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Distinct Responses of Lung and Spleen Dendritic Cells to the TLR9 Agonist CpG Oligodeoxynucleotide¹

Li Chen^*, Meenakshi Arora^*, Manohar Yarlagadda^*, Timothy B. Oriss^*, Nandini Krishnamoorthy^*, Anuradha Ray^*, and Prabir Ray^2,*,

^* Department of Medicine, Pulmonary, Allergy, and Critical Care Division, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Dendritic cells (DCs) sense various components of invading pathogens via pattern recognition receptors such as TLRs. CpG oligodeoxynucleotides (ODNs), which mimic bacterial DNA, inhibit allergic airways disease and promote responses in the spleen to bacterial components. Because many TLR agonists are currently being tested for potential therapeutic effects, it is important to characterize the expression and function of TLRs in different tissues. We show that both myeloid and plasmacytoid DCs in the spleen express TLR9, the receptor for CpG ODNs, but lung DCs show no detectable expression in either subset. TLR4 expression in contrast was detected on both lung and spleen DCs. LPS was superior to CpG ODN in increasing the allostimulatory potential of lung DCs and their expression of CD40. However, both agonists efficiently stimulated spleen DCs. CpG ODNs administered to mice efficiently inhibited Th2 cytokine production both in the lung draining lymph node and in the spleen. Surprisingly, inhibition of Th2 cytokine production was evident despite high levels of expression of GATA-3 and additional transcription factors that regulate Th2 responses. Although in the spleen CpG ODNs induced IL-6, a key cytokine induced via TLR9-MyD88 signaling, no IL-6 was detectable in lung LN cells. These studies show for the first time that lung DCs lack TLR9 expression, but, despite this deficiency, CpG ODNs induce potent inhibitory effects on Th2 cytokine production in the lung without inducing expression of the proinflammatory cytokine, IL-6, which has been linked to chronic diseases in the lung and the gut.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Dendritic cells (DCs)³ play an important role in Ag presentation in different tissues (1, 2), including the lung (3, 4). Research since the late 1980s has shown an important role for DCs in pulmonary immune responses (3, 5, 6, 7, 8, 9, 10, 11, 12, 13). DCs are present in high density in the proximal airways and are well positioned to capture Ags. Overall, pulmonary DCs are immature in that they express low levels of costimulatory molecules (CD80, CD86, and CD40). During inflammation, increased numbers of DCs are rapidly recruited from bone marrow progenitors to the airway epithelium, where these immature DCs efficiently capture Ags due to their high phagocytic ability (6, 12, 14). Subsequent to Ag capture, the DCs undergo maturation, which is influenced by the microenvironment, and the mature DCs traffic to local draining lymph nodes (LNs). The transition from immature to mature state is accompanied by the production of various cytokines and chemokines by the DCs that regulate their ability to interact with naive T cells to direct T cell differentiation (1, 2, 6, 15, 16, 17, 18, 19). Their strategic distribution, and their ability to capture and process Ag, and present them to T cells in the LNs make DCs the key APCs in the lung and in other mucosal surfaces.

Lung DCs are constantly exposed to a myriad of inhaled agents. However, the lung maintains homeostasis in the face of constant provocation by these multiple stimuli. Interestingly, although LPS and CpG oligodeoxynucleotides (ODNs) are both bacterial components, LPS induces severe inflammation in the lung precipitating sepsis (20, 21), while CpG ODNs suppress allergic airway inflammation (22, 23, 24, 25) and also growth of Mycobacterium tuberculosis in the lungs (25). Like the lung, the spleen also encounters pathogens being the principal filter unit for pathogens that enter the bloodstream. Administration of CpG ODNs to mice has been shown to cause transient splenomegaly (26), and CpG ODNs potentiate effects of TLR agonists in the spleen (27, 28). Bacterial CpG motifs have been shown to promote chronic intestinal inflammation via secretion of proinflammatory cytokines such as IL-6 and IFN- (29). CpG ODNs are increasingly being used in various clinical trials (30). Therefore, it is important to better understand the effects of CpG ODNs in different tissues and the expression characteristics of TLR9, which is the best-studied receptor for CpG ODNs. Because little is known about TLR expression in lung DCs, which are the key APCs in the lung, we investigated whether CpG ODNs exert similar or differential effects on DCs from the lung vs the spleen, which serve distinct functions in the body.

We show that while LPS had similar effects on lung and spleen DCs and the DCs in turn stimulated CD4⁺ T cells in a comparable fashion, CpG ODNs exerted differential effects on lung and spleen DCs both in in vitro assays as well as when administered to mice. Studies of the expression profile of the two TLRs at the two locations showed that while TLR4 expression is comparable between lung and spleen DCs, only the latter express TLR9 with no detectable expression in lung DCs. Despite the lack of TLR9 expression in lung DCs, CpG ODN administration caused potent inhibition of allergen-induced Th2 cytokine production in both the spleen and the lung LN. However, only in the spleen, production of IL-6, a key cytokine downstream of TLR9 (31, 32, 33) and MyD88 (34), was noted. Interestingly, this inhibition of cytokine production was evident in the presence of high levels of expression of GATA-3, which we and others (35, 36) previously demonstrated to be the master regulator of Th2 differentiation. With the recent identification of TLR9-independent mechanisms of CpG action (37, 38), our data suggest that CpG ODNs can exert both TLR9-dependent (IL-6) and -independent effects to regulate immune responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

BALB/cByJ, CD40^–/– mice (backcrossed to BALB/c background), and C57BL/6 male mice, obtained from The Jackson Laboratory, and DO11.10 TCR transgenic mice, originally provided by K. Murphy (Washington University School of Medicine, St. Louis, MO), were housed under pathogen-free conditions, were generally used at 6–8 wk of age, and were used under appropriate institutional guidelines.

