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Review

Phosphatidylcholine in Intestinal Mucus Protects against Mucosal Invasion of Microbiota and Consequent Inflammation

by
Wolfgang Stremmel
1,* and
Ralf Weiskirchen
2
1
Medical Center Baden-Baden, D-76530 Baden-Baden, Germany
2
Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany
*
Author to whom correspondence should be addressed.
Livers 2024, 4(3), 479-494; https://doi.org/10.3390/livers4030034
Submission received: 4 July 2024 / Revised: 21 August 2024 / Accepted: 30 August 2024 / Published: 23 September 2024
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Abstract

:
Intestinal mucus serves as the first line barrier within the mucosa to protect against microbiota attack due to its water-repellent properties, which are the result of the high abundance of phosphatidylcholine bound to mucins. A deficiency of mucus phosphatidylcholine predisposes it to mucosal inflammation by the attack of commensal microbiota, as it is intrinsically low in ulcerative colitis. However, for precipitation of an acute inflammatory episode, mucus phosphatidylcholine has to fall below the critical level required for mucosal protection. Bacterial ectophospholipase could be a candidate for further thinning of the mucus phosphatidylcholine shield as shown, for example, with the ectophospholipase containing Helicobacter pylori bacterium. Despite supporting evidence for this mechanism in the intestine, the responsible ectophospholipase-carrying bacteria species are still to be defined. Applying phosphatidylcholine to the lumen can serve to fill up empty mucin-binding sites in ulcerative colitis as well as provide a substrate for the ectophospholipase-carrying bacteria preventing their attacks on the mucus phosphatidylcholine layer. Evidence supporting this concept comes from clinical trials in humans with ulcerative colitis as well as from colitis mouse models where phosphatidylcholine was substituted in the lumen. An alternative strategy could involve adding non-absorbable phospholipase inhibitors to the intestinal lumen, which has been shown to be effective in a mouse model of ulcerative colitis. Bacterial phospholipase should be considered a pathogenetic factor of the intestinal microbiota and therapeutic strategies should be developed to prevent their hyperactivity for clinical improvement of intestinal inflammation.

1. Introduction

The liver and gut are closely connected because they share the same embryonic origin within the endoderm, and both serve for the absorption of nutrients and protection against aggressors such as the microbiota or chemical exposure. They both have mucosal epithelia facing the luminal compartments of the biliary and intestinal systems, as well as a mucus layer, and share diseases of unknown etiology such as primary sclerosing cholangitis (PSC) or inflammatory bowel diseases (IBD). In fact, most cases of PSC are associated with ulcerative colitis, suggesting a common underlying pathogenesis that has recently been addressed by our group [1,2,3]. Our focus of interest in the liver and gut is the hydrophobic mucus shield. What we have learned about the interaction of the gut mucosa and intestinal lumen may also be applicable to the liver and bile ensemble. In both cases, it is the mucus phosphatidylcholine (PC) content that exerts hydrophobicity. In the biliary system, the mucus PC prevents the exposure of bile acids from the aqueous biliary compartment to the surface of biliary epithelial cells. This helps to avoid a bile acid-mediated detergent-like destruction that could potentially also lead to bacterial invasion. Among the microbiota, we postulate that there are also PC-consuming bacteria that further break PC out of the mucus layer.
The fact is that several bacteria have been shown to utilize ectophospholipase to consume phospholipids as nutrients [4,5,6,7,8,9]. The type of phospholipase (PL), whether secretory, cytosolic, or membrane-associated, and its activity varies towards their respective target structures among different bacterial species. Phospholipase A hydrolyzes the fatty acid esters from either the sn-1 (PLA1) or sn-2 (PLA2) position of the glycerol backbone. Phospholipase C (PLC) hydrolyzes the glycerophosphate bond of the polar head group, while phospholipase D (PLD) hydrolyzes the head group itself [10]. In the intestine, they all lead to a reduction in phospholipids at mucosal surfaces. They also exert metabolic function when phospholipid breakdown products (trimethylamine) are absorbed and may also induce an inflammatory response as virulence factors. The different phospholipase species and their impact on intestinal integrity and metabolism are summarized elsewhere [11].
It was also shown that the main phospholipid in the protective mucus shield of the intestines is indeed PC (Figure 1), which is not only an important component of the cell membrane lipid bilayers [12,13].
In the lungs, PC is found as a surfactant within the mucus, protecting alveolar surfaces and facilitating gas exchange [14,15]. In the gastrointestinal tract, PC is bound within the mucus to mucins, creating a hydrophobic barrier against the aqueous luminal space and, thus, preventing the invasion of microbiota [16,17,18]. It was shown that PC-degrading phospholipase is present on membrane surfaces, including bacterial membranes [19,20,21].
The observation that bacterial ectophospholipase disturbs the barrier function by reducing the repelling mucus PC content initially came from studies with Helicobacter pylori in gastric mucosa [6,9]. In this case, it was the phospholipase A2 activity that was responsible for thinning the mucus PC barrier. Studies have shown that bacteria can penetrate the normally impermeable inner colonic mucus layer in both, murine models and patients with ulcerative colitis [16,22]. The thinner mucus layer allowed microbiota to penetrate, although in these studies mucus PC content was not quantified and the role of ectophospholipase-containing microbiota was not addressed. The presence of ectophospholipase in various intestinal bacterial species, the number of ectophospholipase-carrying bacteria, their predominance within the microbiota species, and their enzymatic activity on the microbiota surface have not been systematically explored, despite potentially being important pathogenicity factors. Ectophospholipase has been neglected as a virulence factor. The lack of mucus PC can be therapeutically compensated by adding PC to the luminal space to fill up the empty PC-binding sites of mucins. Moreover, feeding ectophospholipase-carrying bacteria with luminally provided PC helps to neutralize their appetite for mucus PC. Alternatively, the application of non-absorbable phospholipase inhibitors can prevent ectophospholipase-carrying bacteria from attacking mucus PC [23]. These are experimental approaches to indirectly prove the significance of mucus PC in preventing microbiota invasion and subsequent mucosal inflammation.

