Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Sputum from patients with primary ciliary dyskinesia contains high numbers of dysfunctional neutrophils and inhibits efferocytosis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far, the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD, and compare them to blood neutrophils. METHODS: Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n = 32) and a control group of patients with non-PCD, non-cystic fibrosis bronchiectasis (n = 5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization, production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS: Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production, actin polymerization and the expulsion of NETs. In addition, PCD sputum displayed high activity of neutrophil elastase, and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS: Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD.

Absence of CCR2 Promotes Proliferation of Alveolar Macrophages That Control Lung Inflammation in Acute Respiratory Distress Syndrome in Mice.

Acute respiratory distress syndrome (ARDS) consists of uncontrolled inflammation that causes hypoxemia and reduced lung compliance. Since it is a complex process, not all details have been elucidated yet. In a well-controlled experimental murine model of lipopolysaccharide (LPS)-induced ARDS, the activity and viability of macrophages and neutrophils dictate the beginning and end phases of lung inflammation. C-C chemokine receptor type 2 (CCR2) is a critical chemokine receptor that mediates monocyte/macrophage activation and recruitment to the tissues. Here, we used CCR2-deficient mice to explore mechanisms that control lung inflammation in LPS-induced ARDS. CCR2-/- mice presented higher total numbers of pulmonary leukocytes at the peak of inflammation as compared to CCR2+/+ mice, mainly by enhanced influx of neutrophils, whereas we observed two to six-fold lower monocyte or interstitial macrophage numbers in the CCR2-/-. Nevertheless, the time needed to control the inflammation was comparable between CCR2+/+ and CCR2-/-. Interestingly, CCR2-/- mice presented higher numbers and increased proliferative rates of alveolar macrophages from day 3, with a more pronounced M2 profile, associated with transforming growth factor (TGF)-ß and C-C chemokine ligand (CCL)22 production, decreased inducible nitric oxide synthase (Nos2), interleukin (IL)-1ß and IL-12b mRNA expression and increased mannose receptor type 1 (Mrc1) mRNA and CD206 protein expression. Depletion of alveolar macrophages significantly delayed recovery from the inflammatory insult. Thus, our work shows that the lower number of infiltrating monocytes in CCR2-/- is partially compensated by increased proliferation of resident alveolar macrophages during the inflammation control of experimental ARDS.

COOH-terminal SAA1 peptides fail to induce chemokines but synergize with CXCL8 and CCL3 to recruit leukocytes via FPR2.

A natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4 Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.

Insufficient IL-10 Production as a Mechanism Underlying the Pathogenesis of Systemic Juvenile Idiopathic Arthritis.

Systemic juvenile idiopathic arthritis (sJIA) is a childhood-onset immune disorder of unknown cause. One of the concepts is that the disease results from an inappropriate control of immune responses to an initially harmless trigger. In the current study, we investigated whether sJIA may be caused by defects in IL-10, a key cytokine in controlling inflammation. We used a translational approach, with an sJIA-like mouse model and sJIA patient samples. The sJIA mouse model relies on injection of CFA in IFN-γ-deficient BALB/c mice; corresponding wild type (WT) mice only develop a subtle and transient inflammatory reaction. Diseased IFN-γ-deficient mice showed a defective IL-10 production in CD4+ regulatory T cells, CD19+ B cells, and CD3-CD122+CD49b+ NK cells, with B cells as the major source of IL-10. In addition, neutralization of IL-10 in WT mice resulted in a chronic immune inflammatory disorder clinically and hematologically reminiscent of sJIA. In sJIA patients, IL-10 plasma levels were strikingly low as compared with proinflammatory mediators. Furthermore, CD19+ B cells from sJIA patients showed a decreased IL-10 production, both ex vivo and after in vitro stimulation. In conclusion, IL-10 neutralization in CFA-challenged WT mice converts a transient inflammatory reaction into a chronic disease and represents an alternative model for sJIA in IFN-γ-competent mice. Cell-specific IL-10 defects were observed in sJIA mice and patients, together with an insufficient IL-10 production to counterbalance their proinflammatory cytokines. Our data indicate that a defective IL-10 production contributes to the pathogenesis of sJIA.

Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case.

The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.

Anti-inflammatory effects of the GAG-binding CXCL9(74-103) peptide in dinitrofluorobenzene-induced contact hypersensitivity in mice.

BACKGROUND: To recruit leucocytes to an inflammatory site, chemokine binding to glycosaminoglycans (GAGs) is critical. Therefore, strategies to interfere with this interaction, aiming at the production of anti-inflammatory agents, were developed. These include production of modified chemokines without affinity for G protein-coupled receptors but with enhanced affinity for GAGs. Such modified chemokines compete with functional chemokines for GAG binding, prevent chemokine immobilization and presentation, and inhibit leucocyte migration. In addition to modified chemokines, a GAG-binding peptide consisting of the 30 COOH-terminal residues of CXCL9, that is CXCL9(74-103), inhibited CXCL8- and monosodium urate crystal-induced neutrophil migration. OBJECTIVE: We wanted to explore whether interference with chemokine-GAG interactions by CXCL9(74-103) reduces inflammation in neutrophil-dependent dinitrofluorobenzene-induced contact hypersensitivity. METHODS: For this study, we evaluated several inflammatory parameters, including ear swelling and the levels of chemokines, cytokines, proteases and neutrophils in the ears of dinitrofluorobenzene-induced mice treated with CXCL9(74-103) or buffer. RESULTS: One intravenous injection of CXCL9(74-103), just before painting with dinitrofluorobenzene on the ear, did not affect protein levels of the major murine neutrophil attractant, that is CXCL6, in this contact hypersensitivity model. However, IL-6, CXCL1, CCL2 and matrix metalloproteinase-9 (MMP-9) protein concentrations and peroxidase activity in challenged ears were reduced. In addition, intravenous injection of the CXCL9-derived peptide led to a reduced ear swelling response, indicating that the locally produced chemokines were hindered to attract leucocytes. The inhibiting potential of CXCL9(74-103) was explained by its competition for GAG binding with CXCL1, CXCL6 and CCL3 and inhibition of transendothelial migration of neutrophils to CXCL6. CONCLUSIONS AND CLINICAL RELEVANCE: The CXCL9(74-103) peptide inhibited dinitrofluorobenzene-induced infiltration of neutrophils and neutrophil-dependent inflammation in ears. Therefore, CXCL9(74-103) may be a lead molecule for the development of therapeutic peptides or peptide derivatives that compete with functional chemokines for GAG binding.

Mouse Cytomegalovirus Infection in BALB/c Mice Resembles Virus-Associated Secondary Hemophagocytic Lymphohistiocytosis and Shows a Pathogenesis Distinct from Primary Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the ß-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8(+) T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV-infected IFN-γ-deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ-deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.

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

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.

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 mm(3) in COAM-treated mice compared to 755.2 mm(3) 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.

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.

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment.

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.

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

BACKGROUND: Neutrophil 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. METHODS: EAE 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. RESULTS: We 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. CONCLUSIONS: These 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.

Role for Granulocyte Colony-Stimulating Factor in Neutrophilic Extramedullary Myelopoiesis in a Murine Model of Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is a systemic inflammatory disease with childhood onset. Systemic JIA is associated with neutrophilia, including immature granulocytes, potentially driven by the growth factor granulocyte-colony stimulating factor (G-CSF). This study was undertaken to investigate the role of G-CSF in the pathology of systemic JIA. METHODS: Injection of Freund's complete adjuvant (CFA) in BALB/c mice induces mild inflammation and neutrophilia in wild-type (WT) mice and a more pronounced disease, reminiscent to that of JIA patients, in interferon-γ-knockout (IFNγ-KO) mice. Extramedullary myelopoiesis was studied in CFA-immunized mice by single-cell RNA sequencing, and the effect of G-CSF receptor (G-CSFR) blockage on neutrophil development and systemic JIA pathology was evaluated. Additionally, plasma G-CSF levels were measured in patients. RESULTS: Both in systemic JIA patients and in a corresponding mouse model, plasma G-CSF levels were increased. In the mouse model, we demonstrated that G-CSF is responsible for the observed neutrophilia and extramedullary myelopoiesis and the induction of immature neutrophils and myeloid-derived suppressor-like cells. Administration of a G-CSFR antagonizing antibody blocked the maturation and differentiation of neutrophils in CFA-immunized mice. In IFNγ-KO mice, treatment was associated with almost complete inhibition of arthritis due to reduced neutrophilia and osteoclast formation. Disease symptoms were ameliorated, but slight increases in interleukin-6 (IL-6), tumor necrosis factor, and IL-17 were detected upon G-CSFR inhibition in the IFNγ-KO mice, and were associated with mild increases in weight loss, tail damage, and immature red blood cells. CONCLUSION: We describe the role of G-CSF in a mouse model of systemic JIA and suggest an important role for G-CSF-induced myelopoiesis and neutrophilia in regulating the development of arthritis.

