Chemokine Receptor CXCR4 Research Articles

CXCR3-expressing myeloid cells recruited to the hypothalamus protect against diet-induced body mass gain and metabolic dysfunction

Microgliosis plays a critical role in diet-induced hypothalamic inflammation. A few hours after a high-fat diet (HFD), hypothalamic microglia shift to an inflammatory phenotype, and prolonged fat consumption leads to the recruitment of bone marrow-derived cells to the hypothalamus. However, the transcriptional signatures and functions of these cells remain unclear. Using dual-reporter mice, this study reveals that CX3CR1-positive microglia exhibit minimal changes in response to a HFD, while significant transcriptional differences emerge between microglia and CCR2-positive recruited myeloid cells, particularly affecting chemotaxis. These recruited cells also show sex-specific transcriptional differences impacting neurodegeneration and thermogenesis. The chemokine receptor CXCR3 is emphasized for its role in chemotaxis, displaying notable differences between recruited cells and resident microglia, requiring further investigation. Central immunoneutralization of CXCL10, a ligand for CXCR3, resulted in increased body mass and decreased energy expenditure, especially in females. Systemic chemical inhibition of CXCR3 led to significant metabolic changes, including increased body mass, reduced energy expenditure, elevated blood leptin, glucose intolerance, and decreased insulin levels. This study elucidates the transcriptional differences between hypothalamic microglia and CCR2-positive recruited myeloid cells in diet-induced inflammation and identifies CXCR3-expressing recruited immune cells as protective in metabolic outcomes linked to HFD consumption, establishing a new concept in obesity-related hypothalamic inflammation.

NIMG-34. CXCR4-TARGETED PET IMAGING OF CENTRAL NERVOUS SYSTEM LYMPHOMA AND GLIOMA USING [68GA]GA-PENTIXAFOR

Abstract BACKGROUND Initial results suggest that PET imaging of the chemokine receptor CXCR4 is of considerable interest for differential diagnosis and theranostic approaches. Here, we summarized findings of CXCR4 PET imaging in an institutional series of lymphoma of the central nervous system (CNSL) and glioma. METHODS Twenty-three patients with CNSL and 16 patients with glioma (IDH-wildtype, n=9) underwent PET imaging using the CXCR4-directed tracer [68Ga]Ga-Pentixafor. In 18 patients, serial PET imaging was performed (range, 2-14 scans), resulting in a total of 104 PET scans. For evaluation, PET scans were classified as [68Ga]Ga-Pentixafor-positive and -negative visually, and the maximum standardized uptake value (SUVmax) was obtained from [68Ga]Ga-Pentixafor-PET. RESULTS Twenty-four of all 39 patients (62%) had at least one [68Ga]Ga-Pentixafor-positive PET (CNSL, 57%; glioma, 69%). [68Ga]Ga-Pentixafor-positive PET scans were more common in patients with IDH-wildtype gliomas (89%) than in IDH-mutant gliomas (43%). In patients with [68Ga]Ga-Pentixafor-positive PET, the average SUVmax was 6.4±4.0 in CNSL, and 3.4±1.0 in gliomas (P=0.155). Besides visualization of tumors, [68Ga]Ga-Pentixafor uptake was observed in two patients with radionecrosis. In four CNSL patients who had undergone methotrexate/cytarabine-based first-line therapy, serial PET revealed a significant reduction of SUVmax after chemotherapy completion (average decrease of SUVmax from 4.5±3.8 to 0.6±0.7; P=0.044). In three of those four patients, tumor progression has not been reached (range of time after the follow-up PET, 4-55 months); one patient deceased 44 months after the follow-up PET. In contrast, another patient with an increase in SUVmax from 6.9 to 11.0 after first-line treatment died one month after the follow-up PET. DISCUSSION [68Ga]Ga-Pentixafor PET seems to be of value for non-invasively visualizing and quantifying CXCR4 receptor binding. In CNSL, changes in [68Ga]Ga-Pentixafor uptake following therapy may indicate potential for response assessment. CXCR4-targeting radioligand therapies might be more promising in CNSL than in glioma.

