Chemokine Receptor Expression Research Articles

PF4 Silencing Promotes Trophoblast Cell Proliferation, Migration, Invasion and EMT by Regulating SOCS3/STAT3 Signaling Pathway.

Recurrent Miscarriage (RM) is a chronic and heterogeneous autoimmune disease. Platelet factor 4 (PF4) has been found to be involved in the pathogenesis of RM. We aimed to explore the role and mechanism of PF4 on trophoblasts in RM in vitro. In this study, the expression of PF4 and PF4 receptor CXC chemokine receptor 3 (CXCR3) in the placentas of patients with RM were analyzed by RT-qPCR and western blotting. Serum PF4 level was tested by ELISA. Following PF4 silencing and SOCS3 overexpression in HTR-8/SVneo cells, cell proliferation was detected by CCK-8, colony formation, and EDU assays. Wound healing and transwell assays separately evaluated cell migration and invasion. Immunofluorescence assay determined E-cadherin expression. Tube formation assay was used to measure the angiogenesis. Western blotting examined the expression of metastasis, epithelial-mesenchymal transition (EMT) and suppressor of cytokine signaling 3 (SOCS3)/signal transducer and activator of transcription 3 (STAT3) signaling-associated proteins. The results revealed that PF4 displayed increased expression in placental villus tissues of RM patients. Serum PF4 level was also elevated in RM patients. PF4 silencing promoted the proliferation, migration, invasion, EMT, and angiogenesis of HTR-8/SVneo cells. Additionally, PF4 knockdown downregulated SOCS3 expression to activate STAT3 signaling. SOCS3 overexpression countervailed the effects of PF4 deficiency on HTR-8/SVneo cells. In summary, PF4 participated in the proliferation, migration, invasion, EMT and angiogenesis of trophoblast cells by modulating the SOCS3/STAT3 signaling pathway, indicating that targeting the PF4/SOCS3/STAT3 pathway could be a novel therapy for RM.

Monocyte transcriptome signatures of inflammation and enhanced neutrophil recruitment characterize immunopathology in the blood of TB patients

Tuberculosis (TB) is characterized by immunopathology in the blood and monocytes have been shown to be highly sensitive to plasma environment changes in TB patients. Here, we investigated TB plasma effects on ‘reference monocytes’ using RNA sequencing to characterize a potential immunomodulatory role of monocytes in TB. Candidate pathways induced by plasma samples from TB patients (n=99) compared to healthy controls (n=62) were analyzed for changes in signal transduction, phenotype and secreted cytokines by flow cytometry. Finally, potential implications were characterized in blood samples from corresponding patients and controls.Reference monocytes treated with TB plasma showed an enrichment of pathways involved in inflammation and chemotaxis. Inflammatory cytokines were accompanied by enhanced phosphorylation of STAT molecules (i.e., STAT1/3/5) and strong positive correlations were detected for Interleukin (IL)-6 only in TB plasma-treated monocytes. Moreover, monocyte chemokine receptors (i.e., CCR-1, CCR-5) and pro-inflammatory chemokines (i.e., CXCL-1, CXCL-2, CXCL-8, G-CSF, CCL-2) that attract granulocytes and monocytes were significantly higher in TB plasma-treated monocytes. Notably, corresponding clinical samples also showed higher plasma levels for a subset of inflammatory cytokines/chemokines and, in particular, high IL-6 levels correlated positively with accumulation of neutrophil granulocytes in the blood of TB patients. Finally, monocytes from TB patients were characterized by increased chemokine receptor expression, higher proportions of a CCR-2+ subpopulation and aberrant high SOCS3 expression.These results suggest that monocytes may play a significant role in amplifying plasma immunopathology, leading to sustained mobilization and accumulation of neutrophil granulocytes and chronic inflammation in the blood of TB patients.

