CCR5 Research Articles

COVID-19 in pediatrics: Genetic susceptibility.

The uptick in SARS-CoV-2 infection has resulted in a worldwide COVID-19 pandemic, which has created troublesome health and economic problems. We performed case–control meta-analyses in both African and European ethnicity COVID-19 disease cases based on laboratory test and phenotypic criteria. The cases had laboratory-confirmed SARS-CoV-2 infection. We uniquely investigated COVID infection genetics in a pediatric population. Our cohort has a large African ancestry component, also unique to our study. We tested for genetic variant association in 498 cases vs. 1,533 controls of African ancestry and 271 cases vs. 855 controls of European ancestry. We acknowledge that the sample size is relatively small, owing to the low prevalence of COVID infection among pediatric individuals. COVID-19 cases averaged 13 years of age. Pediatric genetic studies enhance the ability to detect genetic associations with a limited possible environment impact. Our findings support the notion that some genetic variants, most notably at the SEMA6D, FMN1, ACTN1, PDS5B, NFIA, ADGRL3, MMP27, TENM3, SPRY4, MNS1, and RSU1 loci, play a role in COVID-19 infection susceptibility. The pediatric cohort also shows nominal replication of previously reported adult study results: CCR9, CXCR6, FYCO1, LZTFL1, TDGF1, CCR1, CCR2, CCR3, CCR5, MAPT-AS1, and IFNAR2 gene variants. Reviewing the biological roles of genes implicated here, NFIA looks to be the most interesting as it binds to a palindromic sequence observed in both viral and cellular promoters and in the adenovirus type 2 origin of replication.

Allelic Variations in the Human Genes TMPRSS2 and CCR5, and the Resistance to Viral Infection by SARS-CoV-2.

During the first wave of COVID-19 infection in Italy, the number of cases and the mortality rates were among the highest compared to the rest of Europe and the world. Several studies demonstrated a severe clinical course of COVID-19 associated with old age, comorbidities, and male gender. However, there are cases of virus infection resistance in subjects living in close contact with infected subjects. Thus, to explain the predisposition to virus infection and to COVID-19 disease progression, we must consider, in addition to the genetic variability of the virus and other environmental or comorbidity conditions, the allelic variants of specific human genes, directly or indirectly related to the life cycle of the virus. Here, we analyzed three human genetic polymorphisms belonging to the TMPRSS2 and CCR5 genes in a sample population from Sicily (Italy) to investigate possible correlations with the resistance to viral infection and/or to COVID-19 disease progression as recently described in other human populations. Our results did not show any correlations of the rs35074065, rs12329760, and rs333 polymorphisms with SARS-CoV-2 infection or with COVID-19 disease severity. Further studies on other human genetic polymorphisms should be performed to identify the major human determinants of SARS-CoV-2 viral resistance.

Transcriptome Analysis Reveals Gene Expression Changes during Repair from Mechanical Wounding in Aquilaria sinensis

Plants repair their mechanical wounds by reprogramming secondary metabolism. However, which genes are reprogrammed during this repair process in Aquilaria sinensis has rarely been studied. Here, we used high-throughput RNA sequencing to explore the changes in the transcriptome of Aquilaria’s xylem, six months after the stem was subjected to mechanical wounding. In total, 1165 transcripts were differentially accumulated, of which 1002 transcripts were increased and 163 were decreased in their abundances (|log2 (fold change)| ≥ 1 and FDR ≤ 0.05). The majority of these genes encode products involved in plant secondary metabolism, transcription regulation, and phytohormone metabolism and signaling. The up-regulated genes were classified into 15 significantly enriched GO terms and were involved in 83 pathways, whereas the down-regulated genes were classified into 5 significantly enriched GO terms and represented 43 pathways. Gene annotation demonstrated that 100 transcripts could encode transcription factors (TFs), such as WRKY, AP2, MYB, and Helix-loop-helix (HLH) TFs. We inferred that the differential expression of TFs, genes associated with plant hormones, phenylpropanoid biosynthesis, and sesquiterpenoid biosynthesis may contribute to the repair of the stem after mechanical wounding in A. sinensis. Using co-expression analysis and prediction of TF binding sites, a TF–gene regulatory network for Aquilaria lignin biosynthesis was constructed. This included the MYB, HLH, WRKY, and AP2 TFs, and the COMT1, 4CLL7, and CCR1 genes. The changes in 10 candidate genes were validated by quantitative reverse-transcription PCR, indicating significant differences between the treated and untreated areas. Our study provides global gene expression patterns under mechanical wounding and would be valuable to further studies on the molecular mechanisms of plant repair in A. sinensis.

