Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus - PubMed (original) (raw)

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

L M Haynes et al. J Virol. 2001 Nov.

Abstract

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

PubMed Disclaimer

Figures

FIG. 1

Decreased pulmonary infiltration of NK cells and CD14+ cells in RSV-infected TLR4null mice. Flow-cytometric analysis of BAL cell subsets from TLR4-deficient (TLR4null) and TLR4-expressing (TLR4wt) mice infected i.n. with the A2 strain of RSV virus (RSV) or the HKx31 influenza A virus (FLU). BAL samples were stained with antibodies against NK cells (A and D), CD14+ monocytes/macrophages (B and E), and PMNs (C and F). Data are presented as the mean number of cells/lung ± standard error of the mean at days 5, 7, and 11 p.i. from three independent experiments. Asterisks indicate a significant difference (P < 0.05) between TLR4null and TLR4wt mice.

FIG. 2

Decreased expression of IL-12 by BAL cells after RSV infection. BAL samples from RSV- or influenza virus-infected TLR4wt and TLR4null mice were harvested and examined for intracellular IL-12 expression (x axis). IL-12 expression at day 5 post-RSV infection for TLR4wt and TLR4null samples is shown in panels A and B, respectively. IL-12 expression at day 5 post-influenza virus infection for TLR4wt and TLR4null samples is shown in panels E and F, respectively. IL-12 expression at day 11 post-RSV infection for TLR4wt and TLR4null is shown in panels C and D, respectively. IL-12 expression at day 11 post-influenza virus infection for TLR4wt and TLR4null samples is shown in panels G and H, respectively_._

FIG. 3

Impaired NK cell activation in TLR4null mice after RSV infection. BAL samples from TLR4null and TLR4wt mice were harvested 7 days post-RSV or -influenza virus (FLU) infection. NK lytic activity against YAC-1 target cells was determined. Results are expressed as the mean of three independent experiments ± the standard error of the mean. Asterisks indicate a significant difference (P < 0.05) between TLR4null and TLR4wt mice. E:T Ratio, effector-to-target ratio.

FIG. 4

RSV-induced NK cytotoxicity is perforin dependent. Seven days p.i., BAL samples from TLR4null (open bar) and TLR4wt (closed bar) mice were examined following RSV infection. Samples were purified for NK cells (DX5+) (80 to 90% enrichment) by positive selection using the MACS separation system. NK lytic activity against YAC-1 target cells was assessed in the presence or absence of EGTA-MgCl2+ at effector-to-target ratios of 40:1 (presented), 20:1, 10:1, and 5:1 (data not shown). The asterisk indicates a significant difference (P < 0.05) between TLR4null and TLR4wt mice. The results are representative of two independent experiments.

FIG. 5

Addition of IL-12 enhances NK-mediated cytotoxicity in RSV-infected TLR4null mice. BAL samples from TLR4null and TLR4wt mice were examined for cytotoxicity 7 days post-RSV infection. Effector BAL cells were cultured in the presence of 2 ng of IL-12/ml for 24 h. NK lytic activities against YAC-1 target cells at effector-to-target ratios of 40:1 (presented), 20:1, 10:1, and 5:1 (data not shown) were analyzed. A representative experiment is shown.

FIG. 6

Delayed RSV clearance in TLR4null mice. The lungs of TLR4null and TLR4wt mice were harvested at days 3, 5, 7, and 11 p.i. with RSV (A) or influenza virus (FLU) (B). The asterisk indicates a significant difference (P < 0.01) between TLR4null and TLR4wt mice. The results are expressed as mean log10 PFU/g ± the standard error of the mean from four separate experiments. In each experiment, three to five mice per group at each time point were analyzed.

FIG. 7

Kinetics of CTL activity in RSV-infected TLR4null mice. TLR4wt and TLR4null mice were sacrificed on days 5 and 11 post-RSV infection. Data are values for percent specific lysis at an effector-to-target ratio of 50:1 and are representative of two independent experiments.

References

1. Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, Akira S. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 1998;9:143–150. - PubMed
1. Aliprantis A O, Yang R B, Mark R B, Suggett S, Devaux B, Radolf J D, Klimpel G R, Godowski P, Zychlinsky A. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science. 1999;285:732–736. - PubMed
1. Chang L, Gusewitch G A, Chritton D B W, Folz J C, Lebeck L K, Nehlsen-Cannarella S L. Rapid flow-cytometric assay for the assessment of natural killer cell activity. J Immunol Methods. 1993;166:45–54. - PubMed
1. Connors M, Collins P L, Firestone C Y, Murphy B R. Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived. J Virol. 1991;65:1634–1637. - PMC - PubMed
1. Duke R C. Methods of analyzing chromatin changes accompanying apoptosis of target cells in killer cell assays. In: Campbell K, Colonna M, editors. Natural killer cell protocols: cellular and molecular methods. Totowa, N.J: Humana Press; 2000. pp. 125–145. - PubMed

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus - PubMed (original) (raw)