TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4
Toshihiro Ito, Matthew Schaller, Cory M. Hogaboam, Theodore J. Standiford, Matyas Sandor, Nicholas W. Lukacs, Stephen W. Chensue, Steven L. Kunkel
Toshihiro Ito, Matthew Schaller, Cory M. Hogaboam, Theodore J. Standiford, Matyas Sandor, Nicholas W. Lukacs, Stephen W. Chensue, Steven L. Kunkel
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Research Article Infectious disease

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

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.

Authors

Toshihiro Ito, Matthew Schaller, Cory M. Hogaboam, Theodore J. Standiford, Matyas Sandor, Nicholas W. Lukacs, Stephen W. Chensue, Steven L. Kunkel

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Figure 3

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

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BM-derived DCs but not BM-derived macrophages from Tlr9–/– mice exhibit ...
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.