Background: Attack by commensal microbiota is one component of induction of inflammatory episodes in ulcerative colitis (UC). In UC, the mucus layer is intrinsically devoid of phosphatidylcholine (PC) resulting in low hydrophobicity which facilitates bacterial invasion. Colonic ectophospholipase-carrying bacterial strains are likely candidates to further thinning the PC mucus barrier and to precipitate inflammatory episodes. Objective: To evaluate the effect of phospholipase A2 (PLA2) inhibitors on inflammation in a genetic UC mouse model. Methods: As PLA2 inhibitor, we applied the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) or as control 5% Tween 80 by oral gavage to intestine-specific kindlin 2 knockout mice. Results: Luminal UDCA-LPE reduced the PLA2 activity in stool by 36 ± 8%. Concomitantly no inflammatory phenotype was observed when compared to kindlin 2(–/–) mice not treated with UDCA-LPE. The improvement was documented in regard to stool consistency, calprotectin levels in stool, and macroscopic/endoscopic as well as histologic features of the mucosa. The pattern of colonic microbiota distribution obtained in the UC phenotype mice was reversed by UDCA-LPE to the control mice pattern. Conclusion: The inhibition of the bacterial ectophospholipase A2 activity improves mucosal inflammation in a genetic mouse model of UC. It is assumed that the remaining mucus PC shield is better preserved when luminal PLA2 is suppressed.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of unknown etiology, for which only symptomatic anti-inflammatory therapy is available. A defective mucosal barrier is postulated as one possible pathogenetic mechanism [1]. Indeed in patients with UC, low intrinsic mucus phosphatidylcholine (PC) content (reduction by 70% of control levels) even in remission has been documented [2, 3], which reduces surface hydrophobicity [4] and, thus, facilitates commensal bacteria to invade and induce inflammation [5]. PC is mainly secreted in the ileum and only marginally in the colon [6]. It was shown that PC is translocated from systemic sources across tight junctions (TJ) to the luminal side of mucosal cells driven by a negative electrochemical gradient generated by the cystic fibrosis transmembrane conductance regulator (CFTR) [7]. After TJ translocation, PC is bound to membrane-associated mucin 3, from where it is handled to secretory mucin 2 [5, 8]. The PC-mucin 2 complex moves within the upper mucus layer along the intestinal wall distally to the colon and rectum to provide sufficient hydrophobic protection against colonic microbiota.

Paracellular PC transport to the mucus was prohibited by TJ disruption in genetic mouse models with tamoxifen-sensitive intestinal deletion of kindlin 1 and 2 [5] with consequent induction of a UC phenotype, due to impaired surface hydrophobicity facilitating the invasion of commensal bacteria [5].

As shown in human UC patients in remission, as little as 30% remaining mucus PC content may be sufficient to maintain a hydrophobic barrier [2, 3]. Acute inflammatory episodes occur when PC is further reduced below a critical level. This is most likely caused by a colonic microbiota with enhanced ectophospholipase activity. The pathogenetic potential of such bacterial species is known from Helicobacter pylori, which carries an ectophospholipase to break the gastric mucus PC barrier for invasion and colonization of the surface of gastric mucosal cells [9]. In the gut, we postulate that the conversion to an ectophosholipase-rich microbiota by changes of the hormonal/metabolic state or by systemic inflammatory stress conditions is capable of further reducing PC-mediated hydrophobicity below a critical threshold which opens the mucus barrier for invasion by the full spectrum of commensal bacteria, resulting in mucosal inflammation. Because of its extended exposure with phospholipase-containing bacteria, the rectum is prone to mucosal inflammation in UC with its intrinsic mucus PC depletion. From there, the colitis extends to upper colonic segments depending on the degree of mucus PC reduction and thus, enhanced susceptibility of the mucosa to bacterial invasion. Thus, the hypothesis of a double-edged sword pathogenesis of UC was generated: (1) an intrinsic, genetically inherited low mucus 2-bound PC content, which reduces protective surface hydrophobicity but, per se, does not induce inflammation; (2) reduction of mucus PC level below a critical level by enhanced ectophospholipase activity of the microbiota allowing further bacterial invasion and consequent inflammation.

