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1.
Nature ; 606(7912): 146-152, 2022 06.
Article in English | MEDLINE | ID: mdl-35614219

ABSTRACT

Real-world memories are formed in a particular context and are often not acquired or recalled in isolation1-5. Time is a key variable in the organization of memories, as events that are experienced close in time are more likely to be meaningfully associated, whereas those that are experienced with a longer interval are not1-4. How the brain segregates events that are temporally distinct is unclear. Here we show that a delayed (12-24 h) increase in the expression of C-C chemokine receptor type 5 (CCR5)-an immune receptor that is well known as a co-receptor for HIV infection6,7-after the formation of a contextual memory determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed expression of CCR5 in mouse dorsal CA1 neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dorsal CA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, and therefore closes the temporal window for memory linking. Our findings also show that an age-related increase in the neuronal expression of CCR5 and its ligand CCL5 leads to impairments in memory linking in aged mice, which could be reversed with a Ccr5 knockout and a drug approved by the US Food and Drug Administration (FDA) that inhibits this receptor, a result with clinical implications. Altogether, the findings reported here provide insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.


Subject(s)
CA1 Region, Hippocampal , Memory , Neurons , Receptors, CCR5 , Animals , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/physiology , Memory/physiology , Mental Recall/physiology , Mice , Neurons/metabolism , Receptors, CCR5/deficiency , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Time Factors
2.
Front Immunol ; 12: 722320, 2021.
Article in English | MEDLINE | ID: mdl-34950130

ABSTRACT

The inhibition of anabolic pathways, such as aerobic glycolysis, is a metabolic cornerstone of memory T cell differentiation and function. However, the signals that hamper these anabolic pathways are not completely known. Recent evidence pinpoints the chemokine receptor CCR5 as an important player in CD4+ T cell memory responses by regulating T cell antigen receptor (TCR) nanoclustering in an antigen-independent manner. This paper reports that CCR5 specifically restrains aerobic glycolysis in memory-like CD4+ T cells, but not in effector CD4+ T cells. CCR5-deficient memory CD4+ T cells thus show an abnormally high glycolytic/oxidative metabolism ratio. No CCR5-dependent change in glucose uptake nor in the expression of the main glucose transporters was detected in any of the examined cell types, although CCR5-deficient memory cells did show increased expression of the hexokinase 2 and pyruvate kinase M2 isoforms, plus the concomitant downregulation of Bcl-6, a transcriptional repressor of these key glycolytic enzymes. Further, the TCR nanoclustering defects observed in CCR5-deficient antigen-experienced CD4+ T cells were partially reversed by incubation with 2-deoxyglucose (2-DG), suggesting a link between inhibition of the glycolytic pathway and TCR nanoscopic organization. Indeed, the treatment of CCR5-deficient lymphoblasts with 2-DG enhanced IL-2 production after antigen re-stimulation. These results identify CCR5 as an important regulator of the metabolic fitness of memory CD4+ T cells, and reveal an unexpected link between T cell metabolism and TCR organization with potential influence on the response of memory T cells upon antigen re-encounter.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Immunologic Memory , Receptors, Antigen, T-Cell/metabolism , Receptors, CCR5/physiology , Animals , Antigens/immunology , Cells, Cultured , Gene Expression Regulation , Glucose/metabolism , Glycolysis/genetics , Ligands , Mice , Mice, Transgenic , Mitochondria/metabolism , Nanostructures , Ovalbumin/immunology , Oxygen Consumption , Peptide Fragments/immunology , Proto-Oncogene Proteins c-bcl-6/biosynthesis , Proto-Oncogene Proteins c-bcl-6/genetics , Receptors, CCR5/deficiency , Specific Pathogen-Free Organisms
3.
EMBO J ; 39(15): e104749, 2020 08 03.
Article in English | MEDLINE | ID: mdl-32525588

ABSTRACT

CCR5 is not only a coreceptor for HIV-1 infection in CD4+ T cells, but also contributes to their functional fitness. Here, we show that by limiting transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T-cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 co-stimulatory activity. CCR5-deficient mice showed reduced production of high-affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T-cell response. This study identifies a CCR5 function in the generation of CD4+ T-cell memory responses and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species.


