Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4

Symbiotic intestinal bacteria derived IPA regulates intestinal permeability and inflammation through PXR

To simulate in vivo homeostatic conditions, we activated PXR using a combination of indole with its respective metabolites. Although IPA alone was a weak human PXR (hPXR) agonist (EC₅₀ 120 μM, E_max 6.38 fold over control) (Figure 1A); IPA in combination with indole significantly activated hPXR (Figure 1B). Similar results were observed with indole 3 acetic acid (IAA) (Figures S1A) and supported by in silico docking studies (Figure S1B; Table S1; Figure S1C). In contrast, mouse PXR (mPXR) was potently activated by IPA (EC₅₀ 0.55 μM, E_max 18.84 fold over control) in vitro (Figure 1A) and induced PXR target gene transcription in vivo (Figure 1C; Figure S1D). More importantly, as specific indoles have been shown to activate the AhR (Denison and Nagy, 2003), we were unable to demonstrate activation of AhR by IPA (Figure S1E).

We next examined effect of indoles on enterocyte inflammatory signals and barrier function. Importantly, differences between Nr1i2^−/− and Nr1i2^+/+ mice were maintained when specifically assaying small intestinal permeability in vivo (Figures S1F and S1G) as well as using an in vivo multi-photon intravital microscopy (Figure S1H and supplemental movies S1 and S2).

For critical validation of the ex vivo experiments demonstrating IPA effects on junctional regulators, we co-administered C. sporogenes to germ-free mice in the presence or absence of L-tryptophan (Figure 1D). We verified that C. sporogenes inoculation led to production of IPA in vivo (thus, it was assumed that indoles were present) (Figure 1E). Germ-free mice exposed to C. sporogenes had a significant reduction in FITC-dextran recovery from the serum and this was further reduced in the presence of L-tryptophan dosing (Figure 1F). The mice intestinal mucosa exposed to C. sporogenes demonstrated significant induction of PXR target genes (Mdr1, Cyp3a11, Ugt1a1), which was further augmented in the presence of L-tryptophan (Figure 1G).

IPA regulates intestinal inflammation through PXR

To validate that IPA was driving the anti-inflammatory response in vivo directly via PXR, we exposed intestinal commensal-depleted Nr1i2^+/+ and Nr1i2^−/− mice to live or heat-killed C. sporogenes. All mice were subsequently exposed to indomethacin (Figure 2A). We verified that only live C. sporogenes, but not the heat-killed bacterial inoculation, led to production of IPA in vivo (Figure 2B). There was a significant reduction in the histologic injury and in mucosal myeloperoxidase (MPO) enzyme activity in Nr1i2^+/+ but not in Nr1i2^−/− mice (Figures 2C and 2D). Furthermore, in these mice, intestinal mucosa exposed to the C. sporogenes had significant induction of PXR target gene (Ugt1a1) when compared to mice exposed to the heat-killed strain (Figure 2E). No such effects were observed in Nr1i2^−/− mice (Figures 2B-2E).

The effects of C. sporogenes in vivo was directly validated using IPA administration by the oral route in both Nr1i2^+/+ and Nr1i2^−/− mice. Although IPA effects could be non-target dependent based on the concentrations administered (i.e. non-specificity of molecular targets based on the concentration of IPA), we chose to study at fixed dose of IPA using an inflammation-based barrier defect (indomethacin) model. In this model, Nr1i2^+/+ and Nr1i2^−/− mice were administered IPA followed by indomethacin and intestinal permeability assessed. The rationale was that a defect in permeability was required in order to show the effect of IPA in both the wild-type and Nr1i2^−/− mice. IPA dosing significantly reduced FITC-dextran permeability in Nr1i2^+/+ (Figure 2F) but not in Nr1i2^−/− mice (Figure 2G).

