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Toward Homeostasis

Regulatory Dendritic Cells from the Bone Marrow of Mice with Inflammation of the Airways and Peritoneal Cavity
      Inflammatory mediators from peripheral tissues may control dendritic cell (DC) development in the bone marrow. In this study, DCs (CD11c+ cells) differentiated from the bone marrow of mice with inflammation of the airways, or the peritoneal cavity had poor priming ability resulting in reduced, long-lived responses to that antigen in vivo. This indicates enhancement of regulatory mechanisms of immune responses through a peripheral tissue-bone marrow axis. If CD11c+ cells, expanded from the bone marrow of mice with tissue inflammation were antigen pre-loaded and injected into mice already sensitized to that antigen, then subsequent contact hypersensitivity responses were significantly reduced. The effects of inflammation were imprinted in vivo and were independent of in vitro culture conditions for DC differentiation. The effect of tissue inflammation on the bone marrow DC precursors was not detected in mice treated subcutaneously with slow-release indomethacin pellets, suggesting a role for prostanoids, including prostaglandin E2, in differentiation of regulatory CD11c+ cells from bone marrow. Our study represents an important homeostatic process with potential for therapeutic use in the future.
      Dendritic cells (DCs) from the bone marrow continuously replace DCs in the spleen, lymph nodes, and other tissues, where their half-life is approximately 2 to 3 days. DCs are heterogeneous, and their differentiation proceeds from myeloid- and lymphoid-committed progenitors within the bone marrow.
      • Wu L.
      • Liu Y.-J.
      Development of dendritic-cell lineages.
      • Shortman K.
      • Naik S.H.
      Steady-state and inflammatory dendritic-cell development.
      • Liu K.
      • Nussenzweig M.C.
      Origin and development of dendritic cells.
      • Kushwah R.
      • Hu J.
      Complexity of dendritic cell subsets and their function in the host immune system.
      Lineage development of different subsets of DCs in the bone marrow and spleen is influenced by factors in the local microenvironment, including cytokines, transcription factors, and signals from stromal cells. During inflammation, there is a greater demand for differentiation of hemopoietic progenitor cells into fully mature blood cells.
      • King K.Y.
      • Goodell M.A.
      Inflammatory modulation of HSCs: viewing the HSC as a foundation for the immune response.
      It is proposed that self-tolerance with steady-state conditions is maintained by immature DCs, which typically present low numbers of self-peptide major histocompatibility complexes, express minimal co-stimulatory molecules, produce reduced amounts of pro-inflammatory cytokines, such as IL-12 (balanced by increased amounts of the regulatory cytokine, IL-10), and cause T-cell anergy, apoptosis, and induction of regulatory cells. With other conditions, pro-inflammatory cytokines can activate DCs, and as it may be relevant during inflammation, these DCs become tolerogenic and unable to induce effector T-cell responses.
      • Joffre O.
      • Nolte M.A.
      • Sporri R.
      • Reis e Sousa C.
      Inflammatory signals in dendritic cell activation and the induction of adaptive immunity.
      There have been several reports that stromal cells (ie, fibroblasts, endothelial cells, stromal macrophages) can play an active role in supporting the differentiation of bone marrow hemopoietic stem and progenitor cells into IL-10–producing tolerogenic DCs.
      • Svensson M.
      • Maroof A.
      • Ato M.
      • Kaye P.M.
      Stromal cells direct local differentiation of regulatory dendritic cells.
      • Zhang M.
      • Tang H.
      • Guo Z.
      • An H.
      • Zhu X.
      • Song W.
      • Guo J.
      • Huang X.
      • Chen T.
      • Wang J.
      • Cao X.
      Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells.
      We have previously reported that exposure of the shaved dorsal skin of mice to erythemal ultraviolet (UV) radiation affects DC precursors in the bone marrow, such that when bone marrow cells from these mice are expanded in vitro with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4, the CD11c+ cells that develop have poor antigen priming ability.
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      The poor priming ability was still demonstrated after maturation by exposure to lipopolysaccharide (LPS).
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      In the study reported here, we questioned whether the production of poorly priming DCs from the bone marrow was a consequence of skin inflammation per se, or could be detected in response to inflammatory processes in other tissues. In addition, the culture conditions for the bone marrow cells were addressed as different growth factors would stimulate the differentiation of various DC precursor populations.
      • Xu Y.
      • Zhan Y.
      • Lew A.M.
      • Naik S.H.
      • Kershaw M.H.
      Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.
      • Gilliet M.
      • Boonstra A.
      • Paturel C.
      • Antonenko S.
      • Xu X.L.
      • Trinchieri G.
      • O'Garra A.
      • Liu Y.J.
      The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor.
      Bone marrow cells were cultured in GM-CSF (+ IL-4) to reflect the increased serum GM-CSF in mice with tissue inflammation. Alternatively, DC precursors were differentiated in medium with the hemopoietic growth factor FLT3 ligand (FLT3-L). Finally, we investigated the potential regulatory capacity of the bone marrow-derived CD11c+ cells from mice with tissue inflammation; in these studies, the control of established memory responses in antigen-presensitized mice were investigated. The results indicate that prostanoids from inflammatory tissues affect early DC precursors in the bone marrow, such that when differentiated in GM-CSF or FLT3-L, they are not only poor at inducing antigen-specific immune responses, but importantly they can direct reduced recall immune responses in mice already sensitized to the challenge antigen.

      Materials and Methods

      Mice

      Female BALB/c mice were obtained from the Animal Resources Centre (Murdoch, Western Australia) and were used when they were 6 to 10 weeks of age. All experiments were performed with the approval of the Telethon Institute for Child Health Research Animal Ethics Committee, according to the guidelines of the National Health and Medical Research Council of Australia.

      Induction of Experimental Allergic Airways Disease and Alum-Induced Peritoneal Inflammation

      For the induction of experimental allergic airways disease (EAAD), ovalbumin (OVA) (Sigma-Aldrich, St. Louis, MO) in alum (aluminum hydroxide suspension, Serva, Heidelberg, Germany) was injected on day 0 (10 μg OVA with 2 mg alum in 200 μL saline i.p. per mouse) and again on day 14.
      • McGlade J.P.
      • Gorman S.
      • Zosky G.R.
      • Larcombe A.N.
      • Sly P.D.
      • Finlay-Jones J.J.
      • Turner D.J.
      • Hart P.H.
      Suppression of the asthmatic phenotype by UVB-induced, antigen-specific regulatory cells.
      • McGlade J.P.
      • Strickland D.H.
      • Lambert M.J.M.
      • Gorman S.
      • Thomas J.A.
      • Judge M.A.
      • Burchell J.T.
      • Zosky G.R.
      • Hart P.H.
      UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells.
      Mice were then challenged on day 21 with a 1% OVA-in-saline aerosol that was administered using an ultrasonic nebulizer (UltraNeb, DeVibiss, Somerset, PA) for 30 minutes. Twenty-four hours later, mice were euthanized, and the bone marrow was harvested.
      For induction of alum-induced peritoneal inflammation, the same suspension of alum (as described in the model of EAAD) was used. Alum was administered on day 0 (2 mg alum in 200 μL saline i.p. per mouse). Control mice (-alum PI) were injected with 200 μL saline i.p. Three days later, the mice were euthanized and bone marrow harvested.

      Isolation and Culture of Bone Marrow Cells

      As previously reported,
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      tibias and femurs of mice were removed and flushed using a glucose-potassium-sodium buffer containing 10% foetal calf serum (SAFC Biosciences, Brooklyn, Australia). Disaggregated bone marrow was passed through cotton wool to remove bone debris and cultured in RPMI 1640 medium (Hyclone, Thermo Scientific, Waltham, MA) containing 10% foetal calf serum, 2 mmol/L L-glutamine (Sigma-Aldrich), 50 μmol/L 2-mercaptoethanol, and 5 μg/mL gentamicin (Sigma-Aldrich) (RPMI 10) at a density of 8 × 105 cells/mL in 24-well plates. Cells were cultured in the presence of 10 ng/mL GM-CSF (PeproTech, Inc, Rocky Hill, NJ), with or without 10 ng/mL IL-4 (PeproTech) for 7 days to promote CD11c+ cell differentiation. The GM-CSF and IL-4 supplemented medium was replaced on days 2 and 4. Alternatively, bone marrow cells were cultured for 9 days without disturbance with 100 ng/mL FLT3-L (PeproTech). The addition of indomethacin (10 μmol/L) (Sigma-Aldrich) throughout the culture was also investigated. At the end of culture, nonadherent cells were harvested, and unless otherwise stated, they were enriched to >95% CD11c+ cells (confirmed by flow cytometry), using anti-CD11c magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and AutoMACS (Miltenyi Biotec) separation.
      Aliquots of freshly isolated bone marrow cells were also incubated for 24 hours with 1 μg/mL LPS (Sigma-Aldrich). In addition, 24 hours before the end of the 7- or 9-day cultures, portions of the bone marrow cells were re-plated at 106 cells/mL and incubated with 1 μg/mL LPS, 5 μg/mL CpG (InvivoGen, San Diego, CA), or 5 μg/mL negative control CpG (InvivoGen) for 24 hours. Supernatants were collected for cytokine measurement, and the cells were analyzed by flow cytometry.