2. Phosphatidylcholine as a Key Component of the Mucus Barrier

Our skin, the outer body surface, is covered by a keratin layer that protects us from mechanical injury and irradiation. The inner body surface is much larger and covers the airways, urogenital system, and gastrointestinal tract. This mucosal surface protects against bacterial invasion without hindering gas exchange, secretion, or absorption. This is especially important in the intestine, where there are 1 trillion bacteria present in just 1 g of stool, and we release about 250 g of stool daily. Stool on the skin would cause an ulcer in a day, but in the intestine, ulcers do not develop under normal circumstances, except in inflammatory conditions, particularly in ulcerative colitis. The key component is the mucus that coats the mucosal surfaces. It is a lubricating film made up of mucins, which are long-stretched, negatively charged proteins designed to bind to positively charged molecules, such as the choline head group of PC [17,22,24]. PC is the physiologic phospholipid that enters the mucus from the vascular system, either through exocytosis as in the alveoli [15,25] or through monomeric translocation of PC through the tight junction (TJ) barrier in the intestine [26,27]. This establishes a hydrophobic barrier [28]. When radiolabeled PC is intravenously administered to rats, it has been shown to be excreted in bile, as it is well known. However, surprisingly it is also excreted to a similar extent in the small intestine and only marginally in the colon. This underscores the proposed close connection between the liver and gut (Figure 2) [29].
Since >90% of phospholipids in mucus are PC and lysoPC (LPC) (Table 1) [12], a specific accumulation process during passage to mucus has to be postulated.
PC travels within the blood in lipoproteins and is in equilibrium with a small-sized lipoprotein-free fraction (LPFF) containing monomeric PC and LPC [30]. The LPFF can pass the endothelial cell layer via intercellular gaps and distribute in the interstitial space. Accordingly, in the intestine, PC and LPC are localized between mucosal cells, into which PC cannot enter due to its complex molecular structure. Therefore, PC has to be translocated across the TJ barrier to be enriched in the luminally attached mucus layer. This apical translocation of PC was identified in experiments using transwell tissue culture dishes [26]. The intestinally differentiated tumor cell line CaCo2 was cultured on these dishes for 21 days to become polarized and establish an apical TJ barrier with a transepithelial resistance (TER) of 450 Ohm (Figure 3).
In control settings, CaCo2 cells were grown in culture for only 3 days, representing unpolarized cell systems without TJ and low TER. Apical and basal transport of the non-cell permeable compounds PC and inulin were determined by applying each substance to the opposite compartment at a concentration of 10 mM for 1 h. Unlike inulin, PC effectively moves to the apical compartment in polarized tissue cultures of CaCo2 cells (Figure 3A,B). Under equilibrium conditions with 1 h incubation of substrates to the upper and lower compartments of the transwell tissue culture systems, PC prefers dose-dependent accumulation in the apical compartment in contrast to oleate with basal preference and inulin with equal distribution (Figure 3C). The hypothesis that TJs are responsible for apical translocation of PC is supported by an experiment where PC transport is significantly reduced when TJs are disrupted by 150 µM acetaldehyde (ACA) or the peroxisome proliferator-activated receptor γ inhibitors T0070907 (10 µM) or GW9002 (1 µM) for 1 h. Additionally, TJ disruption was achieved through knockdown experiments using siRNAs (78 pmol) targeting Claudin 1, 2, or 4, ZO-1, Occludin, and Jam-1. siRNA to kindlin-1 or -2 (both serving as TJ adapter proteins) were also examined because the knockout of these genes were utilized to generate mice with an ulcerative colitis phenotype [31,32,33]. Scrambled siRNA was used as a control. After the pretreatment procedure, apical translocation of PC was determined by basal application of 10 mM (3H)PC for 1 h. In all cases, TJ disruption was observed compared to controls, as evidenced by a drop in TER and fading of the respective knocked-down TJ proteins. The data indicate that it is the disruption of TJs as a whole and not the alteration of one of the many proteins involved that is responsible for PC translocation to the apical side. Accordingly, the apical translocation of PC was significantly reduced (Figure 3D).
At the luminal side, PC is initially bound to membrane-associated mucin 3 from where it is transferred to mucin 2, both of which have an affinity for PC [26]. Although mucin 2 has a slightly more lower affinity for PC, it is the primary mucin secreted by goblet cells, making it abundant in mucus [26]. This allows it to take over PC bound to mucin 3 and transport it distally along the intestinal/colonic wall to the rectum for release in the stool. The vectorial transport to the apical compartment of mucosal cells is effective for choline-containing phospholipids such as PC, LPC, and sphingomyelin. This is due to the positively charged choline head group, which enables binding to negatively charged TJ proteins, followed by mucin 3 and mucin 2 binding, all of which are involved in the apical translocation process (Figure 4, left) [27].
However, the choline-containing sphingomyelin is not found in the interstitial space of the mucosa because it is not part of the LPFF. Additionally, transport requires a negative charge on the apical side of the mucosal surface as a driving force. This negative charge is generated by the cystic fibrosis transmembrane conductance regulator (CFTR), which transports Cl− and HCO3− externally (Figure 4, left). The mechanism by which PC moves from the basal side of TJ to the apical side, driven by a negatively charged environment, remains unclear. Theoretically, a flip-flop or large-channel TJ pathway could be considered [34].
PC bound to secretory mucin 2 moves within the mucus along the mucosal surface of the intestine/colon, creating a hydrophobic repellent that prevents bacterial invasion from the aqueous lumen of the gut [16,35,36]. The PC molecules covering the surface of mucins arrange themselves as a lipid double layer in an aqueous environment or as a monolayer in a non-aqueous (gas) surrounding. Within this hydrophobic shield there is space between the lipophilic mucus strands for fluid and electrolyte exchange, allowing for the absorption of water and salts in the colon and the absorption of nutrients in the upper small intestine.
Due to the proposed close connection between the liver and gut, it is not surprising that a similar transport mechanism for PC also exists in the biliary epithelium. This was observed in the model of the polarized biliary tumor cell line Mz-ChA-1 [1]. The presence of a protective PC-containing mucus layer in the biliary system is essential for shielding against the corrosive effects of bile acids and potential bacterial invasion.

