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

Results

Treatment With Broad-spectrum Antibiotics Reduces Endometriotic Lesion Growth, Proliferation and Inflammation

To determine whether antibiotics affect early endometriotic lesion growth, we treated mice with the broad-spectrum antibiotics VNMA in drinking water containing aspartame to mask the antibiotics taste. Control mice received drinking water containing aspartame alone. We then performed endometriosis-induction surgery (Figure 1A). Mice that consumed VNMA (VNMA-endo) had smaller endometriotic lesions than those that consumed vehicle alone (vehicle-endo) (Figure 1B–D). In a second experiment aimed at assessing progression of established endometriotic lesions, we treated mice with antibiotics after endometriosis surgery (Figure 1E). Lesions were smaller in mice that consumed VNMA (endo-VNMA) than in those that consumed vehicle (endo-vehicle) (Figure 1F–H). These two experiments indicated that antibiotic treatment reduced both early growth and progression of endometriotic lesions.

Figure 1.

Treatment with broad-spectrum antibiotics prevents early endometriotic lesion growth and progression. (A and E) Schematic of experimental timeline and procedures. (B–D and F–H) Representative gross images (B and F), volumes (C and G), and masses (D and H) of ectopic endometriotic lesions from the indicated treatment groups 21 days after surgical induction of endometriosis. Data are presented as mean ± SE(n = 5) *P < 0.05, **P < 0.01.

To begin to uncover the mechanism by which antibiotics affected endometriotic lesion progression, we treated mice with antibiotics immediately after endometriosis surgery and performed a series of analyses (Figure 2). First, we confirmed that neither surgery nor antibiotic treatment had any effect on water consumption or body weight (Supplementary Figure S1). Second, hematoxylin and eosin staining revealed that whereas lesions from endo-vehicle mice had typical endometriosis-like structures, including a thick epithelial layer and glandular areas, lesions from endo-VNMA mice had thinner epithelial areas and no glands (Figure 2E). Additionally, consistent with reports that stromal tissue volume correlates with lesion growth (Korbel et al., 2010), lesions from endo-VNMA mice had smaller stromal areas than lesions from endo-vehicle mice (Figure 2E). Importantly, the eutopic uteri had similar epithelial, glandular and stromal areas in both endo-vehicle and endo-VNMA mice (Figure 2E). Third, we stained the lesions with an antibody specific to estrogen receptor alpha (ERα), which is thought to promote proliferation and inflammation and thus drive endometriotic lesion growth and expansion (Huhtinen et al., 2012). However, ERα expression was similar between lesions from endo-vehicle and endo-VNMA mice (Figure S2). Furthermore, consistent with a report that stage of estrous had no impact on lesion growth (Fainaru et al., 2008; Schreinemacher et al., 2012; Machado et al., 2016; Kiani et al., 2018), lesion volumes did not appear to correlate with the stage of estrous at sacrifice (data not shown).

Figure 2.

Treatment with broad-spectrum antibiotics reduces endometriotic lesion proliferation and inflammation. (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 21 days after surgical induction of endometriosis. Data are presented as mean ± SE; endo-vehicle (n = 15) and endo-VNMA (n = 14). (E) Representative Hematoxylin and Eosin-stained cross-section images of the eutopic uteri and ectopic lesions from the indicated treatment groups. The scale bar (0.5 μm) applies to all images; n = 5. (FG) Representative cross-sectional images (left) of the eutopic uteri and ectopic lesions stained for Ki-67 (F) and Iba1 (G); respective graphs on the right show positively stained cells counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total cells. The scale bar (0.5 μm) applies to all images; n = 5. 'E', 'G' and 'S' denote epithelia, glands and stroma, respectively. (H) ELISA-based quantification of IL-1β, TNF-α, IL-6, IL-10 and TGF-β1 levels in peritoneal fluid from the indicated treatment groups. Data are presented as mean ± SE (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, non-significant.

Supplementary Figure S1.

Surgery and antibiotic treatment have no effect on water consumption or body weight. Graphs show the water consumption (A) and body weight (B) of mice (n = 5 per group) in the indicated treatment groups on the indicated days after surgery. Differences in water consumption or body weights of the different groups of mice at any given time point were not statistically significant.

Supplementary Figure S2.

The effect of antibiotic treatment on endometriotic lesion progression is independent of estrogen receptor alpha. (A) Representative ER-α immunohistochemistry images of the eutopic uteri and ectopic lesions from the indicated treatment groups. The scale bar (0.5 μm) applies to all images; n = 5.