Reduced expression of the tumor necrosis factor gene in blood mononuclear cells in radiologically isolated syndrome – validation of transcriptome analysis results

Radiologically isolated syndrome (RIS) is a nosological form in which magnetic resonance imaging (MRI) reveals lesions of the white matter ofthe brain and/or spinal cord characteristic of multiple sclerosis (MS) in individuals in the absence of clinical symptoms of the disease. Amongthe studies devoted to RIS, the number of works aimed at studying the molecular mechanisms underlying its formation is very small. Previously,using next generation sequencing (NGS), we for the first time revealed significant differences in the expression profiles of several genes in peripheral blood mononuclear cells (MNCs) of individuals with RIS and healthy controls.Objective:to conduct a validation analysis of changes in the expression of the CCR2, CX3CR1, and TNF genes that were observed during NGSin the MNCs of individuals with RIS compared with healthy individuals.Patients and methods.Analysis of the expression of the CCR2, CX3CR1, and TNF genes was performed on independent validation cohorts (inMNCs of 14 subjects with RIS and 14 without RIS) by reverse transcription followed by real-time PCR.Results and discussion.In MNCs of subjects with RIS, the TNF gene expression was significantly reduced compared to healthy controls(p=0.035; FC=0.78). No significant differences in expression levels were found for other genes.Conclusion.The obtained data shows that disturbances of TNF gene expression preceding the clinical manifestations of MS, at least in individuals with RIS, which can lead to further dysregulation of several processes.

STAT1-mediated induction of Ly6c-expressing macrophages are involved in the pathogenesis of an acute colitis model.

The development of inflammatory bowel diseases is thought to be multifactorial, but the exact steps in pathogenesis are poorly understood. In this study, we investigated involvement of the activation of STAT1 signal pathway in the pathogenesis of an acute colitis model. A dextran sulfate sodium-induced acute colitis model was established by using wild-type C57BL/6 mice and STAT1-deficient mice. Disease indicators such as body weight loss and clinical score, induction of cytokines, chemokines, and inflammatory cells were evaluated in the acute colitis model. Disease state was significantly improved in the acute colitis model using STAT1-deficient mice compared with wild-type mice. The induction of Ly6c-highly expressing cells in colorectal tissues was attenuated in STAT1-deficient mice. IL-6, CCL2, and CCR2 gene expressions in Ly6c-highly expressing cells accumulated in the inflamed colon tissues and were significantly higher than in Ly6c-intermediate-expressing cells, whereas TNF-α and IFN-α/β gene expression was higher in Ly6c-intermediate-expressing cells. Blockade of CCR2-mediated signaling significantly reduced the disease state in the acute colitis model. Two different types of Ly6c-expressing macrophages are induced in the inflamed tissues through the IFN-α/β-STAT1-mediated CCL2/CCR2 cascade and this is associated with the pathogenesis such as onset, exacerbation, and subsequent chronicity of acute colitis.

Inferred Causal Mechanisms of Persistent FMDV Infection in Cattle from Differential Gene Expression in the Nasopharyngeal Mucosa.