Subject(s)
Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Ceramides/immunology , Immunologic Memory , Receptors, CCR5/deficiency , Animals , Antigens/genetics , CD4-Positive T-Lymphocytes/cytology , Ceramides/genetics , HEK293 Cells , Humans , Mice , Mice, Knockout , Receptors, CCR5/immunology
4.
Math Med Biol ; 37(2): 212-242, 2020 05 29.
Article in English | MEDLINE | ID: mdl-31265056

ABSTRACT

The use of gene-editing technology has the potential to excise the CCR5 gene from haematopoietic progenitor cells, rendering their differentiated CD4-positive (CD4+) T cell descendants HIV resistant. In this manuscript, we describe the development of a mathematical model to mimic the therapeutic potential of gene editing of haematopoietic progenitor cells to produce a class of HIV-resistant CD4+ T cells. We define the requirements for the permanent suppression of viral infection using gene editing as a novel therapeutic approach. We develop non-linear ordinary differential equation models to replicate HIV production in an infected host, incorporating the most appropriate aspects found in the many existing clinical models of HIV infection, and extend this model to include compartments representing HIV-resistant immune cells. Through an analysis of model equilibria and stability and computation of $R_0$ for both treated and untreated infections, we show that the proposed therapy has the potential to suppress HIV infection indefinitely and return CD4+ T cell counts to normal levels. A computational study for this treatment shows the potential for a successful 'functional cure' of HIV. A sensitivity analysis illustrates the consistency of numerical results with theoretical results and highlights the parameters requiring better biological justification. Simulations of varying level production of HIV-resistant CD4+ T cells and varying immune enhancements as the result of these indicate a clear threshold response of the model and a range of treatment parameters resulting in a return to normal CD4+ T cell counts.


Subject(s)
HIV Infections/therapy , HIV-1 , Models, Biological , Basic Reproduction Number/statistics & numerical data , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , CRISPR-Cas Systems , Computational Biology , Computer Simulation , Gene Editing/methods , HIV Infections/immunology , HIV Infections/virology , HIV-1/immunology , HIV-1/pathogenicity , Hematopoietic Stem Cell Transplantation/methods , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Mathematical Concepts , Models, Immunological , Receptors, CCR5/deficiency , Receptors, CCR5/genetics
5.
Nature ; 576(7787): 397-405, 2019 12.
Article in English | MEDLINE | ID: mdl-31853080

ABSTRACT

Despite considerable global investment, only 60% of people who live with HIV currently receive antiretroviral therapy. The sustainability of current programmes remains unknown and key incidence rates are declining only modestly. Given the complexities and expenses associated with lifelong medication, developing an effective curative intervention is now a global priority. Here we review why and where a cure is needed, and how it might be achieved. We argue for expanding these efforts from resource-rich regions to sub-Saharan Africa and elsewhere: for any intervention to have an effect, region-specific biological, therapeutic and implementation issues must be addressed.


Subject(s)
Combined Modality Therapy , HIV Infections/therapy , Health Resources , Health Services Needs and Demand , Needs Assessment , Anti-HIV Agents/administration & dosage , Anti-HIV Agents/therapeutic use , Global Health , HIV Infections/drug therapy , HIV Infections/epidemiology , HIV Infections/immunology , HIV-1/drug effects , HIV-1/genetics , HIV-1/immunology , Humans , Receptors, CCR5/deficiency , Receptors, CCR5/genetics , T-Lymphocytes, Cytotoxic/immunology
6.
Iran J Allergy Asthma Immunol ; 18(3): 310-319, 2019 Jun 08.
Article in English | MEDLINE | ID: mdl-31522438

ABSTRACT

C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the "Loss of a primer binding site" technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The "Loss of a primer binding site" technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.


