How does the human metapneumovirus regulate neutrophil infiltration into the airways?

“Overall, these findings demonstrate that the attachment protein of HMPV contributes to the recruitment of neutrophils during the viral infection.”

Since its discovery in 2001, human metapneumovirus (HMPV) has been recognized as a respiratory pathogen of clinical significance that infects children, the elderly and immunocompromised patients [1,2]. Infection by this virus is characterized by acute lower respiratory tract infection and symptoms range from common cold-like syndrome to more severe manifestations such as bronchiolitis and pneumonia, which require hospitalization. Thus, HMPV can cause severe pulmonary inflammation. Recent clinical evidence indicates that neutrophil infiltration is induced within the airways of children with HMPV-induced bronchiolitis [3]. This phenomenon has been reproduced in experimental mouse models of infection by several groups [4–7]. However, limited information has been reported regarding the interaction between HMPV and neutrophils in the lungs of infected mice.

In understanding that interaction, we have previously demonstrated that depletion of neutrophils led to an increased disease severity and lung inflammation, indicating a protective role of these cells during HMPV infection [4]. Furthermore, we have also explored the mechanisms by which HMPV infection contributes to the recruitment of neutrophils in the lung. In this context, we have investigated the role of HMPV attachment protein in contributing to the recruitment of neutrophils into the airways [8]. The attachment glycoprotein (G) of HMPV is one of the three putative membrane proteins expressed by the virus. Though, the attributed function for the attachment protein is to aid in the attachment of the virus to the target cells, several studies have demonstrated that cell attachment and fusion can happen in the absence of HMPV-G protein [9,10]. Apart from its roles in viral infection, the G protein is also known to modulate several aspects of the innate immune response [11–13]. However, the contribution of this protein in pulmonary inflammatory responses during HMPV infection remains poorly understood.

Therefore, we speculated on two previous findings in which it was indicated that interferon has a regulatory effect on neutrophil recruitment [14–17], and that G protein of HMPV suppresses interferon responses, as we and other groups have previously reported [11,13]. Based on those observations, we hypothesized that the viral G regulates neutrophil infiltration in HMPV-infected animals. In order to test that, we used a recombinant virus lacking the G protein (rHMPV-∆G), as described previously [11], and infected BALB/c mice intranasally with rHMPV-∆G or with the full-length recombinant HMPV (rHMPV), as a reference control. Recruitment of neutrophils into the airways was quantified by differential cell counts, and the production of interferon and additional cytokines was measured by ELISA and multiplex cytokine detection assays, respectively. Analysis of the interferon production by the rHMPV-∆G in BALB/c mice indicated that the absence of the attachment protein increased the levels of IFN-α in the lungs of the infected mice. Furthermore, those mice infected with the rHMPV-∆G showed decreased neutrophil infiltration into the airways compared with the ones infected with the rHMPV, but did not have an effect on macrophages or lymphocytes, indicating that the G protein was found to have a specific effect on neutrophils. Those findings led us to explore the effect of G protein on additional cytokines/chemokines. We observed that the absence of G protein also decreased the lung cytokine/chemokine profile such as TNF-α, VEGF, IL-17, CCL3, CCL4 and CXCL2, all known to have a chemotactic effect on neutrophils and to contribute to the recruitment of these cells to the site of infection.

Overall, these findings demonstrate that the attachment protein of HMPV contributes to the recruitment of neutrophils during the viral infection. This happens potentially through a mechanism that involves the inhibition of interferon responses by the HMPV-G protein, which in turn would allow the induction of high levels of neutrophil-chemotactic cytokines, and consequently higher numbers of neutrophils present in the airways of the infected animals. Whether other HMPV proteins could also play a role in the neutrophil recruitment is unknown. But it is likely that proteins that modulate the interferon response could have an effect on neutrophilic chemokines, based on the effect of interferon on neutrophil responses [14–17].

In conclusion, our study highlights the important role of viral proteins in neutrophil recruitment, which is not trivial, given the critical contribution neutrophils have in the inflammatory outcome of HMPV-infected individuals [3]. That is, excessive recruitment of these cells during the infection can have a negative outcome, leading to exacerbated inflammatory responses, so their recruitment during HMPV needs to be regulated. In this context, our work demonstrates the contribution of the viral attachment protein to the recruitment of these cells into the airways. On the other hand, we have also reported that the absence of neutrophils leads to an increased inflammatory response during HMPV infection, mediated by γδ T cells [4]. Thus, upon HMPV infection, a fine balance of neutrophil response is necessary for an optimal activation of the immune system and controlled inflammatory response. Hence, these findings help further our understanding of the complex interplay between the beneficial and detrimental roles of neutrophils in the inflammatory responses during HMPV infection.