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Objectives To investigate the prognostic utility of 28 serum biomarkers in systemic sclerosis (SSc), SSc-associated interstitial lung disease (SSc-ILD) and clinically relevant disease subgroups. Methods Participants with sera, high-resolution CT and lung function within 12 months of baseline were identified from the Australian Scleroderma Cohort Study. Baseline was the time of serum collection. Twenty-seven of the prespecified 28 serum biomarkers were analysed and biomarker associations with mortality and ILD progression were investigated in univariable and multivariable analyses, including within disease subgroups and combined with established risk factors for poorer prognosis in SSc. Results A total of 407 participants were identified, 252 (61.9%) with SSc-ILD. The median (interquartile range) follow-up after biomarker measurement was 6.31 (3.11–9.22) years. Sixteen biomarkers were associated with increased mortality. High levels of VCAM-1 were most strongly associated with mortality [hazard ratio (HR) 3.55; 95% CI 2.37–5.33; P <  0.001]. Five additional biomarkers had an HR >2: SP-D (2.28, 1.57–3.31; P <  0.001), E-selectin (2.19, 1.53–3.14; P <  0.001), IL-6 (2.15, 1.50–3.09; P <  0.001), MMP-3 (2.05, 1.42–2.95; P <  0.001) and ET-1 (2.03, 1.40–2.92; P <  0.001). Eleven biomarkers were independently associated with mortality following adjustment for sex, age and baseline forced vital capacity (FVC%predicted). Three biomarkers were associated with ILD progression at 1-year follow-up: CXCL4 (odds ratio 2.67, 1.46–4.88; P  = 0.001), MMP-1 (2.56, 1.43–4.59; P =  0.002) and ET-1 (2.18, 1.24–3.83; P =  0.007). Conclusion Multiple biomarkers, especially VCAM-1, E-selectin, SP-D and CXCL4, provide prognostic utility beyond that of established risk factors for patients with SSc. [ABSTRACT FROM AUTHOR]

Introduction: Systemic sclerosis (SSc) is an autoimmune disease characterized by antinuclear antibody production, which has been linked to an excess of apoptotic cells, normally eliminated by macrophages through efferocytosis. Additionally, circulating levels of CXCL4, a novel SSc biomarker, correlate with more severe fibrotic manifestations of the disease. Considering the defective efferocytosis of macrophages in SSc and the CXCL4-related M4 macrophage phenotype, we hypothesized that CXCL4 could be involved in the alteration of phagocytic functions of macrophages in SSc, including LC3-associated phagocytosis (LAP), another phagocytic process requiring autophagy proteins and contributing to immune silencing. Methods: In this study, CXCL4 levels were measured by ELISA in vitro in the serum of SSc patients, and also in vivo in the serum and lungs of C57BL/6J SSc mice induced by intradermal injections of hypochloric acid (HOCl) or Bleomycin (BLM), with evaluation of M4 markers. Circulating monocytes from healthy donors were also differentiated in vitro into M4 monocytes-derived macrophages (MDMs) in the presence of recombinant CXCL4. In M4-MDMs, phagocytosis of fluorescent beads and expression level of efferocytic receptors were evaluated by flow cytometry in vitro, while efferocytosis of pHrodo-stained apoptotic Jurkat cells was evaluated by real-time fluorescence microscopy. LAP quantification was made by fluorescence microscopy in M4-MDMs exposed to IgG-coated beads as well as apoptotic Jurkat cells. Results: Our results demonstrated that efferocytosis was significantly reduced in M0-MDMs from healthy donors exposed to the CXCL4-rich plasma of SSc patients. In vivo, CXCL4 expression was increased in the lungs of both SScmouse models, along with elevated M4 markers, while efferocytosis of BLM-mice alveolar macrophages was decreased. In vitro, M4-MDMs exhibited reduced efferocytosis compared to M0-MDMs, notably attributable to lower CD36 receptor expression and impaired phagocytosis capacities, despite enhanced LAP. Autophagic gene expression was increased both in vitro in SSc MDMs and in vivo in BLM mice, thus acting as a potential compensatory mechanism. Discussion: Altogether, our results support the role of CXCL4 on the impaired efferocytosis capacities of human macrophages from SSc patients and in SSc mice. [ABSTRACT FROM AUTHOR]

There are a limited number of effective vaccines against dengue virus (DENV) and significant efforts are being made to develop potent anti-virals. Previously, we described that platelet-chemokine CXCL4 negatively regulates interferon (IFN)-α/β synthesis and promotes DENV2 replication. An antagonist to CXCR3 (CXCL4 receptor) reversed it and inhibited viral replication. In a concurrent search, we identified CXCR3-antagonist from our compound library, namely 7D, which inhibited all serotypes of DENV in vitro. With a half-life of ~2.85 h in plasma and no significant toxicity, 7D supplementation (8 mg/kg-body-weight) to DENV2-infected IFNα/β/γR−/−AG129 or wild-type C57BL6 mice increased synthesis of IFN-α/β and IFN-λ, and rescued disease symptoms like thrombocytopenia, leukopenia and vascular-leakage, with improved survival. 7D, having the property to inhibit Sirt-1 deacetylase, promoted acetylation and phosphorylation of STAT3, which in-turn increased plasmablast proliferation, germinal-center maturation and synthesis of neutralizing-antibodies against DENV2 in mice. A STAT3-inhibitor successfully inhibited these effects of 7D. Together, these observations identify compound 7D as a stimulator of IFN-α/β/λ synthesis via CXCL4:CXCR3:p38:IRF3 signaling, and a booster for neutralizing-antibody generation by promoting STAT3-acetylation in plasmablasts, capable of protecting dengue infection. Synopsis: Effective vaccines and anti-viral drugs against dengue are limited. Our study identifies a small molecule, namely 7D as a stimulator of IFNα/β/λ synthesis, and also booster for neutralizing-antibody generation, capable of protecting dengue infection in 3 different mice models. Schematic describes the CXCL4-mediated activation of CXCR3:p38:IRF3 signaling, in turn suppression of IRF3 phosphorylation and IFNα/β/λ synthesis in monocytes and macrophages. Conversely, 7D supplementation reverses the above signaling and improves IFNα/β/λ synthesis. Besides, 7D increases acetylation and phosphorylation of STAT3, in turn promotes proliferation of plasmablasts and plasma cells, in turn increases IgG synthesis via suppression of deacetylase activity of Sirt-1. 7D is a potent anti-viral drug against dengue. Effective vaccines and anti-viral drugs against dengue are limited. Our study identifies a small molecule, namely 7D as a stimulator of IFNα/β/λ synthesis, and also booster for neutralizing-antibody generation, capable of protecting dengue infection in 3 different mice models. [ABSTRACT FROM AUTHOR]