      Measurement of IL-10, IL-12, IL-23, and TNF-α

      IL-10, IL-12, IL-23, and tumor necrosis factor-α (TNF-α) levels in the culture supernatants were determined using a capture enzyme-linked immunosorbent assay with a europium detection label (Wallac OY, Turku, Finland) (sensitivity, 50 pg/mL IL-10; 20 pg/mL IL-12; 6 pg/mL IL-23; and 4 pg/mL TNFα; BD Biosciences, San Jose, CA).

      Characterization of Bone Marrow-Derived Cells by Surface Marker Expression

      Freshly isolated bone marrow cells were classified as myeloid (Gr1+CD11b+), lymphoid (B220+CD11b), and erythroid (Ter119+CD11b) populations
      • O'Connell R.M.
      • Rao D.S.
      • Chaudhuri A.A.
      • Boldin M.P.
      • Taganov K.D.
      • Nicoll J.
      • Paquette R.L.
      • Baltimore D.
      Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder.
      after staining with biotinylated anti-Gr1 (eBioscience, San Diego, CA), fluorescein isothiocyanate-anti-Ter119 (eBioscience), phycoerythrin-anti-CD11b (BD Pharmingen), and allophycocyanin-anti-B220 (Biolegend, San Diego, CA). To prevent nonspecific antibody binding, cells were pre-incubated for 5 minutes with anti-CD16/CD32 Fc receptor antibody (BD Pharmingen). Cells were incubated with antibodies or appropriate isotype controls for 30 minutes before washing and incubation with streptavidin-phycoerythrin-Cy5 for 30 minutes (BD Pharmingen) as required. All incubations and cell washes were performed at 4°C. Data were collected on a LSRII or FACSCalibur flow cytometer (BD Biosciences). FlowJo software (Treestar, version 9.2, Ashland, OR) was used for the flow cytometric analysis.
      Bone marrow cells that were differentiated in culture with the different growth factors and stimulated with LPS for the last 24 hours were analyzed after incubation with phycoerythrin-anti-CD11c (Biolegend), fluorescein isothiocyanate-anti-I-A/I-E (mouse major histocompatibility complex class II; BD Pharmingen), biotinylated anti-CD80, biotinylated anti-CD86, biotinylated anti-B220, allophycocyanin-Cy7-anti-CD11b, allophycocyanin-anti-CD4 (BD Pharmingen), or fluorescein isothiocyanate-anti-CD8 (eBioscience).

      Adoptive Transfer of Bone Marrow CD11c+ Cells into the Ears of Mice and Contact Hypersensitivity Assay

      After culturing the bone marrow cells for 7 days with GM-CSF + IL-4, enriched CD11c+ cells at 106 cells/mL in RPMI 10 were pulsed with 1 mmol/L 2,4-dinitrobenzene sulfonic acid sodium salt (DNBS) (MP Biomedicals LLC, Solon, OH) for 30 minutes at 37°C. CD11c+ cells were washed and resuspended to 5 × 107 cells/mL in 0.9% saline. CD11c+ cells (106 cells in 20 μL) were injected into the ear pinnae of naïve BALB/c mice (n = 8 ears per group). The ears of additional mice were injected with 20 μL 0.9% saline. In a previous study,
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      injection of non-antigen-loaded CD11c+ cells had the same immunological effect as the injection of saline alone. For cells cultured for 9 days with FLT3-L, as the yields of the bone marrow-derived cells were lower, they were not enriched according to CD11c+ expression before pulsing with DNBS and injection into the ears. After 7 days, each side of the ears was painted with 10 μL 0.2% 2,4-dinitro-1-fluorobenzene (DNFB) (Sigma-Aldrich) in acetone; DNFB is the nonwater soluble form of DNBS, but with the same antigenic moiety as DNBS. Ear thicknesses were determined before and 24, 30, and 48 hours after challenge with DNFB, using a spring-loaded micrometer (Mitutoyo, Aurora, IL). The contact hypersensitivity (CHS) response (increased ear thickness associated with swelling and cell influx) was determined by subtracting the ear thicknesses before painting. The mean swelling of ears injected with 20 μL saline (volume of cell injection) was subtracted from values for swelling of ears injected with CD11c+ cells (all ears were challenged with DNFB 7 days after saline or cell injection). For estimation of differences in ear swelling in multiple experiments (Figure 1C, and 2C), the swelling of ears injected with DNBS-pulsed CD11c+ cells from control mice (-EAAD, -alum PI) was normalized as 100% in each experiment. When the ear swelling had subsided (at least 2 weeks after painting with DNFB), the ears of mice were repainted with 0.2% DNFB or 1% 2,4,6-trinitrochlorobenzene (TNCB) dissolved in acetone, and the ear swelling was measured after 24, 30, and 48 hours.
      Figure thumbnail gr1
      Figure 1CD11c+ cells with poor antigen priming ability differentiated from the bone marrow of mice with experimental allergic airways disease (EAAD). A: Expression of the cell surface markers Gr1, CD11b, B220, and Ter119 by bone marrow cells isolated from mice ± EAAD. B: Increased levels of IL-10 in the supernatant of bone marrow cells isolated from mice with EAAD after stimulation for 24 hours with 1 μg/mL LPS. C: Reduced priming ability of CD11c+ cells expanded from the bone marrow of mice with EAAD. Open bars represent cells from mice without EAAD, or ears injected with cells from mice without EAAD (normalized to 100% for each experiment). Solid bars represent cells from mice with EAAD, or ears injected with CD11c+ cells from mice with EAAD. Data for (A–C) represent mean + SEM of 5 independent experiments, *P < 0.05. D: Antigen-specific, long-lasting effect. For one of the experiments in C, ear swelling was measured 24, 30 and 48 hours after DNFB ear painting (challenge 1). After the ear swelling had subsided (2 to 3 weeks), the ears were painted again with either 0.2% DNFB (Sigma-Aldrich) [challenge 2 – DNFB (Sigma-Aldrich)] or 1% TNCB (challenge 2 – TNCB). The ear swelling was measured during the next 48 hours. As a control, swelling is shown for ears injected with saline instead of DNBS-loaded cells before challenge with DNFB (challenges 1 and 2) or TNCB (challenge 2). Data represent ear swelling ± SEM for 8 ears/group. The −EAAD and +EAAD are for ears injected with CD11c+ cells differentiated from the bone marrow of mice without or with EAAD, respectively; Saline shows the swelling of ears injected with saline (no cells), but still hapten challenged. *P < 0.05 for comparison between ears injected with bone marrow-derived CD11c+ cells from mice ± EAAD.
      Figure thumbnail gr2
      Figure 2CD11c+ cells with poor antigen priming ability differentiated from the bone marrow of mice with alum-induced peritoneal inflammation (alum PI). A: Expression of the cell surface markers Gr1, CD11b, B220, and Ter119 by bone marrow cells isolated from mice ± alum PI. B: Increased IL-10 in the supernatant of bone marrow cells isolated from mice with alum PI after stimulation for 24 hours with 1 μg/mL lipopolysaccharide (LPS). C: Reduced priming ability of CD11c+ cells expanded from the bone marrow of mice with and without alum PI, measured 24 hours after 2,4-dinitro-1-fluorobenzene (DNFB) (Sigma-Aldrich) application. AC: Data represent mean + SEM of four independent experiments. Open bars represent cells from mice without alum PI, or ears/mice injected with CD11c+ cells from mice without alum PI (normalized to 100% in C). Hatched bars represent bone marrow cells from mice with alum PI, or ears injected with CD11c+ cells from mice with alum PI. *P < 0.05. D: Long-lasting reduced response. For one of the experiments in C, ear swelling was measured 24, 30, and 48 hours after DNFB ear painting (challenge 1). As a control, swelling is shown for ears injected with saline instead of cells before challenge with DNFB. After the ear swelling had subsided (2 to 3 weeks), the ears were painted again with 0.2% DNFB (challenge 2). The ear swelling was measured during the next 48 hours. Data represent ear swelling ± SEM for 8 ears/group. The -alum PI and + alum PI are for the ears of mice injected with CD11c+ cells differentiated from the bone marrow of mice ± alum PI. Saline shows the swelling of ears injected with saline (no cells), but still hapten challenged. *P < 0.05 for comparison between ears injected with bone marrow-derived CD11c+ cells from mice ± alum PI.
      For primary and secondary CHS responses in the same mice, DNBS-loaded CD11c+ cells were injected into the left ears and a CHS response was measured during the subsequent 48 hours. After 2 weeks, the mice were sensitized on their shaved abdomen with 50 μL of 0.2% DNFB. Seven days later, the right ears were painted with DNFB in acetone. Ear thicknesses were again determined before and then 24, 30, and 48 hours after challenge with DNFB.
      In some experiments, the DNBS-loaded CD11c+ cells were injected into the ears of mice that had been presensitized to DNFB 7 days previously (50 μL of 0.2% DNFB applied to a 2 cm2 shaved section of abdomen). CD11c+ cells (106 cells in 20 μL) were injected into the right ear of the presensitized mice and 20 μL saline was injected into the left ear. After a further 7 days, a CHS assay was conducted with each side of the ears painted with 10 μL 0.2% DNFB.