Foot-and-mouth disease virus (FMDV) can persistently infect pharyngeal epithelia in ruminants but not in pigs. Our previous studies demonstrated that persistent FMDV infection in cattle was associated with under-expression of several chemokines that recruit immune cells. This report focuses on the analysis of differentially expressed genes (DEG) identified during the transitional phase of infection, defined as the period when animals diverge between becoming carriers or terminators. During this phase, Th17-stimulating cytokines (IL6 and IL23A) and Th17-recruiting chemokines (CCL14 and CCL20) were upregulated in animals that were still infected (transitional carriers) compared to those that had recently cleared infection (terminators), whereas chemokines recruiting neutrophils and CD8+ T effector cells (CCL3 and ELR+CXCLs) were downregulated. Upregulated Th17-specific receptor, CCR6, and Th17-associated genes, CD146, MIR155, and ThPOK, suggested increased Th17 cell activity in transitional carriers. However, a complex interplay of the Th17 regulatory axis was indicated by non-significant upregulation of IL17A and downregulation of IL17F, two hallmarks of TH17 activity. Other DEG suggested that transitional carriers had upregulated aryl hydrocarbon receptor (AHR), non-canonical NFκB signaling, and downregulated canonical NFκB signaling. The results described herein provide novel insights into the mechanisms of establishment of FMDV persistence. Additionally, the fact that ruminants, unlike pigs, produce a large amount of AHR ligands suggests a plausible explanation of why FMDV persists in ruminants, but not in pigs.

Kukoamine A inhibits C‐C motif chemokine receptor 5 to attenuate lipopolysaccharide‐induced lung injury

The aim of this study was to elucidate the mechanism underlying the effects of Kukoamine A (KuA) treatment on endotoxin-induced lung injury/inflammation. The study was performed in lipopolysaccharide (LPS)-exposed mouse models of lung injury and LPS-induced alveolar epithelial cell model. Relevant kits were used to detect levels of inflammation-related indicators, oxidative stress indicators, and mitochondrial function. Hematoxylin and eosinstaining was to detect lung injury. Then, C-C motif chemokine receptor 5 (CCR5) overexpression plasmid was transfected into alveolar epithelial cells to investigate the mechanism of KuA in lung injury. The results showed that LPS induction increased the expression of inflammatory factors, oxidative stress markers, and mitochondrial dysfunction in both animal and cellular models. In the mouse model, KuA treatment improved lung tissue injury, decreased wet-to-dry ratio and MPO levels, reduced the expression of inflammatory factors, and ameliorated oxidative stress and mitochondrial dysfunction. The protective effect of KuA in the cell model remained whereas was markedly reversed after CCR5 overexpression. Taken together, KuA might improve LPS-induced lung injury by inhibiting CCR5. This might also provide a novel theory for KuA in the treatment of lung injury.

123 Impact of local iron overload on crosstalk and phenotypes of immune and tissue cells in skin

This project focuses on elucidating the pathogenic effects of iron overload due to erythrocyte extravasation in the skin on the immune-tissue cell crosstalk leading to lipodermatosclerosis and leg ulcer in patients with chronic venous insufficiency. We generated a new mice model with local iron-overload in the skin, via ID-injection of iron-dextran (Fedx). Prussian blue staining proved that iron accumulates in the dermis and in the dWAT of these mice, like in patients’ skin. Skin of Fedx mice shows signs of lipodermatosclerosis; an increase of total cell count (p=0.001) in the dermal layer, particularly of F4/80+ macrophages (p=0.019) and PDGFRa+ fibroblasts (p<0.001), accompanied with less collagen in the deeper dermis and a reduction in the size of the dWAT (p<0.001) and of the adipocytes (p<0.001). Microscopic and gene expression analysis highlight lipolysis, the consequent reduction of lipids droplets (p=0.04), and loss of plin-1 (p=0.004) on adipocytes membrane, associated with downregulation of pro-adipogenic genes in the dWAT of Fedx mice. FACS analysis reveals a shift in F4/80+ Macrophage subtypes with the induction of TNFa (p=0.0163) and reduction of CD301b and Relm-a (both p<0.001). Upregulation of CCL2 (p=0.02), IL1ß, HMOX-1 (p=0.0001 dermis, p=0.01 dWAT) and CCR2 (p=0.01) genes, indicate for a pro-inflammatory activation in the dermal compartment. Mechanistic analysis with human cells in vitro shows that erythrocyte-uptake induces ROS and a pro-inflammatory phenotype in Macrophages. Fibroblasts in response to erythrocyte-fed macrophages resemble those phenotypes found in Fedx mice. The impact of erythrocytes on the differentiation and activation of adipocytes and their crosstalk to Macrophages and Fibroblasts is ongoing. Our data suggest that local iron-overload causes a complex skin phenotype with a maintained inflammatory status in the dermis, proliferative and less fibrogenic fibroblasts, impaired adipogenesis, and increased lipolysis.

