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Small bowel
Intestinal alkaline phosphatase preserves the normal homeostasis of gut microbiota
  1. M S Malo1,
  2. S Nasrin Alam1,
  3. G Mostafa1,
  4. S J Zeller2,
  5. P V Johnson2,
  6. N Mohammad1,
  7. K T Chen1,
  8. A K Moss1,
  9. S Ramasamy1,
  10. A Faruqui1,
  11. S Hodin1,
  12. P S Malo1,
  13. F Ebrahimi1,
  14. B Biswas1,
  15. S Narisawa3,
  16. J L Millán3,
  17. H S Warren4,
  18. J B Kaplan5,
  19. C L Kitts6,
  20. E L Hohmann2,
  21. R A Hodin1
  1. 1Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Massachusetts, USA
  2. 2Infectious Disease Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Massachusetts, USA
  3. 3Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, California, USA
  4. 4Infectious Disease Unit, Departments of Pediatrics and Medicine, Massachusetts General Hospital, Harvard Medical School, Massachusetts, USA
  5. 5Department of Oral Biology, New Jersey Dental School, New Jersey, USA
  6. 6Environmental Biotechnology Institute, California Polytechnic State University, California, USA
  1. Correspondence to M S Malo, Department of Surgery, Massachusetts General Hospital, Jackson 812, 55 Fruit Street, Boston, MA 02114, USA; mmalo{at}partners.org

Abstract

Background and aims The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated.

Methods Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria.

Results IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium).

Conclusions IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

  • Antibiotic-associated diarrhoea
  • gut mucosal defence
  • inflammation
  • lipopolysaccharides
  • microbes
  • Salmonellosis
  • antibiotic therapy
  • bacterial pathogenesis
  • gut inflammation
  • IBD
  • Salmonella

https://doi.org/10.1136/gut.2010.211706

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    Significance of this study

    What is known about the normal homeostasis of intestinal microbiota?

    • Intestinal microbiota is involved in maintaining human health and well-being.

    • Imbalances of normal intestinal microbiotal homeostasis (dysbiosis) are associated with various disease conditions.

    • Molecular mechanisms regulating the normal homeostatic number and composition of intestinal microbes are largely unknown.

    What are the new findings?

    • Compared with wild-type animals, mice deficient in intestinal alkaline phosphatase (IAP knockout) harbour fewer and different types of intestinal bacteria.

    • Oral supplementation of IAP rapidly restores commensal gut microbiota lost due to antibiotic treatment.

    • Oral supplementation of IAP dramatically reduces colonisation of Salmonella typhimurium.

    How this may impact future clinical work?

    • The endogenous brush border enzyme IAP appears to maintain the normal homeostasis of intestinal microbiota.

    • IAP might be an effective therapeutic agent against dysbiosis.

    • IAP might represent a novel therapy against pathogenic bacterial infections.

    Introduction

    Through millions of years of evolution metazoans have developed mechanisms that maintain a mutually beneficial symbiotic relationship with commensal microbiota—for example, intestinal microbes play a pivotal role in maintaining human health and well-being.1–4 The human gastrointestinal tract harbours approximately 1014 bacteria composed of 300–1000 different species.5 Dysbiosis, defined as dysregulation of the normal homeostasis of the intestinal microbiota, has been implicated in the pathogenesis of various disease conditions including (but not limited to) antibiotic-associated diarrhoea (AAD),6 7 Clostridia difficile-associated disease (CDAD),8 inflammatory bowel disease (IBD),9 AIDS10 and obesity.2

    The fundamental mechanisms that govern the normal homeostatic number and composition of the intestinal microbiota remain poorly understood, although a few factors have been implicated in influencing the gut microbiota including antimicrobial peptides, age, immune status, luminal pH, available fermentable materials and general living conditions.11 However, no specific endogenous factor has been identified that functions either directly or indirectly to preserve the normal homeostatic number and composition of the intestinal microbiota.

    Over the last decade, intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, has been recognised as a gut mucosal defence factor. IAP has the ability to detoxify lipopolysaccharides (LPS) from Gram-negative bacteria and exogenous IAP has been shown to attenuate LPS-mediated toxicity.12 13 Bates et al14 demonstrated in zebrafish that, by preventing LPS-mediated inflammatory responses, IAP plays a major role in promoting mucosal tolerance to the commensal gut bacteria. Recently, we found that mice deficient in IAP (IAP knockout, IAP-KO mice) had increased bacterial translocation to mesenteric lymph nodes when the intestine was subjected to a local or distant ischaemic injury.15 This property of IAP as a gut mucosal defence factor led us to investigate its potential interaction with the intestinal microbiota. Here we report that IAP acts to preserve the normal homeostasis of the gut microbiota and it may have therapeutic potential to prevent/treat dysbiosis as well as infections due to pathogenic bacteria.

