Rapid Communication: Ileal alkaline phosphatase is upregulated following functional amino acid supplementation in Salmonella Typhimurium-challenged pigs

We recently showed that functional amino acid (FAA) supplementation improves growth performance and immune status of Salmonella Typhimurium (ST)-challenged pigs which was further improved by a longer adaptation period. It is expected that the effects are associated with increased activity of intestinal alkaline phosphatase (IAP). The objective of this study was to evaluate the effects of FAA supplementation and adaptation period on the ileal, cecal, and colonic activity of IAP in weaned pigs challenged with ST. In Exp. 1, 32 mixed-sex weanling pigs were randomly assigned to dietary treatments in a 2 × 2 factorial arrangement with low (LP) or high protein (HP) content and basal (FAA–) or FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements) as factors. In Exp. 2, a total of 32 mixed-sex weanling pigs were randomly assigned to 1 of 4 dietary treatments, being FAA- fed throughout the experimental period (FAA−) or a FAA profile fed only in the post-inoculation (FAA+0), for 1 wk pre- and post-inoculation (FAA+1), or throughout the experimental period (FAA+2). In Exp. 1 and 2, after a 7- and 14-d adaptation period, respectively, pigs were inoculated with saline solution containing ST (3.3 and 2.2 × 10 9 CFU/mL, respectively). Plasma alkaline phosphatase was measured on d 0 and 7 post-inoculation in Exp. 1, and IAP (ileum, cecum, and colon) was measured in Exp. 1 and 2. Correlations among ileal IAP and serum albumin and haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), and reduced:oxidized glutathione (GSH:GSSG), ileal myeloperoxidase (MPO), ST shedding and ileal colonization, and post-inoculation average daily gain (ADG), feed intake (ADFI), and gain:feed (G:F) were also analyzed. In Exp. 1, plasma alkaline phosphatase was decreased with ST inoculation and overall content was increased in LP-FAA+ compared to LP-FAA- (P < 0.05). Moreover, ileal IAP was increased in FAA+ compared to FAA- pigs in both studies (P < 0.05) regardless of adaptation time (P > 0.05). Intestinal alkaline phosphatase was positively correlated with MDA and ADFI and negatively correlated with SOD and ST shedding in Exp. 1 (P < 0.05). These results demonstrate a positive effect of FAA supplementation, but not adaptation period, on ileal alkaline phosphatase activity in Salmonella-challenged pigs, which may be associated with improvements in antioxidant balance.

Role of Intestinal Alkaline Phosphatase in Innate Immunity

Intestinal alkaline phosphatase (IAP) is a multi-functional protein that has been demonstrated to primarily protect the gut. The role of IAP in maintaining intestinal homeostasis is underscored by the observation that IAP expression is defective in many gastrointestinal-related disorders such as inflammatory bowel disease IBD, necrotizing enterocolitis, and metabolic syndrome and that exogenous IAP supplementation improves the outcomes associated with these disorders. Additionally, studies using transgenic IAP-knock out (IAP-KO) mouse models further support the importance of the defensive role of IAP in the intestine. Supplementation of exogenous IAP and cellular overexpression of IAP have also been used in vitro to dissect out the downstream mechanisms of this protein in mammalian cell lines. Some of the innate immune functions of IAP include lipopolysaccharide (LPS) detoxification, protection of gut barrier integrity, regulation of gut microbial communities and its anti-inflammatory roles. A novel function of IAP recently identified is the induction of autophagy. Due to its critical role in the gut physiology and its excellent safety profile, IAP has been used in phase 2a clinical trials for treating conditions such as sepsis-associated acute kidney injury. Many excellent reviews discuss the role of IAP in physiology and pathophysiology and here we extend these to include recent updates on this important host defense protein and discuss its role in innate immunity via its effects on bacteria as well as on host cells. We will also discuss the relationship between IAP and autophagy and how these two pathways may act in concert to protect the gut.

