CXCR3 Ligands Research Articles

Potential therapeutic targets discovery by transcriptome analysis of an in vitro human gastric signet ring carcinoma model.

Loss of E-cadherin expression is frequently observed in signet ring carcinoma (SRCC). People with germline mutations in CDH1, which encodes E-cadherin, develop diffuse gastric cancer at a higher rate. Loss of E-cadherin expression is thus assumed to trigger oncogenic development. To investigate novel therapeutic targets for gastric SRCC, we engineered an E-cadherin-deficient SRCC model in vitro using a human gastric organoid (hGO) with CDH1 knockout (KO). CDH1 KO hGO cells demonstrated distinctive morphological changes similar to SRCC and high cell motility. RNA-sequencing revealed up-regulation of matrix metalloproteinase (MMP) genes in CDH1 KO hGO cells compared to wild type. MMP inhibitors suppressed cell motility of CDH1 KO hGO cells and SRCC cell lines in vitro. Immunofluorescent analysis with 95 clinical gastric cancer tissues revealed that MMP-3 was specifically abundant in E-cadherin-aberrant SRCC. In addition, CXCR4 molecules translocated onto the cell membrane after CDH1 KO. Addition of CXCL12, a ligand of CXCR4, to the culture medium prolonged cell survival of CDH1 KO hGO cells and was abolished by the inhibitor, AMD3100. In clinical SRCC samples, CXCL12-secreting fibroblasts showed marked infiltration into the cancer area. E-cadherin deficient SRCCs might gain cell motility through upregulation of MMPs. CXCL12-positive cancer-associated fibroblasts could serve to maintain cancer-cell survival as a niche. MMPs and the CXCL12/CXCR4 axis represent promising candidates as novel therapeutic targets for E-cadherin-deficient SRCC.

Leveraging STING, Batf3 Dendritic Cells, CXCR3 Ligands, and Other Components Related to Innate Immunity to Induce A "Hot" Tumor Microenvironment That Is Responsive to Immunotherapy.

Simple SummaryThe conceptualization of a “hot” tumor microenvironment has been characterized by mainly T cell infiltration, whereas, “cold” or “immune-deserted” tumors were seen as lacking T cells. However, the presence of antigen-presenting myeloid cells is equally important. In particular, Batf3-lineage DCs are highly efficient in priming effector T cells. Additionally, they can recruit T cells with CXCR3 ligands and re-activate dysfunctional T cells with co-stimulatory molecules in the tumor microenvironment.In a T-cell-inflamed phenotype, tumor eradication works best and is potentiated by immunotherapy such as checkpoint blockade. However, a majority of patients die despite receiving immunotherapy. One reason is insufficient T cell priming and infiltration in the tumor. Nature provides us with innate immune mechanisms in T-cell-inflamed tumors that we can adopt for more personalized immunotherapy strategies. Tumor sensing through innate signaling pathways and efficient antigen-presenting possess a significant role in bridging innate and adaptive immunity and generating a T-cell-inflamed tumor. One approach to strengthen these innate immune mechanisms is to deliver innate immune factors such as STING or activated DCs into the tumor microenvironment, in particular in patients resistant to checkpoint blockade. The low number of DCs in the tumor bed could potentially be increased with the growth factor FMS-like tyrosine kinase 3 ligand (Flt3L). CD103+ DCs are integral for three phases of anti-tumor immunity: priming, recruiting, and re-invigoration of effector T cells. Re-activation of dysfunctional T cells is achieved via co-stimulatory molecules such as the 4-1BB ligand. The presence of myeloid-cell-derived CXCL9 and CXCL10 in the tumor microenvironment can predict response to immunotherapy. We outline recent preclinical and clinical approaches to deliver these crucial components bridging innate and adaptive immunity into the tumor microenvironment.