Therefore, therapeutically two concepts can be employed: (1) supplementation of PC to mucin 2 by delayed-released oral PC application to fill the gaps and reestablish a fully operative hydrophobic barrier. It serves for the prevention of acute inflammatory episodes and is an ideal therapy for maintenance of clinical remission. (2) Inhibition of the microbiota ectophospholipase activity as pathogenicity factor to allow mucus and mucosa bacterial invasion with consequent inflammation. This predominant anti-inflammatory concept is therefore meant for therapy of inflammatory states of UC as well as any other IBD. It may even function when the mucus is destroyed as a consequence of inflammation. As nontoxic, poorly absorbable phospholipase A2 (PLA2) inhibitor, we chose the phospholipid-bile acid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE), which in previous studies was shown to be effective in the liver [10]. The aim of the present study was to prove this hypothesis. As experimental animal model we chose intestine-specific kindlin 2(–/–) mice kept under non-germ, free conditions [11, 12]. This is an in-house generated genetic mouse model with disrupted lateral tight junctions which causes an UC phenotype [5].

Induction of a Mouse Colitis Phenotype and Its Prevention

In this non-human-based study we employed intestine-specific kindlin 2(–/–) mice and handled them as described recently [5] (online suppl. material; for all online suppl. material, see www.karger.com/doi/10.1159/000486858).

For the induction of the phenotype, female control wild-type and mutant mice (12 weeks old, body weight of 31 ± 2 g) were intraperitoneally injected with 0.2 mg tamoxifen daily for up to 3 days before being sacrificed 1 day later. Western blotting of isolated mucosal cells [5, 13] obtained from kindlin 2(–/–) mice verified depletion of the targeted kindlin 2 in comparison to wild-type controls [5]. As PLA2 inhibitor we chose UDCA-LPE (Chemi, Milan, Italy) which in vivo is degraded to UDCA and LPE [14]. Over the time of tamoxifen injection, the kindlin 2(–/–) mice were daily orally gavaged (tube of 6.5 cm in length, 9918, Codence Science, Cranston, RI, USA) either with a 100-µL bolus of 5% Tween 80 (control) or with 50 mg/kg UDCA-LPE in 5% Tween 80 (n = 6 mice for each group) [5].

Analysis of Absorption Capacity of UDCA-LPE in vitro and in vivo

For in vitro determination of transcellular movement of UDCA-LPE (apical application of 100 µM in PBS), the confluent and polarized 21-day cultured intestinal tumor cell line CaCo2 in transwell culture dishes was employed with a transmembrane electrical resistance (TER) of > 300 [7]. The basal recovery of UDCA-LPE over 1 h was determined by ESI-MS/MS [7].

For in vivo UDCA-LPE recovery studies wild-type mice were starved overnight before administration of a 100-µL bolus of 50 mg/kg UDCA-LPE. After 4 h, the mice were sacrificed and UDCA-LPE was determined by ESI-MS/MS in serum, liver homogenate, and collected luminal content of the intestine.

Clinical Evaluation of the UC Activity

After 3 days of tamoxifen treatment, ileal endoscopy was retrogradely performed as described in detail [5]. Endoscopic alterations were evaluated by the modified MEICS score [15]. In addition, ileum and colon were excised and analyzed by the macroscopic and histological colitis score [16] (online suppl. material). The calprotectin concentration in stool was determined as a marker of mucosal inflammation [17, 18] using the S100A9/calprotectin, mouse, ELISA kit (Hycult Biotech, Uden, The Netherlands).

Impact of UDCA-LPE on Microbiota

We analyzed first the PLA2 activity in stool under control wild-type, colitis and additional UDCA-LPE pretreatment conditions [10, 19].

Microbiota analysis was performed by bacterial 16S rRNA-sequencing (variable V3-V6 region) of the collected stool samples (MicroBIOMix GmbH, Regensburg, Germany) (online suppl. material).

Statistical Analysis

Each experiment was repeated 6-fold. Statistical analysis was performed using Prism 4.0 software (GraphPad Software Inc., La Jolla, CA, USA). Differences between groups were evaluated using the Mann-Whitney U test. Multiple groups were compared by one-way ANOVA with a Dunnett post hoc test. For the microbiota analysis, the statistical software SPSS was used (IBM Corp., Armonk, NY, USA). Data are presented as means ± SD or medians with range, and p < 0.05 was considered statistically significant.

Previously it was shown that UC is due to an intrinsic low PC content of the mucus, which impairs surface hydrophobicity and allows microbiota invasion followed by mucosal inflammation [2-5]. PC passes from systemic sources along a paracellular route between enterocytes across the TJ barrier to the luminal side for incorporation into mucus [7]. When the PC translocation mechanism is disturbed, UC can develop [5]. The predisposition to inflammatory episodes is enhanced by the phospholipase activity of the microbiota because of the further thinning of the mucus PC layer. The rationale of this study was to prove this hypothesis.