Inhibition of renal fibrosis with a human CXCL9-derived glycosaminoglycan-binding peptide.

OBJECTIVES: Renal fibrosis accompanies all chronic kidney disorders, ultimately leading to end-stage kidney disease and the need for dialysis or even renal replacement. As such, renal fibrosis poses a major threat to global health and the search for effective therapeutic strategies to prevent or treat fibrosis is highly needed. We evaluated the applicability of a highly positively charged human peptide derived from the COOH-terminal domain of the chemokine CXCL9, namely CXCL9(74-103), for therapeutic intervention. Because of its high density of net positive charges at physiological pH, CXCL9(74-103) competes with full-length chemokines for glycosaminoglycan (GAG) binding. Consequently, CXCL9(74-103) prevents recruitment of inflammatory leucocytes to sites of inflammation. METHODS: CXCL9(74-103) was chemically synthesised and tested in vitro for anti-fibrotic properties on human fibroblasts and in vivo in the unilateral ureteral obstruction (UUO) mouse model. RESULTS: CXCL9(74-103) significantly reduced the mRNA and/or protein expression of connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA) and collagen III by transforming growth factor (TGF)-ß1-stimulated human fibroblasts. In addition, administration of CXCL9(74-103) inhibited fibroblast migration towards platelet-derived growth factor (PDGF), without affecting cell viability. In the UUO model, CXCL9(74-103) treatment significantly decreased renal α-SMA, vimentin, and fibronectin mRNA and protein expression. Compared with vehicle, CXCL9(74-103) attenuated mRNA expression of TGF-ß1 and the inflammatory markers/mediators MMP-9, F4/80, CCL2, IL-6 and TNF-α. Finally, CXCL9(74-103) treatment resulted in reduced influx of leucocytes in the UUO model and preserved tubular morphology. The anti-fibrotic and anti-inflammatory effects of CXCL9(74-103) were mediated by competition with chemokines and growth factors for GAG binding. CONCLUSIONS: Our findings provide a scientific rationale for targeting GAG-protein interactions in renal fibrotic disease.

The Chemokine-Based Peptide, CXCL9(74-103), Inhibits Angiogenesis by Blocking Heparan Sulfate Proteoglycan-Mediated Signaling of Multiple Endothelial Growth Factors.

Growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are important angiogenesis-mediating factors. They exert their effects not only through their respective receptor tyrosine kinases (RTKs), but they also require molecular pairing with heparan sulfate proteoglycans (HSPGs). Angiogenic growth factors and their signaling pathways are commonly targeted in current anti-angiogenic cancer therapies but have unfortunately insufficient impact on patient survival. Considering their obvious role in pathological angiogenesis, HS-targeting drugs have become an appealing new strategy. Therefore, we aimed to reduce angiogenesis through interference with growth factor-HS binding and downstream signaling using a CXCL9-derived peptide with a high affinity for glycosaminoglycans (GAGs), CXCL9(74-103). We showed that CXCL9(74-103) reduced EGF-, VEGF165- and FGF-2-mediated angiogenic processes in vitro, such as endothelial cell proliferation, chemotaxis, adhesion and sprouting, without exerting cell toxicity. CXCL9(74-103) interfered with growth factor signaling in diverse ways, e.g., by diminishing VEGF165 binding to HS and by direct association with FGF-2. The dependency of CXCL9(74-103) on HS for binding to HMVECs and for exerting its anti-angiogenic activity was also demonstrated. In vivo, CXCL9(74-103) attenuated neovascularization in the Matrigel plug assay, the corneal cauterization assay and in MDA-MB-231 breast cancer xenografts. Additionally, CXCL9(74-103) reduced vascular leakage in the retina of diabetic rats. In contrast, CXCL9(86-103), a peptide with low GAG affinity, showed no overall anti-angiogenic activity. Altogether, our results indicate that CXCL9(74-103) reduces angiogenesis by interfering with multiple HS-dependent growth factor signaling pathways.

Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes.

The inflammatory human chemokine CXCL5 interacts with the G protein-coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1-78), truncated CXCL5 [CXCL5(9-78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and ß-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids.

Objectives: To explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis. Methods: Latent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines. Results: All MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort. Conclusion: Synovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

Truncation of CXCL8 to CXCL8(9-77) enhances actin polymerization and in vivo migration of neutrophils.

CXCL8 is the principal human neutrophil-attracting chemokine and a major mediator of inflammation. The chemokine exerts its neutrophil-chemotactic and neutrophil-activating activities via interaction with glycosaminoglycans (GAGs) and activation of the G protein-coupled receptors (GPCRs) CXCR1 and CXCR2. Natural CXCL8 displays an exceptional degree of amino (NH2 )-terminal heterogeneity. Most CXCL8 forms result from proteolytic processing of authentic CXCL8(1-77). Here, we compared the potencies to activate and recruit neutrophils of the 3 most abundant natural CXCL8 forms: full-length 77 amino acid CXCL8 and the 2 major natural truncated forms lacking 5 or 8 NH2 -terminal amino acids. NH2 -terminal truncation hardly affected the capacity of CXCL8 to induce shedding of CD62L or to up-regulate the expression of the adhesion molecules CD11a, CD11b, or CD15 on human neutrophils. In addition, the potency of CXCL8 to induce neutrophil degranulation and its effect on phagocytosis remained unaltered upon removal of 5 or 8 NH2 -terminal residues. However, NH2 -terminal truncation strongly potentiated CXCL8-induced actin polymerization. CXCL8(6-77) and CXCL8(9-77) showed a comparable capacity to induce Ca2+ signaling in human neutrophils and to direct in vitro neutrophil migration. Strikingly, the ability of CXCL8(9-77) to recruit neutrophils into the peritoneal cavity of mice was significantly enhanced compared to CXCL8(6-77). These results suggest that NH2 -terminal truncation influences specific biological activities of CXCL8 and indicate that CXCL8(9-77) may be the most potent neutrophil-attracting CXCL8 form in vivo.

Local Cytokine Expression Profiling in Patients with Specific Autoimmune Uveitic Entities.

Purpose: To evaluate expression of cytokines GM-CSF, IL-11, IL-12p40, IL-12p70, IL-27p28, IL-35, APRIL, BAFF, TWEAK, and LIGHT in uveitis.Methods: Aqueous humor samples from patients with active uveitis associated with Behçet's disease (BD), sarcoidosis, HLA-B27-related inflammation, and Vogt-Koyanagi-Harada (VKH) disease and control patients were assayed with a multiplex assay.Results: Comparing all patients to controls, GM-CSF, IL-11, IL-12p40, APRIL, and BAFF were significantly increased, whereas LIGHT was significantly decreased. IL-11 and BAFF were the most strongly upregulated, being elevated 19.7-fold and 14.1-fold, respectively, compared with controls. IL-11 was significantly highest in HLA-B27 uveitis. GM-CSF, IL-11, and IL-12p40 were significantly higher in nongranulomatous uveitis (BD and HLA-B27) than in granulomatous uveitis (sarcoidosis and VKH), whereas APRIL and TWEAK were significantly higher in granulomatous uveitis.Conclusions: IL-11-driven immune responses might be more potent in nongranulomatous uveitis, particularly in HLA-B27 uveitis. BAFF and APRIL might contribute to B cell-driven autoimmune response in uveitis.