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SUMMARYThe December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre-symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved ∼100 copies/μL sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.

The evolutionarily conserved SIRT1–FoxO1 axis plays a new role in human CD8+ T cell metabolism and function. Progression from the naive to the terminally differentiated memory state is accompanied by the loss of SIRT1 and FoxO1 expression, which derepresses glycolytic and cytotoxic capacities of CD8+CD28– T cells under resting conditions., The expansion of CD8+CD28– T cells, a population of terminally differentiated memory T cells, is one of the most consistent immunological changes in humans during aging. CD8+CD28– T cells are highly cytotoxic, and their frequency is linked to many age-related diseases. As they do not accumulate in mice, many of the molecular mechanisms regulating their fate and function remain unclear. In this paper, we find that human CD8+CD28– T cells, under resting conditions, have an enhanced capacity to use glycolysis, a function linked to decreased expression of the NAD+-dependent protein deacetylase SIRT1. Global gene expression profiling identified the transcription factor FoxO1 as a SIRT1 target involved in transcriptional reprogramming of CD8+CD28– T cells. FoxO1 is proteasomally degraded in SIRT1-deficient CD8+CD28– T cells, and inhibiting its activity in resting CD8+CD28+ T cells enhanced glycolytic capacity and granzyme B production as in CD8+CD28– T cells. These data identify the evolutionarily conserved SIRT1–FoxO1 axis as a regulator of resting CD8+ memory T cell metabolism and activity in humans.

HSCs are BM-derived, self-renewing multipotent cells that develop into circulating blood cells. They have been implicated in the repair of inflamed parenchymal tissue, but the signals that regulate their trafficking to sites of inflammation are unknown. As monocytes are recruited to sites of inflammation via chemoattractants that activate CCR2 on their surface, we investigated whether HSCs are also recruited to sites of inflammation through CCR2. Initial analysis indicated that in mice, CCR2 was expressed on subsets of HSCs and hematopoietic progenitor cells (HPCs) and that freshly isolated primitive hematopoietic cells (Lin–c-Kit+ cells) responded to CCR2 ligands in vitro. In vivo analysis indicated that after instillation of thioglycollate to cause aseptic inflammation and after administration of acetaminophen to induce liver damage, endogenous HSCs/HPCs were actively recruited to the peritoneum and liver, respectively, in WT but not Ccr2–/– mice. HSCs/HPCs recovered from the peritoneum successfully engrafted into the BM of irradiated primary and secondary recipients, confirming their self renewal and multipotency. Importantly, administration of exogenous WT, but not Ccr2–/–, HSCs/HPCs accelerated resolution of acetaminophen-induced liver damage and triggered the expression of genes characteristic of the macrophage M2 or repair phenotype. These findings reveal what we believe to be a novel role for CCR2 in the homing of HSCs/HPCs to sites of inflammation and suggest new functions for chemokines in promoting tissue repair and regeneration.

Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.

Two forms of the monocyte chemoattractant protein-1 receptors (the type A monocyte chemoattractant protein 1 (MCP-1) receptor CCR-2A and the type B MCP-1 receptor (CCR-2B) have been recently cloned and found to differ only in their terminal carboxyl tails. Here, we report that the two isoforms are alternatively spliced variants of a single MCP-1 receptor gene. Sequencing of the gene revealed that the 47-amino acid carboxyl tail of CCR2B was located in the same exon as the seven transmembrane domains of the receptor, and the 61-amino acid tail of CCR2A was in a downstream exon. Examination of freshly isolated human monocytes by reverse transcriptase-polymerase chain reaction revealed that CCR2B was the predominant isoform and that message levels of both CCR2A and CCR2B decreased as the monocytes differentiated into macrophages. In stably transfected cell lines, CCR2B trafficked well to the cell surface, but CCR2A was found predominately in the cytoplasm. Equilibrium binding studies revealed that those CCR2A receptors that successfully trafficked to the cell surface bound MCP-1 with high affinity (Kd = 310 pM), similar to CCR2B. In signaling studies, both CCR2A and CCR2B mediated agonist-dependent calcium mobilization, as well as inhibition of adenylyl cyclase. Creation of chimeras between CCR2A and the human thrombin receptor revealed that the cytoplasmic retention of CCR2A was due to its terminal carboxyl tail. Progressive truncation of the carboxyl tail indicated that a cytoplasmic retention signal(s) was located between residues 316 and 349. These data indicate that the alternatively spliced form of the human MCP-1 receptor (CCR2A) binds MCP-1 with high affinity and is a functional receptor and that expression at the cell surface is controlled by amino acid sequences located in the terminal carboxyl tail.

Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene ( db/ db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web., Author Summary Hepatitis C Virus (HCV) belongs to the Flaviviridae family, and is an enveloped, positive, single-stranded RNA virus containing a 9.6 kb genome. Plus-strand RNA viruses induce a highly regulated process of membrane rearrangements and novel vesicle formation in infected cells (the “membranous web” for HCV) to create a suitable environment for RNA replication as well as for the assembly and release of new virions. HCV assembly occurs close to lipid droplets (LDs), cell organelles involved in fat storage and utilization. The viral non-structural protein NS5A plays a critical role in both viral RNA replication that occurs within the membranous web and viral assembly at LDs. We identified the host protein TIP47 as a novel host interaction partner for NS5A. TIP47 is a LD-binding protein also known to function as cargo in late-endosome-to-Golgi vesicular transport. Our data support a model where the recruitment of TIP47 by NS5A is required for viral RNA replication, indicating that LDs play a previously unrecognized role not only in viral assembly but also in RNA replication.

The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.

The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4+ T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4+ T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2–long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.

During human pregnancy, the specialized epithelial cells of the placenta (cytotrophoblasts) come into direct contact with immune cells in several locations. In the fetal compartment of the placenta, cytotrophoblast stem cells lie adjacent to macrophages (Hofbauer cells) that reside within the chorionic villus stroma. At sites of placental attachment to the mother, invasive cytotrophoblasts encounter specialized maternal natural killer (NK) cells (CD56(bright)), macrophages, and T cells that accumulate within the uterine wall during pregnancy. Here we tested the hypothesis that fetal cytotrophoblasts can direct the migration of these maternal immune cells. First, we assayed the chemotactic activity of cytotrophoblast conditioned medium samples, using human peripheral blood mononuclear cells as targets. The placental samples preferentially attracted NK cells (both CD56(dim) and CD56(bright)), monocytes, and T cells, suggesting that our hypothesis was correct. A screen to identify chemokine activity through the induction of a Ca(2)+ flux in cells transfected with individual chemokine receptors suggested that cytotrophoblasts secreted monocyte inflammatory protein (MIP)-1alpha. This was confirmed by localizing the corresponding mRNA and protein, both in vitro and in vivo. MIP-1alpha protein in conditioned medium was further characterized by immunoblotting and enzyme-linked immunosorbent assay. Immunodepletion of MIP-1alpha from cytotrophoblast conditioned medium showed that this chemokine was responsible for a significant portion of the induced monocyte and CD56(bright) NK cell chemotaxis. These data suggest the specific conclusion that cytotrophoblasts can attract monocytes and CD56(bright) NK cells by producing MIP-1alpha and the more general hypothesis that these cells may organize and act on leukocytes at the maternal-fetal interface.

