Chemokine Receptor CCR5 Research Articles

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.

Protective role of Metrnl on macrophage recruitment during the acute inflammatory phase in a mouse model of myocardial infarction

Abstract Introduction Meteorin-like protein (Metrnl) is a cytokine involved in the attenuation of inflammation. Previously, a cardioprotective role of Metrnl against cardiac hypertrophy and ischemia/reperfusion has been described. After myocardial infarction (MI), macrophages recruited from the spleen, and their capacity to shift from inflammatory (M1) to reparative (M2) phases determines the fate of cardiac repair. Purpose The aim of this study was to analyse the function of Metrnl in a mouse model of permanent MI during the acute inflammatory phase. Methods Wild-type (WT), Metrnl Knock-out (Metrnl-/-) and Metrnl-reconstituted Metrnl-/- mice (AAV9-Metrnl, with myocardial-specific tropism) were subjected to MI by coronary artery ligation. After 4 days, cardiac function was assessed using echocardiography at baseline and 4 days post-MI. Monocyte populations were analysed in spleen and blood by flow cytometry, and macrophages infiltrating the infarcted myocardium by gene expression analyses. Results Echocardiographic analysis demonstrated that Metrnl deficiency led to cardiac dysfunction (FAC p=0.02) accompanied by myocardial dilation 4 days post-MI (EDV p=0.02; ESV p=0.01). On the contrary, the overexpression of Metrnl in the myocardium of AVV9-Metrnl mice restored cardiac function compared to Metrnl-/- group (FAC p=0.01, EDV p=0.04, ESV p=0.09). Furthermore, we observed a reduction in splenic monocytes (CD11b+F4/80+) in Metrnl-/- mice compared to the WT group at 4 days post-MI (p=0.06). In particular, a significant decrease in pro-inflammatory Ly6CHigh and CD86+ monocytes was noted in the spleens of Metrnl-/- mice compared to the WT group (p=0.03; p=0.04 respectively), suggesting their mobilization out of the spleen, but not in the AVV9-Metrnl group. Although the total number of Ly6CHigh and Ly6CLow monocytes in the bloodstream was similar in all groups, there was a trend to different expression in the CCR2 and CCR5 chemokine receptors (p=0.07; p=0.06 respectively). At the same time, M1 macrophage recruitment chemokines (CCL2) and inflammatory markers (TNFα) were significantly induced in the ischemic area of the infarcted myocardium compared to the remote zone in the WT and Metrnl-/- groups (CCL2 p=0.03; p=0.01 respectively; TNFα p=0.008;p=0.04 respectively). In contrast, TNFα and CCL2 genes were not induced in the ischemic area of AVV9-Metrnl mice. Regarding M2 macrophages, the Arg1 marker was significantly reduced in the ischemic zone of Metrnl-/- mice compared to the WT group (p= 0.01), while in the AVV9-Metrnl group was recovered (p= 0.04). Conclusions Our results demonstrate that the absence of Metrnl promotes the recruitment of inflammatory macrophages to the ischemic myocardium and is associated to cardiac dysfunction.

Antifibrotic effect of disulfiram on bleomycin-induced lung fibrosis in mice and its impact on macrophage infiltration

The accumulation of monocyte-derived macrophages in the lung tissue during inflammation is important for the pathogenesis of fibrotic lung disease. Deficiencies in chemokine receptors CCR2 and CCR5 and their ligands, which mediate monocyte/macrophage migration, ameliorate bleomycin (BLM)-induced lung fibrosis. Disulfiram (DSF), which is used to treat alcoholism because of its aldehyde dehydrogenase (ALDH)-inhibiting effect, inhibits monocyte/macrophage migration by inhibiting FROUNT, an intracellular regulator of CCR2/CCR5 signalling. Here, we investigated the antifibrotic effect of oral DSF administration in a mouse model of BLM-induced lung fibrosis, focusing on macrophage response and fibrosis progression. The direct inhibitory activity of DSF on monocyte migration was measured using the Boyden chamber assay and compared with that of DSF-related inhibitors with different FROUNT-inhibition activities. Quantitative PCR was used to determine the expression of fibrosis-promoting genes in the lung tissue. DSF significantly suppressed macrophage infiltration into lung tissues and attenuated BLM-induced lung fibrosis. DSF and its metabolites, diethyldithiocarbamate (DDC) and copper diethyldithiocarbamate (Cu(DDC)2), inhibited monocyte migration toward the culture supernatant of primary mouse lung cells mainly comprising CCL2, whereas cyanamide, another ALDH inhibitor, did not. DSF, with higher inhibitory activity against FROUNT than DDC and Cu(DDC)2, inhibited monocyte migration most strongly. In BLM-induced fibrotic lung tissues, profibrotic factors were highly expressed but were reduced by DSF treatment. These results suggest DSF inhibits macrophage infiltration, which might be attributed to its inhibitory effect on FROUNT, and attenuates BLM-induced lung fibrosis. In addition, multiplex immunofluorescence imaging revealed reduced infiltration of S100A4+ macrophages into the lungs in DSF-treated mice and high expression of FROUNT in S100A4+ macrophages in idiopathic pulmonary fibrosis (IPF). These findings underscore the potential of macrophage-targeted therapy with DSF as a promising drug repositioning approach for treating fibrotic lung diseases, including IPF.