Polyphosphate Expression by Cancer Cell Extracellular Vesicles Mediates Binding of Factor XII and Contact Activation

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EV are elevated in cancer, and studies suggest that EV may stimulate thrombosis in cancer patients through expression of tissue factor. However, limited data also implicates EV in activation of the contact pathway of coagulation through activation of factor XII (FXII) to factor XIIa (FXIIa). To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with activation of FXII, EV induced the cleavage of high molecular weight kininogen to cleaved kininogen. We also observed that EV from cancer patients stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or E. coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EV and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by deficiency of FXII, HK, or prekallikrein. Moreover, pre-treatment of EV with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates binding of contact factors to EV, and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

When Activator and Inhibitor of PPARα Do the Same: Consequence for Differentiation of Human Intestinal Cells

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-dependent transcription factor that plays a role in various processes including differentiation of several cell types. We investigated the role of PPARα in the differentiation of intestinal cells using HT-29 and Caco2 cell lines as a model as well as human normal colon and colorectal carcinoma tissues. We detected a significant increase in PPARα expression in differentiated HT-29 cells as well as in normal surface colon epithelium where differentiated cells are localised. Thus, it seems that PPARα may play a role in differentiation of intestinal cells. Interestingly, we found that both PPARα activators (fenofibrate and WY-14643) as well as its inhibitor (GW6471) regulated proliferation and differentiation of HT-29 cells in vitro in the same way. Both compounds led to a decrease in proliferation accompanied by a significant increase in expression of villin, intestinal alkaline phosphatase (differentiation markers). Moreover, the same trend in villin expression was observed in Caco2 cells. Furthermore, villin expression was independent of subcellular localisation of PPARα. In addition, we found similar levels of PPARα expression in colorectal carcinomas in comparison to adjacent normal epithelium. All these findings support the hypothesis that differentiation of intestinal epithelium is PPARα-independent.

Biological response of piglets challenged with Escherichia coli F4 (K88) when fed diets containing intestinal alkaline phosphatase

The aim of the study was to investigate the effect of intestinal alkaline phosphatase (IAP) added to diets on growth performance, diarrhoea incidence (DI), blood metabolites, relative organ weight, and intestinal morphometry of weaned piglets challenged with enterotoxigenic Escherichia coli F4 (K88). A total of 64 crossbred entire male piglets (25-day-old and 7.16 ± 0.28 kg body weight) were allocated into four treatments: control diet (CD^–), CD^– + antimicrobial growth promoter (AGP), CD^– + 15 mg IAP/kg of diet and CD^– + 30 mg IAP/kg of diet, with eight replications. At 15 days, all piglets were orally challenged with 6 ml of a solution containing K88 (10⁶ colony forming units/ml). Microencapsulated IAP in acid solution showed 14.43% solubility and pH values of 1.69, 1.72, 1.51, and 1.52 at the different times measured (0.5 h, 1.0 h, 17.0 h, and 24 h); differently, IAP in basic solution had 4.10% solubility and pH values increased (5.95, 6.10, 6.32 and 6.63) according to the different times, respectively. On days 25–35, piglets that received 30 mg IAP and CD^– showed a better feed conversion ratio (P = 0.075) compared to those fed 15 mg IAP. Piglets that consumed 30 mg IAP or CD^– had higher (P = 0.004) average daily gain on days 35–44. On days 35–44, the piglet average daily feed intake was lower (P = 0.033) with 15 mg IAP compared to AGP. In the entire period, piglets fed 15 mg IAP showed a reduction in average daily gain (P = 0.040) and average daily feed intake (P = 0.092). Piglets on 30 mg IAP showed an improvement (P ≤ 0.05) in DI in the pre-and post-challenge periods. The relative spleen weight of the piglet increased (P = 0.043) in response to 30 mg IAP. Overall, the addition of 30 mg IAP to diets improves the growth performance, attenuates DI, and promotes an increase in spleen relative weight to maintain the healthy state of piglets.