Role of Galectin-3 in resident macrophages and effects on atherosclerosis

Abstract Background Arterial macrophages play an important role in atherosclerosis, exhibiting heterogeneity in their origins as either resident or inflammatory monocyte-derived macrophages. Resident macrophages originate predominantly from the yolk sac, and they are endowed with homeostatic functions and can have anti-inflammatory properties under inflammation [1]. Resident macrophages pre-existing within the tissue differentiate into foamy macrophages before monocytes appear in early atherosclerotic lesions [2]. Galectin-3 (Gal-3) encoded by the gene Lgals3, is a crucial effector molecule that is prominently expressed by macrophages [3]. Studies have provided conflicting evidence regarding the role of Gal-3 in atherosclerosis by affecting macrophage functions and monocyte recruitment [4,5]. Purpose This study aims to investigate the role of Gal-3 in arterial resident macrophages for atherosclerosis and reveal the underlying mechanisms. Methods General Lgals3 knockout (Lgals3-/-) and resident macrophage specific knockout (Lgals3-/-Rank-Cretg) mice on the Apoe-deficient background were generated and atherosclerotic lesion development (8 weeks of western diet) was compared. Results Our study revealed that Lgals3-/- mice exhibited increased numbers of monocytes and neutrophils in the blood, which was associated with the development of larger atherosclerotic plaques. Plasma Gal-3 concentration from Lgals3-/-Rank-Cretg mice was decreased by 60%, indicating that the majority of circulating Gal-3 is derived from resident macrophages. Surprisingly, Lgals3-/-Rank-Cretg mice did not show the observed increase in blood monocytes and neutrophils, suggesting that this effect was independent of circulating Gal-3. Consistent with a causal role of monocytosis and leukocytosis in atherogenesis, plaque formation in Lgals3-/-Rank-Cretg was not significantly affected. Additionally, Gal-3 deficiency promoted M1 polarization and reduced efferocytosis, phagocytosis and chemokine receptor expression in BMDMs, and the addition of exogenous Gal-3 restored the effect on phagocytosis and blocked CXCL12 and CCL5-induced leukocyte migration. Conclusion This study reveals a prominent protective role of Gal-3, primarily through inflammatory macrophages rather than resident macrophages for the development of atherosclerotic lesions. This protective effect is attributed to its homeostatic effects on monocyte and neutrophil numbers, as well as its influence on macrophage functions.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 prevents vascular dysfunction in angiotensin II-induced hypertension and selectively modulates aortic myeloid cell phenotype

Abstract Background Leukocyte migration to the vasculature and the heart plays a critical role in hypertensive immune responses and is a carefully orchestrated process, regulated by the interactions of inflammatory chemokines with their receptors, notably CCR1, CCR2, CCR3, and CCR5 (collectively termed inflammatory chemokine receptors (iCCRs)). However, the inflammatory chemokine/receptor system is characterised by redundancy, ligand sharing and overlapping expression patterns. Consequently, our understanding of the specific and combinatory roles of chemokine receptors in cardiovascular inflammation remains incomplete, thus hindering the development of targeted therapies. To address this challenge, we have utilised two novel mouse models: iREP mice, carrying fluorescent reporters of inflammatory chemokine receptor expression, and TACKO mice, in which the entire iCcr locus containing Ccr1, Ccr2, Ccr3, and Ccr5 has been deleted. Purpose To evaluate the expression pattern of iCCRs in experimental hypertension and understand how the global deletion of iCCRs impacts disease progression. Methods We induced hypertension in mice by subcutaneously implanting osmotic minipumps administering angiotensin II (Ang II) for a duration of 14 days. Single-cell RNA sequencing was utilised for investigating iCCR expression in aortas during hypertension. Additionally, we employed flow cytometry and confocal microscopy to track iCCR expression and localization in aortas and hearts obtained from iREP mice. TACKO mice were utilised to assess the effect of global iCCr deficiency on hypertensive phenotypes. Blood pressure was measured via telemetry and vascular function was assessed using myography. Vascular and heart remodelling were evaluated through histology. Molecular mechanisms were analysed using Western Blotting and RT-qPCR, while immune cell changes in the aorta were evaluated through cytometry of time-of-flight (CyTOF). Results Ang II infusion induced higher iCCRs expression levels in both the aorta and the heart in comparison with sham treatment, particularly in fibrotic areas. After Ang II-treatment, global deletion of iCCRs in TACKO mice resulted in the prevention of endothelial dysfunction (n=5/group, P<0.05) and perivascular fibrosis (n=6-9, P<0.01), and a reduction in cardiomyocyte size (n=12-16, P<0.01) compared to wild type mice. However, no significant improvements were observed in other hypertensive phenotypes, including blood pressure, heart fibrosis, vascular NO synthase, and oxidative stress. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta, accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Conclusion Deficiency in iCCRs confers protective effects against vascular dysfunction in experimental hypertension, primarily by selectively reducing inflammatory myeloid cell homing to the aorta.