In an in vivo model of 3-deoxy-D-manno-octulosonic acid (KDO₂)-lipid A (TLR4 ligand) intubation, which elicits inflammatory signals without disrupting the intestinal tissue architecture (see experimental procedures), there was no overt histologic evidence of inflammation (Figure S2A). However, TNF-α mRNA (Figure S2B), p38-MAPK phosphorylation (Figure S2C), and permeability to FITC-dextran (Figure S2D), were clearly induced after KDO2 treatment. In this model, at IPA concentrations that were achievable through oral gavage (Figure S2E), we found that IPA notably decreased TNF-α mRNA expression more in the Nr1i2^+/+ mice (3.73 fold) intestinal epithelium relative to Nr1i2^−/− mice (1.72 fold) (Figure S2F). Together, these studies and the effects of IPA depletion in vivo (see Table S2; Figures S2G-S2I) establish PXR as an important enterocyte target of IPA.

Nr1i2^−/− mice exhibit enhanced ultrastructural defects that directly correlate with increased intestinal permeability and xenobiotic toxicity

To study the function of PXR under steady-state conditions, we performed detailed histological analysis using 6-8 week old Nr1i2^−/− and Nr1i2^+/+ mouse intestines. Although gross features appeared unchanged, histological examination of the mucosa of Nr1i2^−/− mice small intestines showed significant diminution of the villus-crypt ratio (Figure S3A), marked neutrophil infiltration and increased MPO enzyme activity (Figure S3B). The data shown compare the jejunum (highest expression of PXR in the intestine) of Nr1i2^+/+ and Nr1i2^−/− mice; data pertaining to other regions (i.e. duodenum, ileum) have similar trends (data not shown). Although subtle, these differences indicate that the small intestine of Nr1i2^−/− mice may persist in a state of mild elevated stress that may culminate as overt inflammation when exposed to injurious insults. Indeed, we did not observe any significant changes in unfolded protein response (UPR) genes (UPR RT² ProfilerPCR Array, SABiosciences); however, a single gene, Serp1 mRNA, was induced 27-fold in Nr1i2^−/− as compared with Nr1i2^+/+ mouse intestinal mucosa (data not shown). The induction of Serp1 could act to stabilize membrane glycoprotein and stabilize their signaling (e.g., TLR4) (Yamaguchi et al., 1999). In keeping with the mild state of intestinal stress, defects observed in Nr1i2^−/− mice are not due to any gross changes in proliferation (BrdU) or apoptosis (TUNEL) of the epithelium (Figure S3C).

