Stromal Down-Regulation of Macrophage CD4/CCR5 Expression and NF-κB Activation Mediates HIV-1 Non-Permissiveness in Intestinal Macrophages

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation

Processing of polycistronic guide RNAs is associated with RNA editing complexes in Trypanosoma brucei

In kinetoplastid mitochondrial mRNA editing, post-transcriptional insertion or deletion of uridines is templated by guide RNAs (gRNAs). Pre-mRNAs are encoded by maxicircles, while gRNAs are encoded by both maxicircles and minicircles. We have investigated minicircle transcription and the processing of gRNAs in Trypanosoma brucei. We find that minicircles are transcribed polycistronically and that transcripts are accurately processed by an ∼19S complex. This gRNA processing activity co-purifies with RNA editing complexes, and both remain associated in 19S complexes. Furthermore, we show that RNA editing complexes associate preferentially with a polycistronic gRNA over non-processed RNAs. We propose that the ∼19S complexes initially described as RNA editing complex I are gRNA processing complexes that cleave polycistronic gRNA transcripts into monocistrons

Increased trabecular bone and improved biomechanics in an osteocalcin-null rat model created by CRISPR/Cas9 technology

Osteocalcin, also known as bone γ-carboxyglutamate protein (Bglap), is expressed by osteoblasts and is commonly used as a clinical marker of bone turnover. A mouse model of osteocalcin deficiency has implicated osteocalcin as a mediator of changes to the skeleton, endocrine system, reproductive organs and central nervous system. However, differences between mouse and human osteocalcin at both the genome and protein levels have challenged the validity of extrapolating findings from the osteocalcin-deficient mouse model to human disease. The rat osteocalcin (Bglap) gene locus shares greater synteny with that of humans. To further examine the role of osteocalcin in disease, we created a rat model with complete loss of osteocalcin using the CRISPR/Cas9 system. Rat osteocalcin was modified by injection of CRISPR/Cas9 mRNA into the pronuclei of fertilized single cell Sprague-Dawley embryos, and animals were bred to homozygosity and compound heterozygosity for the mutant alleles. Dual-energy X-ray absorptiometry (DXA), glucose tolerance testing (GTT), insulin tolerance testing (ITT), microcomputed tomography (µCT), and a three-point break biomechanical assay were performed on the excised femurs at 5 months of age. Complete loss of osteocalcin resulted in bones with significantly increased trabecular thickness, density and volume. Cortical bone volume and density were not increased in null animals. The bones had improved functional quality as evidenced by an increase in failure load during the biomechanical stress assay. Differences in glucose homeostasis were observed between groups, but there were no differences in body weight or composition. This rat model of complete loss of osteocalcin provides a platform for further understanding the role of osteocalcin in disease, and it is a novel model of increased bone formation with potential utility in osteoporosis and osteoarthritis research

Down-regulation of NF-κB by S-CM correlates with down-regulation in the cells' ability to support HIV-1 replication.

<p>(<b>A</b>) Monocytes were cultured in media plus M-CSF and then inoculated in triplicate with R5 HIV-1 (NA353 B27; MOI = 1) plus S-CM at the indicated concentration for 2 hours. Cells were evaluated for NF-κB p65 translocation by confocal microscopy and NF-κB intensity by IPLab image analysis software after 2 hours and for viral replication by p24 ELISA on day 12 (n = 3 donors). Histograms are representative of a single experiment and show distribution of NF-κB (green line) in relation to the nucleus (blue line). The p24 value of each treatment was normalized to the media control group with the replication level of media control group defined as 100%. Data shown are the means of relative p24 levels from independent experiments with 3 donors. (<b>B</b>) Anti-TGF-β antibodies reverse the inactivation of NF-κB and S-CM-mediated down-regulation of HIV-1 replication. Experiments were performed as in <b>A</b> except the S-CM (250 µg protein/mL) was pre-incubated for 1 hour with anti-TGF-β antibodies at the indicated concentration (n = 3 donors). (<b>C</b>) Recombinant human TGF-β reduces NF-κB translocation and R5 virus replication. Experiments were performed as in <b>A</b>, except the S-CM was replaced with rhTGF-β at 10 or 50 pg/mL (n = 4 donors).</p