Neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology

Abstract

Polymorphonuclear neutrophils (PMN) infiltrate the respiratory tract early after viral infection and can contribute to both host defence and pathology. Coronaviruses are important causes of respiratory tract infections, ranging from mild to severe depending on the viral strain. This study evaluated the role of PMN during a non-fatal pulmonary coronavirus infection in the natural host. Rat coronavirus (RCoV) causes respiratory disease in adult rats, characterized by an early PMN response, viral replication and inflammatory lesions in the lungs, mild weight loss and effective resolution of infection. To determine their role during RCoV infection, PMN were depleted and the effects on disease progression, viral replication, inflammatory response and lung pathology were analysed. Compared with RCoV infection in control animals, PMN-depleted rats had worsened disease with weight loss, clinical signs, mortality and prolonged pulmonary viral replication. PMN-depleted animals had fewer macrophages and lymphocytes in the respiratory tract, corresponding to lower chemokine levels. Combined with in vitro experiments showing that PMN express cytokines and chemokines in response to RCoV-infected alveolar epithelial cells, these findings support a role for PMN in eliciting an inflammatory response to RCoV infection. Despite their critical role in the protection from severe disease, the presence of PMN was correlated with haemorrhagic lesions, epithelial barrier permeability and cellular inflammation in the lungs. This study demonstrated that while PMN are required for an effective antiviral response, they also contribute to lung pathology during RCoV infection.

Fig. 1.

αPMN treatment depletes circulating PMN and increases disease severity during RCoV infection of rats. (a) Rats were injected with αPMN or normal rabbit serum (NRS) intraperitoneally 1 day prior to intranasal inoculation with RCoV or medium (mock), and every 48 h thereafter. (b) Blood was collected from 3–5 animals per group daily to monitor white blood cell populations. Rats were monitored for mortality (c), body weight (d) and clinical signs of disease (e). Data are means±se from 6–18 rats per treatment per day (see key). Statistically significant differences between αPMN- and NRS-treated rats were identified using one-way ANOVA followed by the Newman–Keuls post-test, **P<0.01; ***P<0.001.

Fig. 2.

αPMN treatment reduces PMN recruitment and prolongs viral replication in the lungs. Rats were injected with αPMN or NRS intraperitoneally 1 day prior to intranasal inoculation with RCoV, and every 48 h thereafter (see Fig. 1a). (a) Cells from bronchoalveolar lavage fluid (BALF) were Giemsa–Wright stained and PMN were quantified morphologically. (b) Lung tissues were homogenized and viral titres were determined by plaque assay. Data are means±se from 3–5 rats per group. Statistically significant differences between αPMN- and NRS-treated rats were identified using an unpaired t-test, **P<0.01, ***P<0.001. nd, not determined.

Fig. 3.

PMN are needed early during RCoV infection to be protective. (a) Rats were injected with αPMN or NRS intraperitoneally 1 day prior to and 2 days after intranasal inoculation with RCoV and analysed on days 4 and 8 (arrows). (b) Cells from BALF of 3–5 rats per group were Giemsa–Wright stained and PMN were quantified morphologically. (c) Lungs from 3–6 rats per group were homogenized and viral titres were determined by plaque assay. The data are means±se. Rats (see key for numbers) were monitored for (d) survival (P = 0.046) and (e) weight loss. Statistically significant differences between αPMN- and NRS-treated rats were identified using (e) two-way ANOVA followed by a Bonferroni post-test or (b and c) unpaired t-test, ***P<0.001.

Fig. 4.

PMN promote cellular inflammation in the lungs upon RCoV infection. Rats were injected with αPMN or NRS intraperitoneally 1 day prior to intranasal inoculation with RCoV, and every 48 h thereafter. On day 4 after RCoV infection, (a) lungs were formaldehyde-fixed and paraffin-embedded, and haematoxylin and eosin-stained sections were analysed for cellular inflammation. Representative tissues from three animals per group are shown at ×2 (top panels), ×20 (middle panels) and ×40 (bottom panels) magnification. Examples of cell types at ×40: neutrophil (#),macrophage (*) and lymphocyte (∧) are indicated. (b) Density indices of inflammatory cells were quantified in lung sections from three animals per group using ImageJ software. (c) White blood cells in BALF were quantified by Wright–Giemsa staining, and (d) lymphocyte subtypes by flow cytometry, using 4–6 animals per group. Statistically significant differences compared with NRS/RCoV-treated rats were identified using one-way ANOVA followed by the Newman–Keuls post-test, *P<0.05, **P<0.01, ***P<0.001.

Fig. 5.

αPMN-treated rats have reduced concentrations of chemokines in the airways during RCoV infection. ELISAs were used to quantify the concentrations of (a) PMN-specific (CXCL-1 and CXCL-3) and (b) monocyte- and lymphocyte-specific (IP-10 and CCL2) chemokines in BALF from αPMN- and NRS-treated rats after infection with RCoV or mock-inoculation. The data are means±se from 3–5 rats per treatment. Statistically significant differences between αPMN- and NRS-treated rats were identified using one-way ANOVA followed by the Newman–Keuls post-test, **P<0.01, ***P<0.001.

Fig. 6.

Differential pathology in PMN-depleted vs. NRS-treated rats during RCoV infection. Images of representative lung sections from three animals per treatment: (a, d) αPMN-treated/mock-inoculated, (b, e) NRS-treated/RCoV-infected and (c, f) αPMN-treated/RCoV-infected showing histopathology on day 4 post-infection. αPMN/mock rats had normal terminal (TBr) and respiratory (RBr) bronchioles and alveoli (A). Lungs of NRS/RCoV rats had peribronchiolar inflammation with sloughing of bronchiolar epithelium (†) and alveoli (A) filled with inflammatory cells and necrotic cell debris. Transudate-filled alveoli indicating alveolar oedema (E) were diffusely distributed in the lesions and congested capillaries (arrows) were found throughout the sections. αPMN/RCoV rats had mild pathology with sloughing of bronchiolar epithelium (†) and congested capillaries (arrows). Bars, 25 µm.