Intestinally Deleted Kindlin 2 Mice Reveal an UC Phenotype

As model we chose adult mice with tamoxifen-inducible, villin-Cre-mediated intestinal deletion of kindlin 2, which is known to activate integrin β1 as control instance of TJ stabilization [11, 12]. Western blot analysis of isolated mucosal cells in mutants revealed the completeness of the kindlin 2 knockout and a decrease in representative proteins constituting the link from kindlin 2 to integrin β1 activation, to adherence junctions (e-cadherin), and finally to TJs (ZO1, occludin, claudin 2) [5]. Preceding freeze-fracture electron microscopy studies [5] showed disturbed TJs which occurred already in the preinflammatory state after 2 days of tamoxifen exposure of the mutant mice. In the present study, also a general morphometric light microscopy analysis of mucosal biopsies after HE staining was performed (Fig. 1). The crypt luminal diameter (ratio of crypt lumen to total crypt diameter) in the ileum (at the same position within the crypt) of wild-type mice was 0.082 ± 0.021 and in kindlin 2(–/–) mice 0.162 ± 0.036 and in kindlin 2(–/–) mice with concomitant UDCA-LPE application 0.187 ± 41. While kindlin 2(–/–) mutants had enlarged lumina compared to wild-type mice (p < 0.01), the treatment with UDCA-LPE had no impact on the diameter (p > 0.05) (Fig. 1).

Fig. 1.

Endoscopic and histologic features of the ileum in wild-type mice (a) kindlin 2(–/–) mice orally gavaged with Tween 80 only (b) and kindlin 2(–/–) mice orally gavaged with UDCA-LPE in Tween 80 (c) over the 3 days of tamoxifen exposure (scale bars = 25 µM).

Fig. 1.

Endoscopic and histologic features of the ileum in wild-type mice (a) kindlin 2(–/–) mice orally gavaged with Tween 80 only (b) and kindlin 2(–/–) mice orally gavaged with UDCA-LPE in Tween 80 (c) over the 3 days of tamoxifen exposure (scale bars = 25 µM).

Close modal

The disturbed TJ barrier due to kindlin 2 deletion results in impaired luminal PC secretion with the pathophysiologic consequences of UC development [5]. Indeed, as also demonstrated in the present study, after 3 days of tamoxifen exposure, intestinally deleted kindlin 2(–/–) mice expressed full-blown UC phenotype which was not observed in control mice exposed to tamoxifen (Fig. 1). The stool was soft, amorphic, sticky, and contained blood. The severe inflammatory phenotype of kindlin 2(–/–) mice appeared with an opaque, thickened granular mucosa, absent vascular pattern with visible fibrin adherence, ulcerations and hemorrhagic lesions. Kindlin 2(–/–) mice exhibited a significantly higher MEICS score compared to the concomitantly UDCA-LPE-treated mice (13.3 ± 1.7 vs. 1.8 ± 1.3; p < 0.001) compared to control wild-type mice (1.3 ± 0.6; p > 0.05). The edema of the intestinal wall resulted in colonic weight gain of 202.2 ± 38.7% and length reductions by 32.3 ± 11.3% in kindlin 2(–/–) mice, respectively, versus control wild-type mice (p < 0.05 for both parameters; n = 6; Fig. 2; Table 1). In mutants, histopathology after HE staining revealed destroyed crypt architecture, mucosal and submucosal inflammation with infiltration of neutrophils and lymphocytes, ulcerations, and edema (Fig. 1; Table 1).

Table 1.

Macroscopic and histopathological evaluation of the inflammatory phenotype in wild-type and kindlin 2(–/–) mice orally gavaged with 5% Tween 80 only or UDCA-LPE in Tween 80 over the 3 days of tamoxifen exposure

Macroscopic and histopathological evaluation of the inflammatory phenotype in wild-type and kindlin 2(–/–) mice orally gavaged with 5% Tween 80 only or UDCA-LPE in Tween 80 over the 3 days of tamoxifen exposure
Macroscopic and histopathological evaluation of the inflammatory phenotype in wild-type and kindlin 2(–/–) mice orally gavaged with 5% Tween 80 only or UDCA-LPE in Tween 80 over the 3 days of tamoxifen exposure
Fig. 2.

Colonic weight (including stool), colonic length, and calprotectin concentration in stool of wild-type control, in kindlin 2(–/–) mice injected with tamoxifen and orally gavaged with Tween 80 only, and in kindlin 2(–/–) mice injected with tamoxifen and concomitantly orally gavaged with UDCA-LPE in Tween 80 (means ± SD, * p < 0.05).

