Published online before print April 27, 2005
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* Department of Cell Biology, Faculties of Biology and
Medicine, Complutense University, Madrid, Spain
1 Correspondence: Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain. E-mail: gomariz{at}bio.ucm.es
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, which synergize with bacterial products, contributing to the amplification of TLR presence in the intestine. The other possible mechanism would involve a VIP-mediated decrease of nuclear factor-
B, which would cause a direct down-regulation of TLR expression. In summary, the resultant physiological effect is the decrease of TLR2 and -4 expressions to homeostatic levels. Our study describes for the first time the role of a peptide present in the gut microenvironment as an effective modulator of the initial steps of acute inflammation, acting at local and systemic levels and leading to the restoration of the homeostasis lost after an established inflammatory/autoimmune disease.
Key Words: immunomodulation • inflammation • homeostasis
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As the stimulation of TLR2 and -4 mediates the expression in a cascade of inflammation-related genes [3 ], as well as the induction of an acquired immune response of the Th1 type [14 ], our hypothesis was that TLRs could be involved in the physiopathological development of CD, and therefore, VIP could down-regulate, at least in part, the inflammatory response by modulating TLR expression. Thus, we chose an acute model of intestinal injury and inflammation based on the intrarectal administration of TNBS, which is known to induce an early production of inflammatory factors, followed by a Th1 response that resembles CD [15 , 16 ]. The constitutive expression and the compartmentalization of TLR2 and TLR4 in the mouse gut as well as their up-regulation in dextran sulfate sodium (DSS)-induced colitis, as a model of human ulcerative colitis, have been reported recently [17 ]. However, there are no studies describing the time course of the expression of these receptors in the development of an experimental model of CD as well as the effect of a treatment with a therapeutic peptide on them.
Therefore, the aim of the present study is to know the possible variation of the expression of TLR2 and -4 receptors along the development of TNBS-induced colitis and to elucidate whether the beneficial effect of VIP involves the regulation of the expression of these receptors as important sensors of the gut homeostasis. Our results showed the constitutive expression and the up-regulation of TLR2 and -4 in a mice model of CD and the homeostatic effect of VIP approaching the expression of these receptors to control levels.
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Induction of colitis and treatment protocol
Colitis was induced by rectal instillation of TNBS (Sigma Chemical Co., St. Louis, MO) as described previously [16
]. Briefly, BALB/c mice were lightly anesthetized, and 120 µl of a mixture containing 3 mg TNBS dissolved in ethanol was administered through a catheter advanced 4 cm in the anus. The animals were then held by the tail during 30 s to ensure uniform contact with colonic mucosa. Following the same protocol, control animals were given 120 µl 50% ethanol. Each mouse received 1 nmol VIP (Neosystem, Strasbourg, France) intraperitoneally (i.p.) every other day after TNBS induction of colitis until the collection of samples.
Measurement of myeloperoxidase (MPO) activity in colon extracts
Tissues were removed on day 3, washed with phosphate-buffered saline (PBS), and cut in small pieces. Colon tissue (50 mg/ml) was homogenized in 50 mM phosphate buffer, pH 6.0, with 0.5% hexadecyltrimethylammonium bromide using a polytron (Kinematica, Littau/Luzerne, Switzerland). Samples were frozen and thawed three times and centrifuged for minutes at 30,000 g, and supernatants were stored at –20°C until assay. MPO activity was determined in 96-well plates using a modification of the method described by Bradley et al. [18
]. Briefly, samples were diluted 1:30 with assay buffer [50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml o-dianisidine (Sigma Chemical Co.) and 0.0005% H2O2], and absorbance at 450 nm was measured in a microtiter reader.
RNA extraction
A tissue tearer was used to homogenize mice colons, and total RNA was extracted with the Ultraspec RNA reagent, as recommended by the manufacturer (Biotecx, Houston, TX). RNA was resuspended in diethylpyrocarbonate water and quantitated at 260/280 nm.
Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
Quantitative RT-PCR analysis was performed using the SYBR® Green PCR Master Mix and RT-PCR kit (Applied Biosystems, Foster City, CA), as suggested by the manufacturer. Briefly, reactions were performed in 20 µl with 20 ng RNA, 10 µl 2x SYBR Green PCR Master Mix, 6.25 U MultiScribe RT, 10 U RNase inhibitor, and 0.1 µM primers. The sequences of primers used were: mouse ß-actin sense 5'-AGAGGGAAATCGTGCGTGAC-3', antisense 5'-CAATAGTGATGACCTGGCCGT-3'; mouse TLR2 sense 5'-GGGACGTTTGCTATGATGCC-3', antisense 5'-AAAGGGCGGGTCAGAGTTCT-3'; mouse TLR4 sense 5'-GTTGCAGAAAATGCCAGGATG-3', antisense 5'- CAGGGATTCAAGCTTCCTGGT-3'; mouse interleukin (IL)-1ß sense 5'-CTTCCCCAGGGCATGTGA-3', antisense 5'-ACCCTGAGCGACCTGTCTTG-3'; and mouse interferon- (IFN-) sense 5'-TGCTGATGGGAGGAGATGTCT-3', antisense 5'-TTTCTTTCAGGGACAGCCTGTT-3'. The GenBank accession numbers and numbers for the 5' and 3' ends of the nucleotides for the PCR products are: ß-actin, NM007393, 694–831; TLR2, NM011905, 2226–2236; TLR4, NM021297, 4–104; IL-1ß, NM 15131, 914–1014; and IFN-, K00083, 929–1029. Amplification conditions were 30 min at 48°C, 10 min at 95°C, 40 cycles of denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min.
For relative quantification, we used a method that compared the amount of target normalized to an endogenous reference. The formula used was 2–
Ct, representing the n-fold differential expression of a specific gene in a treated sample compared with the control sample, where Ct is the mean of threshold cycle (cycle at which the amplification of the PCR product is initially detected). Ct was the difference in the Ct values for the target gene and the reference gene, ß-actin (in each sample assayed), and Ct represents the difference between the Ct from the control and each datum. Before using this method, we performed a validation experiment comparing the standard curve of the reference and the target to demonstrate that efficiencies were approximately equal [19
]. The correct size of the amplified products was checked by electrophoresis.
RT-PCR
Total RNA (5 µg) was added to a RT mixture: 3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl, pH 8.3, 10 mM dithiothreitol, 0.5 mM 2'-deoxynucleoside-5'-triphosphates, 100 U SuperscriptTM II RNase H– RT (Invitrogen, Carlsbad, CA), 25 pmol oligo d(T)16, and 40 U RNase out (Invitrogen). The resulting mixture was incubated for 50 min at 42°C, and the sample was heated to 70°C for 15 min on a GeneAmp® PCR System 2700 (Applied Biosystems) and maintained at 4°C until PCR amplification. To control genomic DNA contamination, the reaction was performed regularly in the absence of RT. cDNA was then amplified using the specific primers for TLR2, TLR4, and ß-actin as a control, the same used for real-time RT-PCR. RT cDNA (2 µL) was amplified in PCR mixtures (total volume 25 µl) containing 50 mM KCl, 20 mM Tris-HCl, pH 8.4, 2.5 U Taq DNA polymerase (Invitrogen), 1.5 mM MgCl2, 0.2 mM deoxy-unspecified nucleoside 5'-triphosphates, and 1 µM primers. PCR conditions were: denaturation at 94°C for 3 min, followed by 25 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C and then a final cycle for 10 min at 72°C. PCR products were analyzed in 2% agarose gels.
Preparation of colon membranes and Western blot
For Western blot of TLR2 and TLR4, colons were removed at the indicated times, washed with PBS, and cut in small pieces. Samples were homogenized in 0.1 M Tris-HCl, pH 7.4, 0.3 M NaCl, 0.25 M sucrose, 2 mM EDTA, and protease inhibitor cocktail tablets (Roche, Mannheim, Germany) using a Polytron (Kinematica) and then centrifuged at 1600 g for 15 min at 4°C to remove debris. Supernatants were centrifuged at 30,000 g for 30 min at 4°C to isolate the membrane fraction and then stored at –80°C until assay. Protein concentration was estimated by using the Bradford method (Bio-Rad, Hercules, CA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed loading 100 µg proteins per lane, followed by transference to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked for 2 h in Tris-buffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk. Horseradish peroxidase-conjugated donkey anti-goat immunoglobulin G (IgG; 1:10.000, Santa Cruz Biotechnology) was used for detection. Proteins were visualized using an enhanced chemiluminescence Western blotting analysis system (Santa Cruz Biotechnology). Incubation with mouse anti-ß-actin antibody (1:10,000, Oncogene, Cambridge, MA) was used as a loading control. Densitometry analysis of proteins bands from scanned-ray films were performed using Scion Image software (Scion Corp., Frederick, MD), and the values were normalized against the intensity of ß-actin.