Background: Exosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood. Methods: Exosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements. Results: Our data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes. Conclusions: The study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation. [ABSTRACT FROM AUTHOR]

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular and immunity alterations and skin/internal organ fibrosis. Aberrant levels of plasma CXCL4, CXCL4–RNA/DNA complexes, type I IFN (IFN-I) and anti-CXCL4 antibodies characterize SSc. These parameters influence each other: CXCL4–self-DNA/RNA complexes are triggers of IFN-I in plasmacytoid dendritic cells (pDCs), and anti-CXCL4 autoantibodies amplify this effect. Here, we assess the modulation over time of plasma CXCL4 and the related parameters of CXCL4–DNA/RNA complexes, anti-CXCL4 antibodies, IFN-α and TNF-α in an SSc cohort under the synthetic analogue of prostacyclin PGI2 (iloprost) treatment to address contribution of these parameters to pathogenesis and their role as biomarkers. Methods: We analyzed immunological parameters at baseline (T0) and after 3 (T3) and 6 (T6) months in 30 SSc patients. Responders were the patients that lowered their disease activity parameters after six months of treatment. Results: Anti-CXCL4 autoantibodies correlated with both IFN-α and TNF-α levels in SSc plasma. Responders significantly down-regulated serum IFN-α. In seven patients with a shorter disease duration, improvement coincides with a decrease in plasma IFN-α, CXCL4 and TNF-α. Iloprost efficiently blocks pDCs IFN-α production induced by CXCL4–DNA/RNA complexes in vitro. Conclusions: The data suggest a possible role of iloprost as a disease-modifying drug, mainly accompanied by down-regulation of plasma IFN-I levels. Since CXCL4, IFN-I and TNF-α down-modulation was evident and significant in improving SSc patients with a shorter disease duration, these results warrant future investigations on the early use of iloprost to slow SSc progression. [ABSTRACT FROM AUTHOR]

Abstract There are a limited number of effective vaccines against dengue virus (DENV) and significant efforts are being made to develop potent anti-virals. Previously, we described that platelet-chemokine CXCL4 negatively regulates interferon (IFN)-α/β synthesis and promotes DENV2 replication. An antagonist to CXCR3 (CXCL4 receptor) reversed it and inhibited viral replication. In a concurrent search, we identified CXCR3-antagonist from our compound library, namely 7D, which inhibited all serotypes of DENV in vitro. With a half-life of ~2.85 h in plasma and no significant toxicity, 7D supplementation (8 mg/kg-body-weight) to DENV2-infected IFNα/β/γR−/−AG129 or wild-type C57BL6 mice increased synthesis of IFN-α/β and IFN-λ, and rescued disease symptoms like thrombocytopenia, leukopenia and vascular-leakage, with improved survival. 7D, having the property to inhibit Sirt-1 deacetylase, promoted acetylation and phosphorylation of STAT3, which in-turn increased plasmablast proliferation, germinal-center maturation and synthesis of neutralizing-antibodies against DENV2 in mice. A STAT3-inhibitor successfully inhibited these effects of 7D. Together, these observations identify compound 7D as a stimulator of IFN-α/β/λ synthesis via CXCL4:CXCR3:p38:IRF3 signaling, and a booster for neutralizing-antibody generation by promoting STAT3-acetylation in plasmablasts, capable of protecting dengue infection.

IntroductionSystemic sclerosis (SSc) is an autoimmune disease characterized by antinuclear antibody production, which has been linked to an excess of apoptotic cells, normally eliminated by macrophages through efferocytosis. Additionally, circulating levels of CXCL4, a novel SSc biomarker, correlate with more severe fibrotic manifestations of the disease. Considering the defective efferocytosis of macrophages in SSc and the CXCL4-related M4 macrophage phenotype, we hypothesized that CXCL4 could be involved in the alteration of phagocytic functions of macrophages in SSc, including LC3-associated phagocytosis (LAP), another phagocytic process requiring autophagy proteins and contributing to immune silencing.MethodsIn this study, CXCL4 levels were measured by ELISA in vitro in the serum of SSc patients, and also in vivo in the serum and lungs of C57BL/6J SSc mice induced by intradermal injections of hypochloric acid (HOCl) or Bleomycin (BLM), with evaluation of M4 markers. Circulating monocytes from healthy donors were also differentiated in vitro into M4 monocytes-derived macrophages (MDMs) in the presence of recombinant CXCL4. In M4-MDMs, phagocytosis of fluorescent beads and expression level of efferocytic receptors were evaluated by flow cytometry in vitro, while efferocytosis of pHrodo-stained apoptotic Jurkat cells was evaluated by real-time fluorescence microscopy. LAP quantification was made by fluorescence microscopy in M4-MDMs exposed to IgG-coated beads as well as apoptotic Jurkat cells.ResultsOur results demonstrated that efferocytosis was significantly reduced in M0-MDMs from healthy donors exposed to the CXCL4-rich plasma of SSc patients. In vivo, CXCL4 expression was increased in the lungs of both SSc-mouse models, along with elevated M4 markers, while efferocytosis of BLM-mice alveolar macrophages was decreased. In vitro, M4-MDMs exhibited reduced efferocytosis compared to M0-MDMs, notably attributable to lower CD36 receptor expression and impaired phagocytosis capacities, despite enhanced LAP. Autophagic gene expression was increased both in vitro in SSc MDMs and in vivo in BLM mice, thus acting as a potential compensatory mechanism.DiscussionAltogether, our results support the role of CXCL4 on the impaired efferocytosis capacities of human macrophages from SSc patients and in SSc mice.

Background: Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. CXCL4 is a chemokine that has been reported to have pro‐inflammatory and profibrotic functions. The exact role of CXCL4 in cardiac fibrosis remains unclear. Methods: Viral myocarditis (VMC) models were induced by intraperitoneal injection of Coxsackie B Type 3 (CVB3). In vivo, CVB3 (100 TCID50) and CVB3‐AMG487 (CVB3: 100 TCID50; AMG487: 5 mg/kg) combination were administered in the VMC and VMC+AMG487 groups, respectively. Hematoxylin and eosin staining, severity score, Masson staining, and immunofluorescence staining were performed to measure myocardial morphology in VMC. Enzyme‐linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) were performed to quantify inflammatory factors (IL‐1β, IL‐6, TNF‐α, and CXCL4). Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase‐myocardial band (CK‐MB) levels were analyzed by commercial kits. CXCL4, CXCR3B, α‐SMA, TGF‐β1, Collagen I, and Collagen III were determined by Western blot and immunofluorescence staining. Results: In vivo, CVB3‐AMG487 reduced cardiac injury, α‐SMA, Collagen I and Collagen III levels, and collagen deposition in VMC+AMG487 group. Additionally, compared with VMC group, VMC+AMG group decreased the levels of inflammatory factors (IL‐1β, IL‐6, and TNF‐α). In vitro, CXCL4/CXCR3B axis activation TGF‐β1/Smad2/3 pathway promote mice cardiac fibroblasts differentiation. Conclusion: CXCL4 acts as a profibrotic factor in TGF‐β1/Smad2/3 pathway‐induced cardiac fibroblast activation and ECM synthesis, and eventually progresses to cardiac fibrosis. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC. [ABSTRACT FROM AUTHOR]