Location Bias Contributes to Functionally Selective Responses of Biased CXCR3 Agonists to Regulate Inflammation

G Protein‐Coupled Receptors (GPCRs) represent the largest and most diverse family of cell receptors in the human genome and are targeted by ~35% of all FDA‐approved drugs. GPCR signaling is mediated by various effectors, including G proteins, β‐arrestins, and GPCR kinases (GRKs). Recent studies have uncovered that different ligands can bind to the same GPCR and differentially activate specific signaling pathways, a phenomenon known as biased agonism. Most research has focused on characterizing biased agonism at the plasma membrane, but GPCRs are also known to traffic to and signal from a variety of subcellular compartments. To determine if GPCR subcellular localization contributes to biased signaling, we studied how the chemokine family GPCR CXCR3 signals from endosomes following stimulation by the three endogenous ligands CXCL9, CXCL10, and CXCL11.We determined that the biased ligands of CXCR3 promote different amounts of receptor mediated endocytosis and also show markedly different G protein and β‐arrestin signaling profiles at both the plasma membrane and the endosome. The biased activation of G proteins and β‐arrestins promotes differential activation of cytoplasmic and nuclear ERK1/2, as well as cellular transcription in both HEK293 cells and primary CD8+ T‐cells. Inhibition of receptor internalization greatly diminishes the biased signaling observed between the endogenous chemokines. Finally, we demonstrate in a murine model of contact hypersensitivity that receptor internalization is a key component of the ability of a synthetic β‐arrestin biased CXCR3 agonist to potentiate the inflammatory response. The present study shows the CXCR3 biased ligands induce unique signaling profiles at the plasma membrane and the endosome, and that signaling from endosomes greatly contributes to the overall biased responses seen at CXCR3. These results demonstrate that signaling from subcellar compartments is a critical component of GPCR signaling. Our findings have implications in the development of pharmaceutical drugs targeting chemokine receptors and other GPCRs.

Location Bias of G Protein‐Coupled Receptor Kinases Promotes Biased Signaling at CXCR3

G protein‐coupled receptors (GPCRs) are the largest family of cell‐surface receptors and are known to canonically signal through two main effector proteins: G proteins and β‐arrestins. Biased agonism at GPCRs refers to the functional selectivity of a ligand towards certain signaling pathways over others. In addition to biased agonists, there are various other sources of bias that influence GPCR signaling, such as receptor and location bias, which affect the downstream pathways of receptor activation, leading to disproportionate levels of signaling between the G protein and β‐arrestin pathway. One of the mechanisms underlying this signaling bias is the phosphorylation barcode hypothesis which states that ligand induced receptor phosphorylation promotes differential engagement with downstream transducers. Across the entire GPCR superfamily, GPCR kinases (GRKs) are known to be primarily responsible for receptor phosphorylation. Of the seven known isoforms in the GRK family, GRK2, GRK3, GRK5, and GRK6 are expressed ubiquitously, whereas GRK1 and GRK7 are mainly found in the eye while GRK4 is predominantly located in the testes. Although there is supportive evidence demonstrating that differential phosphorylation promotes biased signaling, there is relatively little known about the role of GRKs in directing GPCR functional selectivity. To study the role of GRKs in biased signaling, we studied the GPCR CXCR3, a chemokine receptor primarily involved in leukocyte migration, which has three endogenous biased ligands, CXCL9, CXCL10, and CXCL11, and two synthetic biased ligands, VUF10661 and VUF11418. We found that, following ligand stimulation, GRK2 and GRK3 are recruited to CXCR3, with VUF10661 and CXCL11 being the most efficacious ligands. However, we also observed that GRK3 and GRK5 are able to traffic to endosomes following ligand stimulation with VUF10661, even though all ligands are able to promote receptor internalization. Given biased engagement of the GRKs observed at both the receptor and in endosomes, we next studied the functional consequences of ligand and location bias by creating GRK 2, 3, 5 and 6 constructs that are specifically targeted to the plasma membrane or the endosome. Using GRK2/3/5/6 KO cells, we determined that localization of the GRKs impacts receptor internalization, ERK activity, and transcription in a ligand specific manner. Together, we observed that the endogenous and synthetic ligands of CXCR3 demonstrate biased engagement of the GRKs at different subcellular locations and highlight the functional significance of these findings on downstream receptor signaling. These data provide evidence that the functional selectivity of biased signaling extends beyond β‐arrestins and G‐proteins to other effector proteins that interact with GPCRs.