Immunohistochemistry
For immunohistochemistry studies, monoclonal antibodies (mAb) against TLR2 and TLR4 were used (rat anti-mouse TLR2 or TLR4 mAb, eBioscience, San Diego, CA). With this purpose, colons were frozen, and 7 µm-thick sections were obtained and fixed in cold acetone for 20 min. To avoid unspecific labeling, sections were incubated with DAKO® protein block serum-free (Dako Corp., Carpinteria, CA) at room temperature for 30 min, and an avidin/biotin-blocking kit (Vector Laboratories, Burlingame, CA) was used. Subsequently, sections were incubated at 4°C overnight with primary antibody at 1:100 dilution in PBS + 0.1% bovine serum albumin and then with 0.36% ß-glucose, 0.01% glucose oxidase, and 0.013% sodium azide in PBS for 1 h at room temperature to block endogenous peroxidase activity. Biotinylated mouse anti-rat polyclonal antibody (eBioscience) was used as secondary antibody at 1:100 dilution for 1 h at room temperature, followed by an incubation with 0.03 mg/ml avidin-peroxidase labeled for 1 h at room temperature. A solution of 0.05% 3-3' diaminobenzidine and 0.015% H2O2 was used to stain. Toluidine blue was used for counterstaining. As negative controls, sections were treated with rat serum instead of primary antibody.
Fluorescein-activated cell sorter (FACS) analysis
Mesenteric lymph node cell suspensions were prepared by mechanical dissociation using a stainless-steel screen (60 mesh, Sigma Chemical Co.). Cells (3x105) were labeled with phycoerythrin (PE)-conjugated rat anti-mouse TLR4/MD2 (clon MTS510, eBioscience) or PE-conjugated rat anti-mouse TLR2 (clon 6C2, eBioscience). Double labeling was also performed by combining the TLR2 and TLR4 antibodies with fluorescein isothocyanate (FITC)-conjugated rat anti-mouse CD11b (clon M1/70.15, Caltag Laboratories, S. San Francisco, CA) and FITC-conjugated rat anti-mouse CD11c (clon N418, Caltag Laboratories). Lymphocytes were distinguished by their different forward-scatter (FSC) versus side-scatter (SSC) profiles and were electronically gated and scored. Incubation with the antibodies was performed at 4°C for 30 min. Aliquots of lymph node cells were incubated with a single fluorochrome-conjugated antibody or isotype-matched control antibodies to compensate for fluorescence emission overlap and nonspecific fluorescence, respectively. Cells were fixed with 0.3% paraformaldehyde and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) using an appropriate scatter gate to exclude dead cells, cellular debris, and residual erythrocytes.
Statistical analysis
All values are expressed as the mean ± SEM of data obtained from at least three mice. Comparison between groups was made using the Student’s t-test, Mann-Whitney W test, and ANOVA test, and P < 0.05 was the significance level.
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) into the colonic mucosa and submucosa associated with a thickening of the colon wall, loss of globet cells, and finally, the disruption of the epithelial barrier [15
]. Administration of VIP improved these signs, restoring the histological appearance of the mucosa and submucosa. As a quantitative measure of the development of the disease and the anti-inflammatory effect of VIP, we evaluated neutrophil infiltration by determining MPO activity in colon extracts. Our results showed that colon MPO activity in the TNBS animals was increased significantly compared with untreated and ethanol-treated control mice, whereas VIP treatment significantly inhibited this activity (Fig. 1B)
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Figure 1. Development of TNBS-induced colitis and effect of VIP. Colitis was induced by rectal instillation of TNBS in 50% ethanol, and 1 nmol VIP was injected i.p. on alternate days. Controls received PBS or 50% ethanol. (A) Macroscopic analysis of colons obtained 3 days after administration of TNBS showed necrosis, hyperemia, and inflammation compared with control colons. Treatment with VIP prevented hyperemia and inflammation. (B) Colons were removed and homogenized on day 5 to obtain protein extracts, and MPO activity was determined as described in Materials and Methods. Error bars represent ± SEM of eight mice per group. **, P < 0.01, versus TNBS-treated animals.
