Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells.

The retrovirus restriction factor SAMHD1 is the first identified mammalian dNTP triphosphohydrolase that is highly expressed in human myeloid lineage cells and CD4(+) T lymphocytes. Although SAMHD1 expression is variable in human cell lines and tissue types, mechanisms underlying SAMHD1 gene regulation have not been defined. Recent studies showed that SAMHD1 is highly expressed in human primary CD4(+) T lymphocytes, but not in some CD4(+) T cell lines. Here, we report that SAMHD1 expression varies among four CD4(+) T cell lines and is transcriptionally regulated. Cloning and sequence analysis of the human SAMHD1 promoter revealed a CpG island that is methylated in CD4(+) T cell lines (such as Jurkat and Sup-T1), resulting in transcriptional repression of SAMHD1. We also found that the SAMHD1 promoter is unmethylated in primary CD4(+) T lymphocytes, which express high levels of SAMHD1, indicating a direct correlation between the methylation of the SAMHD1 promoter and transcriptional repression. SAMHD1 expression was induced in CD4(+) T cell lines by blocking DNA methyltransferase activity, suggesting that promoter methylation is one of the key epigenetic mechanisms by which SAMHD1 expression is regulated.

SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+ T-lymphocytes cannot be upregulated by interferons.

BACKGROUND: SAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs), macrophages, and resting CD4+ T-cells. Acting as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4+ T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN)-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4+ T-lymphocytes is unknown. RESULTS: Here, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4+ T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool. CONCLUSIONS: Our results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4+ T-lymphocytes is not upregulated by type I IFNs.

Azithromycin alters macrophage phenotype and pulmonary compartmentalization during lung infection with Pseudomonas.

Infection with mucoid strains of Pseudomonas aeruginosa in chronic inflammatory diseases of the airway is difficult to eradicate and can cause excessive inflammation. The roles of alternatively activated and regulatory subsets of macrophages in this pathophysiological process are not well characterized. We previously demonstrated that azithromycin induces an alternatively activated macrophage-like phenotype in vitro. In the present study, we tested whether azithromycin affects the macrophage activation status and migration in the lungs of P. aeruginosa-infected mice. C57BL/6 mice received daily doses of oral azithromycin and were infected intratracheally with a mucoid strain of P. aeruginosa. The properties of macrophage activation, immune cell infiltration, and markers of pulmonary inflammation in the lung interstitial and alveolar compartments were evaluated postinfection. Markers of alternative macrophage activation were induced by azithromycin treatment, including the surface expression of the mannose receptor, the upregulation of arginase 1, and a decrease in the production of proinflammatory cytokines. Additionally, azithromycin increased the number of CD11b(+) monocytes and CD4(+) T cells that infiltrated the alveolar compartment. A predominant subset of CD11b(+) cells was Gr-1 positive (Gr-1(+)), indicative of a subset of cells that has been shown to be immunoregulatory. These differences corresponded to decreases in neutrophil influx into the lung parenchyma and alteration of the characteristics of peribronchiolar inflammation without any change in the clearance of the organism. These results suggest that the immunomodulatory effects of azithromycin are associated with the induction of alternative and regulatory macrophage activation characteristics and alteration of cellular compartmentalization during infection.

Heterogeneity of horizontal cells in the chicken retina.