Chemokine Receptor N-Terminus Charge Dictates Reliance on Post-Translational Modifications for Effective Ligand Capture and Following Boosting by Defense Peptides.

The chemokine receptors CCR1 and CCR5 display overlapping expression patterns and ligand dependency. Here we find that ligand activation of CCR5, not CCR1, is dependent on N-terminal receptor O-glycosylation. Release from O-glycosylation dependency is obtained by increasing CCR5 N-terminus acidity to the level of CCR1. Ligand activation of CCR5, not CCR1, drastically improves in the absence of glycosaminoglycans (GAGs). Ligand activity at both CCR1 and CCR5 is boosted by positively charged/basic peptides shown to interact with acidic chemokine receptor N-termini. We propose that receptors with an inherent low N-terminus acidity rely on post-translational modifications (PTMs) to efficiently compete with acidic entities in the local environment for ligand capture. Although crucial for initial ligand binding, strong electrostatic interactions between the ligand and the receptor N-terminus may counteract following insertion of the ligand into the receptor binding pocket and activation, a process that seems to be aided in the presence of basic peptides. Basic peptides bind to the naked CCR1 N-terminus, not the CCR5 N-terminus, explaining the loss of boosting of ligand-induced signaling via CCR5 in cells incapable of glycosylation.

Microglia and monocyte-derived macrophages drive progression of pediatric high-grade gliomas and are transcriptionally shaped by histone mutations.

Pediatric high-grade gliomas (pHGGs), including hemispheric pHGGs and diffuse midline gliomas (DMGs), harbor mutually exclusive tumor location-specific histone mutations. Using immunocompetent de novo mouse models of pHGGs, we demonstrated that myeloid cells were the predominant infiltrating non-neoplastic cell population. Single-cell RNA sequencing (scRNA-seq), flow cytometry, and immunohistochemistry illustrated the presence of heterogeneous myeloid cell populations shaped by histone mutations and tumor location. Disease-associated myeloid (DAM) cell phenotypes demonstrating immune permissive characteristics were identified in murine and human pHGG samples. H3.3K27M DMGs, the most aggressive DMG, demonstrated enrichment of DAMs. Genetic ablation of chemokines Ccl8 and Ccl12 resulted in a reduction of DAMs and an increase in lymphocyte infiltration, leading to increased survival of tumor-bearing mice. Pharmacologic inhibition of chemokine receptors CCR1 and CCR5 resulted in extended survival and decreased myeloid cell infiltration. This work establishes the tumor-promoting role of myeloid cells in DMG and the potential therapeutic opportunities for targeting them.

Effect of Neutrophil–Platelet Interactions on Cytokine-Modulated Expression of Neutrophil CD11b/CD18 (Mac-1) Integrin Complex and CCR5 Chemokine Receptor in Stable Coronary Artery Disease: A Sub-Study of SMARTool H2020 European Project

