Recent advances in intestinal alkaline phosphatase, inflammation, and nutrition

Abstract

In recent years, much new data on intestinal alkaline phosphatase (IAP) have been published, and major breakthroughs have been disclosed. The aim of the present review is to critically analyze the publications released over the last 5 years. These breakthroughs include, for example, the direct implication of IAP in intestinal tight junction integrity and barrier function maintenance; chronic intestinal challenge with low concentrations of Salmonella generating long-lasting depletion of IAP and increased susceptibility to inflammation; the suggestion that genetic mutations in the IAP gene in humans contribute to some forms of chronic inflammatory diseases and loss of functional IAP along the gut and in stools; stool IAP as an early biomarker of incipient diabetes in humans; and omega-3 fatty acids as direct inducers of IAP in intestinal tissue. Many recent papers have also explored the prophylactic and therapeutic potential of IAP and other alkaline phosphatase (AP) isoforms in various experimental settings and diseases. Remarkably, nearly all data confirm the potent anti-inflammatory properties of (I)AP and the negative consequences of its inhibition on health. A simplified model of the body AP system integrating the IAP compartment is provided. Finally, the list of nutrients and food components stimulating IAP has continued to grow, thus emphasizing nutrition as a potent lever for limiting inflammation.

inflammation, intestinal alkaline phosphatase, intestinal barrier, nutrition

INTRODUCTION

Since the first account of intestinal alkaline phosphatase (IAP),¹ 11 reviews on IAP and other alkaline phosphatase (AP) isoforms have been published,^2–12 illustrating the rapidly expanding interest in this enzyme in various academic (eg, physiology, immunology, chemistry), clinical (eg, gastroenterology, hepatology, nephrology, cardiology, surgery, pharmacy), and nutritional fields. Six of these reviews were specifically dedicated to various aspects of IAP, whereas 1 was specific for another AP isoform (tissue-nonspecific AP: TNAP) in the liver.⁸ However, an exhaustive review of IAP, including nutritional modulation, is presently lacking. Briefly, IAP is massively produced by enterocytes in the small intestine and exerts major anti-inflammatory properties both locally and systemically. The underlying mechanism involves efficient dephosphorylation of microbial (eg, lipopolysaccharide [LPS], flagellin) and other pro-inflammatory molecules (eg, ATP, a potent danger signal to the immune system). Importantly, IAP also displays many other physiological roles, including the regulation of intestinal surface pH and that of intestinal absorption of calcium, phosphorus, and lipids and the control of gut microbiota composition and function (see previous reviews by Lallès).^1,3 Recent data reviewed herein now demonstrate the direct involvement of IAP in intestinal barrier function, another fundamental role. Here, all of the recent data in the field of IAP, inflammation, and nutrition are critically analyzed, and complementary measurements for clarifying which AP isoforms are actually involved and future promising lines of research in particular areas (eg, stool IAP as a biomarker, specific roles of dietary fatty acids, interactions between health state and nutrition) are proposed.

RECENT INSIGHTS IN HUMANS IN REGARDS TO INTESTINAL ALKALINE PHOSPHATASE

Intestinal alkaline phosphatase data in humans are scarce for evident ethical reasons associated with invasiveness of intestinal tissue sampling. Interestingly, IAP was evaluated in serum and stools as alternative noninvasive approaches in recent years.

Perioperative fasting in patients was shown to reduce IAP activity levels by 50% in their ileal fluid,¹³ an observation already made in animals.^1,3 Importantly, many usual confounding factors (eg, age, sex, body mass index [BMI], hypercholesterolemia, diabetes) did not influence ileal IAP in this study.¹³

Genetic deficiency in the IAP gene was shown to be present in 2 patients with inflammatory bowel disease (IBD).¹⁴ All IAP gene mutations tested by bioengineering led to enzyme loss of function linked to altered molecular stability or catalytic activity. Furthermore, IAP activity was reduced in intestinal tissues and undetectable in stools of 1 affected patient. A limitation of this publication is the very low number of cases (n = 2). Another study disclosed that a genetic variant in the C-terminal region of IAP was associated with benign familial hyperphosphatasemia.¹⁵ As a result, glycosylphosphatidylinositol anchorage of IAP in enterocyte apical membrane was reduced and free, soluble IAP content was increased in tissues and gut digesta.¹⁵

Fecal IAP is among the top 3 proteins present in human proteins in stools as reported from a sample of 16 healthy individuals.¹⁶ Fecal IAP appears as a promising early biomarker of inflammatory diseases.¹⁷ The enzyme activity of this IAP isotype represents 70%–80% of total AP activity in human stools,¹⁷ compared with only 6%–7% of total AP in serum.¹⁸ Stool IAP activity was calculated from Malo’s data¹⁷ to decrease linearly with age in both male and female controls, with an average annual drop of 0.7 U/g stool, representing approximately −1% of IAP activity per year. This makes a quite spectacular (−30%) drop in IAP activity between 35 and 65 years of age that may contribute to an age-dependent increase in systemic inflammation. This pioneering work disclosed that high IAP activity in stools protected against type 2 diabetes (T2D) in both males and females in a cohort in Bangladesh (n = 202 diabetic patients and 445 healthy controls).¹⁷ Importantly, it also revealed that obese people with high fecal IAP did not develop T2D, whereas 65% of controls with low IAP levels were predicted to display incipient metabolic syndrome.¹⁷ The author concluded that IAP deficiency may be causally involved in T2D in humans, as elegantly demonstrated previously in mice.¹⁹ However, despite the great interest in these findings, stool IAP data must be interpreted with caution because many factors (eg, intestinal infection, inflammation, antibiotics and other drugs, diet) modulate fecal residual IAP activity.²⁰ Finally, patients with type 1 diabetes displayed signs of intestinal inflammation and lower fecal IAP activity than nondiabetic controls.²¹

Intestinal IAP gene expression was found to be higher in healthy controls, intermediate in overweight individuals, and lower in obese patients in 1 study.²² A strong correlation between IAP expression and BMI could be calculated from these data (Y= −0.0033 X + 0.1571; R² = 0.9275). This is in conflict with fecal proteomic data and fecal or serum IAP activities that did not correlate with BMI.^17,18,23 Also, serum IAP did not significantly (P > 0.05) differ among control, overweight, and obese persons in this study.¹⁸ Clearly, additional work is needed on this matter.

