Antibiotic Therapy With Metronidazole Reduces Endometriosis Disease Progression in Mice

A Potential Role for Gut Microbiota

Sangappa B. Chadchan; Meng Cheng; Lindsay A. Parnell; Yin Yin; Andrew Schriefer; Indira U. Mysorekar; Ramakrishna Kommagani

Disclosures

Hum Reprod. 2019;34(6):1106-1116. 

In This Article

Materials and Methods

Study Approval

Animal studies were performed according to a protocol (number 20160227) approved by the Washington University School of Medicine Institutional Animal Care and Use Committee.

Mouse Surgical Endometriosis Model

We used a well-established endometriosis model in which uterine tissue from estrus-stage mice is autologously transplanted onto the peritoneal wall. After 3 weeks, the resulting endometriotic lesions are composed of a single cyst (Cummings and Metcalf, 1995; Pelch et al., 2012) and resemble those observed in human endometriosis (Fainaru et al., 2008; Umezawa et al., 2009; Korbel et al., 2010). Briefly, one uterine horn from 10-week-old, estrus-stage mice (C57BL/6, Taconic, n= 4 to 15 per group) was excised and cut longitudinally. Next, a dermal biopsy punch was used to isolate a 3-mm endometrial fragment, which was sutured to the peritoneal wall in the same mouse through a midline incision (Fainaru et al., 2008; Schreinemacher et al., 2012; Machado et al., 2016; Kiani et al., 2018). For the sham surgery, a similar procedure was performed except that a thread was sutured onto the peritoneal wall without an endometrial fragment.

Antibiotic Treatment

Twenty-four hours after endometriosis-induction surgery, mice were provided drinking water containing 0.5 g/l vancomycin, 1 g/l neomycin, 1 g/l metronidazole and 1 g/l ampicillin (VNMA) for 21 days as described previously (Rakoff-Nahoum et al., 2004). To mask the taste of the antibiotics, 2 g/l aspartame was added to the VNMA-containing water. Control mice received drinking water containing aspartame alone (Huang et al., 2015). In other experiments, mice received water contatining only 1 g/l metronidazole or 1 g/l neomycin plus aspartame, or water containing only aspartame. Then, mice were euthanised, faecal samples were collected and eutopic endometrium and endometriotic lesions were isolated. Peritoneal fluid was collected by washing the peritoneum with 1ml sterile PBS. Lesions were weighed (mg), and lesion volumes (mm3) were measured with a Vernier Calliper (VCB001, United scientific Supplies Waukegan, IL, USA) by an investigator blinded to treatment groups.

Faecal Transplantation

Faecal pellets were immediately frozen at −80°C as reported previously (Hintze et al., 2014). Faecal pellets were resuspended in phosphate-buffered saline (PBS) (one faecal pellet/0.1 ml of PBS), and 200 μl of the suspension was given by oral gavage to each mouse (Wong et al., 2017) as indicated in Figure 5A. The numbers of mice were as follows: endo-metronidazole + non-endo faeces, n = 4 and endo-metronidazole + endo faeces, n = 4.

Figure 5.

Oral gavage of faeces from endometriotic mice promotes endometriotic lesion growth in antibiotic-treated mice. (A) Schematic of experimental timeline and procedures. (BD) Representative gross images (B), volumes (C) and masses (D) of ectopic endometriotic lesions from the indicated treatment groups 28 days after surgical induction of endometriosis; 'V', 'M' and 'ME' denote endo-vehicle, endo-metronidazole + non-endo faeces, and endo-metronidazole + endo faeces, respectively; n = 4–5. (E) Representative Hematoxylin and Eosin-stained cross-section images of ectopic lesions from the indicated treatment groups; n = 4–5. Scale bars represent 200 μm (upper panel) and 500 μm (lower panel). (F) Representative cross-sectional images of ectopic lesions stained for Iba1. (G) Quantification of Iba1-positive cells counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total cells (left panel) and quantification of IL-1β concentration in peritoneal fluid from the indicated treatment groups (right panel). Data are presented as mean ± SE (n = 4–5). 'E', 'G' and 'S' denote epithelia, glands and stroma, respectively. *P< 0.05, ***P < 0.001, and ns, non-significant.

Bacterial 16S rRNA Gene Sequencing and Diversity Analysis

DNA was extracted from faecal pellets (0.1 gm) by using the QIAmp Power Faecal DNA Kit (12850-50, Qiagen) as per the manufacturer's protocol. The numbers of mice were as follows: non-endo, n = 5; endo-vehicle, n = 5; and endo-VNMA, n = 4. Amplicon generation and sequencing were performed by the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine as described previously (Parnell et al., 2017a). Fastq sequences were uploaded to the NCBI Sequence Read Archive (SUB3753571). Quantitative Insights Into Microbial Ecology analysis of variable region four was used to remove operational taxonomic units (OTUs) that were only observed once and rarefy tables to 600 OTUs. This dataset was used for downstream alpha and beta diversity analysis, and identification of the top 10 OTUs was as described previously (Parnell et al., 2017a).

Histological Analysis

Tissues were fixed in 4% paraformaldehyde, and sections were immunostained (n = 4–5 per group) as described previously (Kommagani et al., 2016). Briefly, tissue sections were blocked with 5% goat serum and then incubated overnight at 4°C in 2% goat serum containing the following primary antibodies from Abcam: Rabbit anti-ER-α (ab75635), anti-Ki-67 (ab16667) or anti-Iba1 (ab178847). Sections were then incubated with biotinylated secondary antibody and counter-stained with hematoxylin. Finally, sections were dehydrated and mounted in Permount histological mounting medium (Fisher Scientific Inc.). Ki-67- and Iba1-positive cells were counted manually in images taken at 400X magnification by two independent investigators blinded to treatment groups. Cells were counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total number of cells as described previously (Kommagani et al., 2013). For hematoxylin and eosin staining, tissue sections were fixed, processed, embedded, deparaffinized and stained as described previously (Kommagani et al., 2016).

Enzyme-linked Immunosorbent Assays

Enzyme-linked immunosorbent assays (ELISA) kits were used to measure the peritoneal concentrations of IL-1β (KMC0011, Invitrogen, Life Technologies), TNF-α (ab208348, Abcam), IL-6 (ab100712, Abcam), IL-10 (ab108870, Abcam) and TGF-β1 (ab119557, Abcam), according to the manufacturer's instructions (n = 10 per group). The intra- and inter-assay coefficients of variation for the IL-1β ELISA were 8.4% and 4.9%, respectively. Peritoneal concentrations were deduced from standard curves, and the final concentration was calculated by normalising with the total protein concentration in peritoneal fluid.

Statistics

A two-tailed paired Student's t-test was used for statistical significance testing for all data except the 16 S sequencing data, which did not follow a normal distribution. The non-parametric Kruskal–Wallis test was used to compare the Shannon diversities of microbiota from non-endo, endo-vehicle and endo-VNMA groups and the Mann–Whitney test was used to compare Shannon diversities between groups. For multidimentional scaling (MDS) plots, the R 'Phyloseq' package was used to perform permutation analysis of variance. P < 0.05 was considered significant.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....