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8–induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt–dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense. RIPK3 modulates CXCL4 and TLR8 costimulation of human monocytes by augmenting inflammasome and inflammatory gene activation, while attenuating the interferon response. [ABSTRACT FROM AUTHOR]

Abstract Background Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. CXCL4 is a chemokine that has been reported to have pro‐inflammatory and profibrotic functions. The exact role of CXCL4 in cardiac fibrosis remains unclear. Methods Viral myocarditis (VMC) models were induced by intraperitoneal injection of Coxsackie B Type 3 (CVB3). In vivo, CVB3 (100 TCID50) and CVB3‐AMG487 (CVB3: 100 TCID50; AMG487: 5 mg/kg) combination were administered in the VMC and VMC+AMG487 groups, respectively. Hematoxylin and eosin staining, severity score, Masson staining, and immunofluorescence staining were performed to measure myocardial morphology in VMC. Enzyme‐linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) were performed to quantify inflammatory factors (IL‐1β, IL‐6, TNF‐α, and CXCL4). Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase‐myocardial band (CK‐MB) levels were analyzed by commercial kits. CXCL4, CXCR3B, α‐SMA, TGF‐β1, Collagen I, and Collagen III were determined by Western blot and immunofluorescence staining. Results In vivo, CVB3‐AMG487 reduced cardiac injury, α‐SMA, Collagen I and Collagen III levels, and collagen deposition in VMC+AMG487 group. Additionally, compared with VMC group, VMC+AMG group decreased the levels of inflammatory factors (IL‐1β, IL‐6, and TNF‐α). In vitro, CXCL4/CXCR3B axis activation TGF‐β1/Smad2/3 pathway promote mice cardiac fibroblasts differentiation. Conclusion CXCL4 acts as a profibrotic factor in TGF‐β1/Smad2/3 pathway‐induced cardiac fibroblast activation and ECM synthesis, and eventually progresses to cardiac fibrosis. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular and immunity alterations and skin/internal organ fibrosis. Aberrant levels of plasma CXCL4, CXCL4–RNA/DNA complexes, type I IFN (IFN-I) and anti-CXCL4 antibodies characterize SSc. These parameters influence each other: CXCL4–self-DNA/RNA complexes are triggers of IFN-I in plasmacytoid dendritic cells (pDCs), and anti-CXCL4 autoantibodies amplify this effect. Here, we assess the modulation over time of plasma CXCL4 and the related parameters of CXCL4–DNA/RNA complexes, anti-CXCL4 antibodies, IFN-α and TNF-α in an SSc cohort under the synthetic analogue of prostacyclin PGI2 (iloprost) treatment to address contribution of these parameters to pathogenesis and their role as biomarkers. Methods: We analyzed immunological parameters at baseline (T0) and after 3 (T3) and 6 (T6) months in 30 SSc patients. Responders were the patients that lowered their disease activity parameters after six months of treatment. Results: Anti-CXCL4 autoantibodies correlated with both IFN-α and TNF-α levels in SSc plasma. Responders significantly down-regulated serum IFN-α. In seven patients with a shorter disease duration, improvement coincides with a decrease in plasma IFN-α, CXCL4 and TNF-α. Iloprost efficiently blocks pDCs IFN-α production induced by CXCL4–DNA/RNA complexes in vitro. Conclusions: The data suggest a possible role of iloprost as a disease-modifying drug, mainly accompanied by down-regulation of plasma IFN-I levels. Since CXCL4, IFN-I and TNF-α down-modulation was evident and significant in improving SSc patients with a shorter disease duration, these results warrant future investigations on the early use of iloprost to slow SSc progression.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

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Simple Summary: Systemic Sclerosis (SSc) is a rare autoimmune disease characterized by scarring of the tissues and organs. This scarring, or fibrosis, has a major impact on a patient's quality of life; for example, lung fibrosis reduces the efficiency of breathing and is a significant cause of death for SSc patients. To develop novel therapeutic strategies, it is important to understand what causes fibrosis to occur and progress. It was recently found that plasmacytoid dendritic cells (pDCs) are key immune cells in the development of fibrosis in SSc. These cells are innate immune cells that are specialized in anti-viral responses, and they are also involved in autoimmune diseases. This review will examine the recent research that has started to uncover the molecular mechanisms through which pDCs impact SSc. Systemic sclerosis (SSc) is a debilitating autoimmune disease that affects multiple systems. It is characterized by immunological deregulation, functional and structural abnormalities of small blood vessels, and fibrosis of the skin, and, in some cases, internal organs. Fibrosis has a devastating impact on a patient's life and lung fibrosis is associated with high morbimortality. Several immune populations contribute to the progression of SSc, and plasmacytoid dendritic cells (pDCs) have been identified as crucial mediators of fibrosis. Research on murine models of lung and skin fibrosis has shown that pDCs are essential in the development of fibrosis, and that removing pDCs improves fibrosis. pDCs are a subset of dendritic cells (DCs) that are specialized in anti-viral responses and are also involved in autoimmune diseases, such as SSc, systemic lupus erythematosus (SLE) and psoriasis, mostly due to their capacity to produce type I interferon (IFN). A type I IFN signature and high levels of CXCL4, both derived from pDCs, have been associated with poor prognosis in patients with SSc and are correlated with fibrosis. This review will examine the recent research on the molecular mechanisms through which pDCs impact SSc. [ABSTRACT FROM AUTHOR]