    Materials and methods

    Details of the experimental Materials and Methods are shown in the online supplement

    Animals

    IAP-KO mice (Mus musculus C57BL/6) construction has been described elsewhere.16 Heterozygous mice were obtained from the Burnham Institute for Medical Research, La Jolla, California, USA. These animals were subsequently bred at the Massachusetts General Hospital (MGH) animal facility to create homozygous IAP-KO, heterozygous and wild-type C57BL/6 (WT) littermates. Confirmation of genotype was performed by PCR analysis.16 The animal experiments were reviewed and approved by the IACUC at MGH. Animals in this study were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School (Boston, Massachusetts, USA) and those prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Department of Health, Education and Human Services, publication no. 85–23 (National Institute of Health), revised 1985).

    Terminal restriction fragment length polymorphism analyses

    Terminal restriction fragment length polymorphism (TRFLP) was performed following the protocol described in Kaplan et al17 (also see online supplement).

    Construction of the library of bacterial 16S small subunit ribosomal RNA genes

    Stool samples of equal weight from 10 individual WT mice were pooled and used for isolation of bacterial DNA using the DNA isolation kit from Qiagen (Valencia, California, USA). Similarly, DNA was isolated from pooled stool samples of 10 IAP-KO animals. The forward and reverse primers17 used in PCR amplification of the 16S rRNA gene fragments for TRFLP analyses (see table 1 in online supplement) were also used to amplify the same approximately 500 bp 16S rRNA gene fragments for cloning. PCR was performed using Taq DNA polymerase in a thermocycler (PTC-200, MJ Research, Waltham, Massachusetts, USA). The PCR conditions were: initial denaturation for 2 min, then 32 cycles of denaturation (94°C for 30 s), annealing (45°C for 30 s) and extension (72°C for 60 s) followed by a final extension step of 5 min at 72°C. The PCR products were verified by electrophoresis through a 2% agarose gel. Using a TOPO TA cloning kit (Invitrogen, Carlsbad, California, USA), fresh PCR products were cloned into pCR2.1 TA cloning vector following the manufacturer's protocol. The transformants were plated on Luria-Bertani (LB) agar plates containing ampicillin (100 μg/ml) and X-gal (40 μg/ml) and incubated at 37°C overnight. Approximately 1000 white transformant colonies from each group (WT and KO) were grown at 37°C overnight in 96-well plates, each well containing 150 μl LB broth with 100 μg/ml ampicillin.

    Phylogenetic analyses

    Cloned 16S rRNA gene sequences were analysed with the Classifier program developed by Michigan State University (http://rdp.cme.msu.edu/). The program produced the name and number of 16S rRNA gene sequences and arranged them in the taxonomical hierarchy. The percentage of each sequence was calculated using the Microsoft Excel program. χ2 analysis was performed to determine statistical significance in the distribution of clones in WT and KO libraries; p<0.05 was considered significant.

    Quantitative real-time PCR

    Semiquantitative limited-cycle PCR (<20 cycles) was performed on WT and KO stool DNA using Taq DNA polymerase with E coli LacZ and subphylum-specific 16S rRNA gene primers (see table 1 in online supplement) in a thermocycler (MJ Research). Quantitative real-time PCR was performed in an IQ5 Thermocycler (Bio-Rad, Hercules, California, USA) using a SYBR Green PCR kit (New England Biolabs, Ipswich, Massachusetts, USA). Primers were synthesised by the MGH Core DNA Synthesis Facility.

    For absolute quantitation of bacterial DNA, serial dilutions of a known amount of E coli DH5α genomic DNA were subjected to qPCR amplification with the 16S rRNA gene universal primers (synthesising 175 bp fragment, see table 1 in online supplement), and a standard curve was generated by plotting CTs against the known amounts of DNA. DNA isolated from WT and KO stools were subjected to qPCR using universal as well as subphylum-specific 16S rRNA gene primers. Quantitation of Eubacterial DNA as well as subphylum-specific bacterial DNA was calculated by comparing the known CT values against the standard values. Each PCR was repeated at least three times.

    Colonisation assay

    For studying colonisation of E coli a commensal E coli was isolated from the stool of a WT mouse and a spontaneous streptomycin-resistant mutant was isolated by culturing the bacterial sample in a MacConkey plate containing streptomycin (100 μg/ml). Spontaneous streptomycin-resistant mutants are frequently a result of mutations in the rpsL gene and are phenotypically stable.18 S typhimurium SL1344 was grown in LB broth and the colony-forming units (CFU) were determined by plating on Hektoen plates. Doses of bacteria for oral gavage varied from 2 × 104 to 2.5 × 106 CFU depending on the experiments. After oral gavage, the presence and quantity of bacteria were determined by stool culture on selective media.

    Restoration of gut microbiota after antibiotic treatment

    Two groups of wild-type (C57BL/6) mice (n=5 for each group) were allowed to drink autoclaved tap water containing 5 mg/ml streptomycin for 3 days. One group also received 200 U/ml calf IAP (cIAP, 20 μl/ml, New England Biolabs) along with streptomycin (cIAP+ group), and cIAP was continued until normal gut microbiota was re-established (usually by day 7). The other group received an equal amount (20 μl/ml) of vehicle for cIAP (see Materials and Methods in online supplement) for the total duration of the experiment (cIAP− group). Water containing cIAP and vehicle for cIAP was replaced daily. The faecal sample from each animal was plated everyday on MacConkey agar plates to determine the restoration of Gram-negative bacteria, especially E coli.