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Figure 2. Real-time RT-PCR analysis of TLR2 and TLR4 mRNA expression in colon extracts in TNBS-induced colitis and their down-regulation after VIP treatment. Colitis was induced by rectal instillation of TNBS in 50% ethanol, and 1 nmol VIP was injected i.p. on alternate days. Controls received PBS or 50% ethanol. mRNA was extracted from colons at days 1, 3, 5, and 7. TLR2 (A) and TLR4 (B) mRNA expressions were measured by real-time RT-PCR, values were rectified by mRNA expression of ß-actin for each sample, and arbitrary units were calculated with respect to control expression, being 1 (2– Ct). Each result is the mean ± SEM of at least three mice performed by duplicate. ++, P < 0.01; +++, P < 0.001, versus control animals, and *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus TNBS-treated animals. RT-PCR for TLR2 (A) and TLR4 (B) at day 5 is shown in lower panels. Negative control in the absence of cDNA and control (ß-actin) are shown. Primers amplified a 100-bp band for TLR2 and TLR4 mRNA and 140 bp for ß-actin.
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Figure 3. Western blot analysis of TLR2 and TLR4 in colon extracts in TNBS-induced colitis down-regulation after VIP treatment. Colon extracts from control and ethanol-, TNBS-, and TNBS/VIP-treated animals were prepared and electrophoresed as described in Materials and Methods. Specific bands for TLR2 (A) or TLR4 (B) were quantified by densitometry and rectified with respect to ß-actin protein levels in each case. Each result is the mean ± SEM of at least four mice. +, P < 0.05; ++, P < 0.01; +++, P < 0.001, versus control animals, and *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus TNBS-treated animals. A representative experiment of three others is shown in the upper panels. O.D. = optical density.
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Figure 4. Immunohistochemical analysis of TLR2 and -4 at day 5 in gut sections. Immunohistochemical study was performed to study TLR2 and TLR4 expressions in colon mucosa as described in Materials and Methods. Mononuclear cells positive to antibody against TLR2 within the lamina propria in control (untreated animals; A), TNBS (B), and TNBS + VIP-treated animals (C; original x400). Gut crypts showing positive staining for TLR2 and TLR4 in control (untreated animals; D and G), TNBS (E and H), and TNBS + VIP-treated animals (F and I). Original magnification, x200. Arrows indicate the positive staining. VIP reduced the TNBS-induced expression of TLR2 and TLR4 in epithelial cells from intestinal crypts and mononuclear cells.
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Figure 5. Flow cytometry analysis of TLR2 expression in TNBS-induced colitis. Effect of VIP treatment. Colitis was induced as described in Materials and Methods, and mice were treated i.p. with 1 nmol VIP on alternate days over 7 days. Cell suspensions from mesenteric lymph node extracted from control and treated animals were collected at different time-points and processed for immunofluorescence staining by using a PE-conjugated TLR2 mAb. (A) Aliquots of control lymph node cells were incubated with isotype control antibodies or antibodies to TLR2 (IgG2b), as described. (B) Total cells subjected to single immunofluorescence staining were analyzed by FACSCalibur. (C and D) Double immunofluorescence staining for TLR2 and a subpopulation marker (C, FITC-conjugated CD11c; D, FITC-conjugated CD11b). (E) Lymphocytes were identified by a FSC versus SSC gate and subjected to TLR2 immunofluorescence staining (F). Each result is the mean ± SEM of two separated experiments (three mice/group/experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (ANOVA), with respect to TNBS-treated mice. ++, P < 0.01; +++, P < 0.001 (ANOVA), with respect to control animals.