To investigate whether PXR deficiency sensitizes mice to toxic insults, we subjected Nr1i2^−/− and Nr1i2^+/+ mice to four toxic intestinal injury models. In the indomethacin-induced (85 mg/kg body weight, ip, single dose) small intestine inflammation model, the Nr1i2^+/+ mice exhibited modest inflammation by histology (Figure 3A) and in jejunal MPO activity (Figure 3B). In contrast, the Nr1i2^−/− mice showed significantly enhanced basal MPO activity, which increased upon indomethacin treatment (Figure 3B). In evaluating the histology in Nr1i2^−/− mice, the histologic score increased by 6.2 fold over that observed in Nr1i2^+/+ mice (Figure 3C). In the anti-CD3 antibody induced intestinal inflammation-edema model, mice were exposed to anti-CD3 (clone 2C11, 200 μg ip) and tissues harvested 3 hours later for enteropooling. Notably, 100% of Nr1i2^−/− mice given this dose of anti-CD3 antibody succumb to intestinal edema and died within 72 hours of dosing. Indeed, small bowel isolated after 24 hours of dosing with anti-CD3 antibody showed the mean values of enteropooling did not exceed 30 mg/cm (maximum value obtained ~ 31.5 mg/cm), suggesting that enteropooling values saturate around ~ 30-31 mg/cm (Musch et al., 2002). Although the changes in enterpooling values were not significantly different, it is clear that the Nr1i2^−/− mice have increased susceptibility to anti-CD3 mediated injury based on the observation that the mean enteropooling values have maximized ~ 29.5 mg/cm (maximum value ~ 31.5 mg/cm) (Figure 3D). Therefore, the true increase in enteropooling is capped by saturated values in the anti-CD3 treated Nr1i2^−/− mice. In addition, there was near complete blunting of crypt-villus architecture in Nr1i2^−/− as compared to Nr1i2^+/+ mice (Figure 3E). In the gastrointestinal ischemia-reperfusion model (I/R), the endogenous lectin complement pathway is a key initiator of intestinal inflammation in which the intestinal epithelium plays an important role in mediating such damage (Hart et al., 2005). An early finding that persists and then worsens with prolonged ischemia-reperfusion is intestinal barrier dysfunction as manifested by changes in intestinal permeability (Hart et al., 2005). We assessed FITC-dextran recovery after oral gavage of Nr1i2^+/+ and Nr1i2^−/− mice. The change in permeability as assessed by a change in recovery of mean levels of FITC-dextran in the serum of mice for Nr1i2^+/+ and Nr1i2^−/− was 193% and 488.9% (Figure 3F), respectively. These results suggest that the Nr1i2^−/− mice are significantly more susceptible to ischemia-reperfusion mediated changes in intesinal barrier function. Finally, we used an endotoxic shock model, whereby systemic LPS induces a sepsis like syndrome which prominently features intestinal barrier dysfunction (Roger et al., 2009). Nr1i2^−/− mice treated with LPS had significantly worse survival than its Nr1i2^+/+ counterpart suggesting that the loss of PXR significantly worsens survival in LPS-mediated septic syndrome, in which intestinal dysfunction plays a major role (Figure 3G). The LPS findings corroborate previous observations that PXR protects against LPS mediated liver damage (Wang et al., 2010). Together, Nr1i2^−/− mice demonstrate heightened sensitivity towards all four direct and indirect toxic intestinal injury models (Figure 3) (Clayburgh et al., 2005; Ettarh and Carr, 1996; Zhou et al., 2006). The lack of significant steady-state gross inflammatory pathology in Nr1i2^−/− mice, contrasts with this heightened sensitivity towards xenotoxic challenge. To reconcile these findings, we performed ultra-structural and biochemical analysis of Nr1i2^+/+ and Nr1i2^−/− mice jejunal epithelium. Transmission electron microscopy (TEM) of Nr1i2^−/− mice intestinal epithelial cells showed loosely packed shorter microvilli relative to Nr1i2^+/+ mice (Figures 4A and 4B). Additionally, Nr1i2^−/− mice microvilli show significantly diminished digestive enzyme activities compared to Nr1i2^+/+ microvilli (Figure 4C), and the activity (Figure 4D) and abundance (Figure 4E) of alkaline phosphatase is also notably reduced in the Nr1i2^−/− mice intestine. Together, these data demonstrate that key aspects of the fine structure and enzyme expression in the Nr1i2^−/− mice intestine would explain its enhanced sensitivity towards xenobiotic challenge (Matsui et al., 2011).

We next examined the cell-cell junctional complex, an essential structural component of the epithelial barrier. TEM showed that the tight-junction (Tj) and adherens-junction (Aj) complex (black arrow) in Nr1i2^−/− mice intestinal epithelium to be significantly more electron dense, diffuse with dense interconnected stranding (Figure 4F). The mRNA expression of these key junctional-complex markers were markedly diminished in the Nr1i2^−/− mice, except Claudin-2 which is known to induce barrier defects (Figure 4G) (Van Itallie and Anderson, 2006). Immunofluorescence showed similar trends (Figure S4A and S4B). Furthermore, in comparison with Nr1i2^+/+ mice, there is increased FITC-dextran recovery in serum (Figure 4H). Together, these ultra-structural and functional assays implicate PXR as a physiologic regulator of intestinal barrier function.

