CCL4/CCR5 regulates chondrocyte biology and OA progression.

BACKGROUND: Osteoarthritis (OA) is a common musculoskeletal disorder characterized by chondrocyte apoptosis and extracellular matrix degradation. This study aimed to investigate the role of CCL4/CCR5 in regulating chondrocyte apoptosis and reactive oxygen species (ROS) levels in OA progression. METHODS: Bioinformatics analysis was employed to identify CCL4 as the target gene, following which primary chondrocytes were treated with varying concentrations of CCL4. Apoptosis rate of chondrocytes and ROS levels were assessed using flow cytometry. The mechanism by which CCL4 regulated the extracellular matrix was investigated through Western blot and Immunofluorescence analyses. Additionally, maraviroc, a CCR5 inhibitor, was administered to chondrocytes in order to explore the potential signaling pathway of CCL4/CCR5. RESULTS: Our study found that CCL4 was predominantly up-regulated among the top 10 hub genes identified in RNA-sequencing analysis. Validation through quantitative polymerase chain reaction (qPCR) confirmed elevated CCL4 expression in patients with Hip joint osteoarthritis, knee joint osteoarthritis, and facet joint osteoarthritis. The upregulation of CCL4 was associated with an increase in chondrocyte apoptosis and ROS levels. Mechanistically, CCL4, upon binding to its receptor CCR5, triggered the downstream phosphorylation of P65 in the nuclear factor-κB (NF-κB) signaling pathway. In vitro experiments demonstrated that treatment with maraviroc mitigated chondrocyte apoptosis, reduced intracellular ROS levels, and attenuated extracellular matrix degradation. CONCLUSION: The study highlights the critical role of CCL4/CCR5 in modulating chondrocyte apoptosis and ROS levels in OA progression. Targeting this pathway may offer promising therapeutic interventions for mitigating the pathogenic mechanisms associated with OA.

FcRn-enhancing mutations lead to increased and prolonged levels of the HIV CCR5-blocking monoclonal antibody leronlimab in the fetuses and newborns of pregnant rhesus macaques.

Prenatal administration of monoclonal antibodies (mAbs) is a strategy that could be exploited to prevent viral infections during pregnancy and early life. To reach protective levels in fetuses, mAbs must be transported across the placenta, a selective barrier that actively and specifically promotes the transfer of antibodies (Abs) into the fetus through the neonatal Fc receptor (FcRn). Because FcRn also regulates Ab half-life, Fc mutations like the M428L/N434S, commonly known as LS mutations, and others have been developed to enhance binding affinity to FcRn and improve drug pharmacokinetics. We hypothesized that these FcRn-enhancing mutations could similarly affect the delivery of therapeutic Abs to the fetus. To test this hypothesis, we measured the transplacental transfer of leronlimab, an anti-CCR5 mAb, in clinical development for preventing HIV infections, using pregnant rhesus macaques to model in utero mAb transfer. We also generated a stabilized and FcRn-enhanced form of leronlimab, termed leronlimab-PLS. Leronlimab-PLS maintained higher levels within the maternal compartment while also reaching higher mAb levels in the fetus and newborn circulation. Further, a single dose of leronlimab-PLS led to complete CCR5 receptor occupancy in mothers and newborns for almost a month after birth. These findings support the optimization of FcRn interactions in mAb therapies designed for administration during pregnancy.

Protein phosphatase 2Cm-regulated branched-chain amino acid catabolic defect in dorsal root ganglion neurons drives pain sensitization.

Maladaptive changes of metabolic patterns in the lumbar dorsal root ganglion (DRG) are critical for nociceptive hypersensitivity genesis. The accumulation of branched-chain amino acids (BCAAs) in DRG has been implicated in mechanical allodynia and thermal hyperalgesia, but the exact mechanism is not fully understood. This study aimed to explore how BCAA catabolism in DRG modulates pain sensitization. Wildtype male mice were fed a high-fat diet (HFD) for 8 weeks. Adult PP2Cmfl/fl mice of both sexes were intrathecally injected with pAAV9-hSyn-Cre to delete the mitochondrial targeted 2 C-type serine/threonine protein phosphatase (PP2Cm) in DRG neurons. Here, we reported that BCAA catabolism was impaired in the lumbar 4-5 (L4-L5) DRGs of mice fed a high-fat diet (HFD). Conditional deletion of PP2Cm in DRG neurons led to mechanical allodynia, heat and cold hyperalgesia. Mechanistically, the genetic knockout of PP2Cm resulted in the upregulation of C-C chemokine ligand 5/C-C chemokine receptor 5 (CCL5/CCR5) axis and an increase in transient receptor potential ankyrin 1 (TRPA1) expression. Blocking the CCL5/CCR5 signaling or TRPA1 alleviated pain behaviors induced by PP2Cm deletion. Thus, targeting BCAA catabolism in DRG neurons may be a potential management strategy for pain sensitization.