Chemokine CCL2 and its receptor CCR2 in different age groups of patients with COVID-19

BackgroundDespite the development of various antiviral drugs, most of them are not effective in the treatment of coronavirus disease 2019 (COVID-19) as a hyperinflammatory disorder. Chemokine (C-C motif) ligand 2 (CCL2) is one of the critical CC chemokines involved in the pathogenesis and severity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This study aimed to investigate the expression of CCL2 and CC chemokine receptor 2 (CCR2) in COVID-19 patients.MethodsPeripheral blood samples were collected from 60 confirmed COVID-19 patients and 60 age-matched healthy subjects. The ages of the subjects were categorized as follows: up to 20 years, 20 to 40 years, 40 to 60 years, and more than 60 years. CCL2 serum levels were measured using the enzyme-linked immunosorbent assay (ELISA). CCR2 gene expression in peripheral blood mononuclear cells (PBMCs) was measured employing real-time polymerase chain reaction (PCR).ResultsIn all age groups, CCL2 serum levels were significantly elevated in patients compared to healthy controls (P < 0.0001). CCL2 levels were higher in severe patients than in moderate patients. Moreover, CCR2 expression by PBMCs was higher in patients compared to control subjects. However, a significant difference between patients and controls over 60 years of age was identified (P = 0.0353). There was no significant difference in CCR2 expression between moderate and severe COVID-19 patients.ConclusionsTaken together, the findings demonstrate that CCL2 and CCR2 are upregulated in COVID-19 patients at protein and mRNA levels, respectively. Therefore, the CCL2/CCR2 axis may be a potential therapeutic target in order to improve patient outcomes.

Severe myelotoxicities related to Th1, Th2, Th17 and Treg CD4+ T cells subpopulations and their cytokine profiles in Mexican children with acute lymphoblastic leukemia during induction of chemotherapy

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer in the world, and the myelotoxicities due to chemotherapy can lead to severe infections and hemorrhages with high morbidity and mortality. The immune response has implications in the severity of cancer or survival. This immune response is associated with the predominant subpopulations CD4+ T cells and their effector cytokines. However, it is not known if the immune responses are associated with severe myelotoxicities and mortality in children with ALL. Methods To evaluate the contributions of immunological factors on ALL severe myelotoxicities, we identified the Th1, Th2, Th9, Th17, Th22 and Tregs subpopulations of CD4+ T cells according to their chemokine receptor expression patterns in peripheral blood mononuclear cells by flow cytometry. We evaluated also their cytokines levels by Milliplex in serum from 10 children with ALL at diagnosis (day 0), during induction (day 19) and at the end of induction (day 30) between April 2022 and July 2023. Clinical characteristics such age, sex, type and risk of ALL, hemoglobin and platelets levels, absolute neutrophil count, grade of severity and type of myelotoxicities, infections, and hemorrhages were collected. Clinical outcomes as hospitalization care requirements, ICU admission, leukemia remission or relapsed and mortality were followed and documented. Results The age of children was 8.9 years, (SD,5.6; range 2-16), 60% were male sex, all children had pre- B ALL diagnosis, and 80% were intermediate risk-ALL. Th1 cells were predominant at diagnosis (8.1%) and at the end of induction (7.8%), and Th2 cells at day 19 (7.4%), whereas Th17 were significantly decreased at day 30 (2.6%) (p&amp;lt;0.05). Four patients presented bloodstream infection, two by Gram-positive bacteria (Micrococcus, and Streptococcus), one by Klebsiella pneumoniae antimicrobial resistant, and one by Trichosporum asahii. Severe myelotoxicities were presented in 20% of the patients at day19, and in 30% at day 30. Remission was documented in all patients at day 30 and one was identified with early relapse in the follow-up. Three patients (30%) presented poor outcomes, requiring care in ICU and one died (10%) due to fungal infection. Treg cells were significantly predominant in the children who died by fungal infection (p&amp;lt;0.05), with IL-10 level of 84.6 pg/mL but not detectable IL-35 levels. Conclusion Th17 and Treg subpopulations of CD4+ T cells can play an essential role in immune microenvironment during induction related with the severity of the myelotoxicities and infections, and cytokine IL-10 could be used as marker of infection outcome.

