The present study demonstrated that combination therapy of laninamivir octanoate with artificial surfactant increased survival of mice suffering from influenza virus-induced severe pneumonia, while monotherapy of laninamivir octanoate did not improve survival. This increased survival was determined to be due to artificial surfactant inhibiting alveolar collapse and DAD formation in the lungs, thereby preserving lung function for oxygenation.
We previously showed a serially pathological process in the lungs that led to death in PR8-infected mice 
. Interstitial pneumonia due to viral infection was found histopathologically within 2 days postinfection, and became severe during 2–6 days postinfection. In addition, DAD with hyaline membranes rapidly formed at 24–48 h before death. This rapid progression of DAD formation in the infected mice is consistent with the clinical time course for progression of existing diseases to ARDS in humans 
. It has also been found that the histopathological characteristics of DAD, hyaline membrane formation, inflammatory cell accumulation and pulmonary edema, in PR8-infected mouse lungs are identical to the characteristics of DAD in human cases of mortality involving severe viral pneumonia and ARDS induced by highly pathogenic avian influenza (H5N1) and A(H1N1)09 viruses 
. Therefore, we consider that progression from interstitial pneumonia to DAD induced by influenza virus infection in the mouse resembles pulmonary changes in human cases. Moreover, our previous study demonstrated that interstitial pneumonia was observed in the lungs of live PR8-infected mice and DAD was only observed in dying and dead mice, suggesting that DAD formation was involved in the death of infected mice 
. Based on these findings, we expected that inhibition of DAD formation by any medical intervention would improve survival of mice infected with influenza virus.
Neonatal respiratory distress syndrome (NRDS), also called infant respiratory distress syndrome and previously called hyaline membrane disease, is a syndrome in premature infants caused by developmental insufficiency of pulmonary surfactant production in the lungs 
. NRDS affects approximately 1% of newborn infants and is the leading cause of death in preterm infants with alveolar collapse and hyaline membranes 
. Current therapy of NRDS involves administration of exogenous artificial surfactant, which dramatically decreases mortality. In addition, a hyaline membrane is known to be composed of a mixture of cellular debris, immunoglobulin, fibrin and plasma proteins 
. This indicates an influx of plasma components, which are originally present in blood, to the alveolar lumen. Therefore, disruption of pulmonary surfactant is thought to be involved in the formation of alveolar collapse and hyaline membranes.
Our previous study demonstrated that the leakage of surfactant proteins to capillaries gradually increases in the infected mice in association with the aggravation of pneumonia up to 6 days postinfection 
. This leakage continued to 20 days postinfection in the infected mice with treatment of oseltamivir (our unpublished data). Based on these results, we speculated that the amount of pulmonary surfactant in the alveolar lumen may decrease in infected lungs for at least 20 days after infection. A previous report found that when artificial surfactant was administered to mice infected with influenza virus only once at 3 days postinfection, there was an adverse effect on survival rate 
. Therefore, in the present study, we administered artificial surfactant multiple times during the observation period. When multiple administration of artificial surfactant alone was investigated with a low viral dose inoculation, death in mice was delayed by a few days compared with that in normal saline controls (). Although it is likely that multiple administration of artificial surfactant prevents a decrease in lung function by viral infection, damage of lung cells may continue because artificial surfactant does not inhibit viral proliferation, and then all infected mice eventually die. In the administration of artificial surfactant in the presence of laninamivir octanoate, the two agents acted synergistically, resulting in an improvement in survival of mice.
Our previous study found a delay of a few days between the peak of viral proliferation and death in mice 
. In addition, in our present histopathological examination, the virus had already disappeared from the alveoli and alveolar ducts of living mice in the combination therapy and monotherapy groups at 7 days postinfection (). Furthermore, in the presence of an NAI, artificial surfactant increased survival rate in mice but did not diminish viral proliferation ( and ). These observations suggest that viral replication itself is not directly correlated with death of infected mice. Moreover, as shown in our previous study, DAD formation is involved in the death of mice 
. Therefore, it is hypothesized that progression from viral pneumonia to DAD formation is a multistep process. Infection and replication of influenza virus destroys type I/II pneumocytes in pulmonary parenchyma, leading to inflammation in the microenvironment, and this leads to destruction of the alveolar-capillary structure. Accumulation of infected cell debris in the lumen of alveoli, alveolar ducts and bronchioles, influx of protein-rich plasma components from capillaries into the alveolar lumen, and efflux of pulmonary surfactant from the alveolar lumen into the bloodstream then leads to quantitative and qualitative loss of pulmonary surfactant. The loss of pulmonary surfactant is associated with a decline in interfacial activity, resulting in low alveolar compliance, which accelerates formation of DAD with alveolar collapse and hyaline membrane. These pathological situations in pulmonary parenchyma finally disrupt gas exchange between the alveoli and capillaries, and ultimately lead to death. As mentioned above, at least in influenza virus infection, one of the factors responsible for increasing severity of viral pneumonia is thought to be the loss of pulmonary surfactant.
Treatment using exogenous SP-A and SP-D to influenza virus-infected mice has been previously attempted because of the efficacy of their anti-pathogenic functions in cooperation with alveolar macrophages 
. However, in the present study, we did not consider this self-defense function by SP-A and SP-D. Artificial surfactant used in the present study consisted of lipoprotein, SP-B and SP-C only, because SP-A and SP-D were removed during the production process of artificial surfactant 
. Therefore, artificial surfactant administration itself did not affect virus proliferation, inhibition of inflammation and a host immunological response. However, artificial surfactant administration inhibits formation of alveolar collapse and hyaline membranes, resulting in aeration of the lungs and constant blood O2
levels. Our results showed that interfacial activity of artificial surfactant improved alveolar gas exchange in severe pneumonia induced by influenza virus infection.
In humans, administration of artificial surfactant has been previously attempted for treatment of ARDS, but it was not effective 
. However, a recent case report described that combination therapy with oseltamivir and artificial surfactant in a child infected with influenza virus produced favorable results 
. Our previous study showed that alveolar collapses occurred at 2 days postinfection in mice 
. In addition, the present study showed that commencement of artificial surfactant administration was crucial for treatment of severe pneumonia in the mouse infection model. Taken together, our studies and previous clinical reports suggest that for severe pneumonia in relation to influenza virus infection, the timing of artificial surfactant administration is important for successful treatment. Future studies are required to determine the timeline for successful administration of artificial surfactant to circumvent pathological decline. In humans, combination of NAIs and artificial surfactant could be a candidate for effective and preventive treatment of influenza virus-induced severe pneumonia, which does not improve with monotherapy of NAIs.