TLR9 signaling is required for generation of the adaptive immune protection in Cryptococcus neoformans-infected lungs

Abstract

To determine whether TLR9 signaling contributes to the development of the adaptive immune response to cryptococcal infection, wild-type (TLR9+/+) and TLR9 knockout (TLR9-/-) BALB/c mice were infected intratracheally with 10(4) C. neoformans 52D. We evaluated 1) organ microbial burdens, 2) pulmonary leukocyte recruitment, 3) pulmonary and systemic cytokine induction, and 4) macrophage activation profiles. TLR9 deletion did not affect pulmonary growth during the innate phase, but profoundly impaired pulmonary clearance during the adaptive phase of the immune response (a 1000-fold difference at week 6). The impaired clearance in TLR9-/- mice was associated with: 1) significantly reduced CD4(+), CD8+ T cell, and CD19+ B cell recruitment into the lungs; 2) defects in Th polarization indicated by altered cytokine responses in the lungs, lymphonodes, and spleen; and 3) diminished macrophage accumulation and altered activation profile, including robust up-regulation of Arg1 and FIZZ1 (indicators of alternative activation) and diminished induction of inducible nitric oxide synthase (an indicator of classical activation). Histological analysis revealed defects in granuloma formation and increased numbers of intracellular yeast residing within macrophages in the lungs of TLR9-/- mice. We conclude that TLR9 signaling plays an important role in the development of robust protective immunity, proper recruitment and function of effector cells (lymphocytes and macrophages), and, ultimately, effective cryptococcal clearance from the infected lungs.

Figure 1

Effect of TLR 9 deletion on fungal burdens in the lungs and spleens. TLR9+/+ and TLR9−/− mice were inoculated intratracheally with 10⁴ C. neoformans 52D. A: The pulmonary fungal burden was evaluated at weekly intervals from 1 to 6 wpi. B: Spleen fungal burden was evaluated at 3, 4, 5, and 6 wpi. C. neoformans loads were evaluated via CFU assays performed on serially diluted samples. Data, pooled from three separate matched experiments, are shown as the mean log₁₀ CFU per organ ± SEM. N = 10 and above for each of the analyzed time points; *P < 0.05 in comparison with the matching TLR9+/+ mouse result.

Figure 2

Effect of TLR9 deletion on magnitude of inflammatory response and recruitment of leukocyte subsets into the C. neoformans-infected lungs. Lungs were collected from uninfected (week 0) and C. neoformans-infected TLR9+/+ and TLR9−/− mice at 1, 2, 3, and 5 wpi and dispersed enzymatically. Leukocytes were isolated from individual mice and enumerated by a hemacytometer. Lung leukocyte subsets were assessed by microscopic evaluation of relative frequencies on stained cytospun slides analyzed under the microscope and calculated (see Materials and Methods). Data, pooled from three parallel experiments, are shown as the mean number ± SEM. N = 3 for uninfected controls and at least ten for infected mice at each of the analyzed time points; *P < 0.05 in comparison with the matching TLR9+/+ mouse result.

Figure 3

Effect of TLR9 deletion on lymphocyte subsets in the C. neoformans-infected lungs. Lung leukocytes were isolated from C. neoformans-infected TLR9+/+ and TLR9−/− mice at 3 wpi. A: Frequency of lymphocyte subsets were determined by staining samples with fluorochrome-conjugated antibodies specific for CD4⁺, CD8+, and CD19+ lymphocytes. B: Mean frequencies of lymphocytes in total lung leukocyte isolate (CD45+) population and CD4⁺, CD8+, and CD19+ in a lymphocyte subsets. C: Total numbers of CD4⁺, CD8⁺, and CD19⁺ lymphocytes/lung. Representative plots out of two separate experiments were shown. N = 7 and above for the quantitative data; *P < 0.05 in comparison with the matching TLR9+/+ mouse result.

Figure 4

Effect of TLR9 deletion on cytokine mRNA expression pattern in leukocytes isolated from C. neoformans infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total leukocyte RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative gene expression ± SEM. Data were pooled from two parallel experiments. N = 3 for uninfected controls and at least seven for infected mice at each of the analyzed time points. For the comparisons between TLR9+/+ versus TLR9−/− mice, *P < 0.05, or the appropriate P value is listed above the bars; **P < 0.05 in comparison with the respective uninfected control mouse result.

Figure 5

Effect of TLR9 deletion on cytokine protein production by leukocytes isolated from C. neoformans-infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected mice at 3 wpi and cultured for 24 hours at 5 × 10⁶ cells/ml. Cytokine levels were evaluated by ELISA in cell culture supernatants. Bars represent mean cytokine concentration ± SEM (pg/ml). Data were pooled from two parallel experiments. N = 3 for uninfected controls and at least 6 for infected mice at each of the analyzed time points. For the comparisons between TLR9+/+ versus TLR9−/− mice, *P < 0.05, or the appropriate P value is listed above the bars; **P < 0.05 in comparison with the respective uninfected control mouse result.

Figure 6

Effect of TLR9 deletion on pulmonary lymph node and spleen polarization. A: Pulmonary lymph nodes were collected from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative mRNA expression ± SEM. Data were pooled from two parallel experiments. N = 2 for uninfected controls and 7 for infected mice; *P < 0.05 in comparison with the respective TLR9+/+ result. B: Splenocytes from each infected mouse were isolated, diluted to 5 × 10⁶ cells/ml, and cultured with/without heat killed C.neoformans in a ratio of 1:2 in 24-well plates with 2 ml of complete RPMI medium at 37°C and 5% CO₂ for 48 hours. Cytokine levels were evaluated by ELISA in cell culture supernatants. The antigen-specific cytokine production was calculated as a net gain of cytokine level compared with unstimulated control of the same sample. The values were shown as cytokine concentration ± SEM (pg/ml). N = 5 for infected mice. *P < 0.05 in comparison with the matching TLR9+/+ mouse result; **P < 0.05 in comparison with the respective uninfected control mouse result.

Figure 7

Effect of TLR9 deletion macrophages activation profile in C. neoformans infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected TLR9+/+ and TLR9−/− mice at 3 wpi. Macrophage population was enriched by 90-minute adherence and removal of nonadherent cells. RNA was extracted and analyzed as described above to quantify alternative (Arg1 and Fizz1) versus classical (inducible nitric oxide synthase) macrophage activation gene expression. The values were normalized to GAPDH mRNA levels and were expressed as relative gene expression ± SEM. Data were pooled from three separate matched experiments. N = 3 for uninfected mice and at least nine for each of the analyzed time points. *P < 0.05 in comparison with the matching TLR9+/+ mouse result; **P < 0.05 in comparison with the respective uninfected control mouse result.

Figure 8

Effect of TLR9 deletion on morphological pattern of pulmonary inflammation and pathological lesions in C. neoformans infected lungs. Lungs from infected TLR9−/− and TLR9+/+ mice were perfused with buffered formalin, fixed, and processed for histology at 6 wpi. Representative photomicrographs of H&E + mucicarmine stained slides taken at 10× (A, B) and 40× (C, D) objective power. Note that numerous C. neoformans organisms are widespread in the lungs of TLR9−/− mice loose leukocyte infiltrate extended into the wide areas (A) and fewer cryptococcal cells contained in dense inflammatory infiltrates and a clear margin between healthy and sick section (arrows, B). The high power images (bottom) show enlarged macrophages harboring multiple cryptococcal cells (orange pointers) in TLR9−/− mice lungs (C) and smaller macrophages with remnants of ingested/degraded yeasts (yellow pointers) in TLR9+/+ mice (D).