3. Low Mucus Phosphatidylcholine as Feature of Ulcerative Colitis

The tight lipid layer of PC in mucus repels fluid, but a compromised lipid layer could allow fluid to enter the mucus, leading to the invasion of bacteria from the aqueous intestinal lumen. This theory was supported when the surface tension as a measure of hydrophobicity of mucus was examined on tissue samples from normal controls, patients with ulcerative colitis, and Crohn’s disease (both patient groups in clinical remission) [28]. Only in ulcerative colitis, the surface tension was significantly reduced. This corresponds to the finding that the mucus PC content was markedly reduced by 70% in ulcerative colitis, even in remission, but not in controls or Crohn’s disease [13]. It was indeed shown that in UC a TJ defect is present with a reduced paracellular translocation of PC to the luminal side, compromising the water-repelling competence of the mucus layer (Figure 4, right) [27].
A defect in TJs as a pathogenetic feature of UC had already been proposed by other investigators [37,38,39,40,41,42]. The electron micrograph of a human UC specimen shown in Figure 5A reveals the widening of the intercellular cleft. Indirect signs of a disturbed TJ architecture were shown by hematoxylin and eosin staining, where wider crypt luminal diameters and more cuboidal cell shapes of enterocytes were obvious in UC, indicating a disturbed crypt architecture. The finding that this results in diminished PC translocation into mucus is novel [27]. In Figure 5B, fluorescent PC movement to the apical surface of crypts is disturbed and phospholipid staining with PAS reveals empty apical spaces in UC. This corresponds to the biochemical finding of low mucus PC content in UC, which has been shown before. PC in mucus is the predominant protective mechanism against gut microbiota by establishing a hydrophobic barrier [24,28].
The fact that patients with ulcerative colitis remain in remission despite having a low level of PC in mucus suggests that a 30% residual content of mucus PC is sufficient to maintain a barrier against bacterial invasion [13]. This also implies that a low mucus PC level may be the primary event, likely inherited. However, further reduction in mucus PC may lead to inflammation and could perpetuate the disease activity. In other gut inflammatory conditions, diminished PC secretion may occur as a secondary event, as could be assumed in Crohn’s or Celiac disease [44,45]. When the concentration of PC falls below the critical value of 30%, the hydrophobic barrier may be compromised, allowing bacteria to invade and cause subendothelial inflammation with ulceration (Figure 5C) [35,46]. The proposed underlying mechanism of mucosal PC secretion and its disturbance by TJ disruption in UC is illustrated above (cf. Figure 4). Bloody diarrhea is the final prominent clinical symptom of ulcerative colitis. How can it happen that the critically low level of 30% PC in mucus, which just prevents bacterial invasion, is further reduced to induce inflammation? Possible conditions that can lower mucus PC are listed in Table 2.
It has been previously explained that low mucus PC in UC is a result of a TJ defect. However, it is possible that leaky gut syndrome could also be due to a TJ defect, although this has not been studied yet. Inflammatory conditions, which may also be present in some types of IBS, can impair epithelial function including PC translocation. A reduced mucosal surface after intestinal resection, mucosa bypassing due to surgical procedures or entero-enteric fistulas, or when the fecal stream is diverted as part of a colostomy (leading to the development of diversion colitis) can also contribute to lower mucus PC levels. It is known that non-steroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid or ibuprofen can cause acute episodes of intestinal inflammation in inflammatory bowel disease due to disturbed microcirculation and consequent tissue ischemia. This mechanism may also be applicable in cases of intestinal vascular ischemic diseases. All of these can impair transport of PC to the mucus.

