CXCR4 Expression Research Articles

Oligoclonal CD4+CXCR5+ T cells with a cytotoxic phenotype appear in tonsils and blood

In clinical situations, peripheral blood accessible CD3+CD4+CXCR5+ T-follicular helper (TFH) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of TFH cells in peripheral blood versus tonsils, CD3+CD4+CD45RA–CXCR5+ cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3+CD4+CXCR5+ T-cell subpopulations also appear in tonsils, there with some supplementary TFH characteristics, while peripheral blood-derived TFH cells display markers of proliferation and migration. Three further subsets of TFH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4+CXCR5+ subpopulation presents pronounced cytotoxic properties. Those ‘killer TFH (TFK) cells’ can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other TFH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4+CXCR5+ T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts.

Macrophage Migration Inhibitory Factor (MIF) Upregulates CXCR7 and Contributes to Chemotherapy Resistance in Colorectal Cancer.

Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates making it a focus of research. Chemotherapy is a primary treatment modality for colon cancer, but chemotherapy resistance severely impacts treatment efficacy. MIF has been found to promote tumor progression and resistance in various cancers. This study aims to investigate the role of MIF in chemotherapy resistance in colon cancer and its potential mechanisms, particularly through the upregulation of CXCR7 expression, affecting the metabolism and drug sensitivity of colon cancer cells. The expression levels of MIF in colon cancer tissues and its association with patient prognosis were evaluated by analyzing TCGA and HPA data. Subsequently, the expression levels of MIF in colon cancer cell lines and resistant cell lines were detected by qRT-PCR and immunohistochemistry, and the effect of MIF on oxaliplatin sensitivity was assessed. The impact of MIF on the metabolic activity of colon cancer cells was measured using a cellular energy metabolism analyzer. Further experiments explored the mechanism by which MIF affects the metabolic activity of colon cancer cells through the upregulation of CXCR7 expression, and the role of CTCF in regulating CXCR7 transcription was validated by silencing CTCF. Finally, the effect of MIF on drug sensitivity of colon cancer cells was verified in a mouse xenograft tumor model. In this study, we found that the expression of MIF in colon cancer tissues was significantly higher than in normal tissues, and high MIF expression was associated with poor prognosis in patients. The expression levels of MIF in resistant colon cancer cell lines were significantly higher than in parental cell lines, and MIF overexpression significantly increased the resistance of colon cancer cells to oxaliplatin. Conversely, silencing MIF significantly reduced the IC50 value of resistant cells and increased apoptosis. MIF overexpression significantly increased the ECAR and OCR levels of colon cancer cells, while MIF knockdown significantly reduced these metabolic indicators. Further studies indicated that MIF affects the metabolic activity of colon cancer cells by upregulating CXCR7 expression. CTCF binding peaks at the CXCR7 promoter region and luciferase activity assays indicated that CTCF regulates CXCR7 transcription, and silencing CTCF significantly enhanced the sensitivity of colon cancer cells to oxaliplatin. In vivo experiments in mice showed that MIF silencing combined with oxaliplatin treatment significantly inhibited tumor growth and increased the necrotic area of tumor tissues. In conclusion, this study reveals the crucial role of MIF in chemotherapy resistance in colon cancer through the upregulation of CXCR7 expression, with CTCF playing an important regulatory role in this process. Our findings provide new theoretical insights and potential therapeutic targets for overcoming chemotherapy resistance in colon cancer. Future research should further explore the roles of MIF and CXCR7 in other types of cancers and the potential of MIF and CXCR7 as therapeutic targets.

Long-term changes in the phenotype and cytokine production of monocytes in COVID-19 recovered and vaccinated individuals.