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Figure 6. Flow cytometry analysis of TLR4 expression in TNBS-induced colitis. Effect after VIP treatment. Colitis was induced as described in Materials and Methods, and mice were treated i.p. with 1 nmol VIP on alternate days over 7 days. Cell suspensions from mesenteric lymph node extracted from control and treated animals were collected at different time-points and processed for flow cytometry analysis by using a PE-conjugated TLR4 mAb. (A) Aliquots of control lymph node cells were incubated with isotype control antibodies or antibodies to TLR4 (IgG2a), as described. (B) Total cells subjected to single immunofluorescence staining were analyzed by FACSCalibur. (C and D) Double immunofluorescence staining for TLR4 and a subpopulation marker (C, FITC-conjugated CD11c; D, FITC-conjugated CD11b). (E) Lymphocytes were identified by a FSC versus SSC gate and subjected to TLR4 immunofluorescence staining (F). Each result is the mean ± SEM of two separated experiments (three mice/group/experiment). *, P < 0.05; **, P< 0.01; ***, P < 0.001 (ANOVA), with respect to TNBS-treated mice. ++, P < 0.01; +++, P < 0.001 (ANOVA), with respect to control animals.
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, as the main cellular type expressing TLR2 and –4, and lymphocytes, as the predominant cell type in the lymph node. CD11c is a cell-surface molecule predominantly expressed on DC and thus, is being used as a marker of this cell type, although it can be also found on natural killer (NK) cells, granulocytes, and M. Besides, CD11b is frequently used to distinguish M, although it’s also expressed on NK, granulocytes, and DC. Therefore, we used two rat anti-mouse CD11c and CD11b mAb to label mainly DC and M, whereas lymphocytes were identified by their FSC versus SSC profiles, as the FSC/SSC plot shows (Figs. 5E
and 6E) . The results are summarized in Table 1
. First, we detected a significant decrease of absolute cell numbers on days 3 and 5 after TNBS treatment, more drastic on day 5. Administration of VIP restored cell count, suggesting that this peptide could modulate the cellular traffic. Next, we analyzed the different populations into mesenteric lymph nodes during TNBS colitis development. VIP treatment significantly reduces the TNBS increased proportion of CD11c- and CD11b-positive cells on days 5 and 7, restoring the cellular number to values of control animals. In addition, TNBS treatment significantly reduces the percentage of lymphocytes at these time-points, whereas VIP counterbalances this effect. |
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Table 1. Effect of VIP on Cellular Traffic in Mesenteric Lymph Nodes of TNBS-Treated Animals
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, DC, and lymphocytes in mesenteric lymph nodes.
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Figure 7. Representative example at day 5 of the TLR2 and -4 expressions in CD11c and CD11b cells and lymphocytes after TNBS-induced colitis and VIP effect. Flow cytometry analysis of CD11c and CD11b cells and lymphocytes from mesenteric lymph nodes of control and treated animals. Cell suspensions were processed for double immunofluorescence staining for TLR2 and TLR4 and a subpopulation marker (CD11c and CD11b). Lymphocytes were identified by a FSC versus SSC gate and subjected to TLR2 and TLR4 immunofluorescence staining. Cell suspensions were incubated with isotype control antibodies or antibodies to TLR2 (IgG2b) or TLR4 (IgG2a), as described. The values shown in right quadrants and histograms indicate the percentage of CD11c/CD11b or lymphocytes and TLR2/4-positive cells. The results presented are representative of two independent experiments using three mice per group.
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expression in colon extracts in TNBS-induced colitis and their decrease after VIP treatment in colon extracts. In correlation with TLR2 and -4 expressions, TNBS treatment induces an increase of IL-1ß and IFN- mRNA expression, peaking on day 5 and decreasing more markedly than TLRs on day 7. Ethanol-treated animals showed only a mild increase (Fig. 8A
).
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Figure 8. Real-time RT-PCR analysis of IL-1ß and IFN- mRNA expression in colon extracts in TNBS-induced colitis and their regulation after VIP treatment. mRNA was extracted from colons at days 1, 3, 5, and 7. IL-1ß (A) and IFN- (B) mRNA expressions were measured by real-time RT-PCR, values were rectified by mRNA expression of ß-actin for each sample, and arbitrary units were calculated with respect to control expression, being 1 (2– Ct). Each result is the mean ± SEM of at least three mice performed by duplicate. +++, P < 0.001, versus control animals, and *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus TNBS-treated animals.