SNX9 family mediates βarrestin-independent GPCR endocytosis

Agonist-stimulated GPCR endocytosis typically occurs via the multi-faceted adaptor proteins known as βarrestins. However, endocytosis of several GPCRs occurs independently of β-arrestins, suggesting an additional mode of GPCR endocytosis, but the mechanisms remain unknown. Here we provide evidence that sorting nexin 9 (SNX9), a previously described endocytic remodeling protein, functions as a novel cargo adaptor that promotes agonist-stimulated GPCR endocytosis. We show that SNX9 and SNX18, but not β-arrestins, are necessary for endocytosis of the chemokine receptor CXCR4. SNX9 is recruited to CXCR4 at the plasma membrane and interacts directly with the carboxyl-terminal tail of the receptor in a phosphorylation-dependent manner. We also provide evidence that some receptors do not require SNX9 and SNX18 nor β-arrestins for endocytosis, suggesting additional modes for GPCR endocytosis. These results provide novel insights into the mechanisms regulating GPCR trafficking and broaden our overall understanding of GPCR regulation.

Chemokine receptor CXCR7 antagonism ameliorates cardiac and renal fibrosis induced by mineralocorticoid excess

Cardiorenal fibrosis is a common feature of chronic cardiovascular disease and recent data suggests that cytokines and chemokines may also drive fibrosis. Here we tested the hypothesis that CXCR7, a highly conserved chemokine receptor, contributes to cardiac and renal fibrosis. We generated an anti-mouse CXCR7-specific monoclonal antibody (CXCR7 mAb) and tested its anti-fibrotic actions in cardiorenal fibrosis induced using the deoxycorticosterone acetate/uni-nephrectomy (DOCA-UNX) model. CXCR7 mAb treatment (10 mg/kg, twice weekly for 6 weeks) significantly attenuated the development of cardiac and renal fibrosis, and reduced fibrotic and inflammatory gene expression levels, in the absence of an effect on blood pressure. Immunohistochemical analysis demonstrated an increase in the vascular expression of CXCR7 in DOCA-UNX-treated mice. This study demonstrated that a CXCR7 mediated pathway plays a significant role in cardiac and renal fibrosis induced by DOCA-UNX treatment. Accordingly, antagonism of CXCR7 may provide a therapeutic opportunity to mitigate against fibrosis in the setting of mineralocorticoid excess.

The VEGF-Mediated Cytoprotective Ability of MIF-Licensed Mesenchymal Stromal Cells in House Dust Mite-Induced Epithelial Damage.

Enhancing mesenchymal stromal cell (MSC) therapeutic efficacy through licensing with proinflammatory cytokines is now well established. We have previously shown that macrophage migration inhibitory factor (MIF)-licensed MSCs exerted significantly enhanced therapeutic efficacy in reducing inflammation in house dust mite (HDM)-driven allergic asthma. Soluble mediators released into the MSC secretome boast cytoprotective properties equal to those associated with the cell itself. In asthma, epithelial barrier damage caused by the inhalation of allergens like HDM drives goblet cell hyperplasia. Vascular endothelial growth factor (VEGF) plays a pivotal role in the repair and maintenance of airway epithelial integrity. Human bone marrow-derived MSCs expressed the MIF receptors CD74, CXCR2, and CXCR4. Endogenous MIF from high MIF expressing CATT7 bone marrow-derived macrophages increased MSC production of VEGF through the MIF CXCR4 chemokine receptor, where preincubation with CXCR4 inhibitor mitigated this effect. CATT7-MIF licensed MSC conditioned media containing increased levels of VEGF significantly enhanced bronchial epithelial wound healing via migration and proliferation in vitro. Blocking VEGFR2 or theuse of mitomycin C abrogated this effect. Furthermore, CATT7-MIF MSC CM significantly decreased goblet cell hyperplasia after the HDM challenge in vivo. This was confirmed to be VEGF-dependent, as the use of anti-human VEGF neutralising antibody abrogated this effect. Overall, this study highlights that MIF-licenced MSCs show enhanced production of VEGF, which has the capacity to repair the lung epithelium.

N-[4-(Benzyloxy)-3-methoxybenzyl)]adamantane-1-amine (DZH2), a dual CCR5 and CXCR4 inhibitor as a potential agent against triple negative breast cancer.