Subject(s)
Immune System Diseases/etiology , Immune System Diseases/therapy , Induced Pluripotent Stem Cells/metabolism , Receptors, CCR5/deficiency , Stem Cell Transplantation , Flow Cytometry/methods , Gene Editing , Gene Expression Regulation , Genes, Reporter , Genetic Loci , Genetic Vectors/genetics , Humans , Immune System Diseases/metabolism , RNA, Guide, Kinetoplastida , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Stem Cell Transplantation/methods
7.
Mucosal Immunol ; 12(6): 1391-1403, 2019 11.
Article in English | MEDLINE | ID: mdl-31551493

ABSTRACT

Our current study focused on elucidating the role of specific chemokine-receptor interactions in antigen (Ag)-specific immune cell migration from nasal to genital mucosal tissues. This cellular migration is critical to induce effective Ag-specific immune responses against sexually transmitted genital infections. In this study, nasal immunization with live attenuated HSV-2 TK- induced the upregulation of CCR5 expression in effector immune cells, including CD4+ T cells, in Ag-priming sites and vaginal tissue. The CCR5 ligands CCL3, CCL4, and CCL5 all showed upregulated expression in vaginal tissue; in particular, CCL5 expression was highly enhanced in the stromal cells of vaginal tissue after nasal immunization. Intravaginal blockade of CCL5 by using neutralizing antibody diminished the number of HSV-2-specific effector cells in the vagina. Furthermore, loss of CCR5, a receptor for CCL5, impaired the migration of nasally primed Ag-specific effector cells from the airway to vagina. Effector cells adoptively transferred from CCR5-deficient mice failed to migrate into vaginal tissue, consequently increasing recipient mice's susceptibility to HSV-2 vaginal infection. These results indicate that the CCR5-CCL5 chemokine pathway is required for the migration and retention of nasally primed Ag-specific effector cells in vagina for providing protective immunity against HSV-2 infection.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Chemokine CCL5/metabolism , Herpes Genitalis/prevention & control , Herpesvirus 2, Human/pathogenicity , Immunity, Mucosal , Mucous Membrane/virology , Receptors, CCR5/metabolism , Vagina/virology , Viral Vaccines/administration & dosage , Administration, Intranasal , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/transplantation , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Chemotaxis, Leukocyte , Disease Models, Animal , Female , Herpes Genitalis/immunology , Herpes Genitalis/metabolism , Herpes Genitalis/virology , Herpesvirus 2, Human/genetics , Herpesvirus 2, Human/immunology , Immunization , Mice, Inbred C57BL , Mice, Knockout , Mucous Membrane/immunology , Mucous Membrane/metabolism , Receptors, CCR5/deficiency , Receptors, CCR5/genetics , Receptors, CXCR3/genetics , Receptors, CXCR3/metabolism , Signal Transduction , Vaccines, Attenuated/administration & dosage , Vagina/immunology , Vagina/metabolism , Virulence
8.
BMC Genomics ; 20(1): 705, 2019 Sep 11.
Article in English | MEDLINE | ID: mdl-31506064

ABSTRACT

BACKGROUND: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. RESULTS: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. CONCLUSIONS: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.


Subject(s)
Brain/cytology , Brain/parasitology , Gene Expression Profiling , Receptors, CCR5/deficiency , Toxoplasma/physiology , Animals , Astrocytes/metabolism , Astrocytes/parasitology , Brain/metabolism , Gene Knockout Techniques , Mice , Microglia/metabolism , Microglia/parasitology , Receptors, CCR5/genetics
9.
Nature ; 568(7751): 244-248, 2019 04.
Article in English | MEDLINE | ID: mdl-30836379

ABSTRACT

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression.


Subject(s)
HIV Infections/therapy , HIV Infections/virology , HIV-1 , Hematopoietic Stem Cell Transplantation/methods , Receptors, CCR5/chemistry , Receptors, CCR5/genetics , CD4-Positive T-Lymphocytes/immunology , Cytomegalovirus/chemistry , Cytomegalovirus/immunology , HIV Antibodies/immunology , HIV Infections/complications , HIV-1/chemistry , HIV-1/immunology , Hodgkin Disease/complications , Hodgkin Disease/drug therapy , Humans , Receptors, CCR5/deficiency , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Transplantation, Homologous , gag Gene Products, Human Immunodeficiency Virus/immunology
11.
J Zhejiang Univ Sci B ; 20(1): 32-38, 2019.
Article in English | MEDLINE | ID: mdl-30614228