Reagents

Purified preparations of LPS from Escherichia coli (O55:B5) were purchased from List Biological Laboratories. The CpG ODN 1826 (5'-TCCATGACGTTCCTGACGTT-3'), which is known to be optimal for stimulation of murine cells, and the control ODN 1911 (5'-TCCAGGACTTTCCTCAGGTT-3') were used. The LPS level in the ODN preparations was very low (<0.1 ng/mg DNA). The following mAbs were purchased from BD Pharmingen: PE-labeled Ab against CD11c (clone HL3), allophycocyanin-labeled Ab against CD11c (clone HL3), biotinylated Ab against CD40 (clone 3/23), PE-conjugated anti-CD11b (clone M1/70), anti-CD19 (clone 1D3), anti-Gr-1 (clone RB6-8C5), anti-CD80 (clone16-10A1), anti-CD86 (clone GL1), and PerCP-labeled anti-CD3e (clone 145-2C11). PE-labeled anti-MHC class II (clone NIMR-4) was purchased from Southern Biotechnology Associates. Biotinylated anti-DEC 205 was a gift from R. Hendricks (University of Pittsburgh, Pittsburgh, PA). Biotinylated Abs against TLR4 (clone MTS510) and TLR9 (clone 5G5) were obtained from HyCult Biotechnology. Streptavidin-conjugated allophycocyanin was obtained from Caltag Laboratories. The appropriate isotype controls used were: hamster IgG1 PE (clone G235-2356), hamster IgG1 allophycocyanin (clone G235-2356), hamster IgG1 PerCP (clone A19-3), rat IgG2a PE (clone R35-95), rat Ig2a biotin (clone R35-95), mouse IgG2a biotin (clone G155-178) (BD Pharmingen), and rat IgG2b PE (clone KLH/G2b-1-2) (Southern Biotechnology Associates).

Isolation and purification of DCs from lungs of mice

Lung DCs were isolated by a modification of previously published methods (24, 39). Briefly, BALB/cByJ mice were anesthetized with ketamine/xylazine mixture. After exsanguination via the abdominal aorta, the pulmonary vasculature was perfused with sterile 10 U of heparin/ml PBS to remove peripheral blood cells. The perfused lungs were removed, cut into small pieces, and incubated in an enzyme solution containing 0.7 mg/ml collagenase A (Boehringer Mannheim) and 30 µg/ml type IV bovine pancreatic DNase I (Sigma-Aldrich) in serum-free RPMI 1640 for 90 min at 37°C. Digested lung tissue was ground on a cell strainer (70 µm), particulate matter was removed by rapid filtration through a new cell strainer, and the filtered cells were washed in complete RPMI (cRPMI) (RPMI 1640 supplemented with 10% FBS, 5 x 10^–5 M 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 µg/ml gentamicin). After washing with 0.5% BSA, 2 mM EDTA in PBS, lung cells were resuspended in 2% FBS in PBS, overlaid on the same volume of Nycodenz (density 1.068 g/ml), and centrifuged at 600 x g for 15 min at room temperature. After centrifugation, cells at the interphase were collected and washed with 0.5% BSA, 2 mM EDTA in PBS. Lung DCs were further purified by positive selection using CD11c microbeads (Miltenyi Biotec).

Isolation and purification of spleen DCs

Spleen DCs were isolated by minor modifications of a previously published method (40). Briefly, spleen cell suspensions were prepared by collagenase A and DNase I digestion, and single cells were cultured for 2 h at 37°C in plastic culture plates, followed by removal of nonadherent cells. Adherent cells were collected and washed with PBS containing 2% FBS three times. CD11c⁺ DCs were purified from these cells with anti-CD11c magnetic beads (Miltenyi Biotec).

Flow cytometry

Cells were first incubated for 5 min with Fc block (10 µg/ml; BD Biosciences) to minimize nonspecific Ab binding and were then incubated with saturating concentrations of appropriate Abs for 5 min on ice in 2% FBS/PBS, after which the cells were washed in the same buffer. Before intracellular TLR9 staining, DCs were stained with dye-conjugated Abs against the cell surface molecules CD11c, MHC class II, and B220. The cells were washed, fixed for 30 min using freshly prepared Fix/Perm solution (eBioscience), and were washed in PBS and 1x permeabilization buffer (eBioscience) successively to permeabilize the cells. The cells were incubated with biotinylated mAb against murine TLR9 (clone 5G5) or with isotype control (mouse IgG2a), as described (41). Streptavidin-PerCP (BD Biosciences) was used for detection. TLR9 staining was also investigated using another anti-TLR9 Ab IMG-431 (Imgenex). All samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences). Dead cells were excluded using forward and side light scatter properties. Cell sorting was conducted using a FACSAria cell sorter and FACS Diva software (BD Biosciences).

Microscopy

For scanning electron microscopy, freshly isolated DCs were plated onto 12-mm glass coverslips in 24-well plates and allowed to attach for 15 min at 37°C. The cells were then fixed with 2.5% glutaraldehyde and prepared for electron microscopy using standard techniques.

For light microscopy, DCs were cytospun onto glass slides (800 rpm, 10 min) and air dried. The cells were stained with Hema-3 reagent (Fisher Scientific), according to the manufacturer’s recommendations.