DZH2, a dual inhibitor of the chemokine receptors CCR5 and CXCR4, was discovered from virtual screening for CCR5 antagonists. In specific Ca2+ chemokine signaling assays, DZH2 displayed low micromolar IC50 values at both chemokine receptors. Its binding to intracellular allosteric binding sites of CCR5 and CXCR4 was confirmed by MD simulations and binding free-energy calculations. DZH2 is superior to the CCR5 antagonist maraviroc in terms of its inhibitory activity on the growth of two breast cancer cell lines. In MCF7 and MDA-MB-231 cells, DZH2 was a >100-fold more potent inhibitor of cell viability compared to maraviroc. DZH2 (6.7 µM) reduced migration of MDA-MB-231 cells to 4% compared to 50% inhibition of migration caused by maraviroc (780 µM). Also, DZH2 was a significantly more potent inhibitor of colony formation in MDA-MB-231 cells than maraviroc. In MCF10 cells, DZH2 caused no alteration in the gene expression with respect to cellular pathways mediating cell death, indicating its selectivity to breast cancer cells.

CXCR4 signaling determines the fate of hematopoietic multipotent progenitors by stimulating mTOR activity and mitochondrial metabolism

Both cell-intrinsic and niche-derived, cell-extrinsic cues drive the specification of hematopoietic multipotent progenitors (MPPs) in the bone marrow, which comprise multipotent MPP1 cells and lineage-restricted MPP2, MPP3, and MPP4 subsets. Patients with WHIM syndrome, a rare congenital immunodeficiency caused by mutations that prevent desensitization of the chemokine receptor CXCR4, have an excess of myeloid cells in the bone marrow. Here, we investigated the effects of increased CXCR4 signaling on the localization and fate of MPPs. Knock-in mice bearing a WHIM syndrome–associated CXCR4 mutation ( CXCR4 1013 ) phenocopied the myeloid skewing of bone marrow in patients. Whereas MPP4 cells in wild-type mice differentiated into lymphoid cells, MPP4s in CXCR4 1013 knock-in mice differentiated into myeloid cells. This myeloid rewiring of MPP4s in CXCR4 1013 knock-in mice was associated with enhanced signaling mediated by the kinase mTOR and increased oxidative phosphorylation (OXPHOS). MPP4s also localized further from arterioles in the bone marrow of knock-in mice compared with wild-type mice, suggesting that the loss of extrinsic cues from the perivascular niche may also contribute to their myeloid skewing. Chronic treatment with the CXCR4 antagonist AMD3100 or the mTOR inhibitor rapamycin restored the lymphoid potential of MPP4s in knock-in mice. Thus, CXCR4 desensitization drives the lymphoid potential of MPP4 cells by dampening the mTOR-dependent metabolic changes that promote myeloid differentiation.

Receptor determinants for ß-arrestin functional specificity at C-X-C chemokine receptor 5 (CXCR5).

ß-arrestins are multi-faceted adaptor proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and signaling. It is emerging that receptor-specific determinants specify these divergent functions at GPCRs, yet this remains poorly understood. Here, we set out to identify the receptor determinants responsible for ß-arrestin-mediated regulation of the chemokine receptor CXCR5. Using bioluminescence resonance energy transfer (BRET) we show that ß-arrestin1 and ß-arrestin2 are dose-dependently recruited to CXCR5 by its cognate ligand CXCL13. The carboxy-terminal tail of CXCR5 contains several Ser/Thr residues that can be divided into 3 discrete phospho-site clusters based on their position relative to transmembrane domain 7. Mutagenesis experiments revealed that the distal and medial phospho-site clusters, but not the proximal, are required for agonist-stimulated ß-arrestin1 or ß-arrestin2 recruitment to CXCR5. Consistent with this, we provide evidence that the distal and medial, but not proximal, phospho-site clusters are required for receptor desensitization. Surprisingly, the individual phospho-site clusters are not required for agonist-stimulated internalization of CXCR5. Further, we show that CXCL13-stimulated CXCR5 internalization and ERK1/2 phosphorylation, but not desensitization, remain intact in HEK293 cells lacking ß-arrestin1 and ß-arrestin2. Our study provides evidence that b-arrestins are recruited to CXCR5 and are required for desensitization but are dispensable for internalization or signaling, suggesting that discrete receptor determinants specify the divergent functions of ß-arrestins. Significance Statement CXCL13 and CXCR5 are important in the immune system and are linked to diseases, yet regulation of CXCR5 signaling remains poorly understood. We provide evidence that a phospho-site cluster located at the extreme distal carboxyl-terminal tail of the receptor is responsible for ß-arrestin recruitment and receptor desensitization. ß-arrestins are not required for CXCL13-stimulated internalization or signaling, indicating that ß-arrestins perform only one of their functions at CXCR5 and that discrete receptor determinants specify the divergent functions of ß-arrestins.