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kinetics, it correlated to TLR2 expression on days 5 and 7 but to TLR4 expression only on day 5 (Fig. 8)
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from human inflamed mucosa [23
] as well as the association of a TLR4 polymorphism with CD and ulcerative colitis in patients [24
]. In mice, Ortega Cava et al. [17
] have recently reported that TLR2 and -4 were constitutively expressed in intestinal mucosa, showing different expression patterns along the gut segments, all focused in the colon but in a compartmentalized manner. TLR2 was expressed mainly in the proximal colon and TLR4 in the distal colon, providing evidence that TLR4 was up-regulated in DSS-induced colitis, an animal model of human ulcerative colitis. In agreement with these findings, our results show the constitutive expression of TLR4 in colon extracts at protein and messenger levels, in lamina propria mononuclear cells and epithelial cells from the gut crypts, as well as their up-regulation in another model of inflammatory bowel disease, TNBS-induced colitis, characterized by a Th1-driven autoimmune component. The same pattern and behavior were found for TLR2 expression. Moreover, we evaluated for the first time the expression of these receptors through the development of the disease. Both receptors showed the highest levels of messenger and protein expression on day 5, when most of the inflammatory parameters of the disease peak, and the late phase of the disease starts, whereas a decrease was seen on day 7, when the chronic phase starts. In addition, we demonstrated the constitutive and the TNBS-induced TLR2 and -4 expressions in immune cells from mesenteric lymph nodes. These secondary lymphoid organs play an important role as checkpoints controlling the traffic of antigen-presenting cells and lymphocytes and constitute organized centers, where the antigen presentation and lymphocyte activation occur. Our data showed that after TNBS treatment, there is a significant increase in TLR-positive cells from days 3 to 5, reaching the 30–40% of total lymph node cells. It is well known that M and DC are important players on innate immunity, and DC have evolved specifically to translate innate recognition into acquired immunity. Our results demonstrate that approximately 60% of M and 70% of DC, characterized by CD11c and CD11b surface expression, expressed TLR2 and TLR4 in untreated mice, increasing the percentages after TNBS treatment. Although TLRs are mainly expressed on myelomonocytic cells, relatively little is known about TLR expression on lymphocytes. Recent studies using real-time PCR have demonstrated that murine mRNA TLR2 and mRNA TLR4 are expressed in mouse T cells [25 ] and T and B cells from human peripheral blood mononuclear cells [26 ]. However, the modulation of TLR2 and TLR4 cell-surface expression after an inflammatory stimulus has not been well-characterized to date. Our data showed that approximately 10% of lymphocytes constitutively expressed TLRs and their up-regulation after TNBS treatment. Thus, this inflammatory agent causes the up-regulation of TLR-positive cells of innate and acquired immunity.
Medzhitov and his group [6 ] have recently postulated a new meaning of TLR presence in the gut mucosa, suggesting their protective role in the intestinal homeostasis through two possible mechanisms: the steady-state induction of protective factors, via the constitutive detection of TLR ligands on commensals by TLRs expressed on colon epithelium, and/or the induction by commensal-derived TLR ligands of the production of protective factors upon epithelial damage.
As the inflammatory disorders may break intestinal homeostasis up-regulating TLR expression, a close control of the initial proinflammatory response is crucial to restore the host homeostasis.
In the last 5 years, VIP has emerged as an endogenous agent for the treatment of inflammatory/autoimmune diseases as septic shock, RA, and CD, inhibiting the expression and release of proinflammatory cytokines and chemokines, enhancing the production of anti-inflammatory cytokines, and regulating the Th1/Th2 cytokine profile [11
12
13
]. Moreover, VIP effects are mediated by the presence of pituitary adenylate cyclase type 1, VIP type 1 (VPAC1), and VPAC2 receptors on lymphocytes and M. The therapeutic effect of VIP in TNBS-induced colitis was associated to the down-regulation of inflammatory and Th1-driven, autoimmune responses, including tumor necrosis factor 
(TNF-), IL-1ß, and IL-6 in colon extracts and serum as well as IFN- by splenic and lamina propria CD4 T cells [13
].