ABSTRACT

The rapid developments of science and technology in China over recent decades, particularly in biomedical research, have brought forward serious challenges regarding ethical governance. Recently, Jian-kui HE, a Chinese scientist, claimed to have "created" the first gene-edited babies, designed to be naturally immune to the human immunodeficiency virus (HIV). The news immediately triggered widespread criticism, denouncement, and debate over the scientific and ethical legitimacy of HE's genetic experiments. China's guidelines and regulations have banned germline genome editing on human embryos for clinical use because of scientific and ethical concerns, in accordance with the international consensus. HE's human experimentation has not only violated these Chinese regulations, but also breached other ethical and regulatory norms. These include questionable scientific value, unreasonable risk-benefit ratio, illegitimate ethics review, invalid informed consent, and regulatory misconduct. This series of ethical failings of HE and his team reveal the institutional failure of the current ethics governance system which largely depends on scientist's self-regulation. The incident highlights the need for urgent improvement of ethics governance at all levels, the enforcement of technical and ethical guidelines, and the establishment of laws relating to such bioethical issues.


Subject(s)
Gene Editing/ethics , CRISPR-Cas Systems , China , Consent Forms/ethics , Ethics, Medical , Female , Gene Editing/legislation & jurisprudence , Gene Knockout Techniques/ethics , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/prevention & control , Human Experimentation/ethics , Human Experimentation/legislation & jurisprudence , Humans , Infant, Newborn , Pregnancy , Professional Misconduct/ethics , Receptors, CCR5/deficiency , Receptors, CCR5/genetics
12.
Biol Blood Marrow Transplant ; 25(3): 515-521, 2019 03.
Article in English | MEDLINE | ID: mdl-30315941

ABSTRACT

Graft-versus-host disease (GVHD) remains the most common treatment-related complication after allogeneic hematopoietic cell transplantation (allo-HCT). Lymphocyte migration plays a critical role in the pathogenesis of GVHD. A previous phase I/II trial demonstrated that CCR5 blockade with maraviroc in the first 30days after allo-HCT resulted in a low incidence of early acute GVHD, primarily in visceral organs, but with no impact on late acute or chronic GVHD. We conducted a phase II trial to examine the efficacy of an extended course of maraviroc, administered through post-transplantation day +90 in addition to standard prophylaxis in 37 recipients of reduced-intensity-conditioned unrelated donor allo-HCT performed to treat hematologic malignancies. Extended maraviroc treatment was safe and feasible. The primary study endpoint, day +180 rate of grade II-IV acute GVHD, was 22 ± 7%, liver GVHD was not observed, and gut GVHD was uncommon. The day +180 rate of grade III-IV acute GVHD was 5 ± 4%. The 1-year rate of moderate to severe chronic GVHD was 8 ± 5% and that of disease relapse was 30 ± 8%. Overall survival at 1 year was 70 ± 8%. Compared with the previously studied short course of maraviroc, the extended course resulted in a significantly higher GVHD-free, relapse-free survival (adjusted hazard ratio [HR], .45; 95% confidence interval [CI], .25 to .82; P = .009) and overall survival (adjusted HR, .48; 95% CI, .24 to .96; P = .037). A combined analysis of both trials showed that high maraviroc trough concentrations on the day of hematopoietic cell infusion were associated with lower rates of acute GVHD. An extended course of maraviroc after reduced-intensity-conditioned unrelated donor allo-HCT is safe and effective in preventing acute and chronic GVHD and is associated with favorable survival.


Subject(s)
CCR5 Receptor Antagonists/therapeutic use , Graft vs Host Disease/prevention & control , Hematopoietic Stem Cell Transplantation/methods , Maraviroc/therapeutic use , Receptors, CCR5/deficiency , Transplantation Conditioning/methods , Adult , Aged , Female , Hematologic Neoplasms/complications , Hematologic Neoplasms/therapy , Hematopoietic Stem Cell Transplantation/mortality , Hematopoietic Stem Cell Transplantation/standards , Humans , Male , Maraviroc/pharmacology , Middle Aged , Survival Analysis , Treatment Outcome , Unrelated Donors
13.
Clin Infect Dis ; 68(4): 684-687, 2019 02 01.
Article in English | MEDLINE | ID: mdl-30020413

ABSTRACT

Allogeneic stem cell transplantation (alloSCT) of homozygous CCR5 Δ32 stem cells once resulted in the cure of human immunodeficiency virus (HIV) infection. We have recently reported a viral breakthrough in a similar setting. Here, we demonstrate that the rapid rebound after alloSCT was related to a highly replicative CXCR4-tropic HIV variant, which could already be detected before alloSCT.


