IκK-16 decreases miRNA-155 expression and attenuates the human monocyte inflammatory response

Abstract

Excessive inflammatory responses in the surgical patient may result in cellular hypo-responsiveness, which is associated with an increased risk of secondary infection and death. microRNAs (miRNAs), such as miR-155, are powerful regulators of inflammatory signalling pathways including nuclear factor κB (NFκB). Our objective was to determine the effect of IκK-16, a selective blocker of inhibitor of kappa-B kinase (IκK), on miRNA expression and the monocyte inflammatory response. In a model of endotoxin tolerance using primary human monocytes, impaired monocytes had decreased p65 expression with suppressed TNF-α and IL-10 production (P < 0.05). miR-155 and miR-138 levels were significantly upregulated at 17 h in the impaired monocyte (P < 0.05). Notably, IκK-16 decreased miR-155 expression with a corresponding dose-dependent decrease in TNF-α and IL-10 production (P < 0.05), and impaired monocyte function was associated with increased miR-155 and miR-138 expression. In the context of IκK-16 inhibition, miR-155 mimics increased TNF-α production, while miR-155 antagomirs decreased both TNF-α and IL-10 production. These data demonstrate that IκK-16 treatment attenuates the monocyte inflammatory response, which may occur through a miR-155-mediated mechanism, and that IκK-16 is a promising approach to limit the magnitude of an excessive innate inflammatory response to LPS.

Fig 1

(A) Supernatant TNF-α concentrations and (B) IL-10 concentrations comparing naïve (no LPS pre-treatment) and impaired (10 ng/mL LPS pre-treatment) conditions. Time points are shown for the end of the pre-treatment prior to the LPS challenge (17 h), and then the response 12 h after the LPS 100 ng/mL challenge (29 h). Levels were determined by ELISA. N = 8. Mean ± S.E.M. are shown. *P < 0.05, paired T-test. Time course of TNF-α (C) and IL-10 (D) gene expression in the impaired monocyte. Monocytes were treated with 10 ng/mL of LPS given after 1h for a duration of 16 h. At 17 h, cells were washed and resuspended in fresh media, then received 100 ng/mL of LPS at 17 h. Levels were measured by qRT-PCR, and expressed as fold change (2^-ΔΔCT) relative to the naïve monocyte. Mean ± S.E.M. are shown. N = 4. *P < 0.05. # P = 0.09, paired T-test.

Fig 2. Expression of NFκB (p65) as measured by Western blot analysis.

Cells given pre-treatment at 1 h after isolation until 17 h, and then were washed, suspended in fresh media, and given a further treatment (challenge) for 60 min. Quantitative data are expressed as relative density units, using beta-actin as a loading control. Naïve conditions (media only / 100 ng/mL LPS) and impaired (10 ng/mL LPS / 100 ng/mL LPS) were compared by paired T-test. N = 6. Mean ± S.E.M. *P < 0.05.

Fig 3. Volcano plot showing toll-like receptor (TLR) gene expression profiles, expressed the impaired monocyte relative to the naïve monocyte.

Monocytes were cultured in media only (naïve) or 10 ng/mL LPS (impaired) until 17 h and then stimulated with an LPS 100 ng/mL challenge for 2 h. RNA was extracted from cell pellets, and 44 TLR genes were measured by qRT-PCR. Fold changes (FC) calculated by ΔΔCT method are expressed on the x-axis, with significance (-log10 p-value) demonstrated on the y-axis. Data were normalized to housekeeper genes 18S, GAPDH, and B2M. Green dots represent genes that are downregulated by a fold change of 0.66 (equivalent to fold regulation of -1.5). Dots in the blue boxes represent genes that were significantly dysregulated compared with the naïve monocyte (P < 0.05), paired T-test. N = 4. GAPDH = glyceraldehyde 3-phosphate dehydrogenase. B2M = beta-2 macroglobulin.

Fig 4. Volcano plot of miRNA screening of microRNA expression in the impaired monocyte relative to naïve conditions.

Monocytes were cultured in media only (naïve) or 10 ng/mL of LPS until 17 h. RNA was collected at 17 h, immediately prior to a 100 ng/mL LPS challenge. Data were normalized to housekeeping genes RNU6B, RNU44, and RNU48. Green dots represent miRNAs that were downregulated by a log2 fold change of -1.0. Red dots represent miRNA’s that were upregulated by a log2 fold change of +1.0. miRNAs in the blue boxes represent those that reached statistical significance (P < 0.01), paired T-test. N = 5.

Fig 5. Verification of miRNA differences in the impaired monocyte relative to naïve conditions at 17 h.

The most significant miRNAs selected from screening were used for single assay qRT-PCR confirmation. Data were normalized to RNU6B. N = 8. Mean ± S.E.M. *P < 0.05, paired T-test.

Fig 6

Effect of IκK-16 on miR-155 (A), SOCS1 (B) and SHP1 (C) expression. Monocytes were cultured with 100 nM of IκK-16 or DMSO (control) for 1 h and then 100 ng/mL of LPS was given for 17 h. Levels of miR-155, SOCS1, and SHP1 expression in IκK-16 treated monocytes are expressed as fold change relative to the DMSO (control) monocytes, as determined by qRT-PCR. MiRNA data were normalized to RNU6B, and mRNA data were normalized to 18S. Mean ± S.E.M. N = 6. * P<0.05, paired T-test. Western blot was similarly used determine protein levels at 17 h for SOCS1 (D) and SHP1 (E), with vinculin and beta-actin used as loading controls, respectively.

Fig 7. Effect of IκK-16 on cytokine production.

Monocytes were treated with DMSO (control) or IκK-16 for 1 h and then stimulated with 100 ng/mL of LPS for an additional 16 h. Supernatant levels of (A) TNF-α and (B) IL-10 were measured by ELISA at 17 h. Mean ± S.E.M. are shown. N = 4. *P < 0.05 vs. DMSO only. †P = 0.06 vs. DMSO only. #P<0.05 vs. 1 nM. ‡P = 0.08 vs. 1 nM. Repeated Measures ANOVA with Tukey test for post-hoc analysis.

Fig 8. Effect of miR-155 on cytokine production.

Monocytes were treated either with DMSO (as control) or 100 nM IκK-16 for 1 h and then transfected with scrambled RNA (sham) or miR-155 mimics or antagomirs for 16 h. Cells were then stimulated with 100 ng/mL of LPS for 6 (A & B) and 24 h (C & D). Supernatant protein levels of TNF-α and IL-10 were determined by ELISA. N = 4, Paired t-test. * P < 0.05. † p = 0.050. ‡ p = 0.090.