Monocytes play a crucial role in the immune response against pathogens. Here, we sought to determine COVID-19 and the vaccine Gam-COVID-Vac induce long-term changes in the phenotype and cytokine production of circulating monocytes. Monocytes were purified from peripheral blood mononuclear cells of healthy donors who had not had COVID-19 or vaccination, who had received two doses of Gam-COVID-Vac, and who had mild/moderate COVID-19 in the last 6 months and evaluated by flow cytometry. To investigate the effect of SARS-CoV-2 proteins, monocytes were cultured for 2 days with or without stimulation with recombinant SARS-CoV-2 S1 and N peptides. Monocytes obtained from vaccinated and recovered individuals showed increased basal expression of HLA-DR, CD63, CXCR2, and TLR7. We also observed an increased frequency of CD63+ classical monocytes in both groups, as well as an increased frequency of HLA-DR+ non-classical monocytes in the COVID-19-recovered group compared to the control group. Monocytes from vaccinated and recovered donors produced higher basal levels of IL-6, IL-1β, and TNF-α cytokines. Ex vivo stimulation with SARS-CoV-2 antigens induced increased expression of HLA-DR and TLR7 on monocytes obtained from the control group. The challenge with SARS-CoV-2 antigens had no effect on the production of IL-6, IL-1β, and TNF-α cytokines by monocytes. The acquired data offer compelling evidence of enduring alterations in both the phenotype and functional status of circulating monocytes subsequent to vaccination with Gam-COVID-Vac and mild/moderate COVID-19 infection. At least some of these changes appear to be a consequence of exposure to SARS-CoV-2 S1 and N antigens.

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

CD74 is a functional MIF receptor on activated CD4+ T cells

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

Microvesicles from quiescent and TGF-β1 stimulated hepatic stellate cells: Divergent impact on hepatic vascular injury.

This study evaluated the effect of microvesicles(MVs) from quiescent and TGF-β1 stimulated hepatic stellate cells (HSC-MVs, TGF-β1HSC-MVs) on H2O2-induced human umbilical vein endothelial cells (HUVECs) injury and CCl4-induced rat hepatic vascular injury. HUVECs were exposed to hydrogen peroxide (H2O2) to establish a model for vascular endothelial cell injury. HSC-MVs or TGF-β1HSC-MVs were co-cultured with H2O2-treated HUVECs, respectively. Indicators including cell survival rate, apoptosis rate, oxidative stress, migration, invasion, and angiogenesis were measured. Simultaneously, the expression of proteins such as PI3K, AKT, MEK1+MEK2, ERK1+ERK2, VEGF, eNOS, and CXCR4 was assessed, along with activated caspase-3. SD rats were intraperitoneally injected with CCl4 twice a week for 10 weeks to induce liver injury models. HSC-MVs or TGF-β1HSC-MVs were injected into the tail vein of rats. Liver and hepatic vascular damage were also detected. In H2O2-treated HUVECs, HSC-MVs increased cell viability, reduced cytotoxicity and apoptosis, improved oxidative stress, migration, and angiogenesis, and upregulated protein expression of PI3K, AKT, MEK1/2, ERK1/2, VEGF, eNOS, and CXCR4. Conversely, TGF-β1HSC-MVs exhibited opposite effects. CCl4- induced rat hepatic injury model, HSC-MVs reduced the release of ALT and AST, hepatic inflammation, fatty deformation, and liver fibrosis. HSC-MVs also downregulated the protein expression of CD31 and CD34. Conversely, TGF-β1HSC-MVs demonstrated opposite effects. HSC-MVs demonstrated a protective effect on H2O2-treated HUVECs and CCl4-induced rat hepatic injury, while TGF-β1HSC-MVs had an aggravating effect. The effects of MVs involve PI3K/AKT/VEGF, CXCR4, and MEK/ERK/eNOS pathways.

Emodin suppresses mast cell migration via modulating the JAK2/STAT3/JMJD3/CXCR3 signaling to prevent cystitis.

