Dysfunction of low-density neutrophils in peripheral circulation in patients with sepsis

Low-density neutrophils (LDNs) have been described in tumors and various autoimmune diseases, where they exhibit immune dysfunction and alter disease progression. Nevertheless, LDNs have been rarely reported in sepsis. We studied sepsis patients admitted to the intensive care unit. Wright-Giemsa stain assay and Transmission electron microscopy were performed to detect the morphology of neutrophils. Flow cytometry was used to analyze the number and function of LDNs. Concentration of cytokines was measured using ELISA. Neutrophil chemotaxis was examined using an under-agarose chemotaxis model. We found that LDNs were significantly elevated in patients with sepsis. Phenotypes and morphological characteristics suggest that LDNs may be formed by mixtures of neutrophils at various maturation stages. In vitro experiments showed that LDN formation was closely associated with neutrophil degranulation. We preliminarily discussed changes in immune function in LDNs. Compared with high-density neutrophils, expression levels of CXC chemokine receptor 4 on LDN surfaces were increased, phagocytotic capacity was decreased, and life span was prolonged. The chemotactic ability of LDNs was significantly reduced, possibly related to the increased expression of P2X1. These data suggest that LDNs are essential components of neutrophils in sepsis. To clarify the source and dysfunction mechanism of LDN in sepsis may be helpful for the diagnosis and treatment of sepsis in the future.

In Vivo Targeting of CXCR4—New Horizons

Given its pre-eminent role in the context of tumor cell growth as well as metastasis, the C-X-C motif chemokine receptor 4 (CXCR4) has attracted a lot of interest in the field of nuclear oncology, and clinical evidence on the high potential of CXCR4-targeted theranostics is constantly accumulating. Additionally, since CXCR4 also represents a key player in the orchestration of inflammatory responses to inflammatory stimuli, based on its expression on a variety of pro- and anti-inflammatory immune cells (e.g., macrophages and T-cells), CXCR4-targeted inflammation imaging has recently gained considerable attention. Therefore, after briefly summarizing the current clinical status quo of CXCR4-targeted theranostics in cancer, this review primarily focuses on imaging of a broad spectrum of inflammatory diseases via the quantification of tissue infiltration with CXCR4-expressing immune cells. An up-to-date overview of the ongoing preclinical and clinical efforts to visualize inflammation and its resolution over time is provided, and the predictive value of the CXCR4-associated imaging signal for disease outcome is discussed. Since the sensitivity and specificity of CXCR4-targeted immune cell imaging greatly relies on the availability of suitable, tailored imaging probes, recent developments in the field of CXCR4-targeted imaging agents for various applications are also addressed.

Investigating CXCR4 expression of tumor cells and the vascular compartment: A multimodal approach

The C-X-C chemokine receptor 4 (CXCR4) is G protein-coupled receptor that upon binding to its cognate ligand, can lead to tumor progression. Several CXCR4-targeted therapies are currently under investigation, and with it comes the need for imaging agents capable of accurate depiction of CXCR4 for therapeutic stratification and monitoring. PET agents enjoy the most success, but more cost-effective and radiation-free approaches such as ultrasound (US) imaging could represent an attractive alternative. In this work, we developed a targeted microbubble (MB) for imaging of vascular CXCR4 expression in cancer. A CXCR4-targeted MB was developed through incorporation of the T140 peptide into the MB shell. Binding properties of the T140-MB and control, non-targeted MB (NT-MB) were evaluated in MDA-MB-231 cells where CXCR4 expression was knocked-down (via shRNA) through optical imaging, and in the lymphoma tumor models U2932 and SuDHL8 (high and low CXCR4 expression, respectively) by US imaging. PET imaging of [18F]MCFB, a tumor-penetrating CXCR4-targeted small molecule, was used to provide whole-tumor CXCR4 readouts. CXCR4 expression and microvessel density were performed by immunohistochemistry analysis and western blot. T140-MB were formed with similar properties to NT-MB and accumulated sensitively and specifically in cells according to their CXCR4 expression. In NOD SCID mice, T140-MB persisted longer in tumors than NT-MB, indicative of target interaction, but showed no difference between U2932 and SuDHL8. In contrast, PET imaging with [18F]MCFB showed a marked difference in tumor uptake at 40–60 min post-injection between the two tumor models (p<0.05). Ex vivo analysis revealed that the large differences in CXCR4 expression between the two models are not reflected in the vascular compartment, where the MB are restricted; in fact, microvessel density and CXCR4 expression in the vasculature was comparable between U2932 and SuDHL8 tumors. In conclusion, we successfully developed a T140-MB that can be used for imaging CXCR4 expression in the tumor vasculature.

Reduced CXCR4 expression in associated with extramedullary and predicts poor survival in newly diagnosed multiple myeloma

Multiple myeloma (MM), a bone marrow-resident hematological malignancy of plasma cells, has remained largely incurable despite the advancement of novel therapies in recent years. Because of the heterogeneity of myeloma cells, risk stratification of MM is important for making therapeutic regimens. Nevertheless, no immunohistochemical (IHC) predictive and prognostic marker of MM has been constructed yet. Herein, the prognostic value of C-X-C motif chemokine receptor 4 (CXCR4) expression in 48 newly diagnosed MM patients was explored using IHC. Correlations between CXCR4 expression and clinical features of MM were analyzed. CXCR4-positive patients significantly outperformed CXCR4- negative patients in both 3-year estimated overall survival (93.8% vs 45.8%) and progression-free survival (57.1% vs 40.9%). The incidence of extramedullary lesions in CXCR4-negative patients increased significantly compared with CXCR4-positive patients. Plasma cells that reduce CXCR4 expression have poor prognosis and increase the incidence of extramedullary lesions.