SMAD4 Suppresses Colitis-associated Carcinoma Through Inhibition of CCL20/CCR6-mediated Inflammation

Background & AimsWe previously reported that colon epithelial cell silencing of Smad4 increased epithelial expression of inflammatory genes, including the chemokine c-c motif chemokine ligand 20 (CCL20), and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/c-c motif chemokine receptor 6 (CCR6) in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss. MethodsIn silico analysis of SMAD4, CCL20, and CCR6 messenger RNA expression was performed on published transcriptomic data from human ulcerative colitis (UC), and colon and rectal cancer samples. Immunohistochemistry for CCL20 and CCR6 was performed on human tissue microarrays comprising human UC-associated cancer specimens, Mice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to colitis and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining. ResultsIn human UC-associated cancers, loss of epithelial SMAD4 was associated with increased CCL20 expression and CCR6+ cells. SMAD4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of immune cells to the mouse colon epithelium and stroma, particularly T regulatory, Th17, and dendritic cells. Loss of CCR6 abrogated these immune responses and significantly reduced the incidence of colitis-associated tumors observed with loss of SMAD4 alone. ConclusionsRegulation of mucosal inflammation is central to SMAD4 tumor suppressor function in the colon. A key downstream node in this regulation is suppression of epithelial CCL20 signaling to CCR6 in immune cells. Loss of SMAD4 in the colon epithelium increases CCL20 expression and chemoattraction of CCR6+ immune cells, contributing to greater susceptibility to colon cancer.

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV.

Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.

Effect of Polymeric Nanoparticles with Entrapped Fish Oil or Mupirocin on Skin Wound Healing Using a Porcine Model.

The utilization of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) with entrapped fish oil (FO) loaded in collagen-based scaffolds for cutaneous wound healing using a porcine model is unique for the present study. Full-depth cutaneous excisions (5 × 5 cm) on the pig dorsa were treated with pure collagen scaffold (control, C), empty PLGA NPs (NP), FO, mupirocin (MUP), PLGA NPs with entrapped FO (NP/FO) and PLGA NPs with entrapped MUP (NP/MUP). The following markers were evaluated on days 0, 3, 7, 14 and 21 post-excision: collagen, hydroxyproline (HP), angiogenesis and expressions of the COX2, EGF, COL1A1, COL1A3, TGFB1, VEGFA, CCL5 and CCR5 genes. The hypothesis that NP/FO treatment is superior to FO alone and that it is comparable to NP/MUP was tested. NP/FO treatment increased HP in comparison with both FO alone and NP/MUP (day 14) but decreased (p < 0.05) angiogenesis in comparison with FO alone (day 3). NP/FO increased (p < 0.05) the expression of the CCR5 gene (day 3) and tended (p > 0.05) to increase the expressions of the EGF (day 7, day 14), TGFB1 (day 21) and CCL5 (day 7, day 21) genes as compared with NP/MUP. NP/FO can be suggested as a suitable alternative to NP/MUP in cutaneous wound treatment.

FRI018 - Inhibition of C-C motif chemokine receptor 2 (CCR2) using a novel in silico designed peptide attenuates macrophage migration in vitro and intrahepatic monocyte recruitment in vivo

FRI018 - Inhibition of C-C motif chemokine receptor 2 (CCR2) using a novel in silico designed peptide attenuates macrophage migration in vitro and intrahepatic monocyte recruitment in vivo

