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

Alkaline phosphatase (ALP) is an important marker in primary biliary cholangitis (PBC) associated with risk for disease progression. In the pivotal, Phase 3, placebo-controlled RESPONSE trial (NCT04620733), seladelpar, a first-in-class delpar (selective PPAR-delta agonist), led to a significantly higher percentage of PBC patients achieving the composite biochemical endpoint (ALP <1.67 x upper limit of normal [ULN], ALP decrease ≥15%, and total bilirubin [TB] ≤ULN) compared with placebo (61.7% vs 20%) and decreased mean ALP levels after one year (-42.4% vs -4.3%, respectively). Here, we report additional data on ALP changes in the RESPONSE trial.

Aims & Methods

PBC patients who received ursodeoxycholic acid (UDCA) for ≥12 months or were UDCA intolerant and had ALP ≥1.67 x ULN and TB ≤2 x ULN were randomised 2:1 to daily seladelpar 10 mg or placebo. We assessed ALP changes across demographic subgroups, baseline ALP quartiles, and in patients who did not meet the composite biochemical endpoint. Worsening of ALP (increase >0 U/L from baseline) and safety by baseline ALP subgroup were also evaluated.

Results

128 patients were randomised to seladelpar and 65 to placebo with mean baseline ALP 314.6 U/L and 313.8 U/L, respectively; 53 (27.5%) patients had baseline ALP ≥350 U/L. Seladelpar reduced ALP at month 12 similarly across all subgroups, including in patients with cirrhosis, aged <50 years at PBC diagnosis, and of Hispanic/Latino ethnicity, vs placebo. For patients with baseline ALP ≥350 U/L, seladelpar led to a greater decrease (-44.8%; -216.1 U/L) in ALP vs placebo (-11.6%; -56.4 U/L); similar percent decreases were seen in patients with baseline ALP <350 U/L. Seladelpar reduced ALP similarly across baseline ALP quartiles. Among patients who did not meet the composite biochemical endpoint at month 12, ALP was decreased by -129.9 U/L for seladelpar vs -6.4 U/L for placebo. Seladelpar prevented worsening of ALP, with 7 (5.5%) seladelpar patients vs 19 (29.2%) placebo with increases in ALP from baseline at month 12. Adverse events were reported in 86.4% of patients with baseline ALP <350 U/L and 84.9% of patients with ALP ≥350 U/L, with similar proportions in seladelpar vs placebo patients.

Conclusion

Seladelpar led to robust and consistent ALP decreases across all subgroups studied. Substantial ALP decreases were also observed in patients who did not meet the composite endpoint criteria at month 12. Seladelpar was overall safe and well tolerated, regardless of baseline ALP level.

Disclosure

KVK reports receiving grants or contracts from 89Bio; Boston Scientific; Corcept Therapeutics; CymaBay Therapeutics; Genfit; Gilead Sciences, Inc.; GSK; Hanmi Pharmaceuticals; Intercept Pharmaceuticals; Ipsen; Janssen; Madrigal Pharmaceuticals; Mirum Pharma; NGM Bio; Novo Nordisk; Pfizer; Pliant Therapeutics; Terns Pharmaceuticals; Viking Therapeutics; and Zydus Pharmaceuticals; royalties or licences from UpToDate; consulting fees from 89Bio; CymaBay Therapeutics; Genfit; Gilead Sciences, Inc.; GSK; HighTide Biopharma; Inipharm; Intercept Pharmaceuticals; Ipsen; Madrigal Pharmaceuticals; Mirum Pharma; NGM Bio; and Zydus Pharmaceuticals; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from AbbVie; Gilead Sciences, Inc.; and Intercept Pharmaceuticals; participation on a data safety monitoring board or advisory board with CTI and Medpace; and stock or stock options with Inipharm. KKY reports receiving honoraria from Gilead Sciences, Inc. SK reports receiving grants or contracts from Gilead Sciences, Inc.; and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Gilead Sciences, Inc. EM reports receiving payment or honoraria for speakers bureaus from AbbVie; Eli Lilly; Gilead Sciences, Inc.; and Ipsen; and support for attending advisory boards from AbbVie; Eli Lilly; Gilead Sciences, Inc.; and Ipsen. AB reports receiving grants or contracts and consulting fees from Chemomab; CymaBay Therapeutics; Gilead Sciences, Inc.; GSK; Intercept Pharmaceuticals; Ipsen; and Mirum Pharma; royalties or licences from UpToDate; payment for expert testimony from UMass; and participation on a data safety monitoring board or advisory board with Pfizer. KGR reports receiving grants or contracts from COUR Pharmaceuticals; CymaBay Therapeutics; Gilead Sciences, Inc.; and Pliant Therapeutics; and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Mallinckrodt Pharmaceuticals and Gilead Sciences, Inc. CC reports consulting for CymaBay Therapeutics and Ipsen. JMS reports consultant honoraria from 89Bio; Alentis; Alexion; Altimmune; AstraZeneca; Bionorica; Boehringer Ingelheim; Gilead Sciences, Inc.; GSK; Inventiva Pharma; Ipsen; Lilly; Madrigal Pharmaceuticals; MSD; NorthSea Therapeutics; Novartis; Novo Nordisk; Pfizer; Roche; Sanofi; and Siemens Healthineers; speaker honoraria from AbbVie; Boehringer Ingelheim; Echosens; Gilead Sciences, Inc.; Madrigal Pharmaceuticals; Medscape; and Novo Nordisk; and stockholder options with AGED Diagnostics and Hepta Bio. SC, KY, and DBC are employees of Gilead Sciences, Inc., and may own stock in Gilead Sciences, Inc. CAM reports former employment with CymaBay Therapeutics, a Gilead Sciences, Inc., company, from 2007 to 2024; receiving consulting fees from Gilead Sciences, Inc.; being listed as an inventor on seladelpar patents; and a leadership or fiduciary role (paid or unpaid) with 89Bio (independent director).

