TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4 - PubMed (original) (raw)

TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4

Toshihiro Ito et al. J Clin Invest. 2009 Jan.

Abstract

TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficient mice challenged with a Mycobacterium antigen display an altered Th17 cytokine profile, decreased accumulation of granuloma-associated myeloid DCs, and profoundly impaired delta-like 4 (dll4) Notch ligand expression. Mechanistic analysis revealed that WT bone marrow-derived DCs but not macrophages promoted the differentiation of Th17 cells from bacillus Calmette-Guérin-challenged (BCG-challenged) lung CD4+ T cells. Both lung and bone marrow DCs isolated from Tlr9-deficient mice inoculated with Mycobacterium antigen expressed lower levels of dll4 Notch ligand than the same cells isolated from WT mice. Passively immunizing WT mice with neutralizing antibodies specific for dll4 during granuloma formation resulted in larger granulomas and lower levels of Th17-related cytokines. In addition, dll4 specifically regulated Th17 activation in vitro. Together, these results suggest dll4 plays an important role in promoting Th17 effector activity during a mycobacterial challenge. Furthermore, TLR9 seems to be required for optimal dll4 expression and the regulation of Mycobacterium antigen-elicited granuloma formation in mice.

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Figures

Figure 1

Figure 1. Tlr9–/– mice showed larger pulmonary granulomas and impaired Th17 cytokine levels.

All panels compare parameters between WT and Tlr9–/– mice. (A) Lung tissues were histologically analyzed by H&E at day 4 after initiation of lung granuloma. Original magnification, ×100. (B) Kinetic analysis of the development of lung granulomas using morphometric analysis of the evolving lung lesions in WT and Tlr9–/– mice. Dotted line represents mean granuloma cross-sectional area (μm2) ± SEM. *P < 0.05; **P < 0.001. (C) Protein levels of IL-17 measured in whole lungs using a Luminex system. Data shown are mean ± SEM and are from a representative experiment of 4 independent experiments. †P < 0.01. (D–F) Quantitative real-time PCR (TaqMan) was performed to measure the transcript levels of IL-17A (D), IL-17F (E), IL-6, IL-21, IL-23p19, TGF-β, TNF-α, and IL-10 (F) in whole lungs. Data shown are mean ± SEM and are from a representative experiment of 4 independent experiments. Each time point indicates at least 4–6 mice per group.

Figure 2

Figure 2. Impaired Th17 cell phenotype and migration of mDCs in Tlr9–/– mice during pulmonary granuloma formation.

(A) FACS analysis of intracellular staining of CD4+ cells for IFN-γ, IL-4, and IL-17. Data represent mean ± SEM. *P < 0.01 compared with WT mice. Experiments were performed 3 times with similar results. (B) FACS profile of CD4+ T cells from lungs was analyzed by intracellular staining of IL-17. Displayed in the dot plots are cells gated on CD4+ lymphocyte population by forward scattering and side scattering. The numbers in the dot plots are percentages of IL-17– cells (left gate) and IL-17+ cells (right gate) in the CD4+ lymphocyte population. Shown are representative FACS plots from a total of 4 mice per group. (C) FACS analysis of lung DCs, macrophages, and neutrophils isolated from day 4 PPD-bead challenged mice (n = 4). †P < 0.02 compared with WT mice. Data shown are mean ± SEM and are from a representative experiment of 2 independent experiments. (D) Confocal immunofluorescent examination of pulmonary granulomas showed fewer CD11c+ cells (red) and increased F4/80+ cells (green) in Tlr9–/– mice. Shown are representative sections from 1 mouse of 4 per group. Dotted lines indicate the areas containing a PPD bead and the granuloma. Original magnification, ×200. (E) Quantitative real-time PCR (TaqMan) was performed to measure the transcript levels of chemokine in whole lung at day 4 after the initiation of lung granuloma. #P < 0.05; *P < 0.01. Data shown indicate mean ± SEM and are from a representative experiment of 3 independent experiments. Each time point indicates at least 4–6 mice per group.

Figure 4

Figure 4. BM-derived DCs but not BM-derived macrophages from Tlr9–/– mice exhibit decreased expression levels of Th17-related cytokines and dll4.