Intestinal Barrier Dysfunction Exacerbates Neuroinflammation via the TLR4 Pathway in Mice With Heart Failure

AimsThe present study aimed to investigate alterations in neuroinflammation after heart failure (HF) and explore the potential mechanisms.MethodsMale wild-type (WT) and Toll-like receptor 4 (TLR4)-knockout (KO) mice were subjected to sham operation or ligation of the left anterior descending coronary artery to induce HF. 8 weeks later, cardiac functions were analyzed by echocardiography, and intestinal barrier functions were examined by measuring tight junction protein expression, intestinal permeability and plasma metabolite levels. Alterations in neuroinflammation in the brain were examined by measuring microglial activation, inflammatory cytokine levels and the proinflammatory signaling pathway. The intestinal barrier protector intestinal alkaline phosphatase (IAP) and intestinal homeostasis inhibitor L-phenylalanine (L-Phe) were used to examine the relationship between intestinal barrier dysfunction and neuroinflammation in mice with HF.ResultsEight weeks later, WT mice with HF displayed obvious increases in intestinal permeability and plasma lipopolysaccharide (LPS) levels, which were accompanied by elevated expression of TLR4 in the brain and enhanced neuroinflammation. Treatment with the intestinal barrier protector IAP significantly attenuated neuroinflammation after HF while effectively increasing plasma LPS levels. TLR4-KO mice showed significant improvements in HF-induced neuroinflammation, which was not markedly affected by intestinal barrier inhibitors or protectors.ConclusionHF could induce intestinal barrier dysfunction and increase gut-to-blood translocation of LPS, which could further promote neuroinflammation through the TLR4 pathway.

Faecal biomarkers in type 1 diabetes with and without diabetic nephropathy

Gastrointestinal dysbiosis is common among persons with type 1 diabetes (T1D), but its potential impact on diabetic nephropathy (DN) remains obscure. We examined whether faecal biomarkers, previously associated with low-grade gastrointestinal inflammation, differ between healthy controls and T1D subjects with and without DN. Faecal samples were analyzed for levels of calprotectin, intestinal alkaline phosphatase (IAP), short-chain fatty acids (SCFA) and immunoglobulins in subjects with T1D (n = 159) and healthy controls (NDC; n = 50). The subjects with T1D were stratified based on albuminuria: normoalbuminuria (< 30 mg/g; n = 49), microalbuminuria (30–299 mg/g; n = 50) and macroalbuminuria (≥ 300 mg/g; n = 60). aecal calprotectin, IAP and immunoglobulin levels did not differ between the T1D albuminuria groups. However, when subjects were stratified based on faecal calprotectin cut-off level (50 µg/g), macroalbuminuric T1D subjects exceeded the threshold more frequently than NDC (p = 0.02). Concentrations of faecal propionate and butyrate were lower in T1D subjects compared with NDC (p = 0.04 and p = 0.03, respectively). Among T1D subjects, levels of branched SCFA (BCFA) correlated positively with current albuminuria level (isobutyrate, p = 0.03; isovalerate, p = 0.005). In our study cohort, fatty acid metabolism seemed to be altered among T1D subjects and those with albuminuria compared to NDC. This may reflect gastrointestinal imbalances associated with T1D and renal complications.

Neu3 neuraminidase induction triggers intestinal inflammation and colitis in a model of recurrent human food-poisoning

Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Salmonella enterica Typhimurium (ST), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4–dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.

Spasmodic Abdominal Pain and Other Gastrointestinal Symptoms in Pontocerebellar Hypoplasia Type 2

Introduction Pontocerebellar hypoplasia type 2 (PCH2) is a rare neurodevelopmental disease with a high disease burden. Besides neurological symptoms, somatic symptoms, such as gastroesophageal reflux (GERD) and failure to thrive, are major contributors to this burden. Methods We report three patients with genetically confirmed PCH2A and significant gastrointestinal (GI) symptoms. Results Apart from impaired swallowing and GERD, which are frequently reported in patients with PCH2, all three patients suffered from episodes of spasmodic abdominal pain and restlessness. In one severely affected patient, lack of intestinal alkaline phosphatase (IAP) is demonstrated. Conclusion GI symptoms are common in PCH2. We draw attention to episodes of spasmodic abdominal pain seriously, aggravating the condition of the patients, especially their movement disorder, and discuss the role of IAP.