Neutrophils regulate the lung inflammatory response via γδ T cell infiltration in an experimental mouse model of human metapneumovirus infection

Abstract

Neutrophils are the most abundant leukocytes in human circulation. They are the first immune cell population recruited to the sites of infection. However, the role of neutrophils to regulate host immune responses during respiratory viral infections is largely unknown. To elucidate the role of neutrophils in respiratory antiviral defense, we used an experimental mouse model of human metapneumovirus (HMPV) infection. HMPV, a member of the Paramyxoviridae family, is a leading respiratory pathogen causing severe symptoms, such as bronchiolitis and pneumonia, in young, elderly, and immunocompromised patients. We demonstrate that neutrophils are the predominant population of immune cells recruited into the lungs after HMPV infection. This led us to hypothesize that neutrophils represent a key player of the immune response during HMPV infection, thereby regulating HMPV-induced lung pathogenesis. Specific depletion of neutrophils in vivo using a mAb and simultaneous infection with HMPV exhibited higher levels of inflammatory cytokines, pulmonary inflammation, and severe clinical disease compared with HMPV-infected, competent mice. Interestingly, the lack of neutrophils altered γδ T cell accumulation in the lung. The absence of γδ T cells during HMPV infection led to reduced pulmonary inflammation. These novel findings demonstrate that neutrophils play a critical role in controlling HMPV-induced inflammatory responses by regulating γδ T cell infiltration to the site of infection.

Keywords: HMPV; immune response; paramyxovirus; pulmonary; respiratory.

Figure 1.. Neutrophil recruitment following inoculation of mice with HMPV.

BALB/c mice were infected with 10⁷ PFU of HMPV. BALF was collected at different time points, and neutrophils were quantified using flow cytometry. (A) Differential cell images stained by Wright-Giemsa showing cellular influx in BAL on d 1 from mock and HMPV-infected mice (original magnification, 40×). (B) Flow cytometry analysis of neutrophil influx in BAL at d 1 after infection. (C) Percentage of neutrophils in BAL at d 1, 4, and 7 after infection. (D) Total number of neutrophils in BAL at d 1, 4, and 7 after infection. Means ± sem are shown; n = 6 mice/group. Control mice were mock infected with PBS. *P < 0.05, **P < 0.01.

Figure 2.. Efficiency of neutrophil depletion in vivo during HMPV infection.

BALB/c mice were treated with isotype (IgG) or anti-Ly6G antibody before and p.i. with HMPV. (A) Treatment plan for depletion of neutrophils. (B) Depletion of neutrophils in BAL was confirmed by flow cytometry on d 7 after infection. Total neutrophils in infected mice after depletion in (C) BAL and (D) lung; n = 8 mice/group. Control mice were mock infected with PBS. ***P < 0.001.

Figure 3.. Neutrophil depletion enhances body weight loss and augments disease severity and lung pathology in HMPV infection.

BALB/c mice were treated with isotype or anti-Ly6G antibody before and p.i. with HMPV. (A) Infected mice were scored for illness based on disease severity. (B) Signs of sickness in isotype or anti-Ly6G-treated and HMPV-infected mice on d 7 after infection. (C) Body weight loss. (D) Pathology score from lung sections obtained at d 7 after HMPV infection. Means ± sem are shown; n = 8 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.. Depletion of neutrophils does not affect lung viral titer in HMPV-infected mice.

Whole lungs and lung tissues from depleted or competent mice infected with HMPV were collected on d 4 and 7 p.i. (A) HMPV N gene relative expression in lung quantified by qRT-PCR . (B) Viral titer was determined in lung homogenates by plaque assay. Means ± sem are shown; n = 6 mice/group.

Figure 5.. Pulmonary inflammation in neutrophil-depleted mice infected with HMPV.

BALB/c mice were treated with isotype (IgG) or anti-Ly6G antibody before and p.i. with HMPV or mock-infected with PBS. BAL samples from each group of mice were assessed for cytokine/chemokine production by a multiplex cytokine detection system; n = 8–18 mice/group. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 6.. Neutrophil depletion alters lung T cell responses during HMPV infection.

BALB/c mice were treated with isotype (IgG) or test antibody (anti-Ly6G) before and p.i. with HMPV. Lung single-cell suspensions were stained for (A) myeloid populations (macrophages, dendritic cells) and (B) lymphocyte populations (CD4⁺, CD8⁺, B cells, and γδ T cells). Means ± sem are shown; n = 4–15 mice/group. ***P < 0.001.

Figure 7.. Recruitment and role of γδ T cells during HMPV infection.

(A) C57BL/6 mice were infected i.n. with HMPV and lungs harvested at d 1, 3, 5, 7, and 15 after infection. Lung single-cell suspensions were stained for γδ T cells and quantified using flow cytometry. (B) TCR-δ^−/− or WT C57BL/6 mice were infected with HMPV and lung sections obtained at d 7 after infection for histopathology analysis. Lung pathology is represented as percentage pathology score. (C) Representative scans of lung tissue corresponding to the pathology score from the indicated treatment. Arrows indicate cells infiltrating the perivascular spaces. Original scale bars, 0.4× = 5 mm; 20× = 50 μm. *P < 0.05, **P < 0.01.