This study aimed to determine the preventive effects of emodin on cyclophosphamide (CYP)-induced cystitis and to explore the molecular mechanism. In vivo, mice were modeled by CYP. Before a half hour of CYP treatment, Jumonji domain-containing protein-3 (JMJD3)inhibitors (GSK-J4) and emodin were used to treat CYP model mice. Bladder samples were stained for hematoxylin-eosin and toluidine blue. Next, JMJD3 was quantified by immunofluorescence staining, RT-PCR, and Western blot. CXCR3 was quantified by Western blot and ELISA. In vitro, before stimulated by lipopolysaccharide (LPS), human bladder smooth muscle cells (hBSMCs) were transfected with pcDNA3.1-JMJD3 plasmids, shRNA-JMJD3 plasmids or pretreated with emodin. Collected cells to detect JMJD3 and CXCR3 ligands again; collected supernatant of culture for Transwell assay. Finally, as the JAK2 inhibitor, AG490 was used to pretreat LPS-induced hBSMCs. Western blot was performed to quantify proteins. Emodin inhibited mast cell migration and suppressed the expression of JMJD3, CXCR3, and CXCR3 ligands, not only in vivo but also in vitro. The pharmacological effects of emodin were similar to GSK-J4 or JMJD3 inhibition. In addition, emodin significantly downregulated the phosphorylation of JAK2 and STAT3, and inhibited JMJD3/CXCR3 axis transduction like AG490. Emodin has a preventive effect on cystitis by inhibiting mast cell migration through inhibition of the JAK2/STAT3/JMJD3/CXCR3 signaling pathway.

Role of the IL-8/CXCR2 axis in promoting vasculogenic mimicry in triple-negative breast cancer through epithelial-mesenchymal transition

Vasculogenic mimicry (VM) — a phenomenon where tumor cells form de novo vascular networks independent of endothelial cells — supports the growth of highly aggressive tumors, including triple-negative breast cancer (TNBC). Evidence indicates that the interleukin 8 (IL-8)/CXCR2 axis and epithelial-mesenchymal transition (EMT) independently promote VM in TNBC. This study tested the hypothesis that the IL-8/CXCR2 axis promotes VM via EMT. Tissue samples from 71 female breast cancer cases (age range: 50 – 55 years), comprising 37 TNBC and 34 non-TNBC cases, were retrieved from the Department of Pathology at the University of Ghana Medical School. Out of the 37 TNBC tissues, 10 expressed either IL-8, CXCR2, or vimentin exclusively; three expressed all three markers; and 14 did not express any of these markers. For the non-TNBC tissues, 31 expressed CXCR2 (alone or co-expression with IL-8 and/or vimentin), 27 co-expressed IL-8 with CXCR2 and/or vimentin, and two expressed all three markers. Notably, 24 of the 37 non-TNBC tissues co-expressed IL-8 and CXCR2. A phi coefficient analysis displayed an inverse relationship between CXCR2 or IL-8 expression and TNBC and a positive correlation between vimentin expression and TNBC. Inhibiting the IL-8/CXCR2 axis with the CXCR2 antagonist SB225002 suppressed VM in MDA-MB-231 breast cancer cells. In addition, inhibiting the IL-8/CXCR2 axis using SB225002 and siRNA-mediated CXCR2 knockdown suppressed EMT by upregulating E-cadherin expression and downregulating N-cadherin and vimentin expression. Our findings suggest that the IL-8/CXCR2 signaling axis promotes VM in TNBC by facilitating EMT. Therefore, therapies targeting IL-8/CXCR2 may be used to suppress EMT and VM in TNBC simultaneously.