cGAS-activated endothelial cell-T cell cross-talk initiates tertiary lymphoid structure formation.

Aberrant activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway causes autoimmunity in humans and mice; however, the exact mechanism by which the cGAS-STING pathway initiates adaptive immunity and tissue pathology is still not fully understood. Here, we used a cGAS knockin (KI) mouse model that develops systemic autoimmunity. In the lungs of cGAS-KI mice, blood vessels were enclosed by organized lymphoid tissues that resemble tertiary lymphoid structures (TLSs). Cell-intrinsic cGAS induction promoted up-regulation of CCR5 in CD8+ T cells and led to CCL5 production in vascular endothelial cells. Peripheral CD8+ T cells were recruited to the lungs and produced CXCL13 and interferon-γ. The latter triggered endothelial cell death, potentiated CCL5 production, and was essential for TLS establishment. Blocking CCL5 or CCR5, or depleting CD8+ T cells, impaired TLS formation. cGAS-mediated TLS formation also enhanced humoral and antitumor responses. These data demonstrate that cGAS signaling drives a specialized lymphoid structure that underlies autoimmune tissue pathology.

Hematopoietic Growth Factors Regulate the Entry of Monocytes into the Adult Brain via Chemokine Receptor CCR5.

Monocytes are circulating macrophage precursors generated from bone marrow hematopoietic stem cells. In adults, monocytes continuously replenish cerebral border-associated macrophages under physiological conditions. Monocytes also rapidly infiltrate the brain in pathological settings. The mechanisms of recruiting monocyte-derived macrophages into the brain under pathological conditions have been extensively studied. However, it remains unclear how monocytes enter the brain to renew border-associated macrophages under physiological conditions. Using both in vitro and in vivo approaches, this study reveals that a combination of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), complementarily and synergistically enhances the adhesion of monocytes to cerebral endothelial cells in a dose-dependent manner. Cysteine-cysteine chemokine receptor 5 (CCR5) in brain endothelial cells, but not the cell adhesion molecules mediating neuroinflammation-related infiltration of monocyte-derived macrophages, modulates SCF+G-CSF-enhanced monocyte-endothelial cell adhesion. Blocking CCR5 or genetically deleting CCR5 reduces monocyte-endothelial cell adhesion induced by SCF+G-CSF. The SCF+G-CSF-enhanced recruitment of bone marrow-derived monocytes/macrophages into the cerebral perivascular space is also reduced in adult CCR5 knockout mice. This study demonstrates the role of SCF and G-CSF in regulating the entry of monocytes into the adult brain to replenish perivascular macrophages.

ITGB2 related to immune cell infiltration as a potential therapeutic target of inflammatory bowel disease using bioinformatics and functional research.

Inflammatory bowel disease (IBD) is a chronic systemic inflammatory condition regarded as a major risk factor for colitis-associated cancer. However, the underlying mechanisms of IBD remain unclear. First, five GSE data sets available in GEO were used to perform 'batch correction' and Robust Rank Aggregation (RRA) to identify differentially expressed genes (DEGs). Candidate molecules were identified using CytoHubba, and their diagnostic effectiveness was predicted. The CIBERSORT algorithm evaluated the immune cell infiltration in the intestinal epithelial tissues of patients with IBD and controls. Immune cell infiltration in the IBD and control groups was determined using the least absolute shrinkage selection operator algorithm and Cox regression analysis. Finally, a total of 51 DEGs were screened, and nine hub genes were identified using CytoHubba and Cytoscape. GSE87466 and GSE193677 were used as extra data set to validate the expression of the nine hub genes. CD4-naïve T cells, gamma-delta T cells, M1 macrophages and resting dendritic cells (DCs) are the main immune cell infiltrates in patients with IBD. Signal transducer and activator of transcription 1, CCR5 and integrin subunit beta 2 (ITGB2) were significantly upregulated in the IBD mouse model, and suppression of ITGB2 expression alleviated IBD inflammation in mice. Additionally, the expression of ITGB2 was upregulated in IBD-associated colorectal cancer (CRC). The silence of ITGB2 suppressed cell proliferation and tumour growth in vitro and in vivo. ITGB2 resting DCs may provide a therapeutic strategy for IBD, and ITGB2 may be a potential diagnostic marker for IBD-associated CRC.