    The experiment was repeated six times and the duration of the experiments varied from 7 to 35 days. Data on the day of first appearance of E coli in an animal's stool were compiled for 39 animals in each group. Statistical significance of the difference in the number of animals with E coli at a specific point of time (day) was determined by the two-tailed Fisher exact test and p<0.05 was considered significant.

    Results

    Gram-negative aerobic bacteria are absent from the stools of IAP-KO mice

    Because IAP is a gut mucosal defence factor that detoxifies LPS, the toxic outer membrane component of Gram-negative bacteria, we investigated the effects of IAP on the intestinal microbiota in WT and IAP-KO mice.16 We plated stool samples on a variety of microbiological media, including MacConkey agar, which primarily allows the growth of Gram-negative bacteria (see Materials and Methods in online supplement). Stool samples from WT animals grew numerous colonies on MacConkey plates (figure 1A); however, faecal samples from KO animals grew no colonies on these plates (figure 1B). We plated numerous faecal cultures from KO mice and never observed aerobic Gram-negative bacteria on MacConkey plates. The predominant aerobic faecal bacterium isolated from the stools of WT animals grown on MacConkey plates was E coli; however, occasionally we observed the growth of Enterobacter, Citrobacter, Proteus, Alcaligenes, Stenotrophomonas and Acinetobacter spp.

    Figure 1
    Figure 1

    Number of bacteria in the stools of wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO) mice. Stool samples from individual mice were separately collected in Brain Heart Infusion (BHI) medium on ice, weighed and homogenised followed by serial dilution of samples and plating on selective plates under aerobic and anaerobic conditions. For PCR analyses, DNA was isolated from an equal amount (weight) of individual stool samples. Bacterial counts were expressed as mean±SEM colony forming units (CFU)/g stool. Each experiment was repeated at least five times and similar results were obtained. (A) Growth of Gram-negative bacteria from the stools of WT mice on MacConkey agar plates (0.01 mg stool plated). (B) Growth of Gram-negative bacteria from the stools of KO mice on MacConkey agar plates (10 mg stool plated). (C) Total count of bacteria from the stools of WT and KO mice (n=7) grown in aerobic conditions on Luria-Bertani (LB), BHI, MacConkey (Mac) and Brucella agar plates. (D) Total count of bacteria from the stools of WT and KO mice (n=7) grown in anaerobic conditions on Brucella agar plates. (E) Semiquantitative limited-cycle PCR (<20 cycles) amplifying bacterial DNA from equal amounts of WT and KO mice stools. **p<0.01, ***p<0.001 (two-tailed Student t test of all data points).

    To ascertain whether the absence of E coli in IAP-KO mice stools was a cohort or cage effect, we performed mixed housing experiments in which adult WT animals were caged with KO littermates. Even when housed together for 60 days, KO animals failed to acquire an aerobic Gram-negative microbiota from the WT littermates as measured by plating of the stool samples on MacConkey plates. This observation that Gram-negative bacteria are absent in the stools of IAP-KO mice suggests that IAP is involved in regulating the intestinal microbiota.

    Total number of aerobic bacteria is greatly reduced in the stools of IAP-KO mice

    Quantitative cultures of WT and IAP-KO stools were performed under aerobic conditions using a variety of rich solid agar media (see Materials and Methods in online supplement). Figure 1C shows that, when plated aerobically on LB, Brain Heart Infusion (BHI) and Brucella agar plates, the stools of KO mice yielded dramatically fewer bacterial colonies than the stools of WT animals (1×105 vs 5×106 CFU/g stool). Stool culture on the MacConkey media showed approximately 106 Gram-negative bacteria/g stool of WT animals and again, as expected, there was no bacterial growth from the stools of KO animals (figure 1B).

    Anaerobes are moderately reduced in the stools of IAP-KO mice

    Stool samples from WT and KO mice were cultured on Brucella agar plates in an anaerobic chamber. The number of anaerobic bacteria in the stools of KO mice was approximately half of that present in WT littermates (4.4×1010 vs 9.7×1010 CFU/g stool) and the difference was statistically significant (p<0.01, figure 1D).

    We next used limited-cycle PCR (<20 cycles) to compare the amount of bacterial DNA in equal amounts (weight) of stools from WT and KO animals. Two pairs of 16S rRNA gene-specific universal primers were used (see table 1 in online supplement), amplifying 523 and 175 bp fragments, respectively. Figure 1E (top two gel photographs) shows that the quantity of 16S rRNA gene fragments is relatively higher in the stools of WT animals compared with KO animals. These data confirmed the stool culture data that the number of bacteria is higher in the stools of WT animals than of KO animals.

