TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection

Abstract

Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8(+) T cells in the infected lung, because blocking their recruitment in CCR2(-/-) mice decreases the numbers of CD8(+) effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8(+) T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.

Fig. 1.

Increased pathology correlates with greater tipDC recruitment. (A and B) Host morbidity presented as mean of percentage original body weight ± SEM after infection with PR8 (lethal H1N1) or x-31 (sublethal H3N2) (A) and VN1203 (lethal H5N1) or HK213 (sublethal H5N1) (B). In general, mice infected with x-31 lose less weight than mice infected with PR8 (asterisks, simple effects ANOVA, P < 0.05). For the H5N1 viruses, HK213-infected mice lose less weight than VN1203-infected mice (virus effect, P < 0.0001). Results are representative of at least 3 independent experiments (n ≥ 5). (C) Representative FACS plots for total lung homogenate stained with mAbs to Ly6c and CD11b on day 5 after infection with PR8, x-31, VN1203, or HK213. The gated population (circled in red) from each plot represents tipDCs. (D and E) Mean number of tipDCs ± SEM recovered from total lung homogenate over time after infection with PR8 or x-31 (D) and VN1203 or HK213 (E). Sublethal infections resulted in fewer tipDCs in the lungs on days marked with an asterisk (simple effects ANOVA, P < 0.03). Results are representative of at least 3 independent experiments (n ≥ 5).

Fig. 2.

tipDCs fail to accumulate in the lungs of CCR2^−/− animals. (A) Representative FACS plots of total lung homogenate from either CCR2^+/+ (B6) or congenic CCR2^−/− animals stained with mAbs to Ly6c and CD11b on day 5 after infection with 10² pfu of PR8 (sublethal infection). (B) Host morbidity presented as mean of percentage original body weight ± SEM after infection with 10³ pfu of PR8 (lethal infection). Regression analysis did not reveal a significant difference in weight loss between the two strains of mice (strain effect, P = 0.22; interaction effect, P = 0.07). Results are representative of two independent experiments (n ≥ 5). (C) Elimination of tipDC recruitment to the lungs does not protect against lethal influenza challenge. Results of the Mantel–Cox test showed no statistically significant difference between groups (P = 0.66). Results are representative of 3 independent experiments (n ≥ 5).

Fig. 3.

tipDCs present antigen to CD8⁺ T cells in the lungs. (A) CCR2^−/− animals have significantly fewer CD8⁺ T cells specific for the influenza D^bPA_224–233, K^bPB1_703–711, and D^bPB1-F2_62–70 epitopes. Results are representative of 2 independent experiments (n ≥ 5) and were analyzed by using the two-tailed Student's t test (P values are indicated). (B) There is no statistically significant difference in the number of antigen-specific CD8⁺ T cells between CCR2^+/+ and CCR2^−/− animals in the draining lymph node on day 7 after infection with 10⁵ pfu of x-31. Results are representative of two independent experiments (n ≥ 5) and were analyzed by using the two-tailed Student's t test (P > 0.05 for each comparison). (C) tipDCs isolated from the BAL wash on day 6 after infection present the equivalent of 40 pmol per cell of D^bPB1-F2_62–70 epitope to the D^bPB1-F2_62–70 epitope-specific hybridoma. Bars represent the mean ± SEM of triplicate wells, and the results were analyzed by using the two-tailed Student's t test (P value indicated). (D) tipDCs isolated from the BAL wash of animals infected with the intact x-31 virus (but not the D^bNP_366–374- and D^bPA_224–233-disrupted DKO virus) were sufficient to rescue the D^bNP_366–374 and D^bPA_224–233 epitope-specific CD8⁺ T cell response when transferred into the lungs of CCR2^−/− animals on day 1 after infection (n = 5). Results were analyzed by using a single-factor ANOVA and Tukey's posthoc test (same letter, P > 0.05; different letters, P < 0.05).

Fig. 4.

Pioglitazone protects from lethal influenza challenge. Prophylactic treatment with pioglitazone significantly decreases host morbidity, presented as mean percentage original body weight ± SEM and representative of 3 independent experiments (n ≥ 5) (interaction effect, P < 0.001; asterisks mark significant simple effects, P < 0.001) (A), increases host survival (Mantel–Cox, P = 0.005) after lethal influenza challenge (10³ pfu of PR8) of the pioglitazone-treated group (n = 16) versus PBS-treated controls (n = 23) (B), decreases MCP-1 (CCL2) and MCP-3 accumulation in the BAL wash on days 3 (C) and 6 (D) after infection with 10³ pfu of PR8, and decreases tipDC accumulation in the lungs (E). Results are representative of 2 independent experiments (n ≥ 5) and were analyzed with the two-tailed Student's t test (P values are indicated).