CCR5 Δ32 and CTLA-4 +49 A/G Gene Polymorphisms and Interferon-ß Treatment Response in Croatian and Slovenian Multiple Sclerosis Patients.

The aim of the present study was to investigate the impact of CCR5 Δ32 and CTLA-4 polymorphisms on the response to IFN-ß treatment in our cohort of MS patients from Croatia and Slovenia. Genomic DNA was obtained from 295 MS patients (230 female; 65 male) classified as responders (n = 173) and non-responders (n = 122) based on clinical criteria for treatment efficacy. Genotyping was performed via PCR/PCR-RFLP. No significant differences in the genotype/allele frequencies of CCR5Δ32 and CTLA-4 +49 A/G were detected between male responders and non-responders. A significantly higher prevalence (p = 0.039) of the CTLA-4 +49 AA genotype was found in female responders (42.1%) compared to non-responders (28.9%). Using multiple forward regression analysis, the CTLA-4 +49 AA genotype significantly predicted a positive response to IFN-ß therapy in females (p = 0.011) and contributed to 4.5% of response variability. Furthermore, the combined presence of the CCR5Δ32 wtwt/CTLA-4 +49 AA genotype significantly predicted a positive response to treatment in females (p = 0.025). The age at disease onset, pretreatment relapse rate, and baseline EDSS score were not reliable predictors of treatment response in MS patients. Our results indicate that the presence of the CCR5Δ32 polymorphism was not associated with the response to IFN-ß treatment, whereas the CTLA-4 +49 polymorphism showed a positive correlation with an optimal response in female patients.

Impact of HIV-1 genetic diversity on disease progression: a prospective cohort study in Guangxi.

The high proportion of AIDS cases and mortality rates in Guangxi underscores the urgency to investigate the influence of HIV-1 genetic diversity on disease progression in this region. Newly diagnosed HIV-1 patients were enrolled from January 2016 to December 2021, and the follow-up work and detection of CD4+T lymphocytes were carried out every six months until December 2022. Multivariate logistic regression was used to analyze the factors affecting pre-treatment CD4+T lymphocyte counts, while local weighted regression models (LOESS) and generalized estimating equation models (GEE) were conducted to assess factors influencing CD4+T Lymphocyte Recovery. Cox regression analysis was utilized to examine the impact of subtypes on survival risk. Additionally, HIV-1 env sequences were utilized for predicting CXCR4 and CCR5 receptors. The study encompassed 1867 individuals with pol sequences and 281 with env sequences. Our findings indicate that age over 30, divorced/widowed, peasant, heterosexual infection, CRF01_AE, long-term infection, and Pre-treatment Viral load >10000 copies/ml were factors associated with higher risk for pre-treatment CD4+T lymphocyte decline. Specifically, male gender, age over 30, heterosexual infection (HETs), long-term infection, CRF01_AE, and Pre-treatment CD4 T cell counts below 350/µL were identified as risk factors impeding CD4+T lymphocyte recovery. Pre-treatment CD4+T lymphocyte counts and recovery in individuals infected with CRF01_AE were lower compared to CRF07_BC and CRF55_01B. Additionally, CRF01_AE and CRF08_BC subtypes exhibited higher mortality rates than CRF07_BC, CRF55_01B, and other subtypes. Notably, CRF01_AE demonstrated the highest percentage of CXCR4 affinity ratios. This research unveils the intricate influence of HIV-1 gene diversity on CD4+T lymphocyte dynamics and clinical outcomes. It highlights the multifaceted nature of HIV infection in Guangxi, providing novel insights into subtype-specific disease progression among HIV-infected individuals in this region.