Atherosclerosis is an inflammatory disease wherein neutrophils play a key role in plaque evolution. We observed that neutrophil CD11b was associated with a higher necrotic core volume in coronary plaques. Since platelets modulate neutrophil function, we explored the influence of neutrophil–platelet conjugates on the cytokine-modulated neutrophil complex CD11b/CD18 and CCR5 receptor expression. In 55 patients [68.53 ± 7.95 years old (mean ± SD); 71% male], neutrophil positivity for CD11b, CD18 and CCR5 was expressed as Relative Fluorescence Intensity (RFI) and taken as a dependent variable. Cytokines and chemokines were assessed by ELISA. Following log-10-based logarithmic transformation, they were used as independent variables in Model 1 of multiple regression together with Body Mass Index and albumin. Model 1 was expanded with the RFI of neutrophil CD41a+ (model 2). The RFI of neutrophil CD41a+ correlated positively and significantly with CD11b, CD18, and CCR5. In Model 2, CCR5 correlated positively only with the RFI of neutrophil CD41a+. Albumin maintained its positive effect on CD11b in both models. These observations indicate the complexity of neutrophil phenotypic modulation in stable CAD. Despite limitations, these findings suggest there is a role played by neutrophil–platelet interaction on the neutrophil cytokine-modulated expression of adhesive and chemotactic receptors.

Chemokine Receptor CCR2 Is Protective toward Outer Hair Cells in Chronic Suppurative Otitis Media.

Chronic suppurative otitis media (CSOM) is a neglected disease that afflicts 330 million people worldwide and is the most common cause of permanent hearing loss among children in the developing world. Previously, we discovered that outer hair cell (OHC) loss occurred in the basal turn of the cochlea and that macrophages are the major immune cells associated with OHC loss in CSOM. Macrophage-associated cytokines are upregulated. Specifically, CCL-2, an important member of the MCP family, is elevated over time following middle ear infection. CCR2 is a common receptor of the MCP family and the unique receptor of CCL2. CCR2 knockout mice (CCR2-/-) have been used extensively in studies of monocyte activation in neurodegenerative diseases. In the present study, we investigated the effect of CCR2 deletion on the cochlear immune response and OHC survival in CSOM. The OHC survival rate was 84 ± 12.5% in the basal turn of CCR2+/+ CSOM cochleae, compared with was 63 ± 19.9% in the basal turn of CCR2-/- CSOM cochleae (p ≤ 0.05). Macrophage numbers were significantly reduced in CCR2-/- CSOM cochleae compared with CCR2+/+ CSOM cochleae (p ≤ 0.001). In addition, CCL7 was upregulated, whereas IL-33 was downregulated, in CCR2-/- CSOM cochleae. Finally, the permeability of the blood-labyrinth barrier in the stria vascularis remained unchanged in CCR2-/- CSOM compared with CCR2+/+ CSOM. Taken together, the data suggest that CCR2 plays a protective role through cochlear macrophages in the CSOM cochlea.

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.

Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.

Acute natural killer cells response to a continuous moderate intensity and a work-matched high intensity interval exercise session in metastatic cancer patients treated with chemotherapy

BackgroundIt has been suggested that the acute natural killer (NK) cell response to aerobic exercise might contribute to the tumor suppressor effect of regular exercise observed in preclinical studies. Moreover, because this response is modulated by exercise intensity, high-intensity intervals exercise (HIIE) might represent an interesting therapeutic approach in cancer patients. However, this immune response remains unstudied in cancer patients currently undergoing chemotherapy. ObjectiveTo characterize the acute NK cell response following a moderate-intensity continuous aerobic exercise session (MOD), and a HIIE session in metastatic cancer patients treated with chemotherapy. MethodsTwelve cancer patients (45–65 years old) underwent a MOD and a duration and work-matched HIIE trial, in a block-randomized order. Peripheral blood mononuclear cells (PBMC) were isolated before, after and 1h after each trial. NK cell subsets were enumerated using flow cytometry and complete blood counts. The surface expression of the cytotoxic NK cell (cNK; CD56dimCD16+) subset was evaluated for its expression of the differentiation markers CD57 and CD158a, the activating receptor NKG2D, the immune checkpoints TIM-3 and PD-1, and the chemokine receptors CXCR3, CXCR4 and CCR2. ResultscNK cell blood counts increased immediately following MOD (p < 0.001) and decreased back to pre-exercise values 1 h after exercise cessation (p < 0.001). The most responsive cNK cell subsets were expressing CD57, CD158a, NKG2D, TIM-3 and CXCR3. The HIIE trial elicited a similar biphasic response, without any difference between trials (all p ≥ 0.38). However, significant changes in the MFI values of CXCR4 and NKG2D were observed in the cNK cell subset following HIIE (all p ≤ 0.038), but not MOD. ConclusionIn metastatic cancer patients undergoing chemotherapy, both MOD and HIIE can elicit an acute mobilisation and egress of NK cells exhibiting phenotypic characteristics associated with high cytotoxicity and tumor homing. Future longitudinal trials are needed to determine if combining aerobic exercise training and chemotherapy will translate towards favorable immune and clinical outcomes.

