Activation of Peroxisome Proliferator-Activated Receptor-γ in Dendritic Cells Inhibits the Development of Eosinophilic Airway Inflammation in a Mouse Model of Asthma
2004; Elsevier BV; Volume: 164; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63116-1
ISSN1525-2191
AutoresHamida Hammad, Hendrik Jan de Heer, Thomas Soullié, Véronique Angeli, François Trottein, Henk C. Hoogsteden, Bart N. Lambrecht,
Tópico(s)Immune cells in cancer
ResumoPeroxisome proliferator-activated receptors (PPARs) are activated by an array of polyunsaturated fatty acid derivatives, oxidized fatty acids, and phospholipids and are proposed to be important modulators of immune and inflammatory responses. Recently, we showed that activation of PPAR-γ alters the maturation process of dendritic cells (DCs), the most potent antigen-presenting cells. In the present report, we investigated the possibility that, by targeting DCs, PPAR-γ activation may be involved in the regulation of the pulmonary immune response to allergens. Using a model of sensitization, based on the intratracheal transfer of ovalbumin (OVA)-pulsed DCs, we show that rosiglitazone, a selective PPAR-γ agonist, reduces the proliferation of Ag-specific T cells in the draining mediastinal lymph nodes but, surprisingly enough, dramatically increases the production of the immunoregulatory cytokine interleukin (IL)-10 by T cells, as compared to control mice sensitized with OVA-pulsed DCs. After aerosol challenge, the recruitment of eosinophils in the bronchoalveolar lavage fluids was strongly reduced compared to control mice. Finally, T cells from the mediastinal lymph nodes produced higher amounts of IL-10 and interferon-γ. Inhibition of IL-10 activity with anti-IL-10R antibodies partly restored the inflammation. The specificity of the phenomenon was confirmed by treating OVA-pulsed DCs with ciglitazone, another PPAR-γ agonist, and by using GW9662, a PPAR-γ antagonist. Our data suggest that PPAR-γ activation prevents induction of Th2-dependent eosinophilic airway inflammation and might contribute to immune homeostasis in the lung. Peroxisome proliferator-activated receptors (PPARs) are activated by an array of polyunsaturated fatty acid derivatives, oxidized fatty acids, and phospholipids and are proposed to be important modulators of immune and inflammatory responses. Recently, we showed that activation of PPAR-γ alters the maturation process of dendritic cells (DCs), the most potent antigen-presenting cells. In the present report, we investigated the possibility that, by targeting DCs, PPAR-γ activation may be involved in the regulation of the pulmonary immune response to allergens. Using a model of sensitization, based on the intratracheal transfer of ovalbumin (OVA)-pulsed DCs, we show that rosiglitazone, a selective PPAR-γ agonist, reduces the proliferation of Ag-specific T cells in the draining mediastinal lymph nodes but, surprisingly enough, dramatically increases the production of the immunoregulatory cytokine interleukin (IL)-10 by T cells, as compared to control mice sensitized with OVA-pulsed DCs. After aerosol challenge, the recruitment of eosinophils in the bronchoalveolar lavage fluids was strongly reduced compared to control mice. Finally, T cells from the mediastinal lymph nodes produced higher amounts of IL-10 and interferon-γ. Inhibition of IL-10 activity with anti-IL-10R antibodies partly restored the inflammation. The specificity of the phenomenon was confirmed by treating OVA-pulsed DCs with ciglitazone, another PPAR-γ agonist, and by using GW9662, a PPAR-γ antagonist. Our data suggest that PPAR-γ activation prevents induction of Th2-dependent eosinophilic airway inflammation and might contribute to immune homeostasis in the lung. Dendritic cells (DCs) are powerful antigen-presenting cells with a unique capacity to stimulate naïve T cells.1Banchereau J Steinman RM Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (12389) Google Scholar In the airways, immature lung DCs are ideally placed to sample inhaled antigens.2Lambrecht BN Allergen uptake and presentation by dendritic cells.Curr Opin Allergy Clin Immunol. 