Introduction

Sepsis is defined as organic dysfunction related to a disordered response to infection. Alkaline phosphatases catalyze the hydrolysis of phosphate groups from different substrates, such as bacterial lipopolysaccharide (LPS), an important mediator in the pathophysiology of sepsis (1). Different studies have confirmed that intestinal alkaline phosphatase (IAP) reduces intestinal permeability, intestinal inflammation, bacterial translocation and, as a consequence, systemic inflammation (2, 3). However, several studies have demonstrated the importance of the tissue-nonspecific alkaline phosphatase (TNAP) isoenzyme in inflammatory processes (4).

Aims & Methods

We aimed to investigate the effect of TNAP in polymicrobial sepsis induced by cecal slurry (CS) injection using TNAP heterozygous mice (TNAP^+/-) and an inhibitor of TNAP (SBI-425). Mice underwent intraperitoneal injection of 1 mg CS/kg and were sacrificed after 24 h. Body weight loss and mice behavioral outcomes were determined. LPS level in plasma was determined. In addition, permeability to oral fluorescein isothiocyanate-dextran 4 kD (FITC) was assessed. Finally, RNA extraction and RT-qPCR were carried out for gene expression analysis.

Results

CS-induced sepsis resulted in dramatic weight loss and a significant impact on mice behavioral outcomes, with no differences between groups. The polymicrobial infection was associated to high presence of LPS in septic mice plasma, which triggered a systemic inflammatory response. Namely, sepsis caused intestinal barrier defect (higher concentration of FITC-dextran in plasma) and increased inflammatory cytokines in the colon. In this context, TNAP haplodeficiency or inhibition diminished the expression of certain inflammatory markers (S100a8 and Cxcl1, the latter only in heterozygous mice). The same dampening of inflammatory marker expression was observed in the liver, kidney and particularly in the lung (Cxcl1, Cxcl2, Il6 and Il10). SBI-425 had a less pronounced effect at this level.

Conclusion

These preliminary results show that TNAP haplodeficiency or inhibition modulate CS-induced sepsis, resulting in attenuated inflammatory response.

References

1. Millán, J.L. Alkaline phosphatases: Structure, substrate specificity and functional relatedness to othermembers of a large superfamily of enzymes. Purinergic Signal, 2006. 2(2): p.335-41.
2. W. Wang et al. Intestinal alkaline phosphatase inhibits the translocation of bacteria of gut-origin in mice with peritonitis: mechanism of action. PLoS One, vol. 10, no. 5, p. e0124835, 2015, doi: 10.1371/journal.pone.0124835.
3. P. Plaeke et al. Effects of intestinal alkaline phosphatase on intestinal barrier function in a cecal ligation and puncture (CLP)-induced mouse model for sepsis. Neurogastroenterol. Motil. Off. J. Eur. Gastrointest. Motil. Soc., vol. 32, no. 3, p. e13754, Mar. 2020, doi: 10.1111/nmo.13754.
4. Graser, Stephanie et al. TNAP as a New Player in Chronic Inflammatory Conditions and Metabolism. International journal of molecular sciences vol. 22,2 919. 18 Jan. 2021, doi:10.3390/ijms22020919

Disclosure

This abstract is pending approval at EMIG2023.

Introduction

Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. A defining feature of sepsis is the presence of bacteria in the bloodstream or the translocation of intestinal microbes to distant organs such as the lungs. Many of these pathogens originate from the intestinal microbiota. The intestinal mucosa serves as a critical barrier, and its damage or dysfunction is considered a central factor enabling bacterial invasion and systemic spread during sepsis.

Aims & Methods

This study aims to characterize sepsis-induced dysfunction of the intestinal barrier. Specifically, we investigate whether, and how, the functions of the gastrointestinal epithelial barrier are modulated by different sepsis-causing bacterial strains. To this end, an in vitro model was developed using intestinal epithelial cell lines (HCT-116 and Caco-2) co-cultured with bacterial isolates. These isolates, representing diverse sepsis etiologies, were obtained from the blood of septic patients. The impact of bacterial interaction on epithelial integrity was assessed by quantifying the protein levels of E-cadherin and Occludin using Western blot analysis.

