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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Gut-activated T cells migrating into the liver can cause extraintestinal manifestations of inflammatory bowel disease. T cells acquire a gut-homing phenotype dependent on retinoic acid (RA) provided by intestinal dendritic cells (DC). We investigated whether liver antigen-presenting cells can induce gut tropism supporting an enterohepatic lymphocyte circulation. Priming of CD4+ T cells by liver sinusoidal endothelial cells (LSEC) supported migration into gut and gut-associated lymphoid tissue. As observed for T cells primed by intestinal DCs, this gut tropism depended on α4β7 integrin and CC chemokine receptor 9 (CCR9) expression by LSEC-primed CD4+ T cells. The induction of gut-homing molecules was mediated by RA, a derivate of vitamin A that is stored in large amounts within the liver. LSECs expressed functional retinal dehydrogenases and could convert vitamin A to RA. Conversely, the lack of signaling via the RA receptor prevented the expression of α4β7 integrin and CCR9 on LSEC-primed CD4+ T cells, consequently reducing their in vivo migration to the intestine. Other liver antigen-presenting cells failed to support high expression of α4β7 integrin on CD4+ T cells, thus, the potential to induce gut homing is restricted to LSECs. Conclusion: The capacity to promote gut tropism via vitamin A use is not unique for intestinal DCs but is also a feature of LSECs. Our data support the assumption that CD4+ T cells can migrate from the liver to the gut as one branch of a postulated enterohepatic lymphocyte circulation. (HEPATOLOGY 2012;55:1976–1984)

Migration of leukocytes to defined tissues is closely regulated by the coordinated interaction of adhesion molecules and chemokine receptors on T cells with their specific counterparts on the surface of endothelial cells. The concept of homing implies that T cells become imprinted with distinct homing molecules that enable them to preferentially recirculate into the tissue of their initial activation.1, 2 As exemplified for the gut, upon priming by dendritic cells (DCs) from the gut-associated lymphoid tissue (GALT), particularly by CD103+ DC from Peyer's patches (PP) and lamina propria, T cells acquire high levels of α4β7 integrin and of CC chemokine receptor 9 (CCR9), thereby gaining the ability to home into the small intestine.3, 4 Binding of α4β7 integrin to the mucosal vascular addressin cell adhesion molecule-1 directs lymphocytes into PP and the intestinal lamina propria. CC chemokine ligand 25 (CCL25), the ligand for CCR9, supports the translocation of CCR9+ T cells into the mucosa of the small intestine.5, 6 In addition to the classical concept of homing as outlined above, migration of lymphocytes primed by GALT-DC into the liver has been described,7, 8 suggesting an enterohepatic circulation of lymphocytes.9

In particular, all-trans retinoic acid was found to be necessary to enhance the expression of both α4β7 integrin and CCR9 on T cells during primary activation imprinting the gut tropism.10 GALT-DC convert vitamin A to retinoic acid (RA) in two enzymatic steps. Ubiquitous alcohol dehydrogenases mediate the first metabolization step, the oxidation to retinal. The conversion to RA requires specific retinal dehydrogenases (RALDH)11 that are only present in distinct cell populations, thus generally limiting provision of RA to tissues containing RALDH-expressing cells.

We earlier observed that naive CD4+ T cells primed by liver sinusoidal endothelial cells (LSEC) not only home into the liver but also migrate into the intestine.12 With the liver as a large reservoir of vitamin A,13, 14 we hypothesized that the capacity of CD4+ T cells primed by LSECs (TLSEC) to enter the intestine depends on RA provided by LSECs. To this end, we investigated the molecular mechanisms for the gut tropism of TLSEC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

More detailed descriptions of mouse models, techniques, primers, and fluorescence-activated cell sorting analyses are provided in the Supporting Information.

Mice.

