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Gut microbiota is critical for the induction of chemotherapy-induced pain

Abstract

Chemotherapy-induced pain is a dose-limiting condition that affects 30% of patients undergoing chemotherapy. We found that gut microbiota promotes the development of chemotherapy-induced mechanical hyperalgesia. Oxaliplatin-induced mechanical hyperalgesia was reduced in germ-free mice and in mice pretreated with antibiotics. Restoring the microbiota of germ-free mice abrogated this protection. These effects appear to be mediated, in part, by TLR4 expressed on hematopoietic cells, including macrophages.

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Figure 1: Temporary eradication of gut microbiota prevents oxaliplatin-induced mechanical hyperalgesia (ac) Impacts of antibiotic water feeding on mice gut microbiota.
Figure 2: Gut microbiota is critical for DRG inflammatory responses (a,b) DRG flow cytometry staining for macrophages.
Figure 3: TLR4 on hematopoietic cells is critical for oxaliplatin-induced mechanical hyperalgesia.

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Acknowledgements

We thank P. Waghorn and O. Pinkhasov for assistance with the ICP-MS study, MGH COX-7 animal facility and CNY149 animal facility for animal husbandry, L. Chen for genomic data analysis, Z. Zhang and J. Moon for bone marrow transplantation, Y. Dong and J. Lan for their assistance with Tlr4-knockout animals and Q. Chen for critical comments on the manuscript. S.S. received support from NIH grant 5T32GM007592, a Foundation of Anesthesia Research and Education grant, and departmental research funds. This work was supported by NIH grant R01DE022901 (to J.M.). W.D. was supported by Hangzhou Science and Technology Plan No. 20130633B02 and Zhejiang Medical Science and Technology Plan No. 2011KYB064. The ICP-MS equipment was purchased with support from NIH grant S10OD010650.

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S.S. and J.M. conceived the project and wrote the manuscript. S.S., G.L., Z.Y., W.D., S.T., H.K. and M.M. conducted the experiments. C.R. conducted the bioluminescence study. P.C. carried out the mass spectrometry study. P.H. performed part of the animal study. J.D. contributed to manuscript preparation. K.H. assisted the bone marrow chimera analysis. Z.X. contributed to the Tlr4-knockout mice experiment. D.K. contributed to the analysis of gene sequencing data. B.H. and L.C. contributed intellectually to the project.

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Correspondence to Shiqian Shen or Jianren Mao.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gut fecal microbiota phylogenetic analysis

Fecal samples were collected after three weeks of antibiotic treatment. Data from fecal microbial bacterial 16s rRNA gene sequencing were analyzed: a) OUT rank curve; b)Bray-Curtis cluster tree; and c) Class, Order, Family, Genus histogram for control (H2O) and antibiotic treatment (abx) groups. Each column in the history gram represents one animal.

Supplementary Figure 2 Gut microbiota eradication by oral antibiotic treatment prevents oxaliplatin-induced mechanical hyperalgesia in female mice

a) Female C56BL/7 mice were started on oral antibiotics or regular water followed by oxaliplatin or normal saline i.p. injection (N=8 each group). Hindpaw withdrawal threshold (HWT) to Von Frey filaments was examined at indicated time points. As expected, mice received regular water and oxaliplatin injection developed mechanical hyperalgesia as indicated by reduction of mechanical withdrawal threholds. However, this reduction of HWT was not seen in mice that received oral antibiotics and oxalipatin (* p<0.05, H2O/oxaliplatin vs. abx/oxaliplatin). b) Gut microbiota eradication by oral antibiotic treatment prevents oxaliplatin-induced mechanical hypergalgesia in rats (N=12 each group).Male rats were started on antibiotics (abx) or regular water (H2O) for three weeks, followed by either oxaliplatin or normal saline injection. Oral antibiotics did not change baseline mechanical thresholds in rats (** p>0.05, abx/saline vs. H2O/saline). As expected, rats receiving regular water followed by saline injection did not develop mechanical hyperalgesia, whereas rats receiving regular water followed by oxaliplatin injection developed mechanical hyperalgesia. In contrast, rats receiving oral antibiotics did not develop mechanical hyperalgesia after oxaliplatin injection (* p< 0.05, H2O/oxaliplatin vs. abx/oxaliplatin). c) Mice (male) were started on antibiotics (abx) or regular water (H2O) for three weeks, followed by either oxaliplatin or normal saline injection (N=8 each group, except for N=6 for H2O, saline group). Facial grooming behaviors were counted for 10 minutes. Abx mice exhibited less facial grooming when compared to H2O mice at day 14 post-oxaliplatin therapy (* p<0.05 abx,oxaliplatin vs H2O,oxaliplatin). d) Mere exposure to antibiotics did not alter the development of oxaliplatin-induced mechanical hyperalgesia. Mice received antibiotics or saline injection intrathecally (N=8 each group) prior to oxaliplatin treatment. HWT was examined at indicated time points. * P>0.05 intrathecal antibiotics vs. intrathecal saline.