Results

Co-culture with sepsis-associated bacterial strains led to the disruption of epithelial cell-cell junctions. Specifically, Escherichia coli strains BK10478, BK01267, and BK06223, at a multiplicity of infection (MOI) of 5, induced a marked downregulation of both Occludin (a tight junction protein) and E-cadherin (an adherens junction protein) in HCT-116 cells. Notably, only a subset of these strains elicited similar effects in Caco-2 cells. Some bacterial isolates had no detectable impact on junctional protein expression in either cell line, highlighting strain-specific and cell-type-dependent responses.

Conclusion

Sepsis is a heterogeneous syndrome characterized by complex host-pathogen interactions. Our findings reveal that the ability of sepsis-associated E. coli strains to compromise epithelial cell-cell junctions is both strain-specific and dependent on the epithelial cell type. Given the essential role of barrier integrity in preventing bacterial translocation, these results provide novel insights into sepsis pathogenesis and underscore the therapeutic potential of strategies aimed at preserving or restoring epithelial barrier function.

Introduction

Sepsis is defined as an organic dysfunction that threatens the life of patients due to an abnormally regulated response to infection [1]. The initial phase of sepsis is dominated by an increased production of proinflammatory cytokines, which leads to augmented capillary permeability, extravasation, hypercoagulability and myelopoiesis. One of the main sources of infection in sepsis is believed to be the intestinal microbiota via traslocation through the mucosa to the bloodstream. Systemic inflammation weakens intestinal barrier function (IBF) in animal models, resulting in increased bacterial traslocation [2]. Even if the management of sepsis has advanced in the last decades, mortality is still high and there is room for improvement. Thus, the search for effective treatments is clearly justified. Glucocorticoids (GC) constitute a therapeutic modality in sepsis, but they have only shown moderate benefit. This may be accounted for by the harmful effects of GCs on IBF, which may facilitate translocation of luminal microorganisms and molecules. Besides, GC treatment impairs epithelial healing in experimental colitis in mice [3]. Previous results of our research group have shown that mice with induced deletion of the GC receptor (GR) in intestinal epithelial cells (i.e. NR3C1^ΔIEC mice) are protected against dextran sulphate sodium (DSS)-induced colitis [4].

Aims & Methods

Our aim is to understand the role of the intestinal epithelial GR and its involvement in IBF regulation in experimental sepsis, with the ultimate goal of improving the management of sepsis with GCs.
The cecal ligation and puncture (CLP) model of sepsis was applied to WT C57BL/6J and NR3C1^ΔIEC mice. Caecum-exposed mice were used as control (sham). Mice were sacrificed 24 hours after surgery. Four hours before sacrifice, mice were administered 4 kD FITC-dextran, a fluorescent marker of permeability. Colon, jejunum, adrenes, kidney and liver RT-qPCRs were performed as well as determination of corticosterone plasma levels.

Results

After 24 h, CLP mice exhibited hypoglycemia and splenomegaly with elevated corticosterone plasma levels, which were associated to increased Cyp11a1 in the adrenes. Of note, both parameters were less pronounced in KO mice. Intestinal barrier function was weakened, as indicated by downregulation of Tjp1 and increased FITC-dextran plasma levels, which were similar in WT and KO mice. A modest increase in inflammatory markers was noted in the colon and jejunum. However, KO mice exhibited dampened inflammatory response in the colon, with lower expression of inflammatory markers such as S100a8 and S100a9, but not in the jejunum. Similarly, the colonic expression of Cyp11a1 and Lrh1, involved in local steroidogenesis, was lower in CLP mice, regardless of genotype. WT but not KO mice showed an increased in of tissue non-specific alkaline phosphatase (TNAP) in liver, an enzyme involved in detoxification of phosphorylated molecules such as bacterial lipopolysaccharide, but lower expression of Tsc22d3, whose role is described to be protective against sepsis [5]. Inflammation markers were also augmented in the lung and kidney.

Conclusion

In the early stages of the CLP model of sepsis the colon and jejunum are inflamed, and epithelial deletion of the glucocorticoid receptor appears to modulate colonic inflammation, but not barrier function or systemic status.

References

1. Singer, M, et al., Jama, 2016. 315(8): p. 801-10.
2. Carrico, CJ, et al., Arch Surg, 1986. 121(2): p. 196-208.
3. Ocon, B, et al., Biochem Pharmacol, 2016. 116: p. 73-88.
4. Arredondo-Amador, M, et al., Br J Pharmacol, 2021. 178(12): p. 2482-2495.
5. Ballegeer, M, et al., Shock, 2019. 52(2): p. 208-214.