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.

Topical application of a CCL22-binding aptamer suppresses contact allergy

Allergic contact dermatitis is a prevalent occupational disease with limited therapeutic options. The chemokine CCL22, a ligand of the chemokine receptor CCR4, directs the migration of immune cells. Here, it is shown that genetic deficiency of CCL22 effectively ameliorated allergic reactions in contact hypersensitivity (CHS), a commonly used mouse model of allergic contact dermatitis. For pharmacological inhibition of CCL22, DNA aptamers specific for murine CCL22 were generated by systematic evolution of ligands by exponential enrichment (SELEX). Nine CCL22-binding aptamers were initially selected and functionally tested in vitro. The 29-nucleotide DNA aptamer AJ102.29m profoundly inhibited CCL22-dependent T cell migration and did not elicit undesired Toll-like receptor-dependent immune activation. AJ102.29m efficiently ameliorated CHS in vivo after systemic application. Moreover, CHS-associated allergic symptoms were also reduced following topical application of the aptamer on the skin. Microscopic analysis of skin treated with AJ102.29m ex vivo demonstrated that the aptamer could penetrate into the epidermis and dermis. The finding that epicutaneous application of the aptamer AJ102.29m in a cream was as effective in suppressing the allergic reaction as intraperitoneal injection paves the way for therapeutic use of aptamers beyond the current routes of systemic administration.

Effect of Maraviroc and/or Favipiravir plus systemic steroids versus systemic steroids only on the viral load of adults with severe COVID-19: clinical trial

Background Coronavirus disease 2019 (COVID-19) has created the need to evaluate drugs such as favipiravir (FPV), an antiviral inhibitor of RNA-dependent RNA-polymerase (RdRp), and Maraviroc (MVC), an antiretroviral that antagonizes the chemokine receptor CCR5, which could affect the modulation of inflammation and viral replication in the treatment of COVID-19. We sought to evaluate the effect of MVC and/or FPV plus systemic steroid (SS) vs. SS alone on the viral load and progression to critical disease. Methods Sixteen patients with severe COVID-19 were evaluated in three treatment arms: 1) SS only (n=6), 2) SS plus one test drug MVC or FPV (n=5), and 3) SS plus both test drugs (MVC and FPV, n=5). The viral load was determined for N, E, and RdRp viral genes. Results A significant decrease in viral load was observed in the three treatment groups, with a larger effect size in the group that combined SS with both test drugs. The E, N, and RdRp genes with Cohen’s d were 120%, 123%, and 50%, respectively. Conclusions The largest effect on viral load reduction, as measured by effect size, was observed in the combination treatment group; however, no statistical significance was found, and it did not prevent progression to critical illness.

Cell shape sensing licenses dendritic cells for homeostatic migration to lymph nodes

Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKβ–NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.

Preliminary results of a phase 1, first-in-human, dose escalation study of the anti-CCR8 cytolytic antibody, CHS-114 (formerly SRF114) in patients with advanced solid tumors.