Abstract 6008: Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICIs) have generated durable clinical responses in multiple tumor types, but their therapeutic efficacy is limited by development of primary or acquired resistance. The tumor microenvironment (TME) contains many factors influencing ICI responses. The ratio of effector T-cells (Teffs) and regulatory T-cells (Tregs) in tumors has been shown to be associated with the efficacy of ICIs in murine models, indicating that increasing Teffs or decreasing Tregs in tumor may prevent development of resistance to immunotherapy. There is a high and restricted expression of CC motif chemokine receptor 8 (CCR8) on tumor-infiltrating Tregs, whereas they are negligibly expressed on Teffs. Deletion of tumor-infiltrating Tregs via murine anti-CCR8 surrogates has shown effective anti-tumor activity in tumor models.LM-108 is a novel humanized anti-CCR8 monoclonal antibody with engineered IgG1 that enhances antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of CCR8 and Foxp3 on tumor-infiltrating lymphocytes (TILs) was analyzed using published single-cell sequencing data. The binding affinity of LM-108 was assessed by flow cytometry using CCR8 high- and low-expressing cell lines. In vitro measurements of ADCC and antibody-dependent cellular phagocytosis (ADCP) mediated by LM-108 were conducted in CCR8 overexpressing HEK293, CHO-K1, and Jurkat cells. Furthermore, in vivo anti-tumor activity of LM-108 as monotherapy and in combination with anti-PD-1 therapy was evaluated in syngeneic models and PD-1-resistant tumor models with human CCR8 knock-in mice. Analysis of published single-cell sequencing data showed that in comparison with CCR4, CCR8 gene expression was specifically overlapped with Foxp3 in TILs of breast cancer. Fluorescence-activated cell sorting (FACS) assay revealed that LM-108 specifically binds with high affinity to human CCR8 in a dose-dependent manner. ADCC and ADCP effects induced by LM-108 were observed in HEK293 and CHO-K1 cells with different levels of CCR8 expression. In vivo data of CT26 syngeneic model showed that LM-108 murine surrogate antibody alone or in combination with anti-PD-1 antibody significantly inhibited tumor growth. Treatment with LM-108 effectively depleted tumor-infiltrating Tregs accompanied by increase in CD8/Treg ratio in tumors, leading to significant anti-tumor activity in MC38 model. Finally, LM-108 showed a synergistic therapeutic efficacy in PD-1 resistant models in combination with anti-PD-1 antibody. To conclude, LM-108 is a novel Fc-optimized CCR8 antibody that selectively depletes tumor infiltrating Tregs thereby improving anti-tumor immune response either as monotherapy or combination therapy. Thus, LM-108 can be a promising therapeutic approach to overcome ICI resistance in cancer patients. Citation Format: Jie Luo, Wentao Huang, Junwei Yang, Jin Li, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6008.

Tumor-Derived C-C Motif Ligand 2 Induces the Recruitment and Polarization of Tumor-Associated Macrophages and Increases the Metastatic Potential of Bladder Cancer Cells in the Postirradiated Microenvironment

Radiation therapy (RT) is mainly used for bladder preservation in patients with muscle-invasive bladder cancer. The response of urothelial tumors to RT remains unsatisfactory. We investigated the interaction of RT and tumor-associated macrophages (TAMs) in the context of bladder cancer radioresistance. We evaluated the therapeutic effects of RT and TAM distribution by establishing an ectopic allograft mouse model. A Transwell coculture system was used to simulate the interaction between TAMs and MB49 bladder cancer cells in the tumor microenvironment. Cytokines and chemokines were analyzed in irradiated MB49 cells. Colony formation and Boyden chamber assays were used to assess the cytotoxic effects and the effects of TAMs on MB49 cell invasion, respectively. Local RT delayed primary tumor growth but promoted pulmonary metastases in C57BL/6 mice. Increased secretion of C-C motif chemokine ligand (CCL2) by irradiated MB49 cells, especially in the presence of M1-type TAMs, contributed to the infiltration of bone marrow-derived C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and the polarization of M1-type TAMs toward the M2 type to promote MB49 cell invasion. Blockade of CCL2-CCR2 activation by a CCR2 antagonist reversed the phenotypic TAM transformation and suppressed pulmonary metastases. Bladder cancer cells responded to RT by producing CCL2, which recruited TAM precursors from bone marrow and polarized M1-type TAMs toward the M2 type. This phenotypic TAM transformation promoted the pulmonary metastasis of bladder cancer cells after RT. Disrupting the CCL2-CCR2 signaling axis in combination with RT holds promise for improving RT efficacy in bladder cancer.