CD4 Tcells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection.

HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals have an increased risk of tuberculosis prior to loss of peripheral CD4 Tcells, raising the possibility that HIV co-infection leads to CD4 Tcell depletion in lung tissue before it is evident in blood. Here, we use rhesus macaques to study the early effects of simian immunodeficiency virus (SIV) co-infection on pulmonary granulomas. Two weeks after SIV inoculation of Mtb-infected macaques, Mtb-specific CD4 Tcells are dramatically depleted from granulomas, before CD4 Tcell loss in blood, airways, and lymph nodes, or increases in bacterial loads or radiographic evidence of disease. Spatially, CD4 Tcells are preferentially depleted from the granuloma core and cuff relative to B cell-rich regions. Moreover, live imaging of granuloma explants show that intralesional CD4 Tcell motility is reduced after SIV co-infection. Thus, granuloma CD4 Tcells may be decimated before many co-infected individuals experience the first symptoms of acute HIV infection.

The CXCR4-CXCL12 axis promotes T cell reconstitution via efficient hematopoietic immigration

T cells play a critical role in immunity to protect against pathogens and malignant cells. T cell immunodeficiency is detrimental, especially when T cell perturbation occurs during severe infection, irradiation, chemotherapy, and age-related thymic atrophy. Therefore, strategies that enhance T cell reconstitution provide considerable benefit and warrant intensive investigation. Here, we report the construction of a T cell ablation model in Tg(coro1a:DenNTR) zebrafish via metronidazole administration. The nascent T cells are mainly derived from the hematopoietic cells migrated from the kidney, the functional homolog of bone marrow and the complete recovery time is 6.5 days post-treatment. The cxcr4b gene is upregulated in the responsive hematopoietic cells. Functional interference of CXCR4 via both genetic and chemical manipulations does not greatly affect T lymphopoiesis, but delays T cell regeneration by disrupting hematopoietic migration. In contrast, cxcr4b accelerates the replenishment of hematopoietic cells in the thymus. Consistently, Cxcl12b, a ligand of Cxcr4, is increased in the thymic epithelial cells of the injured animals. Decreased or increased expression of Cxcl12b results in compromised or accelerated T cell recovery, respectively, similar to those observed with Cxcr4b. Taken together, our study reveals a role of CXCR4-CXCL12 signaling in promoting T cell recovery and provides a promising target for the treatment of immunodeficiency due to T cell injury.

SARS-CoV-2-specific CD4+ T cells are associated with long-term persistence of neutralizing antibodies

Understanding the decay and maintenance of long-term SARS-CoV-2 neutralizing antibodies in infected or vaccinated people and how vaccines protect against other SARS-CoV-2 variants is critical for assessing public vaccination plans. Here, we measured different plasm antibody levels 2 and 12 months after disease onset, including anti-RBD, anti-N, total neutralizing antibodies, and two neutralizing-antibody clusters. We found that total neutralizing antibodies declined more slowly than total anti-RBD and anti-N IgG, and the two neutralizing-antibody clusters decayed even more slowly than total neutralizing antibodies. Interestingly, the level of neutralizing antibodies at 12 months after disease onset was significantly lower than that at 2 months but more broadly neutralized SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Lambda (C.37). Significant immune escape by the Omicron variant (B.1.1.529) was also observed 2 months post-recovery. Furthermore, we revealed that a high percentage of virus-specific CD4+ T cells and cTfh1 were associated with a slower decline in humoral immunity, accompanied by higher levels of CXCR3 ligands such as CXCL9 and CXCL10, higher frequency of cTfh1, and lower levels of cTfh2 and cTfh17. Our data highlight the importance of coordinating T-cell and humoral immunity to achieve long-term protective immunity.

