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07.10.2016 | Original Article | Ausgabe 5-6/2016

Immunologic Research 5-6/2016

HIV-1-derived single-stranded RNA acts as activator of human neutrophils

Zeitschrift:
Immunologic Research > Ausgabe 5-6/2016
Autoren:
Diana M. Giraldo, Juan C. Hernandez, Silvio Urcuqui-Inchima
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s12026-016-8876-9) contains supplementary material, which is available to authorized users.

Abstract

Neutrophils are key effector cells of the innate immune system and are involved in the host defense against invading pathogens such as viruses. Recently, it was reported that HIV-1–neutrophil interaction triggers neutrophil activation and promotes expression of Toll-like receptors (TLRs). Here, we assessed the role of single-stranded RNA40 (ssRNA40) derived from HIV-1 in neutrophil activation. We observed functional activation of neutrophils in response to HIV-1-derived ssRNA40 based on the expression of TLR7/8, RIG-I, and MDA5, induction of cytokines (IL-6 and TNF-α), and the production of reactive oxygen species (ROS). Additionally, ssRNA40 promoted the expression of CD62L and TNF-α and the production of ROS in the presence of the TLR2 agonist Pam2CSK4. ssRNA40 together with R848 (a TLR7/8 agonist) increased CD11b expression but decreased CD62L expression. Furthermore, decreased IL-6 expression was observed in the presence of the TLR4 agonist LPS. Finally, we found that ssRNA40 promotes RIG-I and MDA5 expression in the presence of the TLR2, TLR4 and TLR7/8 agonists. This study demonstrates a functional response of TLRs in neutrophils challenged with ssRNA40, suggesting that TLRs could be involved in the innate immune response observed during HIV infection, which might be mediated by its genome.

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Zusatzmaterial
Online resource 1 Protein expression of TLR2 and TLR4 in neutrophils stimulated in vitro with ssRNA40 neutrophils (2 × 105) purified from healthy donors (n = 6) were stimulated in vitro with 3 μg/ml of ssRNA40 or 3 μg/ml of control ssRNA41. The specific agonists for each TLR were used as positive controls as follows: Pam2CSK4 (20 ng/μl) for TLR2, and LPS (0.1 ng/μl) for TLR4. Stimulations were performed for 8 h prior to determining TLR2 (a) and TLR4 (b) expression by flow cytometry. The data are presented as the MFI of each specific TLR. Comparisons were performed using the Wilcoxon test. The level of significance was p < 0.05 (*) and p < 0.01 (**) (TIFF 535 kb)
12026_2016_8876_MOESM1_ESM.tif
Online resource 2 Functional activation of neutrophils by viral RNA. Neutrophils (2.5 × 105) purified from healthy donors (n = 5) were exposed to viral RNA obtained and purified from viral particles present in H9 HTLVIII chronically infected cell supernatants using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany). As a negative control, H9 cell supernatant was used; it was processed and treated the same way as the supernatant H9 HTLVIII cells. IL-6 secretion was quantified by ELISA (a). In addition, ROS production (b) was measured by flow cytometry and is reported as the percentage of neutrophils producing ROS. Comparisons were performed using the Wilcoxon test. The median and range are shown. The level of significance was p < 0.05 (*) and p < 0.01 (**) (TIFF 710 kb)
12026_2016_8876_MOESM2_ESM.tif
Online resource 3 Protein expression of TLR2 and TLR4 in neutrophils co-stimulated with TLR agonists and ssRNA40. A total of 2.5 × 105 neutrophils were stimulated simultaneously with TLR agonists (20 ng/μl Pam2CSK4, 0.1 ng/μl LPS, or 1 μg/μl R848) and ssRNA40 (3 µg/ml) for 8 h. Protein expression of TLR2 (a) and TLR4 (b) was determined by flow cytometry. The data are presented as the MFI of each specific TLR. Comparisons were performed using the Wilcoxon test. The level of significance was p < 0.05 (*) and p < 0.01 (**) (TIFF 520 kb)
12026_2016_8876_MOESM3_ESM.tif
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