Fig. 2.

Colonic weight (including stool), colonic length, and calprotectin concentration in stool of wild-type control, in kindlin 2(–/–) mice injected with tamoxifen and orally gavaged with Tween 80 only, and in kindlin 2(–/–) mice injected with tamoxifen and concomitantly orally gavaged with UDCA-LPE in Tween 80 (means ± SD, * p < 0.05).

Close modal

We postulated that an intrinsic low mucus PC constitutes a risk factor for bacterial invasion [20]. Accordingly, predominance of ectophospholipase carrying bacteria in stool can critically reduce the PC barrier. Indeed, the PLA2 activity in stool was increased, either due to lack of PC or, more likely, due to changes of luminal environment or the applied stress for the animals (Fig. 3). Thus, inhibition of PLA2 could be of therapeutic benefit.

Fig. 3.

Effect of UDCA-LPE on PLA2 activity in mouse stool (means ± SD, * p < 0.05, ** p < 0.01).

Fig. 3.

Effect of UDCA-LPE on PLA2 activity in mouse stool (means ± SD, * p < 0.05, ** p < 0.01).

Close modal

UDCA-LPE as Poorly Absorbable PLA2 Inhibitor

To ensure that UDCA-LPE as nontoxic PLA2 inhibitor [14] exerts its activity mainly on the luminal microbiota, we examined the 1-h absorption capacity in vitro employing polarized CaCo2 cells as well as in vivo by determination of its concentration in stool, serum and liver 4 h after oral gavage application. The in vitro absorption capacity was 5.3 ± 2.4% per hour. In vivo, 4 h after oral gavage, 85.0 ± 20.7% of the UDCA-LPE was recovered in the intestinal lumen, whereas in total serum only 14.8 ± 7.7% and in total liver 0.75 ± 0.11% of applied UDCA-LPE were recorded. The experiments indicate that absorption of orally applied UDCA-LPE to the system is poor and the bulk of UDCA-LPE stays in the gut lumen. The postulated action of UDCA-LPE as PLA2 inhibitor in stool was verified in stool samples of control mice. Incubation of 1 g mouse stool with 100 µM UDCA-LPE over 1 h reduced the PLA2 activity by 34 ± 7% (n = 6; p < 0.001).

Efficacy of UDCA-LPE on Stool PLA2 Activity and Clinical Presentation in Mutant Mice with an UC Phenotype

Assuming that the commensal colonic bacteria contain ectophospholipases which are activated by stress conditions with consequent further reduction of the mucus PC content, we treated the kindlin 2(–/–) mice by oral gavage with the poorly absorbed PLA2 inhibitor UDCA-LPE [10, 14]. Indeed, PLA2 activity in stool could be reduced compared to nontreated and even the wild-type mice (Fig. 3). Also demonstrated was the prevention of colitis, which was confirmed by ileoscopy, the macroscopic and histologic features described by the major colitis score (Fig. 1, 2; Table 1). Moreover, as most sensitive inflammatory read-out for intestinal inflammation the calprotectin concentration in stool was determined [18]. The value dropped from 2.101 ± 0.276 to 1.172 ± 0.341 mg/g stool in UDCA-LPE-treated mutants (wild-type control 0.976 ± 0.225 mg/g stool) (p < 0.05).

Impact of UDCA-LPE on Intestinal Microbiota

A comparison of the colonic microbiota of kindlin 2(–/–) mice with and without UDCA-LPE treatment showed that mice without treatment experienced an expanded quantitative distribution of the various phyla, which was not observed in mice with UDCA-LPE. Indeed, the microbiota observed in the presence of UDCA-LPE was identical to that observed in control wild-type mice (Fig. 4; online suppl. Table S1). The most significant change was observed in regard to bacteroides. These phyla are considered as a cytoprotective microbiota [21], which is suppressed in mutant mice with a colitis phenotype and reversed to control levels by UDCA-LPE pretreatment. In addition, the S24-7 bacterial family was significantly reduced in the mutant mice and reversed to control values by UDCA-LPE (p < 0.05). All of the remaining bacterial phyla remained within the control range (Table 1).

Fig. 4.

Effect of UDCA-LPE on the diversity of bacterial phyla in mouse stool without and with UDCA-LPE application in Tween 80 by oral gavage to kindlin 2(–/–) mice in comparison to controls. The measured percentage distribution at the top is illustrated by cake diagrams at the bottom (means ± SD, * p < 0.05).