C57BL/6, BALB/c, DO11.10, or CD90.1+ ovalbumin (OVA) T cell receptor transgenic OT-II mice were obtained from Bundesamt für Risikobewertung (Berlin, Germany) or Charles River (Wilmington, MA). Transferrin receptor OVA (TF-OVA) mice expressing membrane-bound OVA in hepatocytes and OT-I mice were bred at the Charité animal facility (Berlin, Germany). Vitamin A–deficient mice were generated as described.10 All animals received humane care according to the Tierschutzgesetz (permission number G0336/08).

Cell Isolation.

LSECs were isolated using anti-CD146 antibody. Prior to in vitro coculture, LSECs were allowed to adhere overnight, increasing the purity to more than 99%. Nonparenchymal liver cells were depleted from CD146+ LSECs and enriched for major histocompatibility complex class II (MHCII)+ cells using anti-MHCII antibody resulting in 99% MHCII+LSEC liver antigen-presenting cells (APC). For messenger RNA (mRNA) analysis, ex vivo isolated LSECs were enriched by FACSAria cell sorter (BD Biosciences, Heidelberg, Germany) to a purity of 99%. APCs were isolated from spleen and lymph nodes and depleted from CD90+ cells using anti-CD90.2 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Peripheral lymph nodes (pLN) and mesenteric lymph nodes (mLN) were digested with collagenase D (Roche, Grenzach-Wyhlen, Germany), and the cell suspension was enriched for DCs using anti-CD11c MicroBeads (Miltenyi Biotec). Naive CD4+CD62L+ T cells were prepared using anti-CD4 antibody and anti-CD62L MicroBeads (Miltenyi Biotec), resulting in a purity of 99%. Naive CD8+CD44 T cells were purified using anti-CD8 and anti-CD44 antibody (Miltenyi Biotec).

In Vitro Polarization of Naive CD4+ T Cells.

To generate TLSEC or T cells primed by APC from the spleen (TSAPC), 5 × 105 naive CD4+ T cells from OT-II mice were cocultured with 1 × 106 LSECs or spleen-derived APCs (SAPCs) in the presence of 5 μg/mL OVA peptide (ISQAVHAAHAEINEAGR; Humboldt-Universität, Berlin, Germany) for 6 days. To demonstrate comparable proliferation, naive CD4+ T cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; MoBiTec, Göttingen, Germany) (Supporting Fig. 1). DCs isolated from mLNs pulsed for 2 hours with 1.8 μg/mL OVA (1 × 105) were cocultured with 2 × 105 naive CD4+ T cells from OT-II mice for 4 days. T helper 1 (Th1) cells were polarized in vitro from OVA-specific, naive CD4+ T cells with 5 μg/mL OVA peptide, 5 μg/mL interleukin-4–depleting antibody (11B11; Deutsches Rheumaforschungszentrum, Berlin, Germany), 10 ng/mL interleukin-12 and 20 ng/mL interferon-γ resulting in 70%-80% interferon-γ+ cells.

In Vitro and In Vivo Induction of α4β7 Integrin.

Naive CD4+ T cells or Th1 cells (both 5 × 105) were cocultured with LSECs, MHCII+LSEC liver APCs, and SAPCs (all 1 × 106) in the presence of 5 μg/mL OVA peptide. Naive CD4+ T cells were harvested after 6 days, Th1 cells after 3 days. Naive CD8+ T cells (2.5 × 105) from OT-I mice were cocultured for 6 days with 1 × 106 LSECs in the presence of 20 ng/mL OVA peptide (SIINFEKL, Humboldt-Universität). For retinoic acid receptor (RAR) blockage, LE 540 (1 μM; Wako, Richmond, VA) was added. To study the effect of vitamin A, TLSEC were generated in the presence of 50 nM all-trans retinol (Sigma Aldrich, Steinheim, Germany).

CFSE-labeled, OVA-specific, naive CD4+ T cells (1 × 107) or Th1 cells (5 × 106) were adoptively transferred into TF-OVA or wild-type (WT) mice. Naive CD4+ T cells were re-isolated after 3 days, and Th1 cells were re-isolated after 2 days. CD90.1+ donor cells were analyzed for proliferation and expression of α4β7 integrin.