    We then amplified E coli DNA from the stool DNA samples from WT and KO animals. While multiple pairs of external primers failed to amplify E coli LacZ-specific fragments, we were able to amplify a target 280 bp LacZ fragment only from WT stools using internal primers (see Materials and Methods in online supplement) as shown in figure 1E (bottom gel photograph). These data confirm the stool culture data that stools of WT animals contain E coli, whereas stools of KO animals contain no detectable E coli.

    Taken together, these stool culture and PCR results demonstrate a generalised decrease in bacterial microbiota in IAP-KO mice. The most dramatic finding was a complete absence of E coli in the KO animals. These observations suggest that the endogenous brush border enzyme IAP plays a role in the regulation of the gut microbiota.

    Terminal restriction fragment length polymorphism (TRFLP) of 16S rRNA genes reveals differential intestinal microbiotal profile in the stools of IAP-KO mice

    Faecal bacterial differences between WT and KO mice were further refined using TRFLP analyses of the bacterial 16S rRNA genes (see Materials and Methods in online supplement). DNA was isolated from the stool samples of eight mice in each group (WT and KO) and from each sample we amplified approximately 500 bp of the 5′ end of 16S rRNA genes using fluorescent PCR primers universal to bacteria.17 The PCR products were then digested with specific restriction endonucleases and the profile of the resulting terminal restriction fragments (TRF) was analysed by electrophoresis through a sequencing gel and detected as a peak in fluorescence (see Materials and Methods in online supplement). Figure 2A shows the number of TRF peaks per sample after Dpn II, Hae III and Hpa II digests as well as TRF peaks from the combination of all three restriction digests. There were significantly more TRF peaks in KO samples (p<0.05), indicating a difference in the types of bacteria present compared with WT. Figure 2B shows the ordination of Dpn II, Hae III and Hpa II TRF peaks using multidimensional scaling (MDS). The TRFLP data were similar among the animals within each group and significantly different between the groups (p≤0.05).

    Figure 2
    Figure 2

    Terminal restriction fragment length polymorphism (TRFLP) of the 16S rRNA gene sequences of the bacteria obtained from the stools of wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO) mice. Bacterial DNA was isolated from pooled or individual stool samples (n=8 per group) and 16S rRNA gene fragments were amplified using PCR with dye-labelled primers followed by restriction digestion with a specific enzyme, electrophoresis through a sequencing gel and counting of terminal restriction fragment (TRF) peaks. TRF peaks were ordinated using multidimentional scaling (MDS). (A) TRF peaks per sample of restriction digests (Dpn II, Hae III and Hpa II and all combined). (B) Ordination using MDS showing the similarity in the TRF profiles of individual as well as all combined samples of Dpn II, Hae III, and Hpa II restriction digests of 16S rRNA gene fragments. *p<0.05 (ANOSIM).

    Phylogenetic analyses reveal an altered profile of intestinal microbiota in the stools of IAP-KO mice

    We used the pCR2.1 vector to construct two libraries carrying the same 16S rRNA gene fragments from the WT and IAP-KO mice as described above (also see Materials and Methods in online supplement). Approximately 1000 clones from each library were sequenced, resulting in 805 and 877 sequences of 16S rRNA genes from bacteria in WT and KO mice stools, respectively. Sequences were subjected to phylogenetic analyses and a distribution of the bacterial types to the level of family is shown in table 1. The results show that the Bacteroidetes constitute more than 50% of bacterial populations in either group followed by the Firmicutes (25%) and Proteobacteria (1%); there was no statistically significant difference at the phylum level between the WT and KO groups. Interestingly, we found that a higher number of Clostridia species was present in the stools of KO mice than in the stools of WT animals (4.56% vs 2.36%, p<0.05). For Unclassified Firmicutes, the WT stools had a greater number than the KO stools (11.8% vs 8.78%, p<0.05). About 21% of the bacteria could not be classified in the stools of WT animals, whereas the number of Unclassified Bacteria made up to 25% of the microbiota in the stools of KO mice; this difference was statistically significant (p<0.05). It should be noted that, because <1000 clones were sequenced from each library, we could not expect to find statistically significant differences in aerobic Gram-negative bacteria (eg, E coli) present in relatively low numbers (Proteobacteria, <1%).

    View this table:
    Table 1

    Phylogenetic profile of bacteria in the stools of wild-type (WT) and intestinal alkaline phosphatase knockout (KO) mice as determined by analyses of 16S rRNA gene sequences

    Quantitative PCR reveals subphylum-specific differences between intestinal microbiotas of WT and IAP-KO mice

    To confirm the phylogenetic data we performed semiquantitative limited-cycle PCR (<20 cycles) as well as quantitative real-time PCR (qPCR) on stool DNA samples from WT and IAP-KO mice. For semiquantitative PCR, equal amounts of DNA from each sample were used and, as expected, we observed similar band intensity of the 175 bp 16S rRNA gene fragments (amplified by universal primers) in both WT and KO stools (figure 3). We then used subphylum-specific 16S rRNA gene primers (see table 1 in online supplement) to amplify Clostridiales, Lactobacillaceae, Enterococcus and Bacteroidetes. The results indicate that, when the ratio of Bacteroidetes in WT and KO stools is not changed, Clostridiales are increased and Lactobacillaceae and Enterococcus are decreased in KO animals (figure 3).