      Loading of Bone Marrow-Derived Cells with OVA Alexa488 and Their Tracking to Auricular Lymph Nodes

      Bone marrow-derived cells harvested after 7 days in culture with GM-CSF + IL-4 were incubated at 106 cells/mL for 2 hours at 37°C with OVA Alexa Fluor488 (Invitrogen, Carlsbad, CA) at 1 μg/mL. After washing, an aliquot of the cells was incubated for 5 minutes with anti-CD16/CD32 Fc receptor antibody (BD Pharmingen) before addition of phycoerythrin-anti-CD11c (Biolegend) for 30 minutes at 4°C, followed by flow cytometry to determine the extent of loading. The loaded cells (2 × 106 cells in 20 μL saline) were injected into the ear pinnae of naïve BALB/c mice, as previously described, and the number in the auricular lymph nodes was determined 18 hours later by flow cytometry.

      Indomethacin Administration

      Indomethacin pellets containing 0.05 mg, with a constant release of 2.38 μg/day for 21 days (Innovative Research of America, Sarasota, FL) were subcutaneously implanted into the upper dorsum of mice 4 days before alum injection. As a control, mice were subcutaneously implanted with placebo pellets.

      Real-Time PCR Analysis of CD11c+ Cells Generated in Culture

      After enrichment using anti-CD11c magnetic microbeads, CD11c+ cells (>106 cells) harvested from bone marrow cells in culture were snap-frozen in 350 μL Buffer RLT Plus (Qiagen, Doncaster, Australia). RNA was extracted using the RNAeasy Plus Mini Kit (Qiagen), according to the manufacturer's instructions. Complementary DNA was reverse transcribed from the RNA samples using the QuantiTect Reverse Transcription Kit (Qiagen). Using primers designed in-house, a real-time polymerase chain reaction was then performed as previously described
      • Gorman S.
      • Judge M.A.
      • Hart P.H.
      Gene regulation by 1,25-dihydroxyvitamin D3 in CD4+CD25+ cells is enabled by IL-2.
      using 2x RT2 SYBR Green ROX qPCR MasterMix (Qiagen). The polymerase chain reaction was performed using the standard two-step cycling conditions on the ABI 7900HT SDS (Applied Biosystems, Foster City, CA). Melting curve analysis was used to assess the specificity of the assay. Fold change was determined by using the 2−ΔΔCt method,
      • Gorman S.
      • Judge M.A.
      • Hart P.H.
      Gene regulation by 1,25-dihydroxyvitamin D3 in CD4+CD25+ cells is enabled by IL-2.
      with reference gene eukaryotic translation elongation factor 1α. Primer pairs were as follows: eukaryotic translation elongation factor 1α, 5′-CTGGAGCCAAGTGCTAATATGCC-3′ and 5′-GCCAGGCTTGAGAACACCAGTC-3′; indoleamine dioxygenase (IDO), 5′-AGGCTGGCAAAGAATCTCCT-3′ and 5′-AATGACAAACTCACGGACTGG-3′; cyclooxygenase 2 (COX2), 5′-GGCCATGGAGTGGACTTAAA-3′ and 5′-ACCTCTCCACCAATGACCTG-3′; nitric oxide synthase 2, 5′-ATGTGACATCGACCCGTCCACA-3′ and 5′-TGGACCCCAAGCAAGACTTGGA-3′; and IL-10, 5′-GGTTGCCAAGCCTTATCGGA-3′ and 5′-ACCTGCTCCACTGCCTTGCT-3′.

      Statistical Analysis

      Statistical analysis was performed using the Student's t-test, with Graphpad Prism for Mac (version 5; Graphpad Software, La Jolla, CA). Differences were considered statistically significant when P < 0.05. For comparison of data from n independent experiments, the mean from each experiment of >3 mice/cell suspensions/supernatants per group was used to calculate the mean ± SEM for n experiments.

      Results

      Myelopoiesis in the Bone Marrow of Mice with Experimental Allergic Airways Disease

      Mice were sensitized to OVA-in-alum on days 0 and 14, and were then challenged with aerosolized OVA on day 21. Bone marrow was harvested 24 hours later on day 22 when increased inflammatory cells have been measured in the airways, airway draining lymph nodes, and bronchoalveolar lavage fluid.
      • McGlade J.P.
      • Gorman S.
      • Zosky G.R.
      • Larcombe A.N.
      • Sly P.D.
      • Finlay-Jones J.J.
      • Turner D.J.
      • Hart P.H.
      Suppression of the asthmatic phenotype by UVB-induced, antigen-specific regulatory cells.
      • McGlade J.P.
      • Strickland D.H.
      • Lambert M.J.M.
      • Gorman S.
      • Thomas J.A.
      • Judge M.A.
      • Burchell J.T.
      • Zosky G.R.
      • Hart P.H.
      UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells.
      At this time point, serum OVA-specific IgE levels are significantly increased.
      • McGlade J.P.
      • Gorman S.
      • Zosky G.R.
      • Larcombe A.N.
      • Sly P.D.
      • Finlay-Jones J.J.
      • Turner D.J.
      • Hart P.H.
      Suppression of the asthmatic phenotype by UVB-induced, antigen-specific regulatory cells.
      • McGlade J.P.
      • Strickland D.H.
      • Lambert M.J.M.
      • Gorman S.
      • Thomas J.A.
      • Judge M.A.
      • Burchell J.T.
      • Zosky G.R.
      • Hart P.H.
      UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells.
      Yields of cells from the bone marrow were not significantly altered by inflammation in the airways (+EAAD, 51.9 + 0.9 × 106/mouse, mean + SEM, n = 5 independent experiments; −EAAD, 51.4 + 4.2 × 106/mouse). There were significantly more Gr1+CD11b+ myeloid cells in the bone marrow of mice with EAAD than in control mice without EAAD (Figure 1A); although trends, there were no significant decreases in the percentage of B220+CD11b lymphoid and Ter119+CD11b erythroid cells. When freshly isolated bone marrow cells were incubated with 1 μg/mL LPS for 24 hours, those from mice with EAAD secreted significantly greater levels of IL-10, a cytokine generally associated with homeostatic regulation
      • de Waal Malefyt R.
      • Yssel H.
      • Roncarolo M.G.
      • Spits H.
      • de Vries J.E.
      Interleukin-10.
      (n = 4 independent experiments, Figure 1B), but this may be due to IL-10 production by the increased number of Gr1+CD11b+ myeloid cells (Figure 1A).

      CD11c+ Cells Differentiated from the Bone Marrow of Mice with EAAD Have Reduced Priming Ability

      Cells generated by bone marrow cell culture were enriched for CD11c expression, loaded with DNBS for 30 minutes and washed. The ears of naïve mice injected with DNBS-loaded CD11c+ cells cultured from bone marrow of mice with EAAD had a reduced CHS response (Figure 1C). The swelling of the ears of mice receiving CD11c+ cells from the bone marrow of mice with inflammation in the airways was reduced by 58 ± 17% (mean + SEM for five independent experiments).
      To determine whether bone marrow-derived CD11c+ cells from mice with EAAD directed long-lasting, antigen-specific reduced responses, ear swelling from the initial CHS response with 0.2% DNFB (challenge 1) (Figure 1D) was allowed to abate for at least 2 weeks. The ears of every second mouse were then repainted with 0.2% DNFB (challenge 2, DNFB) (Figure 1D), and ear swelling was measured during the next 48 hours. There was an increased inflammatory response to DNFB (swelling) in the ears on their re-exposure. However, the ears of mice that had been injected with bone marrow-derived CD11c+ cells from mice with EAAD had a significantly reduced CHS response at challenge 2, compared with the ears of mice injected with bone marrow-derived CD11c+ cells from mice without EAAD (challenge 2, DNFB) (Figure 1D). Saline injection into the ears of naïve mice at the time of CD11c+ cell injection provided a measure of the nonspecific effect of injection of 20 μL saline; the ears of these mice were challenged with DNFB at both challenges 1 and 2. In this experiment (Figure 1D), the cells differentiated from the bone marrow of mice with EAAD were very poor at antigen priming and there was a minimal difference measured in thickness between ears injected with DNBS-loaded CD11c+ cells or saline. To investigate whether the CHS response was antigen-specific, the ears of the remaining mice from challenge 1 were painted with 1% TNCB (challenge 2, TNCB) (Figure 1D), and ear swelling measured during the next 48 hours. The swelling associated with the CHS response was similar in all groups after the challenge with this unrelated antigen and equal to that of mice injected with saline, and it was suggested that the reduced responses detected after injection of DNBS-loaded CD11c+ cells were antigen-specific.