Introduction

Platelet-Activating Factor Receptor (Ptafr /PAF-R) is hypomethylated in experimental cirrhosis. Its ligand, the Platelet Activating Factor (PAF) is a proinflammatory phospholipid with multiple biological activities, as aggregation of platelets and modulation of blood pressure^1,2. PAF/PAF-R interaction has been shown to be relevant in pulmonary³ and renal⁴ fibrosis progression. However, the contribution of this pathway to chronic liver disease (CLD) pathophysiology remains unknown.

Aims & Methods

To characterize the contribution of the PAF/PAF-R pathway in CLD progression. A methylation array was performed in hepatic macrophages sorted from control and cirrhotic mice by intragastric administration of CCl₄. Ptafr gene (qPCR) and protein (WB, IHC) expression was evaluated in the livers of cirrhotic and control mice and in primary non-parenchymal liver cells by flow cytometry; as well as in immortalized Kupffer cells (imKCs) in response to different proinflammatory stimuli by qPCR and WB. The presence of the PAF ligand was evaluated in serum, and the supernatant of immortalized hepatic sinusoidal endothelial cells (imLSECs) treated with proinflammatory stimuli by ELISA. The expression of enzymes involved in PAF synthesis and degradation was studied in liver samples. The consequences of treating imKCs with PAF in vitro were evaluated by qPCR and WB. The effect of PAF/PAF-R pathway hampering was tested both in vitro and in vivo by use of a PAF antagonist BN-52021.

Results

The Pafr promoter is hypomethylated in primary hepatic macrophages isolated from cirrhotic mice. We detected a significant increase in PAFR gene and protein expression in the liver of these animals. Liver cells that express PAFR during experimental cirrhosis are primarily hepatic macrophages. In vitro treatment of imKCs with the inflammatory ligands CpG and TNFα induces PAFR expression. Furthermore, stimulation of imKCs with PAF induces a polarization of imKCs toward a proinflammatory profile, increasing gene expression of proinflammatory cytokines (TNF, p≤0.01; IL1β, p≤0.01; TGFβ1, p≤0.01) and chemokines (CCL3, p≤0.05; CCL20, p≤0.0001; CXCL15, p≤0.05; and CXCL16, p≤0.01;), as well as reducing the expression of anti-inflammatory cytokines (IL4, p≤0.01; IL10, p≤.0.0001; and IL-13, p≤0.01 ). This polarization was blocked by the administration of BN-52021 to cell culture. We observed an increase in gene expression of Lpcat1, the enzyme responsible for PAF synthesis, and a decrease in Pafah2, the enzyme that degrades PAF, in liver samples from cirrhotic mice compared to controls, which explains the increase in PAF detected in the serum of cirrhotic mice. All inflammatory stimuli used (IL1b, TNFa, CpG, and LPS) induced PAF secretion by imLSECs in vitro. Cirrhotic mice treatment with BN-52102 resulted in fibrosis and liver damage amelioration (measured by sirius red, aSMA and Vimentin immunostaining), improvement of biochemical markers (hepatic transferases and bile acids) and a reduction of gene expression of proinflammatory and profibrogenic markers (Timp1, Acta2, Col1a1, IL-4, 13, 1b, Tnf, Tgfb).

Conclusion

We have detected the activation of the PAF/PAF-R pathway in experimental cirrhosis. PAF signalling through its receptor induces proinflammatory polarization of hepatic macrophages in vitro. The blockade of receptor activation with a ligand antagonist BN-52021 reduces liver damage and proinflammatory gene expression in cirrhotic mice. These data suggest the value of blocking the PAF/PAF-R pathway to reduce the progression of experimental cirrhosis and its potential application as a therapeutic target.

References

1- Yin et al. Diabetes Metab Syndr Obes. 2022 Jul 8:15:2003-30.
2- Yang et al. Gut. 2004 Jun; 53(6): 877-83.
3- Shahzad et al. J Clin Med. 2024 Apr 6;13(7):2126.
4- Jiang et al. Ren Fail. 2022; 44(1): 660-71.