Association between TP53, PAR1 and CCR5 gene polymorphisms and non-small cell lung cancer susceptibility in the Amazon.

e20538 Background: Lung cancer is one of the most frequent neoplasms in the world, representing 11.4% of all registered cancers, and is responsible for 18% of cancer deaths. It is divided into two categories, small cell lung cancer, responsible for 15% of cases; and non-small cell lung cancer (NSCLC), which represents 85% of cases. The most well-known risk factor for the development of lung cancer is smoking, due to substances contained in tobacco associated with inflammation and carcinogenesis. Pulmonary carcinogenesis is a complex and gradual process, with synergistic and complex interactions between environmental risk factors and individual genetic susceptibility. The aim of this study was to investigate possible associations between TP53, PAR1, and CCR5 gene polymorphism for susceptibility to NSCLC in the Amazon. Methods: This is a pilot, case-control study, which included 263 subjects, 67 patients with NSCLC and 196 healthy subjects. The samples were analyzed for TP53 (rs17880560), PAR1 (rs11267092), and CCR5 (rs333) gene polymorphism, genotyped in PCR, followed by fragment analysis. To avoid misinterpretation due to population substructure, we applied a previously developed set of 61 informative ancestral markers that were genotyped by multiplex PCR. We used logistic regression to identify differences in genotypic frequencies between individuals with and without lung cancer. Results: We observed that some genotypes were associated with protection for NSCLC: TP53 gene LED/DEL genotype (p = 0.041, OR: 0.510, 95%CI: 0.267-0.974); DEL/DEL genotype of the PAR1 gene (p = 0.023, OR: 0.471, 95%CI: 0.247-0.971); and also the INS/INS genotype of the CCR5 gene (p = 0.033, OR: 0.331, 95%CI: 0.120-0.917). Conclusions: TP53 (rs17880560), PAR1 (rs11267092) and CCR5 (rs333) gene variants were significantly associated with NSCLC in patients from the Amazon. The validation of these findings may favor, in the future, the screening of individuals, facilitating the institution of preventive measures personalized, early diagnosis, consequently reducing the cost for health services and mortality from this malignant neoplasm. Keywords: Genetic Polymorphism; TP53; PAR1; CCR5; Biomarker; Non-Small Cell Lung Cancer.

Type 1 Angiotensin Receptors on CD11c-Expressing Cells Protect Against Hypertension by Regulating Dendritic Cell-Mediated T Cell Activation.

Type 1 angiotensin (AT1) receptors are expressed on immune cells, and we previously found that bone marrow-derived AT1 receptors protect against Ang (angiotensin) II-induced hypertension. CD11c is expressed on myeloid cells derived from the bone marrow, including dendritic cells (DCs) that activate T lymphocytes. Here, we examined the role of AT1 receptors on CD11c+ cells in hypertension pathogenesis. Mice lacking the dominant murine AT1 receptor isoform, AT1a, on CD11c+ cells (dendritic cell [DC] AT1aR knockout [KO]) and wild-type (WT) littermates were subjected to Ang II-induced hypertension. Blood pressures were measured by radiotelemetry. DC AT1aR KO mice had exaggerated hypertensive responses to chronic Ang II infusion with enhanced renal accumulation of effector memory T cells and CD40+ DCs. CCL5 (C-C motif chemokine ligand 5) recruits T cells into injured tissues, and CCR7 (C-C motif chemokine receptor 7) facilitates DC and T cell interactions in the kidney lymph node to allow T cell activation. DCs from the hypertensive DC AT1aR KO kidneys expressed higher levels of CCL5 and CCR7. mRNA expressions for CCR7 and tumor necrosis factor-α were increased in CD4+ T cells from the renal lymph nodes of DC AT1aR KO mice. During the second week of Ang II infusion when blood pressures between groups diverged, DC AT1aR KO mice excreted less sodium than WTs. Expressions for epithelial sodium channel subunits were increased in DC AT1aR KO kidneys. Following activation of the renin angiotensin system, AT1aR stimulation on DCs suppresses renal DC maturation and T cell activation with consequent protection from sodium retention and blood pressure elevation.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Exploration of novel ligands to target C-C Motif Chemokine Receptor 2 (CCR2) as a promising pharmacological treatment against traumatic brain injury