Fourth, we assessed epithelial proliferation, which is a hallmark of endometriosis in women and is widely used to assess disease progression in rodent models of endometriosis (Wu et al., 2006; Celik et al., 2008; Burney and Giudice, 2012; Han et al., 2012, 2015; Ozer et al., 2013; Song et al., 2014; Zhao et al., 2015). Consistent with their larger size, lesions from endo-vehicle mice had significantly more epithelial cells that were positive for the proliferation marker Ki-67 than did lesions from endo-VNMA mice (Figure 2F). Fifth, we examined macrophage infiltration in lesions because macrophages drive lesion growth and vascularisation in a mouse model of endometriosis. As illustrated by the macrophage marker Iba1 (Lin et al., 2006; Bacci et al., 2009; Capobianco et al., 2011), lesions from endo-vehicle mice contained significantly more macrophages than did lesions from endo-VNMA mice (Figure 2G). Finally, we measured peritoneal concentrations of IL-1β, as this cytokine is elevated in the peritoneal fluid and peritoneal macrophages of women with endometriosis (Mori et al., 1992; Lebovic et al., 2000). Endo-vehicle mice had higher peritoneal IL-1β than did endo-VNMA mice (Figure 2H left panel). Similarly, endo-vehicle mice had higher peritoneal concentrations of TNF-α, IL-6 and TGF-β1 than did endo-VNMA mice (Figure 2H). Together, these data indicate that treatment with broad-spectrum antibiotics reduces endometriotic lesion proliferation and peritoneal inflammation.

Composition of the gut Microbiota is Altered in Mice With Endometriotic Lesions

To determine the effect of broad-spectrum antibiotics on gut microbial composition, we performed 16 S rRNA gene sequencing of DNA isolated from faecal samples from endo-vehicle and endo-VNMA mice. Additionally, we included mice that did not undergo endometriosis-inducing surgery (non-endo). As shown in Supplementary Figure S3A, microbial diversity (alpha, or Shannon, Diversity) was higher in faeces from non-endo and endo-vehicle mice than in faeces from endo-VNMA mice. MDS analysis uniquely clustered each group, suggesting distinct bacterial community profiles in non-endo, endo-vehicle and endo-VNMA faecal samples (Supplementary Figure S3B). We calculated three metrics of between-group diversity (beta diversity) and noted the greatest microbial diversity in endo-vehicle mice and lowest diversity in endo-VNMA mice (Supplementary Figure S3C). Furthermore, the faecal bacterial composition of endo-VNMA mice was broadly dissimilar from that of either non-endo or endo-vehicle mice (Supplementary Figure S3B–C). This analysis demonstrated that antibiotic treatment altered the enteric bacterial diversity.

To determine whether the unique enteric bacterial profiles were attributed to specific taxa, we profiled the phyla across samples in each group. Faecal samples from endo-vehicle mice contained a higher abundance of Bacteroidetes and lower abundance of Firmicutes than samples from non-endo mice (Figure 3A). In contrast, faecal samples from endo-VNMA mice contained negligible abundance of Bacteroidetes and Firmicutes but had increased abundance of Proteobacteria (Figure 3A). We confirmed these findings by analysing the 10 most abundant OTUs in the datasets (Figure 3B). We next examined bacteria at the genus level and detected Bacteroides genera in the endo-vehicle mice but not in non-endo or endo-VNMA mice (Figure 3 C–D). The Bacteroides genus are gram-negative, non-spore-forming, anaerobic bacteria that are part of the endogenous microbiota of humans and other mammals (Brook, 1989). Finally, to assess whether surgery altered faecal microbial composition, we performed sham surgery on a group of mice. After 3 weeks, the abundances of Bacteroidetes and Firmicutes in these mice were similar to those in non-endo mice (Supplementary Figure S4A–B), indicating that surgery had no effect on gut bacteria composition. We conclude that the gut microbial composition was altered in mice with endometriosis.

Figure 3.

Bacteroides are enriched in faeces from mice with endometriotic lesions. (A) Heat map representation of relative abundances of the phyla in faecal samples from non-endo (n = 5), endo-vehicle (n = 5) and endo-VNMA (n = 4) mice. (B) Stacked bar plots of the phyla belonging to the 10 most abundant OTUs. (C) Heat map depiction of the relative abundances of the genera in each faecal sample. (D) The genera belonging to the 10 most abundant OTUs across each group are shown as stacked bar plots.

Supplementary Figure S3.

Microbial diversity analysis. (A) Intra-group Shannon diversity analysis of faecal samples from the indicated groups. (B) MDS/PCoA analysis of faecal microbiota from the indicated treatment groups. PERMANOVA: bray curtis (bray) P = 0.001; unweighted UniFrac (unifrac) P = 0.001; weighted UniFrac (wunifrac) P = 0.001. (C) Average pairwise intra-group and intergroup distances were calculated with the indicated metrics and presented as box-and-whiskers plots (n = 4–5).