2001; 1: 51-59Crossref PubMed Scopus (79) Google Scholar After they acquire antigens in the periphery, DCs migrate to the draining lymph nodes (LNs) where they localize in the T cell-rich area and initiate immune responses. 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PPAR-γ agonists such as the thiazolidinedione class of anti-diabetic drugs have been shown to suppress experimental 2,4,6-trinitro-benzene sulfonic acid (TNBS) induced colitis and experimental allergic encephalitis.27Desreumaux P Dubuquoy L Nutten S Peuchmaur M Englaro W Schoonjans K Derijard B Desvergne B Wahli W Chambon P Leibowitz MD Colombel JF Auwerx J Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. 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After challenge, a marked shift in the immune response [increased interferon (IFN)-γ and IL-10] was observed in the MLNs and was accompanied by a significant decrease in airway eosinophilia. These data suggest that activation of PPAR-γ in lung DCs might be important in the regulation of airway inflammatory diseases such as asthma. OVA was from Worthington Biochemical Corp (Lakewood, NJ). At the dose we used in our experiments, the endotoxin level of OVA measured by a limulus-amebocyte lysate assay (Biowhittaker, Verviers, Belgium) was 99% as assessed by trypan blue exclusion. The phenotype of DCs was determined by staining for 30 minutes with CD11c-APC, MHCII-FITC, in combination with CD40-PE, CD80-PE, and CD86-PE dissolved in PBS containing 0.5% bovine serum albumin and 0.01% sodium azide. To detect CCR7 expression, DCs were first stained with CCL19-Fc for 30 minutes, washed, and incubated for another 30 minutes with anti-human IgG. DCs were washed and analyzed by flow cytometry on a FACScalibur (BD). Mice were anesthetized with avertin (2% v/v in PBS) and 80 μl of the cell suspension (1 × 106 DCs) was instilled through the opening vocal cords. At day 10 of the culture, unpulsed DCs and OVA-pulsed DCs treated or not with RSG were collected, washed, and labeled with CFSE as previously described.33Lambrecht BN Pauwels RA Fazekas De St Groth B Induction of rapid T cell activation, division, and recirculation by intratracheal injection of dendritic cells in a TCR transgenic model.J Immunol. 2000; 164: 2937-2946PubMed Google Scholar One million DCs were instilled into the trachea of naïve BALB/c mice. Twenty-four hours later, MLNs were collected and minced using scissors. The LNs were then incubated for 1 hour at 37°C in RPMI 1640 containing 5% fetal calf serum, 1 mg/ml collagenase type 2 (Worthington), and 0.02 mg/ml DNase I (Sigma, Zwijndrecht, The Netherlands), according to a modified protocol.36Vremec D Shortman K Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes.J Immunol. 1997; 159: 565-573PubMed Google Scholar LN cells were resuspended in PBS containing 10 mmol/L of ethylenediaminetetraacetic acid and centrifuged. CFSE+ DCs were detected by flow cytometry on a FACScalibur. Cell viability was determined by trypan blue and was >95%. Because the frequency of OVA-specific T cells is very low in immunized animals, the primary activation of a naïve T cell is difficult to detect. To avoid this problem, a detectable number of naïve T cells purified from DO11.10 mice were adoptively transferred into BALB/c mice. Briefly, LNs and spleen were collected from DO11.10 mice and smashed. After red blood cell lysis, cells were labeled with CFSE. Cells were enumerated and dead cells, stained for trypan blue, were excluded. Cells (10 × 106) were injected intravenously in the lateral tail vein of BALB/c mice (day −2). On day 0, the mice received an intratracheal injection of OVA-DC, RSG/OVA-DC, or control unpulsed DCs. On day 4, MLNs were collected, homogenized, and stained for the presence of