    Figure 3
    Figure 3

    Semiquantitative limited-cycle PCR (<20 cycles) amplifying subphylum-specific bacteria in equal amounts of stool DNA from wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO) mice. Sequences of subphylum-specific primers are shown in table 1 in the online supplement. The experiment was repeated three times and similar results were obtained. M, 100 bp DNA markers; P, control PCR with primers only (no template DNA).

    The absolute amounts of Eubacterial DNA and subphylum-specific bacterial DNA/g stool, as determined by qPCR, are shown in table 2. The calculated fold changes of the respective bacterial groups are also shown in table 2. The ratio of Clostridiales is increased by 3.47-fold and Lactobacillaceae and Enterococcus are reduced by approximately 70% in IAP-KO animals compared with WT animals (table 2). On the other hand, although the ratio of Bacteroidetes increased by 1.62-fold in the IAP-KO animals, this difference was not statistically significant. It should be noted that, because amplification of E coli DNA even from WT mouse stools requires use of internal primers (see figure 1E), we could not generate any qPCR data on E coli from mouse stool DNA. These qPCR data also confirm the semiquantitative PCR data shown above (figure 1E and figure 3).

    View this table:
    Table 2

    Absolute amounts of DNA of specific groups of bacteria in the stools of wild-type (WT) and intestinal alkaline phosphatase knockout (IAP-KO) mice determined by quantitative real-time PCR

    The culture, quantitative PCR, TRFLP and phylogenetic data together establish a specific role for IAP in the regulation of gut microbiota. While bacterial counts are decreased in the IAP-KO animals, TRF peaks are increased. There are populations (Clostridia and Unclassified Bacteria) that appear to be present in small numbers in WT mice but are enhanced in KO animals, whereas some organisms that predominate in WT animals (Unclassified Firmicutes) are depressed in KO animals.

    Commensal E coli fails to colonise IAP-KO mice

    The absence of detectable E coli in the stools of IAP-KO mice prompted us to investigate whether the luminal environment was unfavourable for E coli colonisation in these animals. In order to study E coli colonisation we introduced a streptomycin-resistant mutant of commensal E coli (6.0×105 CFU) to WT and IAP-KO animals (n=8) by oral gavage and monitored its presence in the stool over time (see Materials and Methods in online supplement). The inoculated organism was initially present in only 6/8 KO animals 1 day after inoculation, and it was subsequently eliminated by all but one animal after 15 days when the experiment was terminated (figure 4A), suggesting that IAP-KO animals have an intraluminal environment that inhibits colonisation by exogenous E coli. As expected, WT mice carried the inoculated streptomycin-resistant E coli in their stools along with the pre-existing commensal E coli.

    Figure 4
    Figure 4

    Effects of oral intestinal alkaline phosphatase (IAP) supplementation on colonisation and survival of E coli in IAP knockout (IAP-KO) and wild-type (WT) mice. The streptomycin-resistant E coli was a spontaneous derivative of a commensal E coli isolated from a WT mouse stool (see Materials and Methods in online supplement). Groups of IAP-KO and WT mice (n=5) were allowed to drink water with or without calf IAP (cIAP, 200 U/ml) in the presence or absence of streptomycin and/or ciprofloxacin. After oral gavage of streptomycin-resistant E coli, stool samples were collected every day, homogenised in phosphate-buffered saline and plated on Luria-Bertani (LB) and/or MacConkey agar plates with or without streptomycin. (A) Commensal E coli failed to colonise IAP-KO mice. (B) Oral supplementation of IAP favoured the survival of E coli in IAP-KO mice. (C) Temporal enhancement of colonisation of E coli by IAP in WT mice treated with two antibiotics (streptomycin and ciprofloxacin). *p<0.05, **p<0.01 (two-tailed Fisher exact test of all data points).

    Oral supplementation of IAP favours the survival of commensal E coli in IAP-KO mice

    We next tested whether the unfavourable intraluminal environment for E coli in IAP-KO mice could be reversed by oral supplementation with IAP. We first assayed the activity and stability of cIAP in the drinking water itself as well as in the stools of five animals receiving cIAP in their water. The activity of cIAP in drinking water was determined by following the protocol described in Baykov et al19 (see Materials and Methods in online supplement). We found that cIAP is reasonably stable in water and approximately 80% of cIAP enzymatic activity remains after 24 h at room temperature (see figure S1 in online supplement). Faecal IAP enzyme activity increased in a dose-dependent fashion in mice drinking water with the added cIAP (see figure S2 in online supplement).