MLRs

DCs were gamma irradiated at 2000 rad, washed with fresh cRPMI, and seeded in triplicate in round-bottom 96-well plates for use as stimulator cells at 1.2–20 x 10³ cells/well. Allogeneic responder CD4⁺ T cells were obtained from freshly isolated splenocytes from C57BL/6 mice by magnetic bead positive selection methods. Purified CD4⁺ T cells (1 x 10⁵ cells/well) were added to the DCs in a total volume of 200 µl of cRPMI, and then cultured for 96 h. For the final 18 h of culture, the cells were pulsed with 1 µCi of [³H]thymidine/well (catalog no. NET-027; PerkinElmer). Each sample was assayed in triplicate. Cells were harvested using a cell harvester, and incorporation of radioactivity was assessed by liquid scintillation counting. Results are expressed as mean cpm.

RT-PCR

Total RNA was extracted from freshly isolated lung and spleen DCs with TRIzol reagent and RNeasy Mini Kit (Qiagen).

Semiquantitative RT-PCR

First strand cDNA was synthetized using 300 ng of total RNA using oligo(dT) (catalog no. Y01212; Invitrogen Life Technologies) and Superscript II reverse transcriptase (Invitrogen Life Technologies). PCR was performed with the primers 5'-AGTGGGTCAAGGAACAGAAGCA-3' and 5'-CTTTACCAGCTCATTTCTCACC-3' for TLR4, 5'-CCAGACGCTCTTCGAGAACC-3' and 5'-GTTATAGAAGTGGCGGTTGT-3' for TLR9, and 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and 5'-GAGGGTAGGCTGGCCTATAGGCT-3' for hypoxanthine phosphoribosyltransferase. The PCR products were separated by electrophoresis in 2% agarose gels and were visualized by ethidium bromide staining.

Quantitative PCR

Predesigned gene-specific Taqman probe and primer sets were used for quantitative RT-PCR of TLR-4, TLR-9, and -glucuronidase (GUS). First strand cDNA was synthesized using High Capacity cDNA Archive kit (Applied Biosystems). Samples were then subjected to real-time PCR analysis using the ABI PRISM 7700 Sequence System (Applied Biosystems). Relative mRNA abundance of each transcript was normalized against GUS, calculated as 2^{(Ct[GUS] – Ct[gene])}, where Ct represents the threshold cycle for each transcript and the resulting numbers were multiplied by a factor of 10³.

Mouse immunizations and culture of isolated cells

Intranasal (i.n.) immunization and isolation of lung-draining LNs. Mice were lightly anesthetized by isoflurane inhalation, and then they received by i.n. route 5 µg of CpG ODN, 100 µg of OVA plus 1 µg of cholera toxin (CT), or 5 µg of CpG ODN first and followed by 3 h later with 100 µg of OVA plus 1 µg of CT for 3 consecutive days. The mice were sacrificed on day 5 post last immunization. The lung-draining LNs were harvested, and single-cell suspensions were used, unless otherwise stated.

Intraperitoneal immunization and isolation of spleens. Mice were immunized by i.p. injection of 25 µg of CpG ODN alone or 10 µg of OVA plus 2 mg of aluminum hydroxide (alum), or 25 µg of CpG ODN and followed by 3 h later with 10 µg of OVA plus 2 mg of alum. Following 5 days of rest, mice were given 1 boost (i.p.) with the same amount of Ag and adjuvants. Mice were sacrificed 24 h later, and spleens were harvested and single splenocytes were isolated. RBC were lysed by using ammonium chloride lysing reagent (BD Biosciences).

Lung LN or spleen cells from each group were cultured in cRPMI (2.5 x 10⁶ cells/well) in the presence of OVA protein (100 µg/ml) for 5 days. Supernatants were collected for cytokine profile analysis.

Cytokine assays

Cytokine assays were performed by ELISA (R&D Diagnostic) or using a multiplex system (Luminex; Bio-Rad) using commercially available kits, according to the manufacturer’s instructions.

Preparation of nuclear extracts and Western blotting

Lung DCs were incubated for 40 h in cRPMI with or without LPS, control ODN 1911, or CpG ODN 1826. The cells were washed with fresh medium and cultured for another 5 days with CD4⁺ T cells from spleens of DO11.10 TCR transgenic mice, in the presence of the specific OVA peptide (pOVA^323–339) at 5 µg/ml and IL-2 (10 U/ml) in fresh medium.

Nuclear extracts were prepared from CD4⁺ T cells following culture with differentially treated lung DCs, as described previously (35, 42). Nuclear protein content was determined by protein assay (Bio-Rad). Equal amounts of nuclear protein were subjected to 4.5–15% gradient SDS-PAGE, and the resolved proteins were transferred to polyvinylidene difluoride membrane. The blots were probed with anti-GATA3 mAb, anti-T-bet polyclonal Ab, or anti-CREB-1 Ab (Santa Cruz Biotechnology), followed by HRP-conjugated secondary Ab. Bands were visualized by ECL (Amersham).

Statistical analysis

Results are presented as mean ± SD. Statistical comparison between two different groups was performed using Student’s t test. Statistical significance was defined as p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Isolation of lung CD11c⁺ DCs and characterization

A combination of density-gradient centrifugation and CD11c-positive selection was used to recover lung and spleen DCs (24, 39). The density-gradient-positive selection combination approach consistently resulted in the recovery of a population of cells that was >97% CD11c⁺ with the characteristics of DCs as observed by scanning electron microscopy and light microscopy (Fig. 1, A and B). Also, this population was largely devoid of CD3⁺, CD19⁺, or Gr-1⁺ cells (Fig. 1C). This approach resulted in a typical recovery of 1.01.2 x 10⁵ CD11c⁺ DCs cells per mouse lung. The cells were briefly cultured in GM-CSF and analyzed for expression of relevant cell surface molecules. As shown in Fig. 1D, these cells expressed CD11b, MHC class II, and DEC205.