Fig. 4.

Effect of UDCA-LPE on the diversity of bacterial phyla in mouse stool without and with UDCA-LPE application in Tween 80 by oral gavage to kindlin 2(–/–) mice in comparison to controls. The measured percentage distribution at the top is illustrated by cake diagrams at the bottom (means ± SD, * p < 0.05).

Close modal

A new option for UC treatment targets the replenishment of the genetically determined depleted mucus PC content by oral application of a delayed-released PC preparation [22]. It was shown to restore the mucus PC content, which reestablished the barrier against microbiotic invasion and, thus, fighting mucosal inflammation [5, 22-24]. Assuming that genetically low mucus PC content is capable of maintaining barrier function up to a borderline concentration of 30% compared to normal subjects [2, 20], it could be postulated that a drop below this critical level allows bacterial invasion with consequent mucosal inflammation. Accordingly, inflammatory episodes in UC require an impaired mucosal barri er – low mucus PC content – and the invasion of bacteria: the double-edged sword for the pathogenesis of UC. Bacterial invasion requires threshold PC levels to be broken, which can be accomplished by ectophospholipase-containing bacteria. Consequently, the commensal stool flora can invade and cause inflammation.

The ectophospholipase-containing group of the gut microbiota species is not well defined and was not further evaluated in this study. However, it would be interesting to evaluate whether it is a feature of few specific species or abundant in most of the species with different levels of activity. Also of interest is whether specific microbiota – known to be of therapeutic value (e.g., Escherichia coli Nissle) – have low ectophospholipase activity and are able to displace those species with high enzymatic abundance. In our study, we observed a suppression of bacteroides and S24-7 bacteria together with an increase in stool phospholipase activity in the inflammatory state of the colitis phenotype. It is likely that they are secondarily suppressed by the applied stress for the animals, thus leaving space for more aggressive phospholipase-rich microbiota. However, when PLA2 is inhibited by the nontoxic, poorly absorbed UDCA-LPE, both species reverse to normal distribution. The PLA2 activity of total microbiota is significantly reduced, thereby mitigating the pathogenetic potential of bacterial-induced mucus PC consumption, and therefore the consequent mucus invasion by the commensal microbiota leading to mucosal inflammation. Accordingly, colitis/ileitis was significantly abrogated.

The concept of manipulating the microbiota for prevention/treatment of acute episodes of IBDs represents a defensive and rather safe therapeutic strategy. It is already supported by application of the topical acting antibiotic rifaximin which revealed impressive clinical improvement in Crohn’s disease [25]. In UC, a dual therapeutic strategy could be considered: (1) increasing the mucus barrier by supplementation of a delayed-released PC preparation, as maintenance of remission and (2) for inflammatory states the application of a PLA2 inhibitor such as UDCA-LPE to induce remission. Indeed reducing the pathogenetic potential of the gut microbiota by UDCA-LPE or rifaximin is supported by the observation that UC does not occur without the gut microbiota, a phenomenon which is indeed proven in many genetic IBD models under germ-free conditions [26, 27]. The principle anti-inflammatory activity of PLA2 inhibitors could also be utilized for other inflammatory diseases of the intestine. Therefore, we plan to test UDCA-LPE also in future mouse colitis models.

UDCA-LPE is an example of an intestinally working luminal PLA2 inhibitor. Whether other phospholipases (C or D) may also be of relevance has yet to be determined. It is a first in class antimicrobial directed PLA2 inhibitor with absent toxicity. Derivatives of this bile acid-lysophospholipid compound could be equally effective. There may be other structurally different PLA2 inhibitors, but their potential toxicity after absorption has to be considered.

In this study, the proof of principle was supported, i.e., that luminally acting PLA2 inhibitors may be of potential therapeutic use for intestinal inflammation by reduction of the pathogenetic ectophospholipases of microbiota.

We thank R. Faessler (MPI, Munich) for providing the conditional kindlin 2 knockout mice and F. Lasitschkaa (Department of Pathology, Heidelberg) for biopsy preparation.

Animal studies followed the “ARRIVE” guidelines and were approved by the Heidelberg ethical committee (ref. No. 35-9185.81/6123/10 and 6284/11). This is a non-human-based study.

The authors have no competing interest. W.S. is the inventor of the use of UDCA-LPE as an antimicrobial drug in the intestine. The University of Heidelberg holds this patent.

This study was supported by the German Research Foundation (ref. No. STR 216/15-4).

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