Targeting myeloid derived suppressor cells reverts immune suppression and sensitizes BRAF-mutant papillary thyroid cancer to MAPK inhibitors

MAPK signaling inhibitor (MAPKi) therapies show limited efficacy for advanced thyroid cancers despite constitutive activation of the signaling correlates with disease recurrence and persistence. Understanding how BRAF pathway stimulates tumorigenesis could lead to new therapeutic targets. Here, through genetic and pathological approaches, we demonstrate that BRAFV600E promotes thyroid cancer development by increasing myeloid-derived suppressor cells (MDSCs) penetrance. This BRAFV600E-induced immune suppression involves re-activation of the developmental factor TBX3, which in turn up-regulates CXCR2 ligands in a TLR2-NFκB dependent manner, leading to MDSCs recruitment into the tumor microenvironment. CXCR2 inhibition or MDSCs repression improves MAPKi therapy effect. Clinically, high TBX3 expression correlates with BRAFV600E mutation and increased CXCR2 ligands, along with abundant MDSCs infiltration. Thus, our study uncovers a BRAFV600E-TBX3-CXCLs-MDSCs axis that guides patient stratification and could be targeted to improve the efficacy of MAPKi therapy in advanced thyroid cancer patients.

The phosphoinositide-3-kinase (PI3K)-delta inhibitor seletalisib impairs monocyte-derived dendritic cells maturation, APC function, and promotes their migration to CCR7 and CXCR4 ligands.

PI3K pathway plays a crucial role in dendritic cells (DCs) functions, as it regulates different cellular processes, such as maturation and cytokines production. However, the specific role of PI3K p110δ isoform in human DCs has not been thoroughly addressed. In this study, we analyze the effects of seletalisib, a potent and specific inhibitor of PI3K p110δ, on phenotype and antigen-presenting functions of monocyte-derived DCs undergone maturation via LPS. Seletalisib treatment reduced membrane HLA-DR as well as CD83 and CD40 costimulatory molecules, whereas CD80 and CD86 expression was only partially affected. Additionally, DCs cultures showed reduced TNF-α, IL-10, and IL-12 and increased IL-23 secretion levels. This resulted ina reduced capacity of DCs to prime allogeneic T cells, with a strong decrease of Th1 differentiation. On the other hand, PI3K p110δ inhibitor seletalisib increased CXCR4 and CCR7 expression and augmented the DCs migration toward CCL19 and CXCL12 ligands. At molecular level, inhibition of PI3K p110δ isoform by seletalisib significantly down-regulated the phosphorylation of AKT and other downstream signaling molecules, such as ribosomal protein S6, 4E-BP1, and NF-κB p65. In contrast, seletalisib did not affect p38 MAP kinase phosphorylation or TLR-associated adapter molecule TIRAP in DCs. Our results indicate that PI3K p110δ can serve as an important regulatory signalfor DCs, and selective inhibition of PI3K p110δisoform by seletalisib could be used for the prevention of exaggerated and harmful immune responsesoccurring in pathologic conditions, such as autoimmune disorders.

Phase I Trial Combining Chemokine-Targeting with Loco-Regional Chemoimmunotherapy for Recurrent, Platinum-Sensitive Ovarian Cancer Shows Induction of CXCR3 Ligands and Markers of Type 1 Immunity