Identification of immune-associated biomarkers of diabetes nephropathy tubulointerstitial injury based on machine learning: a bioinformatics multi-chip integrated analysis

BackgroundDiabetic nephropathy (DN) is a major microvascular complication of diabetes and has become the leading cause of end-stage renal disease worldwide. A considerable number of DN patients have experienced irreversible end-stage renal disease progression due to the inability to diagnose the disease early. Therefore, reliable biomarkers that are helpful for early diagnosis and treatment are identified. The migration of immune cells to the kidney is considered to be a key step in the progression of DN-related vascular injury. Therefore, finding markers in this process may be more helpful for the early diagnosis and progression prediction of DN.MethodsThe gene chip data were retrieved from the GEO database using the search term ' diabetic nephropathy ‘. The ' limma ' software package was used to identify differentially expressed genes (DEGs) between DN and control samples. Gene set enrichment analysis (GSEA) was performed on genes obtained from the molecular characteristic database (MSigDB. The R package ‘WGCNA’ was used to identify gene modules associated with tubulointerstitial injury in DN, and it was crossed with immune-related DEGs to identify target genes. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on differentially expressed genes using the ‘ClusterProfiler’ software package in R. Three methods, least absolute shrinkage and selection operator (LASSO), support vector machine recursive feature elimination (SVM-RFE) and random forest (RF), were used to select immune-related biomarkers for diagnosis. We retrieved the tubulointerstitial dataset from the Nephroseq database to construct an external validation dataset. Unsupervised clustering analysis of the expression levels of immune-related biomarkers was performed using the ‘ConsensusClusterPlus ‘R software package. The urine of patients who visited Dongzhimen Hospital of Beijing University of Chinese Medicine from September 2021 to March 2023 was collected, and Elisa was used to detect the mRNA expression level of immune-related biomarkers in urine. Pearson correlation analysis was used to detect the effect of immune-related biomarker expression on renal function in DN patients.ResultsFour microarray datasets from the GEO database are included in the analysis : GSE30122, GSE47185, GSE99340 and GSE104954. These datasets included 63 DN patients and 55 healthy controls. A total of 9415 genes were detected in the data set. We found 153 differentially expressed immune-related genes, of which 112 genes were up-regulated, 41 genes were down-regulated, and 119 overlapping genes were identified. GO analysis showed that they were involved in various biological processes including leukocyte-mediated immunity. KEGG analysis showed that these target genes were mainly involved in the formation of phagosomes in Staphylococcus aureus infection. Among these 119 overlapping genes, machine learning results identified AGR2, CCR2, CEBPD, CISH, CX3CR1, DEFB1 and FSTL1 as potential tubulointerstitial immune-related biomarkers. External validation suggested that the above markers showed diagnostic efficacy in distinguishing DN patients from healthy controls. Clinical studies have shown that the expression of AGR2, CX3CR1 and FSTL1 in urine samples of DN patients is negatively correlated with GFR, the expression of CX3CR1 and FSTL1 in urine samples of DN is positively correlated with serum creatinine, while the expression of DEFB1 in urine samples of DN is negatively correlated with serum creatinine. In addition, the expression of CX3CR1 in DN urine samples was positively correlated with proteinuria, while the expression of DEFB1 in DN urine samples was negatively correlated with proteinuria. Finally, according to the level of proteinuria, DN patients were divided into nephrotic proteinuria group (n = 24) and subrenal proteinuria group. There were significant differences in urinary AGR2, CCR2 and DEFB1 between the two groups by unpaired t test (P < 0.05).ConclusionsOur study provides new insights into the role of immune-related biomarkers in DN tubulointerstitial injury and provides potential targets for early diagnosis and treatment of DN patients. Seven different genes ( AGR2, CCR2, CEBPD, CISH, CX3CR1, DEFB1, FSTL1 ), as promising sensitive biomarkers, may affect the progression of DN by regulating immune inflammatory response. However, further comprehensive studies are needed to fully understand their exact molecular mechanisms and functional pathways in DN.

Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B

The changes in HBV-specific B cells in patients with chronic hepatitis B (CHB) undergoing pegylated interferon-α (PEG-IFNα) treatment and achieving functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B cells. We included 39 nucleos(t)ide analogue-treated patients with CHB who received sequential combination therapy with PEG-IFNα and eight treatment-naïve patients. HBV-specific B cells were characterized exvivo using fluorescently labeled hepatitis B surface and core antigens (HBsAg and HBcAg). The frequency, phenotype, and subsets of HBV-specific B cells and follicular helper T cells (Tfh cells) were detected using flow cytometry. The functionality of HBV-specific B cells was quantified through ELISpot assays. During treatment, the fraction of activated memory B cells (MBCs) among HBsAg-specific B cells and the expression of IgG, CXCR3, and CD38 increased. The antibody-secretion capacity of HBsAg-specific B cells was only restored in patients achieving a functional cure after treatment and it positively correlated with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts among HBsAg-specific B cells. HBcAg-specific B cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBC after treatment, irrespective of functional cure. The number of CD40L+ Tfh cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. After PEG-IFNα treatment, HBsAg- and HBcAg-specific B cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh cells is associated with the active recovery of HBsAg-specific B cells. HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of patients with chronic hepatitis B offers a promising therapeutic strategy for viral clearance, e.g. via therapeutic vaccination. We analyzed the alterations in HBV-specific B cells in patients treated with pegylated interferon-α and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Foodborne toxin aflatoxin B1 induced glomerular podocyte inflammation through proteolysis of RelA, downregulation of miR-9 and CXCR4/TXNIP/NLRP3 pathway

Aflatoxin B1 (AFB1) is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin, particularly causing damages to kidney. Glomerular podocytes are terminally differentiated epithelial cells. AFB1 induces podocyte inflammation, proteinuria and renal dysfunction. Studying the mechanism of AFB1-induced podocyte inflammation and murine kidney dysfunction, we detected that AFB1 increased ubiquitin-dependent degradation of the transcription factor RelA through enhanced interaction of RelA with E3 ubiquitin ligase tripartite motif containing 7 (TRIM7) in mouse podocyte clone-5 (MPC-5) and mouse glomeruli. Reduction of RelA resulted in decreasing microRNA-9 (miR-9) and activating the chemokine receptor 4 (CXCR4), thioredoxin interacting protein (TXNIP), and NOD-like receptor pyrin domain-containing 3 (NLRP3) signaling axis (CXCR4/TXNIP/NLRP3 pathway), leading to podocyte inflammation. We also determined that downregulation of miR-9 led to CXCR4 expression and the downstream TXNIP/NLRP3 pathway activation. Overexpression of miR-9 or deletion of CXCR4 suppressed AFB1-induced CXCR4/TXNIP/NLRP3 pathway, resulting in alleviating podocyte inflammation and kidney dysfunction. Our findings indicated that ubiquitin-dependent proteolysis of RelA, downregulation of miR-9, and activation of CXCR4/TXNIP/NLRP3 pathway played an essential role in AFB1-induced glomerular podocyte inflammation. Our study revealed a novel mechanism, via RelA, for the control of AFB1’s nephrotoxicity, leading to an effective protection of food safety and public health.

New Emerging Chemokine Receptors: CCR5 or CXCR5 on Tumor Is Associated with Poor Response to Chemotherapy and Poor Prognosis in Locally Advanced Triple-Negative Breast Cancer.