[Identification of potential biomarkers and immunoregulatory mechanisms of rheumatoid arthritis based on multichip co-analysis of GEO database].

OBJECTIVE: To identify the biomarkers for early rheumatoid arthritis (RA) diagnosis and explore the possible immune regulatory mechanisms. METHODS: The differentially expressed genesin RA were screened and functionally annotated using the limma, RRA, batch correction, and clusterProfiler. The protein-protein interaction network was retrieved from the STRING database, and Cytoscape 3.8.0 and GeneMANIA were used to select the key genes and predicting their interaction mechanisms. ROC curves was used to validate the accuracy of diagnostic models based on the key genes. The disease-specific immune cells were selected via machine learning, and their correlation with the key genes were analyzed using Corrplot package. Biological functions of the key genes were explored using GSEA method. The expression of STAT1 was investigated in the synovial tissue of rats with collagen-induced arthritis (CIA). RESULTS: We identified 9 core key genes in RA (CD3G, CD8A, SYK, LCK, IL2RG, STAT1, CCR5, ITGB2, and ITGAL), which regulate synovial inflammation primarily through cytokines-related pathways. ROC curve analysis showed a high predictive accuracy of the 9 core genes, among which STAT1 had the highest AUC (0.909). Correlation analysis revealed strong correlations of CD3G, ITGAL, LCK, CD8A, and STAT1 with disease-specific immune cells, and STAT1 showed the strongest correlation with M1-type macrophages (R=0.68, P=2.9e-08). The synovial tissues of the ankle joints of CIA rats showed high expressions of STAT1 and p-STAT1 with significant differential expression of STAT1 between the nucleus and the cytoplasm of the synovial fibroblasts. The protein expressions of p-STAT1 and STAT1 in the cell nuclei were significantly reduced after treatment. CONCLUSION: CD3G, CD8A, SYK, LCK, IL2RG, STAT1, CCR5, ITGB2, and ITGAL may serve as biomarkers for early diagnosis of RA. Gene-immune cell pathways such as CD3G/CD8A/LCK-γδ T cells, ITGAL-Tfh cells, and STAT1-M1-type macrophages may be closely related with the development of RA.

Adaptation of SIVmac to baboon primary cells results in complete absence of in vivo baboon infectivity.

While simian immunodeficiency virus (SIV) infection is non-pathogenic in naturally infected African nonhuman primate hosts, experimental or accidental infection in rhesus macaques often leads to AIDS. Baboons, widely distributed throughout Africa, do not naturally harbor SIV, and experimental infection of baboons with SIVmac results in transient low-level viral replication. Elucidation of mechanisms of natural immunity in baboons could uncover new targets of antiviral intervention. We tested the hypothesis that an SIVmac adapted to replicate in baboon primary cells will gain the capacity to establish chronic infections in vivo. Here, we generated SIVmac variants in baboon cells through serial passage in PBMC from different donors (SIVbn-PBMC s1), in PBMC from the same donors (SIVbn-PBMC s2), or in isolated CD4 cells from the same donors used for series 2 (SIVbn-CD4). While SIVbn-PBMC s1 and SIVbn-CD4 demonstrated increased replication capacity, SIVbn-PBMC s2 did not. Pharmacological blockade of CCR5 revealed SIVbn-PBMC s1 could more efficiently use available CCR5 than SIVmac, a trait we hypothesize arose to circumvent receptor occupation by chemokines. Sequencing analysis showed that all three viruses accumulated different types of mutations, and that more non-synonymous mutations became fixed in SIVbn-PBMC s1 than SIVbn-PBMC s2 and SIVbn-CD4, supporting the notion of stronger fitness pressure in PBMC from different genetic backgrounds. Testing the individual contribution of several newly fixed SIV mutations suggested that is the additive effect of these mutations in SIVbn-PBMC s1 that contributed to its enhanced fitness, as recombinant single mutant viruses showed no difference in replication capacity over the parental SIVmac239 strain. The replicative capacity of SIVbn-PBMC passage 4 (P4) s1 was tested in vivo by infecting baboons intravenously with SIVbn-PBMC P4 s1 or SIVmac251. While animals infected with SIVmac251 showed the known pattern of transient low-level viremia, animals infected with SIVbn-PBMC P4 s1 had undetectable viremia or viral DNA in lymphoid tissue. These studies suggest that adaptation of SIV to grow in baboon primary cells results in mutations that confer increased replicative capacity in the artificial environment of cell culture but make the virus unable to avoid the restrictive factors generated by a complex multicellular organism.