Increased prevalence of cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) predicts positive outcomes in patients with epithelial ovarian cancer (EOC), whereas the regulatory T cells (Treg) predict poor outcomes. Guided by the synergistic activity of TLR3 ligands, IFNα, and COX-2 blockers in selectively enhancing CTL-attractants but suppressing Treg-attractants, we tested a novel intraperitoneal chemoimmunotherapy combination (CITC), to assess its tolerability and TME-modulatory impact in patients with recurrent EOC. Twelve patients were enrolled in phase I portion of the trial NCT02432378, and treated with intraperitoneal cisplatin, intraperitoneal rintatolimod (dsRNA, TLR3 ligand), and oral celecoxib (COX-2 blocker). Patients in cohorts 2, 3, and 4 also received intraperitoneal IFNα at 2, 6, and 18 million units (MU), respectively. Primary objectives were to evaluate safety, identify phase 2 recommended dose (P2RD), and characterize changes in the immune TME. Peritoneal resident cells and intraperitoneal wash fluid were profiled via NanoString and Meso Scale Discovery (MSD) multiplex assay, respectively. The P2RD of IFNα was 6 MU. Median progression-free survival and overall survival were 8.4 and 30 months, respectively. Longitudinal sampling of the peritoneal cavity via intraperitoneal washes demonstrated local upregulation of IFN-stimulated genes (ISG), including CTL-attracting chemokines (CXCL-9, -10, -11), MHC I/II, perforin, and granzymes. These changes were present 2 days after chemokine modulation and subsided within 1 week. The chemokine-modulating intraperitoneal-CITC is safe, tolerable, and associated with ISG changes that favor CTL chemoattraction and function. This combination (plus DC vaccine) will be tested in a phase II trial. See related commentary by Aranda et al., p. 1993.

Chemokine Receptor Activation Enhances Memory B Cell Class Switching Linked to IgE Sensitization to Alpha Gal and Cardiovascular Disease.

Background: Recent studies have suggested that IgE sensitization to α-gal is associated with coronary artery disease (CAD). However, the B cell subtype(s) responsible for production of IgE to α-gal and mechanisms mediating this production remain elusive.Methods: Single cell multi-omics sequencing, was utilized to phenotype B cells obtained from 60 subjects that had undergone coronary angiography in whom serum IgE was evaluated by ImmunoCAP. Bioinformatics approaches were used to identify B cell subtype(s) and transcriptomic signatures associated with α-gal sensitization. In vitro characterization of chemokine/chemokine receptor pairs on switched memory B cells associated with IgE to α-gal was performed.Results: Of the 60 patients, 17 (28%) were positive for IgE to α-gal. CITESeq identified CCR6+ class-switched memory (SWM) B cells and CXCR4 expresssion on these CCR6+ SWM B cells as significantly associated with IgE sensitization to α-gal but not to other common allergens (peanut or inhalants). In vitro studies of enriched human B cells revealed significantly greater IgE on SWM B cells with high CCR6 and CXCR4 expression 10 days after cells were treated with IL-4 and CD40 to stimulate class switch recombination. Both CCL20 (CCR6 ligand) and CXCL12 (ligand for CXCR4) increased the expression of IgE on SWM B cells expressing their receptors. However, they appeared to have unique pathways mediating this effect as only CCL20 increased activation-induced cytidine deaminase (AID), while CXCL12 drove proliferation of CXCR4+ SWM B cells. Lastly, correlation analysis indicated an association between CAD severity and the frequency of both CCR6+ SWM and CXCR4+ SWM B cells.Conclusions: CCR6+ SWM B cells were identified as potential producers of IgE to α-gal in CAD patients. Additionally, our findings highlighted non-chemotaxis roles of CCL20/CCR6 and CXCL12/CXCR4 signaling in mediating IgE class switching and cell proliferation of SWM B cells respectively. Results may have important implications for a better understanding and better therapeutic approaches for subjects with IgE sensitization to α-gal.

Ursolic acid inhibits proliferation, migration and invasion of human papillary thyroid carcinoma cells via CXCL12/CXCR4/CXCR7 axis through cancer-associated fibroblasts.

As a pentacyclic triterpenoid compound, Ursolic acid (UA) broads range of biological effects. CXCL12 is a ligand for CXCR4 and CXCR7 proteins on thyroid cancer cells. Here we examined the effects of UA on the proliferation, migration and invasion of papillary thyroid carcinoma (PTCs) in a dose-manner. In addition, UA can reduce the expression levels of CXCR4 and CXCR7 in PTCs. In addition to this direct anticancer pathway, studies have shown that UA can play an anticancer role by affecting the secretion of CXCL12 in cancer-associated fibroblasts (CAFs). After treated with UA, normal fibroblasts and CAFs culture medium (CM) showed differential CXCL12 expression levels. We prepared fibroblast conditioned medium according to the intervention of UA, then cultured TPC-1 and B-CPAP cells with differential CM, and detected significant differences in the proliferation, migration and invasion of cancer cells. Our findings uncovered an indirect anticancer mechanism of UA. This cancer chemopreventive properties is expected to make UA a clinically useful chemopreventive agent.