Subject(s)
HIV Infections/therapy , HIV/isolation & purification , Stem Cell Transplantation/methods , Transplantation, Homologous/methods , Viral Load , Viral Tropism , HIV/physiology , Humans , Receptors, CCR5/deficiency , Receptors, CXCR4/physiology , Treatment Outcome
14.
Immunol Cell Biol ; 97(3): 317-325, 2019 03.
Article in English | MEDLINE | ID: mdl-30536991

ABSTRACT

Human genetic studies demonstrate a link between the 32-bp deletion that produces a nonfunctional CCR5 receptor and enhanced recovery from acute hepatitis B virus (HBV) infection. To investigate the role of CCR5 in immune responses to acute HBV, we intravenously infected Ccr5+/+ (WT) and Ccr5-/- (KO) mice with a replication-incompetent adenovirus containing the overlapping HBV1.3 construct (AdHBV), or vector control. At day 3 following AdHBV infection, analysis of intrahepatic leukocytes (IHL) showed KO mice had increased CD11b+ NK cells compared to WT (18.2% versus 7.6% of live IHL, P < 0.01). These CD11b+ NK cells were nonresident (CD49a- ) and had capacity to degranulate and produce IFN-γ following stimulation. At day 3, plasma CXCL10 was significantly increased in KO, but not WT, mice receiving AdHBV as compared to vector control, while CXCR3 expression on hepatic CD11b+ NK cells in AdHBV-treated KO mice was significantly lower than that in uninfected mice, suggesting these NK cells are recruited along the CXCL10-CXCR3 axis. At days 7 and 14, no differences between genotypes were observed in number, or HBV-specific function, of intrahepatic CD8+ T cells. Instead, at day 14, KO mice had increased intrahepatic proinflammatory monocytes compared to WT mice (17.56% versus 6.57% of live IHL, P = 0.014), corresponding with an increase in plasma alanine aminotransferase and intrahepatic IL-1ß observed in KO mice. Taken together, these findings demonstrate that loss of CCR5 signaling drives a more robust inflammatory liver microenvironment early in acute HBV infection via enrichment of hepatic innate immune cells.


Subject(s)
Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Hepatitis B virus , Hepatitis B/etiology , Immunity, Innate/genetics , Receptors, CCR5/deficiency , Animals , Biomarkers , Cell Degranulation/immunology , Disease Models, Animal , Disease Susceptibility , Gene Expression , Hepatitis B/metabolism , Hepatitis B/pathology , Humans , Immunophenotyping , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Leukocytes/immunology , Leukocytes/metabolism , Male , Mice , Mice, Knockout , Spleen/cytology , Spleen/immunology , Spleen/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
15.
ACS Chem Neurosci ; 10(1): 39-41, 2019 01 16.
Article in English | MEDLINE | ID: mdl-30556694

ABSTRACT

The ultimate outcome in genome-editing research stepped into unknown territories last month when two babies were brought into the world with clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) facilitated knockdown of chemokine receptor 5 (CCR5). An immediate outcry by the public and the scientific community followed, which is still ongoing with much apprehensions and criticism of the ethical and scientific aspects of the procedure and its effects on the future of genome editing needed in other stubborn inheritable diseases for which there is no cure at present. With the debate on the consequences of this particular receptor knockdown still going on and the after-shocks in the form of queries expected to continue for some time in the future, we enter the arena of this particular genome editing as first responders with concerns regarding the neurological aftermath of CCR5 knockout in the babies born.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/standards , Genome, Human/genetics , Nervous System Diseases/genetics , Receptors, CCR5/genetics , Gene Editing/legislation & jurisprudence , Gene Knockdown Techniques/adverse effects , Gene Knockdown Techniques/standards , Humans , Nervous System Diseases/diagnosis , Nervous System Diseases/etiology , Receptors, CCR5/deficiency
17.
J Neuroinflammation ; 15(1): 285, 2018 Oct 10.
Article in English | MEDLINE | ID: mdl-30305110

