrophil) fashion with secondary chemokines induced in stromal cells.

Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes

BackgroundNeutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.MethodsEAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.ResultsWe discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35-55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.ConclusionsThese results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection

Insight into molecular and cellular mechanisms of innate immunity is critical to understand viral pathogenesis and immunopathology and might be exploited for therapy. Whereas the molecular mechanisms of the IFN defense are well established, cellular mechanisms of antiviral immunity are only emerging, and their pharmacological triggering remains unknown. COAM is a polysaccharide derivative with antiviral activity but without comprehension about its mechanism of action. The COAM mixture was fractionated, and prophylactic treatment of mice with COAM polymers of high MW resulted in a conversion from 100% lethal mengovirus infection to an overall survival rate of 93% without obvious clinical sequelae. Differential and quantitative analysis of peritoneal leukocytes demonstrated that COAM induced a profound influx of neutrophils. Selective cell depletion experiments pointed toward neutrophils and macrophages as key effector cells in the rescue of mice from lethal mengovirus. COAM was able to induce mRNA and protein expression of the mouse neutrophil chemokine GCP‐2. Binding of GCP‐2 to COAM was demonstrated in solution and confirmed by SPR technology. Although COAM was not chemotactic for neutrophils, COAM‐anchored muGCP‐2 retained chemotactic activity for human and mouse neutrophils. In conclusion, this study established that COAM rescued mice from acute and lethal mengovirus infection by recruiting antiviral leukocytes to the site of infection, as proposed through the induction, binding, and concentration of endogenous chemokines. These findings reinforce the role of neutrophils and macrophages as critical cells that can be manipulated toward antiviral defense.

CXCL9-derived peptides differentially inhibit neutrophil migration in vivo through interference with glycosaminoglycan interactions

Several acute and chronic inflammatory diseases are driven by accumulation of activated leukocytes due to enhanced chemokine expression. In addition to specific G protein-coupled receptor-dependent signaling, chemokine–glycosaminoglycan (GAG) interactions are important for chemokine activity in vivo. Therefore, the GAG–chemokine interaction has been explored as target for inhibition of chemokine activity. It was demonstrated that CXCL9(74-103) binds with high affinity to GAGs, competed with active chemokines for GAG binding and thereby inhibited CXCL8- and monosodium urate (MSU) crystal-induced neutrophil migration to joints. To evaluate the affinity and specificity of the COOH-terminal part of CXCL9 toward different GAGs in detail, we chemically synthesized several COOH-terminal CXCL9 peptides including the shorter CXCL9(74-93). Compared to CXCL9(74-103), CXCL9(74-93) showed equally high affinity for heparin and heparan sulfate (HS), but lower affinity for binding to chondroitin sulfate (CS) and cellular GAGs. Correspondingly, both peptides competed with equal efficiency for CXCL8 binding to heparin and HS but not to cellular GAGs. In addition, differences in anti-inflammatory activity between both peptides were detected in vivo. CXCL8-induced neutrophil migration to the peritoneal cavity and to the knee joint were inhibited with similar potency by intravenous or intraperitoneal injection of CXCL9(74-103) or CXCL9(74-93), but not by CXCL9(86-103). In contrast, neutrophil extravasation in the MSU crystal-induced gout model, in which multiple chemoattractants are induced, was not affected by CXCL9(74-93). This could be explained by (1) the lower affinity of CXCL9(74-93) for CS, the most abundant GAG in joints, and (2) by reduced competition with GAG binding of CXCL1, the most abundant ELR+ CXC chemokine in this gout model. Mechanistically we showed by intravital microscopy that fluorescent CXCL9(74-103) coats the vessel wall in vivo and that CXCL9(74-103) inhibits CXCL8-induced adhesion of neutrophils to the vessel wall in the murine cremaster muscle model. Thus, both affinity and specificity of chemokines and the peptides for different GAGs and the presence of specific GAGs in different tissues will determine whether competition can occur. In summary, both CXCL9 peptides inhibited neutrophil migration in vivo through interference with GAG interactions in several animal models. Shortening CXCL9(74-103) from the COOH-terminus limited its GAG-binding spectrum.

Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function

Chlorite‐oxidized oxyamylose (COAM), a glycosaminoglycan mimetic and potent antiviral agent, provided significant growth reduction of syngeneic murine B16‐F1 melanoma tumors. A single early dose (100 μg, into the site of tumor cell inoculation) was sufficient to establish a persistent effect over 17 days (resected tumor volume of 78.3 mm3 in COAM‐treated mice compared to 755.2 mm3 in the control cohort, i.e., 89.6% reduction of tumor volumes). COAM was a much better antitumoral agent than the polyanionic glycosaminoglycan heparin. COAM retained its antitumoral effect in lymphopenic mice, reinforcing the idea of myeloid cell involvement. Massive recruitment of myeloid cells into dermal air pouches in response to COAM and their increased presence in early‐treated tumors indicated that mainly CD11b+ GR‐1+ myeloid cells were attracted by COAM to exert antitumoral effects. Leukocyte chemotaxis was mediated by the chemokine system through the induction in B16‐F1 cells of mouse granulocyte chemotactic protein‐2/CXCL6 upon COAM treatment. Thus, COAM constitutes a novel tool to study the role of innate immune cells in the initial stages of tumor development and an example that innate immunostimulating glycosaminoglycan mimicry may be exploited therapeutically.