2664 Background: Depletion of intratumoral regulatory T cells (itTregs) represents an attractive therapeutic strategy to enhance antitumor responses. The chemokine receptor CCR8 is preferentially expressed on itTregs compared to peripheral Tregs and other immune cell types, and CCR8 expressing itTregs are highly immune suppressive. Our preclinical studies have demonstrated that treatment of a PD-1 resistant mouse tumor model with an anti-CCR8 monoclonal antibody (mAb) depletes itTregs, reduces tumor growth, and enhances anti-PD-1 antitumor activity. CHS-114 an afucosylated mAb that binds CCR8, depletes itTregs, and enhances toripalimab- (tori; anti-PD-1 mAb) mediated T cell activation. Here, we present preliminary results of the first-in-human phase 1 study of CHS-114. Methods: This phase 1, multicenter, single agent and combination (combo) dose escalation study (NCT05635643) of CHS-114 is enrolling patients (pts) ≥18 years of age with advanced solid tumors who progressed during or after standard therapy. CHS-114 dose escalation guided by the Bayesian optimal interval (BOIN) design will evaluate doses ranging from 5 to 1500 mg q3w. Five additional pts with advanced head and neck squamous cell carcinoma (HNSCC) will be enrolled at each of 2 selected dose levels (DLs) with required biopsies. Combo dose escalation will evaluate 2 DLs of CHS-114 combined with tori 240 mg q3w in pts with HNSCC using a standard 3+3 design. Primary objectives are to determine the recommended dose(s) for expansion of CHS-114 and the safety and tolerability of CHS-114 + tori. Secondary objectives are to evaluate the safety, tolerability, pharmacokinetics (PK), and preliminary antitumor activity of CHS-114 alone and in combination with tori. Tumor and immune biomarkers are being evaluated as exploratory endpoints. Results: As of 15Dec2023, 15 pts received CHS-114 at doses ranging from 5 to 700 mg q3w. No dose-limiting toxicities (DLTs) were reported. Treatment-related adverse events occurred in 33% of pts; all were CTCAE grade 1-2, with pyrexia (13%) being the most common. Preliminary PK analysis indicates that CHS-114 exposure increases with dose with an elimination half-life between 9-17 days across the dose range of 10-700 mg. Preliminary receptor occupancy analysis using an immune profiling assay in peripheral blood at 100, 300, and 700 mg DLs revealed minimal detection of CCR8+ Tregs and a modest concomitant decrease in overall Treg frequency at the end of cycle 1, consistent with strong receptor occupancy. Conclusions: CHS-114 has an acceptable safety profile to date in heavily pretreated pts at doses up to 700 mg. CHS-114 administration at 100 to 700 mg shows robust decreases in peripheral CCR8+ Tregs following the first dose (cycle 1). Dose escalation is ongoing. Clinical trial information: NCT05635643 .

Discovery of a CCR2-targeting pepducin therapy for chronic pain

Targeting the CCL2/CCR2 chemokine axis has been shown to be effective at relieving pain in rodent models of inflammatory and neuropathic pain, therefore representing a promising avenue for the development of non-opioid analgesics. However, clinical trials targeting this receptor for inflammatory conditions and painful neuropathies have failed to meet expectations and have all been discontinued due to lack of efficacy. To overcome the poor selectivity of CCR2 chemokine receptor antagonists, we generated and characterized the function of intracellular cell-penetrating allosteric modulators targeting CCR2, namely pepducins. In vivo, chronic intrathecal administration of the CCR2-selective pepducin PP101 was effective in alleviating neuropathic and bone cancer pain. In the setting of bone metastases, we found that T cells infiltrate dorsal root ganglia (DRG) and induce long-lasting pain hypersensitivity. By acting on CCR2-expressing DRG neurons, PP101 attenuated the altered phenotype of sensory neurons as well as the neuroinflammatory milieu of DRGs, and reduced bone cancer pain by blocking CD4+ and CD8+ T cell infiltration. Notably, PP101 demonstrated its efficacy in targeting the neuropathic component of bone cancer pain, as evidenced by its anti-nociceptive effects in a model of chronic constriction injury of the sciatic nerve. Importantly, PP101-induced reduction of CCR2 signaling in DRGs did not result in deleterious tumor progression or adverse behavioral effects. Thus, targeting neuroimmune crosstalk through allosteric inhibition of CCR2 could represent an effective and safe avenue for the management of chronic pain.

Functional Heterogeneity of Umbilical Cord Blood Monocyte-Derived Dendritic Cells.

Human umbilical cord blood (UCB) represents a unique resource for hematopoietic stem cell transplantation for children and patients lacking suitable donors. UCB harbors a diverse set of leukocytes such as professional APCs, including monocytes, that could act as a novel source for cellular therapies. However, the immunological properties of UCB monocytes and monocyte-derived dendritic cells (MoDCs) are not fully characterized. In this study, we characterized the phenotype and functions of UCB-MoDCs to gauge their potential for future applications. UCB exhibited higher frequencies of platelets and lymphocytes as well as lower frequencies of neutrophils in comparison with adult whole blood. Leukocyte subset evaluation revealed significantly lower frequencies of granulocytes, NK cells, and CD14+CD16- monocytes. Surface marker evaluation revealed significantly lower rates of costimulatory molecules CD80 and CD83 while chemokine receptors CCR7 and CXCR4, as well as markers for Ag presentation, were similarly expressed. UCB-MoDCs were sensitive to TLR1-9 stimulation and presented quantitative differences in the release of proinflammatory cytokines. UCB-MoDCs presented functional CCR7-, CXCR4-, and CCR5-associated migratory behavior as well as adequate receptor- and micropinocytosis-mediated Ag uptake. When cocultured with allogeneic T lymphocytes, UCB-MoDCs induced weak CD4+ T lymphocyte proliferation, CD71 expression, and release of IFN-γ and IL-2. Taken together, UCB-MoDCs present potentially advantageous properties for future medical applications.

