Indeed, unlike the results in their study, at the peak of lesions development we observed, by quantitative PCR, a higher expression of IFN-γ mRNA in the draining lymph nodes of C57BL/6 mice than in TLR9−/− mice. Similar differences in the IFN-γ responses between C57BL/6 and TLR9−/− mice were also seen by enumerating the IFN-γ-producing CD4+ cells by intracellular staining; 9% of CD4+ cells produced IFN-γ in C57BL/6 mice vs 3% in TLR9−/− mice 17 days after infection. However, after that time, a similar increase in IFN-γ transcripts, compared with uninfected mice of the corresponding strains, was seen in C57BL/6 and TLR9−/− mice.
We agree that a drastic impairment of Th1 cell development in TLR9-deficient mice should have resulted in a failure to ultimately control L. major infection, which was not the case in both studies (1, 2). Nevertheless, the transient inhibition of the CD4+ Th1 response observed in our study could, at least in part, account for the delayed healing of lesions in TLR9−/− mice observed in both studies (1, 2). The enhanced expression of arginase 1 was assumed by Liese et al. to account for the aggravated course of disease in TLR9−/− mice. However, the arginase 1 mRNA expression was still 10-fold higher in the infected footpads of TLR9−/− mice than in WT mice, even at the time of complete healing of lesions in these mice.
To test for a possible effect of TLR9 signaling on IFN-γ production by CD4+ cells, we used an in vitro system in which we compared the capacity of bone marrow dendritic cells (BMDCs) from either C57BL/6 or TLR9−/− mice to induce IFN-γ production by specifically activated CD4+ cells. The addition of ligands of TLR9 in cultures resulted in enhanced production of IFN-γ by CD4+ cells only in the presence of wild-type BMDCs. Although, as mentioned by Bogdan and Schleicher, “this in vitro system essentially tests the adjuvanticity of TLR9,” the results clearly showed a significant enhancement of IFN-γ production by CD4+ cells as a result of TLR9 signaling on dendritic cells (DCs). In addition, they indicate that this effect is unlikely to require NK cells. It is clear that we did not pretend to “imitate cutaneous leishmaniasis” using this system, which was designed only to test whether or not TLR9 signaling on DCs, including through L. major DNA, could directly act on IFN-γ production by CD4+ cells.
- Copyright © 2009 by The American Association of Immunologists, Inc.