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

Abstract

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. We show that morphine analgesic tolerance was significantly attenuated in germfree (GF) and in pan-antibiotic-treated mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in Bifidobacteria and Lactobacillaeae. Probiotics, enriched with these bacterial communities, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotic therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine's efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of proinflammatory cytokines, which drives morphine tolerance.

Keywords: germfree mice; gut dysbiosis; gut−immune−brain axis; morphine tolerance.

Fig. 1.

Gut microbiota are essential for morphine analgesic tolerance. (A and B) Antinociceptive tolerance was attenuated in GF mice. F_{tail flick} (3, 10) = 869.4, and F_{hot plate} (3, 10) = 110.9. (C and D) GF mice recapitulated morphine tolerance after FMT with naïve mouse microbiota. F_{tail flick} (3, 10) = 882.8, and F_{hot plate} (3, 10) = 147.8; n_SPF = 4; n_GF = 3. (E and F) Mice exhibited attenuated analgesic tolerance after gut microbiota depletion; n = 12 to 20. F_{tail flick} (3, 56) = 118.8, and F_{hot plate} (3, 56) = 58.19. (A–F) Data were analyzed by one-way ANOVA followed by Bonferroni correction. **P < 0.01; ****P < 0.0001. (G and H) Time course of the effects of different microbiota on morphine tolerance; n = 6 to 8. F_{Treatment x time} (56, 343) = 73.15 for tail flick. F_{Treatment x time} (56, 343) = 59.66 for hot plate. Two-way ANOVA followed by Tukey’s multiple comparison was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 for Morphine vs. Saline microbiome + Morphine. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 for Morphine vs. Morphine microbiome + Morphine. Each dot represents one mouse. Mean ± SD.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

Probiotics VSL#3 pretreatment attenuates morphine tolerance and prevents morphine-induced gut microbiota alterations. (A and B) Morphine analgesic effect was determined after escalating doses of morphine; n = 10 to 20. F_{tail flick} (3, 51) = 46.78. F_{hot plate} (3, 51) = 82.50. One-way ANOVA followed by Bonferroni’s correction was used to analyze data. (C) Multidimensional scaling was used to visualize the Bray−Curtis distance of different groups; n = 9 to 15. Data were subjected to PERMANOVA test along with Bonferroni correction. (D) Taxonomic distribution of different groups at phylum level. (E and F) Dot plots show changes in abundance of bacteria in Water+Morphine-treated and VSL#3+Morphine-treated mice at family and genus level using Water+Saline mean proportion as reference; n = 9 to 15. (G) Representative H&E stained intestinal sections from different treatment groups; n = 6 to 10. (H) (i) Representative figure of florescent signal distribution and (ii) summary of serum FITC-dextran concentration from different treatment groups; n = 6 to 10. (I) CFU from liver homogenate of each mouse in different treatment groups; n = 6 to 20. (J−L) Probiotics pretreatment inhibits TLR2 up-regulation by morphine on epithelial and immune cells; n = 11 to 14. (M and N) RT-PCR of the IL-6 gene expression; n = 9 to 19. (E–N) The results 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.