#1511 The immune cell ‘signature’ of incident hypertension

Abstract Background and Aims Hypertension results from complex genetic and environmental interactions, mediated by cardiac, vascular, endocrine, and renal systems. The immune system may modulate all of these, though the exact mechanisms linking immune activation with hypertension and organ damage remain poorly defined. We aimed to identify the immune cell ‘signature’ in patients with newly diagnosed, untreated hypertension (HTN; n=76) compared to normotensive controls (NTN; n=67), as well as in relation to nocturnal dipping status. Method Flow cytometry of peripheral blood mononuclear cells (Attune, Thermo Fisher) characterising B cell, T cell, monocyte and dendritic cells (DC) subsets, including activation signature with intracellular staining, in an exploratory comparative analysis. Results The proportion of CD4+ naïve T cells (CD45RA+CCR7+) was lower in hypertension, with CD45RO+CD62L+ cells attaining nominal statistical significance between NTN and HTN groups (1.3 ± 1.1 vs 1.2 ± 1.0, p=0.039). CD4+ T central memory cells were expanded (TCM, 23.1 ± 7.2 vs 27.7 ± 10.0, p=0.002, Figure) in HTN along with T effector memory (TEM) cells lacking CD62 in an adjusted model, (TEM, CD4+CD45RA-CCR7-CD62l-; 31.4 ± 7.5 vs 33.7 ± 10.3, p0.035; CD8+CD45RA-CCR7-CD45RO+CD62l− 48.9 ± 10.5 vs 52.2 ± 13.3, p=0.011). Hypertensive group had proportionally fewer CD4+CD28+ cells (95.1 ± 9.8 vs 92.0 ± 13.5, p=0.034) and CD8+CD28+ (68.0 ± 19.7 vs 62.0 ± 21.0, p=0.011), as well as CD8+ TEMRA cells (Figure). In a fully adjusted model, the CCR7-CD45RO-CD62L+ (52.2 ± 14.5 vs 50.2 ± 16.1, p=0.036), and CD8+CD28+CD45RA+CCR7- significantly differed between groups (23.1 ± 14.7 vs 25.0 ± 17.8, p=0.008). On the molecular level, T cells were polarised in HTN towards a Th1/Tc1 and Th17.1/Tc17.1 pro-inflammatory milieu (Figure). No differences were detected in B cell subsets. The innate immune ‘signature’ was characterised by intermediate monocytes with a differing pattern of CCR2 and CCR5 chemokine receptor expression (Table 1), and alterations in STAT1 and STAT6 phosphorylation cascades. NK cell CD56+Dim expression and fewer CD122-expressing NKT (0.13 ± 0.0 vs 0.08 ± 0.1, p=0.046) and CD122+ T lymphocytes (1.3 ± 3.4 vs 0.6 ± 0.4, p=0.019) further supported pro-inflammatory milieu in hypertension. Finally, in an exploratory nominal statistical analysis, nocturnal non-dipping was associated with particular changes in immune cell signature, including expanded CD4+TCM (CD4+CD45RO+CD62L-, 9.4 ± 4.3 vs 8.0 ± 3.3, p=0.04) and reduced CD4+TEM and TEMRA compartments; polarisation towards Th17 and Tc1; and altered Monocyte CCR2 and CCR5 expression. Additionally, non-dipping was linked to dendritic cell mannose receptor downregulation e.g. mDC non-dipper 10.9 ± 5.8 vs dipper 13.6 ± 9.7, p=0.03. Conclusion These exploratory data support alterations of circulating innate (monocytes) and adaptive (T cells) immune compartments in newly diagnosed untreated, and uncomplicated hypertension.