Intestinal alkaline phosphatase deficiency has been evidenced in animal models of necrotic enterocolitis (NEC) by the group of D. Gourlay in Milwaukee.³ Recent data disclosed that infant serum IAP levels were higher 4 weeks before the average time to NEC development (36 days).²⁴ The probability for infants with high serum IAP to be at higher risk of NEC did not reach significance in this study,²⁴ probably because of the low number of observations in the NEC group (n = 9, in a total of 177 infants).

Collectively, these data confirm the view gained from animal models revealing IAP as a potent anti-inflammatory enzyme inactivating microbial pro-inflammatory molecules and down-regulating cellular inflammatory pathways and inflammation.

INTESTINAL ALKALINE PHOSPHATASE AND INTESTINAL BARRIER FUNCTION

At the time of the last IAP review, there was no evidence for a direct involvement of IAP in the modulation of intestinal barrier function.³ This gap has been filled in by the group of R. Hodin at Harvard. IAP gene up- and down-expression experiments in Caco-2 and T84 cell lines revealed IAP to regulate the levels of key tight junction proteins (zonula occludens, claudin, occludin) and their cellular localization (Table 1).^13,25 Exogenous IAP added to cell cultures also prevented LPS-induced permeability increase and inflammation in these models.²⁵ The precise underlying molecular mechanisms of IAP action on tight junction proteins are still unknown.

Table 1

Influence of intestinal alkaline phosphatase gene expression modulation and exogenous intestinal alkaline phosphatase addition on intestinal epithelial tight junction protein relative gene expression

	Tight junction protein mRNA relative levels
Treament	ZO-1	ZO-2	ZO-3	Occludin	Claudin-1	Claudin-3	Reference
IAP gene deletion in MEF cells from IAP-KO mice	↘	↘	nd	↘	nd	nd	Liu et al. (2016)25
IAP gene overexpression in Caco-2 and T84 cells	↗↗	↗↗	=	=	=	nd	Liu et al. (2016)25
Exogenous IAP added to Caco-2 or T84 cells	↗	=	↗	=	↗	=	Hamarneh et al. (2014)13
Exogenous IAP given to fed WT mice	↗	=	↗	=	↗	=	Hamarneh et al. (2014)13
Exogenous IAP given to fasted WT mice	↗	↗	↗	↗↗	↗	=	Hamarneh et al. (2014)13

A downward-facing arrow (↘) indicates a decrease, a forward-facing arrow (↗) indicates an increase, and the equal sign (=) indicates no change compared with controls. A single arrow in either direction indicates a significance level of P < 0.05, whereas a double arrow in either direction indicates a significance levels of P < 0.01.

Abbreviations: IAP, intestinal alkaline phosphatase; KO, knockout; MEF, mouse embryonic fibroblasts; nd, not determined; WT, wild-type.

In vivo, transgenic mice expressing human chimeric (intestinal–placental) IAP displayed both reduced intestinal permeability and fecal zonulin tight junction protein compared with wild-type (WT) mice when submitted to a high-fat diet (HFD) for 16 weeks.²⁶ Whereas ZO-1 is a tight junction protein encoded by TJP1 in humans, zonulin is pre-haptoglobin 2, and increased secretion of zonulin into intestinal lumen affects tight junction assembly and increases intestinal permeability.²⁷ Therefore, high fecal zonulin is considered a marker for impaired intestinal barrier function. An association between IAP and intestinal tight junction protein gene expression was also reported in the context of the oral pathogen Porphyromonas gingivalis,²⁸ thus strengthening the link between IAP and barrier function.

Functional gastrointestinal disorders are associated with stress and the nervous system, with corticotropin-releasing factors (CRFs) as mediators.²⁹ Recent data indicate that CRF2 expression is inversely correlated to the intestinal epithelial cell differentiation state.³⁰ CRF2 receptor activation (with urocortin 3) was shown to down-regulate IAP expression and protein in cell lines, and this was mediated through the transcription factor KLF4.³⁰ By contrast, enteric nerves, which are known to regulate intestinal stem cell differentiation, did not modulate IAP activity in enterocyte–nerve cell co-cultures.³¹

Earlier data indicated that IAP was able to reduce bacterial translocation in various models.³ Recent work in a model of Escherichia coli–induced peritonitis in mice showed that IAP was able to inhibit gene expressions of the cation channel-forming protein claudin-2 and of inflammation proteins (eg, vascular endothelial growth factor [VEGF]) via the extracellular signal–regulated kinase (ERK) pathway.³²

In summary, these recent data point to the pivotal role of IAP in controlling intestinal barrier function.

INTESTINAL ALKALINE PHOSPHATASE AND THE COLON

In the previous review focused on IAP, the attention of readers was drawn to the differential origins of AP activity in the small intestine versus the colon and, therefore, on data interpretation.³ Indeed, most (approximately 90%) of AP activity in the small intestine is from the IAP isoform, is highly anti-inflammatory, and is protective. By contrast, in colonic inflammation states, colonic AP activity is high and often reflects tissue infiltration with neutrophils that contain and produce TNAP. This should be interpreted as inflammation and not as a beneficial/protective response,³³ unless convincing evidence is provided that the IAP isoform (gene expression and/or specific activity) in the colon is increased.^1,3 For illustrating that colonic AP activity is synonymous with inflammation, correlations were calculated between (mean) colonic AP activity and colonic tissue density of myeloperoxidase (MPO)–positive cells (neutrophils) using 2 sets of data from inflammation experiments published by a single laboratory.^34,35 These 2 variables were indeed highly correlated (r = 0.843; P < 0.0001) (Figure 1). Some authors have interpreted increased AP activity in the colon as beneficial (eg, Chen et al),³⁶ but AP activity origin (enterocyte vs other cells [eg, neutrophils]) and isoform (IAP vs TNAP) were not ascertained. This could be solved by assaying AP activity without (= total AP) and with relatively specific inhibitors of IAP (L-Phe) or TNAP (eg, levamisole).³ This approach is well illustrated, for example, in the work by Capitán-Cañadas et al.³⁷ Alkaline phosphatase concentrations in control, colitis, and colitis plus fructooligosaccharide (FOS) groups were estimated to be 59, 64, and 73 U/g protein and 8, 136, and 60 U/g protein for IAP and TNAP activities, respectively, based on the combination of AP activities and percentage inhibition with levamisole. Thus, TNAP appears highly correlated with total AP, which is not the case for IAP. In a study with rats, IAP activity was not modulated by glucomannan in the small intestine but it was in colonic tissues.³⁸ Inhibition studies suggested colonic IAP activity to be 4-fold higher than TNAP in controls and treated animals and activities of both AP isoforms to be increased more or less similarly with glucomannan supplementation.³⁸ Alkaline phosphatase gene expression analysis revealed a dietary effect on alpi-1 but not on alpi-2 or Alp1 genes (coding for rat IAP enzyme isoforms I and II and TNAP isoform, respectively). Fecal AP also increased with glucomannan supplementation but did not appear to correlate with colonic tissue data.

