Activation of interferon response through toll-like receptor 3 impacts viral pathogenesis and pulmonary toll-like receptor expression during respiratory syncytial virus and influenza infections in the cotton rat Sigmodon hispidus model

Abstract

Interferon (IFN) therapy in humans often causes flu-like symptoms by an unknown mechanism. Poly ICLC is a synthetic dsRNA and a Toll-like receptor 3 (TLR3) agonist with a strong IFN-inducing ability. In this work, we analyzed the effect of poly ICLC on pulmonary responses to influenza and respiratory syncytial virus (RSV) infections in the cotton rat (Sigmodon hispidus) model. Viral replication, pulmonary inflammation, and expression of IFN, TLR, and chemokines were monitored and compared. Antiviral effect of poly ICLC against influenza virus and RSV was best achieved at high poly ICLC concentrations that, in the absence of virus infection, induced a strong IFN response. The antiviral doses of poly ICLC, however, also increased lung inflammation, an unexpected finding because of the reported poly ICLC safety in BALB/c mice. Similarly, in contrast to murine model, pathology of RSV infection was increased in cotton rats treated with poly ICLC. Augmented lung inflammation was accompanied by an earlier induction of IFN and TLR responses and a stronger chemokine expression. Overall, these findings indicate significant association between antiviral IFN action and pulmonary inflammation and highlight important animal model-specific variations in the potential of IFN to cause pathology.

FIG. 1.

Interferon (IFN)-inducing and proinflammatory effects of poly ICLC in the cotton rat. (A) Expression of IFN and IFN-associated genes in the lungs of cotton rats after poly ICLC treatment. Cotton rats were inoculated i.n. with 1–40 μg poly ICLC per animal. Cytokine mRNA expression in the lungs was analyzed at various times after poly ICLC treatment by RT-PCR and normalized to the β-actin mRNA level in the same sample. Control animals were inoculated with saline. (B) Histopathology analysis of lungs following poly ICLC treatment. Lungs from cotton rats treated with saline or 1–40 μg poly ICLC were collected at various time points after poly ICLC inoculation. Histopathological changes were expressed as cumulative score reflecting 4 principal parameters of inflammation: peribronchiolitis, perivasculitis, interstitial inflammation, and alveolitis (Prince and others 2001). Results represent the mean ± SEM for 3 cotton rats per group. *P < 0.05 when compared to saline-treated animals.

FIG. 2.

Pulmonary histopathology of influenza or respiratory syncytial virus (RSV) infection following saline or poly ICLC treatment. Animals were inoculated i.n. with saline (left panel of images) or poly ICLC (40 μg per animal) (right panel of images) and 24 h later infected with influenza virus, RSV A/Long, or left uninfected. Lungs were collected for histopathology analysis on day 4 post-infection (H&E, 100×). Inserts in the RSV panels highlight neutrophilic alveolitis in poly ICLC-treated animals (H&E, 200×).

FIG. 3.

Bronchoalveolar lavage changes of influenza- or respiratory syncytial virus (RSV)-infected animals following saline or poly ICLC treatment. Animals were inoculated i.n. with saline or poly ICLC (40 μg per animal) and 24 h later infected with influenza virus, RSV A/Long, or left uninfected. BAL was collected on day 4 post-infection and cellular composition of BAL was estimated by differential staining (A). Lymphocytes (Lymph), monocytes/macrophages (Monoc/Macr), and neutrophils (Neutr) were quantified. Results represent the mean ± SEM for 3–5 animals per group. (B) BAL of RSV-infected animals treated with saline (left panel) or with poly ICLC (right panel). Single arrows in the left panel point to macrophages (M) or neutrophils (N). Double arrows in the right panel point to enlarged macrophages visible in the BAL of all poly ICLC-treated animals (Diff-Quik, 400×).

FIG. 4.