H19 Overexpression Improved Efficacy of Mesenchymal Stem Cells in Ulcerative Colitis by Modulating the miR-141/ICAM-1 and miR-139/CXCR4 Axes

Overexpression of C-X-C motif chemokine receptor 4 (CXCR4) and intercellular cell adhesion molecule-1 (ICAM-1) may promote homing of mesenchymal stem cells (MSC). In this study, we treated ulcerative colitis animals with MSC preconditioned with or without H19 and compared the therapeutic effect of MSC and MSC-H19. We evaluated the regulatory relationship of H19 vs. miR-141/miR-139 and miR-141/miR-139 vs. ICAM-1/CXCR4. We established an ulcerative colitis mouse model to assess the effect of MSC and MSC-H19. H19 was found to bind to miR-141 and miR-139. The activity of H19 was strongly decreased in cells c-transfected with miR-141/miR-139 and WT H19. ICAM-1 was confirmed to be targeted by miR-141 and CXCR4 was targeted by miR-139. The H19 expression showed a negative regulatory relationship with the miR-141 and miR-139 expression but a positive regulatory relationship with the ICAM-1 and CXCR4 expression. In summary, the overexpression of H19 in MSC downregulated miR-139 and miR-141, thus increasing the activity of their targets ICAM-1 and CXCR4, respectively, to exhibit therapeutic effects in ulcerative colitis.

Peptide Nanoparticle-Mediated Combinatorial Delivery of Cancer-Related siRNAs for Synergistic Anti-Proliferative Activity in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is one of the deadliest types of cancer for women of different age groups. Frequently this cancer does not respond to conservative treatment. Combinatorial RNAi can be suggested as an advanced approach to TNBC therapy. Due to the fact that TNBC cells overexpress chemokine receptor 4 we used modular L1 peptide-based nanoparticles modified with CXCR4 ligand for combinatorial delivery of siRNAs suppressing major transduction pathways. TNBC cell line MDA-MB-231 was used as a cellular model. Genes encoding the AQP3, CDC20, and COL4A2 proteins responsible for proliferative activity in TNBC cells were selected as RNAi targets. The siRNA binding ability of the carrier was studied at different charge ratios. The silencing specificity was demonstrated for all siRNAs studied. Alamar Blue proliferation assay has shown significant reduction in the anti-proliferative activity after combinatorial siRNA transfection compared to single siRNA delivery.The most significant synergistic effect has been demonstrated for combinatorial transfection of anti-COL4A2and anti-CDC20 siRNAs what resulted in 1.5–2 fold inhibition of proliferation and migration of TNBC cells. Based on our findings, we have concluded that combinatorial treatment by CXCR4-ligand modified L1-polyplexes formed with AQP3, CDC20, and COL4A2 siRNAs effectively inhibits proliferation of TNBC cells and can be suggested as useful tool for RNAi-mediated cancer therapy.

Dysfunction of Low-Density Neutrophils in Peripheral Circulation in Patients With Sepsis

Background Low-density neutrophils (LDNs) have been described in tumors and various autoimmune diseases, where they exhibit immune dysfunction and alter disease progression. Nevertheless, LDNs have been rarely reported in sepsis. Methods We studied sepsis patients admitted to the intensive care unit. WrightGiemsa stain assay and Transmission electron microscopy were performed to detect the morphology of neutrophils. Flow cytometry was used to analyze the number and function of LDNs. Concentration of cytokines was measured using ELISA. Neutrophil chemotaxis was examined using an under-agarose chemotaxis model. Results We found that LDNs were significantly elevated in patients with sepsis. Phenotypes and morphological characteristics suggest that LDNs may be formed by mixtures of neutrophils at various maturation stages. In vitro experiments showed that LDN formation was closely associated with neutrophil degranulation. We preliminarily discussed changes in immune function in LDNs. Compared with high-density neutrophils, expression levels of CXC chemokine receptor 4 on LDN surfaces were increased, phagocytotic capacity was decreased, and life span was prolonged. The chemotactic ability of LDNs was significantly reduced, possibly related to the increased expression of P2X1. Conclusions These data suggest that LDNs are essential components of neutrophils in sepsis. To clarify the source and dysfunction mechanism of LDN in sepsis may be helpful for the diagnosis and treatment of sepsis in the future.

Computational modeling and experimental validation of the EPI-X4/CXCR4 complex allows rational design of small peptide antagonists

EPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.

Modifying strategies for SDF-1/CXCR4 interaction during mesenchymal stem cell transplantation

Mesenchymal stem cell (MSC) transplantation is regarded as a promising candidate for the treatment of ischaemic heart disease. The major hurdles for successful clinical translation of MSC therapy are poor survival, retention, and engraftment in the infarcted heart. Stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4) constitutes one of the most efficient chemokine/chemokine receptor pairs regarding cell homing. In this review, we mainly focused on previous studies on how to regulate the SDF-1/CXCR4 interaction through various priming strategies to maximize the efficacy of mesenchymal stem cell transplantation on ischaemic hearts or to facilitate the required effects. The strengthened measures for enhancing the therapeutic efficacy of the SDF-1/CXCR4 interaction for mesenchymal stem cell transplantation included the combination of chemokines and cytokines, hormones and drugs, biomaterials, gene engineering, and hypoxia. The priming strategies on recipients for stem cell transplantation included ischaemic conditioning and device techniques.