The chemokine MIP1alpha/CCL3 determines pathology in primary RSV infection by regulating the balance of T cell populations in the murine lung

Abstract

Background: CD8 T cells assist in the clearance of respiratory syncytial virus (RSV) infection from the lungs. However, disease after RSV infection is in part caused by excessive T cell activity, and a balance is therefore needed between beneficial and harmful cellular immune responses. The chemokine CCL3 (MIP1alpha) is produced following RSV infection and is broadly chemotactic for both T cells and natural killer (NK) cells. We therefore investigated its role in RSV disease.

Methodology/principal findings: CCL3 was produced biphasically, in both the early (day 1) and late (day 6-7) stages of infection. CCL3 depletion did not alter the recruitment of natural killer (NK) cells to the lungs during the early stage, but depletion did affect the later adaptive phase. While fewer T cells were recruited to the lungs of either CCL3 knockout or anti-CCL3 treated RSV infected mice, more RSV-specific pro-inflammatory T cells were recruited to the lung when CCL3 responses were impaired. This increase in RSV-specific pro-inflammatory T cells was accompanied by increased weight loss and illness after RSV infection.

Conclusions/significance: CCL3 regulates the balance of T cell populations in the lung and can alter the outcome of RSV infection. Understanding the role of inflammatory mediators in the recruitment of pathogenic T cells to the lungs may lead to novel methods to control RSV disease.

Figure 1. CCL3 is produced biphasically following RSV infection.

Mice were infected intranasally (i.n.) with 5×10⁵ PFU RSV. Weight change after RSV infection (A). Time course of cell recruitment (B), CCL3 RNA (C) protein (D) in the lung and RSV M2 (_82-90) peptide stimulated CD3 T cells stained for intracellular CCL3 (E). Points represent mean of n≥4 mice ± SEM, p<0.05, ** p<0.01, *** p<0.001.

Figure 2. CCL3 is not involved in the recruitment of NK cells during RSV infection.

Chemotaxis of enriched NK cells to recombinant CCL3 or supernatant (supe) from RSV, UV-RSV, or control exposed macrophages; anti-CCL3, anti-TNF or control antibody was used to assess the chemotactic effect of specific mediators (A). BALB/c mice were treated on day −1 and +1 of RSV infection with anti-CCL3 (white bars) or control Ig (black bars). Lung cell number (B), percentage lung NK cells (C) and lung viral load (D) on day 4 post infection. Bars represent n≥3 ± SEM, ** p<0.01.

Figure 3. CCL3^−/− knockout mice have reduced total cellular recruitment without altering RSV specific cell number.

CCL3^−/− (white bars) or wild type C57BL/6 control (black bars) mice were infected i.n. with RSV. Lung cell number (A) and percentage of lung CD4 and CD8 + cells on day 7 p.i. (B). RSV specific IFNγ secretion measured by lung cell ELISPOT at day 7 p.i. (C). Points represent n≥4 mice ± SEM, * p<0.05, ** p<0.01.

Figure 4. CCL3 depletion reduces cell recruitment without changing RSV specific cell number.

BALB/c mice were treated on day −1 and +1 of RSV infection with anti-CCL3 (white bars) or control Ig (black bars). Lung cell numbers (A) and percentage of lung CD4 and CD8+ T cells on day 7 p.i. (B). Proportion (C) and total number (D) of RSV specific T cells in lung measured using RSV (M2) specific pentamer. Points represent n≥4 mice ± SEM, * p<0.05, ** p<0.01.

Figure 5. CCL3 depletion increases pathology following RSV infection.

BALB/c mice were treated on day −1 and +1 of RSV infection with anti-CCL3 (white bars/squares) or control Ig (black bars/triangles). Weight change after RSV infection (A). Lung cells were stimulated with the RSV M2 (_82-90) peptide to determine RSV specific responses on day 7 p.i.; percentage (B) and total (C) TNF producing CD8 cells, percentage (D) and total (E) IFNγ producing CD8 cells. Points represent n≥4 mice ± SEM, * p<0.05, ** p<0.01, *** p<0.001.

Figure 6. TNF depletion decreases pathology following RSV infection.

Weight change after RSV infection (A). BAL cell number (B). Percentage of lung CD4⁺ and CD8⁺ cells (C), RSV M2 pentamer specific lung CD8 T cells (D), measured on day 7 p.i. using flow cytometry. Lung RSV titer on day 4 p.i. measured by plaque assay (E). Points represent n≥4 mice ± SEM, * p<0.05, ** p<0.01, *** p<0.001.