Role of deubiquitinase USP47 in cardiac function alleviation and anti-inflammatory immunity after myocardial infarction by regulating NLRP3 inflammasome-mediated pyroptotic signal pathways.

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.

CXCL4/CXCR3 axis regulates cardiac fibrosis by activating TGF-ß1/Smad2/3 signaling in mouse viral myocarditis.

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-1ß, 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-ß1, 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-1ß, IL-6, and TNF-α). In vitro, CXCL4/CXCR3B axis activation TGF-ß1/Smad2/3 pathway promote mice cardiac fibroblasts differentiation. CONCLUSION: CXCL4 acts as a profibrotic factor in TGF-ß1/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.

CXCL4:NLRP3-mediated pyroptosis product that regulates 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/ß-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/ß-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.

Hyperactivation of ß-catenin signal in hepatocellular carcinoma recruits myeloid-derived suppressor cells through PF4-CXCR3 axis.

The high mutation rate of CTNNB1 (37 %) and Wnt-ß-catenin signal-associated genes (54 %) has been notified in hepatocellular carcinoma (HCC). The activation of Wnt-ß-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 ß-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 ß-catenin on recruiting MDSCs. Chromatin immunoprecipitation and luciferase reporter assay demonstrated that ß-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 ß-catenin, PF4 and MDSCs and CD8+ T-cells infiltration were verified in HCC clinical samples. Our results unveiled HCC tumor cell intrinsic hyperactivation of ß-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. SIGNIFICANCE: This study identifies PF4-CXCR3-MDSCs as a downstream mechanism underlying CTNNB1 mutation associated immune "desert" phenotype.

Multiple serum biomarkers associate with mortality and interstitial lung disease progression in systemic sclerosis.

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 computed tomography, and lung function within 12 months of baseline were identified from the Australian Scleroderma Cohort Study. Baseline was the time of serum collection. 27 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: 407 participants were identified, 252 (61.9%) with SSc-ILD. The median follow up after biomarker measurement was 6.31 (3.11-9.22) years. 16 biomarkers were associated with increased mortality. High levels of VCAM-1 were most strongly associated with mortality (HR 3.55; 95%CI 2.37-5.33; p< 0.001). Five additional biomarkers had a 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), MMP3 (1.42-2.95; p< 0.001) and ET-1 (2.03, 1.40-2.92; p< 0.001). 11 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 one year follow up: CXCL4 (OR 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.

Platelet factor 4 improves survival in a murine model of antibiotic-susceptible and methicillin-resistant Staphylococcus aureus peritonitis.

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.

PLATELET FACTOR 4 (PF4) IMPROVES SURVIVAL IN A MURINE MODEL OF ANTIBIOTIC-SUSCEPTIBLE AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS PERITONITIS.

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.

Dichotomous roles of RIPK3 in regulating the IFN response and NLRP3 inflammasome in human monocytes.

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.

CXCR2 (rs3890158 and rs2230054) and CXCL4 (rs352008 and rs1801572) gene polymorphisms in patients with thoracic aortic aneurysm.

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.

Platelet-instructed SPP1+ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner.

Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.

Molecular Mechanisms Behind the Role of Plasmacytoid Dendritic Cells in Systemic Sclerosis.

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.

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.

Role of CXCR3 in fibrotic tissue responses.

Development of fibrosis leads to end stage diseases that defy treatments across all organs. This ensues as chronic inflammation is not dampened by physiologic processes that issue in the resolution phase of wound healing. Thus, these conditions can be considered diseases of "failure to heal". In the absence of broadly viable treatments, it is proposed to examine key switches in wound healing resolution to seek insights into novel approaches. Signaling through the GPCR CXCR3 has been shown to be one such critical player in this physiologic transition that limits and even reverses early fibrosis. As such, a number of investigators and early stage technology companies have posited that triggering this signaling network would limit fibrosis. While there are some conflicting results, a consensus is emerging that pharmacologic interventions that promote signaling through this pathway represent innovative ways to limit fibrotic diseases.

Utility of cytokines CXCL4, CXCL8 and GDF15 as biomarkers in systemic sclerosis

Background and objectives: Systemic sclerosis (SSc) is an autoinmune disease that can affect several organs and its mortality is fundamentally related to its pulmonary involvement. There are some cytokines with high serum levels of patients with SSc. Our goal is to determine the role of CXCL4, CXCL8 and GDF15 in the physiopathology of SSc and whether they can be considered organic damage biomarkers.Patients and methodsObservational case–control study of SSc patients (ACR/EULAR 2013 criteria). Demographic, clinical, analytical, activity, severity, health perception, and disability variables were collected. Moreover, Videocapillaroscopy, Echocardiography and Respiratory Function Test were made. Serum levels of CXCL4, CXCL8 and GDF15 were measured both in SSc patients and in healthy controls.ResultsA total of 42 patients were included (95.4% women), with an average age of 59.2 years and a median of 4 years from diagnosis. We also included 42 healthy controls. We found significantly higher levels of GDF15 in SSc patients than in controls (p<0.001), but no higher CXCL4 or CXCL8 levels. GDF15 was associated with Diffuse SSc, pulmonary arterial hypertension, interstitial lung disease, less forced vital capacity, high titles of antiScl70, disease activity, and dilated loops in capillaroscopy. CXCL4 levels were associated to a higher Rodnan punctuation, while CXCL8 was associated to C4 fraction consumption and tortuosities in capillaroscopy. (AU)