Our results showed that in acute inflammatory conditions, as TNBS-induced colitis, VIP treatment exerts a time-course inhibition of TLR2 and -4 expressions at mRNA and protein levels from days 1 to 7, producing a general down-regulation at the colon local microenvironmental level with the involvement of epithelial and mononuclear cells. Furthermore, VIP exerts its action at a systemic level in lymph nodes, regulating the cellular traffic, reducing significantly the TNBS-increased proportion of DC and M, and restoring the number of cells at the level of control animals. The arrival of M and DC from inflamed tissues to regional lymph nodes has been described to perform antigen processing and presentation to T cells, which results in a migration of lymphocytes to sites of inflammation. TNBS-induced inflammation is characterized by a massive infiltration of neutrophils and M in the early stages of the disease, producing high levels of proinflammatory cytokines; these events are followed by an infiltration of T cells producing high levels of IFN- [13
]. Our results reveal that VIP could selectively modulate cell recruitment into lymph nodes, which could prevent the pathological effects of TNBS. In addition, VIP exerts its effect by modulating the number of TLR2- and TLR4-positive DC, M, and lymphocytes. Although further studies are needed to deepen a potential, direct action of VIP on TLR expression, we do not rule out this hypothesis.
The stimulation of TLR2 and -4 mediates a cascade that finally leads to activation of nuclear factor (NF)-B, contributing to the expression of inflammation-related genes such as TNF- or IL-1ß [2
]. In this sense, TLR4 activation activates two cascades: the myeloid differentiation primary-response protein 88 (MyD88)-dependent and MyD88-independent pathways. The MyD88-dependent pathway involves the early phase of NF-B activation, which leads to the production of inflammatory cytokines. The MyD88-independent pathway activates the late phase of NF-B activation and IFN regulatory factor 3 (IRF3), which lead to the production and expression of IFN-inducible genes [5
].
The VIP-mediated down-regulation of TLR2 and -4 could be explained through two possible ways that are not mutually exclusive. The first one would be a primary through the VIP-mediated decrease of NF-B, which would cause a down-regulation of TLR expression, as a binding site for NF-B in the murine TLR2 has been described [27
]. In this sense, it has been shown that VIP decreases NF-B binding in vitro and in vivo. In vitro, a cyclic adenosine monophosphate-independent pathway associated with the VPAC1 receptor leads to in the inhibition of inhibitor of B (IB) phosphorylation [28
]. In vivo, data from our laboratory in a murine model of RA (manuscript in preparation) revealed that the beneficial effect of VIP treatment is mediated by the inhibition of IB phosphorylation/degradation. Thus, VIP prevents NF-B nuclear translocation and inhibits the transcription of several inflammatory factors [29
]. Furthermore, we have described VIP-mediated inhibition of IFN- at the transcriptional level [30
], and the promoter for TLR4 was shown to contain an IRF-binding site that regulates expression of TLR4 in myeloid cells [31
].
Conversely, different studies have suggested that inflammatory cytokines such as TNF-, IL-1ß, and IFN- could act synergistically, together with gram-positive and gram-negative bacterial components, up-regulating TLR2 and -4 expressions [20
, 21
, 32
, 33
]. Thus, TLR2 and -4 would be activated by molecular products of bacteria, leading to the production of proinflammatory cytokines, and many of these cytokines would be able to amplify TLR expression, establishing a positive feedback that may lead to the perpetuation of inflammatory conditions, favoring the chronic phase of the disease. Therefore, the other possible mechanism to explain VIP-induced TLR2 and -4 modulations is the secondary reduction of the Toll expression caused by the decrease of inflammatory mediators such as TNF-, IL-1ß, and IFN-, which amplify TLR signaling. In this sense, we have previously demonstrated in this model of colitis that VIP treatment down-regulates the protein levels of different proinflammatory cytokines [13
]. In this study, we report that the expressions of IL-1ß and IFN- are time-correlated to TLR2 and TLR4 in TNBS-nontreated and VIP-treated animals. Thus, through the reduction of IL-1ß and to a minor extent, of IFN- VIP could break the positive feedback that perpetuates inflammation, reducing the presence and availability of both TLRs, maintaining their homeostatic levels. Through a VIP primary reduction of NF-B, which causes a decrease of TLR expression, or through a secondary, VIP-mediated decrease of the proinflamatory cytokines, which synergize with bacterial products to up-regulate TLR expression or acting in both ways, the resultant physiological effect is the approach of TLR2 and -4 expressions to constitutive and thus, homeostatic levels (Fig. 9
).
