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The hepatitis C virus (HCV) is a single-strand RNA virus that infects millions of people worldwide. Recent advances in therapy have led to viral cure using two- and three- drug combinations of direct acting inhibitors of viral replication. CCR5 is a chemokine receptor that is expressed on hepatocytes and represents a key co-receptor for HIV. We evaluated the effect of CCR5 blockade or knockdown on HCV replication in Huh7.5JFH1 cells. Cells were exposed to varying concentrations of maraviroc (CCR5 inhibitor), cenicriviroc (CCR2/CCR5 inhibitor), sofosbuvir (nucleotide polymerase inhibitor), or raltegravir (HIV integrase inhibitor). HCV RNA was detected utilizing two qualitative strand-specific RT-PCR assays. HCV core antigen and NS3 protein was quantified in the supernatant and cell lysate, respectively. siRNA was utilized to knockdown CCR5 gene expression in hepatocytes. Alternatively, anti-CCR5 antibodies were employed to block the receptor. Supernatant levels of HCV RNA (expressed as fold change) were not reduced in the presence of raltegravir but were reduced 8.55-fold and 12.42-fold with cenicriviroc and maraviroc, respectively. Sofosbuvir resulted in a 16.20-fold change in HCV RNA levels. HCV core and NS3 protein production was also reduced in a dose-dependent manner. Two distinct anti-CCR5 antibodies also resulted in a significant reduction in HCV protein expression, as did siRNA knockdown of CCR5 gene expression. These data provide evidence that CCR5 modulation could have a significant effect on HCV replication in an in vitro system. Further evaluation of the role of CCR5 inhibition in clinical settings may be warranted.

Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood–brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions. Although it is known that microglia respond to injury and systemic disease in the brain, it is unclear if they modulate blood–brain barrier (BBB) integrity, which is critical for regulating neuroinflammatory responses. Here authors demonstrate that microglia respond to inflammation by migrating towards and accumulating around cerebral vessels, where they initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 before switching, during sustained inflammation, to phagocytically remove astrocytic end-feet resulting in impaired BBB function

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago 1 , 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. An adult infected with HIV-1 who underwent allogeneic haematopoietic stem-cell transplantation for Hodgkin’s lymphoma using cells from a CCR5Δ32/Δ32 donor achieved full remission of HIV-1 for 18 months after transplantation and 16 months after cessation of antiretroviral therapy.

HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160) 3 cleaved to (gp120 and gp41) 3 , interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120–coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents. The cryo-electron microscopy structure of the gp120 component of the HIV-1 envelope glycoprotein, in complex with the primary receptor CD4 and coreceptor CCR5, provides insight into the cell-entry mechanism of HIV-1.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals 1 . Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages ‘dwell’ within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease. Specific macrophage populations provide a transient niche that activates muscle stem cells after muscle injury and supply proliferation-inducing cues that govern the repair process mediated by these cells in both zebrafish and mouse injury models.

ObjectivesWhile various monocyte chemokine systems are increased in expression in osteoarthritis (OA), the hierarchy of chemokines and chemokine receptors in mediating monocyte/macrophage recruitment to the OA joint remains poorly defined. Here, we investigated the relative contributions of the CCL2/CCR2 versus CCL5/CCR5 chemokine axes in OA pathogenesis.MethodsCcl2-, Ccr2-, Ccl5- and Ccr5-deficient and control mice were subjected to destabilisation of medial meniscus surgery to induce OA. The pharmacological utility of blocking CCL2/CCR2 signalling in mouse OA was investigated using bindarit, a CCL2 synthesis inhibitor, and RS-504393, a CCR2 antagonist. Levels of monocyte chemoattractants in synovial tissues and fluids from patients with joint injuries without OA and those with established OA were investigated using a combination of microarray analyses, multiplexed cytokine assays and immunostains.ResultsMice lacking CCL2 or CCR2, but not CCL5 or CCR5, were protected against OA with a concomitant reduction in local monocyte/macrophage numbers in their joints. In synovial fluids from patients with OA, levels of CCR2 ligands (CCL2, CCL7 and CCL8) but not CCR5 ligands (CCL3, CCL4 and CCL5) were elevated. We found that CCR2+ cells are abundant in human OA synovium and that CCR2+ macrophages line, invade and are associated with the erosion of OA cartilage. Further, blockade of CCL2/CCR2 signalling markedly attenuated macrophage accumulation, synovitis and cartilage damage in mouse OA.ConclusionsOur findings demonstrate that monocytes recruited via CCL2/CCR2, rather than by CCL5/CCR5, propagate inflammation and tissue damage in OA. Selective targeting of the CCL2/CCR2 system represents a promising therapeutic approach for OA.

The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of G i1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation. The chemokine receptor CCR5 plays multiple roles in the immune system. Here, structures of G i1 protein-coupled CCR5 with or without a chemokine bound and of the CCR5- chemokine MIP-1 α complex offer insight into the distinct binding modes of the ligands and into the mechanism of CCR5 activation.

Real-world memories are formed in a particular context and are often not acquired or recalled in isolation 1 – 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 not 1 – 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 infection 6 , 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 Cc r5 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. A molecular mechanism involving CCR5 and CCL5 determines the temporal window in which a memory can be linked with subsequent memories, and in aged mice an increase in CCR5 is associated with defects in memory linking.

T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8 + T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for G αi -coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8 + effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8 + effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy. Tumours secrete many lymphocyte-attracting chemokines. Here the authors show that despite the abundance of their ligands, CCR2 and CCR5 do not mediate trafficking of effector CD8 T cells into the tumour, whereas CXCR3 is essential for this process and for T-cell-based elimination of melanoma in mice.

The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom-resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor—gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.