Antecedentes y objetivos: La esclerosis sistémica (ES) es una enfermedad autoinmunitaria que afecta a diferentes órganos y cuya mortalidad se relaciona fundamentalmente con su afectación pulmonar. Los pacientes con ES presentan niveles séricos elevados de algunas citocinas. Nuestro objetivo es determinar el papel de CXCL4, CXCL8 y GDF15 en la fisiopatología de la ES, y si pueden considerarse biomarcadores de daño orgánico.Pacientes y métodosEstudio observacional de casos-controles, con pacientes afectados de ES (criterios ACR/EULAR 2013) y controles sanos. Se determinaron los niveles séricos de CXCL4, CXCL8 y GDF15 en ambos grupos, y se registraron variables demográficas, clínicas, analíticas, de actividad, gravedad, percepción de salud y discapacidad de pacientes con ES, a quienes, además, se les realizó videocapilaroscopia, ecocardiograma y espirometría.ResultadosSe incluyeron 42 pacientes (95,4% mujeres), con una edad media de 59,2 años y una mediana de 4 años desde el diagnóstico, con 42 controles sanos. Se hallaron niveles significativamente mayores de GDF15 en pacientes con ES que en controles (p<0,001), pero no de CXCL4 ni CXCL8. GDF15 se asoció a ES difusa, hipertensión pulmonar, enfermedad pulmonar intersticial, menor capacidad vital forzada, títulos altos de anti-Scl70, actividad de ES y dilataciones capilares. Asimismo, los niveles de CXCL4 se asociaron a mayor afectación cutánea (Rodnan), mientras que CXCL8 se asoció a consumo de la fracción C4 del complemento y tortuosidades en la capilaroscopia. (AU)

CXCL4L1 May Help Differentiate Benign from Malignant Pulmonary Lesions and Predicts Prognosis of Patients with Lung Cancer.

Background: Lung cancer (LC) is the leading type of cancer worldwide, yet it's challenging to detect early LC. Therefore, it is valuable to explore diagnostic biomarker that can distinguish malignant pulmonary lesions from benign diseases. The potential role of plate factor-4 variant (CXCL4L1) will be investigated in detecting early LC. Methods: A consecutive of 174 patients with single pulmonary nodule and 50 healthy controls were enrolled. Serum CXCL4L1 expression level was evaluated using ELISA. Survival curves were generated to analyze survival outcomes. Receiver operating characteristic curves were used to calculate diagnostic accuracy. Results: Serum CXCL4L1 was downregulated in patients with LC when compared with those with lung benign lesions (LBL) or healthy controls. Meanwhile, lower serum CXCL4L1 expression was associated with advanced TNM stage and lymph node metastasis. Furthermore, a low expression of CXCL4L1 resulted in worse survival outcomes in LC patients. Serum CXCL4L1 expression obtained an area under curve (AUC) of 0.81 (95% CI: 0.74-0.88), a sensitivity of 70.6%, and a specificity of 85.8% for discriminating patients with LC form patients with LBL. In addition, serum CXCL4L1 expression achieved an AUC of 0.82 (95% CI, 0.74-0.90), a sensitivity of 72.0%, and a specificity of 85.9% for distinguishing patients with LC form healthy controls. Conclusion: This study suggests that CXCL4L1 may prove to be a potential non-invasive diagnostic and prognostic biomarker for early LC patients.

Differential Effects of Platelet Factor 4 (CXCL4) and Its Non-Allelic Variant (CXCL4L1) on Cultured Human Vascular Smooth Muscle Cells.

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.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Utility of cytokines CXCL4, CXCL8 and GDF15 as biomarkers in systemic sclerosis.

BACKGROUND AND OBJECTIVES: Systemic sclerosis (SSc) is an autoinmune disease that can affect several organs and its mortality is fundamentally related to its pulmonary involvement. There are some cytokines with high serum levels of patients with SSc. Our goal is to determine the role of CXCL4, CXCL8 and GDF15 in the physiopathology of SSc and whether they can be considered organic damage biomarkers. PATIENTS AND METHODS: Observational case-control study of SSc patients (ACR/EULAR 2013 criteria). Demographic, clinical, analytical, activity, severity, health perception, and disability variables were collected. Moreover, Videocapillaroscopy, Echocardiography and Respiratory Function Test were made. Serum levels of CXCL4, CXCL8 and GDF15 were measured both in SSc patients and in healthy controls. RESULTS: A total of 42 patients were included (95.4% women), with an average age of 59.2 years and a median of 4 years from diagnosis. We also included 42 healthy controls. We found significantly higher levels of GDF15 in SSc patients than in controls (p<0.001), but no higher CXCL4 or CXCL8 levels. GDF15 was associated with Diffuse SSc, pulmonary arterial hypertension, interstitial lung disease, less forced vital capacity, high titles of antiScl70, disease activity, and dilated loops in capillaroscopy. CXCL4 levels were associated to a higher Rodnan punctuation, while CXCL8 was associated to C4 fraction consumption and tortuosities in capillaroscopy. CONCLUSIONS: GDF15 high levels were associated with diffuse SSc, lung impairment, disease activity and changes in capillaroscopy. Moreover, CXCL4 was only associated with skin impairment, while CXCL8 was not related to organic damage.

Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α.

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.

Platelet Activation and Chemokine Release Are Related to Local Neutrophil-Dominant Inflammation During Hyperacute Human Stroke.

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.