(A) BM-derived DCs from WT and Tlr9–/– mice were stimulated with CpG (1 μM) for 6 hours, and then quantitative real-time PCR was performed and expression levels of Notch ligands were evaluated. #P < 0.01. (B) BM-derived DCs and (C) BM-derived macrophages from Tlr9–/– and WT mice were stimulated with BCG (MOI = 0, 0.1, or 1.0) for 6 hours, then quantitative real-time PCR was performed to assess mRNA expression for indicated cytokines. *P < 0.05. (D) BM-derived DCs from WT or Tlr9–/– mice were stimulated with BCG (MOI = 1.0) or CpG (1 μM) for 24 hours. Flow cytometry was performed using a specific polyclonal Ab against dll4. Bars within histogram spans indicate cells gated on CD11c+ populations. Significant differences in MFI were seen when dll4 protein expression in DCs from WT mice was compared with that in DCs from Tlr9–/– mice at day 4. Data shown indicate mean ± SEM and are from a representative experiment of 3 independent experiments. Each time point represents at least 4 mice per group.

Figure 3

Figure 3. Lung DCs from Tlr9–/– mice exhibit decreased expression levels of both Th17-related cytokine and dll4.

Lung mDCs (CD11b+CD11c+) were purified from granulomatous lungs using cell sorter at days 0 and 4 after PPD-bead challenge. mRNA levels were quantitated as described in Methods. (A) Gene expression levels of IL-6, IL-10, IL-12p40, and IL-23p19 in lung DCs. *P < 0.03; **P < 0.01 compared with lung DCs from WT mice. (B) Gene expression levels of Notch ligands in lung DCs. *P < 0.03 compared with lung DCs from WT mice. (C) Gene expression levels of Notch ligands (dll1, dll4, Jagged-1, and Jagged-2) in whole lungs from WT mice during granuloma formation. (D) Gene expression of dll4 in whole lungs from WT and Tlr9–/– mice during granuloma formation. †P < 0.05. (E) The level of dll4 in lung mDCs (CD11b+CD11c+) from WT or Tlr9–/– mice was determined with flow cytometry using a specific polyclonal Ab against dll4. Significant differences in MFI were seen when dll4 protein expression in DCs from WT mice was compared with Tlr9–/– at day 4. Data shown indicate mean ± SEM and are from a representative experiment of 3 independent experiments. Each time point represents at least 4 mice per group.

Figure 5

Figure 5. Blockage of IL-6 increases granuloma size and abrogates Th17 phenotype.

(A) Histological appearance of pulmonary granulomas from WT mice treated with either control Abs (Cont Ab) or anti–IL-6 Abs at day 4 after granuloma development. H&E staining. Original magnification, ×40; ×400. (B) Morphometric analysis of pulmonary granuloma size from mice treated with either control Abs or anti–IL-6 Abs 4 days after initiation of lung granuloma development. *P < 0.01 compared with control. (C) Protein levels of IL-17 measured in whole lungs using a Luminex system. (D) Quantitative real-time PCR (TaqMan) was performed to measure the transcript levels of IL-17A, IL-17F, and IL-21 in whole lungs. †P < 0.03; ‡P < 0.01 compared with control. (E) FACS profile of CD4+ T cells from lungs was analyzed by intracellular staining of IL-17. Displayed in the dot plots are cells gated on CD4+ lymphocyte population by forward and side scattering. Numbers are percentages of IL-17– cells (left gate) and IL-17+ cells (right gate) in the CD4+ lymphocyte population. Shown are representative FACS plots from a total of 4 mice per group. (F) FACS analysis of intracellular staining of CD4+ cells for IFN-γ, IL-4, and IL-17. §P < 0.02 compared with control. Data represent mean ± SEM. Experiments were performed 3 times with similar results.

Figure 6

Figure 6. BM-derived DCs but not BM-derived macrophages promote the differentiation of Th17 cells from T cells restimulated with BCG.

T cells were isolated from lungs of either WT or Tlr9–/– mice and restimulated with DCs (A and B) or macrophages (C and D) isolated from WT or Tlr9–/– mice, as indicated. (A) IFN-γ production. **P < 0.01. (B) IL-17 production. (C) IFN-γ production. *P < 0.05. (D) IL-17 production. Data shown indicate mean ± SEM and are from a representative experiment of 3 independent experiments. Each point had 4 mice per group.

Figure 7

Figure 7. Passive immunization using Abs against dll4 increases granuloma size, abrogates the migration of DCs, and alters the expression of lung Th17-related cytokines and of CCL20.