      CD11c+ Cells Differentiated from the Bone Marrow of Mice with Alum-Induced Peritoneal Inflammation Are Similar in Function to Those from Mice with Respiratory Inflammation

      In a different inflammatory model, mice were injected with alum alone and bone marrow harvested 3 days later. Yields of bone marrow cells were not significantly altered by alum injection (+alum PI, 52.1 + 3.9 × 106/mouse, mean + SEM, n = 6 independent experiments; -alum PI, 52.1 + 4.0 × 106/mouse). There were significantly more Gr1+CD11b+ myeloid cells in the bone marrow, which reflects an inflammatory response in the peritoneum (Figure 2A). There was a significant compensatory decrease in B220+CD11b lymphoid cells. When freshly isolated bone marrow cells were incubated with 1 μg/mL LPS for 24 hours, those from mice with alum PI secreted significantly greater levels of IL-10 (n = 4 independent experiments) (Figure 2B).
      Using the methodology previously described for mice with or without EAAD, the naïve mice that had been injected with CD11c+ cells differentiated from the bone marrow of mice with alum-PI had a reduced CHS response (Figure 2C). For four independent experiments, the swelling of the ears of mice receiving CD11c+ cells generated from the bone marrow of mice with alum PI was reduced by 65 ± 9% (mean + SEM) (Figure 2C). Maintenance of a reduced response to DNFB was confirmed 3 weeks later by a re-challenge of the ears with 0.2% DNFB (Figure 2D).

      CD11c+ Cells Differentiated from the Bone Marrow of “Inflammatory” Mice Are Not Altered in Antigen Uptake or Migration to Draining Lymph Nodes

      Poor priming ability may be a reflection of reduced antigen uptake by CD11c+ cells or fewer cells migrating to the draining lymph nodes. To investigate this question, cells differentiated with GM-CSF + IL-4 from the bone marrow of mice with or without alum PI (but not CD11c+-cell enriched) were incubated with OVA Alexa488 for 2 hours at 37°C before their injection into the ears of naïve mice; we have previously reported negligible OVA Alexa488 uptake at 4°C.
      • McGlade J.P.
      • Strickland D.H.
      • Lambert M.J.M.
      • Gorman S.
      • Thomas J.A.
      • Judge M.A.
      • Burchell J.T.
      • Zosky G.R.
      • Hart P.H.
      UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells.
      The in vitro uptake of OVA Alexa488 was predominantly by CD11c+ cells and did not differ for cells from the bone marrow of mice with or without alum PI (Figure 3, A and B). Eighteen hours after injection of the labeled cells into the ears, the auricular lymph nodes were disaggregated and the CD11c+ cells containing OVA Alexa488 were enumerated. This provided a measure of the trafficking of antigen-loaded CD11c+ cells under steady-state conditions into the draining lymph nodes. The CD11c+ cells in the auricular lymph nodes comprised approximately 1% of the total cells. Of these, the percentage containing OVA Alexa488 was not different between the mice receiving cells from the bone marrow of mice with alum PI (2.21 + 1.35%, mean + SEM, n = 3 independent experiments) or without (1.75 + 0.83%) and was greater than the nonspecific staining measured in CD11c+ cells in the nodes of mice injected with saline instead of cells (0.69 + 0.28%) (Figure 3C).
      Figure thumbnail gr3
      Figure 3Antigen uptake and migration to the auricular lymph nodes are not altered in CD11c+ cells differentiated from the bone marrow of mice with alum-induced peritoneal inflammation (alum PI). A: Cells before, and post-ovalbumin (OVA)-Alexa488 (B) loading in vitro. Bone marrow-derived cells that take up OVA Alexa488 are predominantly CD11c+ cells, with similar uptake by cells differentiated from the bone marrow of mice with and without alum PI. C: OVA Alexa488-containing CD11c+ cells in the auricular lymph nodes 18 hours after injection. Bone marrow cells from mice without (−alum PI) or with PI (+alum PI) were differentiated for 7 days with granulocyte-macrophage colony stimulating factor (GM-CSF) + IL-4 before incubation (without enrichment for CD11c+ cells) for 2 hours with OVA Alexa.488 The ears of naïve mice were injected with saline or 2 × 106 of these cells. After 18 hours, auricular lymph nodes were harvested, CD11c+ cells gated and OVA Alexa488 measured in these cells. Representative plots of OVA Alexa488-containing cells are shown.

      IL-4 Reduces the I-A/I-E and CD86 Expression on CD11c+ Cells Differentiated from Bone Marrow but Does Not Alter the Immunogenic Properties of CD11c+ Cells Differentiated from Bone Marrow of Mice with EAAD

      Although IL-4 is important for the differentiation of human DCs from blood monocytes, there has been variable use of IL-4 in the culture medium for differentiation of DCs from murine bone marrow.
      • Xu Y.
      • Zhan Y.
      • Lew A.M.
      • Naik S.H.
      • Kershaw M.H.
      Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.
      IL-4 has anti-inflammatory actions on human monocytes and other myeloid cells, and can reduce the expression of major histocompatibility complex class II and co-stimulatory molecules.
      • Hart P.H.
      • Vitti G.F.
      • Burgess D.R.
      • Whitty G.A.
      • Piccoli D.S.
      • Hamilton J.A.
      Potential anti-inflammatory effects of interleukin-4 Suppression of human monocyte TNFα, IL-1 and PGE2 levels.
      • Hart P.H.
      • Bonder C.S.
      • Jones C.A.
      • Finlay-Jones J.J.
      Control of major histocompatibility complex class II expression on human monocytes by interleukin-4: regulatory effect of lipopolysaccharide.
      Thus, IL-4 may reduce the antigen-presenting ability of DCs during culture. To test this hypothesis, bone marrow cells from mice (+EAAD) were cultured in the presence of GM-CSF, with or without IL-4. Within each culture condition, there was no difference in the expression of CD11c (Figure 4A and B), or I-A/I-E, CD80, and CD86 on CD11c+ cells from mice ± EAAD (Figure 4C). However, inclusion of IL-4 in the culture medium altered the phenotype of the harvested cells (±EAAD). A small population of CD11clo cells were harvested (Figure 4A), but their function was not examined as priming was only examined on cells enriched for CD11c expression (Figure 4, B, F, and G). With IL-4, the cell surface expression of I-A/I-E and CD86 on CD11c+ cells was reduced (Figure 4, D and E). However, the lower levels of I-A/I-E and CD86 on cells exposed to IL-4 did not alter the significantly reduced priming ability of the CD11c+ cells that differentiated from the bone marrow of mice with EAAD (Figure 4, F and G). Thus, differences in co-stimulatory molecule expression were not responsible for the less immunogenic, more regulatory properties of the CD11c+ cells differentiated from the bone marrow of the “inflammatory” mice.
      Figure thumbnail gr4
      Figure 4Addition of IL-4 does not alter the in vitro development of poorly priming CD11c+ cells from the bone marrow of mice with experimental allergic airways disease (EAAD). Cells were harvested from 7-day bone marrow cell cultures containing granulocyte-macrophage colony stimulating factor (GM-CSF), with or without IL-4. A: CD11c+ expression on harvested cells pre-CD11c cell enrichment (±EAAD). B: Post-CD11c+ cell enrichment (±EAAD). CE: Expression of I-A/I-E, CD80 and CD86 on CD11c+ cells (±EAAD). C: Cells from mice without EAAD are shown by the shaded area, the nonshaded area indicates cells from mice with EAAD. D and E: Inclusion of IL-4 (solid black line) resulted in less expression by CD11c+ cells of I-A/I-E and CD86 than measured in the absence of IL-4 (gray dashed line). F and G: Bone marrow cells were cultured for 7 days with GM-CSF, with or without IL-4, before CD11c+ cells were purified, pulsed with 1 mmol/L 2,4-dinitro-1-fluorobenzene (DNFB) (Sigma-Aldrich) for 30 minutes, and 106 cells injected subcutaneously into the ears of naïve mice. After 7 days, the ears of the mice were painted with 0.2% DNFB and increased ear swelling was measured after 24, 30, and 48 hours. As a control, swelling is shown for ears injected with saline instead of cells before challenge with DNFB. After ear swelling had subsided, the ears were painted again with 0.2% DNFB, and the ear swelling measured during the next 48 hours. Data represent mean ear swelling ± SEM for 8 ears/group. The -EAAD and +EAAD are for the ears of mice injected with CD11c+ cells differentiated from bone marrow of mice ± EAAD. Saline shows swelling of the ears of mice injected with saline (no cells), but still hapten challenged. *P < 0.05 for comparison between ears injected with CD11c+ cells differentiated from the bone marrow of mice ± EAAD.