View larger version (55K): [in this window] [in a new window]   FIGURE 1. Purity and morphology of lung DCs. A, CD11c+ cells were collected from the lung of mice using a combination of density-gradient centrifugation and positive selection methods. The purity of the CD11c+ cells was >97%. B, Scanning electron microscopy and light microscopy of freshly isolated CD11c+ DCs. C, Lack of CD3-, CD19-, and Gr-1-expressing cells in the purified DCs. D, Expression of CD11b, MHC II, and DEC205 on CD11c+ DCs after brief incubation with GM-CSF (10 ng/ml). Light lines indicate staining with isotype control Ab. Shown is a representative of three independent experiments.

To study the response of lung DCs to TLR4 and TLR9 agonists, we first examined expression of the TLRs in the purified lung DCs and compared with expression in spleen DCs. The expression of both TLRs was examined by semiquantitative and quantitative RT-PCR. Using both techniques, lung DCs were found to express slightly higher levels of TLR4 compared with spleen DCs, while the converse was true for TLR9 expression (Fig. 2A). At the protein level, as determined by flow cytometry, TLR4 expression was clearly evident on the cell surface of both lung and spleen DCs (Fig. 2B). Because the RT-PCR data showed low expression of TLR9 in lung tissue DCs, we investigated TLR9 expression in DCs isolated from lung-draining LNs and spleens. This approach was undertaken based on the knowledge that in mice both plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) express TLR9, and pDCs typically reside in LNs draining respective tissues unless recruited into the tissues due to specific infections. Because it is not feasible to isolate enough DCs from lung-draining LNs of naive mice, we immunized mice with Ag + adjuvant to examine TLR9 expression in DCs isolated from lung-draining LNs and spleens. CpG ODN, known to promote Th1 responses in different tissues including the lung, and the best known TLR9 agonist, was used with Ag (OVA) and administered either i.n. to induce immune responses in the lung (43), or i.p. to stimulate responses in the spleen (44, 45). DCs were purified from lung-draining LNs or spleens of mice, and TLR9 expression was examined by flow cytometry. As shown in Fig. 2C, TLR9 expression was readily detected in both mDCs and pDCs isolated from spleens with greater expression in mDCs. Surprisingly, TLR9 protein expression could not be appreciated in lung DCs in either subset. We have also observed a high level of TLR9 expression in thioglycolate-elicited peritoneal macrophages (data not shown). Additionally, we have confirmed this differential TLR9 expression profile between lung and spleen DCs using a different anti-TLR9 Ab (data not shown).

View larger version (42K): [in this window] [in a new window]   FIGURE 2. TLR4 and TLR9 expression by lung and spleen DCs. A, Expression of TLR4 and TLR9 mRNA in CD11c+ lung and spleen DCs. mRNA was isolated from freshly isolated lung or spleen CD11c+ DCs. TLR4 and TLR9 expression was determined by both semiquantitative and quantitative RT-PCR. Hypoxanthine phosphoribosyltransferase expression was assessed as a control for loading. The results for quantitative RT-PCR are expressed as relative TLR4 or TLR9 mRNA expression (mean ratio ± SD) normalized to expression of GUS. B, Cell surface TLR4 expression in freshly isolated CD11c+ lung and spleen DCs as assessed by flow cytometry. The open histograms show staining by the specific Abs, and the filled histograms are staining by isotype controls. C, TLR9 is expressed in spleen, but not lung-draining LN DCs. Mice were immunized with OVA plus adjuvant i.n. (i.n. OVA plus CpG) or systemically (i.p. OVA plus CpG) to activate DCs in the lung-draining LNs and spleen, respectively. DCs were partially purified by density-gradient centrifugation, then were surface stained to identify pDCs and mDCs using directly labeled anti-CD11c FITC, anti-MHC class II PE, and anti-B220 allophycocyanin mAbs. Appropriate isotype control Abs for each of these markers were used to establish the level of background staining for each. Expression of intracellular TLR9 was then determined by incubation of fixed, permeabilized cells with either biotinylated anti-TLR9 Ab or relevant biotinylated isotype control Ab, followed by incubation with PerCP-labeled streptavidin. Flow cytometric analysis was performed using multiple gating techniques to identify pDCs and mDCs according to the parameters listed above. For spleen cells, the expression of MHC class II was similar (data not shown) and CD11c expression varied slightly such that pDCs were CD11c low, MHC class II+, B220+, and mDCs were CD11c high, MHC class II+, B220–. For lung-draining LN cells, CD11c expression was similar and MHC class II varied (data not shown) such that pDCs were CD11c+, MHC class II low, B220+, and mDCs were CD11c+, MHC class II high, B220–. TLR9 expression is shown relative to CD11c expression for pDCs and mDCs in lung-draining LNs and spleens. The isotype control Ab staining for TLR9 is shown in the upper panels. The percentages listed represent the proportion of TLR9 expression on the multiple-gated cell population shown. This is a representative of two independent experiments.