    IAP-KO animals were then treated or not treated with cIAP (n=6) and each animal was gavaged twice (2 days apart) with low doses of streptomycin-resistant E coli (20 000 CFU). The presence of E coli in the stools was monitored each day. We observed that only a few of the KO animals from either group carried E coli in their stools on any day, and on day 6 most of the animals carried no E coli in their stools (figure 4B). We then stopped cIAP supplementation and treated the animals with streptomycin for 3 days, thereby killing most of the native microbiota but allowing proliferation of the surviving streptomycin-resistant E coli. We observed that all animals in the group receiving cIAP had a very high number of E coli in their stools after streptomycin treatment (days 10 and 15), whereas animals receiving no cIAP had no E coli in their stools (p<0.01). The experiment was repeated twice and similar results were obtained. These data indicate that exogenous cIAP administration promoted the survival of E coli in IAP-KO animals, further supporting a role for this enzyme in the maintenance of commensal bacteria.

    Temporal enhancement of colonisation of E coli in WT mice receiving oral IAP supplementation

    To determine the temporal effects of IAP on the restoration of gut microbiota, groups of WT animals (n=6) were treated with streptomycin and ciprofloxacin for 3 days and the eradication of aerobic Gram-negative microbiota in their stools was documented. This was followed by oral gavage of small doses (20 000 CFU) of streptomycin-resistant E coli for two alternate days in the presence or absence of cIAP (see Materials and Methods in online supplement). The results (figure 4C) show that animals receiving cIAP had more rapid recolonisation (p<0.015) with E coli, which suggests that IAP could be used to help restore the gut microbiota after antimicrobial therapy.

    Oral administration of the IAP protein enhances recovery of the endogenous gut microbiota in antibiotic-treated WT mice

    The experiments described above show that cIAP enhances colonisation with re-fed enteric bacteria after antimicrobial eradication. To further investigate a possible therapeutic use of IAP, we examined whether treatment with exogenous cIAP might promote the restoration of endogenous enteric microbiota and therefore potentially be useful in disorders like C difficile colitis and AAD. Accordingly, experiments were performed in which groups of WT mice were treated orally with the antibiotic streptomycin in the presence or absence of cIAP. All animals had enteric Gram-negative organisms present at the outset of the experiment and these organisms were eradicated by streptomycin. After discontinuation of the antibiotic, stools were cultured daily and the day of appearance of Gram-negative bacteria (usually E coli, occasionally Proteus mirabilis) was recorded for each animal (see Materials and Methods in online supplement). The experiment was repeated six times (total n=39 per group), but the duration of each experiment varied (7–35 days). The data from all animals were combined and presented as the number of animals with faecal cultures positive for E coli in each group by day. Faecal culture data are shown for day −4 to day +3, at which point all animals receiving cIAP had E coli in their stools (figure 5A). On each given day the number of animals with Gram-negative bacteria was much higher in the cIAP+ group than in the cIAP− group, and the differences were statistically significant for all time points (p<0.00004).

    Figure 5
    Figure 5

    Oral intestinal alkaline phosphatase (IAP) supplementation enhances the restoration of antibiotic-associated loss of gut microbiota and inhibits Salmonella typhimurium infection. (A) Groups of wild-type (WT) mice were treated with streptomycin with or without calf IAP (cIAP, 200 U/ml drinking water). Stool culture was performed daily and time of appearance (day) of Gram-negative (E coli) bacteria recorded for each animal. Data from six independent experiments (for each group n=39 in total) were compiled to calculate the number of animals with E coli in each group on a specific day. ***p<0.001 (two-tailed Fisher exact test) difference in number of animals with E coli between the two groups at a specific point of time (day). (B) Oral IAP administration inhibits growth of S typhimurium in WT mice. Groups of animals (n=5) were treated with streptomycin with or without cIAP as described above. Each animal received an oral gavage of 500 000 colony-forming units (CFU) streptomycin-resistant S typhimurium. Stool culture was performed on Hektoen plates containing streptomycin. Bacterial counts were expressed as mean±SEM CFU/g stool. The experiment was repeated twice. *p<0.05, **p<0.01, ***p<0.001 (two-tailed Student t test of all data points).

    IAP inhibits the growth of Salmonella typhimurium in vivo

    It is well known that enteric pathogenic bacteria compete with the endogenous microbiota and that enteric infections are more common in settings where the normal intestinal microbiota is lost or disrupted. Given our findings with regard to the restoration and/or maintenance of the normal microbiota, we speculated that cIAP could work to inhibit infection by pathogenic bacteria. Groups of WT mice (n=5) were therefore treated with streptomycin for 3 days in the presence or absence of of cIAP. Four days after discontinuation of streptomycin, when all animals in the cIAP+ group but none in the cIAP− group had E coli in their stools, oral gavage of 500 000 CFU streptomycin-resistant virulent S typhimurium SL1344 was performed and the presence of S typhimurium was monitored by plating stools on Hektoen plates (see Materials and Methods in online supplement). The results (figure 5B) show that the number of S typhimurium was dramatically lower in animals in the cIAP+ group (4 log fewer CFU, p<0.001). It is worth noting that E coli also returned 2 days after Salmonella infection in the cIAP− group, and both the cIAP+ and cIAP− groups maintained E coli in their stools during salmonellosis (data not shown). We believe that the delayed return of E coli in the cIAP− group is the normal consequence of antibiotic treatment and not related to Salmonella infection. In addition to the E coli species, we also occasionally observed a few P mirabilis colonies. The experiment was repeated with oral gavage of 2.5×106 CFU S typhimurium and similar results were obtained. Combined data from two experiments showed that, after 7 days of infection, 70% of animals from the cIAP+ group survived compared with only 20% of the animals from the cIAP− group.