      Differentiation with GM-CSF+IL-4 or FLT3-L Does Not Alter the Poor Priming Ability of CD11c+ Cells from the Bone Marrow of Mice with Peritoneal Inflammation

      Under homeostatic conditions, FLT3-L is the major growth factor for DC precursors, in contrast to GM-CSF, which stimulates differentiation of DC precursors when inflammatory conditions exist.
      • Wu L.
      • Liu Y.-J.
      Development of dendritic-cell lineages.
      • Shortman K.
      • Naik S.H.
      Steady-state and inflammatory dendritic-cell development.
      • Liu K.
      • Nussenzweig M.C.
      Origin and development of dendritic cells.
      • Kushwah R.
      • Hu J.
      Complexity of dendritic cell subsets and their function in the host immune system.
      Parallel bone marrow cell cultures from mice with or without alum PI were established with FLT3-L (100 ng/mL) as the sole growth factor to expand the DC precursors (9 day culture, no interference
      • Xu Y.
      • Zhan Y.
      • Lew A.M.
      • Naik S.H.
      • Kershaw M.H.
      Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.
      ), and cultures in which the cells were incubated for 7 days with GM-CSF + IL-4 and cytokine replacement on days 2 and 4.
      A higher number of cells (by eightfold) was harvested from cultures of bone marrow cells incubated with GM-CSF + IL-4 than with FLT3-L but the percentage of cells that were CD11c+ was lower (Figure 5A). There were no differences in the cells developing from the bone marrow of mice with or without alum PI. However, culture in the presence of FLT3-L stimulated the development of 10% to 13% CD11c+ cells that were CD4 CD8+ (Figure 5B). The CD11c+ cells from culture in GM-CSF + IL-4 were CD4CD8. The CD11c+ cells cultured with FLT3-L were 9% to 13% CD11bB220+ (ie, likely plasmacytoid DCs),
      • Gilliet M.
      • Boonstra A.
      • Paturel C.
      • Antonenko S.
      • Xu X.L.
      • Trinchieri G.
      • O'Garra A.
      • Liu Y.J.
      The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor.
      • Brawand P.
      • Fitzpatrick D.R.
      • Greenfield B.W.
      • Brasel K.
      • Maliszewski C.T.
      • DeSmedt T.
      Murine plasmacytoid pre-dendritic cells generated from Flt3 Ligand-supplemented bone marrow cultures are immature APCs.
      compared with 1% to 3% for cells cultured with GM-CSF + IL-4 (Figure 5C). Cells that were cultured with FLT3-L were all visually smaller, and produced less TNFα, IL-23, and IL-12 (but similar IL-10) in response to LPS than those expanded in GM-CSF + IL-4 (Table 1). Cells that were cultured with FLT3-L produced more IL-10, TNFα, IL-23, and IL-12 in response to the Toll-like Receptor 9 ligand, CpG, compared to those cultured with GM-CSF + IL-4 (Table 1), and suggests that FLT3-L induced Toll-like receptor 9 expression on at least some of the cells. The levels of LPS-induced cytokines, did not vary significantly if bone marrow was taken from mice with or without alum PI (n = 5 independent experiments). For each culture condition, alum PI did not alter the surface expression of CD86 on CD11c+ cells (Figure 5D). The CD11c+ cells differentiated from bone marrow with GM-CSF + IL-4 had a higher expression of CD86 before treatment with LPS compared to bone marrow cells cultured with FLT3-L (Figure 5D).
      Figure thumbnail gr5
      Figure 5CD11c+ cells with poor priming ability develop from the bone marrow of mice with alum-induced peritoneal inflammation (alum PI) after culture with granulocyte-macrophage colony stimulating factor (GM-CSF) + IL-4 or with FLT3 ligand (FLT3-L). A: Surface expression of CD11c on cells from the bone marrow cultures. The surface expression of CD4 and CD8 (B), and CD11b and B220 on the CD11c+ cells (C) (gated as shown in A). D: The surface expression of CD86 on CD11c+ cells after culture for 24 hours with lipopolysaccharide (LPS). Cells cultured with LPS are indicated by no shading and a broken black line, the shaded area represents cells cultured without LPS. E: Culture with GM-CSF + IL-4 or with FLT3-L causes the differentiation of CD11c+ cells with poor priming ability from the bone marrow of mice with alum PI. Bone marrow cells were cultured with GM-CSF + IL-4 for 7 days, or FLT3-L for 9 days before pulsing with 1 mmol/L 4-dinitro-1-fluorobenzene (DNFB) (Sigma-Aldrich) for 30 minutes, and 106 cells (not CD11c+ cell enriched) injected subcutaneously.into the ears of naïve mice. After 7 days, ears were painted with 0.2% DNFB and increased ear swelling measured after 24, 30, and 48 hours. As a control, swelling is shown for ears injected with saline instead of cells before challenge with DNFB. After ear swelling due to the first application of DNFB had subsided (>2 weeks), the ears were painted with 0.2% DNFB again, and the ear swelling was re-measured during the next 48 hours. Data represent mean ear swelling ± SEM for 8 ears/group. Closed symbols represent ears injected with cells cultured for 7 days with GM-CSF + IL-4. Open symbols represent ears injected with cells cultured for 9 days with FLT3-L. The -alum PI and +alum PI are for the ears of mice injected with CD11c+ cells expanded from bone marrow of mice ± alum PI. Saline shows the swelling of the ears injected with saline (no cells), but still hapten challenged. *P < 0.05 for comparison between ears injected with bone marrow-derived CD11c+ cells from mice ± alum PI.
      Table 1Cytokine Production by Bone Marrow Cells from Control Mice Cultured for 7 Days with GM-CSF + IL-4 or 9 Days with FLT3-L
      Cytokine (pg/mL)TreatmentGM-CSF + IL-4FLT3-L
      IL-1069 ± 46
      Data presented as means ± SEM (n = 3 replicate cultures).
      <50
      LPS1465 ± 1321642 ± 108
      Negative control CpG<50<50
      CpG<501831 ± 43
      Significantly greater stimulation by CpG for cells cultured with FLT3-L than GM-CSF+IL-4, P < 0.05.
      TNF-α129 ± 5<4
      LPS10,308 ± 125
      Significantly greater stimulation by LPS for cells cultured with GM-CSF + IL-4 than FLT3-L, P < 0.05.
      1799 ± 45
      Negative control CpG244 ± 1219 ± 5
      CpG1913 ± 415599 ± 115
      Significantly greater stimulation by CpG for cells cultured with FLT3-L than GM-CSF+IL-4, P < 0.05.
      IL-23<6<6
      LPS69 ± 8
      Significantly greater stimulation by LPS for cells cultured with GM-CSF + IL-4 than FLT3-L, P < 0.05.
      <6
      Negative control CpG<6<6
      CpG17 ± 545 ± 3
      Significantly greater stimulation by CpG for cells cultured with FLT3-L than GM-CSF+IL-4, P < 0.05.
      IL-12<20<20
      LPS634 ± 16
      Significantly greater stimulation by LPS for cells cultured with GM-CSF + IL-4 than FLT3-L, P < 0.05.
      358 ± 15
      Negative control CpG<20<20
      CpG308 ± 432,282 ± 1242
      Significantly greater stimulation by CpG for cells cultured with FLT3-L than GM-CSF+IL-4, P < 0.05.
      FLT3-L, FLT3 ligand; GM-CSF, granulocyte-macrophage colony stimulating factor; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α.
      low asterisk Data presented as means ± SEM (n = 3 replicate cultures).
      Significantly greater stimulation by LPS for cells cultured with GM-CSF + IL-4 than FLT3-L, P < 0.05.
      Significantly greater stimulation by CpG for cells cultured with FLT3-L than GM-CSF+IL-4, P < 0.05.
      Regardless of culture in GM-CSF + IL-4 or FLT3-L, the CD11c+ cells differentiated from the bone marrow of mice with alum PI were inefficient at priming antigen responses in naïve mice (Figure 5E). When the properties of cells differentiated from the bone marrow of control mice (-alum PI) were examined, cells from FLT3-L cultures were less efficient at antigen priming, despite the higher concentration of CD11c+ cells in the 106 cells injected per ear (Figure 5E). In summary, despite changes in culture conditions for the DC precursors and induction of 10% to 14% putative plasmacytoid DCs with FLT3-L, cells differentiated from the bone marrow of mice with peritoneal inflammation remained poorly immunogenic.