We tested the allostimulatory capacity of DCs isolated from lung tissue and spleen after treating with either LPS, a TLR4 agonist, or CpG ODN, a TLR9 agonist, to assess the functional consequence of differential expression of TLRs on lung and spleen DCs. We first used spleen DCs to establish a dose response to the agonists. Based on the results of the dose-response study, as shown in Fig. 3A, 20 µg/ml CpG ODN and 10 µg/ml LPS were used to stimulate DCs from the two tissues. Lung DCs do not fare well in low serum medium or with other supplements, and therefore we have used serum-containing medium in all of our experiments. Using different end points in different experiments, the DCs were found not to undergo maturation when maintained in serum containing medium unless specific stimuli were added. As shown in Fig. 3B, between the two TLR agonists, LPS and CpG ODN, the former induced stronger allostimulatory potential in the lung DCs. However, the overall level of T cell proliferation was not particularly high. In the case of spleen DCs in contrast, pretreatment with either LPS or CpG ODN promoted comparable allostimulatory potential of the DCs. Furthermore, T cell proliferation achieved with splenic DCs was greater compared with that obtained with lung DCs (Fig. 3B). Similarly, LPS- or CpG ODN-treated bone marrow-derived DCs also induced vigorous T cell proliferation (data not shown).

View larger version (40K): [in this window] [in a new window]   FIGURE 3. The effect of TLR agonists on allostimulatory function of lung and spleen DCs. A, Dose response of TLR agonists on allostimulatory function of spleen DCs. Freshly isolated spleen DCs were cultured for 40 h with different concentrations of LPS or CpG ODN, as indicated, or ODN control 1911 (20 µg/ml). The ratio of DC:T cell was 1:5. A dose of 10 µg/ml LPS and 20 µg/ml CpG ODN was used to stimulate DCs in all subsequent experiments. B, Allostimulatory ability of lung and spleen DCs stimulated by LPS or CpG ODN. Freshly isolated lung and spleen DCs were maintained in medium alone or stimulated with the indicated agents for 40 h. DCs were then washed with cRPMI and cocultured with allogeneic CD4+ T cells for 96 h. [3H]Thymidine was added during the final 18 h of culture. +, p < 0.05 LPS vs CpG treatment; *, p < 0.05 LPS vs medium; and #, p < 0.05 CpG ODN vs control ODN control. Shown is a representative experiment of four. C, Sorting of CD11c+, low autofluorescent cells and allostimulatory function after stimulation by LPS or CpG ODN. CD11c+ cells were isolated from lungs by positive selection methods (see Presort population), and the less abundant low autofluorescent CD11c+ cells were enriched by sorting (>80% purity in the Postsort low autofluorescent population). The sorted cells were then used in MLRs. The data shown are mean ± SD. +, p < 0.05 LPS vs CpG treatment; *, p < 0.05 LPS vs medium. The experiment was repeated twice with similar results.

Phenotypic maturation of DCs in response to chemokines and TLR agonists

The results of the MLRs led us to examine the ability of the TLR agonists LPS and CpG ODN to cause maturation of lung and spleen DCs (Fig. 4A). With the lung DCs, CpG ODN 1826, but not the control ODN 1911, induced a small increase in CD40- and CD86-expressing cells. LPS, in contrast, increased a higher percentage of CD40-expressing cells (Fig. 4A). Consistently, simply culturing the lung DCs, whether in medium alone or with the two agonists, resulted in down-regulation of CD80 expression on the lung DCs.

View larger version (57K): [in this window] [in a new window]   FIGURE 4. Effects of TLR agonists on phenotypic maturation of lung and spleen DCs. Freshly isolated lung (A) or spleen (B) DCs were cultured with the indicated stimuli for 40 h. Cells were stained with PE-conjugated CD80, CD86, or MHC II or biotin-conjugated CD40, followed by incubation with streptavidin-conjugated allophycocyanin. The percentages indicate positive staining relative to control Ab staining (filled histograms), and the numbers in parentheses indicate mean fluorescence intensity. Similar results were obtained in two independent experiments.

LPS-stimulated lung DCs selectively induce Ag-specific responses in CD4⁺ T cells, resulting in GATA-3 and T-bet activation