4. The Microbiota as a Game Changer

A more important factor in reducing mucus PC content may be related to a change in the microbiota. Bacteria with phospholipase on their surface (ectophospholipase) are part of the normal microbiota [4,5]. When these bacteria become more prevalent, they can become harmful and are considered a virulence factor [7]. Ectophospholipase-containing bacteria consume PC by breaking it down in the mucus layer, further thinning the mucus PC content and impairing hydrophobicity to a level below what the 30% PC level in patients with ulcerative colitis in remission can provide [6,7,9]. This allows for bacterial invasion and causes inflammation. The hypothesis that the enrichment of phospholipase-carrying bacteria in the colon is the precipitating factor to induce an inflammatory episode could in the future be proven by determination of the phospholipase activity in stool samples. It would be even more interesting to determine the microbiota species in regard to their phospholipase activity under these conditions. A lower mucus PC content may increase the likelihood of inflammatory episodes in ulcerative colitis. Although not experimentally proven, it seems reasonable to assume that a relationship exists between a low mucus PC content, due to the impaired ileal secretion and the extent of colonic manifestation up to pancolitis, as well as the severity of the inflammatory phenotype with bloody diarrhea and even a septic-like condition. Indeed, in a previous study, mucin 2 and 3 expression in inflammatory bowel diseases was shown to be markedly reduced, particularly in UC, but the relation to the mucus PC content has not been analyzed there [47]. Experimentally, the relationship between the extent of disease and mucus PC depletion is hard to verify because blood contains high amounts of PC and, thus, the actual mucus PC content in a bleeding episode of UC may not be a representative measure. In a condition with little reduction in the mucus PC content, only ulcerative proctitis may be observed. It has also been considered that determining the mucus PC content for clinical routine use is technically challenging.
A pathogenetically interesting aspect is the development of pouchitis, which occurs in the ileoanal pouch after colectomy in 23–44% of patients with ulcerative colitis within 10 years after the procedure [48,49]. When the same surgical procedure is performed in familial adenomatous polyposis (FAP) for cancer prevention, pouchitis does not develop as frequently (0–11%) and with lower inflammatory activity and later onset [48,49]. This difference is due to the fact that the ileal mucus in ulcerative colitis is depleted of PC because impaired PC secretion originates in the ileum [29] and, thus, PC is already depleted in mucus when it enters the ileoanal pouch. Moreover, the TJ defect in the UC pouch epithelium interferes with the paracellular pathway, prohibiting PC secretion and allowing an unprotected attack of bacteria [50]. In contrast to the physiologic situation in the terminal ileum with reduced bacterial colonization, the ileoanal pouch contains a high load of microbiota. Furthermore, the type of bacterial colonization with potential enrichment of ectophospholipase-containing microbiota may be another contributing factor.

5. Helicobacter pylori as an Example of Phosphatidylcholine-Consuming Bacteria

Helicobacter pylori is a prime example of an ectophospholipase-positive bacterium. It carries the phospholipid-degrading enzyme on its “nose” to consume mucus PC in the gastric and duodenal mucosa [6,9]. By doing so, it creates holes in the mucus shield to hide within and protect itself from gastric acid. Over time, Helicobacter can penetrate the mucus layer until it reaches the gastric/duodenal mucosal wall below, causing type B gastritis through the release of antigens that trigger an immune reaction [51]. It has also been reported that Helicobacter disturbs the TJ barrier in the gastric mucosa [52] with a proposed consequent reduction in PC release by the mucosa. This causes further thinning of the PC mucus layer, allowing gastric acid to enter, which leads to erosion and ulceration.
It is interesting to note that the application of green bananas has been shown to heal gastric ulcers [53,54]. This may be due to the fact that bananas contain high amounts of PC, among other mucoprotective compounds like pectins [53,54]. The prolonged stay of unripe bananas in the stomach allows sufficient exposure time of PC to gastric mucus, filling up empty PC-binding sites on Helicobacter-exposed mucins and aiding in the recovery of the mucus shield. Additionally, gastric luminal PC serves as easily accessible food for Helicobacter pylori, eliminating the need for it to consume mucus PC and, thus, protects against mucosal injury [54]. Even the application of PC directly was shown to prevent and cure ulcers induced by NSAIDs, which indicates its mucoprotective potential [18]. Although not experimentally proven, we would like to suggest that such PC-consuming bacteria also exist in the colon, thus thinning the mucus PC barrier. In UC with its intrinsic low PC content, a phospholipase-rich microbiota could undercut a critical mucus PC level and induce an inflammatory episode.

6. Topical Substitution of Phosphatidylcholine in Distal Ileum as Treatment for Ulcerative Colitis

The observation of mucosal improvement with green bananas was the reason for developing an intestinal release PC preparation to supply PC from the luminal side to mucus and increase the deficient mucus PC levels in ulcerative colitis [55]. This endeavor was successful. A recent meta-analysis of three phase 2 trials and one maintenance of remission study demonstrated that intestinally released PC induces dose-dependent remission or significantly reduces mucosal inflammation (Table 3, adapted from [36]).
One of these phase 2 trials focused on patients with steroid-refractory ulcerative colitis, showing that most patients treated with PC could discontinue steroids, while the remaining group saw a significant reduction in steroid dosage [56]. Maintenance of remission was also prolonged with PC therapy [58]. According to the beneficial effect on intestinal inflammation, the quality of life also improved significantly, which includes bowel symptoms as well as systemic, emotional, and social aspects. A more than 50% improvement was reported in all categories [36,59]. These positive results were achieved without serious side effects, as adverse events were as frequent as in the placebo-controlled group [35]. Unfortunately, a follow-up phase 3 trial failed due to the simultaneous use of mesalazine, which acts as a detergent. This could result in a superficially layered PC foam that prevented PC incorporation into the PC-deficient mucus layer [60].