MiR-147-3p in pathogenic CD4 T cells controls chemokine receptor expression for the development of experimental autoimmune diseases

Incomplete Freund's adjuvant (IFA) has long been used to trigger autoimmune diseases in animal models, such as experimental autoimmune encephalitis and collagen-induced arthritis. However, the molecular mechanisms that control CD4 T cell effector functions and lead to the development of autoimmune diseases are not well understood. A self-antigen and heat-killed Mycobacterium tuberculosis emulsified in IFA augmented the activation of CD4 T cells, leading to the differentiation of pathogenic CD4 T cells in the draining lymph nodes. In contrast, IFA emulsification did not elicit Foxp3+ regulatory T cell expansion. We found that pathogenic Th1 cells expressed miR-147-3p, which targets multiple genes to affect T cell function. Finally, miR-147-3p expressed in CXCR6+SLAMF6– Th1 cells was required for the onset of neurological symptoms through the control of CXCR3 expression. Our findings demonstrate that miR-147-3p expressed in pathogenic CD4 T cells regulates the migratory potential in peripheral tissues and impacts the development of autoimmune diseases.

Blockade of the CCR3 receptor reduces neutrophil recruitment to the lung during acute inflammation.

Neutrophils represent one of the host's first lines of defense against invading pathogens. However, an aberrant activation can cause damage to the host. In the case of respiratory infections with viral or bacterial pathogens, one of the most common complications is the development of acute respiratory distress syndrome, in which neutrophil infiltration into the lung is a hallmark. Neutrophils gain expression of chemokine receptors under inflammatory conditions, and their activation can amplify the neutrophil responses. Earlier studies showed that neutrophils recruited to the lung mucosa during bacterial infection upregulate expression of CCR3 and ex vivo stimulation of CCR3 results in an increased neutrophil activation. Therefore, the modulation of effector functions or migration of neutrophils to target sites through chemokine receptors constitutes an opportunity for pharmacological intervention. We aimed to determine whether the blockade of the CCR3 using the specific antagonist SB-328437 reduces neutrophil recruitment and inflammation in the lung in the lipopolysaccharide (LPS)-induced lung injury model and influenza infection in mice. We found that neutrophils acquire CCR3 expression in the lung alveolar space. The intraperitoneal administration of SB-328437 reduced neutrophil recruitment to the lung alveolar space and reduced tissue damage in both the LPS-induced lung injury model and influenza infection. Moreover, treatment with SB-328437 reduced the percentage of neutrophils producing TNFα and neutrophil activation in the alveolar space. Together, these data suggest that CCR3 blockade might be a pharmacological strategy to prevent the aberrant neutrophil activation that results detrimental for the host but preserves sufficient effector response to control the pathogen.

Cytokine and chemokine receptor profiles in adipose tissue vasculature unravel endothelial cell responses in HIV.

Chronic systemic inflammation significantly increases myocardial infarction risk in people living with HIV (PLWH). Endothelial cell dysfunction disrupts vascular homeostasis regulation, increasing the risk of vasoconstriction, inflammation, and thrombosis, contributing to cardiovascular disease. We aimed to characterize endothelial cell (EC) chemokines, cytokine, and chemokine receptors of PLWH, hypothesizing that in our cohort, glucose intolerance contributes to their differential expression implicated in endothelial dysfunction. Using single-cell transcriptomic analysis, we phenotyped chemokine and cytokine receptor expression on arterial ECs, capillary ECs, venous ECs, and vascular smooth muscle cells (VSMCs) in subcutaneous adipose tissue of 59 PLWH with and without glucose intolerance. Our results show that arterial and capillary ECs express significantly higher interferon and tumor necrosis factor (TNF) receptors than venous ECs and VSMCs. Venous ECs exhibited more interleukin (IL)1R1 and ACKR1 receptors, and VSMCs showed significant IL6R expression than arterial and capillary ECs. When stratified by group, arterial ECs from PLWH with glucose intolerance expressed significantly higher IL1R1, IL6R, CXCL12, CCL14, and ICAM2 transcripts than arterial ECs from PLWH without diabetes. Of the different vascular cell types studied, arterial ECs as a proportion of all ECs in adipose tissue were positively correlated with plasma fasting blood glucose. In contrast, venous ECs and VSMCs were positively correlated with plasma IL6. To directly assess the effect of plasma from PLWH on endothelial function, we cultured human arterial ECs (HAECs) in plasma-conditioned media from PLWH and performed bulk RNA sequencing. Plasma from PLWH stimulated ECs with the upregulation of genes that enrich for the oxidative phosphorylation and the TNF-α via NFK-β pathways. In conclusion, ECs in PLWH show heterogeneous cytokine and chemokine receptor expression, and arterial ECs were the most influenced by glucose intolerance. Further research must explicate cytokine and chemokine roles in EC dysfunction and identify biomarkers for disease progression and therapeutic response.

