Regulation of TLR2-mediated tolerance and cross-tolerance through IRAK4 modulation by miR-132 and miR-212

Abstract

Innate immune response is the first defense against pathogens via recognition by various conserved pattern recognition receptors, such as TLRs, to initiate a rapid and strong cytokine alarm. TLR signaling-mediated cytokine production must be properly regulated to prevent pathological conditions deriving from overproduction of cytokines. In this study, the role of specific microRNAs in TLR-signaling pathway was investigated to reveal the cross-interaction and -regulation in the MyD88 pathway. In peptidoglycan (PGN)/TLR2-stimulated THP-1 monocytes, PBMCs, and primary macrophages showed rapid and dramatic miR-132 and miR-212 (miR-132/-212) upregulation. This newly identified response appeared earlier in time than the characteristic miR-146a response in LPS-TLR4 stimulation. The rapid induction of miR-132/-212 was transcription factor CREB dependent, and the sustained expression of miR-132/-212 was responsible for inducing tolerance to subsequent PGN challenge. Cross-tolerance was observed by TLR5 ligand flagellin and heat-killed or live bacteria resulting from miR-132/-212 upregulation. Mechanistically, IRAK4 was identified and validated as a target of miR-132/-212 by luciferase reporter assay and seed-sequence mutagenesis of the reporter. Transfection of miR-132 or miR-212 alone mimicked PGN tolerance in monocytes, whereas transfected specific miRNA inhibitors tampered the tolerance effect. During bacterial infection, PGN-mediated TLR2 signaling induces miR-132/-212 to downregulate IRAK4, an early component in the MyD88-dependent pathway, whereas LPS/TLR4-induced miR-146a downregulates downstream components of the same MyD88-dependent pathway. The identification of miR-132/-212 and miR-146a together to prevent damaging consequences from the overproduction of proinflammatory cytokines by targeting a common signaling pathway is significant and will provide insights into future design and development of therapeutics.

Figure 1

PGN induction of TNF-α and miRNA expression kinetics in monocytes/macrophages. (A) Dose-response and time-course analysis of TNF-α secretion in culture supernatant by THP-1 monocytes stimulated with 0–5 μg/ml PGN for 2–48 h (horizontal axis). TNF-α in culture supernatants at the indicated time points was measured by ELISA. (B) Quantitative RT-PCR (qRT-PCR) analysis of miR-146a, miR-132, miR-212, and miR-155 expression kinetics in respective PGN-treated THP-1 cells. (C,D) Dose- and time-dependent secretion of TNF-α and induction of miR-132 in human PBMCs stimulated with 0–5 μg/ml PGN for 6–24 h. At least 3 PBMCs from different healthy subjects were analyzed. (E) qRT-PCR analysis of miR-132 expression kinetics in mouse RAW264.7 cells stimulated for 2–24 h with 1 μg/ml PGN, Pam, or LPS. (F) TNF-α secretion by mouse primary macrophages treated for 6–24 h with 0–10 μg/ml PGN. Mouse macrophages were pooled from multiple animals and assays were analyzed in triplicate. (G) qRT-PCR analysis of miR-132 expression. Human and mouse miRNA expression was normalized with control RNU44 and SnoRNA202, respectively. Data are from three independent experiments (A–E, mean ± s.d.) or experiments were performed three times and a set of representative data is shown (F,G; mean ± s.d.). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with untreated control.