7. Would Phosphatidylcholine Also Be Effective in Other Intestinal Inflammatory Conditions?

It has been shown that PC acts directly as an anti-inflammatory phospholipid in the mucosa, while other phospholipids, such as ceramide, can induce inflammation when topically applied [61]. Therefore, PC could potentially improve inflammatory bowel diseases, such as Crohn’s disease or Celiac disease (Table 2). Some investigators also consider irritable bowel syndrome to be an inflammatory condition [62,63]. In these conditions, TJ may be destructed leading to impaired PC secretion into mucus and perpetuating disease activity [64]. Even the entity of a leaky gut syndrome may be associated with a TJ defect and patients may benefit from topical PC substitution [63]. This deduction is still speculative and requires experimental support. However, studies in mice with intestinal deletion of TJ by knockout of the TJ adapter proteins mucosal kindlin-1 or -2 have shown that PC secretion into mucus is significantly reduced, leading to severe mucosal inflammation [27].
Besides diseases of the gut, extra-intestinal disorders may also be due to the absorption of pathogenetic factors originating from disturbed microbiota, as suggested for pancreatic disorders or intestinal-borne dermatoses [65,66,67].
For the liver, it would be most interesting to compensate for the postulated defect of PC translocation across the biliary epithelium with consequent deficiency of mucus PC as key pathogenicity in PSC. Direct PC substitution by luminal application is impossible. Therefore, we propose the following approach based on the pathophysiology of hepatobiliary transport mechanisms: PC is actively secreted at the canalicular pole of hepatocytes via ABCD4 into bile in conjunction with bile acids, which are biliary excreted through ABCB11. Both form micelles traveling down the biliary system. Bile acids with low affinity to coenzyme A synthetase are not conjugated and, thus, more lipophilic to be readily taken up by cholangiocytes from the biliary lumen, leaving the associated PC behind in bile. After passage through cholangiocytes, these protonated bile acids are recirculated via the periductular plexus to the hepatocytes. The perpetuation of hepatocellular secretion and biliary reabsorption enhances bile flow and accumulation of free PC in bile [68]. From there, it is able to fill up empty PC-binding sites on mucin 2 to reestablish a hydrophobic mucus shield. Indeed, the application of NorUDCA as a bile acid with enhanced cholehepatic shunting revealed therapeutic efficacy in initial clinical trials in PSC patients [69].

8. Further Experimental Evidence from Genetic Mouse Models

The role of ectophospholipase-carrying microbiota was further analyzed in the mentioned genetic mouse model (intestinal kindlin-2 deleted) resembling an ulcerative colitis phenotype [27]. An initial study showed that inflammatory activity could be reversed by luminal application of PC through an intestinally placed tube, a procedure that prevents degradation by pancreatic enzymes and enables intestinal luminal provision [27]. In another study, a newly developed non-absorbable phospholipase inhibitor, the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE), was tested [23]. It was shown to act by binding to calcium-independent membrane phospholipase A2 type β (iPLA2β) [70]. In mouse models of non-alcoholic steatohepatitis, it inhibited intracellular LPC generation, a key mediator of inflammation [71]. UDCA-LPE was provided to the ileum again by tube feeding, and thus was protected from intestinal degradation by pancreatic phospholipase. Figure 6 (composite data of [23]) illustrates the beneficial effect of the phospholipase inhibitor UDCA-LPE on mouse colitis.
This includes the inhibitory effect on phospholipase activity in stool, which was reduced to a normal level. The concomitant improvement in inflammatory activity is demonstrated by the reduction in calprotectin levels in stool, as well as the amelioration of endoscopic and histology appearance. Finally, the change in bacterial composition in stool after UDCA-LPE application revealed a quantitative distribution pattern similar to that observed in wild-type mice. Moreover, the drop in mucoprotective Bacteroides concentration under kindlin-2−/− conditions was reversed to the wild-type pattern [23]. This may suggest that Bacteroides can suppress the colonization of ectophospholipase-carrying bacterial species. Alternatively, it could just indicate a concomitant shift of the microbiota adapted to the local inflammatory, more aerobic conditions, which suppresses anaerobic Bacteroides [11].
Both setups support the hypothesis that PC supplementation to the intestinal lumen and inhibition of phospholipase activity protects from intestinal inflammation. UDCA-LPE is an effective example of a phospholipase A2 inhibitor that is poorly absorbable, exerting its activity in the distal ileum and colon. However, it may also inhibit secretory pancreatic and Paneth cell-derived phospholipases A2, which are responsible for the absorption of breakdown products or signaling by LPC and fatty acids. This still needs to be experimentally evaluated. The question of whether a replacement of ectophospholipase-carrying bacteria could be achieved by microbiota replacement with little or no phospholipase activity remains open unless such probiotic colonization can be successfully demonstrated. The secondary effect of an environmental change in inflammation with a more aerobic milieu has also been considered.