(A) Histological appearance of pulmonary granulomas from WT mice treated with either control Ab or Abs directed against dll4 at day 4 after granuloma development. H&E staining. Original magnification, ×40; ×400. Experiments were performed 3 times with similar results. (B) Morphometric analysis of pulmonary granuloma size from mice treated with either control Abs or dll4 Abs after 4 days of lung granuloma development. *P < 0.01 compared with control. (C) FACS profiles for mDCs (CD11b+CD11c+) recovered from granulomatous lung cells isolated from day 4 lesions. †P < 0.03 compared with control. (D) Protein level of IL-17 was measured in whole lungs using a Luminex system. (E) Quantitative real-time PCR was performed on lung mRNA for the cytokines IFN-γ, IL-4, IL-17A, IL-17F, IL-6, and IL-21. *P < 0.01 compared with control. (F) Quantitative real-time PCR was performed on lung mRNA for the indicated chemokines. ‡P < 0.02 compared with control. Data shown are mean ± SEM from 5 mice and are from a representative experiment of 3 independent experiments. Each time point represents 5 mice per group.

Figure 8

Figure 8. The differentiation of Th17 cells from T cells restimulated with BCG was specifically regulated in a dll4-dependent manner.

(A–D) Lung CD4+ T cells were isolated from WT mice and stimulated with BCG-pulsed BM-derived DCs from either WT or Tlr9–/– mice. (E and F) Splenic CD4+ T cells were isolated from DO11.10 mice and stimulated with OVA-pulsed BM-derived DCs from either WT or Tlr9–/– mice. Cells were cocultured with recombinant dll4 (rdll4) or PBS controls (A and B) or with anti-dll4 Ab or control IgG (C–F). Supernatants were assayed for IFN-γ production (A, C, and E) or IL-17 production (B, D, and F). *P < 0.05 compared with WT DC. #P < 0.01 compared with PBS (A and B) or control Ab (C and D). Data shown are mean ± SEM and are from a representative experiment of 3 independent experiments. Each time point represents 4 mice per group.

Figure 9

Figure 9. Schematic representation of the TLR9-Notch ligand (dll4) on _Mycobacterium_-dependent granuloma formation.

DCs play an important role in inducing the differentiation of Th17 cells through the TLR9 effector pathway that upregulates the Notch ligand dll4. In vivo granuloma formation induced by BCG/Mycobacterium Ag demonstrates larger granuloma formation in Tlr9–/– mice with decreased numbers of Th17 cells and mDCs in the lungs when compared with lung granulomas from WT mice. Further, Tlr9–/– mice showed an increase in IL-10 with a concomitant decrease in Th17 cell–related cytokines (IL-17, IL-6, IL-21, IL-23, and TNF-α) and a decrease in the levels of the chemokines CCL20 and CCL22, important for DC migration, compared with levels in WT mice. The decreased expression of dll4 and the perturbation of the indicated cytokine and chemokine expression levels led to the abrogation of the Th17 phenotype in the Tlr9–/– mice with the concomitant increase in granuloma size. Accompanying these phenomena, there was a decrease in Th17 cells and mDCs in the lungs of Tlr9–/– mice and an increase in lung macrophages. L, lymphocyte; M, macrophage.

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References

    1. Boros D.L. Granulomatous inflammations. Prog. Allergy. 1978;24:183–267. - PubMed
    1. El-Zammar O.A., Katzenstein A.L. Pathological diagnosis of granulomatous lung disease: a review. Histopathology. 2007;50:289–310. doi: 10.1111/j.1365-2559.2006.02546.x. - DOI - PubMed
    1. Dye, C., et al. 2007. Global tuberculosis control: surveillance, planning, financing. WHO Report 2007. C. Dye, K. Floyd, M. Hosseini, A. Piatek, and C. Watt, editors. WHO. Geneva, Switzerland. 277 pp.
    1. Chensue S.W., Ruth J.H., Warmington K., Lincoln P., Kunkel S.L. In vivo regulation of macrophage IL-12 production during type 1 and type 2 cytokine-mediated granuloma formation. . J. Immunol. 1995;155:3546–3551. - PubMed
    1. Chensue S.W., et al. Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor. Am. J. Pathol. 1994;145:1105–1113. - PMC - PubMed

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