Introduction

Intestinal biofilms are microbiological colonies that colonize biotic and abiotic interfaces, occurring in various adult diseases like IBD, IBS, and CRC.

Aims & Methods

This study investigates the occurrence and characteristics of visible biofilms in the colon of paediatric patients across different disease groups undergoing colonoscopy, aiming to record frequency, location, extent, and adherence in relation to diagnoses and intestinal sections. The prospective, multicentre observational study included 157 paediatric patients undergoing ileocolonoscopy for diagnostic purposes. The presence, extent, and adherence of visible biofilms were documented using high-resolution white light endoscopy. Biofilms, defined as yellow-greenish adherent layers, were classified by extent (small, medium, large) and adherence (weak, medium, strong), then subdivided by diagnostic categories and colonic segments. 43 endoscopies with a Boston Bowel Preparation Score (BBPS) <6 were excluded. Individual diseases that could not be assigned to these patient groups accounted for 7% (n=8).

Results

Biofilms were found in 86.7% of patients with systemic disease and normal colonoscopy, 84.6% with ulcerative colitis, 72.7% with Crohn's disease, 85.7% with IBS, and 87.5% with colorectal polyps. There was a decreasing trend in extent and adherence from proximal to distal bowel segments across all groups. The caecum had the highest adherence and extent in all groups, while the rectum had the lowest. No significant differences in biofilm occurrence and characteristics between disease groups were observed. However, biofilms were significantly more common in proximal bowel segments (ileum, caecum, ascending colon).

Conclusion

Macroscopically visible biofilms are frequently detected in paediatric patients across various diagnoses, showing a predilection for proximal bowel segments. Particularly, patients with IBD, systemic diseases, and IBS are predisposed to biofilms, supporting the hypothesis that biofilms play a pathophysiological role in gastrointestinal diseases. These findings highlight the need for systematic investigation of biofilms in paediatric GI disease diagnosis and treatment.

Introduction

Tissue non-specific alkaline phosphatase (TNAP) is an enzyme that catalyses the removal of phosphate groups from different physiological substrates. The enzyme is encoded in the mouse by the Alpl gene and gives rise to the liver, bone and kidney TNAP isoforms, which differ in their glycosylation and sensitivity to different inhibitors (1). TNAP is widely known for its role in bone mineralization, by hydrolyzing the extracellular mineralization inhibitor inorganic pyrophosphate and allowing normal skeletal/dental mineralization (2). Additionally, TNAP has been related to purinergic signalling pathway, as it can act as an ectonucleotidase and hydrolyse extracellular ATP via ADP and AMP to adenosine (3). Recently, TNAP has been shown to be crucial in many biologic functions, such as, hepatic metabolism (4, 5), thermogenesis (6) or immune cell homeostasis (7, 8). Phosphocholine is a TNAP substrate that could be a link between TNAP activity and liver metabolism (9). TNAP is also expressed in the gut, specifically in the lamina propria leukocytes and in intestinal epithelial cells (IECs). However, the function that TNAP exerts in IECs remains unclear.

Aims & Methods

We aimed to characterize the role of TNAP in intestinal epithelial cells in basal conditions. We studied the role of TNAP using epithelial-specific Alpl deficient mice (Alpl^flox/flox-Villin-CreERT2, referred to as Alpl^IEC-/-). Colon, intestinal epithelial cells and liver were analysed by qRT-PCR. Colon alkaline phosphatase activity was determined spectrophotometrically, using 5.5 mM disodium nitrophenyl phosphate as substrate. Jejunum organoids were generated, and qRT-PCRs were performed.