CXCL4 is an important biomarker of systemic sclerosis (SSc), an incurable autoimmune disease characterized by vasculopathy and skin/internal organs fibrosis. CXCL4 contributes to the type I interferon (IFN-I) signature, typical of at least half of SSc patients, and its presence is linked to an unfavorable prognosis. The mechanism implicated is CXCL4 binding to self-DNA, with the formation of complexes amplifying TLR9 stimulation in plasmacytoid dendritic cells (pDCs). Here, we demonstrate that, upon binding to self-RNA, CXCL4 protects the RNA from enzymatic degradation. As a consequence, CXCL4-RNA complexes persist in vivo. Indeed, we show for the first time that CXCL4-RNA complexes circulate in SSc plasma and correlate with both IFN-I and TNF-α. By using monocyte-derived DCs (MDDCs) pretreated with IFN-α as a model system (to mimic the SSc milieu of the IFN-I signature), we demonstrate that CXCL4-RNA complexes induce MDDC maturation and increase, in particular, pro-inflammatory TNF-α as well as IL-12, IL-23, IL-8, and pro-collagen, mainly in a TLR7/8-dependent but CXCR3-independent manner. In contrast, MDDCs produced IL-6 and fibronectin independently in their CXCL4 RNA-binding ability. These findings support a role for CXCL4-RNA complexes, besides CXCL4-DNA complexes, in immune amplification via the modulation of myeloid DC effector functions in SSc and also during normal immune responses. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) both (Type 1 and Type 2) are one of the common causes for activation of platelet. Inflammation-induced abnormal platelet function contributes to chronic complications, which are the leading causes of death and morbidity among diabetics. Rac1 has been shown to regulate a variety of platelet functions; predicted Rac1could regulate platelet release of CXCL4, which leads to kidney injury in DM. DM' effect on Rac1 activation, a 21kD G-protein implicated in platelet activation, was investigated and platelet induced inflammation and kidney injury. Swiss albino male mice were pretreated with 5 mg/kg of a specific Rac1 inhibitor NSC23766 and injected with (45 mg/kg body wt.) streptozotocin, twice for 5 days. Moreover, the concentration of serum chemokines CXCL4 was assayed using ELISA and histology score for kidney was examined. Our results showed that DM was induced in mice by streptozotocin. In addition, platelet chemokines (CXCL4) were markedly higher in diabetic mice when compared to the sham (control) group. Moreover, pre-treatment with NSC23766 decreased liver and kidney injury assessed by histology score, P < 0.05. Our study reveals that Rac1 has a critical role in platelet chemokines secretion due to diabetes-induced inflammation in the liver and kidneys, targeting Rac1 could be a target for innovative treatment to control inflammation in diabetic individual. Targeting platelets involved in inflammatory pathways could be part of a strategy to control and manage diabetes and its consequences. [ABSTRACT FROM AUTHOR]

Objective SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production. Methods Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays. Results CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P   <  0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P   =  0.0079) leading to stabilization of HIF-2α (P   =  0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs. Conclusion TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4. [ABSTRACT FROM AUTHOR]

Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018–05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39%: 571 vs 410 ng/ml, P =.0095) and CXCL7 (+ 9%: 693 vs 636 ng/ml, P =.013) concentrations were higher compared with self-controls. Local platelet counts were lower (− 10%: 347,582 vs 383,284/µl, P =.0052), whereas neutrophil counts were elevated (+ 10%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P =.034), and between CXCL7 and MPO (r = 0.24, P =.048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r = − 0.2523, P =.0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P =.0014) and platelet counts (r = 0.288, P =.0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization. [ABSTRACT FROM AUTHOR]

• USP47 is highly expressed in both in vivo and ex vivo MI models. • USP47 inhibition improves cardiac function and suppresses inflammation. • USP47 inhibition inhibits infarction-induced cardiomyocyte pyroptosis. • USP47 inhibits the ubiquitination of IRF1 and promotes CXCL4 transcription. • USP47/IRF1/CXCL4 promotes cardiac dysfunction and pyroptosis by the NLRP3 pathway. Myocardial infarction (MI) is an event of heart attack due to the formation of plaques in the interior walls of the arteries. This study is conducted to explore the role of ubiquitin-specific peptidase 47 (USP47) in cardiac function and inflammatory immunity. MI mouse models were established, followed by an appraisal of cardiac functions, infarct size, pathological changes, and USP47 and NLRP3 levels. MI cell models were established in HL-1 cells using anoxia. Levels of cardiac function-associated proteins, USP7, interferon regulatory factor 1 (IRF1), platelet factor-4 (CXCL4), pyroptotic factors, and neutrophil extracellular traps (NETs) were determined. The bindings of IRF1 to USP47 and the CXCL4 promoter and the ubiquitination of IRF1 were analyzed. USP47 was upregulated in myocardial tissues of MI mice. USP47 inhibition alleviated cardiac functions, and decreased infarct size, pro-inflammatory cytokines, NETs, NLRP3, and pyroptosis. The ubiquitination and expression levels of IRF1 were increased by silencing USP47, and IRF1 bound to the CXCL4 promoter to promote CXCL4. Overexpression of IRF1 or CXCL4 in vitro and injection of Nigericin in vivo reversed the effect of silencing USP47 on alleviating pyroptosis and cardiac functions. Collectively, USP47 stabilized IRF1 and promoted CXCL4, further promoting pyroptosis, impairing cardiac functions, and aggravating immune inflammation through NLRP3 pathways. [ABSTRACT FROM AUTHOR]

We aimed to explore the correlations of C-X-C motif chemokine receptor 2 (CXCR2) and chemokine (C-X-C motif) ligand 4 (CXCL4) gene polymorphisms with thoracic aortic aneurysm. A total of 50 patients with thoracic aortic aneurysm (disease group) and 50 healthy people in the physical examination center (control group) in our hospital were selected as the subjects. The CXCR2 and CXCL4 gene polymorphisms were detected by means of blood drawing, DNA extraction, PCR and sequencing. Moreover, the levels of serum CXCR2 and CXCL4 were measured using ELISA, and the levels of C-reactive protein (CRP) and low-density lipoprotein (LDL) were determined. The study found significant differences in the distribution of genotypes and alleles of CXCR2 and CXCL4 gene polymorphisms between the disease group and control group. The frequencies of certain genotypes (AA of rs3890158, CC of rs2230054, AT of rs352008, and CT of rs1801572) were higher in the disease group, as were the frequencies of certain alleles (C of rs2230054 and rs1801572). The distribution of recessive models of rs2230054 was also different, with a lower frequency of CC+CT in the disease group. The haplotype distributions of both gene polymorphisms differed between the groups. CXCR2 rs3890158 and CXCL4 rs352008 were correlated with lower serum levels of their respective proteins, while CXCL4 rs1801572 was associated with CRP levels and CXCR2 rs2230054 with LDL levels in patients ( P  < 0.05). The gene polymorphisms of CXCR2 and CXCL4 probably have apparent correlations with the susceptibility to thoracic aortic aneurysm.

Background: CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis. Methods: Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4+CD25+FOXP3+Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents. Results: Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4+CD25+FOXP3+Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25+FOXP3+ mouse regulatory T cells (Tregs) cells was decreased in mouse CD4+ T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4+ T cells in vitro. Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25+FOXP3+Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4+ T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor (p

Systemic sclerosis (SSc) is a debilitating autoimmune disease that affects multiple systems. It is characterized by immunological deregulation, functional and structural abnormalities of small blood vessels, and fibrosis of the skin, and, in some cases, internal organs. Fibrosis has a devastating impact on a patient’s life and lung fibrosis is associated with high morbimortality. Several immune populations contribute to the progression of SSc, and plasmacytoid dendritic cells (pDCs) have been identified as crucial mediators of fibrosis. Research on murine models of lung and skin fibrosis has shown that pDCs are essential in the development of fibrosis, and that removing pDCs improves fibrosis. pDCs are a subset of dendritic cells (DCs) that are specialized in anti-viral responses and are also involved in autoimmune diseases, such as SSc, systemic lupus erythematosus (SLE) and psoriasis, mostly due to their capacity to produce type I interferon (IFN). A type I IFN signature and high levels of CXCL4, both derived from pDCs, have been associated with poor prognosis in patients with SSc and are correlated with fibrosis. This review will examine the recent research on the molecular mechanisms through which pDCs impact SSc.