Abstract 2547 Poster Board II-524 Hematopoietic stem cells (HSCs) are bone-marrow derived, self-renewing pluripotent cells that give rise to terminally differentiated circulating blood cells. Recent work has implicated HSCs in the repair of parenchymal tissue in the setting of inflammation. In response to the antagonist of the chemokine receptor CXCR4, HSCs and their progenitors migrate from bone marrow to the blood. However, little is known about the signals that mediate their trafficking from the blood into peripheral tissues. Recently, we showed that mice genetically deficient in chemokine receptor CCR2 (CCR2–/– mice) have a marked decrease in the number of circulating “inflammatory” (7/4+, Ly6c+) monocytes, but no decrease in myeloid progenitor cells in the bone marrow (Tsou et al, J Clin Invest, 2007, 902). These data indicated that although CCR2 is not necessary for HSCs to differentiate into mature monocytes, it does play a role in monocyte egress from bone marrow to blood. In the current study, we extend this work and investigate the expression of CCR2 on hematopoietic stem cells and progenitor cells (HSPCs), and tested the hypothesis that CCR2 is critical for the recruitment of circulating HSPCs to sites of inflammation. Here we report that the chemokine receptor CCR2 is expressed on subsets of primitive hematopoietic stem cells as well as some early myeloid progenitors but not on the progenitors dedicated to megakaryocyte and erythroid differentiation. CCR2 mediates the chemotaxis of c-Kit+Lin- bone marrow derived cells to MCP-1 (CCL2) and MCP-3 (CCL7), which are known CCR2 ligands. In unchallenged mice, CCR2 appears to play a minimal role in HSCs trafficking. However, following instillation of thioglycollate to cause aseptic inflammation HSPCs were actively recruited to the peritoneum, as demonstrated by both FACS and functional colony formation assays. That these cells were true HSCs was demonstrated by their engraftment into the bone marrow of irradiated recipients. In contrast, mice genetically deficient in CCR2 (CCR2−/−) were profoundly impaired in the recruitment of HSCs to the inflamed peritoneum. Furthermore, in human disease models of acetaminophen induced liver damages, hematopoietic stem and progenitors were increased dramatically at site of injury in WT mice but not in CCR2−/− mice. CCR2 recruited HSPC play a tissue protective role by increase the numbers of M2 macrophages in injured liver. Taken together, these findings document a novel role for CCR2 in the specific homing of primitive hematopoietic stem and progenitors to sites of inflammation and injury, and suggest a new role for chemokines in increasing the numbers of differentiated leukocytes at sites of inflammation. Recruited HSPCs hasten the resolution of the inflammatory response, and promote repair of injured tissue, at least partially due to locally increase the number of tissue reparative macrophages. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) are bone-marrow derived, self-renewing pluripotent cells that give rise to terminally differentiated circulating blood cells. HSCs have been implicated in parenchymal tissue repair in the setting of inflammation. In response to the antagonist of the chemokine receptor CXCR4, HSCs and their progenitors migrate from bone marrow to the blood. However, little is known about the signals that mediate their trafficking from the blood into peripheral tissues. Recently, we showed that mice genetically deficient in chemokine receptor CCR2 (CCR2−/− mice) have a marked decrease in the number of circulating “inflammatory” (7/4+, Ly6c+) monocytes, but no decrease in myeloid progenitor cells in the bone marrow (Tsou et al, J Clin Invest, 2007, 902). These data indicated that although CCR2 is not necessary for HSCs to differentiate into mature monocytes, it does play a role in monocyte egress from bone marrow to blood. In the current study, we extend this work and investigate the expression of CCR2 on HSCs, and tested the hypothesis that CCR2 is critical for the recruitment of circulating HSCs to sites of inflammation. We found that CCR2 was expressed on subsets of primitive HSCs and myeloid progenitors and mediated HSC movement in response to inflammation. Using traditional transwell chambers, we found that c-Kit+Lin− cells derived from bone marrow underwent chemotaxis in response to the CCR2 ligands MCP-1 (CCL2) and MCP- 3 (CCL7). To determine whether CCR2 mediates HSC movement in vivo, we treated wildtype mice with thioglycollate to induce aseptic inflammation. HSCs were actively recruited to the peritoneum, as shown by fluorescence-activated cell sorting and functional colony formation assays. In contrast, this response was profoundly impaired in CCR2−/− mice. To determine whether the clonogenic cells recruited to peritoneum were true HSCs, we performed competitive transplantation assays. Thioglycollate was instilled into wildtype CD45.2+ mice, and peritoneal Lin− cells were collected, purified, and infused, together with CD45.1+ bone marrow cells, into lethally irradiated CD45.1+ mice. Four months later, up to 12% of the leukocytes in the peripheral blood of these primary recipient mice were CD45.2+. At the time of sacrifice, bone marrow cells were collected from these mice and injected into lethally irradiated secondary CD45.1+ recipient mice. Two months following the transplantation, up to 9% of the blood leukocytes in these secondary recipient mice were CD45.2+, confirming that long-term repopulating HSCs were recruited to the inflamed peritoneum of the donor mice. These findings suggest a novel role for CCR2 in the recruitment of long-term repopulating HSCs to sites of inflammation and injury. We are currently investigating whether recruited HSCs and their progenitors hasten the resolution of the inflammatory response or promote the repair of injured tissue.