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A systems immunology approach identifies the collective impact of 5 miRs in Th2 inflammation
Ayşe Kılıç, … , Amitabh Sharma, Harald Renz
Ayşe Kılıç, … , Amitabh Sharma, Harald Renz
Published June 7, 2018
Citation Information: JCI Insight. 2018;3(11):e97503. https://doi.org/10.1172/jci.insight.97503.
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Research Article Immunology Inflammation

A systems immunology approach identifies the collective impact of 5 miRs in Th2 inflammation

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Abstract

Allergic asthma is a chronic inflammatory disease dominated by a CD4+ T helper 2 (Th2) cell signature. The immune response amplifies in self-enforcing loops, promoting Th2-driven cellular immunity and leaving the host unable to terminate inflammation. Posttranscriptional mechanisms, including microRNAs (miRs), are pivotal in maintaining immune homeostasis. Since an altered expression of various miRs has been associated with T cell–driven diseases, including asthma, we hypothesized that miRs control mechanisms ensuring Th2 stability and maintenance in the lung. We isolated murine CD4+ Th2 cells from allergic inflamed lungs and profiled gene and miR expression. Instead of focusing on the magnitude of miR differential expression, here we addressed the secondary consequences for the set of molecular interactions in the cell, the interactome. We developed the Impact of Differential Expression Across Layers, a network-based algorithm to prioritize disease-relevant miRs based on the central role of their targets in the molecular interactome. This method identified 5 Th2-related miRs (mir27b, mir206, mir106b, mir203, and mir23b) whose antagonization led to a sharp reduction of the Th2 phenotype. Overall, a systems biology tool was developed and validated, highlighting the role of miRs in Th2-driven immune response. This result offers potentially novel approaches for therapeutic interventions.

Authors

Ayşe Kılıç, Marc Santolini, Taiji Nakano, Matthias Schiller, Mizue Teranishi, Pascal Gellert, Yuliya Ponomareva, Thomas Braun, Shizuka Uchida, Scott T. Weiss, Amitabh Sharma, Harald Renz

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Figure 7

Targeting a miR network interferes with cytokine production in Th2 cells in vitro.

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Targeting a miR network interferes with cytokine production in Th2 cells...
(A) The WT and mutant 3′-UTR sequences of the human genes CTCF (target to miR-27b), NCOA3 (miR-206), ELK3 (miR-106b), AHR (miR-203), and ELF2 (miR-23b) were cotransfected with miR mimics or a scrambled sequence (con-miR). Relative light units (RLUs) were normalized to the respective control group and are presented as relative luciferase activity. Results of 3 independent experiments with n = 3 per group are shown. (B) Relative expression of target miRs in in vitro–polarized murine Th2 cells was determined by real-time RT-PCR and normalized to naive CD4+ T cells. Graph summarizing results of 3 independent experiments with n = 2–3 per group. (C) Relative mRNA expression, depicted as fold change, of the selected miR targets Ctcf, Ncoa3, Elk3, Ahr, and Elf2 in murine Th2 cells normalized to naive CD4+ T cells in n = 4–5 samples. (D) Graph summarizing the results for IL-13+ cells after nucleofecting miR inhibitors and miR mimics as indicated in the table below the bar graph and antagonizing the IDEAL (Impact of Differential Expression Across Layers) predictions. In each condition, the con-miR transfection contained the equal amount and composition of control sequences (see Methods section). Values were normalized to the respective con-miR samples and summarize 4 independent experiments with n = 2–3 replicates per experiment. (E) Representative histogram showing the effect of antagonizing the miR expression pattern in Th2 cells on IL-13 expression. A miR cocktail containing anti–miR-27b, miR-206 mimic, miR-106b mimic, miR-203 mimic, and anti–miR-23b was nucleofected. IL-13+ cells were determined by flow cytometry (blue dashed line). The effect was controlled by nucleofecting the respective amount of scrambled miR mimics/inhibitors (con-miR, red line; see Methods). Shaded histogram indicates samples stained with isotype control antibody. Values summarize 4–5 independent experiments with n = 2–3 replicates per experiment. (F) Twenty-four hours after nucleofection, cells were stimulated with anti-CD3/anti-CD28 for 24 hours and cytokines were measured in culture supernatant. Concentrations were normalized to con-miR sample. Graph summarizes results of 4 independent experiments with n = 2–3 replicates per group and experiment. All graphs with quantitative data show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed Mann-Whitney U test (D–G).

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