Figure 1

Correlation between AP and myeloperoxidase (MPO) activities in colonic tissues of rats (calculated from data by Witaicenis et al^34,35)

Collectively, these data reveal the complexity of colonic AP responses between protection (IAP) and inflammation (TNAP) and interpretation of total AP data. This calls for more detailed information brought about by systematic inhibition and gene expression investigations in the future.

INTESTINAL ALKALINE PHOSPHATASE, THE GUT MICROBIOTA, AND IMMUNITY

The microbiota

In the previous IAP review, pivotal cross-talk between IAP and the gut microbiota was reported.³ Recent publications indicate that IAP promotes the growth of various commensal bacteria in the gut by decreasing (through dephosphorylation) luminal concentrations of nucleotide triphosphates.³⁹ This was suggested to be linked with inflammation because these molecules are danger signals to the immune system when present in the extracellular milieu.⁴⁰

Data on IAP, microbiota, and immunity are scarce in humans. No correlation was found between stool IAP protein and microbial composition.¹⁶

Since the last IAP review,³ new studies confirm a modulation of IAP by some antibiotics. An interaction between age and amoxicillin in offspring born to sows treated with this antibiotic around farrowing was reported.⁴¹ These offspring displayed lower ileal IAP expression and activity transiently at 2 weeks of age.⁴¹ The effect was site dependent and not significant (P > 0.05) in the jejunum. A mixture of nonabsorbable antibiotics (neomycin, 100 mg/L; polymyxin B, 10 mg/L) was found to double IAP activity in growing mice (knocked out for LDL receptor: LDLR^-/-) submitted to an HFD.⁴² This was not observed with amoxicillin (100 mg/L) in this model,⁴² suggesting complex relationships with the gut microbiota.

Immunity

The involvement of IAP in innate immunity is now well established,^1,3 contrary to acquired immunity. Intestinal IAP activity was reported to correlate positively with intestinal tissue levels of immunoglobulin A (IgA) in mice and with fecal immunoglobulins (IgA, IgG, IgM) in humans.²¹ Similar conclusions were reported⁴³ or can be drawn from data in pigs.³⁶ In addition, oral IAP supplementation of mice for 11 weeks increased cecal and colonic IgA concentrations by 9-fold and 3-fold, respectively.²¹ The precise underlying mechanisms are presently unknown, but they may involve the links between IAP and the gut microbiota.⁴⁴

Regarding immune cell type, exogenous IAP was shown to inhibit inflammation not only in intestinal epithelial cells but also in peritoneal macrophages.⁴⁵

In summary, there is good agreement between published results on positive correlations between IAP and secretory immunoglobulin A (sIgA) in various animal models and humans, thus highlighting IAP as an important player in acquired immunity. The link may be the gut microbiota.

INTESTINAL ALKALINE PHOSPHATASE AND DISEASES

Diseases and alkaline phosphatase inhibitors

Overexpression of TNAP isoform has been specifically identified in various diseases involving excessive calcium deposition leading to tissue calcification,^2,11 thus justifying the development of TNAP inhibitors. A number of them are specific for this isoform (eg, levamisole derivatives⁴⁶ or some cyclic sulfonamides⁴⁷). But many compounds are nonspecific of TNAP or may even be more specific inhibitors of IAP. This is the case, for example, for some cyclic sulfonamides,⁴⁷ 4-quinolone derivatives,⁴⁸ and coumarin derivatives.⁴⁹ Specific inhibitors of IAP have also been synthesized (eg, different chromone-based sulfonamides and diarylsulfonamides).^50,51 Therefore, caution must be exercised when using AP inhibitors in order not to inhibit IAP, which is highly protective against inflammation.^1,3 Such highly specific AP inhibitors may, however, be used in research for conducting more specific AP inhibition experiments because L-phenylalanine and levamisole are not absolute inhibitors.

Protein digestion and enteropathies

Alterations in gut permeability are suspected to be pivotal in the development of food allergies,⁵² but little is known about the possible implication of IAP. Dephosphorylation of allergenic proteins, including ovalbumin and casein, by IAP increased their hydrolysis and reduced epithelial translocation through Caco-2 cell monolayers in the case of ovalbumin.^53,54 Another food allergen, soybean β-conglycinin, was found to concentration-dependently increase permeability and IAP activity and reduce tight junction protein expression in the porcine IPEC-J2 cell line.⁵⁵ The reason for increased IAP in this study is unclear.

Alterations in IAP may be involved in the pathogenesis of nonceliac/nonallergic gluten/wheat sensitivity,⁵⁶ but experimental evidence is lacking.

Chronic kidney injury

This research has been developed essentially by the group of P. Pickkers in the Netherlands. The recombinant human chimeric intestinal-placental AP (recAP) is produced by a company (AM-Pharma; https://www.am-pharma.com/). This chimeric enzyme combines the efficiency of IAP isoform for dephosphorylating LPS and the stability of the crown domain of placental AP isoform, which has a long half-life (approximately 1 week) in humans.^57,58 Biodistribution and pharmacokinetics of recAP were investigated in rats, mini-pigs, and humans.^57,58 A population pharmacokinetic model was developed for determining optimal dose and duration for patient studies. Tissue distribution studies in rats indicated sequential uptake of recAP by liver, spleen, adrenals heart, lungs, and kidneys, followed by the gut and thyroid.⁵⁸ In terms of mechanisms of action, recAP was found to attenuate renal inflammation in a rat model of LPS-induced injury and in human proximal tubule epithelial cells in vitro.^59,60 More precisely, the production of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin 6 [IL-6], and interleukin 8 [IL-8]) induced by LPS was prevented by recAP in vitro, and this involved dephosphorylation activity of the enzyme.⁵⁹ A study protocol with recAP administration in humans has been published,⁶¹ and results are now expected. Finally, the importance of the gut–kidney axis and putative causal influence of the gut in renal disorders have been reviewed very recently.⁶²