A New Application for Cenicriviroc, a Dual CCR2/CCR5 Antagonist, in the Treatment of Painful Diabetic Neuropathy in a Mouse Model.

The ligands of chemokine receptors 2 and 5 (CCR2 and CCR5, respectively) are associated with the pathomechanism of neuropathic pain development, but their role in painful diabetic neuropathy remains unclear. Therefore, the aim of our study was to examine the function of these factors in the hypersensitivity accompanying diabetes. Additionally, we analyzed the analgesic effect of cenicriviroc (CVC), a dual CCR2/CCR5 antagonist, and its influence on the effectiveness of morphine. An increasing number of experimental studies have shown that targeting more than one molecular target is advantageous compared with the coadministration of individual pharmacophores in terms of their analgesic effect. The advantage of using bifunctional compounds is that they gain simultaneous access to two receptors at the same dose, positively affecting their pharmacokinetics and pharmacodynamics and consequently leading to improved analgesia. Experiments were performed on male and female Swiss albino mice with a streptozotocin (STZ, 200 mg/kg, i.p.) model of diabetic neuropathy. We found that the blood glucose level increased, and the mechanical and thermal hypersensitivity developed on the 7th day after STZ administration. In male mice, we observed increased mRNA levels of Ccl2, Ccl5, and Ccl7, while in female mice, we observed additional increases in Ccl8 and Ccl12 levels. We have demonstrated for the first time that a single administration of cenicriviroc relieves pain to a similar extent in male and female mice. Moreover, repeated coadministration of cenicriviroc with morphine delays the development of opioid tolerance, while the best and longest-lasting analgesic effect is achieved by repeated administration of cenicriviroc alone, which reduces pain hypersensitivity in STZ-exposed mice, and unlike morphine, no tolerance to the analgesic effects of CVC is observed until Day 15 of treatment. Based on these results, we suggest that targeting CCR2 and CCR5 with CVC is a potent therapeutic option for novel pain treatments in diabetic neuropathy patients.

Plasmodium infection induces phenotypic, clonal, and spatial diversity among differentiating CD4+ T cells.

Naive CD4+ T cells must differentiate in order to orchestrate immunity to Plasmodium, yet understanding of their emerging phenotypes, clonality, spatial distributions, and cellular interactions remains incomplete. Here, we observe that splenic polyclonal CD4+ T cells differentiate toward T helper 1 (Th1) and T follicular helper (Tfh)-like states and exhibit rarer phenotypes not elicited among T cell receptor (TCR) transgenic counterparts. TCR clones present at higher frequencies exhibit Th1 skewing, suggesting that variation in major histocompatibility complex class II (MHC-II) interaction influences proliferation and Th1 differentiation. To characterize CD4+ T cell interactions, we map splenic microarchitecture, cellular locations, and molecular interactions using spatial transcriptomics at near single-cell resolution. Tfh-like cells co-locate with stromal cells in B cell follicles, while Th1 cells in red pulp co-locate with activated monocytes expressing multiple chemokines and MHC-II. Spatial mapping of individual transcriptomes suggests that proximity to chemokine-expressing monocytes correlates with stronger effector phenotypes in Th1 cells. Finally, CRISPR-Cas9 gene disruption reveals a role for CCR5 in promoting clonal expansion and Th1 differentiation. A database of cellular locations and interactions is presented: https://haquelab.mdhs.unimelb.edu.au/spatial_gui/.

Evaluation of the maternal systemic immune system during frozen euploid embryo transfer according to cycle outcome.