Figure 2

Rapid induction of miR-132 and miR-212 in PGN-/Pam-stimulated THP-1 monocytes is mediated through CREB-dependent machinery. (A) Immunoblot analysis demonstrating phosphorylation of CREB (Ser 133, pCREB) within 15 min after PGN (top) or Pam (bottom) stimulation of THP-1 cells. Relative intensity is calculated as ratios of pCREB to total CREB in cell lysates. (B) qRT-PCR analysis of CREB mRNA expression in THP-1 cells transfected without (mock) siRNA, or with 100 nM siCREB or siLMNA. (C) Immunoblot analysis showing 70% reduction of CREB expression in THP-1 cells transfected with 100 nM siCREB using tubulin as a loading control. (D) miR-132 expression in siCREB-knockdown THP-1 cells stimulated for 5 h with PGN, Pam, and LPS. (E) Two-hour pre-treatment of synthetic kinase inhibitors PD98059 and U0126 blocked phosphorylation of CREB in THP-1 monocytes stimulated with 1 μg/ml PGN for 30 min. Immunoblot analysis showed 5.22-fold increase in relative intensity of pCREB in PGN-treated versus untreated control (UTX) with addition of DMSO alone and then stimulated for 30 min with 1 μg/ml PGN. (F,G) qRT-PCR analysis of primary- and mature-miR-132, miR-212, and miR-146a expression in THP-1 monocytes pre-treated for 2 h with inhibitors PD98059 and U0126 and then stimulated for 8 h with PGN or Pam. Data points and error bars represent three independent experiments (B,D,F,G, mean ± s.d.). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with mock transfected THP-1 monocytes (D) or corresponding cells not treated with inhibitors (F, G). Western blot data are representatives of 2 independent experiments (A,C,E).

Figure 3

High levels of miR-132 and miR-212 may account for TLR2 ligand-induced tolerance via downregulation of IRAK4. (A) TNF-α production by THP-1 monocytes primed with or without (unprimed) PGN or Pam (500 ng/ml) for 18 h and then challenged with ligands (1 μg/ml) for 3 h. (B) qRT-PCR analysis of TNF-α mRNA in the PGN and Pam primed THP-1 cells as in A. (C) qRT-PCR analysis of miR-132, miR-212, miR-146a, and miR-155 expression in the same tolerized THP-1 cells as in B. (D) TNF-α secretion by human PBMCs primed with or without PGN (1 μg/ml) for 18 h and then challenged with PGN or LPS for 5 h. (E) qRT-PCR analysis of miR-132 in the PGN-primed and unprimed PBMCs as in D. (F) TNF-α production by mouse primary macrophages primed with or without PGN (1 μg/ml) for 18 h and then challenged with PGN, Pam, or LPS for 5 h. (G) qRT-PCR analysis of miR-132 in the same macrophages as in F. (H) Immunoblot analysis of IRAK4 protein levels showing reduction in PGN- or Pam-primed and unprimed THP-1 cells as in A–C and challenged with various TLR ligands for 2 h. Tubulin serves as a loading control. (I) Changes in IRAK4 mRNA detected in THP-1 cells stimulated with 500 ng/ml PGN using the same RNA samples from experiments shown in Figure 1B. Data are expressed as mean ± s.d. of three independent experiments (A–C and I), two independent experiments with three individuals, n=6 (D,E), or experiments were performed three times and a set of representative data is shown (F,G). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with unprimed controls (A–B, F–G) or untreated cells (I). Western blot data (H) are representatives of 2 independent experiments. LPS Pg, LPS from P. gingivalis; LPS Se, LPS from S. enterica.

Figure 4

Flagellin-induced tolerance contributed by high level of miR-132 and miR-212 expression. (A) TNF-α production by THP-1 cells primed with or without flagellin (200 ng/ml) for 18 h and then challenged with flagellin (300 ng/ml), PGN (1000 ng/ml), or Pam (1000 ng/ml) for 3 h. (B,C) qRT-PCR analysis of miR-132 and miR-212 expression in the same THP-1 cells as in A. (D) Immunoblot analysis of IRAK4 in THP-1 monocytes stimulated for 2, 4, or 6 h with flagellin (200 ng/ml). Tubulin serves as a loading control. (E) TNF-α production by THP-1 cells primed with or without PGN or Pam (1000 ng/ml) for 12 h and then challenged with 300 ng/ml flagellin for 3 h. (F) qRT-PCR for miR-132 expression in the PGN or Pam primed THP-1 monocytes as described in E. (G) Immunoblot analysis of IRAK4 and tubulin (loading control) in THP-1 monocytes stimulated for 3 h with PGN and Pam (1000 ng/ml). Data are representative of three independent experiments (A–C,E,F, mean ± s.d.). Western blot data are representatives of 2 independent experiments (D,G). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with unprimed controls.