We next investigated Ag-specific responses in CD4⁺ T cells when stimulated with lung or spleen DCs previously exposed to the TLR agonists. Ag-specific response was examined using CD4⁺ T cells from DO11.10 TCR transgenic mice with specificity for an epitope (aa 323–339) in OVA. We investigated expression of the transcription factors T-bet and GATA-3, which program CD4⁺ T cells to differentiate into the Th1 or Th2 lineage, respectively, as shown previously by us and others (35, 36, 45, 47). LPS-treated lung DCs induced high levels of T-bet and GATA-3 expression in the CD4⁺ T cells (Fig. 5A). However, neither T-bet nor GATA-3 expression was induced in the T cells by CpG ODN-stimulated lung DCs. The ability of LPS-treated DCs to induce a mixed Th1/Th2 response in CD4⁺ T cells was also noted in previous studies (48). When T cells were exposed to spleen DCs, GATA-3 expression was found to be high irrespective of whether the DCs were previously incubated in medium alone or stimulated with LPS or CpG ODN. This may be due to the relatively high level of basal CD86 expression on spleen DCs since CD86 is known to promote Th2 differentiation (49). T-bet expression in the T cells, however, increased if the cells were cocultured with LPS or CpG ODN-treated DCs as compared with incubation with DCs cultured in medium alone. These results showed that CpG-treated lung DCs have low levels of expression of costimulatory molecules and have poor T cell stimulatory ability. LPS stimulation of lung DCs in contrast promotes CD40 expression and renders them competent to up-regulate both T-bet and GATA-3 expression in T cells. Spleen DCs in contrast are highly responsive to both TLR agonists, as reflected in increased expression of costimulatory molecules on the DCs and their ability to activate T cells. We also examined cytokine production by the cocultured CD4⁺ T cells, and the cytokine secretion profile also matched T-bet and GATA-3 expression in the T cells (Fig. 5B). Only LPS-stimulated lung DCs induced high levels of secretion of the cytokines IFN-, IL-4, IL-5, IL-13, and IL-10 secretion from the CD4⁺ T cells. In the case of spleen DCs, both LPS and CpG ODN treatments increased cytokine production from the T cells. Despite comparable levels of IL-5 and IL-13 in the supernatants of cocultures of CD4⁺ T cells with lung DCs and spleen DCs, there was a marked difference in IL-4 levels, with CD4⁺ T cells incubated with LPS-treated lung DCs secreting a higher level of IL-4.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Effects of the TLR agonist-stimulated DCs on Ag-specific CD4+ T cell responses. Lung DCs were cultured with the indicated stimuli for 40 h. The culture supernatant was then removed, the cells were washed, and fresh medium was added containing CD4+ T cells from DO11.10 TCR transgenic mice, the OVA peptide, pOVA (323–339), at 5 µg/ml, and IL-2 (10 U/ml). The cells were cocultured for another 5 days, after which nuclear extracts were made and the culture supernatants were saved for cytokine estimation. A, Expression of transcription factors T-bet and GATA-3 in the CD4+ T cells stimulated by lung or spleen DCs as assessed by Western blot analysis of the nuclear extracts. CREB-1 expression was assessed as a control for protein loading. B, Cytokine levels in the culture supernatants were determined by multiplex assay. The data shown are mean concentrations ± SD of triplicate wells. The experiment was repeated three times with similar results.

Because all of the above experiments showed an attenuated response of lung DCs to CpG ODN, we investigated whether this was also reflected in cytokine production by the stimulated DCs. We investigated IL-12 production by the stimulated DCs because it is known to be induced by both LPS and CpG ODN. In these experiments, we also used DC-T cell cocultures using DCs isolated from either wild-type (wt) or CD40^–/– mice. The reason for the inclusion of DC-T cell cocultures and DCs from both wt and CD40^–/– mice was the known involvement of both microbial and CD40-CD154 interaction in stimulation of IL-12 production by DCs (50). Although our previous experiments suggested that lung DCs are refractory to CpG ODN, this was found to be not entirely true when IL-12p40 production was examined from the treated DCs. IL-12p40 secretion was found to be higher from CpG ODN-stimulated lung DCs than from LPS-stimulated cells (Fig. 6). Interestingly, the level was similar to that produced by CpG ODN-treated spleen DCs (Fig. 6). 1L-12p70 production from the stimulated DCs was not detected at a high level from either lung or spleen DCs. The levels detected were, however, comparable to those reported in other studies using spleen DCs (51). Although CpG ODN induced higher levels of IL-12p40 secretion from lung DCs compared with LPS, the opposite was true with respect to IL-12p70 with LPS stimulating a higher level of IL-12p70 production. Also, consistent with the reported involvement of CD40 stimulation in IL-12 production by DCs, lung or spleen DCs isolated from CD40^–/– mice produced lower levels of IL-12 (p40 and p70) in response to both agonists (Fig. 6). Interestingly, the requirement for CD40 appeared to be more stringent for LPS-induced IL-12p40 production. Collectively, these results showed that lung DCs are not completely unresponsive to the TLR9 agonist CpG ODN.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Cytokine profile of the culture supernatants of lung and spleen DCs from wt and CD40–/– mice cocultured with CD4+ T cells. DCs were isolated from the lungs and spleens of wt and CD40–/– mice and were cocultured with CD4+ T cells for 5 days, as described in the legend to Fig. 5. Cytokine levels were determined by ELISA. The data are shown as mean ± SD and are representative of two independent experiments. * and **, p < 0.01 vs medium or control ODN, respectively; #, p < 0.05 vs LPS stimulation.