9. Conclusions

Low mucus PC content plays a crucial role in the development of inflammatory gastrointestinal diseases. Topically application of PC to the intestinal lumen using specialized delayed-release preparations can help to compensate for a deficiency in mucus PC, commonly seen in ulcerative colitis. One key factor in this process is the presence of ectophospholipase-carrying microbiota. These bacteria consume mucus PC, leading to a breakdown of the mucus barrier and subsequent mucosal inflammation. Their harmful activity can be counteracted by topically applying PC to the intestinal lumen, protecting the mucosa from attacks by ectophospholipase-containing microbiota and preserving mucus PC (Figure 7).
Another potential approach is to use ectophospholipase inhibitors to prevent the degradation of the mucus PC layer. The next step would involve verifying this hypothesis through microbiota analysis to determine if reducing ectophospholipase-containing bacteria could promote a more beneficial intestinal flora.

Author Contributions

Conceptualization, W.S.; resources, R.W.; data curation, W.S. and R.W.; writing—original draft preparation, W.S. and R.W.; writing—review and editing, W.S. and R.W.; visualization, R.W.; supervision, W.S.; project administration, W.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

This review only presents data that has been previously published. No new data was generated.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

IBD inflammatory bowel disease(s)
LPFFlipoprotein-free fraction
PCphosphatidylcholine
NSAID(s)non-steroidal anti-inflammatory drug(s)
PLphospholipase
PSCprimary sclerosing cholangitis
TERtransepithelial resistance
TJtight junction(s)