    Discussion

    Based on the present data, we believe that the endogenous gut enzyme IAP functions in regulating the gut microbiota. Furthermore, it appears that exogenous IAP could be a useful treatment for maintaining and/or restoring the normal microbiota under a variety of disease-related conditions including, for example, in the setting of antibiotic therapy.

    Intestinal alkaline phosphatase has been known to physiologists for more than half a century.20 IAP is a brush border enzyme that is exclusively expressed in villus-associated enterocytes and hence it has been recognised as an enterocyte differentiation marker.21 The human IAP gene maps to chromosome 2q34-37 and produces a 528-amino acid membrane-bound glycoprotein.22 IAP hydrolyses a wide variety of monophosphate esters at high pH optima. The enzyme is thought to be involved in phosphate and fat metabolism, and is known to be a major component of the fat-containing surfactant-like particles observed in enterocytes after high fat feeding and are secreted into the intestinal lumen as well as the interstitial spaces.23 Narisawa et al16 reported on the phenotype of IAP-KO mice, showing that these animals displayed accelerated fat absorption and became obese compared with their pair-fed WT littermates when fed a high-fat diet.

    Over the past decade evidence has accumulated to suggest an important physiological role for the endogenous IAP enzyme in gut mucosal defence.14 15 Based on the interaction of IAP with the luminal microbial environment, we examined the status of the gut microbiota in IAP-KO mice and found dramatic differences compared with their WT littermates. Given that IAP works to dephosphorylate LPS, the toxic cell wall component of Gram-negative bacteria, we initially wondered whether the absence of IAP in KO mice might result in a greater number of intestinal Gram-negative bacteria. However, to our surprise we observed no growth of aerobic Gram-negative bacteria in the stools of KO mice, as determined by culturing the stool samples in MacConkey agar plates (figure 1B). Although MacConkey media preferentially allow the growth of Gram-negative bacteria, there are some Gram-negative bacteria that do not grow in this medium. We are therefore unable to say that Gram-negative bacteria are completely absent in the stools of IAP-KO mice. However, it is clear that the overall bacterial count is greatly reduced in the stools of KO mice (figure 1B–E), as determined by stool culture and semiquantitative PCR. We focused much of our work on the common enteric commensal E coli and observed that the luminal environment of IAP-KO mice is particularly unfavourable to this bacterium. In future studies it will be interesting to determine whether colonisation by other types of Gram-negative bacteria is similarly affected by the absence of IAP.

    The numerous beneficial roles of gut commensal bacteria are well known and include effects on development as well as vitamin and nutrient absorption.4 It will be interesting to investigate whether there are differences between IAP-KO and WT mice with regard to gut development or vitamin and nutrient absorption. However, under controlled laboratory environmental conditions, we and others have found no gross phenotypic differences except for KO mice being obese when fed a high-fat diet.

    Dysregulation of the normal homeostasis of intestinal microbiota is associated with a wide variety of disease states such as AAD,6 7 CDAD,8 IBD,9 AIDS,10 irritable bowel syndrome,24 obesity,2 diabetes,25 colorectal carcinoma26 and rheumatoid arthritis.27 The recent use of probiotics to treat some of these disease conditions is entirely based on the concept of a beneficial role for commensal microbiota.7 11 28

    Tuin et al29 recently reported that IAP attenuates the colonic inflammation in DSS-induced IBD in rats. Compared with controls, animals treated with IAP showed dramatic inhibition of the DSS-induced inflammation. In addition, oral IAP treatment may be of benefit in human IBD.30 The mechanisms responsible for these beneficial effects of IAP have not yet been determined. In light of the present work and the known role of the gut microbiota with regard to IBD, it will be of interest to examine the status of commensal bacteria in these animals.

    We have used TRFLP analyses of 16S rRNA to show evidence that the gut microbiota in WT and KO mice is different (figure 2). However, phylogenetic analysis ultimately determines the composition of bacteria in a given specimen.31 Comparison of phylogenetic data shows that IAP-KO mice have more Clostridia class of bacteria belonging to the Firmicutes phylum than WT mice (table 1). Recent studies have demonstrated a relationship between the gut microbiota and body weight, the Firmicutes being the pro-obesity bacteria whereas the opposite occurs with the Bacteroidetes.2 Interestingly, IAP-deficient mice have been shown to become obese when fed a high-fat diet16 but the mechanism for the weight gain is not entirely clear. The obesity in the IAP-KO mice was initially attributed to enhanced fat absorption due to loss of the Akp3 gene (IAP-deficient)16 but, more recently, to the increased expression of the Akp6 gene.32 Based on our data on the gut microbiota, it is possible that Clostridia spp., which are greatly increased in KO mice (tables 1 and 2 figure 3), may contribute to the obesity seen in the IAP-KO mice. However, Clostridia probably contributes to obesity only in the presence of a high-fat diet as IAP-KO mice on a normal diet and harbouring increased Clostridia do not become obese. Also, because IAP-KO mice on a normal diet do not become obese, we believe that differences in the microbiota observed in IAP-KO mice are not related to fat absorption, fat metabolism or body weight.