Infertility affects 15 % of couples in the US, and many turn to assisted reproductive technologies, including in vitro fertilization and subsequent frozen embryo transfer (FET) to become pregnant. This study aimed to perform a broad assessment of the maternal immune system to determine if there are systemic differences on the day of FET in cycles that result in a live birth compared to those that do not. Women undergoing FET of euploid embryos were recruited and blood was collected on the day of FET as well as at early timepoints in pregnancy. Sixty immune and angiogenic proteins were measured in plasma, and gene expression of 92 immune-response related genes were evaluated in peripheral blood mononuclear cells (PBMCs). We found plasma concentrations of interleukin-13 (IL-13) and macrophage derived chemokine (MDC) were significantly lower on the day of FET in cycles that resulted in a live birth. We also found genes encoding C-C chemokine receptor type 5 (CCR5), CD8 subunit alpha (CD8A) and SMAD family member 3 (SMAD3) were upregulated in PBMCs on the day of FET in cycles that resulted in live birth. Measurements of immune mediators from maternal blood could serve as prognostic markers during FET to guide clinical decision making and further our understanding of implantation failure.

Senescence induces miR-409 to down-regulate CCL5 and impairs angiogenesis in endothelial progenitor cells.

This study explores the impact of senescence on autocrine C-C motif chemokine ligand 5 (CCL5) in human endothelial progenitor cell (EPCs), addressing the poorly understood decline in number and function of EPCs during ageing. We examined the effects of replication-induced senescence on CCL5/CCL5 receptor (CCR5) signalling and angiogenic activity of EPCs in vitro and in vivo. We also explored microRNAs controlling CCL5 secretion in senescent EPCs, its impact on EPC angiogenic activity, and validated our findings in humans. CCL5 secretion and CCR5 levels in senescent EPCs were reduced, leading to attenuated angiogenic activity. CCL5 enhanced EPC proliferation via the CCR5/AKT/P70S6K axis and increased vascular endothelial growth factor (VEGF) secretion. Up-regulation of miR-409 in senescent EPCs resulted in decreased CCL5 secretion, inhibiting the angiogenic activity, though these negative effects were counteracted by the addition of CCL5 and VEGF. In a mouse hind limb ischemia model, CCL5 improved the angiogenic activity of senescent EPCs. Analysis involving 62 healthy donors revealed a negative association between CCL5 levels, age and Framingham Risk Score. These findings propose CCL5 as a potential biomarker for detection of EPC senescence and cardiovascular risk assessment, suggesting its therapeutic potential for age-related cardiovascular disorders.

CD147-K148me2-Driven Tumor Cell-Macrophage Crosstalk Provokes NSCLC Immunosuppression via the CCL5/CCR5 Axis.

Immunosuppression is a major hallmark of tumor progression in non-small cell lung cancer (NSCLC). Cluster of differentiation 147 (CD147), an important pro-tumorigenic factor, is closely linked to NSCLC immunosuppression. However, the role of CD147 di-methylation in the immunosuppressive tumor microenvironment (TME) remains unclear. Here, di-methylation of CD147 at Lys148 (CD147-K148me2) is identified as a common post-translational modification (PTM) in NSCLC that is significantly associated with unsatisfying survival outcomes among NSCLC sufferers, especially those in the advanced stages of the disease. The methyltransferase NSD2 catalyzes CD147 to generate CD147-K148me2. Further analysis demonstrates that CD147-K148me2 reestablishes the immunosuppressive TME and promotes NSCLC progression. Mechanistically, this modification promotes the interaction between cyclophilin A (CyPA) and CD147, and in turn, increases CCL5 gene transcription by activating p38-ZBTB32 signaling, leading to increased NSCLC cell-derived CCL5 secretion. Subsequently, CD147-K148me2-mediated CCL5 upregulation facilitates M2-like tumor-associated macrophage (TAM) infiltration in NSCLC tissues via CCL5/CCR5 axis-dependent intercellular crosstalk between tumor cells and macrophages, which is inhibited by blocking CD147-K148me2 with the targeted antibody 12C8. Overall, this study reveals the role of CD147-K148me2-driven intercellular crosstalk in the development of NSCLC immunosuppression, and provides a potential interventional strategy for PTM-targeted NSCLC therapy.

Immunotherapy-activated T cells recruit and skew late-stage activated M1-like macrophages that are critical for therapeutic efficacy.

