CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection

Abstract

Infection with influenza virus induces severe pulmonary immune pathology that leads to substantial human mortality. Although antiviral therapy is effective in preventing infection, no current therapy can prevent or treat influenza-induced lung injury. Previously, we reported that influenza-induced pulmonary immune pathology is mediated by inflammatory monocytes trafficking to virus-infected lungs via CCR2 and that influenza-induced morbidity and mortality are reduced in CCR2-deficient mice. In this study, we evaluated the effect of pharmacologically blocking CCR2 with a small molecule inhibitor (PF-04178903) on the entry of monocytes into lungs and subsequent morbidity and mortality in influenza-infected mice. Subcutaneous injection of mice with PF-04178903 was initiated 1 d prior to infection with influenza strain H1N1A/Puerto Rico/8/34. Compared with vehicle controls, PF-04178903-treated mice demonstrated a marked reduction in mortality (75 versus 0%) and had significant reductions in weight loss and hypothermia during subsequent influenza infection. Drug-treated mice also displayed significant reductions in bronchoalveolar lavage fluid total protein, albumin, and lactose dehydrogenase activity. Administration of PF-04178903 did not alter viral titers, severity of secondary bacteria infections (Streptococcus pneumoniae), or levels of anti-influenza-neutralizing Abs. Drug-treated mice displayed an increase in influenza nucleoprotein-specific cytotoxic T cell activity. Our results suggest that CCR2 antagonists may represent an effective prophylaxis against influenza-induced pulmonary immune pathology.

Figure 1

Monocytes, dendritic cells, and CD11b⁺Gr-1⁺ DI cells are decreased in the lungs of PF-04178903-treated mice during influenza infection. Mice were treated with PF-04178903 or PBS starting immediately after virus inoculation. On day 5 of influenza infection, mice were sacrificed. Cells from BAL (A, C, E, G) and lung digests (B, D, F, H) were harvested from PF-04178903-treated and PBS-treated mice and analyzed by flow cytometry. Gate labels: DC, dendritic cells; DN, double negative (CD11c^- MHCII^-) cells; DI, double intermediate (CD11c^intMHCII^int); MAC, total macrophages; AM, alveolar macrophages; exMAC, exudate macrophages; mono: Gr-1⁺ monocytes. Results shown are from individual mice representative of two separate experiments. Numbers shown represent the percentage of cells within the gates. (I&J) Total cell numbers (per mouse) of individual cell types obtained from BAL (I) or lung digests (J) of PF-04178903-treated and PBS-treated mice were calculated. Bars represent the mean ± SD for 3 mice per group. *, p < 0.05; **, p<0.005; ***, p<0.0005 by student's t test.

Figure 2

The number of inflammatory monocytes in the blood of control or PF-treated mice after influenza infection. Mice were treated with PF-04178903 (PF) or PBS (Ctrl) starting one day before influenza virus inoculation. On days 3, day 5, and day 7 of influenza infection, mice were sacrificed and their blood collected and subjected to flow cytometric analysis. (A) The profile of CD115 vs. Gr-1 staining of blood CD11b+Ly6G^- cells is shown. (B) Total cell number of blood inflammatory monocytes in PF-treated and Ctrl mice. Data is representative of two independent experiments. Bars represent mean ± SD for 4 mice per group. *, p < 0.05 by student's t test.

Figure 3

Influenza-induced lung injury is reduced in PF-04178903-treated mice. Mice were treated with PF-04178903 (PF) or PBS (Ctrl) starting one day before influenza virus inoculation. On day 5 and 7 of influenza infection, mice were sacrificed and their BAL fluid collected and assayed for LDH activity (A), albumin concentration (B), and total protein concentration (C). The value of albumin in BALF from naïve mice is 150 mu;g/ml. The values of LDH activity and total protein concentration for naïve BALF are both near zero. Data is representative of two independent experiments. Bars represent the mean ± SD for 4 or 5 mice per group. p value is calculated by student's t test.

Figure 4

Influenza-induced morbidity and mortality are reduced by PF-04178903 prophylaxis. Mice were treated s.c. with PF-04178903 or PBS starting one day before (A), or immediately following (B), influenza inoculation and treated twice per day until day 10. Mortality, weight loss, and rectal temperature was monitored daily until day 22. Data is representative of two independent experiments. n = 6∼10 mice. Value of p for the survival curve is calculated by log rank test. P values for overall weight loss and temperature curves were calculated by ANOVA repeated measures. *, p < 0.05 by student's t test comparing individual time points.

Figure 5

Neither influenza viral titers nor bacterial loads during secondary streptococcus pneumonae infection are increased in PF-04178903-treated mice. Mice were treated with PF-04178903 or PBS starting one day before influenza inoculation. (A) On infection days 3, 5, 7, and 9, mice were sacrificed and their lung homogenates harvested and subjected to virus plaque forming assays. (B) On day 5 of influenza infection, control and PF-04178903-treated mice were infected with S. pneumonae intranasally, and sacrificed 2 days later. Lung homogenates were serially diluted and plated on agar with sheep blood, and the number of colonies counted. Data is representative of two independent experiments. Bars represent the mean ± SD for 4∼5 mice per group.

Figure 6

Adaptive immune responses to influenza virus are not impaired in PF-04178903-treated mice. Mice were treated with PF-04178903 or PBS starting one day before influenza inoculation. (A) For virus neutralization assays, mice were treated twice per day until day 10 of infection. Sera from infected mice was collected on days 21 and 28, and assayed for viral neutralizing antibodies. (B&C) For in vivo CTL assays, recipient mice were treated twice per day until day 9 when they received CD45.1 donor splenocytes pulsed with virus nucleoprotein (NP) peptide (labeled CFSE^high) or OVA peptide (labeled CFSE^low). Recipient mice were euthanized 6 hours after adoptive transfer and their spleens and LN harvested. Naïve mice were used as controls. Cells from draining mediastinal LN and spleens were analyzed for the presence of CFSE^high and CFSE^low target cell populations. The flow data from spleens is shown in (B). To quantify in vivo cytotoxicity, the elimination of the NP-pulsed CFSE^high population was monitored and the percentage of specific lysis (C) was determined as described in materials and methods. Data is representative of two independent experiments. Bars represent the mean ± SD for 4∼5 mice per group. *, p < 0.05 by student's t test.