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

Abstract Tu021: Genome-wide CRISPR Screen In Vivo Decodes Monocyte Infiltration Associated With Abdominal Aortic Aneurysm

Introduction: Aortic aneurysm is the second most common aortic disease following atherosclerosis and accounts for the ninth-leading cause of death overall. The inflammatory response has a crucial role in the Abdominal Aortic Aneurysm (AAA) development. The infiltration of monocyte into aortic wall is a hallmark of aortic inflammation and it still requires further understanding of this complicated process. CRISPR screening is a powerful tool to investigate biological process, but it requires large pool of mutant cells and reliable selection procedure, which can usually only be performed in vitro or in xenograft models. To overcome this problem, we developed an unbiased genome-wide CRISPR-Cas Screen for Monocyte Infiltration in vivo (CRISPR-MI) and employed this new method in mouse AAA model to systematically study key genes regulating monocyte infiltration into aortic wall. Hypothesis: Monocyte infiltration is an important process in AAA development and CRISPR-MI could be utilized to study this process in vivo. Methods: To acquire enough monocytes for screen, we used the adoptive transfer method to transplant in vivo differentiated monocytes (bone marrow derived macrophage, BMDM) back into mouse circulation via tail vein injection. We optimized the lentivirus infection protocol, so that sgRNA library delivered by lentivirus can effectively knock out target genes in BMDMs. The BMDMs were infected with sgRNA library before adoptive transfer into AAA-model mice. After transfer, by comparing the sgRNA enrichment in different organs of receipt mice, we could identify key genes inhibiting monocyte infiltration. Results: CRISPR-MI identified novels genes regulating monocyte infiltration into aorta in AAA model, including Trem2 gene. In vitro experiments confirmed that Trem2 knockout increased adhesion molecules and chemokine receptors expression. Conclusions: CRISPR-MI can be used to identify new therapeutical targets for cardiovascular diseases.

The CCL2-CCR2 axis determines whether glomerular endocapillary hypercellularity or wire-loop lesions develop through glomerular macrophage and neutrophil infiltration in lupus nephritis.

The CCL2-CCR2 axis is involved in lupus nephritis, however the precise roles in the mechanisms by which different pathological lesions develop after glomerular immune complex deposition remain elusive. Previously, we demonstrated that genetic CCR2 inhibition induced a histological switch from glomerular endocapillary hypercellularity to wire-loop lesions in murine lupus nephritis. This study aimed to clarify the CCL2-CCR2 axis-mediated cellular mechanism in the formation of these different pathological lesions. We injected MRL/lpr mouse-derived monoclonal IgG3 antibody-producing hybridomas, 2B11.3 or B1, into wild-type (WT) mice to selectively induce glomerular endocapillary hypercellularity or wire-loop lesions. The expression of chemokine and chemokine receptors was analyzed using RT-quantitative PCR and/or immunofluorescence. We found 2B11.3 caused glomerular endocapillary hypercellularity in WT mice with glomerular infiltration of larger numbers of CCR2-expressing macrophages and neutrophils phagocyting immune complex, whereas B1 induced wire-loop lesions. In glomerular endocapillary hypercellularity, CCL2 was identified as the ligand involved in the CCR2-positive cell infiltration; it was expressed by glomerular endothelial cells and macrophages. Notably, 2B11.3-induced glomerular endocapillary hypercellularity converted to wire-loop lesions with reduced glomerular macrophage and neutrophil infiltration in CCL2-deficient (Ccl2-/-) mice similarly observed in Ccr2-/- mice. Moreover, this histological conversion was also observed when both glomerular macrophage and neutrophil infiltration were inhibited in anti-Ly6G antibody-treated Ccr5-/- mice but not when only glomerular macrophage infiltration was inhibited in Ccr5-/- mice or when only glomerular neutrophil infiltration was inhibited in anti-Ly6G antibody-treated WT mice. In contrast, B1 injection caused wire-loop lesions in Ccl2-/- and Ccr2-/- mice, as observed in WT mice. Moreover, 2B11.3 induced CCL2 from glomerular endothelial cells to a larger extent than B1 when injected into Ccr2-/- mice. In conclusion, the CCL2-CCR2 axis determines whether glomerular endocapillary hypercellularity or wire-loop lesions develop by regulating glomerular infiltration of phagocytic cells: macrophages and neutrophils. © 2024 The Pathological Society of Great Britain and Ireland.