Despite numerous reports that different markers are expressed by horizontal cells in the avian retina, it remains unknown whether different types of horizontal cells can be defined by differences in their immunocytochemical profiles. The purpose of this study was to rectify this deficiency. We identified horizontal cells by indirect immunofluorescence with antibodies to calretinin, trkA, GABA, Prox1, AP2alpha, Pax6, islet1, and Lim1 + 2. We found two major groups of horizontal cells, those that express trkA and those that express calretinin. The trkA-immunoreactive (-IR) horizontal cells had small, round somata and robust, bulbous dendritic endings, whereas calretinin-IR horizontal cells had large, polygonal cell bodies and fine, diffuse dendritic endings, both contacting the calbindin-IR pedicles of double cones. Weak gamma-aminobutyric acid (GABA) immunoreactivity was observed only in a few of the trkA-IR horizontal cells, whereas the overlap of calretinin and GABA immunoreactivities was 100%. The majority of trkA-IR horizontal cells expressed islet1, and the majority of calretinin-IR horizontal cells expressed Lim1 + 2, AP2alpha, and Pax6. Islet1 immunoreactivity was observed in a small fraction of calretinin-IR/non-trkA-IR cells. In agreement with previous reports, we detected Prox1 immunoreactivity in all types of horizontal cells. These immunolabeling profiles suggest that there are four immunochemically distinct subtypes of horizontal cells in the postnatal chick retina, which may match the four types that have been observed in Golgi-impregnated pigeon and turtle retinas.

Covert signal disruption: anti-ecdysteroidal activity of bisphenol A involves cross talk between signaling pathways.

Bisphenol A is a key industrial chemical used in the manufacture of polycarbonate plastics and other products. Several recent reports ascribe toxicological properties to this compound that have been attributed to the disruption of endocrine-related processes. In the present study, the toxicity of bisphenol A was definitively characterized in the water flea (Daphnia magna) in an effort to discern whether this compound may elicit endocrine toxicity in an invertebrate species and to establish the mechanism by which this toxicity is elicited. The ability of bisphenol A to interfere with two ecdysteroid-dependent physiological processes--molting and embryonic development--was evaluated. Bisphenol A elicited antiecdysteroidal activity as indicated by its prolongation of the intermolt period and interference with embryonic development. This apparent antiecdysteroidal activity was not due to reduced availability of endogenous ecdysteroid nor due to ecdysteroid-receptor antagonism. The ability of bisphenol A to elicit antiecdysteroidal activity by functioning as a juvenoid hormone was next evaluated. Bisphenol A, alone, did not elicit juvenoid activity. However, bisphenol A did enhance the activity of the crustacean juvenoid hormone methyl farnesoate. A definitive assessment of the effects of bisphenol A on the reproductive capacity of daphnids revealed a concentration-response relationship that extended at least one order of magnitude below exposure levels that were overtly toxic to the maternal organisms. These results demonstrate that bisphenol A is chronically toxic to daphnids, probably through its ability to interfere with ecdysteroid/juvenoid regulated processes. However, effects are elicited at levels that are not likely to pose environmental concern.

Characterization of macrophage activation states in patients with cystic fibrosis.

BACKGROUND: Chronic airway inflammation characterizes patients with cystic fibrosis (CF). The role of alternative macrophage activation in this disease course is unknown. OBJECTIVE: We evaluated markers of alternative and classical macrophage activation in the lungs of patients with CF and evaluated these characteristics in the context of Pseudomonas aeruginosa (PA) infection, immunomodulatory drug therapy and pulmonary function. METHODS: Bronchoalveolar lavage or spontaneously expectorated sputum samples were collected from 48 CF patients. Clinical data were related to macrophage surface expression of mannose receptor (MR) (up-regulated in alternatively activated macrophages) and TLR4 (up-regulated in classically activated macrophages). Also, the activity of the alternatively activated macrophage effector molecule arginase was compared among patient groups, and pro- and anti-inflammatory cytokines produced by alternatively and classically activated macrophages were measured. RESULTS: There were significant differences between PA-infected and -uninfected patients in several clinical measurements. PA-infected patients exhibited increased use of azithromycin, up-regulation of MR on CD11b+ cells and increased arginase activity in their lung samples, and had a strong inverse relationship between MR and arginase activity to FEV(1). Upon further analysis, PA-infected patients who were treated with azithromycin had the highest arginase activity and the highest number of macrophages that were MR+TLR4-, and both of these markers were inversely related to the FEV(1). CONCLUSIONS: Our findings suggest an increase in both MR and arginase expression as pulmonary function declines in PA-infected patients with CF. These markers of an alternatively activated macrophage phenotype give cause for future study to define the function of macrophage activation states in the CF lung.