Association of chemokines IP-10/CXCL10 and I-TAC/CXCL11 with insulin resistance and enhance leukocyte endothelial arrest in obesity.

Obesity is a key contributing factor to incidental type 2 diabetes and cardiovascular disease. CXCR3 receptor and its ligands CXCL 10 and 11 are associated with atherosclerosis and cardiovascular disease. The aim of our study was to analyse the role of the CXCR3 ligands on insulin resistance (IR) and endothelial dysfunction in human obesity. We have studied 45 obese patients (mean age 44±6years, body mass index 45±9kg/m2) who were selected for Roux-Y-gastric bypass surgery and 21 non obese control subjects with similar age and gender distribution. We measured by ELISA the circulating levels of the CXCR3 ligands interferon-γ inducible protein 10 (IP-10/CXCL10) and interferon-γ-inducible T-cell alpha chemoattractant (I-TAC/CXCL11). Using an ex vivo procedure with the flow chamber assay, we have investigated the effect of such chemokines on endothelial leukocytes arrest under dynamic conditions. Peripheral blood levels of CXCL10 and CXCL11 were significantly higher in obese subjects than in controls (p<0.001) and significantly correlated with BMI, waist circunference and HOMA-IR. Obese patients with HOMA-IR index above 75th percentile showed highest increase of circulating CXCL10 and CXCL11 values. Under dynamic flow conditions, the enhanced adhesion of patient leukocytes to TNFα-induced human arterial endothelial cells was partly dependent on CXCR3. The study demonstrates that CXCL10 and CXCL11 are associated with IR and enhance leukocyte endothelial arrest in obese subjects. Blockade of CXCR3 signaling might be a new therapeutic approach for the prevention of obesity-associated cardiovascular co-morbidities.

Abstract 9214: β-arrestin-biased Receptor CXCR7 in Cardiomyocytes Acts on Cell Survival and Inhibits Post-Infarction Remodeling

Introduction: Seven transmembrane receptors (7TMRs) play various important roles in the heart in which β-arrestin-biased agonism has been recently reported to be cardioprotective. Chemokine receptor CXCR7 is a 7TMR expressed in the heart and binds not to G protein but only to β-arrestin, serving as a β-arrestin-biased receptor. The expression levels of CXCL12, the ligand of CXCR7, are increased in myocardial infarction (MI) but the role of CXCL12/CXCR7 in MI remains elusive. Hypothesis: CXCR7 protects the heart from myocardial infarction as a β-arrestin-biased receptor. Methods and Results: First, we performed bulk RNA sequencing (RNAseq) in murine hearts and found Cxcr7 to be the most abundantly expressed 7TMR. We next used single-cell RNAseq and demonstrated that Cxcr7 was strongly expressed in cardiomyocytes and fibroblasts but not in the other type of cells. Second, we generated cardiomyocyte-specific Cxcr7 knockout (cKO) mice and induced MI by left anterior descending artery ligation. Cxcr7 cKO mice showed significantly exacerbated cardiac dilation and dysfunction at 4 weeks after MI compared to controls. In contrast, fibroblast-specific knockout mice showed no such difference. Third, we revealed that CXCL12 recruited β-arrestin to CXCR7 in CXCR7 expressing cells. We stimulated cultured neonatal rat ventricular myocytes with CXCL12 and induced cell death by oxygen glucose deprivation. ERK was activated and cell death was suppressed by CXCR7 stimulation. In the border zone of mouse infarcted hearts, Cxcr7 expression was increased and ERK was activated, which was attenuated in Cxcr7 cKO hearts. Finally, CXCR7 expression was also increased in human failing hearts, suggesting CXCR7 is also associated with human heart failure. Conclusion: CXCR7 is predominantly expressed in cardiomyocytes, acts on cell survival through ERK activation, and inhibits post-infarction remodeling. CXCR7 may be a new therapeutic target for MI.