Peripheral immune landscape of pediatric fulminant myocarditis revealed by single-cell sequencing

Abstract Fulminant myocarditis (FM) is one of the most feared diseases in children because of its fulminant nature and high mortality rate. However, the precise pathological immune subsets and molecular changes in FM are unknown. Here, we present the first comprehensive cellular and transcriptomic profiling of the peripheral blood mononuclear cells of children in FM acute and recovery phases using Single-cell RNA sequencing. 21 cell clusters are identified among 79542 cells. The proportion of T cells and NK cells increase, while myeloid cells and B cells reduce in acute phase, which were consistent with our flow cytometry data of 35 FM patients. Several unique cell types in peripheral blood are identified, including regulatory B cells, MAIT cells, adaptive NK cells and CD8+Tpex cells. The transcriptomic analysis exhibited elevated expression levels of chemokine receptor CXCR4 and S100A family genes almost in all cell types in FM acute phase, as well as MHC-II molecules in antigen-presenting cells. TCR and BCR exhibit remarkable amplification and obvious skewed usages of V genes in FM. Ligand receptor analysis show myeloid cells maintained active communication with other immune cells. Considering that CXCR4 may play an important role in myocarditis, we designed experiments to explore its function in mouse model. Vital myocarditis (VMC) mouse model was constructed using Coxsackie virus type3（CVB3）.We found that myocardial inflammation and myocardial injury of VMC mice were alleviated when treated with CXCR4 blocker. CXCR4 may be a potential therapeutic target for myocarditis. Our study will provide useful insights into key immune cell subsets and transcriptomic profiles implicated in pediatric fulminant myocarditis acute phase and recovery phase, thus providing several potential diagnostic and therapeutic targets for this disease.

Chemo-Click: Receptor-Controlled and Bioorthogonal Chemokine Ligation for Real-Time Imaging of Drug-Resistant Leukemic B Cells.

Drug resistance in B cell leukemia is characterized by the coexpression of CXCR5 and CXCR3 chemokine receptors, making it a valuable biomarker for patient stratification. Herein, we report a novel platform of activatable chemokines to selectively image drug-resistant leukemic B cells for the first time. The C-terminal derivatization of the human chemokines CXCL13 and CXCL10 with bioorthogonal tetrazine-BODIPY and BCN groups retained binding and internalization via their cognate CXCR5 and CXCR3 receptors and enabled rapid fluorescence labeling of CXCR5+ CXCR3+ resistant B cells─but not drug-susceptible leukemic cells─via intracellular chemokine ligation. This modular chemical approach offers a versatile strategy for real-time immunophenotyping of cell populations with distinct chemokine profiles and will accelerate the design of new precision medicine tools to advance personalized therapies in blood tumors.

A CXCR4 partial agonist improves immunotherapy by targeting polymorphonuclear myeloid-derived suppressor cells and cancer-driven granulopoiesis.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that potently impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target1. Here, we fused a secreted CXCR4 partial agonist TFF2 to mouse serum albumin (MSA) and demonstrated that TFF2-MSA peptide synergized with anti-PD-1 to induce tumor regression or eradication, inhibited distant metastases, and prolonged survival in multiple gastric cancer (GC) models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track PMN-MDSC in vivo , we found TFF2-MSA selectively reduced the immunosuppressive Hdc-GFP + CXCR4 hi tumor PMN-MDSCs while preserving proinflammatory neutrophils, thereby boosting CD8 + T cell-mediated anti-tumor response together with anti-PD-1. Furthermore, TFF2-MSA systemically reduced PMN-MDSCs and bone marrow granulopoiesis. In contrast, CXCR4 antagonism plus anti-PD-1 failed to provide a similar therapeutic benefit. In GC patients, expanded PMN-MDSCs containing a prominent CXCR4 + LOX-1 + subset are inversely correlated with the TFF2 level and CD8 + T cells in circulation. Collectively, our studies introduce a strategy of using CXCR4 partial agonism to restore anti-PD-1 sensitivity in GC by targeting PMN-MDSCs and granulopoiesis.