We aim to investigate any possible associations between chemokine receptor expression and responses to neoadjuvant chemotherapy (NAC) along with outcomes in patients with triple-negative breast cancer (TNBC) with locally advanced disease. Expressions of chemokine receptors were examined immunohistochemically after staining archival tissue of surgical specimens (n = 63) using specific antibodies for CCR5, CCR7, CXCR4, and CXCR5. Patients with high CCR5, CCR7, CXCR4, and CXCR5 expression on tumors and high CXCR4 expression on tumor-infiltrating lymphocytes (TILs) were less likely to have a pathological complete response (pCR) or Class 0-I RCB-Index compared to others. Patients with residual lymph node metastases (ypN-positive), high CCR5TM(tumor), and high CXCR4TM expressions had an increased hazard ratio (HR) compared to others (DFS: HR = 2.655 [1.029-6.852]; DSS: HR = 2.763 [1.008-7.574]), (DFS: HR = 2.036 [0.805-5.148]; DSS: HR = 2.689 [1.020-7.090]), and (DFS: HR = 2.908 [1.080-7.829]; DSS: HR = 2.132 (0.778-5.846)), respectively. However, patients without CXCR5TIL expression had an increased HR compared to those with CXCR5TIL (DFS: 2.838 [1.266-6.362]; DSS: 4.211 [1.770-10.016]). High expression of CXCR4TM and CCR5TM was found to be associated with poor prognosis, and CXCR5TM was associated with poor chemotherapy response in the present cohort with locally advanced TNBC. Our results suggest that patients with TNBC could benefit from a chemokine receptor inhibitor therapy containing neoadjuvant chemotherapy protocols.

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Chemokine Receptor-4 Targeted PET/CT Imaging with 68Ga-Pentixafor in Head and Neck Cancer-A Comparison with 18F-FDG and CXCR4 Immunohistochemistry.

Head and neck squamous cell carcinoma (HNSCC) is common, and its incidence is increasing, particularly in HIV-infected individuals who present with more aggressive disease. Despite aggressive treatment, the prognosis remains poor because of resistance to chemoradiation therapy. So far, studies report very low [68Ga]Ga-Pentixafor avidity in HNSCC. This study investigated the diagnostic performance of CXCR4-directed imaging of carcinoma of the oral cavity, oropharynx, and nasopharynx with positron emission tomography/computed tomography (PET/CT) using the radiolabelled chemokine ligand [68Ga]Ga-Pentixafor and explored its ability to quantify CXCR4 expression in vivo. In this prospective cross-sectional study, twenty-three (23) patients aged 52.9 ± 10.4 (19.6), 17 males and 6 females with primarily diagnosed (n = 17) or pre-treated (n = 6) SCC of the oral cavity (OCSCC, n = 11), oropharynx (OPSCC, n = 9), nasopharynx (NPSCC, n = 2) and unknown primary (n = 1) underwent imaging with [68Ga]Ga-Pentixafor-PET/CT. In 16/23 patients 2-[18F]fluoro-2-deoxy-D-glucose ([18F]F-FDG) served as a standard reference. All lesions were visually rated using a 5-point Likert scale. For both tracers, maximum standardized uptake values (SUVmax) and the total lesion uptake (TLU) were recorded and compared using the Wilcox-signed rank test. In addition, the tumor-to-background ratios were derived using the liver (TLR), spleen (TSR), and posterior cervical muscles (TMR) as background. The relationships between the SUVs of the two tracers were assessed using the Spearman correlation. CXCR4 immunohistochemistry (IHC) staining was correlated with 68Ga-Pentixafor-PET/CT in 21/23 patients. Ninety-one percent (21/23) of tumors were visually detected on [68Ga]Ga-Pentixafor; however, [68Ga]Ga-Pentixafor was less intense compared with [18F]F-FDG-PET. Quantitative analysis showed higher [18F]F-FDG SUVmax in comparison with [68Ga]Ga-Pentixafor (16 ± 6.7 vs. 5.8 ± 2.6 g/mL, p = 0.011) and SUVmean (9.3 ± 4.1 vs. 3± 1.6 g/mL, p < 0.001) and TBR 4.9 ± 2.3 vs. 2.36 ± 1.4 p = 0.014. Nasopharyngeal cancer demonstrated more intense tracer accumulation than oropharyngeal and oral cavity malignancies. CXCR4 IHC staining was positive in 15/21 patients, and there was a statistically significant correlation between IHC staining and [68Ga]Ga-Pentixafor SUVmean r = 0.5 p = 0.027, and performance status r = 0.83 p = 0.0104. In conclusion, although [68Ga]Ga-Pentixafor cannot replace [18F]F-FDG as a diagnostic tool because of its lower avidity, the correlation between CXCR4 targeted 68Ga-Pentixafor PET imaging and CXCR4 IHC staining indicates the potential of 68Ga-Pentixafor as an effective tool for selecting patients who may benefit from therapies targeting CXCR4. In addition, [68Ga]Ga-Pentixafor has no physiological brown fat uptake, which often obscures cervical lesions on [18F]F-FDG PET/CT imaging.