Metabolic diseases

Previous data in laboratory rodents concluded that a causal link between reduced IAP and metabolic diseases exists.³ This was well demonstrated by the work of Hodin’s group in various models of mice and metabolic syndrome induction. In a first report, both endogenous and orally supplemented IAP reduced intestinal absorption of LPS induced by an HFD and improved liver injury and metabolic status, including endotoxemia, glucose intolerance, insulin resistance, circulating lipid profiles, adiposity, and body weight in mice.¹⁹ In a model of antibiotic-associated metabolic syndrome, oral consumption of IAP (in drinking water) was, again, able to prevent all facets of metabolic syndrome induced by azithromycin.⁶³ Low-grade inflammation, glucose intolerance, and obesity were also found to be promoted by chronic consumption of the sweetener aspartame in mice.⁶⁴ This was explained by a strong and direct inhibition of IAP by L-phenylalanine released from aspartame during digestion.⁶⁴ Recently, a transgenic mouse (IAPTg model) expressing human chimeric IAP along the small intestine was also shown to be resistant to metabolic syndrome induced by a Western diet, compared with WT mice.²⁶ Protection involved higher IAP-mediated reduction in endotoxemia and subsequent inflammation.

In a rat model of type 1 diabetes induced by intravenous injection of streptozotocin, jejunal IAP activity was 60% higher than in controls.⁶⁵ However, a large part of the observed increase in IAP was probably due to higher food intake (+32%) in the streptozotocin-treated rats because food intake is a major driver of enterocyte IAP activity.³

Finally, metabolic disorders could be induced by repeated administration of the pathogen Porphyromonas gingivalis to mice. This was shown to involve a reduction in Iap (Akp3) gene expression.²⁸

To summarize, all of the available experimental data presented above support the pivotal role of both endogenous and orally administered IAP in controlling endotoxemia and preventing or limiting the development of all components of metabolic syndrome. This is accomplished through detoxification (by dephosphorylation) of pro-inflammatory microbial components and strengthening of the gut barrier function.

Infectious diseases

Salmonella is a leading cause of enterocolitis worldwide.⁶⁶ A major work relating Salmonella infection to IAP was recently published in Science. It demonstrated that recurrent low-dose and nonlethal gut infection with a virulent strain (ATCC 14028) of Salmonella enterica serovar Typhimurium induced a form of long-lasting (5 months) IAP deficiency.⁶⁷ More precisely, repeated infection induced the production of a neuraminidase (neuraminidase-3 isoform) by enterocytes that was responsible for sustained desialylation and premature endocytic degradation of IAP molecules in a TLR4-dependent manner.⁶⁷ Mice deficient in ST3Gal6 sialyltransferase had normal Iap gene expression but displayed a 50% reduction in IAP activity, thus demonstrating the importance of IAP glycosylation on enzyme functionality.⁶⁷ Importantly, both exogenous IAP and the antiviral drug zanamivir (inhibiting neuraminidase activity) successfully protected mice against the effects of repeated Salmonella infection.⁶⁷ This is in agreement with the pioneering work by Hodin’s group in Boston showing the protective effects of exogenous IAP on Salmonella infection in mice.^68,69 However, previous data reported IAP actions on pro-inflammatory microbial components (eg, LPS, flagellin, CpG DNA) with no direct effects on the alive pathogen per se. Finally, Muc2^-/- mice were reported to be highly susceptible to Salmonella infection, and this may be due, at least in part, to the associated reduction in Iap gene expression in this model.⁷⁰

Exogenous IAP was also reported to protect against Clostridium difficile infection in mice,⁶⁹ but underlying molecular mechanisms were not reported.

Collectively, these data support a direct protective effect of IAP against serious pathogens. In most cases, the precise mechanisms are still unknown.

PROPHYLACTIC AND THERAPEUTIC USE OF INTESTINAL ALKALINE PHOSPHATASE AND OTHER ISOFORMS

Many publications in which prophylactic and/or therapeutic effects were evaluated in different models have been previously reviewed.^1,3 Additional results on this matter have been released during the last 5 years.

Safety concerns and route of administration

No particular safety problems have been reported so far with (I)AP administration. This was the case with bovine IAP (bIAP) administered intraduodenally (via a nasogastric tube) daily for 7 days in 21 patients⁷¹ and with recAP after 3 days of intravenous administration in healthy volunteers.^58,59 The route of administration is also important to consider.³ Previous data in animal models pointed to the fact that oral administration of (I)AP had the advantage of being able to both stimulate endogenous IAP in the small intestine and reduce systemic inflammation, whereas nonoral administration (eg, intraperitoneal or intravenously) was able to decrease systemic inflammation only.^1,3 Alkaline phosphatase administered intrarectally was more efficient than oral administration for reducing colitis in rats.⁷² The reason put forward was that oral AP was partially degraded during its transit through the stomach and small intestine, compared with direct AP administration in the rectum, which was closer to the site of inflammation.

Intestinal alkaline phosphatase

In experiments, bIAP is the most frequently used IAP. Bovine IAP (40 U/kg/d) was able to reverse radiation-induced changes in IAP (and TNAP) in rats.⁷³ Intestinal alkaline phosphatase was even proposed as a biomarker for evaluating mucosal damage following total-body irradiation.⁷⁴ Intraperitoneal administration of bIAP to mice during surgery reduced postoperative intraperitoneal adhesions and gene expression of pro-inflammatory cytokines interleukin 1β (IL-1β) and TNF-α.⁷⁵ Administration of bIAP (100 U/mL) in drinking water was able to prevent metabolic syndrome induced by the antibiotic azithromycin (50 mg/kg/day) in mice.⁶³ Exogenous IAP (100–200 U/L in a liquid diet) was found to attenuate endotoxemia, hepatosteatosis, and inflammation caused by acute or chronic alcohol consumption in mice.⁷⁶

Administration of bIAP to pregnant mice that received LPS protected them against LPS-induced complications.⁷⁷ Bovine IAP was also successfully expressed in the yeast Pichia pastoris,⁷⁸ paving the way for large-scale production of this IAP in this system.

Other alkaline phosphatase isoforms

Human recombinant chimeric AP using the intestinal isoform for the catalytic part and the placental isoform for the rest of the enzyme has been developed by Pikkers and AM-Pharma in the Netherlands.^57–59,79 Experimental⁸⁰ and therapeutic (Alexio; http://www.alexion.com/) forms of human recombinant TNAP isoform also exist.