      Indomethacin Prevents the Development of CD11c+ Cells with Poor Priming Ability from the Bone Marrow of Mice with Peritoneal Inflammation

      Altered conditions in vivo were investigated because CD11c+ cells expanded from the bone marrow of mice with alum-PI poorly primed immune responses irrespective of the growth factors used for their differentiation (Figure 4, Figure 5). Prostaglandin E2 (PGE2) is a small lipid molecule produced on activation by many immune and nonimmune cells due to activation of the cyclooxygenase enzymes.
      • Harris S.G.
      • Padilla J.
      • Koumas L.
      • Ray D.
      • Phipps R.P.
      Prostaglandins as modulators of immunity.
      PGE2 can be immunomodulatory by suppressing the maturation of DCs
      • Harizi H.
      • Juzan M.
      • Pitard V.
      • Moreau J.F.
      • Gualde N.
      Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulated dendritic cell functions.
      or by modulating other immunological properties of DCs.
      • Stock A.
      • Booth S.
      • Cerundolo V.
      Prostaglandin E2 suppresses the differentiation of retinoic acid-producing dendritic cells in mice and humans.
      In the bone marrow, inflammation-associated PGE2 may directly or indirectly regulate DC precursors.
      • Nemeth K.
      • Leelahavanichkul S.
      • Yuen P.S.T.
      • Mayer B.
      • Parmelee A.
      • Doi K.
      • Robey P.G.
      • Leelahavanichkul K.
      • Koller B.H.
      • Brown J.M.
      • Hu X.
      • Jelinek I.
      • Star R.A.
      • Mezey E.
      Bone marrow stromal cells attenuate sepsis via prostaglandin E2-dependent reprogramming of host macrophages to increase their interleukin-10 production.
      To investigate the involvement of PGE2 and other prostanoids, slow-release indomethacin (an inhibitor of the cyclooxygenase enzymes) or placebo pellets were inserted subcutaneously into mice 4 days before intraperitoneal injection of alum or saline. Three days after alum injection, bone marrow cells were isolated and cultured with GM-CSF + IL-4. The myelopoiesis in the alum-injected mice was not altered by indomethacin administration (Figure 6A). The IL-10 induced by LPS in freshly isolated bone marrow cells from alum-injected mice was also not changed (Figure 6B). In mice administered with the placebo pellets, the CD11c+ cells cultured from the bone marrow of the mice with alum PI had poor priming ability (Figure 6C). However, administration of indomethacin to the mice before alum injection prevented the differentiation of these cells. In fact, in the indomethacin-treated mice, there was no difference in the immune function of CD11c+ cells differentiated from the bone marrow of mice injected intraperitoneally with alum or with saline (Figure 6C). There was no effect of indomethacin added to bone marrow cells in the culture (Figure 6D). The production of PGE2 and other prostanoids in the mouse, rather than during culture, therefore, was important and suggests that PGE2 and prostanoids imprint an effect on DC precursors in the early stages of differentiation.
      Figure thumbnail gr6
      Figure 6Indomethacin prevents the development of CD11c+ cells with poor antigen priming ability from bone marrow of mice with alum-induced peritoneal inflammation (alum PI), but does not affect myelopoiesis in the bone marrow. A: Expression of the cell surface markers Gr1, CD11b, and B220 by bone marrow cells isolated from mice ± alum PI. Indomethacin or placebo pellets were implanted subcutaneously into the backs of mice 4 days before mice were injected intraperitoneally with alum (hatched bars) or saline (open bars). Bone marrow from these mice was isolated 3 days later. Mean value for cells from 2 experiments. B: Levels of IL-10 in the supernatant of bone marrow cells isolated from mice ± alum PI and stimulated for 24 hours with 1 μg/mL lipopolysaccharide (LPS) (mean + SEM, n = 3 from representative experiment). C: Indomethacin in vivo prevents decreases in the priming ability of CD11c+ cells differentiated from the bone marrow of mice with alum PI. D: Indomethacin in vitro does not reverse the poor priming ability of CD11c+ cells differentiated from the bone marrow of mice with alum PI. Bone marrow cells were cultured for 7 days with granulocyte-macrophage colony stimulating factor (GM-CSF) + IL-4, and with indomethacin (10 μm) in D, before CD11c+ cells were purified, pulsed with 1 mmol/L DNBS, and 106 cells injected subcutaneously into the ears of naïve mice. After 7 days, ears were painted with 0.2% DNFB and increased ear swelling after 24 hours was measured. Data represent mean ear swelling + SEM for 8 ears/group. *P < 0.05. Open bars represent cells/IL-10/ears injected with CD11c+ cells from mice without alum PI. Hatched bars represent cells/IL-10/ears injected with CD11c+ cells from mice with alum PI.

      DCs Generated from Bone Marrow of Mice with EAAD Do Not Express Increased Indoleamine Dioxygenase, Cyclooxygenase 2, or Nitric Oxide Synthase 2 mRNA

      Expression of indoleamine dioxygenase, cyclooxygenase 2, or nitric oxide synthase 2, known mechanisms of immunoregulation
      • Bronte V.
      • Zanovello P.
      Regulation of immune responses by L-arginine metabolism.
      • Mellor A.L.
      • Munn D.H.
      IDO expression by dendritic cells: tolerance and tryptophan catabolism.
      • Sinha P.
      • Clements V.K.
      • Fulton A.M.
      • Ostrand-Rosenberg S.
      Prostaglandin E2 promotes tumor progression by inducing myeloid-derived suppressor cells.
      • Obermajer N.
      • Muthuswamy R.
      • Lesnock J.
      • Edwards R.P.
      • Kalinski P.
      Positive feedback between PGE2 and COX2 redirects the differentiation of human dendritic cells toward stable myeloid-derived suppressor cells.
      were investigated. Bone marrow cells from mice with alum PI were cultured with GM-CSF + IL-4 or FLT3-L as previously described. For CD11c+ cells cultured with all growth factors, despite differences in antigen priming ability, if they were generated from mice with inflammation (Figure 5E), there was no difference in mRNA levels for indoleamine dioxygenase, cyclooxygenase 2, or nitric oxide synthase 2 (Figure 7). mRNA levels for IL-10 were also not changed, which supports the results of Table 1 suggesting that IL-10 was not part of the mechanism by which CD11c+ cells from bone marrow of mice with EAAD or alum PI were poor at priming immune responses.
      Figure thumbnail gr7
      Figure 7Fold change in mRNA levels for indoleamine dioxygenase (IDO), IL-10, cyclooxygenase 2 (COX2), and nitric oxide synthase 2 (NOS2) in CD11c+ cells generated from bone marrow cells from mice with alum-induced peritoneal inflammation (alum PI) and cultured with ligand (FLT3-L) (A) and granulocyte-macrophage colony stimulating factor (GM-CSF) + IL-4 (B). The expression of mRNAs for the four genes was determined relative to the housekeeping gene eukaryotic translation elongation factor 1α (Eef1α). Results are shown as mean + SEM for bone marrow cells from three independent mice. The fold change for cells from mice with alum PI has been expressed relative to measures in cells from mice without alum PI. nd, not detectable.

      Hapten-Loaded CD11c+ Cells from the Bone Marrow of Mice with Peritoneal Inflammation Reduce the CHS Response in Hapten-Presensitized Mice