Having failed to detect TLR9 expression in lung DCs in contrast to readily detectable expression in spleen DCs, and yet detecting a CpG ODN-induced IL-12p40 response in the lung DCs, we were curious to determine the response of lung and spleen cells to CpG ODN in vivo. Toward this end, we used three groups of mice each for the analysis of lung and spleen responses. Mice received either CpG ODN alone, OVA + the Th2-skewing mucosal adjuvant CT to study the response in lung LNs (43), or OVA + alum to study response in the spleen (44, 45), or a combination of OVA + CT/alum + CpG ODN. Although CpG ODN has been shown to inhibit allergic airway inflammation in mice (22, 23, 24, 25), the response of lung LN cells to CpG ODN has not been reported to date. It was also important to check whether CpG ODN triggered IL-12p40 in the lung LNs as observed in vitro. As shown in Fig. 7, CpG ODN alone induced IL-12p40 production from both lung LN and spleen cells. A combination of OVA + CT induced appreciable levels of IL-4, IL-5, IL-10, IL-13, and IFN- from lung LN cells. Similar or slightly lower levels of Th2 cytokines (except for IL-5) were noted from splenic cells when animals were immunized with OVA + alum i.p. Surprisingly, IFN- production from spleen cells was markedly lower than what was observed from lung LN cells. The most notable difference was low/undetectable IL-6 production from lung LN cells compared with modest levels (350–400 pg/ml) from splenic cells. The lack of IL-6 production from lung LN cells parallels the recent results of Sanjuan et al. (38), in which CpG ODN induced tyrosine phosphorylation of target proteins in macrophages in the absence of TLR9 or MyD88, but failed to induce IL-6 secretion in the absence of MyD88. When a combination of CpG ODN and CT/alum was used, the production of Th2 cytokines was drastically reduced. Although IL-4 and IL-5 production was almost abolished, the levels of IL-10 and IL-13 were reduced by half in the lung. However, there was no inhibitory effect on IFN- production, and if anything, IFN- levels increased in the presence of CpG ODN. CpG ODN did not inhibit OVA/alum-stimulated IL-6 production either.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. CpG inhibits Th2 cytokine production in lung-draining LNs and spleens of mice. Mice were immunized i.n. with CpG ODN (5 µg/mouse) alone or OVA (100 µg) + CT (1 µg/mouse), or CpG ODN (5 µg/mouse) followed by OVA (100 µg) + CT (1 µg/mouse). On day 5 after last immunization, lung LN cells were isolated and cultured with OVA protein (100 µg/ml) for 5 days. Mice were also immunized i.p. with CpG ODN (25 µg/mouse) alone or OVA (10 µg) + alum (2 mg/mouse), or CpG ODN (25 µg/mouse) followed by OVA (10 µg) + alum (2 mg/mouse). After 5 days, mice received an i.p. boost with the same dose of Ag and adjuvants. Splenocytes were isolated 24 h after the last booster dose and cultured in the presence of OVA protein (100 µg/ml) for 5 days. Cytokine profiles of the culture supernatants of lung LN and spleen cells were detected by multiplex assay. *, p < 0.05 spleen vs lung LN levels. The results are representative of two independent experiments.

Having observed preferential inhibition of cytokine expression in the presence of CpG ODN in both the lung LN and the spleen, mice were immunized via the i.n. route using the same regimen followed for cytokine assays. After ex vivo stimulation of lung LN cells in the presence of OVA, nuclear extracts were prepared of the total LN cell population, and the extracts were subjected to Western blot analysis to determine activation/induction of specific transcription factors. Although the results of the cytokine assay showed remarkable inhibition of Th2 cytokine expression in the presence of CpG ODN, interestingly, GATA-3 expression was similar to what was observed in the presence of OVA + CT (Fig. 8). T-bet expression in contrast was higher in the presence of CpG ODN, which may explain the higher IFN- levels when mice received OVA plus both adjuvants. We also investigated expression of IFN regulatory factor 8 and NFAT-2, factors known to be important for IL-12p40 gene expression. Although expression of IFN regulatory factor 8 was similar under all three conditions, that of NFAT-2 was appreciably higher in the presence of CpG ODN, whether used alone or in conjunction with OVA and adjuvants. This high level of NFAT-2 expression may underlie the high level of IL-12p40 production in the context of CpG ODN (Fig. 8). Overall, while we did observe the appropriate transcription factor signature to support IL-12p40 gene expression, we did not observe a profile that would explain inhibition of Th2 cytokine production, such as inhibition of expression of GATA-3. We also analyzed expression of additional Th2-dominant factors such as c-maf and JunB (data not shown), none of which was differentially expressed under the three different immunization regimens. Collectively, these results show for the first time the ability of CpG ODN to cause a drastic inhibition of Th2 cytokine production in the absence of TLR9 expression and in the presence of high levels of GATA-3 expression.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Effect of CpG ODN on OVA/CT-induced transcription factor expression. Mice were immunized i.n. with CpG ODN, OVA + CT, or CpG ODN + OVA + CT, as described in the legend to Fig. 7. Lung LN cells were isolated on day 5 and cultured with OVA protein for 5 days. Nuclear extracts were prepared for analysis of transcription factor expression by Western blot techniques. CREB-1 expression was assessed as a control for protein loading. The results are representative of two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

One distinguishing feature of CpG ODN- vs LPS-stimulated lung DCs is the level of CD40 expression on the stimulated cells. Compared with CpG ODN, LPS induced more CD40 expression on the DCs. On spleen DCs, however, both LPS and CpG ODN induced high levels of CD40 expression. Interestingly, although both of these agonists augmented MHC class II, CD80, and CD86 expression on the spleen DCs, they failed to have a significant impact on the expression of these molecules on lung DCs. Clearly, CD40 expression by the DCs is important because its absence reduced/eliminated IL-12p40 or IL-12p70 production by the DCs (Fig. 6). Recently, TLR agonists and CD40 signaling were shown to synergize in stimulation of CD8⁺ T cell proliferation (54). Similarly, studies of Steinman and colleagues (55) have also shown a unique role for CD40-CD40L signaling in inducing functional maturation of DCs. The molecular nature of this distinctive CD40 signaling pathway in DCs is currently unclear. LPS is known to up-regulate costimulatory molecule expression on APCs through activation of the Toll/IL-1 receptor domain-containing adaptor-inducing IFN- (TRIF) pathway that through secretion of IFN- activates the IFN-RI receptor (56). It will be interesting to determine whether LPS can activate the TRIF/IFN-/IFN-RI signaling axis in lung DCs and whether this pathway contributes to the higher level of CD40 expression induced by LPS on lung DCs. However, it is also important to note that LPS-stimulated lung DCs are still inferior to spleen DCs with regard to allostimulation. TLR4 is known to use both MyD88-Mal/Toll-IL-1R domain-containing adapter protein and TRIF/TRIF-related adapter molecule (MyD88-independent) pathways (2), and their relative use in lung and spleen DCs remains to be determined.