Total tumor clearance through immunotherapy is associated with a fully coordinated innate and adaptive immune response, but knowledge on the exact contribution of each immune cell subset is limited. We show that therapy-induced intratumoral CD8+ T cells recruited and skewed late-stage activated M1-like macrophages, which were critical for effective tumor control in two different murine models of cancer immunotherapy. The activated CD8+ T cells summon these macrophages into the tumor and their close vicinity via CCR5 signaling. Exposure of non-polarized macrophages to activated T cell supernatant and tumor lysate recapitulates the late-stage activated and tumoricidal phenotype in vitro. The transcriptomic signature of these macrophages is also detected in a similar macrophage population present in human tumors and coincides with clinical response to immune checkpoint inhibitors. The requirement of a functional co-operation between CD8+ T cells and effector macrophages for effective immunotherapy gives warning to combinations with broad macrophage-targeting strategies.

Involvement of CCR5 on interstitial macrophages in the development of lung fibrosis in severe asthma.

CCR5 may be involved in the pathogenesis of asthma; however, the underlying mechanisms remain unclear. In comparison with a mild asthma model, subepithelial fibrosis was more severe and CCR5 gene expression in the lungs was significantly higher in our recently developed murine model of steroid-resistant severe asthma. Treatment with the CCR5 antagonist, maraviroc, significantly suppressed the development of subepithelial fibrosis in bronchi, whereas dexamethasone did not. On the other hand, increases in leukocytes related to type 2 inflammation, eosinophils, Th2 cells, and group 2 innate lymphoid cells in the lungs were not affected by the treatment with maraviroc. Increases in neutrophils and total macrophages were also not affected by the CCR5 antagonist. However, increases in transforming growth factor (TGF)-ß-producing interstitial macrophages (IMs) were significantly reduced by maraviroc. The present results confirmed increases in CCR5-expressing IMs in the lungs of the severe asthma model. In conclusion, CCR5 on IMs plays significant roles in the development of subepithelial fibrosis in severe asthma through TGF-ß production in the lungs.

CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

In vitro phenotypic susceptibility of HIV-1 non-group M to CCR5 inhibitor (maraviroc): TROPI-CO study.

The susceptibility of genetically divergent HIV-1 strains (HIV-1 non-M) from groups O, N, and P to the CCR5 co-receptor antagonist, maraviroc (MVC), was investigated among a large panel of 45 clinical strains, representative of the viral genetic diversity. The results were compared to the reference strains of HIV-1 group M (HIV-1/M) with known tropism. Among the non-M strains, a wide range of phenotypic susceptibilities to MVC were observed. The large majority of HIV-1/O strains (40/42) displayed a high susceptibility to MVC, with median and mean IC50 values of 1.23 and 1.33 nM, respectively, similar to the HIV-1/M R5 strain (1.89 nM). However, the two remaining HIV-1/O strains exhibited a lower susceptibility (IC50 at 482 and 496 nM), in accordance with their dual/mixed (DM) tropism. Interestingly, the two HIV-1/N strains demonstrated varying susceptibility patterns, despite always having relatively low IC50 values (2.87 and 47.5 nM). This emphasized the complexity of determining susceptibility solely based on IC50 values. Our study examined the susceptibility of all HIV-1 non-M groups to MVC and correlated these findings with virus tropism (X4, R5, or DM). The results confirm the critical significance of tropism determination before initiating MVC treatment in patients infected with HIV-1 non-M. Furthermore, we advocate for the consideration of additional parameters, such as the slope of inhibition curves, to provide a more thorough characterization of phenotypic susceptibility profiles. IMPORTANCE: Unlike HIV-1 group M, the scarcity of studies on HIV-1 non-M groups (O, N, and P) presents challenges in understanding their susceptibility to antiretroviral treatments, particularly due to their natural resistance to non-nucleoside reverse transcriptase inhibitors. The TROPI-CO study logically complements our prior investigations into integrase inhibitors and anti-gp120 efficacy. The largest panel of 45 non-M strains existing so far yielded valuable results on maraviroc (MVC) susceptibility. The significant variations in MVC IC50 reveal a spectrum of susceptibilities, with most strains displaying R5 tropism. Notably, the absence of MVC-resistant strains suggests a potential therapeutic avenue. The study also employs a robust novel cell-based phenotropism assay and identifies distinct groups of susceptibilities based on inhibition curve slopes. Our findings emphasize the importance of determining tropism before initiating MVC and provide crucial insights for selecting effective therapeutic strategies in the delicate context of HIV-1 non-M infections.