      The value of regulatory CD11c+ cells would be enhanced if they could direct reduced recall responses in already sensitized animals. To test this, the ears of both naïve and DNFB-sensitized mice were injected with 106 DNBS-loaded CD11c+ cells differentiated from the bone marrow of mice with or without alum PI. For the DNFB-presensitized mice, the right ear was injected with cells, the left ear with saline. Saline injections into the left ears of DNFB-presensitized mice at the time of CD11c+ cell injection into the right ears provided a measure of the swelling associated on DNFB re-exposure, with presensitization per se (Figure 8A). The DNBS-loaded CD11c+ cells differentiated from the bone marrow of mice with alum PI compared with those from mice without alum PI significantly suppressed the CHS response on challenge with DNFB in both the naïve and DNFB-presensitized mice (Figure 8A). This result demonstrates that the CD11c+ cells differentiated from the bone marrow of inflammatory mice can actively suppress responses in presensitized mice, and thus as regulatory DCs may have therapeutic potential. With this active suppression of an established memory response, the findings of Figure 3, in which the CD11c+ cells differentiated from the bone marrow of mice with alum PI do not have reduced antigen uptake or migration to the auricular lymph nodes, are supported.
      Figure thumbnail gr8
      Figure 8Regulatory CD11c+ cells differentiated from the bone marrow of mice with alum-induced peritoneal inflammation (alum PI). Bone marrow cells were isolated from mice ± alum PI and cultured for 7 days with granulocyte-macrophage colony stimulating factor (GM-CSF) + IL-4. CD11c+ cells were purified and pulsed with 1 mmol/L 2,4-dinitrobenzene sulfonic acid sodium salt (DNBS) (MP Biomedicals LLC, Solon, OH) for 30 minutes. A: Reduced contact hypersensitivity (CHS) response in 4-dinitro-1-fluorobenzene (DNFB) (Sigma-Aldrich) presensitized mice. There were 106 purified and pulsed CD11c+ cells injected into the ears of recipient mice. If the recipient mice were naïve, both ears were injected with cells. As a control, the ears of further mice were injected with saline. If the mice were already sensitized to DNFB (50 μL 0.2% DNFB on a shaved 2 × 2 cm patch of abdomen 7 days previously), DNBS-loaded CD11c+ cells were injected only into the right ears of the recipient mice. An equal volume of saline was injected into the left ears. Seven days after injection, all ears were painted with 0.2% DNFB and ear swelling was measured 24 hours later. Data represent mean ear swelling + SEM for 8 mice/group. *P < 0.05. Open bars are for ears injected with CD11c+ cells differentiated from the bone marrow of mice without alum PI. Hatched bars are for ears injected with CD11c+ cells differentiated from the bone marrow of mice with alum PI. Shaded bars show the swelling by ears injected with 20 μL saline, but are still hapten challenged. B: Active suppression of a CHS response in the alternative ear. The left ears of mice were injected with 106 DNBS-loaded CD11c+ cells, and 7 days later the primary CHS response was measured. After 2 weeks, the mice were sensitized with DNFB on their abdominal skin with DNFB. Later, 1 week further on, the right ears were challenged with DNFB. Ear swelling after 24 hours was measured (secondary CHS) and is shown as mean + SEM for 8 ears/group. Open bars represent ears/mice injected with CD11c+ cells from mice without alum PI. Hatched bars represent ears/mice injected with CD11c+ cells from mice with alum PI. *P < 0.05.
      In another approach to measure the regulatory properties of CD11c+ cells, DNBS-pulsed CD11c+ cells were injected into the left ears only and a CHS to DNFB measured after 7 days (primary CHS) (Figure 8B). The mice were rested for 2 weeks before DNFB sensitization on their abdominal skin, a process that would depend on competent DCs. After a further 7 days, the right ears were painted with DNFB, and ear swelling during the next 48 hours was measured. Significantly reduced responses to DNFB were measured in the right ears of mice that had previously received (into their left ears) CD11c+ cells generated from the bone marrow of alum PI mice (secondary CHS) (Figure 8B). The reduced swelling in the right ears suggested active regulation of responses to DNFB (Figure 8B).