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Figure 9. Hypothesis planned to explain the two possible VIP modes of action. Based on our observations, we hypothesized that TLR2 and -4 modulations, induced after VIP treatment, may be explained through two possible ways that are not mutually exclusive. The first is an indirect through the reduction of TLR2 and -4, caused by the VIP-mediated decrease of inflammatory mediators such as IL-1ß and IFN-, which synergize with bacterial products contributing to the amplification of TLR (blue arrows). The second possible mechanism, which remains to be elucidated in this model, is a primary (black arrows) through the VIP-mediated decrease of NF-B, which would cause a down-regulation of TLR expression. IRAK, IL-1 receptor-activated kinase; TRAF6, TNF receptor-associated factor 6; IKK, IB kinase.
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Received October 5, 2004; revised March 16, 2005; accepted March 30, 2005.
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  V. S. Conlin, X. Wu, C. Nguyen, C. Dai, B. A. Vallance, A. M. J. Buchan, L. Boyer, and K. Jacobson Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis Am J Physiol Gastrointest Liver Physiol, October 1, 2009; 297(4): G735 - G750. [Abstract] [Full Text] [PDF]
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 S. C. Gribar, R. J. Anand, C. P. Sodhi, and D. J. Hackam The role of epithelial Toll-like receptor signaling in the pathogenesis of intestinal inflammation J. Leukoc. Biol., March 1, 2008; 83(3): 493 - 498. [Abstract] [Full Text] [PDF]
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J. S. Bryson, C. D. Jennings, J. A. Brandon, J. Perez, B. E. Caywood, and A. M. Kaplan Adoptive transfer of murine syngeneic graft-vs.-host disease by CD4+ T cells J. Leukoc. Biol., December 1, 2007; 82(6): 1393 - 1400. [Abstract] [Full Text] [PDF]
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 E. Gonzalez-Rey, P. Anderson, and M. Delgado Emerging roles of vasoactive intestinal peptide: a new approach for autoimmune therapy Ann Rheum Dis, November 1, 2007; 66(suppl_3): iii70 - iii76. [Abstract] [Full Text] [PDF]
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H. F. Rosenberg Vasoactive intestinal peptide, periodontal disease, and the innate immune response: an interview with Dr. John J. Taylor J. Leukoc. Biol., April 1, 2007; 81(4): 904 - 906. [Full Text] [PDF]
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N. Foster, S. R. Lea, P. M. Preshaw, and J. J. Taylor Pivotal Advance: Vasoactive intestinal peptide inhibits up-regulation of human monocyte TLR2 and TLR4 by LPS and differentiation of monocytes to macrophages J. Leukoc. Biol., April 1, 2007; 81(4): 893 - 903. [Abstract] [Full Text] [PDF]
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 A. M. Szema, S. A. Hamidi, S. Lyubsky, K. G. Dickman, S. Mathew, T. Abdel-Razek, J. J. Chen, J. A. Waschek, and S. I. Said Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L880 - L886. [Abstract] [Full Text] [PDF]
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 I. Gutierrez-Canas, Y. Juarranz, B. Santiago, A. Arranz, C. Martinez, M. Galindo, M. Paya, R. P. Gomariz, and J. L. Pablos VIP down-regulates TLR4 expression and TLR4-mediated chemokine production in human rheumatoid synovial fibroblasts Rheumatology, May 1, 2006; 45(5): 527 - 532. [Abstract] [Full Text] [PDF]
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 A. Chorny, E. Gonzalez-Rey, A. Fernandez-Martin, D. Pozo, D. Ganea, and M. Delgado Vasoactive intestinal peptide induces regulatory dendritic cells with therapeutic effects on autoimmune disorders PNAS, September 20, 2005; 102(38): 13562 - 13567. [Abstract] [Full Text] [PDF]
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