An Analysis of the Role of the Indigenous Microbiota in Cholesterol Gallstone Pathogenesis

Background and Aims: Cholesterol gallstone disease is a complex process involving both genetic and environmental variables. No information exists regarding what role if any the indigenous gastrointestinal microbiota may play in cholesterol gallstone pathogenesis and whether variations in the microbiota can alter cholesterol gallstone prevalence rates. Methods: Genetically related substrains (BALB/cJ and BALB/cJBomTac) and (BALB/AnNTac and BALB/cByJ) of mice obtained from different vendors were compared for cholesterol gallstone prevalence after being fed a lithogenic diet for 8 weeks. The indigenous microbiome was altered in these substrains by oral gavage of fecal slurries as adults, by cross-fostering to mice with divergent flora at <1day of age or by rederiving into a germ-free state. Results: Alterations in the indigenous microbiome altered significantly the accumulation of mucin gel and normalized gallbladder weight but did not alter cholesterol gallstone susceptibility in conventionally housed SPF mice. Germ-free rederivation rendered mice more susceptible to cholesterol gallstone formation. This susceptibility appeared to be largely due to alterations in gallbladder size and gallbladder wall inflammation. Colonization of germ-free mice with members of altered Schaedler flora normalized the gallstone phenotype to a level similar to conventionally housed mice. Conclusions: These data demonstrate that alterations in the gastrointestinal microbiome may alter aspects of cholesterol gallstone pathogenesis and that in the appropriate circumstances these changes may impact cholesterol cholelithogenesis.National Institutes of Health (U.S.) (Grant T32OD010978)National Institutes of Health (U.S.) (Grant P30ES002109)National Institutes of Health (U.S.) (Grant R01AT004326

Components of the biliary phenotype may be altered by crossfostering mice shortly after birth.

<p>(A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding. (A) Both Tac and Jax fostered to Tac developed significantly larger gallbladders (*P<0.05) compared to Jax mice. (B) Jax mice displayed significant (*P<0.05) elevations in mucin gel accumulation compared to all other groups including Jax fostered to Tac. (D–E) Jax mice and Jax fostered to Tac developed significantly more (P<0.05) cholesterol monohydrate and sandy stones then either group from Taconic. (F) Tac mice developed significantly (P<0.05) less cholesterol gallstones then Jax mice and both fostered groups displayed intermediate and non-significantly different levels of cholesterol gallstone formation.</p

Transfer of Taconic flora to Jackson mice leads to significant increases in ASF457 (A) and 361.

<p>ASF361 (A) and 457 (B) were quantified from a cohort of cross-faunated mice (n = 5) by real-time PCR. There were significant elevations in the colonization of these two bacteria in mice that were of Taconic origin or those which received Taconic flora (*P<0.05). Colonization of Taconic mice with Jackson flora did not significantly diminish the colonization by these organisms.</p

Biliary phenotype of genetically related BALB/cAnNTac (Tac) and BALB/cByJ (ByJ) and BALB/cJBomTac (Bom) and BALB/cJ (Jax) mice.

<p>(A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding. (A) Normalized gallbladder weight significantly differed between Tac mice and both ByJ and Jackson mice (*P<0.05). (B) Mucin gel score for both BALB/cJ and BALB/cJBomTac mice was significantly elevated compared to either Tac or ByJ mice (P<0.05). (C) Liquid crystal phase separation occurred in all animals studied. (D) Cholesterol monohydrate crystal formation occurred significantly more in BALB/cJ mice compared to both ByJ and Tac mice (*P<0.05). (E) Both BALB/cJ and BALB/cJBomTac mice compared to either Tac or ByJ mice displayed significantly increased sandy stone formation (*P<0.05). (F) Cholesterol gallstone formation was significantly increased in BALB/cJ and BALB/cJBomTac mice compared to Tac mice (*P<0.05).</p

Crossfostering of mice leads to significant alterations in ASF361 and 457.

<p>ASF361 (A) and 457 (B) were quantified from a cohort (n = 5) of cross-fostered mice by real-time PCR. There were significant elevations in the colonization of these two bacteria in mice of Jackson origin crossfostered to Taconic mothers and significant decreases in Taconic pups crossfostered to Jackson mothers (*P<0.05) indicating effective alteration of the flora.</p

Germ-free mice are more susceptible to cholesterol cholelithogenesis (A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding.

<p>(A) Germ-free mice developed significantly increased normalized gallbladder weights compared to either conventional or ASF colonized mice (*P<0.05). Mucin gel score did not significantly differ among groups (all conventional and ASF mice developed mucin gel scores of “1”). (D–E) Germ-free mice developed significantly more cholesterol monohydrate (D, *P<0.05) and Sandy stones (E, *P<0.05) and had an non-significant increase in cholesterol gallstones (F).</p

Mucin gene expression is significantly increased in germ-free mice compared to conventionally reared mice.

<p>(A) <i>Muc1</i>, (B) <i>Muc3</i>, and (C) <i>Muc4</i> are all significantly (<i>P</i><0.05) increased (∼2.5–4.5 fold) in germ-free mice when compared to conventionally reared mice. Neither (D) <i>Muc5ac</i> nor (E) <i>Muc5b</i> displayed any differences and expression was highly variable among mice.</p

Components of the biliary phenotype may be altered by transferring flora to adult mice.

<p>(A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding. (A) Both Jax and Tac mice colonized with Jax flora developed significantly smaller gallbladders when compared to Tac mice (*P<0.05) (B) Jackson origin mice regardless of flora produced higher mucin scores compared to control Taconic mice (*P<0.05). (D) Flora transferred Jax mice displayed significant increases in cholesterol monohydrate crystal formation compared to control Tac mice (*P<0.05) and control Jax mice displayed significant increases compared to both Taconic groups (**P<0.05). (E) Both groups of Jackson mice displayed significant increases in sandy stone formation compared to both groups of Taconic mice (P*<0.05). Control Taconic mice displayed significant increases in sandy stones when compared to Taconic mice that underwent crossfaunation (*P<0.05). (F) Jax mice displayed significant increases in cholesterol gallstone formation compared to both groups of Tac mice (*P<0.05).</p

Germ-free mice develop significantly more severe gallbladder histopathological changes compared to ASF colonized mice.

<p>(A, B) Low power and high power image of gallbladders from Germ-free mice demonstrate hyperplasia (arrowhead), inflammation (arrow) and the presence of eosinophilic luminal material which is consistent with mucin gel (asterisk). (C) Overall, gallbladder histopathology score which examines inflammation, edema, and hyperplasia was significantly increased (*P<0.05) in Germ-free mice compared to ASF colonized mice and nearly significant (P = 0.07) when compared to conventional mice. Both ASF colonized (D, low power, E, high power) and conventional (F, high power) mice display minimal inflammation and maintain a normal epithelial lining of a single layer of cuboidal epithelium.</p