ABSTRACT

BACKGROUND: The collective cognitive and motor deficits known as HIV-associated neurocognitive disorders (HAND) remain high even among HIV+ individuals whose antiretroviral therapy is optimized. HAND is worsened in the context of opiate abuse. The mechanism of exacerbation remains unclear but likely involves chronic immune activation of glial cells resulting from persistent, low-level exposure to the virus and viral proteins. We tested whether signaling through C-C chemokine receptor type 5 (CCR5) contributes to neurotoxic interactions between HIV-1 transactivator of transcription (Tat) and opiates and explored potential mechanisms. METHODS: Neuronal survival was tracked in neuronal and glial co-cultures over 72 h of treatment with HIV-1 Tat ± morphine using cells from CCR5-deficient and wild-type mice exposed to the CCR5 antagonist maraviroc or exogenously-added BDNF (analyzed by repeated measures ANOVA). Intracellular calcium changes in response to Tat ± morphine ± maraviroc were assessed by ratiometric Fura-2 imaging (analyzed by repeated measures ANOVA). Release of brain-derived neurotrophic factor (BDNF) and its precursor proBDNF from CCR5-deficient and wild-type glia was measured by ELISA (analyzed by two-way ANOVA). Levels of CCR5 and µ-opioid receptor (MOR) were measured by immunoblotting (analyzed by Student's t test). RESULTS: HIV-1 Tat induces neurotoxicity, which is greatly exacerbated by morphine in wild-type cultures expressing CCR5. Loss of CCR5 from glia (but not neurons) eliminated neurotoxicity due to Tat and morphine interactions. Unexpectedly, when CCR5 was lost from glia, morphine appeared to entirely protect neurons from Tat-induced toxicity. Maraviroc pre-treatment similarly eliminated neurotoxicity and attenuated neuronal increases in [Ca2+]i caused by Tat ± morphine. proBDNF/BDNF ratios were increased in conditioned media from Tat ± morphine-treated wild-type glia compared to CCR5-deficient glia. Exogenous BDNF treatments mimicked the pro-survival effect of glial CCR5 deficiency against Tat ± morphine. CONCLUSIONS: Our results suggest a critical role for glial CCR5 in mediating neurotoxic effects of HIV-1 Tat and morphine interactions on neurons. A shift in the proBDNF/BDNF ratio that favors neurotrophic support may occur when glial CCR5 signaling is blocked. Some neuroprotection occurred only in the presence of morphine, suggesting that loss of CCR5 may fundamentally change signaling through the MOR in glia.


Subject(s)
Analgesics, Opioid/pharmacology , Gene Expression Regulation/genetics , Neuroglia/metabolism , Opiate Alkaloids/pharmacology , Receptors, CCR5/deficiency , tat Gene Products, Human Immunodeficiency Virus/metabolism , AIDS Dementia Complex , Animals , CCR5 Receptor Antagonists/pharmacology , Corpus Striatum/cytology , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Maraviroc/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Neuroglia/drug effects , Neurons/physiology , Opiate Alkaloids/metabolism , Receptors, CCR5/genetics , Receptors, Opioid, mu/metabolism , tat Gene Products, Human Immunodeficiency Virus/genetics
18.
AIDS ; 32(16): 2269-2278, 2018 10 23.
Article in English | MEDLINE | ID: mdl-30005022