Abstract 10144: Xcr1 + Dendritic Cell-Induced Cd4 + T Helper-1 Activation Leads to Exacerbation of Remodeling After Myocardial Infarction

Background and Aim: Immune response including T cell activation after myocardial infarction (MI) is important mechanism in cardiac remodeling. We established a new model of ischemic cardiomyopathy using SR-BI KO/ApoeR61 h/h (SRBI KO) mice in which MI is induced by high fat diet (HFD) for 7 days. We aimed to clarify the precise mechanism leading to post-MI cardiac remodeling: activation of inflammatory CD4 + T-cell subsets and conventional dendritic cells (cDCs), using our non-surgical MI, coronary ligation MI and conditional cCDs KO mice model. Methods and Results: Probucol, a potent antioxidant, was known to improve cardiac remodeling in rat MI model. Using SRBI +/- mice as a non-MI control group, we fed SRBI KO mice HFD to induce MI (non-treatment group) and added probucol (treatment group). With probucol treatment, survival rate and EF were improved, and fibrosis area was reduced. We analyze CD4 + cell subsets in the heart, blood and spleen. Th1 was predominantly increased and activated in border and infarcted area, and decreased with probucol treatment. RNA-seq revealed, specifically Th1 related chemokines, CXCR3 was down-regulated with probucol treatment. Flow data revealed, CXCR3 was highly expressed in Th1 (up to 50%) and decreased with probucol treatment. To elucidate the mechanism of the predominant activation of Th1 after MI, we analyze the dynamics of cDCs. XCR1 + cDCs was activated and decreased with probucol treatment. Furthermore, CXCR3 ligands, CXCL9 and CXCL10, were highly expressed in XCR1 + cDCs (up to 50%). In bone marrow, siglec-H – Ly6C – pre-DCs (XCR1 + cDCs lineage) were increased in non-treatment group and also in coronary ligation MI model, and decreased with probucol treatment. Finally, to confirm the induction of post-MI Th1 type inflammation by XCR1 cDCs, we conducted the conditional depletion of XCR1 + cDCs. In XCR1 KO mice, infiltration of CXCR3 + Th1 in the infarcted area and fibrosis area was decreased, and deterioration of EF, which was evaluated by cardiac MRI, was suppressed after MI. Conclusions: The predominant activation of Th1 after MI exacerbated cardiac remodeling. CXCR3 chemokine system were characteristic of Th1 and XCR1 + cDCs. The modulation of Th1- XCR1 + cDCs interaction could be a novel therapeutic target in post-MI remodeling.

Antibacterial Activity of T22, a Specific Peptidic Ligand of the Tumoral Marker CXCR4.

CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms.

FLT3 Inhibitors Upregulate CXCR4 and E-Selectin Ligands and CD44 Via ERK Suppression in AML Cells, and Blockade of CXCR4 and E-Selectin Signaling with GMI-1359 Overcomes AML Resistance to Quizartinib in Vitro and In Vivo

FLT3 Inhibitors Upregulate CXCR4 and E-Selectin Ligands and CD44 Via ERK Suppression in AML Cells, and Blockade of CXCR4 and E-Selectin Signaling with GMI-1359 Overcomes AML Resistance to Quizartinib in Vitro and In Vivo

A Role for CXCR3 Ligands as Biomarkers of Post-Operative Crohn's Disease Recurrence.