Results

The expected decrease in Alpl expression was shown in the colon of Alpl^IEC-/- mice; the extent of the decrease suggests that the main Alpl source in the colon in basal conditions is IECs. No differences were detected in the expression of colonic inflammatory markers (Il6, Tnf, S100a8, S100a9). To assess the effect of TNAP silencing in IECs, colonic enterocytes were obtained from WT and Alpl^IEC-/- mice. KO cells were found to exhibit reduced Alpl and increased (p=0.08) Akp6 expression, suggesting that the latter may compensate for the loss of function of Alpl. Besides, the lack of Alpl in colonic IEC causes the downregulation in one of the ectonucleotidases related to purinergic signalling pathway, Nt5e, which hydrolyses AMP to adenosine. Jejunal organoids derived from Alpl^IEC-/- mice exhibit higher expression of both intestinal AP isozymes (Akp3 and Akp6), suggesting a mechanism of compensation. Interestingly, knocking down Alpl in jejunum organoids increased the expression of the chemokine Cxcl10 and drastically reduced the expression of Cxcl1. Finally, the effect of intestinal Alpl deletion was analysed in the liver. A large increase in Tff3, Tnf and Tgfb1 expression and modulation of metabolic genes (Fads2 and Sult3a1) were observed in the liver of Alpl^IEC-/- mice. Finally, consistent with recently published data (9), there was a decrease in liver choline (with a similar trend in phosphocholine, methionine, betaine and methylglycine) in mice in which Alpl was depleted in intestinal epithelial cells.

Conclusion

Our data show that the deletion of Alpl in IEC affects normal cell physiology. Furthermore, the expression of intestinal Alpl appears to have a critical role in liver metabolism.

References

1. Millán, J.L. Alkaline phosphatases: Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes. Purinergic Signal, 2006. 2(2): p.335-41.
2. Millán JL, Whyte MP. Alkaline Phosphatase and Hypophosphatasia. Calcif Tissue Int. 2016;98(4):398-416.
3. Zimmermann H, Zebisch M, Sträter N. Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal. 2012;8(3):437-502.
4. Poupon R. Liver alkaline phosphatase: a missing link between choleresis and biliary inflammation. Hepatology. 2015;61(6):2080-2090.
5. Gámez-Belmonte R, et al. Deficiency in Tissue Non-Specific Alkaline Phosphatase Leads to Steatohepatitis in Mice Fed a High Fat Diet Similar to That Produced by a Methionine and Choline Deficient Diet. Int J Mol Sci. 2020;22(1):51. Published 2020 Dec 23.
6. Sun Y, et al. Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine. Nature. 2021;593(7860):580-585.
7. Hernández-Chirlaque C, et al. Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis. J Crohns Colitis. 2017;11(7):857-870.
8. Marquez C, et al. Expression of alkaline phosphatase in murine B lymphocytes. Correlation with B cell differentiation into Ig secretion. J Immunol. 1989;142(9):3187-3192.
9. Bessueille L, et al. Inhibition of alkaline phosphatase impairs dyslipidemia and protects mice from atherosclerosis [published online ahead of print, 2022 Jun 17]. Transl Res. 2022;S1931-5244(22)00144-X.

Introduction

Patients with inflammatory bowel disease, particularly those with colonic inflammation, are at increased risk of colorectal dysplasia and cancer. Colitis-associated carcinogenesis is believed to follow an inflammatory-driven pathway, potentially modulated by microbiome-derived metabolites, especially those related to amino acid and nitrogen metabolism. However, the precise metabolic signatures underlying this transition remain undefined. We aimed to characterize serum metabolomic profiles across five clinical groups - healthy controls (HC), ulcerative colitis (UC), Crohn’s disease (CD), advanced adenoma (AA), and colorectal cancer (CRC) - to identify disease-specific metabolic shifts and metabolite associations across the inflammatory-neoplastic spectrum.

Aims & Methods

Patients were recruited from the Digestive Center of the National University Hospital, Singapore. Serum was collected and profiled using targeted liquid chromatographic tandem mass spectrometry, quantifying 80 metabolites spanning amino acids, biogenic amines, indoles, kynurenines, and nitrogenous bases. Unsupervised principal component analysis (PCA) was used to assess global metabolic validation, followed by pairwise subgroup comparison using Wilcoxon rank-sum test with false discovery (FDR) correction.

Results

A total of 125 participants were enrolled (25 per group, mean age 61.5 years, 41% female). PCA revealed distinct clustering by disease states, with CRC showing the greatest divergence from healthy controls (ANOSIM R = 0.264, p<0.01). Comparing IBD and CRC to healthy control, 40 metabolites were significantly differentially expressed (FDR <0.05) with enrichment patterns suggestive of class-based divergence. Amino acid-related metabolites (eg. glutamine, GABA, spermine) were progressively downregulated in CRC. Biogenic amines and microbial indole derivatives (eg. 1-methyhistamine, indole-3-propionic acid) showed distinct alterations, with 1-methyhistamine elevated in IBD, and reduced in CRC. Kynureine pathway intermediates (eg. kynurenine, xanthurenic acid) also displayed differential expression between inflammatory and neoplastic states.