Structural insights into KSHV-GPCR constitutive activation and CXCL1 chemokine recognition

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a viral G protein-coupled receptor, KSHV-GPCR, that contributes to KSHV immune evasion and pathogenesis of Kaposi's sarcoma. KSHV-GPCR shares a high similarity with CXC chemokine receptors CXCR2 and can be activated by selected chemokine ligands. Like other herpesvirus-encoded GPCRs, KSHV-GPCR is characterized by its constitutive activity by coupling to various G proteins. We investigated the structural basis of ligand-dependent and constitutive activation of KSHV-GPCR, obtaining high-resolution cryo-EM structures of KSHV-GPCR-Gi complexes with and without the bound CXCL1 chemokine. Analysis of the apo-KSHV-GPCR-Gi structure (2.81 Å) unraveled the involvement of extracellular loop 2 in constitutive activation of the receptor. In comparison, the CXCL1-bound KSHV-GPCR-Gi structure (3.01 Å) showed a two-site binding mode and provided detailed information of CXCL1 binding to a chemokine receptor. The dual activation mechanism employed by KSHV-GPCR represents an evolutionary adaptation for immune evasion and contributes to the pathogenesis of Kaposi's sarcoma. Together with results from functional assays that confirmed the structural models, these findings may help to develop therapeutic strategies for KSHV infection.

ACKR3 agonism induces heterodimerization with chemokine receptor CXCR4 and attenuates platelet function.

Platelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardiovascular diseases. Like most chemokine receptors, CXCR4 is a G protein coupled receptor that induces platelet activation. In contrast, the atypical chemokine receptor 3 (ACKR3) lacks the ability to activate heterotrimeric G proteins and its activation leads to platelet inhibition and attenuates thrombus formation. In nucleated cells, heterodimerization of ACKR3 with CXCR4 regulates CXCL12-dependent signalling. The aim of our study was to investigate the formation of ACKR3/CXCR4 heterodimers in platelets and the subsequent consequences for platelet function. Using a proximity ligation assay (PLA, Duolink®) to screen for CXCR4/ACKR3 heterodimerization inducing compounds, we found that ACKR3 agonism but not conventional platelet agonists or endogen ligands lead to heterodimer formation. To further characterize the formation of ACKR3/CXCR4 heterodimers, we studied the CXCL12-dependent platelet activation via CXCR4. Both, CXCL12-dependent platelet aggregation and collagen-dependent exvivo thrombus formation were significantly downregulated by ACKR3 agonism. Moreover, platelet intracellular calcium and Akt signalling were increased by CXCL12 and again suppressed by ACKR3-specific agonists. Previously, CXCL12 was shown to decrease platelet cAMP levels via CXCR4. Treatment with a specific ACKR3 agonist counteracted this CXCL12/CXCR4-dependent cAMP decrease. Our results reveal that the formation of platelet ACKR3/CXCR4 heterodimers is dependent on ACKR3 rather than CXCR4. Furthermore, ACKR3 agonism induced heterodimerization is associated with mitigating CXCL12/CXCR4-dependent platelet activation possibly by modulating CXCR4-dependent G protein signalling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists.