KJ1-26+ CD4+ reactive OVA-specific T cells. Some of the LN cells (200,000 cells/well in triplicates) were resuspended in RPMI 1640 containing 5% fetal calf serum and antibiotics and placed in 96-well plates. Four days later, supernatants were harvested and analyzed for the presence of Th1 (IFN-γ) and Th2 (IL-4, IL-5, and IL-10) cytokines by enzyme-linked immunosorbent assay (BD Pharmingen). On day 0, BALB/c mice were injected intratracheally with unpulsed DCs, OVA-DCs treated or not with ciglitazone (ciglitazone/OVA-DCs), or with RSG (RSG/OVA-DCs). To confirm the specificity of the phenomenon, RSG/OVA-DCs were also treated or not with the antagonist GW9662 (GW9662/RSG/OVA-DCs). In some experiments, mice received 250 μg of blocking anti-IL-10R antibodies or control antibodies (BD) 1 day before secondary challenge. From days 10 to 13, mice were exposed to 30-minute OVA aerosols (Grade III, Sigma). Mice were sacrificed 24 hours after the last aerosol. Bronchoalveolar lavage (BAL) was performed with 3 × 1 ml of Ca2+- and Mg2+-free HBSS (Invitrogen) supplemented with 0.1 mmol/L of sodium ethylenediaminetetraacetic acid. The BAL fluid was centrifuged; the cells were resuspended in HBSS, and enumerated in a hemocytometer. After washing, cells were stained for 30 minutes with anti-I-Ad/I-Ed FITC (macrophages), anti-CCR3 PE (eosinophils), anti-CD3 cy-chrome, anti-B220 cy-chrome (T and B cells, respectively), and anti-CD11c APC (macrophages) in PBS containing 0.5% bovine serum albumin and 0.01% sodium azide. Cells were washed and analyzed by flow cytometry as previously described.37van Rijt LS Prins JB Leenen PJ Thielemans K de Vries VC Hoogsteden HC Lambrecht BN Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31(hi)Ly-6C(neg) bone marrow precursors in a mouse model of asthma.Blood. 2002; 100: 3663-3671Crossref PubMed Scopus (123) Google Scholar After BAL was performed, 1 ml of fixative was gently infused through the catheter. The lungs were resected and embedded in paraffin. Four-μm sections were performed and stained with May-Grunwald Giemsa. MLNs were removed, homogenized, and resuspended in RPMI 1640 containing 5% fetal calf serum and antibiotics before enumeration. Ex vivo production of cytokines by T cells collected from the MLNs was measured after restimulation of 2 × 106 cells/ml with 10 μg/ml of OVA for 4 days. For all experiments, the difference between the groups was calculated using the Mann-Whitney U-test for unpaired data. Differences were considered significant if P was <0.05. We have previously established a model of adoptive transfer of bone marrow-derived DCs (BM-DCs) pulsed with OVA into the trachea of naïve mice. This model generates a primary OVA-specific immune response in the lung draining MLNs33Lambrecht BN Pauwels RA Fazekas De St Groth B Induction of rapid T cell activation, division, and recirculation by intratracheal injection of dendritic cells in a TCR transgenic model.J Immunol. 2000; 164: 2937-2946PubMed Google Scholar that leads to Th2 priming. When mice are challenged with OVA aerosols, a Th2-dependent eosinophilic airway inflammation develops.32Lambrecht BN De Veerman M Coyle AJ Gutierrez-Ramos JC Thielemans K Pauwels RA Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation.J Clin Invest. 2000; 106: 551-559Crossref PubMed Scopus (442) Google Scholar This model was used to test the effect of RSG. We have previously shown that mouse spleen DCs taken from Flt-3L-treated mice express the PPAR-γ.21Faveeuw C Fougeray S Angeli V Fontaine J Chinetti G Gosset P Delerive P Maliszewski C Capron M Staels B Moser M Trottein F Peroxisome proliferator-activated receptor gamma activators inhibit interleukin-12 production in murine dendritic cells.FEBS Lett. 2000; 486: 261-266Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar By staining permeabilized DCs with a polyclonal rabbit anti-PPAR-γ antibody, we confirmed that BM-DCs also express PPAR-γ (Figure 1). We next