Dysregulated miR-34a–SIRT1–Acetyl p65 Axis Is a Potential Mediator of Immune Activation in the Colon during Chronic Simian Immunodeficiency Virus Infection of Rhesus Macaques

Abstract

Persistent gastrointestinal (GI) inflammation, a hallmark of progressive HIV/SIV infection causes disruption of the GI epithelial barrier, microbial translocation and generalized immune activation/inflammation driving AIDS progression. Apart from protein regulators, recent studies strongly suggest critical roles for microRNAs (miRNAs) in regulating and managing certain aspects of the inflammatory process. To examine their immunoregulatory role, we profiled miRNA expression in colon from 12 chronic SIV-infected and 4 control macaques. After applying multiple comparisons correction, 10 (3-up and 7-down) miRNAs showed differential expression. Most notably, miR-34a showed significant upregulation in both epithelial and lamina propria leukocyte (LPL) compartments. Intense γH2AX expression in colonic epithelium and LPL confirmed the contribution of DNA damage response in driving miR-34a upregulation. SIRT1 mRNA and protein decreased significantly in both colonic epithelium and LPL. Luciferase reporter assays validated rhesus macaque SIRT1 as a direct miR-34a target. Decreased SIRT1 expression was associated with constitutively enhanced expression of the transcriptionally active form of the p65 (acetylated on lysine 310) subunit of NFκB exclusively in the LPL compartment. The intensity and number of acetylated-p65+ cells was markedly elevated in LPLs of chronically SIV-infected macaques compared to uninfected controls and localized to increased numbers of IgA+ and IgG+ plasma cells. These findings provide new insights into the potential role of the miR-34a-SIRT1-p65 axis in causing hyperactivation of the intestinal B cell system. Our results point to a possible mechanism where the normal immunosuppressive function of SIRT1 is inhibited by elevated miR-34a expression resulting in constitutive activation of acetylated-p65 (lysine 310).

Longitudinal Examination of the Intestinal Lamina Propria Cellular Compartment of Simian Immunodeficiency Virus-Infected Rhesus Macaques Provides Broader and Deeper Insights into the Link between Aberrant MicroRNA Expression and Persistent Immune Activation

“…Lately, the enhanced expression of miR-21 (13) and miR-142 (14) was correlated with macrophage activation and encephalitis in the brain of SIV-infected rhesus macaques. Focusing on the GI tract, an important site of CD4 ϩ T-cell depletion and HIV/SIV replication, we (15,16) and others (17) reported significant miRNA dysregulation in intact colon during SIV infection. Unlike our two previous studies that utilized intact intestinal tissue (15,16), in the present study, we focused exclusively on the intestinal lamina propria compartment to obtain a deeper understanding of the link between aberrant miRNA expression and chronic immune activation/inflammation in the intestine.…”

“…Focusing on the GI tract, an important site of CD4 ϩ T-cell depletion and HIV/SIV replication, we (15,16) and others (17) reported significant miRNA dysregulation in intact colon during SIV infection. Unlike our two previous studies that utilized intact intestinal tissue (15,16), in the present study, we focused exclusively on the intestinal lamina propria compartment to obtain a deeper understanding of the link between aberrant miRNA expression and chronic immune activation/inflammation in the intestine. In tune with this objective, we collected serial intestinal resection segments from the same animals before and at 21, 90, and 180 dpi and separated the different mucosal compartments (epithelium, intraepithelial lymphocytes, lamina propria leukocytes [LPLs], and fibrovascular stroma).…”

“…Intestinal resections were collected from SIV-infected macaques in our previous studies (15,16,20,21); the animals tolerate this procedure extremely well. The three resections (preinfection and 21 and 90 days post-SIV infection) were spaced wide apart from one another (ϳ2.5 to 3 months), and complete healing occurs within 2 to 3 weeks.…”

“…LPLs from duodenal pinch biopsies were isolated as described in the previous section and adjusted to a concentration of 10 7 /ml. For in vivo T-cell and in vitro CD8 ϩ T-cell immunophenotyping, ϳ100-l aliquots were stained with appropriately diluted, directly conjugated monoclonal antibodies to CD3 (Pacific blue, SP34-2), CD4 (SK3, peridinin chlorophyll protein-Cy5.5), and CD8 (phycoerythrin-Texas red, 3B5), Uninfected controls HT74 NA NA NA NA NA NA IT62 NA NA NA NA NA NA IT16 NA NA NA NA NA NA IT05 NA NA NA NA NA NA IH95 NA NA NA NA NA NA HT73 NA NA NA NA NA NA EJ34 NA NA NA NA NA NA IT13 NA NA NA NA NA NA HD08 NA NA NA NA NA NA HH07 NA NA NA NA NA NA HF54 NA NA NA NA NA NA HR42 NA NA NA NA NA (15,16). Total RNA was extracted from LPL samples using the miRNeasy total RNA isolation kit (Qiagen Inc., CA).…”

“…The expression of a select list of differentially expressed (DE) miRNAs at 21, 90, and 180 dpi was further confirmed in both intestinal LPLs (ϳ100 ng total RNA) and epithelium (ϳ250 ng total RNA) using the TaqMan microRNA predesigned and preoptimized assays (Life Technologies) as described previously (15,16). Briefly, total RNA was reverse transcribed using the stem-loop primers provided in the predesigned kit in a total volume of 15 l. Approximately 4 l of cDNA was subjected to 40 cycles of PCR in a total volume of 20 l on the ABI 7900 HT fast PCR system (Life Technologies) using the following thermal cycling conditions: 95°C for 10 min, followed by 40 repetitive cycles of 95°C for 15 s and 60°C for 1 min.…”