Fluorescence-Activated Cell Sorting Analysis.

Cells were stained with various antibodies to surface antigens. Data were acquired using a FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Reverse-Transcription Polymerase Chain Reaction Analysis.

Polymerase chain reactions (PCR) were set up with 25 ng complementary DNA (cDNA) for RALDH1, RALDH4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or 100 ng cDNA for RALDH2 and RALDH3.

Quantitative Reverse-Transcription PCR Analysis.

PCR reactions were set up with 250 ng cDNA for all RALDH isoforms and GAPDH. Sequences of the forward primers were the same used for reverse-transcription PCR (RT-PCR) analysis. Quantitative PCR was performed using SYBR Green PCR MasterMix (Applied Biosystems, Darmstadt, Germany). Using the 2−ΔΔCT method, the data for liver RALDH expression were presented as the fold change in gene expression normalized to the housekeeping gene GAPDH and relative to the normalized RALDH expression in mLN. Expression of the RALDH isoforms by LSECs was determined in relation to GAPDH expression (ΔCT).

Western Blot Analysis.

Cells were lysed and separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Membranes were incubated with anti-RALDH1 (H-85; Santa Cruz Biotechnology, Santa Cruz, CA) or anti–β-actin (AC-15; Sigma Aldrich). The secondary antibody (Dako, Glostrup, Denmark) was conjugated to horseradish peroxidase.

Analysis of RALDH Activity.

RALDH activity was assessed using the ALDEFLOUR staining kit (StemCell Technologies, Vancouver, Canada).

Transmigration Assay.

TLSEC or TSAPC (5 × 105) were added to the upper chamber of the transwell with or without 300 nM CCL25 (R&D Systems, Wiesbaden, Germany) in the lower chamber, and assays were performed as described.15

In Vivo Homing Assay.

Radioactively labelled cells (1 × 106; 20 μCi/mL 51Cr; GE Healthcare, Braunschweig, Germany) were intravenously transferred into C57BL/6 mice. After 24 hours, radioactivity of the PPs, mLNs, small and large intestines, as well as the remaining body was counted using a Wizard gamma counter (Wallac, Turku, Finland).

Data Analysis.

Data were analyzed using the GraphPad Prism software (GraphPad Software, San Diego, CA). Statistical comparison was performed using a nonparametric two-tailed Mann-Whitney test or a one-sample t test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

LSECs Induce Expression of the Gut-Homing Molecules α4β7 Integrin and CCR9 on CD4+ T Cells.

The migration pattern of TLSEC is distinctly different from that of TSAPC, with TLSEC migrating significantly stronger to the mLN and the intestine than TSAPC.12 Addressing the molecular basis for the migration properties of TLSEC, we analyzed the expression of the gut-homing molecules α4β7 integrin and CCR9 as well as the expression of the P-selectin ligand (P-lig) necessary for migration to skin and inflamed tissues.3, 16 In line with their migration phenotype, TLSEC expressed α4β7 integrin and CCR9. Compared with TSAPC, P-lig expression was strongly reduced on TLSEC. LSECs and mLN DCs were equipotent to induce α4β7 integrin, whereas mLN DCs supported a stronger expression of CCR9 compared with LSECs (Fig. 1A).

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Figure 1. Expression and stability of homing molecules on TLSEC. (A) TLSEC, TSAPC, or T cells primed by mLN DCs (TmLN DC) were stained for α4β7 integrin, CCR9, or P-lig and analyzed by flow cytometry. Plots are representative of at least five independent experiments. Filled graph, unstained control; bold line, specific antibody. Percentages of positive cells are given. (B) TLSEC were restimulated for 6 further days by LSECs or SAPCs and thus were eventually cultured for 12 days in total. Cells were stained for α4β7 integrin, CCR9, or P-lig and analyzed by flow cytometry. Plots are representative of four independent experiments. Filled graph, unstained control; bold line, specific antibody. Percentages of positive cells are given. Bar graphs show the ratios of the percentages of positive cells measured on day 12 to that of day 6 as the mean ± SD of four independent experiments. The dotted line indicates equal expression levels. *P < 0.05.