CXCL4 is an important biomarker of systemic sclerosis (SSc), an incurable autoimmune disease characterized by vasculopathy and skin/internal organs fibrosis. CXCL4 contributes to the type I interferon (IFN-I) signature, typical of at least half of SSc patients, and its presence is linked to an unfavorable prognosis. The mechanism implicated is CXCL4 binding to self-DNA, with the formation of complexes amplifying TLR9 stimulation in plasmacytoid dendritic cells (pDCs). Here, we demonstrate that, upon binding to self-RNA, CXCL4 protects the RNA from enzymatic degradation. As a consequence, CXCL4-RNA complexes persist in vivo. Indeed, we show for the first time that CXCL4-RNA complexes circulate in SSc plasma and correlate with both IFN-I and TNF-α. By using monocyte-derived DCs (MDDCs) pretreated with IFN-α as a model system (to mimic the SSc milieu of the IFN-I signature), we demonstrate that CXCL4-RNA complexes induce MDDC maturation and increase, in particular, pro-inflammatory TNF-α as well as IL-12, IL-23, IL-8, and pro-collagen, mainly in a TLR7/8-dependent but CXCR3-independent manner. In contrast, MDDCs produced IL-6 and fibronectin independently in their CXCL4 RNA-binding ability. These findings support a role for CXCL4-RNA complexes, besides CXCL4-DNA complexes, in immune amplification via the modulation of myeloid DC effector functions in SSc and also during normal immune responses.

• CXCL4 as a biomarker in the VEDOSS and related with peripheral vasculopathy in SSc. • CXCL4 exerts its anti-angiogenesis effects by downregulating Fli-1 via the c-Abl pathway. • CXCL4 blocked the TGF -β or PDGF induced cell proliferation of HUVECs. CXCL4, a chemokine with anti-angiogenic property, is involved in systemic sclerosis (SSc) related pulmonary arterial hypertension (PAH). To investigated the contribution of CXCL4 to SSc development by focusing on the correlation of circulatory CXCL4 levels with their peripheral vasculopathy, and the effect of CXCL4 on endothelial cell dysfunction and the potential signaling. We measured the plasma CXCL4 levels in 58 patients with SSc, 10 patients with the very early diagnosis of SSc (VEDOSS), and 80 healthy controls (HCs). Then, CXCL4 concentrations were correlated with clinical features, especially the peripheral vasculopathy. These observations were further validated in an additional cohort. Moreover, we studied the anti-angiogenic effects of CXCL4 and the underlying downstream signaling in human umbilical vein endothelial cells (HUVECs) in vitro. Circulating CXCL4 levels were 103.62 % higher in patients with SSc and 201.51 % higher in patients with VEDOSS than matched HCs, which were confirmed in two independent cohorts. CXCL4 levels were associated with digital ulcers (DU) and nailfold videocapillaroscopy (NVC) abnormalities in SSc. The proliferation, migration, and tube formation of HUVECs were inhibited by CXCL4 or SSc derived plasma, which reversed by CXCL4 neutralizing antibody, but failed by CXCR3 inhibitor. CXCL4 downregulated the transcription factor Friend leukaemia integration factor‐1 (Fli-1) via c-Abl signaling. Furthermore, CXCL4 blocked the transforming growth factor (TGF) -β or platelet-derived growth factor (PDGF) induced cell proliferation of HUVECs. CXCL4 may contribute to peripheral vasculopathy in SSc by downregulating Fli-1 via c-Abl signaling in endothelial cells and interfering angiogenesis. [ABSTRACT FROM AUTHOR]

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases.

• The pyroptosis of cardiac fibroblasts occurred in the heart of VMC. • NLRP3-mediated pyroptosis of cardiac fibroblasts and production of CXCL4. • CXCL4 activates PI3K/AKT signaling to regulate cardiac fibrosis. Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. NOD-like receptor protein 3 (NLRP3) inflammation is involved in the development of myocarditis and is closely related to the form of cell death. Inhibiting pyroptosis mediated by NLRP3 inflammasome can reduce cardiac fibrosis, although its exact mechanism remains unknown. In this study, we induced Viral myocarditis (VMC) via infection of CVB3 to explore the relationship between pyroptosis and fibrosis. Our results showed that intraperitoneal injection of an NLRP3 inhibitor MCC950 or use of NLRP3−/− mice inhibited cardiac pyroptosis mediated by NLRP3 inflammasome in VMC. CXCL4 is a chemokine that has been reported to have pro-inflammatory and pro-fibrotic functions. In VMC, we further found that pyroptosis of Mouse myocardial fibroblasts (MCF) promoted the secretion of CXCL4 by activating Wnt/β-Catenin signaling. Subsequently, the transcriptome sequencing data showed that CXCL4 could promote cardiac fibrosis by activating PI3K/AKT pathway. In summary, infection of CVB3 induced host oxidative stress to further activate the NLRP3 inflammasome and ultimately lead to heart pyroptosis, in which MCF secreted CXCL4 by activating Wnt/β-Catenin signaling and CXCL4 participated in cardiac fibrosis by activating PI3K/AKT pathway. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC. [ABSTRACT FROM AUTHOR]

