Representative western blot (A,B) and quantification (C) showing a reduction in protein expression of MMP8 (~52 kD) in whole colon tissue of THC/SIV rhesus macaques. of RNA, one animal (FT11) in the SIV-no vehicle group was not included in the RT-qPCR analysis shown in (B). Note the high normalized signal intensity (A) and low delta-CT (B) values (increased mRNA expression) in vehicle treated and vehicle untreated SIV-infected rhesus macaques compared to THC/SIV and uninfected control rhesus macaques suggesting a lack of effect of vehicle on intestinal inflammatory gene expression. Image_1.TIF (698K) GUID:?247D5BF9-8DDD-4E33-B25B-5948FACE9702 Figure S2: Gating strategy of Ki67+ cells in duodenum lamina propria leukocytes from an SIV- infected rhesus macaque. Cells were gated first on singlets, CD45+ cells, followed by live cells and then on CD3+ T cells and subsequently on CD3/CD4++ and CD3/CD8++ T cell subsets. CD4+ T cells were further gated to quantify proliferating Ki67+ cells. The percentages of the total gated population are shown in each box of the plot. Note that the THC/SIV infected macaque had significantly fewer proliferating CD4+ T cells compared to the VEH/SIV infected macaque. Image_2.TIF (165K) GUID:?8618251C-4D1D-4911-94A8-B9002F84F1B0 Figure S3: Plasma lipopolysaccharide binding protein (LBP) levels are relatively lower in THC-SIV (B) compared to VEH/SIV rhesus macaques. Plasma LBP levels were similar in both groups at 60 days post infection (days post infection) (A). Within groups, the increase in plasma LBP concentration from 60 to 180 days post infection was greater in VEH/SIV (Avg 4.7C46 ng/mL) (C) compared to THC/SIV (Avg 4.4C21 ng/mL) (D) group. At both time points, plasma LBP concentrations remained undetectable (below 2 ng/ml) in 5/9 VEH/SIV and 2/7 THC/SIV rhesus macaques. Plasma samples were not available from A2L0694 (THC/SIV group). LBP data were analyzed using the Mann-Whitney Isocarboxazid = 9) or 9-tetrahydrocannabinol (9-THC; THC/SIV; = 8). Pro-inflammatory miR-130a, miR-222, and miR-29b, Isocarboxazid lipopolysaccharide-responsive miR-146b-5p and SIV-induced miR-190b Isocarboxazid were significantly upregulated in VEH/SIV rhesus macaques. Compared to VEH/SIV rhesus macaques, 10 miRNAs were significantly upregulated in THC/SIV rhesus macaques, among which miR-204 was confirmed to directly target MMP8, an extracellular matrix-degrading collagenase that was significantly downregulated in THC/SIV rhesus macaques. Moreover, THC/SIV rhesus macaques failed to upregulate pro-inflammatory miR-21, miR-141 and miR-222, and alpha/beta-defensins, suggesting attenuated intestinal inflammation. Further, THC/SIV rhesus macaques showed higher expression of tight junction proteins (occludin, claudin-3), anti-inflammatory (epithelial proliferation), and anti-HIV studies showed that miR-204, a miRNA upregulated in the colon of THC/SIV rhesus macaques could potentially target and downregulate the expression of = 4) received twice daily injections of vehicle (VEH) Rabbit Polyclonal to IRAK2 (1:1:18 of emulphor: alcohol: saline) and were infected intravenously with 100 times the 50% tissue culture infective dose (100TCID50) of SIVmac251. Group-2 (= 8) received twice daily injections of 9-THC for 4 weeks prior to SIV infection. Group 3 (= 6) served as uninfected controls. To obtain adequate statistical power, five SIV-infected animals (FT11, GH25, HB31, GA19, and HD08) that did not receive VEH treatments were added to the VEH/SIV group increasing the group size to nine. However, vehicle treatment alone is unlikely to influence pro-inflammatory signaling in the colon. The lack of effect of vehicle on inflammatory gene expression is clear from the high normalized signal intensity and significantly (< 0.05) low delta CT (CT) values for inflammation-induced (or in the colon of SIV-infected rhesus macaques that received 9-THC are not different from uninfected controls. Later, an additional eight age-matched male VEH/SIV (= 4) and THC/SIV (= 4) rhesus macaques were used exclusively for T cell and macrophage immunophenotyping studies, using intestinal pinch biopsies collected longitudinally during the course of the infection (Table 1). Chronic administration of 9-THC or VEH was initiated 4 weeks before SIV infection at 0.18 mg/kg as used in previous studies (10, 11). This dose of 9-THC was found to eliminate responding in a complex operant behavioral task in almost all animals (13). The dose was subsequently increased for each subject to 0.32 mg/kg, over a period of ~2 weeks when responding was no longer affected by 0.18 mg/kg on a daily basis (i.e., tolerance developed), and maintained for the duration Isocarboxazid of the study. The optimization of the 9-THC dosing in rhesus macaques accounts for the development of tolerance during the initial period of administration. Because in our previously published studies (10, 11) this dose of 9-THC showed protection, the same dose was used in this study. The 0.32 mg/kg dose was also shown to be effective in SIV-infected rhesus macaques of Chinese origin (14). SIV levels in plasma and intestine were quantified by using the TaqMan One-Step Real-time RT-qPCR assay that targeted the LTR gene (15C18). At necropsy, colon segments were split open and luminal contents were first removed by washing with sterile PBS after which small 1 cm2 pieces were collected in RNAlater (Thermo Fisher Scientific, Waltham, MA) for total RNA extraction. Table 1 Animal IDs, SIV inoculum, duration of infection, viral loads and colon.
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