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Ex vivo isolated LSECs did not contain other liver APCs potentially involved in the induction of homing-receptor expression as demonstrated by staining with CD11c, MHCII, and CD45 in combination with the LSEC-specific marker CD14617 or CD31, which are commonly used to discriminate endothelial cells from, for example, hepatic stellate cells18, 19 (Supporting Fig. 2).

The stability of homing-receptor expression was investigated by using either LSECs or SAPCs. The expression of α4β7 integrin on TLSEC remained stable upon restimulation both with LSECs and with SAPCs. In contrast, CCR9 was lost upon reactivation with SAPCs, whereas P-lig increased significantly (Fig. 1B).

In summary, we report that LSECs as liver-derived APCs induce in vitro gut tropism of CD4+ T cells.

LSECs Support Gut Tropism of CD4+ T Cells In Vivo.

To determine whether LSECs can imprint gut homing of CD4+ T cells in vivo, OVA-specific naive CD4+ T cells or in vitro polarized Th1 cells were adoptively transferred into TF-OVA mice expressing OVA exclusively in the liver.20 After 3 days, naive donor cells proliferated within the livers of TF-OVA mice, but not WT mice (Fig. 2A). Concomitantly, donor cells showed increased expression of α4β7 integrin within liver and mLNs but not pLNs (Fig. 2B). As demonstrated by in vitro coculture, among MHCII+ liver APCs only LSECs induced substantial levels of α4β7 integrin on naive CD4+ T cells (Fig. 2C). Donor Th1 cells became likewise reactivated in TF-OVA mice (Fig. 2D) and up-regulated α4β7 integrin in mLNs, but not in pLNs (Fig. 2E). Again, only LSECs but no other MHCII+ liver APCs or SAPCs promoted in vitro α4β7 integrin expression on Th1 cells (Fig. 2F). Taken together, LSECs induced α4β7 integrin expressing naive CD4+ T cells and Th1 cells that could be detected in vivo in liver and mLNs, but not in pLNs.

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Figure 2. Induction of α4β7 integrin on CD4+ T cells in the liver. (A) Naive CD4+ T cells or (D) in vitro polarized Th1 cells adoptively transferred into TF-OVA or WT mice were reisolated after 3 days or 2 days, respectively. Proliferation of donor cells from the liver was quantified by incremental decrease in cellular CFSE content. Thin line, WT mice; bold line, TF-OVA mice. Geometric mean fluorescence intensity of CFSE is given. Plots are representative for two independent experiments (three mice per group). (B,E) Percentages of α4β7 integrin+ cells within donor cells were determined by flow cytometry. Medians of six mice from two independent experiments are shown. **P < 0.01. (C) Naive CD4+ T cells or (F) Th1 cells activated in vitro by LSECs, MHCII+ liver APCs depleted from LSECs or SAPCs were stained for α4β7 integrin and analyzed by flow cytometry. Data from one out of four independent experiments are shown. Percentages of positive cells are given.

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LSECs Express RALDH1 and RALDH4.

Gut-homing molecules on T cells are induced by RA that is metabolized from vitamin A by RALDH.10 Therefore, we asked whether LSECs (1) also possess RALDH and (2) imprint the potential for gut tropism of TLSEC via RA converted from vitamin A. Analyzing mRNA levels for all RALDH isoforms, we detected stronger RALDH1 expression in liver tissue compared with mLN tissue. Liver tissue also expressed RALDH4 absent in mLN tissue. In contrast, mLN tissue contained high amounts of RALDH2 and RALDH3 mRNA, whereas these isoforms were only weakly expressed in liver tissue (Fig. 3A). Purified LSECs expressed RALDH1 and to a significantly lesser extent RALDH4, whereas RALDH2 and RALDH3 were below detection level (Fig. 3B).