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Figure 1. Phosphatidylcholine (PC). PC is composed of a choline head group, a phosphate group, and two fatty acid chains. It is essential for maintaining the structural integrity and fluidity of cell membranes. PC exists as a zwitterion, possessing both polar (hydrophilic) and apolar (hydrophobic) properties, making it amphiphilic. The various elements—carbon (grey), oxygen (red), nitrogen (blue), and phosphorus (orange)—in phosphatidylcholine are represented. The structure was drawn using Jmol (version 14.2.15_2015.07.09) and information was obtained from PubChem SID: 438472886.
Figure 1. Phosphatidylcholine (PC). PC is composed of a choline head group, a phosphate group, and two fatty acid chains. It is essential for maintaining the structural integrity and fluidity of cell membranes. PC exists as a zwitterion, possessing both polar (hydrophilic) and apolar (hydrophobic) properties, making it amphiphilic. The various elements—carbon (grey), oxygen (red), nitrogen (blue), and phosphorus (orange)—in phosphatidylcholine are represented. The structure was drawn using Jmol (version 14.2.15_2015.07.09) and information was obtained from PubChem SID: 438472886.
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Figure 2. Phosphatidylcholine (PC) secretion into in vivo perfused rat intestinal segments. Respective [3H]-PC secretion rates in jejunum, ileum, colon, and bile (liver) (fmol h−1 g liver−1) of male rats were tested in the presence of 2 mM taurocholate. [3H]-PC was injected intravenously at time 0. Green circles represent ileal secretion (n = 8), red circles represent jejunal secretion (n = 8), white circles respresent colonic secretion (n = 8), and blue circles represent secretion into bile (n = 8). After 30 min, the secretion rates of each of the intestinal segments reached equilibrium and were significantly different from one another. Only secretion in bile and ileum were superimposable. Data are expressed as mean ± S.E. for each 10 min period. The data depicted were taken in modified form from [29].
Figure 2. Phosphatidylcholine (PC) secretion into in vivo perfused rat intestinal segments. Respective [3H]-PC secretion rates in jejunum, ileum, colon, and bile (liver) (fmol h−1 g liver−1) of male rats were tested in the presence of 2 mM taurocholate. [3H]-PC was injected intravenously at time 0. Green circles represent ileal secretion (n = 8), red circles represent jejunal secretion (n = 8), white circles respresent colonic secretion (n = 8), and blue circles represent secretion into bile (n = 8). After 30 min, the secretion rates of each of the intestinal segments reached equilibrium and were significantly different from one another. Only secretion in bile and ileum were superimposable. Data are expressed as mean ± S.E. for each 10 min period. The data depicted were taken in modified form from [29].
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Figure 3. Phosphatidylcholine transport through tight junctions examined in transwell tissue culture systems of CaCo2 cells. (A) Apical and basal transport of 10 mM PC and inulin from the basal compartment over 1 h was measured in unpolarized versus polarized CaCo2 cells. (B) Apical transport of 10 mM PC and inulin from the basal compartment over 1 h was measured as a function of culture time of CaCo2 cells that increases stepwise in cell density and polarity. The expression of the tight junction (TJ) marker ZO-1 in CaCo2 cells was assessed during prolonged culturing by Western blot analysis. Actin expression was used to ensure equal protein loading in each lane. (C) Apical-basal equilibrium distribution of 10 mM PC, inulin, and oleate in polarized CaCo2 transwell tissue culture systems was determined after the application of equal concentrations of the substrates (left) or increasing PC concentration (right) to both compartments for 1 h. (D) Apical PC release was measured in polarized CaCo2 cells after basal application of 10 mM PC for 1 h following TJ disruption by ACA and peroxisome proliferator-activated receptor γ inhibitors (left), or (si)RNA knockdown of claudin-1, -2, -4, ZO-1, occludin, jam-1, kindlin-1 and -2, all involved in TJ formation (right). Scrbl indicates control scrambled siRNA. The figures illustrate apical PC transport rates, TER in Ω, and the reduction in the respective protein expression. Means ± SD. ** p < 0.01, *** p <0.001, ns/n.s.: not significant (n = 6). The image shown was taken in a modified form from [26] and reproduced with permission.
Figure 3. Phosphatidylcholine transport through tight junctions examined in transwell tissue culture systems of CaCo2 cells. (A) Apical and basal transport of 10 mM PC and inulin from the basal compartment over 1 h was measured in unpolarized versus polarized CaCo2 cells. (B) Apical transport of 10 mM PC and inulin from the basal compartment over 1 h was measured as a function of culture time of CaCo2 cells that increases stepwise in cell density and polarity. The expression of the tight junction (TJ) marker ZO-1 in CaCo2 cells was assessed during prolonged culturing by Western blot analysis. Actin expression was used to ensure equal protein loading in each lane. (C) Apical-basal equilibrium distribution of 10 mM PC, inulin, and oleate in polarized CaCo2 transwell tissue culture systems was determined after the application of equal concentrations of the substrates (left) or increasing PC concentration (right) to both compartments for 1 h. (D) Apical PC release was measured in polarized CaCo2 cells after basal application of 10 mM PC for 1 h following TJ disruption by ACA and peroxisome proliferator-activated receptor γ inhibitors (left), or (si)RNA knockdown of claudin-1, -2, -4, ZO-1, occludin, jam-1, kindlin-1 and -2, all involved in TJ formation (right). Scrbl indicates control scrambled siRNA. The figures illustrate apical PC transport rates, TER in Ω, and the reduction in the respective protein expression. Means ± SD. ** p < 0.01, *** p <0.001, ns/n.s.: not significant (n = 6). The image shown was taken in a modified form from [26] and reproduced with permission.
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Figure 4. Scheme of establishment of a protective phosphatidylcholine mucus layer in normal mucosa and pathophysiology of a disturbed phosphatidylcholine mucus layer due to disturbed tight junctions in ulcerative colitis. This image was taken with permission from [27].
Figure 4. Scheme of establishment of a protective phosphatidylcholine mucus layer in normal mucosa and pathophysiology of a disturbed phosphatidylcholine mucus layer due to disturbed tight junctions in ulcerative colitis. This image was taken with permission from [27].
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Figure 5. Widened crypts due to disturbed TJs in human UC (in remission), consequent impairment of luminal PC accumulation, and bacterial invasion of the defective mucus layer. (A) An electron micrograph (EM) of a human UC specimen with epithelial disturbance (arrow shows widening of the intercellular cleft) and hematoxylin and eosin staining of non-inflamed mucosa with wider crypt lumina in UC patients compared to control subjects. (B) (upper panel) Nitrobenzo-oxa-diazole (NBD)-PC live exposure of colonic biopsies showing impaired paracellular and mucus staining only in UC patients, not in healthy controls. (lower panel) Reduced PAS phospholipid staining of samples from UC patients in clinical remission versus control subjects (scale bars = 25 µm). (C) (left) The colonic wall of a healthy human control is covered with mucus that prevents bacteria from coming into contact with the colon mucosa. (right) The epithelial surface of a patient with ulcerative colitis shows bacteria attached to the exposed mucosa. The ulcer ground is marked by arrows in this image. Images (A,B) were taken from [27] with permission. Image (C) was taken from [43] and adapted with permission of the Journal of Physiology and Pharmacology.