Low dose methotrexate impaired T cell transmigration through down-regulating CXCR4 expression in rheumatoid arthritis (RA)

BackgroundCXC chemokine CXCL12 is involved in the pathological development of rheumatoid arthritis (RA) through abnormal migration of peripheral immune cells in the joint. Although low dose methotrexate (MTX) is clinically used to treat RA patients, CXCL12 signaling responses to MTX-mediated treatments is still not well understood.MethodsIn this study, we examined the expression of CXCR4 (cognatic receptor for CXCL12) in peripheral T cells from RA patients and arthritis mice models received from low dose MTX therapies. The effects of low dose MTX on CXCR4 were further determined via both in vitro CD3+ T cells and Cxcr4 conditional knockout (CKO) arthritis mice models.ResultsOur clinical data shows that low dose MTX treatment was clinically associated with down-regulated expression of chemokine receptor CXCR4 on patient peripheral T cells. In vitro, low dose MTX significantly decreased cell transmigration through down-regulated CXCR4’s expression in CD3+ T cells. Consistently, CD3+ T cells treated with low dose MTX demonstrated an increased genomic hypermethylation across the promoter region of Cxcr4 gene. Furthermore, our preclinical studies showed that low dose MTX-mediated downregulation of CXCR4 significantly improved the pathological development in mouse arthritis models. Conditional disruption of the Cxcr4 gene in peripheral immune cells potentially alleviated inflammation of joints and lung tissue in the arthritis mice, though genetic modification itself overall did not change their clinical scores of arthritis, except for a significant improvement on day 45 in CXCR4 CKO arthritis mice models during the recovery phase.ConclusionOur findings suggest that the effect of low dose MTX treatment could serve to eliminate inflammation in RA patients through impairment of immune cell transmigration mediated by CXCR4.

Crosstalk between androgen signaling and the chemokine receptor CXCR4: a novel strategy to promote myelin regeneration.

Crosstalk between androgen signaling and the chemokine receptor CXCR4: a novel strategy to promote myelin regeneration.

Chemokine receptor CXCR4 based positron emission tomography imaging in systemic sclerosis-related interstitial lung disease.

To assess chemokine receptor CXCR4 expression in lung parenchyma and on peripheral immune cells in systemic sclerosis-related interstitial lung disease (SSc-ILD) patients. SSc-ILD patients underwent 68Ga- CPCR4 Trifluoroacetate positron emission tomography (PET) scan, SUVmean in different lung regions and architecturally abnormal areas, and receiver operating characteristic (ROC) curves were analyzed. CXCR4 expression on peripheral immune cells using flow cytometer was studied and correlated with the different lung regions. In addition, subset analysis of CXCR4 expression by clinical subset (early, progressive, stable), ILD pattern and anti-Scl-70 positivity were done. On PET, SSc-ILD patients showed higher median SUVmean uptake of CXCR4 in the whole lung (0.56; p< 0.0001), different lung regions and architecturally abnormal areas than controls. Highest area under curve (AUC) were observed in dorsobasal regions (AUC-0.91; p< 0.0001) and reticular with architecturally distorted areas (AUC-0.95; p< 0.0001). Progressive subset had higher whole lung median SUVmean (0.73) than early (0.49; p< 0.0001) and stable (0.45; p< 0.0001) subsets, and AUC than early and stable subsets. Usual interstitial pneumonia pattern ILD showed higher CXCR4 uptake compared with non-specific interstitial pneumonia (p= 0.0032). Additionally, a trend for higher uptake was noted in anti-Scl70 positive patients as compared with anti-Scl70 negative ones. SSc-ILD patients had higher CD4+CXCR4+T cells (p= 0.0003), and CD8+CXCR4+T cells (p= 0.0013), and showed moderate to strong association on correlation with the lung parenchymal regions. In SSc-ILD, CXCR4 expression is upregulated in both lung parenchyma and peripheral T cells, significantly in progressive and UIP subsets. CXCR4 expression is a potential tool for activity assessment and prognostication.

Structural insights into CXCR4 modulation and oligomerization.