Exogenous AP may be used to support postoperative cardiovascular surgery in infants with congenital heart disease because endogenous/serum AP is reduced in these patients and because this enzyme is the primary soluble ectonucleotidase for controlling circulating pro-inflammatory molecules.⁸¹ Exogenous human liver AP at a physiological concentration (500 U/L) was found to be more effective in dephosphorylating blood serum adenosine monophosphate (AMP) into adenosine than bIAP at the same concentration.⁸² Exogenous human liver AP (1600 U/L) also reduced endotoxin activity in an assay in infant blood in vitro.⁸³ Both studies demonstrated efficient dephosphorylation activity of human liver AP on pro-inflammatory phosphorylated moieties (AMP and LPS) in human blood.

In animal studies, human recombinant TNAP (hrTNAP; 3, 6, 9 U) was successful in counteracting LPS-induced sepsis in mice.⁸⁰ Furthermore, hrTNAP displayed synergistic effects when associated with the anticancer drug methotrexate and had systemic anti-inflammatory effects.⁸⁰ Finally, human recombinant placental AP (200 µg) administered subcutaneously to rats prevented and treated experimental arthritis.⁸⁴

Collectively, these data clearly show that anti-inflammatory properties of different AP isoforms can be exploited to prevent or treat various inflammatory diseases, although AP doses need to be adjusted depending on the respective isoforms and targeted diseases.

SIMPLIFIED MODEL OF THE BODY’S ALKALINE PHOSPHATASE SYSTEM INTEGRATING THE INTESTINAL ALKALINE PHOSPHATASE COMPARTMENT

The literature clearly shows that IAP is part of a whole AP system made for controlling inflammation everywhere in the body. The aim of this section is not to review everything on all of the different AP isoforms in the body (and serum AP). Rather, it is for positioning the gut in this system in a clearer way because it has a special location and role with regard to diet and the microbiota, as already reviewed.³ Recent publications^85,86 prompted the proposal of a simplified schematic representation of the body’s AP system, including IAP, and its broad regulation (Figure 2).

Figure 2

Simplified schematic representation of the alkaline phosphatase system integrating intestinal alkaline phosphatase (IAP) and tissue nonspecific alkaline phosphatase (TNAP) produced by the liver in response to increased circulating concentration of microbial pro-inflammatory components (so-called PAMPs) originating from the gut or brought about by inflammatory cells (eg, neutrophils) in inflamed tissues (eg, in the colon in this representation). A. Physiological state characterized by high IAP concentration and flow in the small intestine and low circulating concentration of PAMPs. B. Low-grade inflammation state characterized by lower IAP concentration and flow in the small intestine and higher gut (colonic) pool and circulating levels of PAMPs. C. Overt inflammation characterized by low IAP concentration and flow in the small intestine and high gut (colonic) pool and circulating levels of PAMPs. The bone alkaline phosphatase compartment is not included in this model.

In brief, IAP production by the small intestine is normally maintained at a relatively high level through a balanced diet consumed in sufficient amount. Intestinal AP contributes to control gut barrier function and the microbiota, thus preventing local (gut) and systemic inflammation (Figure 2A). Colonic expression and activity of IAP and TNAP are very low in the physiological situation. Following any substantial insults, inflammation develops, starting at the point of insult. In a sterile or nonsterile inflammatory situation, IAP is inhibited by pro-inflammatory cytokines, thus resulting in decreased local anti-inflammatory tonus associated with a weakened gut barrier and altered microbiota (often toward increases in Gram-negative bacteria; eg, proteobacteria and pools of pathogen-associated molecular patterns [PAMPs] in the gut). This, in turn, favors increased translocation of PAMPs and possibly the appearance of adenine nucleotides (eg, ATP) released by lytic or nonlytic mechanisms from injured cells into the blood circulation (Figure 2B,C). The liver detects increased concentrations of circulating PAMPs (originating from the gut or from elsewhere) and free adenine nucleotides (eg, ATP) at the level of bile canaliculus (see Figure 2 in Pike et al⁸⁵). Following the consumption of AP molecules for detoxifying PAMPs flooding into the liver, the production of liver TNAP is stimulated,⁸⁵ accounting for increased serum (TN)AP activity. In parallel, macrophages and neutrophils become activated, eventually migrating at the site of inflammation and infiltrating tissues. As mentioned before, neutrophils themselves do produce and release TNAP in inflamed tissues. Thereafter, the inflammation resolves with the progressive decrease in concentrations of active PAMPs and adenine nucleotides in serum. Recent data, which show TNAP to be 100-fold more efficient than bIAP in dephosphorylating ATP, suggest that IAP and TNAP isoforms have different roles in fighting pro-inflammatory compounds.⁸²

Regarding routes of administration, oral (or intragastric or intraintestinal) administration of exogenous AP has 2 consequences. One is detoxifying PAMPs, and the other is stimulating the production of endogenous IAP by the enterocyte. The precise mechanism for the latter is not known, but it may be indirect, resulting, for example, from the reduction of inflammation locally.³ Exogenous AP administered intraperitoneally or intravenously contributes to the detoxification of circulating PAMPs and adenine nucleotides, without a positive feedback effect on IAP in the small intestine.³ It may also stimulate endogenous production of TNAP by the liver, as suggested by a recently published model, although experimental evidence is lacking at present.⁸⁶ The differential regulation of endogenous AP following oral versus nonoral route is also illustrated in Figure 2. Finally, whether the liver can detect the circulating IAP isoform itself is not known at present.

NOVEL INSIGHTS INTO INTESTINAL ALKALINE PHOSPHATASE AND DIET

Dietary regulation of IAP has been documented.^1,3 An update of this section is provided here. In most cases, this regulation may often result from a more general effect of diet composition (or components) on enterocyte differentiation. For example, the transcription factors KLF4 and Cdx1 were shown to be involved in Iap gene expression modulation by early nutrition.⁸⁷ These 2 factors are known to activate Iap gene transcription in intestinal epithelial cell lines.^88,89 However, some recent publications suggest more precise or direct effects of dietary components (eg, vitamins or omega-3 fatty acids [FAs]) on IAP.

