Mechanical stretch induces hair regeneration through the alternative activation of macrophages.

Tissues and cells in organism are continuously exposed to complex mechanical cues from the environment. Mechanical stimulations affect cell proliferation, differentiation, and migration, as well as determining tissue homeostasis and repair. By using a specially designed skin-stretching device, we discover that hair stem cells proliferate in response to stretch and hair regeneration occurs only when applying proper strain for an appropriate duration. A counterbalance between WNT and BMP-2 and the subsequent two-step mechanism are identified through molecular and genetic analyses. Macrophages are first recruited by chemokines produced by stretch and polarized to M2 phenotype. Growth factors such as HGF and IGF-1, released by M2 macrophages, then activate stem cells and facilitate hair regeneration. A hierarchical control system is revealed, from mechanical and chemical signals to cell behaviors and tissue responses, elucidating avenues of regenerative medicine and disease control by demonstrating the potential to manipulate cellular processes through simple mechanical stimulation.

RNA editing enzyme adenosine deaminase is a restriction factor for controlling measles virus replication that also is required for embryogenesis.

Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses.

Functional glycosylation of dystroglycan is crucial for thymocyte development in the mouse.

BACKGROUND: Alpha-dystroglycan (alpha-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, alpha-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins. METHODOLOGY/PRINCIPAL FINDINGS: We report that expression of functionally glycosylated alpha-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4(-)CD8(-) double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4(+)CD8(+) double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.

In vivo conversion of BM plasmacytoid DC into CD11b+ conventional DC during virus infection.

DC are a highly heterogeneous population that plays a critical role in host defense. We previously demonstrated that virus infection induces BM plasmacytoid DC (pDC) differentiation into CD11b(+) conventional DC (cDC) upon in vitro culture with Fms-like tyrosine kinase 3 ligand (Flt3L). Here we use immunoglobulin D-J rearrangements and pDC adoptive transfer to provide definitive proof supporting BM pDC conversion into CD11b(+) cDC during in vivo viral infection. We show that in vivo BM pDC conversion into CD11b(+) cDC relates to enhanced ability to prime virus-specific T cells. Furthermore, we demonstrate that in vivo pDC conversion does not rely on viral infection of BM pDC, but instead is mediated by type I IFN signaling. Finally, by exploiting recently identified pDC-specific Ab, we provide further characterizations of the BM pDC fraction that exhibits this broader developmental plasticity. Collectively, these data indicate that BM pDC actively contribute to the CD11b(+) cDC pool during in vivo viral infection and delineates molecular, functional, and phenotypic features of this novel developmental pathway.

Persistent virus infection inhibits type I interferon production by plasmacytoid dendritic cells to facilitate opportunistic infections.

Emerging studies indicate an association between virus-induced impairment in type I interferon (IFN-I) production and enhanced susceptibility to opportunistic infections, which represent a major health problem. Here, we provide in vivo evidence that lymphocytic choriomeningitis virus (LCMV) infection of its natural murine host dramatically diminishes the unique capacity of plasmacytoid dendritic cells (pDCs) to secrete high levels of systemic IFN-I. While both acute and persistent LCMV infections suppress pDC IFN-I response, only the persistent virus induces a long-lasting diversion of this innate immune pathway. The consequent reduction in IFN-I production serves to impair natural killer cell responses in LCMV-infected mice challenged subsequently with murine cytomegalovirus (MCMV) as an opportunistic pathogen. This innate defect also compromises the host's ability to counteract early MCMV spread. These findings provide a mechanistic explanation for the occurrence of opportunistic infections following viral insults and have important implications for treating such medical complications.

Cyclin T1 but not cyclin T2a is induced by a post-transcriptional mechanism in PAMP-activated monocyte-derived macrophages.

Positive transcription elongation factor b (P-TEFb) is an RNA polymerase II elongation factor which exists as multiple complexes in human cells. These complexes contain cyclin-dependent kinase 9 as the catalytic subunit and different cyclin subunits-cyclin T1, T2a, T2b, or K. Cyclin T1 is targeted by the human immunodeficiency virus (HIV) Tat protein to activate transcription of the HIV provirus. Expression of this P-TEFb subunit is highly regulated in monocyte-derived macrophages (MDMs). Cyclin T1 is induced early during differentiation and is shut off later by proteasome-mediated proteolysis. Cyclin T1 can be reinduced by pathogen-associated molecular patterns (PAMPs) or HIV infection. In this study, we analyzed regulation of P-TEFb in MDMs by examining 7SK small nuclear RNA and the HEXIM1 protein; these factors associate with P-TEFb and are thought to regulate its function. 7SK and HEXIM1 were induced early during differentiation, and this correlates with increased overall transcription. 7SK expression remained high, but HEXIM1 was shut off later during differentiation by proteasome-mediated proteolysis. Significantly, the cyclin T2a subunit of P-TEFb was not shut off during differentiation, and it was not induced by activation. Induction of cyclin T1 by PAMPs was found to be a slow process and did not involve an increase in cyclin T1 mRNA levels. Treatment of MDMs with PAMPs or a proteasome inhibitor induced cyclin T1 to a level equivalent to treatment with both agents together, suggesting that PAMPs and proteasome inhibitors act at a similar rate-limiting step. It is therefore likely that cyclin T1 induction by PAMPs is the result of a reduction in proteasome-mediated proteolysis.