The high mutation rate of CTNNB1 (37 %) and Wnt-β-catenin signal-associated genes (54 %) has been notified in hepatocellular carcinoma (HCC). The activation of Wnt-β-catenin signal pathway was reported to be associated with an immune "desert" phenotype, but the underlying mechanism remains unclear. Here we mainly employed orthotopic HCC models to explore on it. Mass cytometry depicted the immune contexture of orthotopic HCC syngeneic grafts, unveiling that the exogenous expression of β-catenin significantly increased the percentage of myeloid-derived suppressor cells (MDSCs) and decreased the percentage of CD8+ T-cells. Flow cytometry and immunohistochemistry further confirmed the findings. The protein microarray analysis, Western blot and PCR identified PF4 as its downstream regulating cytokine. Intratumorally injection of cytokine PF4 enhanced the accumulation of MDSCs. Knockout of PF4 abolished the effect of β-catenin on recruiting MDSCs. Chromatin immunoprecipitation and luciferase reporter assay demonstrated that β-catenin increases the mRNA level of PF4 via binding to PF4's promoter region. In vitro chemotaxis assay and in vivo administration of specific inhibitors identified CXCR3 on MDSCs as receptor for recruiting PF4. Lastly, the significant correlations across β-catenin, PF4 and MDSCs and CD8+ T-cells infiltration were verified in HCC clinical samples. Our results unveiled HCC tumor cell intrinsic hyperactivation of β-catenin can recruit MDSC through PF4-CXCR3, which contributes to the formation of immune "desert" phenotype. Our study provided new insights into the development of immunotherapeutic strategy of HCC with CTNNB1 mutation. This study identifies PF4-CXCR3-MDSCs as a downstream mechanism underlying CTNNB1 mutation associated immune "desert" phenotype. • Orthotopic HCC models rather than subcutaneous tumor models find more liver-specific alteration. • The abnormal accumulation of MDSCs was found in β-catenin-activating orthotopic HCC models. • PF4 as a downstream target of β-catenin was essential for β-catenin-mediated MDSC accumulation. • PF4 receptor CXCR3 was responsible for the recruitment of MDSCs by PF4 and β-catenin. [ABSTRACT FROM AUTHOR]

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in multiple inflammatory and fibrotic diseases. Here, we used monocyte-derived cells as a model system to study the effects of CXCL4 exposure on dendritic cell development by integrating 65 longitudinal and paired whole genome transcriptional and methylation profiles. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. Importantly, these pro-inflammatory cells directly trigger a fibrotic cascade by producing extracellular matrix molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils that CXCL4 acts as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis.

Platelet factor 4 (CXCL4) is a chemokine abundantly stored in platelets. Upon injury and during atherosclerosis, CXCL4 is transported through the vessel wall where it modulates the function of vascular smooth muscle cells (VSMCs) by affecting proliferation, migration, gene expression and cytokine release. Variant CXCL4L1 is distinct from CXCL4 in function and expression pattern, despite a minor three-amino acid difference. Here, the effects of CXCL4 and CXCL4L1 on the phenotype and function of human VSMCs were compared in vitro. VSMCs were found to constitutively express CXCL4L1 and only exogenously added CXCL4 was internalized by VSMCs. Pre-treatment with heparin completely blocked CXCL4 uptake. A role of the putative CXCL4 receptors CXCR3 and DARC in endocytosis was excluded, but LDL receptor family members appeared to be involved in the uptake of CXCL4. Incubation of VSMCs with both CXCL4 and CXCL4L1 resulted in decreased expression of contractile marker genes and increased mRNA levels of KLF4 and NLRP3 transcription factors, yet only CXCL4 stimulated proliferation and calcification of VSMCs. In conclusion, CXCL4 and CXCL4L1 both modulate gene expression, yet only CXCL4 increases the division rate and formation of calcium-phosphate crystals in VSMCs. CXCL4 and CXCL4L1 may play distinct roles during vascular remodeling in which CXCL4 induces proliferation and calcification while endogenously expressed CXCL4L1 governs cellular homeostasis. The latter notion remains a subject for future investigation.

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in multiple inflammatory and fibrotic diseases. Here, we used monocyte-derived cells as a model system to study the effects of CXCL4 exposure on dendritic cell development by integrating 65 longitudinal and paired whole genome transcriptional and methylation profiles. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. Importantly, these pro-inflammatory cells directly trigger a fibrotic cascade by producing extracellular matrix molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils that CXCL4 acts as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Chemokines and inflammatory response of endothelial cells is crucial in the development and progression of inflammatory disease. Lipopolysaccharide (LPS) is a well‐known factor to trigger inflammatory response and induce damage of endothelial cells. The present study used lipopolysaccharide (LPS)‐treated human vascular endothelial cells (HUVECs) to investigate the function of chemokine CXC chemokine ligand 4 (CXCL4) and its receptor CXC chemokine receptor 3 (CXCR3) in inflammatory‐induced endothelial injury. LPS exposure (50, 100, 200 ng/ml) to HUVECs induced a dose‐ and time‐dependent increase in CXCL4 and CXCR3 expression at both mRNA and protein levels. The LPS‐induced endothelium hyperpermeability was inhibited by the addition of CXCL4 neutralizing antibody. Moreover, the addition of CXCL4 neutralizing antibody abolished the effects of LPS on tight junction (TJ) protein expression (occludin claudin‐4 and Zonula occluden‐1[ZO‐1]) and p38 phosphorylation, which is supported by the observation of increased TJ protein expression and decreased p38 phosphorylation in LPS‐treated HUVECs. SB203580, a p38 inhibitor, protected HUVECs from CXCL4‐stimulated damage. In conclusion, CXCL4/CXCR3, which was enhanced by LPS, may be involved in endothelial proliferation, apoptosis, and permeability via the p38 signaling pathway. [ABSTRACT FROM AUTHOR]

• CXCL4-moDCs display a pro-inflammatory phenotype when compared to conventional moDCs. • Increased IL6 and TNF production in CXCL4-moDCs relies on enhanced mRNA stability. • In CXCL4-moDCs, IL12 and IL23 are regulated at the transcriptional level. • The mRNA degrading protein TTP is inactive in CXCL4-moDCs. • p38 inhibition restores TTP function and decreases cytokine production in CXCL4-moDCs. The chemokine CXCL4 has been implicated in several immune diseases. Exposure of monocyte-derived dendritic cells (moDCs) to CXCL4 potentiates the production of inflammatory cytokines in the presence of TLR3 or TLR7/8 agonists. Here we investigated the transcriptional and post-transcriptional events underlying the augmented inflammatory responses in CXCL4-moDCs. Our results indicate that CXCL4-moDCs display an increased expression and secretion of IL-12, IL-23, IL-6 and TNF upon TLR3 activation. Analysis of the cytokine transcripts for the presence of AU-rich elements (ARE), motifs necessary for ARE-mediated mRNA decay, revealed that all these cytokine transcripts are, at least in silico , possibly regulated at the level of mRNA stability. In vitro assays confirmed that mRNA stability of IL6 and TNF , but not IL12B and IL23A, is increased in CXCL4-moDCs. We next screened the expression of ARE-binding proteins (ARE-BPs) and found that TLR stimulation of CXCL4-moDCs induced tristetraprolin (TTP or ZFP36). Increased TTP mRNA expression was found to be a consequence of TTP phospho-mediated inactivation, which over time causes the protein to degrade its own mRNA. Concomitantly with TTP inactivation, we observed increased MAPK p38 signalling, upstream of TTP, in stimulated CXCL4-moDCs. P38 inhibition restored TTP activation and subsequently reduced the production of inflammatory cytokines. Finally, TTP knockdown in moDCs resulted in an increased production of IL6 and TNF after TLR stimulation. Overall, our study shows that the pro-inflammatory phenotype of CXCL4-moDCs relies in part on enhanced cytokine mRNA stability dictated by TTP inactivation. [ABSTRACT FROM AUTHOR]