Oligosaccharides and polysaccharides

Human milk oligosaccharides (HMOs) are the subject of intense research due to their possible effects on gut microbiota colonization and shaping and for their immunomodulatory properties in neonates and during development.⁹⁰ Both acidic and neutral HMO fractions were shown to increase IAP activities in various lines of preconfluent intestinal epithelial cells (IEC) in vitro.⁹¹ Regarding specific fractions, 2’-fucosyllactose, a neutral HMO, and 3’-sialyllactose and 6’-sialyllactose, acidic HMOs may be responsible for these effects.^91–93 The mechanism may be through modulation of enterocyte differentiation.^91–93

Lactose plus fucose used as control oligosaccharides in 1 of these studies had no effect on the IAP activity of IEC cells in culture.⁹² Fructose consumed in the solid (vs liquid) form by mice was reported to enhance (+57%) intestinal IAP activity.⁹⁴ However, most (86%) of this effect could have been the result of the increased feed intake observed with solid fructose. Short chain-fructooligosaccharides (scFOS, 2–8 fructose units) increased ileal IAP activity in pigs (4.5 g scFOS/kg in the weaner diet)⁴³ and decreased AP activity in the inflamed colon of mice (75 mg scFOS/day/mouse by oral gavage).³⁷ In this case, a large reduction in TNAP activity (AP inhibited by levamisole, a relatively specific TNAP inhibitor) was observed, with IAP activity itself varying very little. A recent work conducted with various oligosaccharides (FOS, galacto-oligosaccharides [GOS], isomalto-oligosaccharides [IMOS], raffinose, and lactulose) revealed that GOS drastically increased (+106%) ileal IAP activity (same numerical trends for FOS and IMOS) in rats fed an HFD.⁹⁵ More important, systematic AP inhibition measurements and AP gene expression revealed a strong effect of FOS (same trends for GOS) on Alpi-1 and Akp3 genes (experiment 1) or the Alpi-1 gene alone (experiment 2) in the colon (with no effects on gene expression along the small intestine).⁹⁵ This work is a clear demonstration of the effects of prebiotics specifically on the intestinal AP isoform in the colon. Chito-oligosaccharides used to supplement growing pigs (30 mg/kg feed) had a beneficial effect on ileal IAP (+30%) without effects in the duodenum or jejunum.⁹⁶ Dietary glucomannan stimulated colonic AP activity but not AP activity in the small intestine.³⁸ However, complementary inhibition and gene expression data in this work were difficult to reconcile, thus making the final interpretation hazardous. Arabinoxylan from wheat nearly doubled the IAP activity in the distal ileum and mid-colon of growing male pigs,³⁶ but detailed interpretation was not provided. Previous work in rats suggested that arabinoxylan produced a “stressful” microenvironment for the colonic epithelium.⁹⁷ Cellulose had no effect at either site.³⁶

Collectively, these data show a role for some dietary saccharides in stimulating intestinal IAP, although part of the observed effects may be secondary to their fermentation by or effects on gut microbiota.

Vitamins and minerals

Four papers have been published on vitamin D and IAP in recent years.^98–101 Vitamin D3 (calcitriol; 1, 25(OH)₂D3) strongly increased gene expression of 2 IAP gene variants and IAP activity in Caco-2 cells in vitro.^98,99 In vivo, a vitamin D-deficient diet reduced IAP activity (−37%) in the proximal small intestine of rats. The inhibition was much stronger (−67%) when the rats were fed an HFD,¹⁰¹ thus demonstrating an interaction between vitamin D level and diet composition. This interaction was observed for both IAP gene variants. A direct effect of vitamin D3 on the (nonclassical) vitamin D receptor element (VDRE) of ALPI/Alpi genes in enterocytes was suggested.¹⁰⁰ Recent data show that vitamin D acts through a large set of VDRE regions localized in promoters of numerous genes, some of which are involved in calcium and phosphorus intestinal absorption and intracellular pathways and others that relate to the intestinal tight junction (eg, in the Cdx1-binding site of the claudin-2 promoter).^102,103 Vitamin D deficiency also exacerbated inflammatory responses to Citrobacter rodentium infection in mice, and LPS-detoxifying capacity (synonymous with IAP activity) was completely abolished in the duodenum.¹⁰⁰

Menaquinone-4 (vitamin K2) also specifically stimulated IAP gene expression and IAP activity in Caco-2 cells.¹⁰⁴ This is consistent with previous observations in rats.¹⁰⁵ Intestinal alkaline phosphatase stimulation by vitamin K2 could involve nuclear steroid xenobiotic receptor SXR.¹⁰⁴

Regarding minerals, calcium was previously shown to modulate IAP at the intestinal level in rats.¹⁰⁶ Recent IAP inhibition and gene deletion (Akp3^-/- mice) experiments show that IAP limits intestinal calcium uptake.^107,108 Finally, zinc (sulfate) was shown to enhance intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells.¹⁰⁹ Conversely, inhibition of the PI3K signaling pathway (by LY294002) in vitro or zinc deficiency in vivo decreased zinc-induced IAP activity.^109,110

These additional data confirm the primary importance of some vitamins and minerals in regulating IAP/Iap gene expression and activity and the role of IAP in controlling intestinal absorption of calcium and phosphorus.

Amino acids, peptides, and proteins

Many protein/peptide factors, including lactoferrin and glucagon-like peptide-2 (GLP-2), are known for their growth-promoting effects on the gut.^111,112 Three papers recently reported data on the effects of these factors on IAP. Both human (hLF) and bovine (bLF) milk lactoferrin stimulated IAP gene expression and activity in Caco-2 cells, the former displaying the largest effects on this human intestinal epithelial cell line.¹¹³ This effect was confirmed with bLF (10 g/kg diet) in mice, with a tremendous (10-fold) stimulation of IAP in the jejunum.¹¹³ Similar results, although less spectacular (+28% in jejunal IAP activity with 285 mg bLF/kg body weight [BW]/d) were reported in young pigs.¹¹⁴ Linear dose–response relationships between IAP and bLF were shown in this work. Finally, exogenous GLP-2 administered to LPS-challenged pigs was able to restore normal jejunal IAP activity without altering feed intake.¹¹⁵

The amino acid L-phenylalanine is a potent specific, a competitive inhibitor of IAP.^1,3 As mentioned before in this review, it composes part of the aspartame molecule, an artificial sweetener often used as a food additive (E951) in “light” food products. Phenylalanine is released from aspartame at basic pH in the intestine.⁶⁴ In acute experiments in mice, it was elegantly demonstrated that aspartame (34 mg/kg BW, that is 0.68 mg/mouse of 20 g BW) strongly inhibits IAP (–50%) in intestinal loops.

Regarding amino acids, L-glutamine and derived oligopeptides have already been reported to stimulate IAP in various settings.³ The mechanisms behind the action of glutamine and derivatives may involve increased cellular production of the antioxidant glutathione and reduced oxidative stress.¹¹⁶ However, the stimulating effect of glutamine on IAP was not confirmed in a recent study with type 1 diabetic rats.⁶⁵ Very recently, branched-chain amino acids (BCAAs) were shown to be uncompetitive inhibitors (Leu > Ile > Val) of bIAP in vitro.¹¹⁷ This observation might be relevant to BCAA supplementation in clinical (BCAA deficiencies, cachexia in cancer) and sports/fitness settings.