TLR4 signaling is an essential causative pathway in the epithelial barrier defects observed in the Nr1i2^−/− mice

To screen for molecular mechanisms that are responsible for barrier dysfunction in Nr1i2^−/− mice, we determined relative abundance of key host mRNAs encoding proteins involved in inflammation and microbial invasion. We performed real-time qPCR for anti-inflammatory, anti-microbial and pro-inflammatory markers using total RNA isolated from Nr1i2^+/+ and Nr1i2^−/− mice villi enterocytes. In the Nr1i2^−/− mice, there is a significant down-regulation of mRNAs involved in anti-inflammatory and anti-microbial function, along with a concomitant increase in pro-inflammatory cytokine mRNAs (Figure 5A). Given that TLRs are critical regulators of intestinal barrier function as well as inflammation, we chose to focus on them (Asquith and Powrie, 2010). We performed real-time qPCR for all ten mammalian (mouse) TLRs. TLR over-expression was modest (1.2 to 1.8 fold) and variable (Figure 5B); however, downstream TLR pathway kinase activation was enhanced in Nr1i2^−/− mice (Figure S5A). Among the TLRs, TLR2 and 4 are expressed on the villi enterocytes and play significant roles in the regulation of intestinal barrier function (Cario, 2010). To identify the TLRs that may affect inflammatory cytokine mediated barrier function in Nr1i2^−/− mice, we incubated Nr1i2^−/− enterocytes ex vivo with TLR2 and 4 inhibitors. TLR2 inhibition increased TNF-α and IL-6 mRNA expression in the intestinal mucosa of Nr1i2^−/− mice, conversely, TLR4 inhibition significantly suppressed cytokine mRNA expression (Figure S5B). Furthermore, we generated Nr1i2^−/−Tlr2^−/− mice and mucosa extracted from these mice show increased Tnf-α and Il-6 mRNA expression when compared to Nr1i2^−/− (Figure S5C). These findings correlates with the observation that the loss of Tlr2 in Nr1i2^−/− increased intestinal permeability (Figures S5D-S5F).

We focused next on TLR4 because it is a critical determinant of LPS signaling in the intestine (Abreu et al., 2001). The basal expression of TLR4 protein was modestly elevated (~1.8 fold) in Nr1i2^−/− (Figure 5C) and we also observed an inverse relationship between Pxr and Tlr4 mRNA expression in an intestinal cell line, Caco-2 (data not shown) and primary human intestinal mucosa (data not shown). To study the role of TLR4 in barrier dysfunction in Nr1i2^−/− mice, we crossed the Nr1i2^−/− with Tlr4^−/− mice. In comparison with Nr1i2^−/− mice, the abundance of mRNAs encoding key Tj, Aj proteins, pro- and anti-inflammatory cytokines were reversed in Nr1i2^−/−Tlr4^−/− mice epithelium to levels comparable to those observed in Nr1i2^+/+ mice (Figure 5D).

Since these markers encode for proteins involved in epithelial junctional complex formation and inflammation, we speculate that TLR4 is indeed responsible for the barrier dysfunction observed in Nr1i2^−/− mice. To validate this hypothesis, we assessed if the magnitude of Tnf-α mRNA expression and FITC-dextran permeability was Tlr4 gene-dose dependent. We generated Tlr4 heterozygotes (at least ~50% reduction in protein) in Nr1i2^−/− mice background (data not shown). Enterocyte Tnf-α mRNA expression and FITC-dextran recovery had similar quantitative reduction (Figures S5G and S5H). To further validate our observations regarding genes encoding junctional complex proteins, we performed TEM and immunofluorescence studies. The Nr1i2^−/− Tlr4^−/− mice have microvilli lengths that were larger than Nr1i2^−/− mice and comparable to that observed in Nr1i2^+/+ mice (Figure 5E). Indeed, the same trend was observed in the ultra-structure of the junctional complex (Figure 5F) and immunofluorescence staining for Zo-1 and E-cad (Figures S5I and S5J). The activities of brush border enzymes (Figure 5G) and FITC-dextran permeability (Figure 5H) in the Nr1i2^−/−Tlr4^−/− mice was also similar to that observed in Nr1i2^+/+ mice. Additionally, Nr1i2^−/−Tlr4^−/− mice displayed significantly attenuated damage response to indomethacin (Figure 6A and 6B). Together, these studies show that Tlr4 is essential to maintain epithelial barrier defects observed in the Nr1i2^−/− mice.