Crohn's disease [CD] recurrence following ileocolic resection [ICR] is common. We sought to identify blood-based biomarkers associated with CD recurrence. CD patients undergoing ICR were recruited across six centres. Serum samples were obtained at post-operative colonoscopy. A multiplex immunoassay was used to analyse 92 inflammation-related proteins [Olink Proteomics]. Bayesian analysis was used to identify proteins associated with increasing Rutgeerts score. Identified proteins were used in receiver operating characteristic [ROC] analysis to examine the ability to identify CD recurrence [Rutgeerts score ≥i2]. Existing single cell data were interrogated to further elucidate the role of the identified proteins. Data from 276 colonoscopies in 213 patients were available. Median time from surgery to first and second colonoscopy was 7 (interquartile range [IQR] 6-9) and 19 [IQR 16-23] months, respectively. Disease recurrence was evident at 60 [30%] first and 36 [49%] second colonoscopies. Of 14 proteins significantly associated with Rutgeerts score, the strongest signal was seen for CXCL9 and MMP1. Among patients on anti-tumour necrosis factor drugs, CXCL9 and CXCL11 were most strongly associated with Rutgeerts score. Both are CXCR3 ligands. Incorporation of identified proteins into ROC analysis improved the ability to identify disease recurrence as compared to C-reactive protein alone: area under the curve [AUC] 0.75 (95% confidence interval [CI]: 0.66-0.82] vs 0.64 [95% CI 0.56-0.72], p = 0.012. Single cell transcriptomic data provide evidence that innate immune cells are the primary source of the identified proteins. CXCR3 ligands are associated with CD recurrence following ICR. Incorporation of novel blood-based candidate biomarkers may aid in identification of CD recurrence.

Melatonin Prevents T Lymphocyte Infiltration to the Kidneys of Hypertensive Rats, Induced by a High-Salt Diet, by Preventing the Expression of CXCR3 Ligand Chemokines.

In a previous study, we demonstrated that melatonin prevents kidney damage in a salt-induced hypertension model by decreasing oxidative stress. We hypothesized that this effect involves melatonin’s immunomodulatory properties. In vivo Study-Dahl salt-sensitive (DSS) rats were fed normal chow, a high-salt diet (HSD), or a HSD and melatonin (30 mg/kg/day) in their water for eight weeks. Kidneys were harvested for immediate lymphocyte isolation and characterization by Flow cytometry (CD3+CD4+ and CD3+CD8+) and for lymphocyte chemoattractant (mainly CXCL chemokines) gene expression studies. In vitro study-rat mesangial cells (RMC) were cultured in a high-salt medium without and with melatonin. A HSD was associated with significant renal infiltration of CD4+ and CD8+ T lymphocytes compared to control. Melatonin significantly reduced renal lymphocyte infiltration. A HSD significantly increased mRNA expression of CXCL chemokines. Adding melatonin to the HSD abolished this effect. Treating RMC cells with salt increased the expression of CXCL10 and CXCL11 but not CXCL9. Adding melatonin to the culture media prevented this increase. Treating HSD-fed rats with melatonin decreased renal lymphocyte chemoattractant mRNA expression and is associated with significantly reducing renal T lymphocyte infiltration. Salt may have a direct effect on chemokine-producing renal cells, which is blunted by melatonin treatment.

CXCR4-CCR7 Heterodimerization Is a Driver of Breast Cancer Progression.

Metastatic breast cancer has one of the highest mortality rates among women in western society. Chemokine receptors CXCR4 and CCR7 have been shown to be linked to the metastatic spread of breast cancer, however, their precise function and underlying molecular pathways leading to the acquisition of the pro-metastatic properties remain poorly understood. We demonstrate here that the CXCR4 and CCR7 receptor ligands, CXCL12 and CCL19, cooperatively bind and selectively elicit synergistic signalling responses in invasive breast cancer cell lines as well as primary mammary human tumour cells. Furthermore, for the first time, we have documented the presence of CXCR4-CCR7 heterodimers in advanced primary mammary mouse and human tumours where number of CXCR4-CCR7 complexes directly correlate with the severity of the disease. The functional significance of the CXCR4-CCR7 association was also demonstrated when their forced heterodimerization led to the acquisition of invasive phenotype in non-metastatic breast cancer cells. Taken together, our data establish the CXCR4-CCR7 receptor complex as a new functional unit, which is responsible for the acquisition of breast cancer cell metastatic phenotype and which may serve as a novel biomarker for invasive mammary tumours.