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Figure 3. Expression of RALDH isoforms in liver and mLNs. (A) mRNA levels of RALDH isoforms in liver and mLN tissue and (B) in purified LSECs were assessed via reverse-transcription and PCR and confirmed via quantitative RT-PCR. One representative RT-PCR result of four independent experiments is shown. The expression of the RALDH isoforms in total liver tissue was quantified in relation to the respective expression in mLNs. RALDH1 and RALDH4 expression in purified LSECs was quantified in relation to GAPDH. Bar graphs show the mean ± SD of six independent experiments. (C) Protein expression of RALDH1 in LSECs and liver tissue was determined via western blot analysis. Data are representative of two independent experiments. (D) The RALDH activity of LSECs and CD11c+ DCs from mLNs was quantified by flow cytometry using ALDEFLOUR. CD11c+ DCs from pLNs served as negative control. Thin line, with RALDH inhibitor DEAB; bold line, without inhibitor. The ratio of the mean fluorescence intensity (MFI) of ALDEFLOUR of untreated versus DEAB-treated APCs is given. Bar graphs show the mean ± SD of four to six independent experiments. *P < 0.05.

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In the next step, the presence of the RALDH1 protein as the only isoform in LSECs capable of converting the all-trans isomer of retinal to all-trans RA was analyzed.21 Using western blot analysis, we confirmed RALDH1 protein in LSECs and liver tissue (Fig. 3C).

Subsequently, the RALDH activity of LSECs was determined using the ALDEFLOUR assay.22 Like mLN DCs, LSECs showed RALDH activity that was significantly higher compared with the control incubated with the RALDH inhibitor 4-diethylaminobenzaldehyde (DEAB). In contrast, pLN DCs not involved in gut-homing induction10 had only marginal RALDH activity (Fig. 3D). Thus, LSECs are equipped with the specific enzyme necessary for the conversion of vitamin A to RA—namely, RALDH1.

LSECs Can Induce Expression of α4β7 Integrin on CD4+ T Cells by Conversion of Vitamin A to RA.

Underlining that vitamin A is essential for gut homing of T cells, T cells are depleted from the lamina propria of the small intestine of vitamin A–deficient mice.10 To investigate whether LSECs equipped with RALDH convert vitamin A to RA, eventually leading to the expression of the gut-homing molecule α4β7 integrin on CD4+ T cells, LSECs from vitamin A–deficient mice were used to generate TLSEC. The proportion of α4β7 integrin+ TLSEC derived from cultures with vitamin A–deficient LSECs was dramatically reduced compared with TLSEC primed by LSECs from WT mice (Fig. 4A). Exogenously added vitamin A significantly increased the α4β7 integrin expression of TLSEC generated from vitamin A–deficient mice underlining the presence of functional RALDH in LSECs. Vitamin A had no effect on the α4β7 integrin expression of CD4+ T cells activated via CD3- and CD28-specific antibodies, excluding unspecific effects of vitamin A itself (Fig. 4A). In summary, the data demonstrate the RALDH-mediated conversion of vitamin A to RA by LSECs.

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Figure 4. Capacity of LSECs to induce α4β7 integrin on TLSEC depending on vitamin A. (A) TLSEC generated using LSECs from vitamin A–deficient (Vit A) or WT mice were stained for α4β7 integrin and analyzed by flow cytometry. Filled graph, unstained control; thin line, TLSEC cultured with LSECs from WT mice; bold line, TLSEC generated from vitamin A–deficient LSECs. Bar graphs show percentages of positive cells as the mean ± SD of five to seven independent experiments. **P < 0.01. (B) TLSEC generated with LSECs from vitamin A–deficient mice in the presence or absence of vitamin A were stained for α4β7 integrin and analyzed by flow cytometry. Filled graph, unstained control; thin line, TLSEC cultured in absence of vitamin A; bold line, TLSEC cultured in the presence of vitamin A. The mean fluorescence intensity (MFI) of α4β7 integrin expression was quantified and the ratio of vitamin A–treated versus untreated cells determined. CD4+ T cells activated with plate-bound CD3- and CD28-specific antibodies served as negative control. Bar graphs show the mean ± SD of four independent experiments. *P < 0.05.