IPA protects against indomethacin induced intestinal injury via PXR and TLR4

To further validate the requirement for TLR4 as a critical determinant of the effects of microbiota-derived PXR ligand, IPA, Tlr4^+/+ and Tlr4^−/− mice were exposed to IPA followed by indomethacin (as previously described for experiments with Nr1i2^+/+ and Nr1i2^−/− mice). The results demonstrate that the presence of Tlr4 is required IPA-induced changes in permeability defects induced by indomethacin (i.e. IPA significantly reduces FITC-dextran permeability induced by indomethacin) (Figure 6C). Indeed, to directly validate that the entire loop driven by microbiota-derived IPA results from modulation of TLR4 which in turn regulates intestinal permeability, Nr1i2^+/+ mice were commensal depleted for 15 days and/or followed by exposure to TLR4 ligand, KDO2 and/or IPA delivered orally (Figure 6D). The results demonstrate that commensal depletion increased intestinal permeability, which was further worsened by ~2.8fold with the addition of the TLR4 ligand, KDO2. Note, in conventional Nr1i2^−/− mice possessing intact intestinal microbiota (i.e. LPS), the FITC-dextran recovery in blood was ~ 856 μg/ml (Figure 4H) which is actually lower than commensal depleted Nr1i2^+/+ mice exposed to KDO2 (~ 1495 μg/ml) (Figure 6D). In commensal depleted Nr1i2^+/+ mice, reconstitution of mice with IPA results in a significant decrease in permeability. These results further validate our hypothesis that loss of microbiota producing IPA can induce a Nr1i2^−/− like (not identical) state, whereby, the presence of a TLR4 ligand, KDO2, worsens intestinal permeability and is only corrected when IPA is reconstituted in Nr1i2^+/+ mice (Figure 6D) but not in Nr1i2^−/− mice (Figure 2F&G). Finally, to understand whether the initial signals leading to disruption of the epithelial barrier originates via hematopoietic or non-hematopoietic cells (e.g., predominantly the epithelium), we performed bone marrow chimera experiments between Nr1i2^−/− and Nr1i2^+/+ mice. These studies clearly show that immune reconstitution with either Nr1i2^+/+ or Nr1i2^−/− hematopoietic cells have no effect on pro-inflammatory markers and intestinal permeability to FITC-dextran (Figures 7A-7C). Indeed, since intestine homing hematopoietic derived cells are major source of cytokines that disrupt barrier function (e.g., TLR4 signaling), it should be stated that PXR negatively regulates TLR4 bone marrow derived macrophages or mononuclear cells (data not shown). We saw no changes in serum FITC-dextran recovery, Tnf-α or Il-6 mRNA in Nr1i2^+/+ and Nr1i2^−/− mice transplanted with either Nr1i2^−/− or Nr1i2^+/+ bone marrow, respectively (Figures 7A-7C). In order to further determine if there was any contribution by TLR4 signals emanating from hematopoietic derived cells within the intestine, we derived combinatorial chimeric strains harboring loss or gain of TLR4 protein in these cells resident in Nr1i2^+/+ or Nr1i2^−/− mice. Nr1i2^+/+ mice harboring either a gain or loss of TLR4 protein in bone marrow derived cells within the intestine (LP cells) showed similar levels of serum FITC-dextran recovery as well as Tnf-α or Il-6 mRNA, respectively. Nr1i2^−/− mice expressing non-hematopoietic TLR4 and harboring either a gain or loss of TLR4 protein in bone marrow derived cells within the intestine (LP cells) showed no significant difference in serum FITC-dextran recovery or Tnf-α or Il-6 mRNA levels. Similar results were obtained in Nr1i2^−/− mice with loss of non-hematopoietic TLR4 and harboring either a gain or loss of TLR4 protein in bone marrow derived cells within the intestine (LP cells) (Figures 7D and 7E). This effectively shows that bone marrow derived TLR4, a major LPS-responsive effector of Tnf-α, is unable to alter the intestinal phenotype induced by non-hematopoietic tissue. Thus, PXR resident in the non-hematopoietic compartment (i.e., epithelium) is important in dictating the initial response to external cues inciting epithelial barrier dysfunction.

