Project description:Human immunodeficiency virus (HIV) is widely recognized for its striking impact on the immune system, and it also significantly impacts the central nervous system (CNS). The primary immune constituents in the brain, microglia and macrophages, are the target for HIV in people and the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological dysfunction, known as HIV-associated neurocognitive disorder (HAND) and pathological entities such as HIV-induced encephalitis (HIVE). Given the gaps in our knowledge on how these cells respond in vivo to CNS infection, we performed single-cell multiomic sequencing, including gene expression and ATAC-seq on myeloid cells from the brains of two rhesus macaques with SIV-induced encephalitis (SIVE) as well as two uninfected controls. We used both RNA expression and chromatin accessibility to identify cell clusters. We found that the myeloid cell populations were significantly changed by SIVE, which differed greatly from those found in the uninfected brains. We categorized myeloid cells in the SIVE brains into two primary phenotypes: microglia-like and CAM-like, which were different from each other regarding trancriptomic profile and chromatin accessibility. We found that these microglia-like cells express high levels of chemoattractants for capable of recruiting highly the activated CAM-like cells to the site of infection/inflammation. Additionally, we observed a dramatic shift of upstream gene regulators and their targets in brain myeloid cells during SIVE. The transcription factors (TFs) highly enriched in SIVE-specific cell clusters were related to IRF/STAT and NF-κB mediated interferon and other proinflammatory cytokines production. Interestingly, some of these TF binding sites were also enriched in the open chromatin identified in the SIV DNA provirus in infected myeloid cells, indicating the immune activation in the host cells might also promote HIV/SIV gene expression and regulation . In summary, this study further uncover the trancriptome, gene regulaory events and potenital roles of differenet brain myeloid phenotypes in SIVE.

Project description:In SIV/HIV infection, the gastrointestinal tissue dominates as an important site due to the impact of massive mucosal CD4 depletion and immune activation-induced tissue pathology. Unlike AIDS-susceptible rhesus macaques, natural hosts do not progress to AIDS and resolve immune activation earlier. Here, we examine the role of dendritic cells in mediating immune activation and disease progression. We demonstrate that plasmacytoid dendritic cells (pDC) in the blood upregulate ?7-integrin and are rapidly recruited to the colorectum following a pathogenic SIV infection in rhesus macaques. These pDC were capable of producing proinflammatory cytokines and primed a Tc1 response in vitro. Consistent with the upregulation of ?7-integrin on pDC, in vivo blockade of ?4?7-integrin dampened pDC recruitment to the colorectum and resulted in reduced immune activation. The upregulation of ?7-integrin expression on pDC in the blood was also observed in HIV-infected humans but not in chronically SIV-infected sooty mangabeys that show low levels of immune activation. Our results uncover a new mechanism by which pDC influence immune activation in colorectal tissue following pathogenic immunodeficiency virus infections. SIV negative controls (n=4) and week 12 post SIV infected (n=4) groups of Rhesus macaques and SIV negative controls (n=4) and week 55 post SIV infected (n=4) groups of Sooty mangabeys colorectal tissue biopsies were collected in to RNA later reagent (Qiagen) and were homogenized with syringe and needle method. RNA was extracted with Rneasy mini kit (Qiagen) and was used for microarray experiments. Rhesus GeneChip assays were performed in the Yerkes Microarray Core Facility (www.microarray.emory.edu) , one of the Affymetrix Microarray Core Labs.The 0.5µg of total RNA sample was analyzed on Rhesus Macaque Genome GeneChip that consists of over 52,000 probe sets (Affymetrix, Santa Clara, CA). Target RNA labeling, hybridization and post-hybridization processing were performed following the Affymetrix GeneChip Expression Analysis standard protocols. In brief, The 5 ?g of RNA sample was first reverse-transcribed using T7-Oligo(dT) Promoter Primer and SuperScript II in the first-strand cDNAs synthesis reaction. Following RNase H-mediated second-stranded cDNA synthesis, the double-stranded cDNAs were purified by use of a GeneChip sample clean-up module and served as templates in the generation of biotinylated complementary RNAs (cRNAs) in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix by in vitro transcription (IVT) reaction. The biotinylated cRNAs were cleaned up, fragmented, and hybridized to the rhesus macaque expression arrays at 45°C for 16 h with constant rotation at 60 rpm. The gene chips were then washed and stained with Affymetrix fluidics stations 450 and scanned on Affymetrix scanner 3000. The images are processed to collect raw data with GeneChip Operating Software (GCOS) 1.4. Tissue: Colorectal tissue Time after SIV infection: 12 weeks for SIV infected Rhesus macaques, 55 weeks for Sooty mangabeys Infection: SIVmac251 infection for Rhesus macaques, SIVsm infection for Sooty mangabeys

Project description:HIV/SIV-associated periodontal disease (gingivitis/periodontitis) (PD) represents a major comorbidity affecting HIV patients on anti-retroviral therapy. Employing a systems biology approach, we report molecular changes underlying PD and its modulation by phytocannabinoids [delta-9-tetrahydrocannabinol (THC)] in uninfected and SIV-infected rhesus macaques (RMs) treated with vehicle (VEH/SIV) or THC (THC/SIV). VEH/SIV but not THC/SIV RMs showed significant enrichment of genes linked to anti-viral defense, interferon-beta, NFkB, RIG-1, and JAK-STAT signaling. We focused on the anti-microbial DUOX1 and immune activation marker IDO1 that were reciprocally regulated in gingiva of VEH/SIV RMs. Both proteins localized to the gingival epithelium and CD163+ macrophages, and showed differential expression in the gingiva of THC/SIV and VEH/SIV RMs. Additionally, inflammation-associated miR-21, miR-142-3p, miR-223, and miR-125a-5p showed significantly higher expression in the gingiva of VEH/SIV RMs. In human primary gingival epithelial cells, miR-125a-5p post-transcriptionally downregulated DUOX1 and THC inhibited IDO1 protein expression through a cannabinoid receptor-2 mediated mechanism. Interestingly, THC/SIV RMs showed relatively reduced plasma levels of kynurenine, kynurenate, and the neurotoxic quinolinate compared to VEH/SIV RMs. Most importantly, THC blocked HIV/SIV-induced depletion of Firmicutes and Bacteroidetes, and reduced Gammaproteobacteria abundance in saliva. Reduced IDO1 protein expression was associated with significantly (p<0.05) higher abundance of Prevotella, Lactobacillus (L. salivarius, L. buchneri, L. fermentum, L. paracasei, L. rhamnosus, L. johnsonii) and Bifidobacteria and reduced abundance of the pathogenic Porphyromonas cangingivalis and Porphyromonas macacae. These translational findings suggest that phytocannabinoids could help reduce gingival/systemic inflammation and salivary dysbiosis in ART naïve and treated HIV individuals.

			Action	DRS
	SRR8800210.fastq.gz	Fastqsanger.gz
	SRR8800211.fastq.gz	Fastqsanger.gz
	SRR8800202.fastq.gz	Fastqsanger.gz
	SRR8800212.fastq.gz	Fastqsanger.gz
	SRR8800203.fastq.gz	Fastqsanger.gz

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