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 5

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

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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.