The soybean seed storage protein β-conglycinin was found to increase IAP activity drastically and reduce the tight junction proteins occludin and ZO-1 in porcine IPEC-J2 intestinal epithelial cells.⁵⁵ These results are somewhat surprising because of the positive role of IAP in intestinal barrier maintenance, which is now well established.^13,25

Effects of energy intake, fat content, and type

Earlier studies have shown that reduced enteral nutrition is deleterious to IAP in the intestine, whereas refeeding restores normal IAP.^3,118 Total parenteral nutrition (TPN) has the same effect in mice, and partial (20%) enteral nutrition was sufficient to restore both Iap gene expression and activity.¹¹⁹ Importantly, intestinal IAP activity was negatively correlated with bacterial translocation.¹¹⁹ In particular, the polymeric formula used in enteral feeding in Crohn’s disease stimulated IAP activity in Caco-2 cells.¹²⁰ This may be due to the presence of supplemental glutamine.

High-fat diets increase IAP expression and/or activity, although this adaptive mechanism appears insufficient to counteract the parallel increase in LPS intestinal translocation stimulated by dietary fat.^1,3 Excess of energy intake and dietary fat content are often confounded in these studies.¹²¹ A recent study investigated them separately using pair-feeding and found that jejunal IAP expression and relative activity were higher in HFD (60% fat) pair-fed than HFD ad libitum–fed (60% fat) mice and was the lowest among mice receiving the low-fat (10%) control diet.¹²² This suggests that IAP activity may be strongly stimulated by an HFD when consumed at normal energy intake but may be partially depressed, possibly through so-called metabolic inflammation associated with obesity, when an HFD is consumed in excess of energy needs. However, this differential response was not confirmed with rats.¹²³

The anti-inflammatory activity of omega-3 FAs may be mediated, at least partly, through IAP. This was elegantly demonstrated using the transgenic fat-1 mouse model, which is able to convert omega-6 FAs to omega-3 FAs without needing dietary omega-3 supplementation.¹²⁴ Among both WT and fat-1 mice being fed the same diet enriched in omega-6 FAs, the latter displayed much higher omega-3 FAs in intestinal tissues and increased production and secretion of IAP.¹²⁴ The underlying mechanisms of IAP stimulation by omega-3 FAs are still not fully understood. They may involve multiple pathways still to be explored (eg, changes in membrane fluidity, local lipid microenvironment, or the lipid mediator resolvin E1 derived from omega-3 FAs).¹²⁴ Interestingly, gut microbiota dysbiosis following antibiotic administration was also less marked in fat-1 than WT mice.¹²⁵ Other studies were carried out with omega-6 and omega-3 FAs and other sources of fat (corn, olive, milk).^126,127 The first paper revealed that a high omega-6 diet was pro-inflammatory to the colon of mice as numbers of neutrophil cells (positive for both TNAP and MPO), macrophages, and prostaglandin E2–positive cells were increased compared with the low omega-6 treatment.¹²⁶ This was also partially reflected in increased LPS-dephosphorylation capacity of colonic tissue. Conversely, using the same criteria, omega-3 FAs (fish oil, 10%) was confirmed in its potent anti-inflammatory properties at the colonic level.¹²⁶ However, LPS-dephosphorylating activity appeared abnormally high with high omega-6 plus omega-3 supplementation, when compared with very low AP-positive cell densities in colonic tissues in this group. High mortality to C. rodentium infection and decreased body weight were also observed with this treatment.¹²⁶ Collectively, these data suggest that omega-3 FAs from fish oil may have favored lethal consequences of infection through mechanisms largely independent from colonic AP. In the second paper by this group, corn oil and milk fat revealed their pro-inflammatory action in the colon, contrary to olive oil, which was strongly anti-inflammatory.¹²⁷ The LPS-dephosphorylating activity and AP staining of colonic tissue were much higher with olive oil than corn oil or milk fat, and AP staining was not restricted to infiltrating cells,¹²⁷ suggesting that the former may have stimulated the endogenous IAP isoform in the colon. This is supported by complementary experiments with the intestinal-like Caco-2 cells, showing that only oleic acid (but not linoleic or palmitic acid) stimulated AP activity (corresponding to IAP isoform) in these cells when challenged with LPS.¹²⁷ Finally, infection experiments without/with added fish oil indicated clearly that corn oil plus fish oil favored mouse lethality to C. rodentium. This was not the case when fish oil was added to olive oil or milk fat,¹²⁷ but the reasons appear unclear. These interesting studies clearly illustrate the complex interactions between dietary FAs and pathogenic environment. Unfortunately, neither enzyme inhibition nor transcriptomic experiments were conducted to discriminate between IAP and TNAP isoforms in these studies. Thus, proper AP isotype characterization is needed in future studies on FAs and their interactions with other factors (eg, inflammation, infection).

Recent data show that both energy intake and fat type have independent effects on IAP. Importantly, omega-3 FAs were shown to have direct effects on IAP, although underlying mechanisms are still unknown. Complex interactions between dietary fat and environment (eg, infection) were also disclosed, thus calling for additional investigations in this domain.

Plant bioactives

Many plant bioactives have been shown to modulate gene expression and/or activity of IAP, although many effects may be due to different molecules present in the compound mixtures often used and may operate at the level of cell differentiation.

