Vincent Vanheule

The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74–103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice

Background: Chemokines, such as CXCL8 and CXCL9, drive leukocyte migration to an inflammation site. Results: CXCL9(74–103), derived from CXCL9, lacks leukocyte-attracting activity but competes with CXCL8 for GAG binding and inhibits neutrophil migration in two murine acute inflammation models. Conclusion: Through inhibition of chemokine-GAG interaction, CXCL9(74–103) blocks neutrophil migration. Significance: CXCL9(74–103) may be a lead molecule for development of anti-inflammatory agents. The ELR−CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74–103)) being the most potent. The COOH-terminal peptide CXCL9(74–103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74–103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74–103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74–103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.

Natural nitration of CXCL12 reduces its signaling capacity and chemotactic activity in vitro and abrogates intra-articular lymphocyte recruitment in vivo

The chemokine CXCL12/stromal cell-derived factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and stimulates tumor development. In vitro, CXCL12 activity through CXCR4 is abolished by proteolytic processing. However, limited information is available on in vivo effects of posttranslationally modified CXCL12. Natural CXCL12 was purified from the coculture supernatant of stromal cells stimulated with leukocytes and inflammatory agents. In this conditioned medium, CXCL12 with a nitration on Tyr7, designated [3-NT7]CXCL12, was discovered via Edman degradation. CXCL12 and [3-NT7]CXCL12 were chemically synthesized to evaluate the biological effects of this modification. [3-NT7]CXCL12 recruited β-arrestin 2 and phosphorylated the Akt kinase similar to CXCL12 in receptor-transfected cells. Also the affinity of CXCL12 and [3-NT7]CXCL12 for glycosaminoglycans, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3 were comparable. However, [3-NT7]CXCL12 showed a reduced ability to enhance intracellular calcium concentrations, to generate inositol triphosphate, to phosphorylate ERK1/2 and to induce monocyte and lymphocyte chemotaxis in vitro. Moreover, nitrated CXCL12 failed to induce in vivo extravasation of lymphocytes to the joint. In summary, nitration on Tyr7 under inflammatory conditions is a novel natural posttranslational regulatory mechanism of CXCL12 which may downregulate the CXCR4-mediated inflammatory and tumor-promoting activities of CXCL12.

Overview of the Mechanisms that May Contribute to the Non-Redundant Activities of Interferon-Inducible CXC Chemokine Receptor 3 Ligands

The inflammatory chemokines CXCL9, CXCL10, and CXCL11 are predominantly induced by interferon (IFN)-γ and share an exclusive chemokine receptor named CXC chemokine receptor 3 (CXCR3). With a prototype function of directing temporal and spatial migration of activated T cells and natural killer cells, and inhibitory effects on angiogenesis, these CXCR3 ligands have been implicated in infection, acute inflammation, autoinflammation and autoimmunity, as well as in cancer. Intense former research efforts led to recent and ongoing clinical trials using CXCR3 and CXCR3 ligand targeting molecules. Scientific evidence has claimed mutual redundancy, ligand dominance, collaboration or even antagonism, depending on the (patho)physiological context. Most research on their in vivo activity, however, illustrates that CXCL9, CXCL10, and CXCL11 each contribute to the activation and trafficking of CXCR3 expressing cells in a non-redundant manner. When looking into detail, one can unravel a multistep machinery behind final CXCR3 ligand functions. Not only can specific cell types secrete individual CXCR3 interacting chemokines in response to certain stimuli, but also the receptor and glycosaminoglycan interactions, major associated intracellular pathways and susceptibility to processing by particular enzymes, among others, seem ligand-specific. Here, we overview major aspects of the molecular properties and regulatory mechanisms of IFN-induced CXCR3 ligands, and propose that their in vivo non-redundancy is a reflection of the unprecedented degree of versatility that seems inherent to the IFN-related CXCR3 chemokine system.

The chemokine fragment CXCL9(74–103) diminishes neutrophil recruitment and joint inflammation in antigen‐induced arthritis

This study investigates if treatment with a peptide corresponding to the 30 C‐terminal amino acids of CXCL9, CXCL9(74–103), ameliorates joint inflammation in a murine model of antigen‐induced arthritis (AIA). AIA was induced in male C57BL/6J mice. Intravenous injection of CXCL9(74–103), simultaneously performed with a tibiofemoral challenge with methylated BSA (mBSA) as antigen in mice immunized with mBSA, diminished the accumulation of leukocytes, in particular neutrophils, in the synovial cavity. The levels of the chemokines CXCL1, CXCL2, and CXCL6 and of the cytokine IL‐6 were decreased in inflamed periarticular tissue of mice treated with the CXCL9‐derived peptide compared to non‐treated AIA mice. In addition, CXCL9(74–103) treatment substantially reduced joint and cartilage damage. CXCL9(74‐103) competes with CXCL6 and CCL3 for binding to the glycosaminoglycans heparan sulfate and chondroitin sulfate in vitro. In vivo, CXCL9(74‐103) quickly binds to blood vessels in joints as observed by confocal microscopy. Next, we evaluated if later treatment with CXCL9(74–103) had a beneficial impact on joint inflammation. CXCL9(74‐103) injection 6 h after mBSA challenge still reduced neutrophil accumulation in the joint, although it did not reduce chemokine and IL‐6 concentrations. However, a delay of treatment until 12 h after challenge had no effect on cell recruitment and chemokine and IL‐6 levels. Taken together, we demonstrated that treatment with a peptide, which interferes with the interaction between chemokines and glycosaminoglycans, from the beginning of the disease controlled the massive accumulation of neutrophils in the joint of AIA mice, greatly impacting on joint inflammation and tissue damage.