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. 2006 Jun;2(6):e53.
doi: 10.1371/journal.ppat.0020053. Epub 2006 Jun 9.

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia

Ronan Le Goffic  1 , Viviane Balloy, Micheline Lagranderie, Lena Alexopoulou, Nicolas Escriou, Richard Flavell, Michel Chignard, Mustapha Si-Tahar
Affiliations

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia

Ronan Le Goffic et al. PLoS Pathog. 2006 Jun.

Abstract

Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3(-/-) mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3-/- animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Time-course of Dynamic Parameters in Wild-Type Mice Infected by a Lethal IAV Challenge
Male C57Bl/6 mice were infected intranasally with 300 pfu of IAV and different parameters were analyzed during the course of infection. (A) Survival of mice. (B) Body weight changes. (C) Leukocyte recruitment into the airways ▪, all leukocytes; •, polymorphonuclear cells; ▴, mononuclear cells). (D and E) RANTES and IL-6 production in BAL fluids. (F) Total protein amount in BAL fluids as an index of alveolocapillar permeability. All these results are the mean ± SD values obtained from three distinct animals and are representative of three independent experiments.
Figure 2
Figure 2. Reduced Lethality in IAV-Infected TLR3−/− Mice
(A) Pulmonary expression and regulation of TLR3 during influenza infection. Mice were either noninfected (Ctrl) or infected with 300 pfu of IAV by intranasal route and whole lungs were harvested at 2, 3, 4, and 9 d postinfection. Total RNA was extracted and TLR3 mRNA was analyzed by RT-PCR; a representative result of two is shown. β-Actin mRNA was assessed as a control for RNA loading. (B) Lethality induced by IAV in TLR3 −/− and MyD88 −/− mice in comparison with wild-type mice. Age-matched TLR3−/−, MyD88−/−, and wild-type male mice received intranasally 300, 100, or 30 pfu of IAV. Wilcoxon test for comparisons of Kaplan-Meier survival curves indicated a significant increase in the survival of TLR3−/− mice compared to that of wild-type animals (****p < 0.0001, *p < 0.05) but not to that of MyD88−/− mice.
Figure 3
Figure 3. Reduced Inflammation in IAV-Infected TLR3−/− Mice
(A) Basal respiratory function of wild-type versus TLR3−/− mice before (day 0) and 3 d post–viral infection. This was measured using a barometric plethysmographic chamber and is expressed as Penh (cf. Materials and Methods for details), the increase of which is an indicator of deterioration changes in airway mechanics (**p < 0.01). (B–D) BAL fluids levels of total protein, RANTES, and IL-6 in wild-type versus TLR3−/− mice before (day 0) and 3 d post–viral infection (*p < 0.05, ***p < 0.001, ****p < 0.0001). Histograms are the mean ± SD values obtained from five animals and are representative of at least three independent experiments.
Figure 4
Figure 4. Distinct Inflammatory Profile in Wild-Type and TLR3−/− Mice Infected by a Lethal IAV Challenge
(A) BAL fluids levels of inflammatory mediators at day 9 postinfection. The data were normalized to internal positive controls spotted on the same protein array membrane and are expressed as relative units. (Inset) Example of a protein array blot probed with BAL collected from wild-type and TLR3−/− mice. Relevant spots are highlighted: (1) G-CSF, (2) IL-12p40/p70, (3) IFN-γ, and (4) RANTES. (B) BAL fluids levels of IFN-γ in wild-type (□) and TLR3−/− (▪) mice before (day 0) and 4, 7, and 9 d postinfection by 300 pfu IAV (*p < 0.05). Histograms are the mean ± SD values obtained from five animals and are representative of three independent experiments. (C) Examination of lungs isolated from noninfected wild-type and TLR3−/− mice (lower panel) and at day 9 post–viral infection (upper panel).
Figure 5
Figure 5. Viral Load in the Lungs of IAV-Infected Mice
Viral load in TLR3−/− and wild-type mice challenged intranasally by 300 pfu of IAV. (A) Results are the mean ± SD values obtained from four animals at days 2 to 4 postinfection and 17 mice at day 9 postinfection. They are expressed as RNA copies normalized to β-actin expression levels, as determined by real-time PCR. (B) Results are the mean ± SD values obtained from nine wild-type and eight TLR3−/− mice at day 9 postinfection. They are expressed as pfu, as determined by standard plaque assay [21].
Figure 6
Figure 6. Wild-Type and TLR3−/− Mice Raise a Contrasted Leukocyte Content in Their Lungs after IAV Challenge
BAL cells were collected from IAV-infected mice at day 9 postinfection. To characterize the recovered leukocyte cell types, polymorphonuclear neutrophils (PMN, Gr1+, CD11b+), macrophages (Mϕ, F4/80+), CD4+ T lymphocytes (CD4), and CD8+ T lymphocytes (CD8) were stained with fluorescently labeled specific antibodies. Far right and far left dot-plots: Representative BAL cell composition of naive wild-type and TLR3−/− mice. Right and left dot-plots: Representative BAL cell composition of wild-type mice and TLR3−/− mice at day 9 postinfection by 300 pfu of IAV. Central histograms: Results are the mean ± SD obtained from 12 wild-type mice and seven TLR3−/− mice (**p < 0.01, ***p < 0.001, ****p < 0.0001).
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