CXCL4 is mainly produced by activated platelets, and certain somatic cells and cancer cells also express CXCL4. However, the physiological function of non-platelet-derived CXCL4 is unclear. Previously, we reported that CXCL4 produced by cancer cells accelerated tumor growth by suppressing the antitumor activities of cytotoxic T lymphocytes (CTLs). To elucidate the mechanism of CXCL4 in tumor immunity, we compared the CTLs and regulatory T cells (Tregs) from CXCL4-/-, CXCR3-/- and C57BL/6 mice overexpressing CXCL4 via intramuscular electroporation. CXCL4 accelerated tumor growth in CXCL4-/- and C57BL/6 mice but not in CXCR3-/- mice. Furthermore, CXCL4 decreased CTLs proliferation and IFN-γ production and enhanced CTLs apoptosis and programmed death 1 (PD-1) expression. Conversely, CXCL4 promoted Tregs proliferation and TGF-β production and downregulated PD-1 expression in Tregs. Notably, these effects of CXCL4 were both observed in the splenic and tumor-infiltrating CTLs and Tregs from wild-type but not CXCR3-/- mice. Thus, we revealed a negative immune regulatory function for non-platelet-derived CXCL4 through CXCR3 that cancer cells could hijack to evade the host immune system, suggesting that the CXCL4/CXCR3 axis may serve as a novel target for colorectal cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Aims Macrophage phagocytosis of dead cells is a prerequisite for inflammation resolution. Because CXCL4 induces macrophage phagocytosis in vitro, we examined the impact of exogenous CXCL4 infusion on cardiac wound healing and macrophage phagocytosis following myocardial infarction (MI). Methods and results CXCL4 expression significantly increased in the infarct region beginning at Day 3 post-MI, and macrophages were the predominant source. Adult male C57BL/6J mice were subjected to coronary artery occlusion, and MI mice were randomly infused with recombinant mouse CXCL4 or saline beginning at 24 h post-MI by mini-pump infusion. Compared with saline controls, CXCL4 infusion dramatically reduced 7 day post-MI survival [10% (3/30) for CXCL4 vs. 47% (7/15) for saline, P  < 0.05] as a result of acute congestive heart failure. By echocardiography, CXCL4 significantly increased left ventricular (LV) volumes and dimensions at Day 5 post-MI (all P  < 0.05), despite similar infarct areas compared with saline controls. While macrophage numbers were similar at Day 5 post-MI, CXCL4 infusion increased Ccr4 and Itgb4 and decreased Adamts8 gene levels in the infarct region, all of which linked to CXCL4-mediated cardiac dilation. Isolated Day 5 post-MI macrophages exhibited comparable levels of M1 and M4 markers between saline and CXCL4 groups. Interestingly, by both e x vivo and in vitro phagocytosis assays, CXCL4 reduced macrophage phagocytic capacity, which was connected to decreased levels of the phagocytosis receptor CD36. In vitro, a CD36 neutralizing antibody (CD36Ab) significantly inhibited macrophage phagocytic capacity. The combination of CXCL4 and CD36Ab did not have an additive effect, indicating that CXCL4 regulated phagocytosis through CD36 signalling. CXCL4 infusion significantly elevated infarct matrix metalloproteinase (MMP)-9 levels at Day 5 post-MI, and MMP-9 can cleave CD36 as a down-regulation mechanism. Conclusion CXCL4 infusion impaired macrophage phagocytic capacity by reducing CD36 levels through MMP-9 dependent and independent signalling, leading to higher mortality and LV dilation. [ABSTRACT FROM AUTHOR]

Chemokines and their receptors participate in the development of cancers by enhancing tumor cell proliferation, angiogenesis, invasion, metastasis and penetration of tumor immune cells. It remains unclear whether CXC chemokine ligand 4 (CXCL4)/CXC chemokine receptor 3-B (CXCR3-B) can be used as an independent molecular marker for establishing prognosis for breast cancer patients. We evaluated CXCL4 and CXCR3-B expression in 114 breast cancer tissues and 30 matched noncancerous tissues using immunohistochemistry and western blot, and determined the correlation between their expression and clinicopathologic findings. We observed that breast cancer tissues express CXCL4 strongly and CXCR3-B weakly compared to noncancerous tissues. Strong CXCL4 expression was detected in 94.7% and weak CXCR3-B expression was detected in 78.9% of the tissues. Therefore, CXCL4/CXCR3-B might play a crucial role in breast cancer progression. We found no significant correlation between CXCL4 and age, tumor stage, tumor grade or TNM stage. CXCR3-B was associated significantly with tumor grade. Moreover, the Chi-square test of association showed that the expression of CXCL4/CXCR3-B might be an independent prognostic marker for breast cancer. Therefore, we suggest that CXCR3-B is an indicator of poor prognosis and may also be a chemotherapeutic target. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an SSc biomarker, predicting unfavorable prognosis and lung fibrosis. CXCL4 binds DNA/RNA and favors interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs), contributing to the type I IFN (IFN-I) signature in SSc patients. However, whether CXCL4 is an autoantigen in SSc is unknown. Here, we show that at least half of SSc patients show consistent antibody reactivity to CXCL4. T-cell proliferation to CXCL4, tested in a limited number of patients, correlates with anti-CXCL4 antibody reactivity. Antibodies to CXCL4 mostly correlate with circulating IFN-α levels and are significantly higher in patients with lung fibrosis in two independent SSc cohorts. Antibodies to CXCL4 implement the CXCL4–DNA complex’s effect on IFN-α production by pDCs; CXCL4–DNA/RNA complexes stimulate purified human B-cells to become antibody-secreting plasma cells in vitro. These data indicate that CXCL4 is indeed an autoantigen in SSc and suggest that CXCL4, and CXCL4-specific autoantibodies, can fuel a harmful loop: CXCL4–DNA/RNA complexes induce IFN-α in pDCs and direct B-cell stimulation, including the secretion of anti-CXCL4 antibodies. Anti-CXCL4 antibodies may further increase pDC stimulation and IFN-α release in vivo, creating a vicious cycle which sustains the SSc IFN-I signature and general inflammation.