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

Increased intestinal permeability is a central factor in the disruption of the gut-liver axis during cirrhosis. Innovative strategies focused on limiting intestinal inflammation are needed to restore the integrity of the mucosal, endothelial, and epithelial barriers in cirrhosis. Modulating the interaction between intestinal fibroblasts (Fib_INT) and cellular immunity in the lamina propria may help to restore intestinal homeostasis in cirrhosis.

Aims & Methods

A prospective experimental study was conducted using mice with advanced liver injury induced by oral administration of CCl₄ for 12 weeks (n=5). Control mice received olive oil as vehicle (n=5). Fib_INT activation, chemokine production, and immune recruitment were assesed in ileum samples through gene (RT-qPCR) and protein expression (immunofluorescence). Activation states of primary Fib_INT (CD45-CD326-CD90+) isolated from the small intestine and colon of control and cirrhotic mice were evaluated, as well as the expression of molecules involved in T-cell activation (MHC-II, CD40, CD80, CD86, OX-40L, PD-L1) by flow cytometry. Co-cultures of Fib_INT (cirrhosis) and T lymphocytes (control), and vice versa, were conducted to determine the modulatory effects of cell-to-cell interactions.

Results

An increase in gene (COL1A1, FAP, PDPN) and protein (⍺-SMA, Pdpn, Vim) expression of stromal cell activation markers was observed in the ileum during experimental cirrhosis. Augmented expression of chemokines (CCL2, CXCL10) in the ileum of cirrhotic mice correlated with increased leukocyte (CD45+) infiltration into the villi. Local increases in intestinal lymphocyte (CD3+) and macrophage (F4/80+) populations were detected. In cirrhosis, the percentages of primary Fib_INT from the small intestine expressing stromal cell activation markers (Vim and Pdpn) and T-cell stimulation molecules (CD40, CD80, CD86) were elevated compared to control mice. Isolated Fib_INT from cirrhotic mice interacted with more CD4+ T lymphocytes in vitro and were more effective at inducing their activation (CD25+) and differentiation towards a Th17 profile (IL-17+). Similarly, CD4+ intestinal T lymphocytes from cirrhotic mice could activate Fib_INT isolated from control animals (Vim+, CD86+, OX40L).

Conclusion

Fib_INT are activated in experimental cirrhosis and acquire the ability to induce an inflammatory Th17 response. These findings suggest that modulating the interaction between intestinal fibroblasts and T lymphocytes may recover intestinal homeostasis in cirrhosis.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) presents as a complex multifactorial disease, in which dysregulation of metabolic homeostasis in the liver, as well as in distant organs, plays a substantial role. Recent evidence places skeletal muscle and the muscle-liver axis at the center of the MASLD pathogenic cascade. In fact, low muscle mass and muscle fat infiltration associates with increased risk of advanced MASLD, namely metabolic-associated steatohepatitis (MASH). In parallel, we showed that microRNA-21 (miR-21) is increased in the liver and skeletal muscle of MASH patients, with its abrogation ameliorating disease progression in vivo.

Aims & Methods

Here, we investigated the effects of miR-21 on muscle function in the context of experimental MASLD.Mir21 whole body knockout (KO); Mir21Alb-Cre KO (hepatocyte-specific KO) and wild-type (WT) mice were fed ad libitum with a high-fat/calorie diet (HFD) with high fructose/glucose corn syrup in drinking water for 20 weeks, or with a standard diet (SD), to recapitulate key, complementary metabolic features of human MASLD. Muscle phenotype and function were evaluated through grip strength and wheel running assays one week before sacrifice. In parallel, glucose tolerance test was also performed. At sacrifice, liver/muscle biopsies and total serum were collected for subsequent analyses.

Results

Both Mir21Alb-Cre KO and Mir21 KO HFD-fed mice displayed significant lower weight increments throughout time, compared to WT HFD-fed mice. Moreover, all HFD-fed mice presented reduced voluntary wheel-running distance, except for Mir21KO HFD-fed mice, which showed SD-like wheel-running distance. Mir21 KO mice also displayed a tendency to have grip strength levels higher than WT mice. Furthermore, both Mir21Alb-Cre KO and Mir21 KO HFD-fed mice had AUC of glucose significantly lower than WT HFD-fed mice, and that was similar to WT SD-fed mice. In agreement, Mir21 KO HFD-fed mice showed increased mRNA levels of MyoD and Pax7, two transcription factors responsible for the development and regeneration of skeletal muscle through the differentiation of myoblasts into myotubes, thereby counteracting the decrease observed in WT HFD-fed mice. Moreover, both Mir21Alb-Cre KO and Mir21 KO HFD-fed mice had significantly decreased mRNA expression levels of Mafbx (atrofin-1), a ubiquitin ligase responsible for marking muscle cell proteins for degradation, when compared with WT mice.

Conclusion

Our results show that Mir21 KO mice on a high-fat diet exhibit higher skeletal muscle function compared to their WT counterparts, further suggesting that the ablation of miR-21 contributes to muscle regeneration while inhibiting muscle degradation. These findings highlight the relevance of the muscle-liver axis in MASLD pathogenesis and, possibly, therapeutic targeting. (2022.08837.PTDC, FCT).