It is widely reported that the overexpression of the C-C Motif Chemokine Receptor 2 (CCR2) has negative implications in neuroinflammatory diseases such as traumatic brain injury (TBI), although promising drugs to tackle this have been less forthcoming. As of 2016, only 2 drugs specifically targeting this receptor have made their way to market, with unsuccessful outcome unfortunately, suggesting that the search for more specific and precise ligands is utterly necessary. In this paper we hypothesized that by targeting Glu291, Met295, Trp98, Leu45 and Val189 amino acids, essential in the binding of CCR2 with C-C Motif Chemokine Ligand 2 (CCL2), the endogenous substrate, mitigates its activity in TBI. We used a pharmacophore model to screen for suitable ligands that may bind to CCR2, which returned 871 ligands. Docking and molecular dynamics results uncovered that two ligands (A102) and (A435) contained several of those important residues and showed a stability and compactness when in complex with CCR2, with these results confirmed by MMGBSA calculations with A102 recording a better interaction compared to A435. Finally, a PPI network was built to explore downstream signaling being regulated by both ligands in TBI, showing amyloid precursor protein (APP) as a key target and neuroactive-ligand receptor interaction (1.80E-27) the top functional annotated category. In conclusion, for the first time we report novel ligands A102 and A435 targeting CCR2 as a potential new pharmacological approach to target inflammation post-TBI.

Evaluation of CCR5-Δ32 Mutation and HIV-1 Surveillance drug-resistance Mutations in Peripheral Blood Mononuclear Cells of long-term Non Progressors of HIV-1-infected Individuals

Aim: This study aimed to evaluate chemokine receptor 5 delta 32 (CCR5-Δ32) mutation and HIV-1 surveillance drug-resistance mutations (SDRMs) in peripheral blood mononuclear cells of long-term non progressors (LTNPs) of HIV-1-infected individuals. Materials &amp; methods: This research was performed on 197 treatment-naive HIV-1-infected patients. After follow-up, it was determined that 15 (7.6%) of these people were LTNPs. The PCR assay was performed to identify the CCR5 genotype and HIV-1 SDRMs. Results: One (6.7%) of the LTNPs was heterozygous (wt/Δ32) for the CCR5 delta 32 (CCR5Δ32). However, none of the individuals was homozygous for this mutation (Δ32/Δ32). Moreover, none of the LTNPs showed HIV-1 SDRMs. The CRF35-AD subtype was the most dominant subtype, with a percentage of 93.3%. Conclusion: Iranian elite controllers are negative for CCR5-delta 32 homozygous genotype and drug resistance against antiretroviral drugs.

C-C motif chemokine ligand 5 confines liver regeneration by down-regulating reparative macrophage-derived hepatocyte growth factor in a forkhead box O 3a-dependent manner.

Liver regeneration (LR) is vital for the recovery of liver function after hepatectomy. Limited regeneration capacity, together with insufficient remnant liver volume, is a risk factor for posthepatectomy liver failure (PHLF) resulting from small-for-size syndrome. Although inflammation plays an important role in controlling LR, the underlying mechanisms still remain obscure. We identified C-C motif chemokine ligand (CCL) 5 as an important negative regulator for LR. CCL5 levels were elevated after partial hepatectomy (PHx), both in healthy donors of living donor liver transplantation (LT) and PHx mouse models. Ccl5 knockout mice displayed improved survival after 90% PHx and enhanced LR 36 h after 70% PHx. However, primary hepatocytes from Ccl5-/- mice exposed to growth factors in vitro showed no proliferation advantage compared to those from wild-type (WT) mice. Flow cytometry analysis showed that proportions of Ly6Clo macrophages were significantly increased in Ccl5-/- mice after 70% PHx. RNA-sequencing analysis revealed that sorted macrophages (CD11b+ Ly6Clo&hi ) manifested enhanced expression of reparative genes in Ccl5-/- mice compared to WT mice. Mechanistically, CCL5 induced macrophages toward proinflammatory Ly6Chi phenotype, thereby inhibiting the production of hepatocyte growth factor (HGF) through the C-C motif chemokine receptor (CCR) 1- and CCR5-mediated forkhead box O (FoxO) 3a pathways. Finally, blockade of CCL5 greatly optimized survival and boosted LR in the mouse PHx model. Our findings suggest that inhibition of CCL5 is a promising strategy to improve regeneration restoration by enhancing HGF secretion from reparative macrophages through the FoxO3a pathway, which may potentially reduce the mortality of PHLF.