Gut feeling: MicroRNA discriminators of the intestinal TLR9-cholinergic links

Abstract

The intestinal tissue notably responds to stressful, cholinergic and innate immune signals by microRNA (miRNA) changes, but whether and how those miRNA regulators modify the intestinal cholinergic and innate immune pathways remained unexplored. Here, we report changes in several miRNA regulators of cholinesterases (ChEs) and correspondingly modified ChE activities in intestine, splenocytes and the circulation of mice exposed to both stress and canonical or alternative Toll-Like Receptor 9 (TLR9) oligonucleotide (ODN) aptamer activators or blockers. Stressful intraperitoneal injection of saline, the anti-inflammatory TLR9 agonist mEN101 aptamer or the inflammation-activating TLR9 aptamer ODN 1826 all increased the expression of the acetylcholinesterase (AChE)-targeting miR-132. In comparison, mEN101 but neither ODN 1826 nor saline injections elevated intestinal miR-129-5p, miR-186 and miR-200c, all predicted to target both AChE and the homologous enzyme butyrylcholinesterase (BChE). In cultured immune cells, BL-7040, the human counterpart of mEN101, reduced AChE activity reflecting inflammatory reactions in a manner preventable by the TLR9 blocking ODN 2088. Furthermore, the anti-inflammatory BL-7040 TLR9 aptamer caused reduction in nitric oxide and AChE activity in both murine splenocytes and human mononuclear cells at molar concentrations four orders of magnitude lower than ODN 1826. Our findings demonstrate differential reaction of cholinesterase-targeting miRNAs to distinct TLR9 challenges, indicating upstream miRNA co-regulation of the intestinal alternative NFκB pathway and cholinergic signaling. TLR9 aptamers may hence potentiate miRNA regulation that enhances cholinergic signaling and the resolution of inflammation, which opens new venues for manipulating bowel diseases.

Keywords: Acetylcholinesterase; Butyrylcholinesterase; Intestinal inflammation; MicroRNA; Non-neuronal acetylcholine; Toll-Like Receptor 9.

Fig. 1

TLR9 aptamers induce differential changes in intestinal miRNA controllers of Cholinesterases. A. To test the TLR9 effects on CholinomiRs targeting two cholinesterase variants, intestinal sections were prepared from mice exposed to 500 μg/kg of the NFκB stimulating ODN 1826 (red), to the anti-inflammatory TLR9 stimulating oligonucleotide mEN101 (blue) or saline (green), to control for the stress impact. The sequences of tested CholinomiRs are shown; all of them are predicted to target AChE-R and either AChE-S or BChE. B. Seven days after exposure, qRT-PCR measurements quantified the intestinal levels of the AChE-S and AChE-R targeting CholinomiR-132 as well as miR-129-5p, − 186 or − 200c, all of which are predicted to target both AChE-R and BChE. The bar graphs show ΔΔC_t values normalized to snRNA U6 (white: control; green: saline; blue: mEN101; red: ODN1826). Bonferroni corrected p-values are noted above. Note mEN101 induced increases in all of the AChE-R and BChE-targeting miRNAs, but not in miR-132. MiRNA-target binding structures (miRNA sequence: red, target mRNA sequence: green) binding energies in kcal/mol are shown. The binding energies of miR-132 towards the two AChE variants are equal, whereas the other three CholinomiRs show lower binding energies towards BChE.

Fig. 2

BL-7040 induces a decrease in murine AChE activity through low affinity TLR9 activation. A. Molecular modeling of complexes between the second exon of human (h) and mouse (m) AChE (orange) and BL-7040 (blue). Mismatches are portrayed in red. B. AChE activity in murine splenocytes 24 h after treatment with BL-7040 (0.01 μM) with or without ODN 2088 (0.1 μM). C. Nitrite production in RAW264.7 macrophages following exposure to 0.01 μM BL-7040 or 0.1 μM ODN 1826, concomitantly with increasing doses of ODN 2088 that block their effects (molar ratio of ODN 2088/agonist shown). * indicates p-value < 0.05 using Mann–Whitney's U-test.

Fig. 3

Anti-inflammatory TLR9-mediated response induces a decline of AChE activity in murine splenocytes and human mononuclear cells. A. Murine splenocytes 24 h after treatment with 1 μg/ml CD40L or 0.01 μM of ODN 1826 show ~ 50% reduction in AChE activity. B. AChE activity in human mononuclear cells, 24 h after treatment with 0.01 μM BL-7040 or 0.1 μM of CpG B (ODN 1826), CpG A (ODN 1585) or TLR9 Inhibitor (ODN 2088), n = 3. * indicates p-value < 0.05 using Mann–Whitney's U-test.

Fig. 4

Oligonucleotide discriminators of the TLR9-cholinergic links. Left: miRNA regulators suppress the production of AChE and BChE, thus modifying ACh levels and regulating the cholinergic anti-inflammatory blockade of the NfκB pathway through the α7 nAChR. Right: Both CpG type B oligonucleotides and the BL-7040 TLR9 aptamer can activate inflammatory modifying responses, whereas the blocking aptamer ODN 2088 prevents TLR9 signaling to the NfκB pathway. Bottom: The canonical activation mode is pro-inflammatory (marked red), whereas the alternative mode is anti-inflammatory (marked blue) and induces immunosuppression, while at the same time suppressing AChE activity, increasing ACh levels and potentiating the cholinergic anti-inflammatory response.