Figure 5

Dose-dependent priming effect of PGN and Pam to induce efficient tolerance are inversely correlated to the levels of miR-132 and miR-212 expression. (A,B) TNF-α levels by THP-1 monocytes primed with 0–1 μg/ml PGN (1'PGN) or Pam (1'Pam) for 18 h and then challenged with high doses of PGN or Pam (1 μg/ml, 2' challenge) for 3 h. (C,D) miR-132 expression in the same PGN-treated THP-1 monocytes as in A and B. (E,F) miR-212 expression in the THP-1 monocytes as mentioned in A and B. (G,H) Immunoblot analyses for IRAK4, and tubulin (loading control) in the THP-1 cell lysates collected 2 h after PGN or Pam challenge (0–1 μg/ml). Data points and error bars represent of three independent experiments (A–F, mean ± s.d.). Western blot data are representatives of 2 independent experiments (G,H). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with unprimed THP-1 cells challenged with 1 μg/ml PGN.

Figure 6

Reduction in miR-132 and miR-212 expression in PGN-tolerized THP-1 cells inversely correlated with TNF-α production. (A) TNF-α production by THP-1 cells cultured with (tolerized) or without (untolerized) PGN (100 ng/ml) for 18 h and then cells were washed with PBS and cultured in complete growth medium for an additional 0, 12, or 22 h (PGN withdrawal). At each time point, 8×10⁵ cells were challenged for 5 h with PGN (500 ng/ml) prior to analysis of TNF-α in culture supernatant. (B) qRT-PCR analysis of miR-132 and miR-212 in the same THP-1 cells as described in A. Values are mean ± s.d. from three independent experiments. *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with unprimed THP-1 cells challenged with 500 ng/ml PGN.

Figure 7

Overexpression of miR-132 or miR-212 alone can mimic TLR2 ligand priming while specific miRNA inhibitors tamper PGN-induced tolerance. (A) Reduction in TNF-α production by THP-1 cells transfected for 24 h with miRNA mimics or non-specific (NS) negative control (total 40 nM) and then challenged for 8 h with 500 ng/ml PGN. (B) Enhanced TNF-α production by THP-1 monocytes transfected in parallel as in A substituted only with miRNA inhibitor(s) (total 40 nM). (C) Effective inhibition of miR-132 and miR-212 expression in THP-1 cells transfected with 40 nM miRNA respective inhibitors (132-inh, 212-inh) for 24 h and then stimulated for 16 h with 100 ng/ml PGN. (D) Recovery of TNF-α production by THP-1 monocytes transfected for 24 h with miRNA inhibitor (total 40 nM) and primed/tolerized with PGN (100 ng/ml) for 8 h, and then challenged with 500 ng/ml PGN for 3 h. There was no apparent change in proliferation and cell survival in cells treated with miRNA-mimics and inhibitors differently from the controls. Data represent three independent experiments (A–C) or experiment with triplicate sample analyzed (D, mean ± s.d.). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with mock transfected controls.