Cell lines and reagents

The human colon cancer cell lines (Caco-2 and LS174T) and 293T cells were obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. For further details refer Supplemental Experiemental Procedures.

Mice

Tlr4^−/− (B6.B10ScN-Tlr4^lps-del/JthJ, stock number 007227), Tlr2^−/− (B6.129-Tlr2tm1Kir/J, stock number 004650) and C57BL/6J (stock number 000664) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Swiss Webster (SW, control) and Swiss Webster Germ Free (SWGF) mice (7-8 weeks old, female) were purchased from Taconic (Hudson, NY). All animal experiments were approved by the animal institute committee (protocol # 20070715, 20100711) of the Albert Einstein College of Medicine and were performed in accordance with institutional and national guidelines. For further details refer Supplemental Experiemental Procedures.

Genotyping

Mouse tail DNAs were used for genotyping according to manufacturer’s instructions (DNeasy Blood and Tissue kit, Qiagen). Primer sequences and methods are in Supplemental Experiemental Procedures.

Histology and Immunofluorescence analysis

Intestinal tissue sections (paraffin and frozen) were prepared and Hematoxylin-eosin staining were performed in the Histology core facility of Albert Einstein College of Medicine. Histological scoring was performed according to previously published methods (Asano et al., 2009). Experimental details in Supplemental Experiemental Procedures.

Isolation of small intestine villi enterocytes

We have employed the modified Weiser method to isolate pure villus enterocytes from the jejunum in all mice experiments as previously published (Weiser, 1973). Details in Supplemental Experiemental Procedures.

In vitro transcription assay

Experimental details in Supplemental Experiemental Procedures.

Oral dosing of IPA

Nr1i2^+/+ and Nr1i2^−/− mice were gavaged with 10, 20 and 40 mg/kg IPA, dissolved in sterile PBS (pH 7.4) in 100 μl volume per mice for four consecutive days. Swiss Webster (SW) and Swiss Webster Germ Free (SWGF) mice were orally gavaged with 20 mg/kg IPA for four consecutive days.

Transmission electron microscopy

Experimental details in Supplemental Experiemental Procedures.

Enzyme activity assay

Experimental details in Supplemental Experiemental Procedures.

Tissue myeloperoxidase (MPO) activity assay

Experimental details in Supplemental Experiemental Procedures.

In vivo intestine permeability assay

Experimental details in Supplemental Experiemental Procedures.

Germ free mice

Experimental details in Supplemental Experiemental Procedures.

Commensal depletion

Experimental details in Supplemental Experiemental Procedures.

Real-Time Quantitative RT-PCR

Experimental details in Supplemental Experiemental Procedures.

Immunoblot

Experimental details in Supplemental Experiemental Procedures.

Tandem mass spectrometry (LC/MS/MS)

Experimental details in Supplemental Experiemental Procedures.

In vivo toxic small intestine injury models

For indomethacin induced mouse jejunitis model, we have modified a protocol published by Ettarh and Carr by using a single intraperitoneal dose of indomethacin to reduce the toxicity in Nr1i2^−/− mice (Ettarh and Carr, 1996). Details in Supplemental Experiemental Procedures.

Bone marrow transplantation

Experimental details in Supplemental Experiemental Procedures.

BrdU and TUNEL staining

Experimental details in Supplemental Experiemental Procedures.

TLR2 and 4 inhibition experiments

Experimental details in Supplemental Experiemental Procedures.

Intravital imaging of the intestines

Experimental details in Supplemental Experiemental Procedures.

Lenti-based shRNA knock-down systems

Details of the procedure can be found in the recent publication by our laboratory (Wang et al., 2011).

Statistical analysis

Data are shown as mean ± s.e.m. The significance of difference was analyzed by two-tailed Student’s t-test or ANOVA with post-hoc tests were used as indicated. The Kaplan-Meier method was used for survival and differences were analyzed by the log rank test (Figure 3G). All analyses were performed using GraphPad PRISM version 6.01 (GraphPad Software). P < 0.05 was considered statistically significant.