Curcumin is a component of the dietary spice curcuma, which is prepared from turmeric (Curcuma longa) root and has renowned anti-inflammatory effects against chronic kidney disease and other disorders, though its bioavailability is low.⁷ Importantly, the anti-inflammatory properties of curcumin (100 mg/kg BW, oral) were recently demonstrated in mice to result from a strong stimulatory action on IAP (+100% activity) in the small intestine, resulting in a 50% decrease in circulating LPS.^7,42 This was associated with a strong reduction (−60%) in the LPS-induced increase in epithelial permeability and normalization of gene expression of key tight junction proteins (ZO-1 and claudin-1) in Caco2 cells.⁴² This strongly suggests that effects of low-available bioactives like curcumin on the body may be indirectly mediated at the lumen–intestinal interface. Curcumin (50 mg/kg BW, oral) also improved colonic inflammation in a model of IBD in rats, although the main effect was primarily against inflammatory cells (eg, neutrophils) and TNAP in this study.¹²⁸

Among other plant bioactives, a Fuzhuan tea water-soluble extract (0.2 g/kg BW/d) and the phenolic compound chlorogenic acid (1 g/kg diet) were found to stimulate IAP (+ 85% and +38%) in the ileum of rats and jejunum of young pigs, respectively.^129,130 Also an ancient Chinese herbal decoction (Yu Ping Feng San) and its individual botanical herbs stimulated IAP at both gene expression (+20%–30%) and activity (5- to 8-fold) in Caco-2 cells.¹³¹

Flavonoids rutin and apigenin K also displayed anti-inflammatory properties in mouse models of colitis.^132,133 It could be estimated from AP inhibition experiments in these studies that only colonic TNAP isoform (inhibited by levamisole) was responsive to rutin supplementation, whereas colonic IAP, which was low in the control group was increased by 70%–80% with apigenin K. This clearly illustrates the need for looking at both IAP and TNAP isoforms for a correct interpretation of the data. Finally, red raspberry (50 g/kg dry food, 10 wk), which contains a complex mixture of phytonutrients and bioactives, doubled colonic Iap gene expression while decreasing inflammation in a chemical model of colitis in mice.¹³⁴

Collectively, these new data confirm and extend the role of various phytonutrients in stimulating endogenous IAP and provide new insights into their underlying mechanisms of action.

Nanoparticles

Concerns have been raised about the possible adverse effects of nanoparticles added to foods on living organisms, including humans,. Cytotoxicity has been documents in both prokaryotic and eukaryotic cells.¹³⁵ Titanium and silicon dioxide nanoparticles both increased IAP in acute and chronic experiments in Caco-2 cells.^136,137 Underlying mechanisms were not reported, and in vivo data are still lacking, thus calling for additional work in this area.

CONCLUSION

The present review complements and extends the observations already available regarding IAP and its fundamental role in protecting the gut and the entire body against inflammation.^1,3 Many important breakthroughs in animal settings and humans have been made in recent years (Table 2). Remarkably, nearly all of the published data are in agreement in regards to the protective effects of IAP. However, interpretation of AP responses in the colon still needs to be improved by complementary measurements—namely, on AP isoform gene expression and AP activity inhibition experiments because 2 AP isoforms are present at that site and may respond differently to the factors under study (Table 3). Investigations on fecal IAP should be further developed to fully validate it as a valuable biomarker. Research on nutrients and other food components has continued to show that nutrition is a potent lever for boosting IAP and preventing or limiting inflammation. In that respect, some FAs appear to have specific effects on IAP and perhaps other AP isoforms,^124–127 thus calling for additional investigations in this field. Finally, the knowledge recently gained in humans on IAP biology is consistent with animal data. Therefore, more clinical trials with exogenous IAP or other AP isoforms for treating metabolic and inflammatory diseases are to be expected in the future.

Table 2

Summary of recent major breakthroughs on intestinal alkaline phosphatase

Species	Breakthrough	Reference
Mouse	IAP regulates tight junction protein (ZO-1, ZO-2, occludin) levels and their cellular localization	Hamarneh et al (2014)13; Liu et al (2016)25
Mouse	Recurrent low-concentration, nonlethal gut infection with a virulent strain of Salmonella enterica serovar Typhimurium induced long-lasting (5 mo) IAP deficiency. Neuraminidase-3 isoform produced by enterocytes upon infection was responsible for sustained desialylation and premature endocytic degradation of IAP in a TLR4-dependent manner	Yang et al (2017)67
Mouse (fat-1 strain)	Omega-3 (but not omega-6) fatty acids specifically induce IAP in intestinal mucosa. Mechanisms involved are probably multiple (eg, changes in membrane fluidity, local lipid microenvironment or the lipid mediator resolvin E1)	Kaliannan et al (2015)124
Human	Genetic deficiency in the IAP gene was shown to be responsible for forms of IBD in 2 patients. Lack of IAP isoform in stools of 1 patient was noted	Parlato et al (2018)14
Human	A genetic variant in the C-terminal region of the IAP gene was associated with benign familial hyperphosphatasemia. This led to reduced glycosylphosphatidylinositol anchorage of IAP in enterocyte apical membrane and increased free, soluble IAP	Ishige et al (2018)15
Human	A high level of IAP isoform in stool is protective against type 2 diabetes mellitus irrespective of obesity. Stool IAP may be an early marker of incipient diabetes in healthy persons	Malo (2015)17

Abbreviations: IAP, intestinal alkaline phosphatase; IBD, inflammatory bowel disease; TLR, Toll-like receptor; ZO, zonula occludens.

Table 3

Suggestions for additional measurements in future studies on intestinal alkaline phosphatase

Site	Information needed for data interpretation	Optional information
Whole body/blood	Data on food/energy intake Data on body weight (initial and final at least)	Marker/s of inflammation (eg, CRP, IL-1β, TNF-α) Serum LPS
Small intestine	IAP (total) enzyme activity IAP gene expression	AP inhibition data (eg, with L-Phe or levamisole) Gene expression of IAP and TNAP isoforms Marker/s of intestinal inflammation (eg, MPO, IL-1β, TNF-α; products/genes) LPS-detoxification capacity
Colon	Total AP enzyme activity AP inhibition data by levamisole (TNAP) or L-Phe (IAP) (preferably in absolute values rather than percentage inhibition) Gene expression (TNAP, IAP isoforms)	Marker/s of colonic inflammation (eg, MPO, IL-1β, TNF-α; products/genes) LPS-detoxification capacity LPS in digesta
Digesta contents or stools	Total AP enzyme activity AP inhibition data by levamisole (TNAP) or L-Phe (IAP) (preferably in absolute values rather than percentage inhibition)	Marker/s of inflammation in digesta or stool (eg, MPO, calprotectin)

Abbreviations: AP, alkaline phosphatase; CRP, C-reactive protein; IAP, intestinal alkaline phosphatase; IL-1β, interleukin 1β; L-Phe, L-phenylalanine; LPS, lipopolysaccharide; MPO, myeloperoxidase; TNAP, tissue-nonspecific alkaline phosphatase; TNF-α, tumor necrosis factor α.

Acknowledgments

Author contributions. J.P.L. gathered and read all of the cited literature, extracted all of the relevant information, wrote the entire manuscript, and created the illustrations.

Funding. No external funding was provided for this work.

Declaration of interest. The author has no relevant interests to declare.