Conclusion

Serum metabolomic profiling distinguishes IBD from CRC with both disease-specific and progressive changes. The marked depletion of amino acids (glutamine, GABA, spermine) and shifts in microbial or immune-associated metabolites (e.g., 1-methylhistamine, indole-3-propionic acid, kynurenines) support a model of colitis-associated carcinogenesis mediated by host–microbial metabolic crosstalk. These metabolites may serve as candidate biomarkers for early detection, disease monitoring, and risk stratification in IBD patients at risk for progression to CRC.

Introduction

The intestinal epithelium serves as a site for digestion and absorption of the nutrients and dietary factors, while forming the selective barrier to separate the body’s internal environment from the external luminal environment. The intestinal barrier is established by a combination of several mechanisms, including the mucous layer, antimicrobial peptides, and the intercellular tight junction (TJ)⁽¹⁾. The TJ structure is a multiprotein complex composed of both transmembrane proteins such as claudins and occludin, and cytosolic plaque proteins such as zonula occludens and cingulin, and regulates the paracellular passage of macromolecules, ions, and water. Our and other research groups have shown that the regulation of the intestinal TJ barrier is closely related to intestinal microbial metabolism^(2-4), however, the precise underlying mechanisms are still unclear.

Aims & Methods

The aim of this study was to investigate the role of intestinal microflora in the regulation of the colonic TJ barrier using mice and Caco-2 and MDCKII epithelial cells. In particular, we focused on the molecular mechanisms of n-butyrate-mediated expression of claudin-23. To investigate the effect of intestinal microflora on the TJ expression, RNA sequencing analysis was performed in the colonic tissues from germ-free (GF) and specific pathogen-free (SPF) mice. Claudin-23 expression was also examined in the colon of experimental colitis mice. Human intestinal Caco-2 cells were treated with various microbial metabolites and components including n-butyrate. The TJ barrier function was assessed by measuring the transepithelial resistance (TER) and FITC-dextran (FD) permeability. Claudin-23 expression and transcriptional activity were examined by qRT-PCR, immunoblot, immunofluorescence, and luciferase reporter analyses. The siRNA targeting SP1 and the selective inhibitor of AMPK were used in combination in some experiments. To investigate the role of claudin-23 in the epithelial barrier function, TER and FD permeability were measured in MDCKII epithelial cells stably expressing human claudin-23.

Results

Among several TJ molecules, the claudin-23 mRNA was lower in the colon of GF mice than that of the SPF mice. Claudin-23 mRNA expression was higher in the lower intestinal segments, such as ileum, cecum, and colon, compared with those in the duodenum and jejunum. Claudin-23 expression in the colitis mice was downregulated compared to the normal mice. n-butyrate, but not other microbial metabolites or components, upregulated the claudin-23 expression at both mRNA and protein levels in intestinal Caco-2 cells. N-butyrate enhanced the intestinal TJ barrier integrity, indicated by increased TER and decreased FD permeability. Luciferase reporter assay showed that n-butyrate increased the claudin-23 transcriptional activity. The SP1 siRNA and AMPK inhibitor reduced the n-butyrate-mediated claudin-23 expression and promoter activity. Exogenously expressed claudin-23 localized to the junctional region of MDCKII cells, but did not affect the TER and FD permeability.

Conclusion

Claudin-23 expression in the mouse colon is regulated by a microbial metabolite, n-butyrate. Our results suggest that the transcription factor, SP1, and the AMPK pathway are involved in its transcriptional regulation. Although the precise role of claudin-23 in the intestine is still unclear, the n-butyrate-mediated claudin-23 expression may contribute to the intestinal homeostasis.

References

(1) Suzuki T. Anim Sci J. 2020;91(1):e13357.
(2) Isayama K et al. Exp Cell Res. 2023;425(2):113528.
(3) Yokoo K et al. FASEB J. 2021;35(11):e21854.
(4) Tinh NTT et al. Foods. 2021;10(2):240.