Effect of poly ICLC on influenza infection in the cotton rat. (A) Dose dependency of the antiviral effect of poly ICLC. Cotton rats were inoculated i.n. with 1–40 μg poly ICLC (or with saline) and infected with H3N2 influenza A/Wuhan/395/95 virus 24 h later. One day after influenza infection, animals were sacrificed and lungs were collected for viral titrations. Results represent the mean ± SEM for 4 animals per group. *P < 0.05 when compared to influenza-infected, saline-treated animals (0 poly ICLC). (B) Effect of high-dose (40 μg per animal) poly ICLC on histopathology of influenza infection in cotton rats. Animals were inoculated i.n. with saline or poly ICLC and 24 h later infected with influenza virus. On various days after infection, animals were sacrificed and lungs collected for the analysis of histopathology. Results represent the mean ± SE for 4 animals per group. Experiment was repeated 3 times with similar results. Results of the representative experiment are shown.

FIG. 5.

Effect of poly ICLC on respiratory syncytial virus (RSV) infection in the cotton rat. (A) Antiviral effect of poly ICLC against RSV infection. Cotton rats were inoculated i.n. with saline (gray bars) or 40 μg per animal poly ICLC (black bars) and 24 h later infected with RSV (A/Long or A/A2). On days 1, 4, and 7 after infection, animals were sacrificed and lungs collected for analysis of pulmonary viral load. (B) Histopathology in the lungs of animals infected with RSV after saline or poly ICLC treatment. Results represent the mean ± SEM for 4 animals per group. *P < 0.05 when infected, poly ICLC-treated animals are compared to infected, saline-treated animals sacrificed on the same day. Experiment was repeated 2 times with similar results. Results of the representative experiment are shown.

FIG. 6.

Effect of poly ICLC on the interferon (IFN) response during influenza and respiratory syncytial virus (RSV) infection. Animals were inoculated i.n. with saline or 40 μg poly ICLC and 24 h later infected with influenza virus or RSV A/Long. On days 1, 4, and 7 after infection, animals were sacrificed and lungs collected for cytokine analysis (open circles: saline-treated, virus-infected animals; closed circles: poly ICLC-treated, virus-infected animals). Control animals were inoculated with poly ICLC (open squares, thick black line) or saline (open triangles) in the absence of viral infection and sacrificed at the same time intervals. Cytokine mRNA expression was measured by RT-PCR and normalized to the β-actin mRNA level. Results represent the mean ± SEM for 4 animals per group.

FIG. 7.

Pulmonary toll-like receptor (TLR) expression during influenza and respiratory syncytial virus (RSV) infection and its modification by poly ICLC treatment. Cotton rats were infected with influenza virus or RSV A/Long 24 h after treatment with saline (Infl+sal, RSV+sal) or with 40 μg/animal poly ICLC (Infl+poly, RSV+poly). On days 1, 4, and 7 after infection, animals were sacrificed and lungs were collected for analysis of TLR expression. Control animals were treated with saline (saline control) or with poly ICLC and sacrificed 1, 2, 5, or 8 days later (corresponds to d0, d1, d4, and d7 of viral infection). TLR levels were measured by qRT-PCR, normalized by the β-actin mRNA level in the corresponding sample, and expressed relative to the mRNA level of the same TLR detected in the lungs of saline-treated animals (assigned a value of 1). The results represent the mean ± SEM values for 4 animals per group. *P < 0.05 when compared to saline control animals; **P < 0.05 when infected poly ICLC-treated animals are compared to infected saline-treated animals.

FIG. 8.

Pulmonary chemokine expression during influenza and respiratory syncytial virus (RSV) infection and its modification by poly ICLC treatment. Cotton rats were infected with influenza virus or RSV A/Long following saline or poly ICLC treatment as described in the legend to Figure 7. Lung samples from days 1 and 4 post-infection (correspond to d2 and d5 for poly ICLC group) were collected for analysis of chemokine expression by qRT-PCR. Chemokine levels were normalized by the β-actin mRNA level in the corresponding sample and expressed relative to the mRNA level of the same chemokine detected in the lungs of saline-treated animals (assigned a value of 1). The results represent the mean ± SEM values for 4 animals per group. *P < 0.05 when compared to saline control animals; **P < 0.05 when infected poly ICLC-treated animals are compared to infected saline-treated animals.