#2899 Role of the Th17.1 cells in IgA nephropathy

Abstract Background and Aims T helper (Th) cells play a crucial role in glomerulonephritis, including IgA nephropathy (IgAN). Recently identified Th17.1 cells, which are elevated in sarcoidosis, lupus, and Takayasu arteritis, express chemokine receptors CCR6 and CXCR3. These receptors are implicated in inflammatory cell infiltration and fibrosis in glomerulonephritis models. Notably, Th17.1 cells express MDR-1 (multidrug resistance protein) and the drug efflux protein p-glycoprotein, associated with corticosteroid resistance. However, the exact role of Th17.1 cells in IgAN remains unclear. Intriguingly, Th17.1 cells produce INF-γ and IL-17, cytokines implicated in IgAN pathogenesis. Method In this retrospective cross-sectional study, we examined circulating Th17.1 cells in 18 confirmed IgAN patients. Antibodies such as anti-CXCR3-FITC and anti-CCR6-PCy7 (Biolegend, U.S.A.) were used to stain Th subpopulations. We also used combinations of the following antibodies: anti-CD45-FITC, anti-CD4-PE, anti-CD8-ECD, anti-CD3-PCy5, anti-CD56-PCy5, anti-CD3-PCy7, anti-CD19-ECD, anti-CD127-FITC, anti-CD25-PCy5, and anti-CD4-PCy7 (Coulter Immunotech, France) to evaluate T, B, NK, and Treg cells. Multicolor flow cytometry was performed using a Navios cytometer, and data were analyzed using Kaluza software (Beckman Coulter, U.S.A.). Th effector subpopulations of CD4+ lymphocytes were identified based on chemokine receptor expression: Th17.1 (CXCR3+CCR6+). Th17.1 cells were quantified as a percentage of CD4+ lymphocytes. Th1, Th2, and Treg cells were also evaluated. We explored correlations between Th17.1 cell counts, estimated glomerular filtration rate (eGFR, mL/min/1.73 m²), and proteinuria (24-hour collection). Among the patients, 5 were classified as refractory (progressive eGFR decline &amp;gt; 5 mL/min/1.73 m² and 24-hour proteinuria &amp;gt; 1g despite steroid and immunosuppressant therapy). Results The study included 18 patients. The mean (95% CI) Th17.1 cell count was 20% (16-23), and the 24-hour proteinuria was 2.1g (1.4-2.7). A significant direct correlation was observed between Th17.1 cells and proteinuria (g/L, adjusted for 24 hours) (r = 0.50, P = 0.03). Refractory patients exhibited higher Th17.1 levels compared to others (25% [95% CI: 17-33] vs. 18% [95% CI: 14-21], P = 0.02) (Fig. 1). Patients with proteinuria &amp;lt; 0.5g/24 h had lower Th17.1 cell counts compared to those with higher proteinuria (16% [95% CI: 12-20] vs. 21% [95% CI: 17-25], P = 0.06. No correlation was found between eGFR, proteinuria, and Th1, Th2, Th17, or Treg cells. Conclusion These findings suggest that Th17.1 cells may contribute to the pathogenesis of IgAN and could potentially indicate the response to steroids or immunosuppressants in treatment. While the direct involvement of Th17.1 cells in kidney tissue remains unexplored, further investigation is warranted due to its therapeutic and prognostic implications.

Discovery of the Clinical Candidate IDOR-1117-2520: A Potent and Selective Antagonist of CCR6 for Autoimmune Diseases.

Migration of immune cells to sites of inflammation is a critical step in the body's response to infections but also during autoimmune flares. Chemokine receptors, members of the GPCR receptors, are instrumental in directing specific cell types to their target organs. Herein, we describe a highly potent small molecule antagonist of the chemokine receptor CCR6, which came out of fine-tuned structural elaborations from a proprietary HTS hit. Three main issues in the parent chemical series-cytotoxicity, phototoxicity, and hERG, were successfully solved. Biological characterization demonstrated that compound 45 (IDOR-1117-2520) is a selective and insurmountable antagonist of CCR6. In vivo proof-of-mechanism studies in a mouse lung inflammation model using a representative compound from the chemical class of 45 confirmed that the targeted CCR6+ cells were efficiently inhibited from migrating into the bronchoalveoli. Finally, ADMET and physicochemical properties were well balanced and the preclinical package warranted progress in the clinic.