Figure 5. Widened crypts due to disturbed TJs in human UC (in remission), consequent impairment of luminal PC accumulation, and bacterial invasion of the defective mucus layer. (A) An electron micrograph (EM) of a human UC specimen with epithelial disturbance (arrow shows widening of the intercellular cleft) and hematoxylin and eosin staining of non-inflamed mucosa with wider crypt lumina in UC patients compared to control subjects. (B) (upper panel) Nitrobenzo-oxa-diazole (NBD)-PC live exposure of colonic biopsies showing impaired paracellular and mucus staining only in UC patients, not in healthy controls. (lower panel) Reduced PAS phospholipid staining of samples from UC patients in clinical remission versus control subjects (scale bars = 25 µm). (C) (left) The colonic wall of a healthy human control is covered with mucus that prevents bacteria from coming into contact with the colon mucosa. (right) The epithelial surface of a patient with ulcerative colitis shows bacteria attached to the exposed mucosa. The ulcer ground is marked by arrows in this image. Images (A,B) were taken from [27] with permission. Image (C) was taken from [43] and adapted with permission of the Journal of Physiology and Pharmacology.
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Figure 6. Comparison of wild-type mice and ulcerative colitis mice (kindlin-2−/− mice induced with tamoxifen) and ulcerative colitis mice treated with the phospholipase inhibitor ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE). (A) PLA2 activity in stool. (B) Inflammatory activity as calprotectin activity in stool. (C) Endoscopic appearance and histology (scale bars = 25 µm) of left wild type, middle colitis mice, right colitis mice after treatment with UDCA-LPE. (D) Change in the diversity of bacterial phyla in stool. In (A,B,D), means ± SD are provided, * p < 0.05, ** p < 0.01. Images were taken from [23] and published with permission.
Figure 6. Comparison of wild-type mice and ulcerative colitis mice (kindlin-2−/− mice induced with tamoxifen) and ulcerative colitis mice treated with the phospholipase inhibitor ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE). (A) PLA2 activity in stool. (B) Inflammatory activity as calprotectin activity in stool. (C) Endoscopic appearance and histology (scale bars = 25 µm) of left wild type, middle colitis mice, right colitis mice after treatment with UDCA-LPE. (D) Change in the diversity of bacterial phyla in stool. In (A,B,D), means ± SD are provided, * p < 0.05, ** p < 0.01. Images were taken from [23] and published with permission.
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Figure 7. Schematic illustration of ectophospholipase-containing bacteria attacking mucus phosphatidylcholine. Healthy individuals form proper mucus and have an intact mucosal cell layer. Bacteria that consume mucus PC provoke breakdown of the mucus barrier and consequent mucosal inflammation (red arrows). The topical application of PC to the intestinal lumen protects the mucosa from attacks by ectophospholipase-containing microbiota. Similarly, ectophospholipase inhibitors can prevent the degradation of the mucus layer.
Figure 7. Schematic illustration of ectophospholipase-containing bacteria attacking mucus phosphatidylcholine. Healthy individuals form proper mucus and have an intact mucosal cell layer. Bacteria that consume mucus PC provoke breakdown of the mucus barrier and consequent mucosal inflammation (red arrows). The topical application of PC to the intestinal lumen protects the mucosa from attacks by ectophospholipase-containing microbiota. Similarly, ectophospholipase inhibitors can prevent the degradation of the mucus layer.
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Table 1. Phospholipid composition in percent of intestinal mucus, surfactant, and intestinal brush border 1.
Table 1. Phospholipid composition in percent of intestinal mucus, surfactant, and intestinal brush border 1.
SubstanceMucusSurfactantMembrane
Lysophosphatidylcholine3302
Sphingomyelin7016
Phosphatidylinositol2014
Phosphatidylcholine588522
Phosphatidylserine0018
Phosphatidylethanolamine0528
Phosphatidylglycerol0100
1 Modified from [12].
Table 2. Potential mucus phosphatidylcholine (PC) deficiencies.
Table 2. Potential mucus phosphatidylcholine (PC) deficiencies.
Impaired PC transport to mucus
  •
Ulcerative colitis;
  •
Leaky gut syndrome;
  •
Cystic fibrosis.
Secondary to inflammation
  •
Crohn’s disease;
  •
Celiac disease;
  •
Irritable bowel syndrome.
Secondary to ischemia
  •
Non-steroidal anti-inflammatory drugs
(NSAID: ibuprofen, acetylsalicylic acid);
  •
Intestinal vascular ischemia.
Secondary to reduced mucosal surface
  •
Intestinal resection;
  •
Surgical bypass;
  •
High fistulas;
  •
Diverted fecal stream.
Overgrowth of ectophospholipase bacteria
Table 3. Trials with intestinal release of phosphatidylcholine (PC) in patients with ulcerative colitis (UC).
Table 3. Trials with intestinal release of phosphatidylcholine (PC) in patients with ulcerative colitis (UC).
AInduction of Remission (n = 60 Patients), 3 Randomized Controlled Trials (RCT)
PhasePC (g/Day)RemissionClinical ImprovementEndoscopic ImprovementHistology ImprovementLife Quality ImprovementCohort
IIa0
2		3/30
16/30		3/30
27/30		0/30
11/30		3/30
13/30		2/30
16/30		Active UC (efficacy testing) [55]
n = 60
IIa0
2		3/30
12/30		3/30
15/30		1/30
21/30		10/30
15/30		5/30
13/30		Steroid refractory UC (efficacy testing) [56]
n = 60
IIB0.5
1.0
3.0
4.0		0/10
3/10
5/10
6/10		0/10
7/10
7/10
7/10		0/0
5/10
6/10
6/10		0/10
3/10
4/10
5/10		1/10
4/10
6/10
6/10		Active UC (dose-finding study)
n = 40 [57]
BMaintenance of Remission (n = 80 Patients)
PC (g/day)8 Weeks Follow-up26 Months Follow-upCohort
Patients
in remission by PC
n = 80 [58]
020/402/20
230/4010/30
Data show numbers of patients responding in relation to total patients included in the respective groups. The analysis was performed with a delayed release phosphatidylcholine (30% PC-containing lecithin encapsulated with Eudragit S-100 1:1). The PC amount provided daily is indicated. In (A), three RTCs are shown. In (B), patients in remission after PC therapy from the RCTs in (A) (n = 42) are included as well as patients in remission after an individual healing attempt with PC for ≥12 weeks (n = 38). PC, phosphatidylcholine; UC, ulcerative colitis.
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MDPI and ACS Style

Stremmel, W.; Weiskirchen, R. Phosphatidylcholine in Intestinal Mucus Protects against Mucosal Invasion of Microbiota and Consequent Inflammation. Livers 2024, 4, 479-494. https://doi.org/10.3390/livers4030034

AMA Style

Stremmel W, Weiskirchen R. Phosphatidylcholine in Intestinal Mucus Protects against Mucosal Invasion of Microbiota and Consequent Inflammation. Livers. 2024; 4(3):479-494. https://doi.org/10.3390/livers4030034

Chicago/Turabian Style

Stremmel, Wolfgang, and Ralf Weiskirchen. 2024. "Phosphatidylcholine in Intestinal Mucus Protects against Mucosal Invasion of Microbiota and Consequent Inflammation" Livers 4, no. 3: 479-494. https://doi.org/10.3390/livers4030034

APA Style

Stremmel, W., & Weiskirchen, R. (2024). Phosphatidylcholine in Intestinal Mucus Protects against Mucosal Invasion of Microbiota and Consequent Inflammation. Livers, 4(3), 479-494. https://doi.org/10.3390/livers4030034

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