The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

PurposeSpontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities, including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma.MethodsWe performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine.ResultsWe established a flow cytometry gating strategy for identifying and isolating FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines, and expression increased when exposed to Tregs but not CD4 + helper T-cells.ConclusionOur study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.

Distinct association patterns of chemokine profile and cardiometabolic status in children and adolescents with type 1 diabetes and obesity.

We compared peripheral blood (PBL) chemokine ligand/receptor profiles in children and adolescents with type 1 diabetes mellitus (T1D) or obesity (OB) (both involving inflammation and vascular complications) to identify their associations with cardiometabolic risk factors. PBL samples from children and adolescents (12-18 years) included: healthy controls (n=29), patients with T1D (n=31) and OB subjects (n=34). Frequency of mononuclear cell populations and chemokine receptor expression (CCR2, CCR4, CXCR3, CXCR4) were determined by flow cytometry. Chemokine levels of CCL2, CCL5, CXCL10 and CXCL11 were measured by bead-based assay and CXCL12 by ELISA. Data were correlated with cardiovascular, metabolic and inflammatory parameters. The proportion of CD14+ monocytes was higher in T1D, whereas the proportion of CD19+ B lymphocytes was higher and CD3+ T lymphocytes was lower in OB. The level of CCL2 was higher in T1D (241.0 (IQR 189.6-295.3) pg/mL in T1D vs 191.5 (IQR 158.0-254.7) pg/mL in control, p=0.033), CXCL11 was lower in OB (6.6 (IQR 4.9-7.7) pg/mL in OB vs 8.2 (IQR 6.9-11.3) pg/mL in control, p=0.018) and CXCL12 was lower in both diseases (2.0 (IQR 1.8-2.5) ng/mL in T1D, 2.1 (IQR 1.9-2.4) ng/mL in OB vs 2.4 (IQR 2.2-2.5) ng/mL in control, p=0.016). Numerous significant associations were found for chemokine ligand/receptor profiles and clinical data. Among these, we are suggesting the most important indicators of cardiometabolic risk in T1D: positive associations of CCR2+ monocytes with blood pressure and CCL12 levels with urine albumin-to-creatinine ratio (ACR), inverse association of CXCR3+ B lymphocytes with AST but positive with triglycerides; and OB: positive associations of CXCL12 levels with triglycerides and AST/ALT, inverse association of CCR4+ and CXCR3+ monocytes with ACR. Both diseases share positive associations for CCR4+ T lymphocytes and blood pressure, inverse associations of CXCR4+ subsets with ACR and CXCR3+ T lymphocytes with lipid profile. Significantly changed chemokine ligand/receptor profiles were found in both T1D and OB even at a young age. Although different associations with cardiometabolic risk factors indicate disease-specific changes, overlapping pattern was found for the associations between CCR4+ T lymphocytes and vascular inflammation, CXCR4+ subsets and albuminuria as well as CXCR3+ T lymphocytes and dyslipidemia. Thus, chemokine axes might present potential therapeutic targets for disease-related morbidity.

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.

Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.

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.

Chemokine-mediated cell migration into the central nervous system in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) has been associated with different forms of immune compromise. This study analyzes the chemokine signals and attracted immune cells in cerebrospinal fluid (CSF) during PML to define immune cell subpopulations relevant for the PML immune response. In addition to chemokines that indicate a general state of inflammation, like CCL5 and CXCL10, the CSF of PML patients specifically contains CCL2 and CCL4. Single-cell transcriptomics of CSF cells suggests an enrichment of distinct CD4+ and CD8+ Tcells expressing chemokine receptors CCR2, CCR5, and CXCR3, in addition to ITGA4 and the genetic PML risk genes STXBP2 and LY9. This suggests that specific immune cell subpopulations migrate into the central nervous system to mitigate PML, and their absence might coincide with PML development. Monitoring them might hold clues for PML risk, and boosting their recruitment or function before therapeutic immune reconstitution might improve its risk-benefit ratio.

The CCL2-CCR4 Axis Promotes Regulatory T Cell Trafficking to Canine Glioma Tissues.

Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high grade glioma and human glioblastomas share many molecular similarities, including accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford targeting the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic model of glioma. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. We established a flow cytometry gating strategy for identification and isolation of FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines and expression increased when exposed to Tregs but not to CD4 + helper T-cells. Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.