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 4

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

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Lung DCs from Tlr9–/– mice exhibit decreased expression levels of both T...
(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.