investigated whether RSG, a PPAR-γ agonist, could alter the OVA-induced maturation of BM-DCs. Compared to unpulsed DCs, the expression of the co-stimulatory molecules CD40, CD80, and CD86, but also of MHC II was increased in OVA-DCs (100 μg OVA/ml), a phenomenon probably caused by trace amounts of LPS in commercially available OVA. However, PPAR-γ activation by RSG treatment did not modify the expression of these markers on the cells (data not shown). Compared to unpulsed DCs, OVA-DCs express a higher amount of CCR7, as assessed by CCL19-Fc binding (Figure 2). However, compared to untreated OVA-pulsed DCs, CCR7 expression was down-regulated in cells treated with RSG. These data suggest that PPAR-γ activation alters DC maturation by affecting the expression of CCR7, a chemokine receptor involved in DC emigration, but not the synthesis of co-stimulatory molecules and MHC II.Figure 2Effect of RSG treatment on CCR7 expression by DCs. BM-DCs were pulsed or not overnight with 100 μg/ml of OVA in the presence or in the absence of 10 μmol/L of RSG. LPS at the dose of 500 ng/ml was also used as a positive control. Cells were incubated with CCL19-Fc for 30 minutes before addition of PE-labeled anti-human IgG (black histograms). White histogram represents fluorochrome-matched isotype control mAbs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the treatment of OVA-pulsed DCs with RSG decreased the expression of CCR7, we next hypothesized that PPAR-γ activation might affect the migratory capacities of DCs to the MLNs. As shown in Figure 3, when injected into the trachea, CFSE-labeled OVA-DCs could be detected in the MLNs 24 hours after the instillation.32Lambrecht BN De Veerman M Coyle AJ Gutierrez-Ramos JC Thielemans K Pauwels RA Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation.J Clin Invest. 2000; 106: 551-559Crossref PubMed Scopus (442) Google Scholar, 37van Rijt LS Prins JB Leenen PJ Thielemans K de Vries VC Hoogsteden HC Lambrecht BN Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31(hi)Ly-6C(neg) bone marrow precursors in a mouse model of asthma.Blood. 2002; 100: 3663-3671Crossref PubMed Scopus (123) Google Scholar However, the treatment of OVA-DCs with RSG reduced their capacity to reach the MLNs, as compared to mice injected with OVA-DCs. These data were confirmed in vitro in chemotaxis assays performed in transwells, where the migration of OVA-DCs treated with RSG at the dose of 10 μmol/L in response to the CCR7 ligand MIP-3β was highly reduced as compared to OVA-DCs. Treatment of OVA-DCs with 1 μmol/L of RSG did not modify the migration as compared to untreated OVA-DCs (data not shown). These data suggest that the activation of PPAR-γ interferes with the CCR7-mediated migratory capacities of DCs. Because only the dose of 10 μmol/L of RSG altered DC migration in vitro, all further experiments were performed with this single dose of RSG. As PPAR-γ activation affects the migration of OVA-pulsed DCs to the MLNs, we next investigated whether it could also impact the activation of naïve T cell by DCs within the MLNs. To this end, naïve T cells from DO11.10 mice were labeled with CFSE and adoptively transferred, on day −2, into BALB/c mice. On day 0, these mice received an intratracheal administration of OVA-DCs, RSG/OVA-DCs, or unpulsed DCs. Flow cytometry was used to track cell division of CFSE-labeled T cells in MLNs. Figure 4 shows that 4 days after transfer of OVA-DCs, some transgenic T cells had already undergone seven divisions. In mice that received RSG/OVA-DC, some T cells also reached the seventh division peak, but the total number of naïve T cells activated and recruited into divisions was lower as compared to the group immunized with OVA-DCs. As expected, in mice immunized with unpulsed DCs, naïve T cells failed to divide (data not shown). As PPAR-γ activation impacts the activation of OVA-specific naïve T cells in the
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