VNN2-expressing circulating monocytes exhibit unique functional characteristics and are decreased in patients with primary Sjögren's syndrome

ObjectiveThis study aims to elucidate the significance of VNN2 expression in peripheral blood monocytes and its clinical relevance in primary Sjögren's syndrome (pSS). MethodsWe investigated VNN2 expression by analyzing single-cell RNA sequencing (scRNA-seq) data from peripheral blood mononuclear cells. Flow cytometry was used to detect and compare VNN2 expression in total monocytes, classical monocytes (cMo), intermediate monocytes (iMo) and non-classical monocytes (ncMo). Additionally, we examined the expression of HLA, ICAM1, CD62L, ITGAM, S100A8, S100A9, CCR2, CCR6, CX3CR1 and CXCR3 in VNN2+ and VNN2- cells. We analyzed the correlation between VNN2 expression and clinical indicators and assessed the clinical utility of VNN2+ monocytes in pSS diagnosis using receiver operating characteristic curves. ResultsWe observed high VNN2 expression in monocytes, with significantly higher levels in CD14++ monocytes compared to ncMo. VNN2+ monocytes exhibited decreased expression of HLA and CD62L and increased expression of ICAM1, ITGAM, S100A8, S100A9, CCR2, CCR6, CX3CR1 and CXCR3 compared to VNN2- monocytes. Although scRNA-seq data showed that VNN2 mRNA was upregulated, cell surface expression of VNN2 was decreased in monocytes from pSS patients compared to healthy controls. The reduced levels of VNN2+ monocyte subpopulations in pSS patients were negatively correlated with anti-ribosome antibody levels and positively correlated with complement 4 levels. Detection of VNN2 expression in monocytes can aid in the auxiliary diagnosis of pSS. ConclusionMonocytes expressing cell surface VNN2 are significantly reduced in pSS patients. This suggests a potential role for VNN2 in pSS development and its potential use as a diagnostic marker for pSS.

Differential alterations of CXCR3, CXCR5 and CX3CR1 in patients with immune thrombocytopenia

As a versatile element for maintaining homeostasis, the chemokine system has been reported to be implicated in the pathogenesis of immune thrombocytopenia (ITP). However, research pertaining to chemokine receptors and related ligands in adult ITP is still limited. The states of several typical chemokine receptors and cognate ligands in the circulation were comparatively assessed through various methodologies. Multiple variable analyses of correlation matrixes were conducted to characterize the correlation signatures of various chemokine receptors or candidate ligands with platelet counts. Our data illustrated a significant decrease in relative CXCR3 expression and elevated plasma levels of CXCL4, 9–11, 13, and CCL3 chemokines in ITP patients with varied platelet counts. Flow cytometry assays revealed eminently diminished CXCR3 levels on T and B lymphocytes and increased CXCR5 on cytotoxic T cell (Tc) subsets in ITP patients with certain platelet counts. Meanwhile, circulating CX3CR1 levels were markedly higher on T cells with a concomitant increase in plasma CX3CL1 level in ITP patients, highlighting the importance of aberrant alterations of the CX3CR1-CX3CL1 axis in ITP pathogenesis. Spearman’s correlation analyses revealed a strong positive association of peripheral CXCL4 mRNA level, and negative correlations of plasma CXCL4 concentration and certain chemokine receptors with platelet counts, which might serve as a potential biomarker of platelet destruction in ITP development. Overall, these results indicate that the differential expression patterns and distinct activation states of peripheral chemokine network, and the subsequent expansion of circulating CXCR5+ Tc cells and CX3CR1+ T cells, may be a hallmark during ITP progression, which ultimately contributes to thrombocytopenia in ITP patients.

