Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome

Gut microbiome is responsible for gut permeability and bacterial translocation induced by morphine. (A–C) Representative H&E stained section of mouse ileum. (Scale bar: 100 μm.) WT and ABX mice: n_SPF = 10, n_GF = 3. (D and E) Representative images and summary of FITC-dextran fluorescent signal distribution in mice; n = 6 to 11. (F) Bacterial colony-forming unit (CFU) in liver homogenates of GF and SPF mice; n_SPF = 10 to 20; n_GF = 3. Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. (G) CFU from GF mice reconstituted with normal gut microbiota and treated with either morphine or saline; n_GF = 3. (H) CFU from GF mice and ABX-treated SPF mice received gut microbiota either from saline or morphine-tolerant donors; n_GF = 3; n_SPF/ABX = 10 to 12. Data were analyzed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Mean ± SD.

Morphine regulates gut and systemic TLR2 expression through gut microbiome. (A−C) The up-regulation of TLR2 expression by morphine was suppressed in GF and ABX mice; n_GF = 3; n_SPF = 12 to 15. (D−F) Morphine induced TLR2 expression in GF mice following gavaging with naïve mouse microbiota; n_GF = 3. (A–F) Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. (G−I) The microbiota FMT from morphine-tolerant mice alone increased TLR2 expression in GF and ABX mice; n_GF = 3; n_ABX = 5. Two-tailed Student’s t test was used for statistical analysis. (J and K) Morphine analgesic tolerance was determined in WT, TLR2KO, and TLR4KO mice. F_{tail flick} (5, 46) = 75.71; F_{hot plate} (5, 46) = 42.04; n = 8 to 10. (L) (i) Representative images and (ii) summary of fluorescent signal distribution in morphine-treated WT, TLR2KO, and TLR4KO mice; n = 6 to 10. (M) Visualization of bacterial colonies in blood agar plates from liver homogenates. (J–M) One-way ANOVA followed by Bonferroni’s multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Mean ± SD.

Gut microbiome is essential for morphine-induced chronic systemic inflammation. (A and B) IL-6 expression in morphine-treated GF and ABX mice was reduced compared with WT controls; n_GF = 3; n_SPF = 10 to 17. (C and D) Morphine effect on IL-6 expression was restored after gut microbiota was reconstituted in GF mice; n_GF = 3. (A–D) Data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparisons test. (E and F) GF and ABX mice were gavaged with microbiota from either saline or morphine-tolerant mice; n_GF = 3; n_SPF/ABX = 13 to 15. Significance was tested by two-tailed Student’s t test. (G and H) IL-6 expression was detected in WT, TLR2KO, and TLR4KO mice; n = 8 to 16. (I and J) WT and IL-6KO mice were treated with escalating morphine dosing for 8 d; n = 13 to 17. F_{tail flick} (3, 56) = 206.1. F_{hot plate} (3, 56) = 268.9. Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Mean ± SD.

Morphine analgesic tolerance induces gut dysbiosis. (A−C) Multidimensional scaling analysis of gut microbiota to visualize the Bray−Curtis distance of WT, TLR2KO, and TLR4KO morphine-tolerant mice and their controls. Red circles depict samples from morphine-tolerant mice; blue triangles represent WT saline-treated mice. β-diversity was found to be significantly different between the WT morphine-tolerant and saline-treated groups (P = 0.00256). (D) Taxonomic distribution of WT, TLR2KO, and TLR4KO morphine-tolerant mice and their controls at phylum level. Each column represents a fecal sample from a treatment group. (E and F) Dot plots show changes in abundance of bacteria with morphine treatment in WT, TLR2KO, and TLR4KO at family and genus level using WT saline mean proportion as reference. Microbial taxa with significant difference in WT mice were selected at false discovery rate < 0.1 and average relative abundance of WT control > 0.1%; n_WT = 6 to 7; n_TLR2KO = 19 to 24; n_TLR4KO = 8 to 11. NMDS, nonmetric multidimensional scaling.