Activation of the chemokine receptor CXCR4 by its chemokine ligand CXCL12 regulates diverse cellular processes. Previously reported crystal structures of CXCR4 revealed the architecture of an inactive, homodimeric receptor. However, many structural aspects of CXCR4 remain poorly understood. Here, we use cryo-electron microscopy to investigate various modes of human CXCR4 regulation. CXCL12 activates CXCR4 by inserting its N terminus deep into the CXCR4 orthosteric pocket. The binding of US Food and Drug Administration-approved antagonist AMD3100 is stabilized by electrostatic interactions with acidic residues in the seven-transmembrane-helix bundle. A potent antibody blocker, REGN7663, binds across the extracellular face of CXCR4 and inserts its complementarity-determining region H3 loop into the orthosteric pocket. Trimeric and tetrameric structures of CXCR4 reveal modes of G-protein-coupled receptor oligomerization. We show that CXCR4 adopts distinct subunit conformations in trimeric and tetrameric assemblies, highlighting how oligomerization could allosterically regulate chemokine receptor function.

Interaction and dynamics of chemokine receptor CXCR4 binding with CXCL12 and hBD-3

Chemokine receptor CXCR4 is involved in diverse diseases. A comparative study was conducted on CXCR4 embedded in a POPC lipid bilayer binding with CXCL12 in full and truncated forms, hBD-3 in wildtype, analog, and mutant forms based on in total 63 µs all-atom MD simulations. The initial binding structures of CXCR4 with ligands were predicted using HADDOCK docking or random-seed method, then μs-long simulations were performed to refine the structures. CXCR4&ligand binding structures predicted agree with available literature data. Both kinds of ligands bind stably to the N-terminus, extracellular loop 2 (ECL2), and ECL3 regions of CXCR4; the C2-C3 (K32-R38) region and occasionally the head of hBD-3 bind stably with CXCR4. hBD-3 analogs with Cys11-Cys40 disulfide bond can activate CXCR4 based on the Helix3-Helix6 distance calculation, but not other analogs or mutant. The results provide insight into understanding the dynamics and activation mechanism of CXCR4 receptor binding with different ligands.

CSF1R Ligands Expressed by Murine Gliomas Promote M-MDSCs to Suppress CD8+ T Cells in a NOS-Dependent Manner.

Glioblastoma (GBM) is the most common malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a subset of myeloid cells, expressing monocytic (M)-MDSC markers and dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate the TME. This study evaluated the mechanism of CCR2+/CX3CR1+ M-MDSC differentiation and T cell suppressive function in murine glioma models. We determined that bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Glioma-secreted CSF1R ligands M-CSF and IL-34 were identified as key drivers of M-MDSC differentiation while adenosine and iNOS pathways were implicated in the M-MDSC suppression of T cells. Mining a human GBM spatial RNAseq database revealed a variety of different pathways that M-MDSCs utilize to exert their suppressive function that is driven by complex niches within the microenvironment. These data provide a more comprehensive understanding of the mechanism of M-MDSCs in glioblastoma.

Immune response and protection efficacy of formalin-killed vaccines against Streptococcus iniae in four-finger threadfin Eleutheronema tetradactylum.

Four-finger threadfin, Eleutheronema tetradactylum farming in southern Taiwan has been facing disease problems caused by Streptococcus iniae since 2018. The development of a vaccine against infectious S. iniae in the cultured threadfin industry is necessary. Thus, this study aimed to examine the efficacy of threadfin immunized formalin-killed cells (FKC) from S. iniae GSI-111 for 42 days post-vaccination (dpv) using two doses of FKC alone (a booster at 14 dpv) as group A, and FKC mixed with ISA763A adjuvant using a single dose as group B or double doses as group C. Immunoglobulin (Ig)-M was purified from threadfin, and rabbit anti-threadfin IgM polyclonal antibodies were used to detect antibody level in immunized fish; the vaccinated group A displayed higher levels at 3 dpv and all vaccinated treatments demonstrated high antibody levels between 14 and 42 dpv. All vaccine groups showed significantly higher values of lysozyme activity at 42 dpv compared with the control group; the vaccinated A group peaked at 14 dpv. The expression profiles of pro-inflammatory and immune-related genes, TNF-α, IL-12A, and C2 were upregulated at 3 dpv, while CD8A and chemokine receptor CXCR4 were upregulated at 42 dpv. Finally, the threadfins were challenged with S. iniae at 42 dpv. The average relative percent survival was 96% for vaccination A and B treatments, and 100% for vaccination C treatment. In summary, this study demonstrated that FKC vaccines whether formulated with an adjuvant could stimulate immune response and effective protect threadfins against S. iniae infection.