Stem cell-like circulating tumor cells identified by Pep@MNP and their clinical significance in pancreatic cancer metastasis.

The circulating tumor cells (CTCs) could be captured by the peptide functionalized magnetic nanoparticles (Pep@MNP) detection system in pancreatic ductal adenocarcinoma (PDAC). CTCs and the CXCR4 expression were detected to explore their clinical significance. The CXCR4+ CTCs, this is highly metastatic-prone stem cell-like subsets of CTCs (HM-CTCs), were found to be associated with the early recurrence and metastasis of PDAC. CTCs were captured by Pep@MNP. CTCs were identified via immunofluorescence with CD45, cytokeratin antibodies, and the CXCR4 positive CTCs were assigned to be HM-CTCs. The over-expression of CXCR4 could promote the migration of pancreatic cancer cell in vitro and in vivo. In peripheral blood (PB), CTCs were detected positive in 79.0% of all patients (49/62, 9 (0-71)/2mL), among which 63.3% patients (31/49, 3 (0-23)/2mL) were HM-CTCs positive. In portal vein blood (PVB), CTCs were positive in 77.5% of patients (31/40, 10 (0-40)/2mL), and 67.7% of which (21/31, 4 (0-15)/2mL) were HM-CTCs positive CTCs enumeration could be used as diagnostic biomarker of pancreatic cancer (AUC = 0.862), and the combination of CTCs positive and CA19-9 increase shows improved diagnostic accuracy (AUC = 0.963). in addition, PVB HM-CTCs were more accurate to predict the early recurrence and liver metastasis than PB HM-CTCs (AUC 0.825 vs. 0.787 and 0.827 vs. 0.809, respectively). The CTCs identified by Pep@MNP detection system could be used as diagnostic and prognostic biomarkers of PDAC patients. We identified and defined the CXCR4 over-expressed CTC subpopulation as highly metastatic-prone CTCs, which was proved to identify patients who were prone to suffering from early recurrence and metastasis.

CXCR4 has a dual role in improving the efficacy of BCMA-redirected CAR-NK cells in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4R334X. Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro. Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites.

Metabolic flexibility ensures proper neuronal network function in moderate neuroinflammation

Microglia, brain-resident macrophages, can acquire distinct functional phenotypes, which are supported by differential reprogramming of cell metabolism. These adaptations include remodeling in glycolytic and mitochondrial metabolic fluxes, potentially altering energy substrate availability at the tissue level. This phenomenon may be highly relevant in the brain, where metabolism must be precisely regulated to maintain appropriate neuronal excitability and synaptic transmission. Direct evidence that microglia can impact on neuronal energy metabolism has been widely lacking, however. Combining molecular profiling, electrophysiology, oxygen microsensor recordings and mathematical modeling, we investigated microglia-mediated disturbances in brain energetics during neuroinflammation. Our results suggest that proinflammatory microglia showing enhanced nitric oxide release and decreased CX3CR1 expression transiently increase the tissue lactate/glucose ratio that depends on transcriptional reprogramming in microglia, not in neurons. In this condition, neuronal network activity such as gamma oscillations (30–70 Hz) can be fueled by increased ATP production in mitochondria, which is reflected by elevated oxygen consumption. During dysregulated inflammation, high energy demand and low glucose availability can be boundary conditions for neuronal metabolic fitness as revealed by kinetic modeling of single neuron energetics. Collectively, these findings indicate that metabolic flexibility protects neuronal network function against alterations in local substrate availability during moderate neuroinflammation.

Notch3-regulated microRNAs impair CXCR4-dependent maturation of thymocytes allowing maintenance and progression of T-ALL.

Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.