    Bacteria of the Clostridia class are mostly commensals but a few of them are responsible for many critical illnesses, most notably C difficile infection which generally occurs following antibiotic exposure and has become a major health problem, especially among hospitalised patients. Many of these hospitalised patients take in little or no enteral nutrition and we have previously shown that starvation leads to villus atrophy and loss of IAP expression.15 33 It is possible that the loss of IAP leads to an increase in the number of Clostridia in the gut and increased susceptibility to subsequent infections. Our data (figure 5A) showing enhanced recovery of the gut microbiota in WT animals treated with an antibiotic in the presence of cIAP suggests that IAP could be an effective therapeutic agent to prevent or treat AAD and CDAD. Along the same lines, inhibition of Salmonella colonisation in the presence of cIAP (figure 5B) suggests a promising possibility of a therapeutic use for IAP in the prevention or treatment of pathogenic infection. We believe that the IAP-mediated increased growth of commensal bacteria limits the ‘food and shelter’ (nutrition, anchorage sites, space, etc) for the invading pathogens and consequently inhibits pathogenic infection.

    The molecular mechanisms of how IAP favours the growth of E coli and other enteric bacteria are unclear. We initially speculated that IAP might exert a direct effect on the growth of certain bacteria. However, we have observed no direct growth-promoting effects of exogenous cIAP on several commensal and pathogenic bacteria (E coli, S typhimurium, S aureus, L monocytogenes) in vitro (see figure S3 in the online supplement), which suggests that the effects of IAP on the gut microbiota occur through a more indirect mechanism.

    Because IAP detoxifies LPS12 and IAP expression is silenced by proinflammatory cytokines,34 we wondered whether the IAP-mediated regulation of the microbiota was related to changes in the inflammatory state of the intestine. However, histological analyses of IAP-KO intestine revealed no inflammatory changes (data not shown). Interestingly, we have found elevated MHC class II expression in the liver of IAP-KO mice (in press), suggesting some degree of chronic inflammation within the portal system of these mice. Therefore, although the changes in the gut microbiota in the IAP-KO mice are probably not due to an altered gut inflammatory state, it remains possible that inflammatory factors may play some role in mediating the effects of IAP.

    Another possible mechanism for the action of IAP with regard to the gut microbiota relates to pH. It is known that pH affects bacterial growth,35 and IAP has been shown to regulate luminal pH through dephosphorylation of ATP (the higher the ATP concentration, the higher is the luminal pH).36 Accordingly, we determined the pH of luminal content and stool. Compared with WT animals, the pH levels of the luminal contents and stools of IAP-KO mice were marginally higher but the difference was not statistically significant (data not shown). It should be recognised, however, that the pH of the microenvironment adjacent to the mucosal membrane (bacterial attachment site) may not be reflected in the assessment of the total luminal contents. We speculate that IAP regulates the pH of the mucosal microenvironment by altering the luminal ATP concentration, since ATP is a known target of IAP.36 A change in pH and/or ATP concentration might then regulate bacterial growth. Future studies will be needed to determine whether changes in pH-specific luminal microenvironments play a role in the IAP regulation of the gut microbiota.

    Bates et al14 showed that IAP plays a major role in promoting mucosal tolerance to commensal microbiota. It would be interesting to colonise newborn WT or IAP-KO mice with E coli to investigate whether the absence of E coli colonisation in IAP-KO adult animals is due to abnormal maturation of the intestinal immune system.

    Taken together, it appears that IAP exerts its effects on the gut microbiota via some indirect mechanism, perhaps related to pH, inflammation, immunity or some other factors. In addition, it is possible that the effects of IAP are confined to certain species and the changes in other species result from the competition effects for nutrients and other factors.

    In summary, our data show that the endogenous brush border enzyme IAP appears to provide a favourable environment for E coli and perhaps other enteric microbes. Furthermore, exogenous administration of oral IAP promotes restoration of the normal gut microbiota following antibiotic exposure and also appears to inhibit the colonisation and infection of the gut pathogen Salmonella. Based on these data, we suggest that orally administered IAP might be an effective treatment for bacterial pathogenesis as well as a variety of disease conditions associated with dysregulated intestinal microbiota.

    Acknowledgments

    We thank our colleagues Drs Ramnik Xavier and Hans-Christian Reinecker for their critical review of the manuscript.

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    Supplementary materials

    Footnotes

    • Funding This work was supported by NIH grants R01DK050623 and R01DK047186 to RAH, a Junior Faculty Award to MSM from the MGH Department of Surgery and a Grand Challenge Exploration Grant from the Bill and Melinda Gates Foundation to MSM.

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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