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Expression of Gut-Homing Molecules on TLSEC Depends on RA Signaling.

To analyze whether the induction of gut-homing molecules on TLSEC directly relates to RA signaling, the RAR was inhibited by the pan-RAR antagonist LE 540.23 Blockage of RAR reduced the percentage of TLSEC expressing α4β7 integrin (Fig. 5A) and CCR9 (Fig. 5B) to levels of TSAPC. The proportion of TLSEC expressing P-lig was simultaneously enhanced to levels of TSAPC (Fig. 5C). Thus, RA is indeed responsible for the induction of gut-homing molecules on TLSEC. In line with findings on CD4+ T cells, LSECs induced expression of α4β7 integrin and CCR9 as well as low levels of P-lig on CD8+ T cells. The expression levels of α4β7 integrin and P-lig depended on RA. In contrast to CD4+ T cells, RAR blockage only slightly affected the percentage of CCR9+ cells among CD8+ T cells (Supporting Fig. 3).

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Figure 5. Effects of RA signaling on the expression of α4β7 integrin, CCR9, and P-lig on TLSEC. TLSEC were generated in the absence or presence of LE 540 blocking RAR. TSAPC served as the control. (A-C) Cells were stained for (A) α4β7 integrin, (B) CCR9, and (C) P-lig and were analyzed by flow cytometry. Filled graph, unstained control; thin line, TLSEC cultured without LE 540; bold line, blockage of RAR by LE 540. Percentages of positive cells without LE 540 (upper number) and with blockage of RAR by LE 540 (lower number) are given. Bar graphs show percentages of positive cells as the mean ± SD of five to seven independent experiments. *P < 0.05; **P < 0.01; ***P <0.001; ns, not significant.

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RA Is the Key Player in the Enterohepatic Circulation of TLSEC.

Having shown that TLSEC express gut-homing molecules, we analyzed their migration properties in more detail. First, we focused in vitro on the migration of TLSEC toward a chemokine gradient. The CCR9TSAPC did not respond to CCL25, but the CCR9+ TLSEC transmigrated toward a gradient of its ligand CCL25 (Fig. 6A), demonstrating functional CCR9. Inhibition of RA signaling by LE 540 suppressed CCR9 expression (see Fig. 5B), and transmigration of TLSEC was consequently reduced to basal migration without chemokine (Fig. 6A).