Until recently, TLR9 was thought to be totally reliant on MyD88-dependent signaling. Recent studies indicate that TLR9 can signal via both TLR9-dependent and -independent mechanisms (37, 38). However, the production of IL-6 has been shown to be TLR9 and MyD88 dependent (33, 38). Our results show that while CpG ODN can induce production of several cytokines in lung LN cells, it is unable to induce IL-6 production, which can be readily detected from splenic cells. The absence of IL-6 production from lung LN cells upon CpG ODN treatment is most likely due to absence of TLR9 protein expression in lung LN DCs. It is surprising that this response is selectively absent in the lung. Although IL-6 is induced in lungs upon infection with pathogens, it is interesting that CpG motifs are unable to induce IL-6 in lung LNs that have many types of resident cells, including DCs, T cells, B cells, and macrophages. It remains to be determined whether all lung cell types lack TLR9. The biological significance of absence of TLR9 in lung LN cells to restrict production of IL-6 may be a protective measure that has evolved to protect the lung from the development of diseases such as pulmonary fibrosis that are associated with high IL-6 production and a high mortality rate (57, 58, 59). CpG motifs of bacterial DNA have been shown to promote chronic inflammation in colitis, in which the two key cytokines implicated are IL-6 and IFN- (29). It will be interesting to examine TLR9 expression in cells present in mesenteric LNs to determine whether within the mucosal tract there exists differential expression of TLR9 between DCs present in draining LNs of the lung vs those draining the gastrointestinal tract. The production of appreciable levels of IL-12p40 by the CpG ODN-stimulated lung DCs shows that the lung actively responds to this agent. IL-12p40 is the common subunit between IL-12p70 and IL-23. Because IL-12p70 is not always detectable at high levels, high IFN- levels in the lung LN cells suggest induction of sufficient IL-12p70 to induce IFN- production.

Multiple studies have shown previously that CpG ODN inhibits Th2-mediated disease in the lung (22, 23, 24, 52). Our data show that the Th2 inhibition is also evident in the spleen. The ability of CpG ODN to inhibit Th2 responses being similar in the lung LN and spleen, the mechanism of Th2 suppression by CpG ODN is most likely distinct from that used for promotion of IL-6 gene expression. Studies published recently have identified Src family kinases Hck, Lyn, and DNA-dependent protein kinase as alternate TLR9-independent mechanisms of CpG ODN action (37, 38). Whether any of these molecules or other pathways are used by CpG ODN in the lung to induce its effects needs to be investigated in the future.

It is interesting that the Th2 inhibition caused by CpG ODN occurs without any apparent inhibition of GATA-3 expression. Because GATA-3 levels are maintained in the presence of CpG ODN, the underlying mechanism is not a simple Th1/Th2 imbalance induced by high IL-12/IFN- levels that typically down-regulate the GATA-3 response. In a recent study, CpG-induced indoleamine 2,3-dioxygenese (IDO) expression in lung cells was implicated in the inhibition of experimental allergic airways disease in mice (24). Although lung epithelial cells were considered to be one source of IDO in this study, lung DCs were also shown to express IDO. Whether IDO induction in a DC imparts a dominant-negative signal to a CD4⁺ T cell via secretion of kynurenine metabolites that cause Th2 inhibition without effects on GATA-3 remains to be determined. Another potential mechanism for Th2 inhibition despite high GATA-3 levels is inhibition of GATA-3 DNA-binding activity by tyrosine-phosphorylated T-bet (60). This could be important because we detected an increase in T-bet expression when CpG was administered in animals before treatment with OVA + CT. Other mechanisms that would also functionally impair GATA-3 despite high levels of expression include expression of repressor of GATA-3, which inhibits GATA-3 function through inhibition of DNA-binding activity, and other as yet to be identified mechanisms (61).

In conclusion, the increase in GATA-3, T-bet, and concomitant cytokine gene expression in T cells induced by LPS-stimulated lung DCs has important ramifications in DC-mediated immunotherapy of lung cancer because the lung is frequently a site of cancer metastasis. It may be useful to stimulate the TLR4 pathway in DCs used in cancer immunotherapy. Recently, expression of a constitutively active mutant of TLR4 was shown to enhance allostimulatory potential of DCs (62). The DCs expressing melanoma Ag and constitutively active mutant of TLR4 elicited significant cytolytic activity in stimulated T cells (62). This approach may also be important to enhance adaptive immune responses in chronic infectious diseases such as tuberculosis to efficiently eliminate pathogens. The ability of CpG ODNs to inhibit Th2 responses in the lung shows that appropriate TLR agonists may have inhibitory or stimulatory effects on lung DCs that could be exploited in DC-mediated immunotherapy of chronic lung diseases.

	Acknowledgments

 
We thank Drs. Donna B. Stolz and Simon C. Watkins for assistance with scanning electron microscopy.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants HL 60207 (to P.R.) and HL 77430 (to A.R.) from the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Prabir Ray, Department of Medicine, Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, 3459 Fifth Avenue, MUH 628 NW, Pittsburgh, PA 15213. E-mail address: rayp{at}pitt.edu

³ Abbreviations used in this paper: DC, dendritic cell; alum, aluminum hydroxide; cRPMI, complete RPMI; CT, cholera toxin; GUS, -glucuronidase; IDO, indoleamine 2,3-dioxygenese; i.n., intranasal; LN, lymph node; pDC, plasmacytoid DC; mDC, myeloid DC; ODN, oligodeoxynucleotide; TRIF, Toll/IL-1 receptor domain-containing adapter-inducing IFN-; wt, wild type.

Received for publication November 17, 2005. Accepted for publication May 26, 2006.

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