ABSTRACT

OBJECTIVES: This study investigated whether Env-mediated fusion levels of R5X4 viruses are associated with long-term survival of an infected CCR5-/- patient. DESIGN: Four R5X4 Envs were cloned from each of two infected homosexual individuals (DR and C2) homozygous for the CCR5Δ32 allele. DR is a long-term survivor chronically infected with HIV-1 and his Envs were cloned 12 years after testing HIV-infected, whereas C2 Envs were isolated 1 year after primary infection. METHODS: The current study sequenced the gp41 subunits and created hybrid Envs that contained exchanged gp41 subunits or V3 loops. The Env-mediated fusion activity of Envs was examined in cell fusion and virus infection assays. RESULTS: Sequence analysis indicated novel polymorphisms in the gp41 subunits of C2 and DR, and revealed sequence homology between DR and certain long-term nonprogressors. The DR Envs consistently showed lower Env-mediated fusion, smaller size, and delayed onset of syncytia formation. Envs containing swapped gp41 regions resulted in the transfer of most of the fusion phenotype and in the shift of the inhibition concentration 50 (IC50) of the inhibitory T20 peptide. In contrast, Envs with swapped V3 domains resulted in the partial transfer of the fusion phenotype and no significant change in the IC50 of T20. CONCLUSIONS: Env sequence polymorphisms identified two distinct fusion phenotypes isolated from infected CCR5-/- patients. Swapping experiments confirmed DR's low fusion phenotype. Env-mediated fusion is a critical factor among others contributing to long-term survival.


Subject(s)
HIV Infections/pathology , HIV Long-Term Survivors , HIV-1/genetics , HIV-1/isolation & purification , Receptors, CCR5/deficiency , Virus Internalization , HIV Envelope Protein gp41/genetics , HIV Infections/virology , HIV-1/physiology , Humans , Male , Polymorphism, Genetic , Sequence Analysis, DNA
19.
Curr Neurovasc Res ; 14(2): 125-131, 2017.
Article in English | MEDLINE | ID: mdl-28294064

ABSTRACT

BACKGROUND: Stroke is the second leading cause of death and a major cause of disability of adults worldwide. Inflammatory processes are known to contribute to the pathophysiology of cerebral ischemia, especially following reperfusion. Chemokines and their receptors are involved in migration of leukocytes and have been implicated in the pathogenesis of ischemic stroke. OBJECTIVE: In the present study, we investigated the effects of C-C chemokine receptor type 5 (CCR5) deficiency on neurological outcome, brain damage and expression of pro-inflammatory chemokines: chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (CC motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5), and the brain-derived neurotrophic factor (BDNF). METHODS: Adult male C57BL/6 (wild-type) (WT) and CCR5 deficient mice were subjected to transient cerebral ischemia induced by 25 min of bilateral common carotid artery occlusion (BCCAO) followed by 24 hours of reperfusion. Mice were divided into four groups: WT sham group, which underwent sham operation; WT ischemic group, which was subjected to transient bilateral common carotid artery occlusion, CCR5-/- sham group, which underwent sham operation, and CCR5-/- ischemic group, which was subjected to transient BCCAO. RESULTS: In CCR5 deficiency, we observed a significant improvement in the neurological deficits associated with decreased brain infarcted area as evaluated by triphenyltetrazolium chloride (TTC). Moreover, CCR5 deficiency revealed decreased percentage of necrotic cavities areas and frequency of ischemic neurons by histometric analysis. In addition, CCR5-/- ischemic animals showed lower brain levels of the chemokine CXCL1 and higher levels of BDNF by ELISA, compared with WT BCCAo mice. CONCLUSION: Taken together, our results suggest a potential neuroprotection in the absence of CCR5 receptor during global brain ischemia and reperfusion injury.


Subject(s)
Brain Ischemia/metabolism , Brain Ischemia/therapy , Gene Expression Regulation/genetics , Receptors, CCR5/deficiency , Reperfusion Injury/metabolism , Reperfusion Injury/therapy , Animals , Brain/pathology , Brain Ischemia/genetics , Brain-Derived Neurotrophic Factor/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Growth Factors/metabolism , Neurologic Examination , Receptors, CCR5/genetics , Reperfusion Injury/genetics
20.
J Acquir Immune Defic Syndr ; 74(2): 229-241, 2017 Feb 01.
Article in English | MEDLINE | ID: mdl-27749600

ABSTRACT

Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.


Subject(s)
Gene Knockout Techniques/methods , HIV-1/physiology , Receptors, CCR5/deficiency , Receptors, HIV/deficiency , Transcription Activator-Like Effector Nucleases/metabolism , Adult , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , HIV-1/growth & development , Humans , Sequence Deletion
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