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Figure 6. Chemotaxis of TLSEC to CCL25 and in vivo migration of TLSEC as well as Th1 cells restimulated by LSECs into distinct mucosal sites. (A) Transmigration assays were performed with TLSEC cultured in the absence or presence of LE 540 blocking RAR. TSAPC served as the control. Cells were assessed for chemotactic activity toward the chemokine CCL25. Bar graphs show percentages of transmigrated cells as the mean ± SD of four independent experiments. *P < 0.05. (B) TLSEC cultured with or without LE 540 and TSAPC were analyzed for their migration patterns by in vivo homing assay. Bar graphs show percentages of total radioactivity within mLNs, PPs, and small and large intestines as the mean ± SD of three independent experiments (three to five mice per group). (C) Th1 cells cocultured with LSECs or SAPCs were analyzed for their migration patterns by in vivo homing assay. Bar graphs show percentages of total radioactivity within mLNs, PPs, small and large intestines as the mean ± SD of two independent experiments (five mice per group). *P <0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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Migration patterns of TLSEC generated in the absence or presence of RA signaling were determined by in vivo homing assays to investigate whether gut-homing molecule expression translated into corresponding recruitment into distinct intestinal sites. TSAPC, devoid of gut-homing molecules (see Fig. 1A; Fig. 5A,B), were not detected in PPs, and only very few cells were found in small and large intestine (Fig. 6B). In contrast, TLSEC migrated into PPs as well as into the small and large intestine (Fig. 6B) according to their α4β7 integrin and CCR9 expression (see Fig. 1A; Fig. 5A,B). Fitting their lack of gut-homing molecules (see Fig. 5A-C), the homing pattern of TLSEC generated with blocked RA signaling was comparable to that of TSAPC (Fig. 6B). As observed in naive CD4+ T cells, LSECs but not SAPCs promoted expression of α4β7 integrin by Th1 cells (see Fig. 2F). Concomitantly, significantly higher numbers of Th1 cells restimulated by LSECs compared with those cocultured on SAPCs migrated into mLNs, PPs, and intestine (Fig. 6C).

Taken together, the results show that the gut tropism of CD4+ T cells primed by LSECs depended on RA derived from vitamin A, a conversion that can be mediated by LSECs.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Priming by extraintestinal, liver-derived APCs, namely LSEC, supports the migration of CD4+ T cells into small and large intestine as well as into GALT. This migration pattern is different from that of CD4+ T cells primed by spleen-derived APCs that are only found in minor numbers in lymph nodes and intestine.12 These differences are based on distinct expression patterns of homing molecules. TSAPC strongly expressed P-lig, a molecule present on activated T cells and necessary for migration into skin and inflamed tissue.3, 16 As a consequence of T cell activation, TSAPC preferentially migrated to liver and spleen, a migration pattern typical for effector T cells.24 Conversely, expression of α4β7 integrin and CCR9 directed TLSEC into the intestine, comparable to T cells primed by GALT-DCs.25 Furthermore, the preferential recruitment of TLSEC into mLN seemed to be mediated by additional expression of the lymph node homing receptors CCR7 and CD62L and low activation reflected by minor levels of CD25 and CXCR3 (Supporting Fig. 4). Underlining the in vivo relevance, antigen expression within the liver resulted in activation and induction of α4β7 integrin on CD4+ naive and effector T cells. Spleen APCs do not have the capacity to induce α4β7 integrin, and ex vivo isolated mLNs from TF-OVA mice were unable to prime naive CD4+ T cells (Supporting Fig. 5). Therefore, we assume that α4β7 integrin+ CD4+ T cells from the liver accumulated within mLNs through interaction with mucosal vascular addressin cell adhesion molecule-1.5 Because LSECs were the only liver APC population able to support high expression of α4β7 integrin, induction of gut-homing CD4+ T cells in vivo could be attributed to the liver endothelium. In summary, we report that LSECs as liver-derived APCs can induce gut tropism, a capacity typically ascribed to gut-derived APCs.3, 4

Recent studies suggest high plasticity of α4β7 integrin and CCR9 expression on CD8+ T cells activated by DCs isolated from pLNs or PPs.26, 27 Upon reactivation of TLSEC with SAPCs, CCR9 decreased, whereas expression of α4β7 integrin was maintained. Conversely, Th1 effector cells acquired α4β7 integrin and partially lost P-lig expression (data not shown) upon restimulation by LSECs accompanied by increased migration into GALT and intestine. Taken together, LSEC-primed CD4+ T cells displayed stability of their gut tropism. In addition, they gain homing receptors for the migration into the skin28 or inflamed tissues29 if restimulated elsewhere. Furthermore, Th1 effector cells can acquire gut-homing properties upon reactivation by LSECs. It is tempting to speculate that antigen re-encounter in vivo might redirect TLSEC into skin and, conversely, Th1 cells into the gut.