      Discussion

      In this study, CD11c+ cells differentiated from the bone marrow of mice with respiratory or peritoneal inflammation had poor priming ability when transferred after antigen-loading into the ears of naïve mice. The persistently reduced response in the recipient mice (Figure 1D and 2D), and the reduced responses in an alternative ear despite resensitization to DNFB by competent DCs of the abdominal skin (Figure 8B), suggested that regulatory CD11c+ cells had been generated. The CD11c+ cells differentiated from the bone marrow of mice with peritoneal inflammation, furthermore, actively reduced the responses to antigens in mice presensitized to that antigen. The effect of inflammation on bone marrow DC precursors involved a prostanoid-dependent mechanism as the effects could be blocked by administration of slow-release indomethacin pellets in mice (Figure 6C), but not by the addition of indomethacin to cells in culture (Figure 6D). When the culture conditions for the bone marrow cells were altered, the differentiated CD11c+ cells varied in their size, phenotype, and ability to produce inflammatory cytokines on LPS and CpG stimulation. However, regardless of the culture conditions used, CD11c+ cells differentiated from the bone marrow of mice with tissue inflammation were less efficient at inducing immunity than those from the control mice. Our interpretation of these results is that inflammation-associated PGE2, and possibly other prostanoids (directly or indirectly), can modulate early DC precursors in bone marrow that are susceptible to differentiation in either GM-CSF or FLT3-L. Importantly, these studies demonstrate that the effects of inflammation are imprinted in vivo and are not dependent on the in vitro conditions for DC differentiation.
      It was hypothesized that in this well-characterized, allergen-driven model of EAAD, the inflammation would be sustained by immunogenic, not regulatory, DCs emerging from the bone marrow.
      • von Garnier C.
      • Wikstrom M.E.
      • Zosky G.
      • Turner D.J.
      • Sly P.D.
      • Smith M.
      • Thomas J.A.
      • Judd S.R.
      • Strickland D.H.
      • Holt P.G.
      • Stumbles P.A.
      Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa.
      Bone marrow was harvested 24 hours after OVA aerosol challenge of mice sensitized to OVA-in-alum. At this point in time, there is a significantly increased infiltration of effector CD4+ T-lymphocytes into the trachea of mice,
      • McGlade J.P.
      • Gorman S.
      • Zosky G.R.
      • Larcombe A.N.
      • Sly P.D.
      • Finlay-Jones J.J.
      • Turner D.J.
      • Hart P.H.
      Suppression of the asthmatic phenotype by UVB-induced, antigen-specific regulatory cells.
      • McGlade J.P.
      • Strickland D.H.
      • Lambert M.J.M.
      • Gorman S.
      • Thomas J.A.
      • Judge M.A.
      • Burchell J.T.
      • Zosky G.R.
      • Hart P.H.
      UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells.
      and increased numbers of myeloid cells
      • O'Connell R.M.
      • Rao D.S.
      • Chaudhuri A.A.
      • Boldin M.P.
      • Taganov K.D.
      • Nicoll J.
      • Paquette R.L.
      • Baltimore D.
      Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder.
      were harvested from the bone marrow (Figure 1A). Similarly, 3 days after intraperitoneal injection of alum, there was increased bone marrow myelopoiesis (Figure 2A). This result confirmed that the inflammation in the periphery had systemic consequences. However, myelopoiesis per se was not responsible for the effects of inflammation on bone marrow DC precursors (ie, lineage-negative cells
      • Wu L.
      • Liu Y.-J.
      Development of dendritic-cell lineages.
      • Shortman K.
      • Naik S.H.
      Steady-state and inflammatory dendritic-cell development.
      • Liu K.
      • Nussenzweig M.C.
      Origin and development of dendritic cells.
      • Kushwah R.
      • Hu J.
      Complexity of dendritic cell subsets and their function in the host immune system.
      ). Indomethacin administration blocked the effect of alum PI on development of regulatory DCs from bone marrow, but it did not affect the extent of myelopoiesis or the increased production of IL-10 by nonfractionated bone marrow cells (Figure 6). Similarly, PGE2 administration, which can alter bone marrow DC precursors such that they differentiate in vitro into regulatory DCs, did not stimulate myelopoiesis.
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      PGE2 produced by bone marrow cells in culture was without effect on the outcomes as indomethacin added to cells in culture did not alter the generation of regulatory DCs; PGE2 has been shown previously to reduce DC differentiation by GM-CSF
      • Sharma S.
      • Stolina M.
      • Yang S.C.
      • Baratelli F.
      • Lin J.F.
      • Atianzar K.
      • Luo J.
      • Zhu L.
      • Lin Y.
      • Huang M.
      • Dohadwala M.
      • Batra R.K.
      • Dubinett S.M.
      Tumor cyclooxygenase 2-dependent suppression of dendritic cell function.
      and survival of DC progenitors cultured with FLT3-L.
      • Singh P.
      • Hoggatt J.
      • Hu P.
      • Speth J.M.
      • Fukuda S.
      • Breyer R.M.
      • Pelus L.M.
      Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development form hematopoietic progenitor cells.
      How alum induces inflammation in the peritoneal cavity is not fully known. Alum can stimulate inflammatory DCs through activation of the NALP3 inflammasome.
      • Kool M.
      • Petrilli V.
      • De Smedt T.
      • Rolaz A.
      • Hammad H.
      • van Nimwegen M.
      • Bergen I.M.
      • Castillo R.
      • Lambrecht B.N.
      • Tschopp J.
      Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome.
      Caspase 1 is activated in the inflammasome complexes with induction of the maturation and secretion of the pro-inflammatory cytokines, IL-1β and IL-18.
      • Lamkanfi M.
      Emerging inflammasome effector mechanisms.
      Alum can also induce increased expression of the gene for cytosolic phospholipase A2,
      • Exley C.
      • Siesjo P.
      • Eriksson H.
      The immunobiology of aluminium adjuvants: how do they really work?.
      and thus increased PGE2 production. Complementary actions of alum that increase its adjuvanticity include alteration of the membrane lipid structures in DCs,
      • Flach T.L.
      • Ng G.
      • Hari A.
      • Desrosiers M.D.
      • Zhang P.
      • Ward S.M.
      • Seamone M.E.
      • Vilaysane A.
      • Mucsi A.D.
      • Fong Y.
      • Prenner E.
      • Ling C.C.
      • Tschopp J.
      • Muruve D.A.
      • Amrein W.W.
      • Shi Y.
      Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.
      and stimulation of cell death and release of host cell DNA, a damage-associated molecular pattern and potent endogenous immunostimulatory signal.
      • Marichal T.
      • Ohata K.
      • Bedoret D.
      • Mesnil C.
      • Sabatel C.
      • Kobiyama K.
      • Lekeux P.
      • Coban C.
      • Akira S.
      • Ishii K.J.
      • Bureau F.
      • Desmet C.J.
      DNA released from dying host cells mediates aluminium adjuvant activity.
      The effects of intraperitoneal alum were not totally responsible for the effects reported for mice with respiratory inflammation. The effect of the aerosol challenge was examined in mice sensitized and boosted intraperitoneally with lower amounts of OVA-in-alum; there was a cumulative effect of inflammation of the airways (due to OVA aerosol) and peritoneal cavity on DC precursors in the bone marrow (data not shown).
      PGE2 and possibly other prostanoids were responsible for modulation of bone marrow DC precursors. Generally PGE2, by binding to E-prostanoid receptors 2 and 4, and elevating cyclic adenosine monophosphate, mediates anti-inflammatory effects.
      • Harris S.G.
      • Padilla J.
      • Koumas L.
      • Ray D.
      • Phipps R.P.
      Prostaglandins as modulators of immunity.
      In the mouse model of multiple sclerosis, PGE2-E-prostanoid receptor 4 signaling provided a facilitative role during immunization but down-regulated the elicitation phase.
      • Esaki Y.
      • Li Y.
      • Sakata D.
      • Yao C.
      • Segi-Nishida E.
      • Matsuoka T.
      • Fukuda K.
      • Narumiya S.
      Dual role of PGE2-EP4 signaling in mouse experimental autoimmune encephalomyelitis.
      PGE2 can have different roles, depending on the maturity of the DCs.
      • Singh P.
      • Hoggatt J.
      • Hu P.
      • Speth J.M.
      • Fukuda S.
      • Breyer R.M.
      • Pelus L.M.
      Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development form hematopoietic progenitor cells.
      • Esaki Y.
      • Li Y.
      • Sakata D.
      • Yao C.
      • Segi-Nishida E.
      • Matsuoka T.
      • Fukuda K.
      • Narumiya S.
      Dual role of PGE2-EP4 signaling in mouse experimental autoimmune encephalomyelitis.
      In our study, the effect of prostanoids may be direct on DC precursors or indirect on cells both external and internal to the bone marrow. In UV-irradiated skin, keratinocytes respond to UV radiation-induced PGE2
      • Soontrapa K.
      • Honda T.
      • Sakata D.
      • Yao C.
      • Hirata T.
      • Hori S.
      • Matsuoka T.
      • Kita Y.
      • Shimizu T.
      • Kabashima K.
      • Narumiya S.
      Prostaglandin E2-prostagandin E receptor subtype 4 (EP4) signaling mediates UV irradiation-induced systemic immunosuppression.
      and similarly, inflammatory and epithelial cells in the inflamed airways and peritoneal cavity express prostaglandin receptors. Within the bone marrow, mature DCs (<2%) may be a target for PGE2 function; in one study, removal of CD11c+ cells before differentiation of bone marrow cells in culture altered the properties of the CD11c+ cells harvested after 1 week.
      • Pastille E.
      • Didovic S.
      • Brauckmann D.
      • Rani M.
      • Agrawal H.
      • Schade F.U.
      • Zhang Y.
      • Flohe S.B.
      Modulation of dendritic cell differentiation in the bone marrow mediates sustained immunosuppression after polymicrobial sepsis.
      Bone marrow-derived stromal cells, otherwise known as mesenchymal stem cells, may be both the target of, or the producer of, the prostanoids of critical importance in our models.
      • Yanez R.
      • Oviedo A.
      • Aldea M.
      • Bueren J.A.
      • Lamana M.L.
      Prostaglandin E2 plays a key role in the immunosuppressive properties of adipose and bone marrow tissue-derived mesenchymal stromal cells.
      • Nauta A.J.
      • Fibbe W.E.
      Immunomodulatory properties of mesenchymal stromal cells.
      PGE2 from bone marrow mesenchymal stem cells can furthermore stimulate IL-10 production by host macrophages and thus attenuate sepsis.
      • Nemeth K.
      • Leelahavanichkul S.
      • Yuen P.S.T.
      • Mayer B.
      • Parmelee A.
      • Doi K.
      • Robey P.G.
      • Leelahavanichkul K.
      • Koller B.H.
      • Brown J.M.
      • Hu X.
      • Jelinek I.
      • Star R.A.
      • Mezey E.
      Bone marrow stromal cells attenuate sepsis via prostaglandin E2-dependent reprogramming of host macrophages to increase their interleukin-10 production.
      Bone marrow mesenchymal stem cells are altered significantly by different Toll-like Receptor ligands during infection and inflammation.
      • Nemeth K.
      • Mayer B.
      • Mezey E.
      Modulation of bone marrow stromal cell functions in infectious diseases by toll-like receptor ligands.
      • Nemeth K.
      • Keane-Myers A.
      • Brown J.M.
      • Metcalfe D.D.
      • Gorham J.D.
      • Bundoc V.G.
      • Hodges M.G.
      • Jelinek I.
      • Madala S.
      • Karpati S.
      • Mezey E.
      Bone marrow stromal cells use TGF-β to suppress allergic responses in a mouse model of ragweed-induced asthma.
      • Spaggiari G.M.
      • Abdelrazik H.
      • Becchetti F.
      • Moretta L.
      MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2.
      It is possible that their proliferation, apoptosis, migration, and production and release of immunomodulatory factors, including PGE2, are altered by the inflammation in our experimental models.
      The immunogenic properties of the CD11c+ cells differentiated from the bone marrow of inflammatory mice were determined using a robust test of DC behavior in vivo. As shown in Figure 1D, the regulatory properties of the transferred CD11c+ cells were antigen-specific. Bone marrow-derived CD11c+ cells were purified to >95% before DNBS pulsing, except after generation in FLT3-L (Figure 5), and as functional responses did not change, the process of enrichment did not appear to qualitatively change cell properties. The mechanism by which the bone marrow-derived CD11c+ cells were regulatory in presensitized mice was not determined, although it is clear that it was not by mechanisms by which myeloid-derived suppressor cells suppress immune responses, predominantly in tumor microenvironments.
      • Bronte V.
      • Zanovello P.
      Regulation of immune responses by L-arginine metabolism.
      • Mellor A.L.
      • Munn D.H.
      IDO expression by dendritic cells: tolerance and tryptophan catabolism.
      • Sinha P.
      • Clements V.K.
      • Fulton A.M.
      • Ostrand-Rosenberg S.
      Prostaglandin E2 promotes tumor progression by inducing myeloid-derived suppressor cells.
      • Obermajer N.
      • Muthuswamy R.
      • Lesnock J.
      • Edwards R.P.
      • Kalinski P.
      Positive feedback between PGE2 and COX2 redirects the differentiation of human dendritic cells toward stable myeloid-derived suppressor cells.
      The CD11c+ cells differentiated from the bone marrow of mice with tissue inflammation did not express lower levels of co-stimulatory molecules (Figure 4) or produce increased IL-10 levels (Figure 7).
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      In addition, the bone marrow-derived CD11c+ cells were not blocked from maturation as the expression of co-stimulatory molecules and production of multiple cytokines were increased in response to LPS (Figure 4, Figure 5) (Table 1). The reduced priming ability of the CD11c+ cells differentiated in vitro from the bone marrow of UV-irradiated mice, furthermore, is not altered/reversed by incubation with LPS for 24 hours before DNBS loading and transfer into the ears of naive mice for assessment of their functional ability.
      • Ng R.L.X.
      • Bisley J.L.
      • Gorman S.
      • Norval M.
      • Hart P.H.
      UV-irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway.
      Confirmation that these regulatory DCs develop in the bone marrow of mice with respiratory and peritoneal inflammation, and that they return to the inflammatory tissues is required. Regulatory DCs develop in mice, however, with UV-induced skin inflammation and are an integral component of UV-induced systemic immunosuppression and reduced immunity to antigens administered to nonirradiated sites (for review, see Hart et al
      • Hart P.H.
      • Gorman S.
      • Finlay-Jones J.J.
      Modulation of the immune system by UV radiation: more than just the effects of vitamin D?.
      and Norval and Halliday.
      • Norval M.
      • Halliday G.M.
      The consequences of UV-induced immunosuppression for human health.
      ). The CD11c+ cells expanded from the bone marrow of mice with tissue inflammation actively reduced a recall immune response (Figure 8A) and reduced the inflammatory response in mice that were subsequently re-sensitized (Figure 8B). These results suggest a mechanism of active suppression by bone marrow-derived DCs from inflammatory mice, which would be unlikely if the CD11c+ cells were poor at uptake of antigen or migration to the auricular lymph nodes. The reduced responses measured in the presensitized mice also highlight the potential therapeutic power of regulatory DCs harvested (and expanded) from bone marrow of inflammatory mice.
      In summary, inflammation-associated, prostanoid-driven development of regulatory DCs from bone marrow may be a previously unrecognized homeostatic mechanism to prevent overzealous inflammatory responses.

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