Human immunodeficiency virus type 1 infection induces cyclin T1 expression in macrophages.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication and activates RNA polymerase II transcriptional elongation through the association with a cellular protein kinase composed of Cdk9 and cyclin T1. Tat binds to this kinase complex through a direct protein-protein interaction with cyclin T1. Monocytes/macrophages are important targets of HIV-1 infection, and previous work has shown that cyclin T1 but not Cdk9 protein expression is low in monocytes isolated from blood. While Cdk9 expression is expressed at a high level during monocyte differentiation to macrophages in vitro, cyclin T1 expression is induced during the first few days of differentiation and is shut off after 1 to 2 weeks. We show here that the shutoff of cyclin T1 expression in late-differentiated macrophages involves proteasome-mediated proteolysis. We also show that cyclin T1 can be reinduced by a number of pathogen-associated molecular patterns that activate macrophages, indicating that up-regulation of cyclin T1 is part of an innate immune response. Furthermore, we found that HIV-1 infection early in macrophage differentiation results in sustained cyclin T1 expression, while infection at late times in differentiation results in the reinduction of cyclin T1. Expression of the viral Nef protein from an adenovirus vector suggests that Nef contributes to the HIV-1 induction of cyclin T1. These findings suggest that HIV-1 infection hijacks a component of the innate immune response in macrophages that results in enhancement rather than inhibition of viral replication.

HIV-1 infection and regulation of Tat function in macrophages.

The macrophage is an important cell type in the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages both support viral replication and are capable of attracting and activating lymphocytes, thus rendering CD4+ T lymphocytes highly permissive for infection. The viral Tat protein, whose function is mediated by the cellular cyclin T1 protein complexed with CDK9, is required for efficient transcription of the integrated HIV-1 provirus by RNA polymerase II. Cyclin T1 expression is highly regulated during macrophage differentiation, and this has important implications for HIV-1 replication. In monocytes isolated from healthy blood donors, cyclin T1 protein expression is low and is induced to high levels within the first few days of differentiation by a post-transcriptional mechanism. After 1-2 weeks of macrophage differentiation, however, cyclin T1 expression is shut off. Treatment of macrophages with lipopolysaccharide (LPS) can re-induce cyclin T1, indicating that the activation status of macrophages can regulate cyclin T1 expression. Recent results indicate that HIV-1 infection is able to induce cyclin T1 expression in macrophages. Future studies of cyclin T1 regulation in macrophages may suggest means of manipulating expression of this crucial cellular co-factor for therapeutic benefit in HIV-1 infected individuals.

Isolation and characterization of the human DC-SIGN and DC-SIGNR promoters.

DC-SIGN is a C-type lectin expressed on the surface of dendritic cells (DCs) that is used by a number of human pathogens to disseminate infection in the host. In the human genome, there is a gene closely related to DC-SIGN, termed DC-SIGNR (also L-SIGN, DC-SIGN2), which likely arose through gene duplication. DC-SIGN protein and RNA expression is largely restricted to DCs and some specialized macrophages in lung and placenta, while DC-SIGNR expression is largely restricted to lymph nodes and liver sinusoidal endothelial cells. To begin to investigate the cell type-restricted expression of these closely related genes, we isolated the human DC-SIGN and DC-SIGNR promoters. They were found to be relatively weak promoters that express similarly in plasmid transfection assays in several transformed cell lines, suggesting that the cis-regulatory elements that confer cell-type restricted expression of these two genes are located outside of the promoters. The DC-SIGN gene contains four major transcriptional start sites at +1, +271, +364, and +435, with the +364 site being the most abundantly expressed in DCs. The DC-SIGN promoter is contained within nucleotides +251 to +487. AP-1, Sp1, Ets-1, and NF-kappaB binding sites in the DC-SIGN promoter appear to be important for function. Thus, despite its highly restricted pattern of expression, the DC-SIGN promoter has features common to promoters that are active in other cell types.

Transient induction of cyclin T1 during human macrophage differentiation regulates human immunodeficiency virus type 1 Tat transactivation function.

The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.