Iwata et al.10 reported that RA induces the expression of α4β7 integrin and CCR9 and suppresses that of ligands for E- and P-selectin on T cells. The ability of GALT-DCs to generate gut-homing T cells lies in their specific expression of RALDH, providing the particular capacity to produce RA from vitamin A. In LSECs, we found strong expression of RALDH1 and low expression of RALDH4. RALDH4 catalyzes oxidation of 9-cis retinal to 9-cis RA but not of all-trans retinal to all-trans RA.21 Because no evidence for the in vivo occurrence of 9-cis RA has been provided,30 we assumed that LSECs supplied all-trans RA during antigen presentation by enzymatic activity of the RALDH1 isoform. The enzymatic functionality of LSEC-expressed RALDH was demonstrated by the ALDEFLOUR assay, earlier described by Yokota et al.22 to assess RALDH activity of mLN-derived DCs and using LSECs from vitamin A–deficient mice. LSECs from this model, virtually devoid of vitamin A, had a significantly reduced capacity to induce gut-homing molecules on TLSEC. Conversely, the addition of exogenous vitamin A restored their ability to induce gut tropism of CD4+ T cells, indicating a RALDH-mediated conversion of vitamin A to RA. Within the liver, dietary vitamin A stored and released by hepatic stellate cells into the space of Disse provides a constant in vivo supply for LSECs in the immediate vicinity14, 31 that can take up and metabolize vitamin A to RA.

Blockage of RAR by LE 54023 during priming by LSECs converted the expression pattern of homing molecules on TLSEC to that of effector T cells, characterized by low expression of α4β7 integrin and CCR9 in conjunction with high P-lig expression.32 According to the expression of homing molecules, the migration pattern of TLSEC cultured in the presence of LE 540 changed to that of TSAPC. In summary, we provide evidence that LSECs have the capacity to metabolize alimentary vitamin A to RA. Other factors (e.g., the antigen dose used for priming of CD4+ T cells) had no significant influence on the expression of α4β7 integrin (data not shown). Thus, we assume that in the end, RA is responsible for the induction of the gut tropism of TLSEC.

Besides LSECs, the liver contains other APC populations that have previously been scrutinized for their potential to induce gut-homing molecules. Eksteen et al.23 recently presented first hints for the capacity of hepatic stellate cells to support low expression levels of CCR9 on CD8+ T cells. However, migration of these cells to the gut remains elusive. Liver DC mediate only weak expression of gut-homing molecules on CD8+ T cells23 and CD4+ T cells (data not shown), leading to the conclusion that this hepatic cell population does not promote extrahepatic tropism.23 Until now, no evidence which liver cell population might induce gut tropism of CD4+ T cells has been provided. Our data support the assumption that LSECs, in contrast to other MHCII+ liver-derived APCs, induce gut-homing receptor expression on CD4+ T cells, as previously only demonstrated for GALT-DCs.10

Grant et al.9 have proposed a model of enterohepatic lymphocyte circulation implying that T cells can traffic between gut and liver. Until now, only one branch of the model, the migration of gut-primed T cells into the liver, has been described. Our data show that liver-primed CD4+ T cells can migrate into the intestine and GALT, thus closing the circle. The model of enterohepatic lymphocyte circulation explains the clinical coincidence of pathogenesis in gut and liver: ectopic expression of mucosal vascular addressin cell adhesion molecule-1 and CCL25 in the liver redirects α4β7 integrin+ and CCR9+ effector T cells into the liver, causing extraintestinal manifestations of inflammatory bowel diseases such as primary sclerosing cholangitis.7, 8, 33 On the other hand, it is conceivable that not only proinflammatory but also regulatory T cells circulate between the gut and the liver. Because liver-primed TLSEC, in particular, exert regulatory functions,12 recruitment into the gut might support local tolerance of food antigens or control intestinal inflammation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are grateful to Katja Blunert for excellent technical assistance. We thank Eckart Schott for providing the TF-OVA mice.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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