Figure 8

IRAK4 mRNA is a molecular target of miR-132 and miR-212 post-transcriptional silencing in TLR signaling. (A) Sequence alignment of miR-212 (top) and miR-132 (bottom) with putative target site in 3'UTR of IRAK4 (IRAK4-wt) and mutant construct (IRAK4-mut). (B) Luciferase activity in HEK293 cells cotransfected for 48 h with either wild type or mutant 3'UTR IRAK4-luciferase reporter and 100 nM miR-132-, miR-212-, or miR-146a-mimic. (C) qRT-PCR analysis of IRAK1, TRAF6, IRAK4, and lamin A/C (LMNA, a negative control) in THP-1 monocytes transfected for 48 h with 40 nM miR-132- and miR-212-mimic. (D) Immunoblot analysis of IRAK4 and tubulin (loading control) in THP-1 monocytes transfected with 40 nM miR-132- and miR-212-mimic for 48 h. Reduction of IRAK4 level was noted for cells transfected with miR-132-and miR-212-mimic, but not miR-146a-mimic (left). The levels of NF-kB p65 and ERK 1/2 were not affected in cells transfected by miR-132-mimic compared to cells transfected by NS control (right). (E) qRT-PCR analysis showing significant and specific elevated level of IRAK4 in THP-1 monocytes transfected with 40 nM miR-132 or miR-212 inhibitor for 48 h. LMNA was monitored as an unrelated negative control. (F) PGN-induced regulation of IRAK4 3'UTR in THP-1 cells. IRAK4-wt or IRAK4-mut reporter constructs were co-transfected with RL luciferase constructs into THP-1 monocytes for 6 h and cell lysates were harvested 12 h after PGN stimulation (2 μg/ml) to monitor for luciferase activities. (G) qRT-PCR analysis showing specific reduction of IRAK4 mRNA in THP-1 monocytes transfected with 100 nM IRAK4-specific siRNA (siIRAK4) for 48 h; IRAK1 mRNA served as a negative control. (H) Immunoblot analysis of IRAK4 and tubulin (loading control) in THP-1 monocytes transfected with 100 nM siIRAK4 for 48 h. (I) TNF-α and IL-6 production by THP-1 monocytes transfected with 100 nM siIRAK4 for 48 h and then treated for 3 to 24 h with PGN, Pam, or LPS (1000 ng/ml). Data points and error bars represent three independent experiments (B,C,E–G,I, mean ± s.d.). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with mock transfected controls (B–C, E, H, I) or with IRAK4-mut (B, F). Western blot data are representatives of 2 independent experiments (D,H).

Figure 9

Heat-killed bacterial stimulation- or live bacterial infection-induced miR-132 and miR-212 contribute to resistance to recurrent bacterial challenge. (A,B) qRT-PCR analysis of miR-132 and miR-212 expression in THP-1 monocytes stimulated for 2–48 h with heat-killed (HK) and live P. gingivalis, T. denticola, and T. forsythia at MOI 100. (C) TNF-α production by THP-1 cells primed with or without HK T. forsythia (MOI 10) for 18 h and then challenged for 2 h with HK bacteria (MOI 100), 1000 ng/ml of LPS, PGN, and Pam and 300 ng/ml flagellin. (D) qRT-PCR of miR-132 expression in the same THP-1 monocytes as described in C. (E) Immunoblot analysis of IRAK4 and tubulin (loading control) in THP-1 monocytes treated for 4 h with HK and live P. gingivalis or T. forsythia (MOI 100). Data are representative of three independent experiments (A–D, mean ± s.d.) or two independent experiments (E). *P < 0.05; **P < 0.01 (two-tailed unpaired t-test) compared with untreated controls at the same time point (A–B) or untolerized controls (C–D).

Figure 10

PGN-induced miR-132 and miR-212 in TLR signaling. (A–B) A schematic summary of PGN-mediated induction of miR-132 and miR-212 targeting IRAK4 in tolerized and untolerized THP-1 monocytes. (C) LPS, PGN, and flagellin from bacterial infection bind to their respective TLRs and activate the MyD88-dependent pathway. This eventually leads to gene transcription with the production of TNF-α and other cytokines. The MyD88 pathway leads to activation of NF-kB and production of miR-146a whereas activation of CREB leads to miR-132/-212 production. miR-132/-212 targets IRAK4 whereas miR-146a targets IRAK2/1 and TRAF6. IRAK4 and IRAK2/1 are critical components of the myddosome which is in turn critical for activation of the MyD88-dependent pathway.