Fig 1.
Phenotype and cytokine pattern of CoPP-treated BM-DCs.
Treated BM-DCs as indicated from BALB/c mice were stimulated with or without LPS for 24 hours. BM-DCs were harvested for surface marker detection using flow cytometry and supernatants for cytokine analysis using ELISA. (A) The histograms shown were gated for live, CD11c+ cells. Dotted line: isotype control; red area: DMSO with LPS; blue line: CoPP with LPS. (B) Percentages of gated cells (M1 as shown in A) from treated BM-DCs were normalized to DMSO with LPS group. The results are shown as mean ± SD of three independent experiments. The number in each graph represents the mean percentage (%) of M1-gated cells in DMSO with LPS group. *p < 0.05 vs. DMSO with LPS. (C) The levels of cytokines are shown as relative expression (%) compared to DMSO with LPS (mean ± SD of three to five experiments). * p < 0.05 vs. DMSO with LPS. The cytokine levels of DMSO with LPS (mean ± SD): IL-10: 252 ± 57 pg/ml; IL-12: 1,393 ± 415 pg/ml; IL-23: 97 ± 28 pg/ml. IL-6: 76 ± 5 ng/ml; TNF-α: 1,806 ± 588 pg/ml.
Fig 2.
CoPP-conditioned BM-DCs display low CD4+ T-cell stimulatory activity and inflammatory cytokine production.
(A) Treated BM-DCs from BALB/c mice were co-cultured with allogenic CD4+ T cells from C57BL/6 mice for 4 days. (B) Treated BM-DCs from BALB/c mice were pulsed with OVA323-339 and cocultured with syngeneic DO11.10 CD4+ T cells for 4 days. Proliferative response was assessed by [3H]-thymidine assay. (C, D) The supernatants from 72-hour DO11.10 CD4+ T-DC coculture were analyzed for cytokine levels using ELISA. Data are mean ± SD of three to four independent experiments. * p < 0.05 vs. vehicle (DMSO). All treated BM-DCs in Fig 2 were stimulated with LPS prior to DC-T coculture.
Fig 3.
BM-DCs with HO-1 induction enhance PD-L1 expression and adaptive regulatory T cell (Treg) differentiation in vitro.
(A) BM-DCs treated with DMSO or CoPP were stained with fluorochrome-labeled antibodies for PD-L1, PD-L2, and ICOSL and analyzed by flow cytometry. Gray area: isotype control; red area: DMSO with LPS; blue line: CoPP with LPS. (B) The histograms shown are the percentages of PD-L1+ or PD-L2+ BM-DCs and the mean fluorescence intensity (MFI) of ICOSL+ cells as analyzed in A. (mean ± SD, n = 3). (C) Treated BM-DCs were co-cultured with naïve CD4+ T cells from Foxp3EGFP mice for 3 days in the presence of TGF-β and anti-CD3 activating mAb. Representative dot-plots show the expression of Foxp3 (EGFP) on gated CD3+CD4+ T cells. The treated BM-DCs in C were stimulated with LPS. (D) The frequency (mean ± SD, n = 5) of Foxp3(EGFP)+ Tregs in CD4+ T cells as analyzed in C. * p < 0.05 vs. DMSO with LPS (Mann-Whitney U test).
Fig 4.
CoPP-conditioned BM-DCs promote Treg differentiation in vivo.
(A) OVA323-339-pulsed BM-DCs and DO11.10 naïve CD4+ T cells from Thy1.2+ mice were intravenously transferred into Thy1.1+ mice for 5 days. Representative dot plots (left) show the expression of CD4 and CD25 on gated Thy1.2+KJ1-26+ (OVA-specific Tregs, lower panel) or on gated Thy1.2+KJ1-26- cells (non-specific Tregs, upper panel). The frequencies (mean ± SD, n = 4) of Tregs are shown (right). (B) OVA323-339-pulsed BM-DCs and DO11.10 naïve CD4+ T cells were intratracheally transferred into BALB/c mice for 5 days. Representative dot plots show the expression of CD4 and Foxp3 on gated CD3+KJ1-26+ T cells (mean ± SD, n = 5). * p < 0.05 vs. DMSO (Mann-Whitney U test). All treated BM-DCs in Fig 4 were stimulated with LPS.
Fig 5.
CoPP-conditioned BM-DCs induce less airway inflammation in a neutrophil-dominant model.
BALB/c mice were intravenously injected with OVA-pulsed DMSO or CoPP-treated BM-DCs on day 0. Ten days later, both groups of mice were received three daily OVA aerosol challenges. The next day after the last challenge, effector cells in BALFs (A), cytokines (B) in BALFs and Foxp3+ regulatory T cells (Tregs) (C) in local lymph nodes (LN) of lung were analyzed by flow cytometry and ELISA, respectively. Data are pooled from one to two independent experiments. The line within the vertical points marks the mean for each group. * p < 0.05 vs. DMSO. DC, dendritic cell. (D) Representative section stained with hematoxylin and eosin. The magnification: 200 ×. All treated BM-DCs in Fig 5 were stimulated with LPS.
Fig 6.
CoPP-conditioned BM-DCs induce less severe eosinophil infiltration in an allergic asthma model.
BALB/c mice were intratracheally injected with OVA-pulsed DMSO or CoPP-treated BM-DCs on day 0 and followed by daily OVA aerosol challenges on day 10–12. The next day after the last challenge, effector cells (A), cytokines (B), and Foxp3+ regulatory T cells (Tregs) (C) in BALFs were analyzed by flow cytometry and ELISA, respectively. Data are pooled from one to three independent experiments. The line within the vertical points marks the mean for each group. * p < 0.05 vs. DMSO. DC, dendritic cell. (D) Representative section stained with hematoxylin and eosin. The magnification: 200 ×. All treated BM-DCs in Fig 6 were stimulated with LPS.