Supplementary Figure S4.

Sham surgery had no effect on endometriosis-associated bacteria. Quantification of relative abundances of Bacteroidetes (A) and Firmicutes (B) in faeces from mice in the indicated groups. Data are presented as mean ± SE (n = 5).

Metronidazole-sensitive gut Bacteria may Promote Endometriotic Lesion Growth

Because members of the Bacteroides genus are highly susceptible to metronidazole and are resistant to neomycin (Ingham et al., 1968; Sutter et al., 1973; Yehya et al., 2013), we examined the effects of metronidazole and neomycin individually on endometriotic lesion growth. Mice treated with metronidazole alone (endo-metronidazole) developed ectopic lesions that were significantly smaller in volume and mass than those that developed in endo-vehicle mice (Figure 4A–C). In contrast, mice treated with neomycin alone (endo-neomycin) developed similarly sized ectopic lesions as endo-vehicle mice (Figure 4A–C). Histological analysis revealed that lesions from endo-metronidazole mice lacked the typical endometriosis-like appearance (e.g. glands and thick epithelial layer) seen in lesions from endo-vehicle and endo-neomycin mice (Figure 4D). Consistent with endometriotic lesion growth, metronidazole-treated mice had fewer macrophages in lesions and less IL-1β in the peritoneal fluid than vehicle- or neomycin-treated mice (Figure 4E–F). Together, these data indicate that metronidazole suppresses endometriotic lesion growth in mice, possibly by reducing Bacteroides growth.

Figure 4.

Metronidazole treatment reduces endometriotic lesion growth. (AC) Representative gross images (A), volumes (B) and masses (C) of ectopic endometriotic lesions from the indicated treatment groups; (n = 5). (D) Representative Hematoxylin and Eosin-stained cross-section images of ectopic lesions from the indicated treatment groups; n = 5. Scale bars represent 200 μm (upper panel) or 500 μm (lower panel). (E) Quantification of IL-1β concentration in peritoneal fluid from the indicated treatment groups. (F) Representative cross-sectional images of ectopic lesions stained for Iba1; graph on the right shows the number of positively stained cells counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total cells. 'E', 'G' and 'S' denote epithelia, glands and stroma, respectively. Data are presented as mean ± SE; (n = 5). *P < 0.05, ***P < 0.001, ****P < 0.0001, and ns, non-significant.

Faeces From Endometriotic Mice Promotes Endometriotic Lesion Progression

Given our observation that faeces from endo-vehicle mice contained more Bacteroides than faeces from non-endo mice, we wondered whether this altered gut bacteria in the faeces from mice with endometriosis was sufficient to drive endometriosis progression. To address this possibility, we performed endometriosis-induction surgery on Day 0, provided mice with metronidazole in drinking water on Days 1 through 5, orally gavaged the mice with PBS containing faeces from mice with or without endometriosis on Days 7 and 14, and examined lesions on Day 28 (illustrated in Figure 5A). Endo-metronidazole mice gavaged with faeces from mice with endometriosis (endo-faeces) developed endometriotic lesions that were similar in mass and volume to those in endo-vehicle mice. In contrast, endo-metronidazole mice gavaged with faeces from mice without endometriosis (non-endo faeces) developed significantly smaller lesions (Figure 5B–D). As a control, we examined endometriotic lesion growth in mice that were not gavaged with faeces but were allowed to recover from metronidazole until Day 28. As expected, endometriotic lesions were significantly smaller in these mice than in those that did not receive metronidazole (Supplementary Figure S5). We observed typical endometriosis-like histology (presence of glands and thick epithelial layer) in lesions from endo-metronidazole mice gavaged with faeces from endo-mice (Figure 5E). In contrast, lesions from endo-metronidazole mice gavaged with faeces from non-endo mice lacked glands and had a thin epithelial layer (Figure 5E). Furthermore, endo-metronidazole mice that received endo-faeces contained more macrophages in lesions and more IL-1β in the peritoneal fluid than endo-metronidazole mice that received non-endo faeces (Figure 5F–G). Taken together, these findings suggest a role for gut microbiota in endometriosis disease progression.

Supplementary Figure S5.

Withdrawal of metronidazole does not restore endometriotic lesion growth. (A) Schematic of experimental timeline and procedures. (B–D) 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. Data are presented as mean ± SE; (n = 4) **P < 0.01 and ***P < 0.001.

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