Supplementary Figure 3 Tissue platinum concentrations in saline-treated mice.

Mice received oral antibiotics (abx) or regular water (H2O) followed by saline treatment. Tissue platinum levels were examined as a control study for the Fig 1g in the main text. As expected, tissues platinum levels were negligible in all tissues for both groups of mice. There was no significant difference between the abx and H2O groups (two-way ANOVA, p=0.6).

Supplementary Figure 4 Il6 and Tnf gene transcripts after oxaliplatin treatment.

Mice received oral antibiotics (abx) or regular water (H2O), followed by oxaliplatin injection. Three days after the last dose of oxaliplatin, DRG and spinal cords were collected and tissue mRNA was used as template for cDNA synthesis. Q-PCR for IL-6 and TNF-a was performed. The results were normalized to levels of H2O group. In the DRG, abx group showed reduced Il-6 and Tnf-αgene transcript levels. However, reduction of gene transcripts for Il-6 and Tnf-a was not present in the spinal cord. *p<0.05 abx vs. H2O.

Supplementary Figure 5 Oral antibiotic treatment does not change major immune cell populations in peripheral blood.

a) Body weight comparison between control (H2O) and antibiotic treatment (abx) groups (N=8 each group). There was no significant difference between the two groups by two-way ANOVA test. However, body weight of the abx group was significantly lower than that of the H2O group at day 7 (p=0.03). This difference did not exist at day 14 and day 21. b) Peripheral blood major immune cell population characterization. Peripheral blood samples were stained for T cells (CD3), B cells (B220), dendritic cells (CD11c), monocytes (CD11b+ Ly6G-), neutrophils (CD11+Ly6G+), NK cells (CD335). Samples were acquired with LSR II flow cytometer followed by data analysis using Flowjo software. Abx: antibiotic treatment group; H2O: control group on regular water. Shown dot plots were gated on white blood cells by dumping red blood cells and dead cells based on FSC, SSC parameters. c) Summary of peripheral cell major immune cell populations in antibiotic treatment group (abx) and regular water control group (H2O). Percentages of immune cell populations include T cells, B cells, dendritic cells, monocytes, neutrophils, and NK cells did not differ between the two groups. NS: non-significant.

Supplementary Figure 6 Apoptosis in primary macrophage cultures.

Macrophages were collected from mice peritoneal cavity and were plated on 24-well plate in the presence of indicated concentrations of LPS and oxaliplatin. a) At the beginning of the culture, and 24 hours post-stimulation, cells were harvested for assessment of apoptosis with flow cytometry staining using Annexin V and 7-AAD. Cells positive for Annexin V are in apoptosis, whereas cells positive for both Annexin V and 7-AAD are in late apoptosis/necrosis. At 0 h, there were only small percentages of cells in apoptosis in all treatment groups. The percentages of apoptotic cells (Annexin V positive, Annexin V and 7-AAD double positive) increased at 24 h. b) There was no significant difference among the groups, indicating that the concentrations of oxaliplatin and LPS used in the experiment were not cytotoxic.

Supplementary Figure 7 Confirmation of successful generation of bone marrow chimera.

Eight to 10 weeks after BM transplantation, peripheral blood samples were collected and stained for CD45.1 and CD45.2 congenic markers. Contour plots were gated on CD11b+ cells. Each panel represents 6 independent stainings.

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Shen, S., Lim, G., You, Z. et al. Gut microbiota is critical for the induction of chemotherapy-induced pain. Nat Neurosci 20, 1213–1216 (2017). https://doi.org/10.1038/nn.4606

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