It has been suggested that CXCR3 is important for nociception. Our experiments were conducted to evaluate involvement of CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11/CCL21) in neuropathic pain. Our studies give new evidence that intrathecal administration of each CXCR3 ligand induces pain-like behaviour in naive mice that occurs shortly after injection due to its location of neurons, which is confirmed by immunofluorescent staining. Moreover, intrathecal administrations of CXCL9, CXCL10, CCL21 neutralizing antibodies diminished pain-related behaviour. RT-PCR/Western blot analysis unprecedentedly showed spinal elevated levels of CXCR3 after chronic constriction injury of the sciatic nerve in rats in parallel with different time-course changes of its endogenous ligands. Initially, on day 2 we observed spinal increased levels of CXCL10 and CXCL11 indicating that these chemokines have important roles in triggering neuropathy. Then, on day 7, we observed increased levels of CXCL4, CXCL9, CXCL10. Interestingly, changes in CXCL9 level persisted until day 28, suggesting that these chemokines are responsible for long-term, persistent neuropathy. Additionally, in DRG the CXCL4, CXCL9 were elevated. The results obtained from primary glial cultures, suggests that all CXCR3 ligands can be produced in microglia, but also, except for CXCL4, in astrocytes. We provide the first evidence that in neuropathy chronic intrathecal administration of CXCR3 antagonist, (±)-NBI-74330, attenuates hypersensitivity with concomitant occurrence of microglial and some of CXCR3 ligands activation observed in the spinal cord and/or DRG level. This paper underlies the significance of CXCR3 in neuropathic pain and shows therapeutic potential of its blockade for enhancement of morphine analgesia as the major novelty of this work. [ABSTRACT FROM AUTHOR]

Surgical stress has been shown to facilitate the tumor growth and metastasis of colon cancer. To unravel the mechanisms underlying surgery induced-colon cancer progression, a syngeneic transplantation tumor model was established with murine colon cancer CT26 cells and the effect of laparotomy on tumor progression was investigated. Especially the expression of several CXC chemokines was assayed, and its roles in regulating myeloid-derived suppressor cells (MDSCs) recruitment were analyzed. We found that laparotomy promoted in vivo tumor growth and angiogenesis. CXCL4 expression was significantly downregulated by laparotomy in the tumor tissue and the peritoneal cavity. Functionally, CXCL4 overexpression significantly reduces tumor volume compared to control. Through analysis of CD11b + /Gr1 + MDSCs cell, we found an upregulated proportion of MDSCs in the tumor tissues and peritoneal cavity following laparotomy, and this enhancement was blocked after CXCL4 overexpression. Further, a negative correlation was found between CXCL4 expression and MDSC amounts in clinical samples. Higher CXCL4 expression and lower MDSCs proportion is positively related to overall survival. Conclusion Surgical trauma contributes to colon cancer progression by downregulating CXCL4 and hence promoting MDSC recruitment, which leads to an immunosuppressive environment. [ABSTRACT FROM AUTHOR]

Abstract: CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4+ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4+ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4+ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4+ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4+ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4+ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4+ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology. [ABSTRACT FROM AUTHOR]

CXCL4L1, a platelet-derived ELR-negative CXC chemokine, is a powerful angiostatic and anti-tumoral chemokine. We developed a mass spectrometric assay for the detection of different natural CXCL4L1 isoforms. Using this assay, we identified 4 different CXCL4L1 isoforms in the supernatant of thrombin-stimulated platelets from healthy volunteers: the classical isoform CXCL4L1(1-70), CXCL4L1(-4-70), which probably arises through alternative signal peptide removal and two COOH-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). CXCL4L1(1-70) was the most abundant isoform, whereas CXCL4L1(-4-70) was detected in 50% of the platelet preparations. Since alterations to the NH 2 -terminus of chemokines can have severe biological consequences, we investigated the impact of the extension with 4 NH 2 -terminal amino acids on the biological activity of CXCL4L1. In vitro , CXCL4L1(-4-70) was as potent as CXCL4L1(1-70) in inhibiting signal transduction and migration of human microvascular endothelial cells towards vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). In a FITC-conjugated dextran cell permeability assay, both splice variants showed a strong but comparable anti-permeable effect upon VEGF stimulation of the endothelial cell monolayer. In vivo angiogenesis induced by FGF-2 was equally reduced by CXCL4L1(1-70) and CXCL4L1(-4-70). In chemotaxis assays with CXCR3A-transfected cells the CXCL4L1 isoforms both induced migration from 125 ng/ml onward. Finally, CXCL4L1(1-70) and CXCL4L1(-4-70) showed the same affinity for heparin. In conclusion, the investigated biological activities of CXCL4L1 are not influenced by the four extra NH 2 -terminal residues present in the alternatively spliced isoform CXCL4L1(-4-70). Therefore, our results suggest that both isoforms equally interact with the CXCR3A and CXCR3B receptor. [ABSTRACT FROM AUTHOR]

First thought to orchestrate exclusively leukocyte trafficking, chemokines are now acknowledged for their multiple roles in the regulation of cell proliferation, differentiation, and survival. Dysregulation of their normal functions contributes to various pathologies, including inflammatory diseases and cancer. The two chemokine receptor 3 variants CXCR3-A and CXCR3-B, together with their cognate chemokines (CXCL11, CXCL10, CXCL9, CXCL4, and CXCL4L1), are involved in the control but also in the development of many tumors. CXCR3-A drives the infiltration of leukocytes to the tumor bed to modulate tumor progression (paracrine axis). Conversely, tumor-driven changes in the expression of the CXCR3 variants and their ligands promote cancer progression (autocrine axis). This review summarizes the anti- and pro-tumoral activities of the CXCR3 variants and their associated chemokines with a focus on the understanding of their distinct biological roles in the tumor microenvironment.

Although hair loss or alopecia is a common disease, its exact mechanisms are not yet well understood. The present study investigated the hypothesis that the homeostatic regulation of genes during hair regeneration may participate in hair loss, based on the cyclicity of hair growth. A cluster of such genes was identified by an expression gene-array from the dorsal skin in a depilated mouse model, and CXCL4 was identified as a significantly regulated gene during the hair regeneration process. To elucidate the function of CXCL4 in hair growth, CXCL4 activity was blocked by the administration of an anti-CXCL4 monoclonal antibody (mAb). Histomorphometric analysis indicated that anti-CXCL4 mAb induced an earlier anagen phase and delayed hair follicle regression, in contrast with that in the control group. Moreover, CXCL4 mAb upregulated the transcription levels of several hair growth-related genes, including Lef1, Wnt10b, Bmp4 and Bmp2. In addition, CXCL4 mAb increased the levels of the proliferation-related protein PCNA and Bcl-2 during the anagen phase, while it reduced the expression of pro-apoptotic protein Bax and cleaved caspase-3 during the catagen phase. These findings reveal that CXCL4 plays an important role in hair growth, and that blockade of CXCL4 activity promotes hair growth. [ABSTRACT FROM AUTHOR]

Background Nickel (Ni) is the most frequent metal allergen and induces a TH1-dependent type- IV allergy. Although Ni2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide ( LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. Objective The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. Methods BALB/ cA mice were sensitized with an i.p. injection of NiCl2 and LPS. Ten days after sensitization, mice were challenged with NiCl2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Results Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni2+. Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. Conclusions These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. [ABSTRACT FROM AUTHOR]