6. DISCUSSION
6.1. Mono-infection of precision-cut lung slices by swine influenza virus or
Precision-cut lung slices (PCLS) were widely used in pharmacological analysis (Kim et al., 2015; Lauenstein et al., 2014; Neuhaus et al., 2013). Recently PCLS were applied to investigate viral infections (Cousens et al., 2015; Kirchhoff et al., 2014a; Kirchhoff et al., 2014b; Punyadarsaniya et al., 2011). So far, no study about the infection of porcine PCLS by bacteria has been reported. One similar study is the analysis of infection of an organ culture from human respiratory mucosa by S. pneumoniae (Rayner et al., 1995). My colleagues and I intended to establish porcine precision-cut lung slices (PCLS) for analyzing the infection of well-differentiated respiratory epithelial cells by swine influenza virus SIV or S. suis.
SIV are important pathogens of the respiratory tract in pigs causing severe acute respiratory disease. In a previous study my colleague has characterized the infection of swine PCLS by influenza A/Bissendorf/IDT1864/2003 (H3N2) strain (Punyadarsaniya et al., 2011). Here I extended my investigation to five additional strains comprising all three subtypes (H1N1, H1N2, H3N2) prevailing in the swine population of Europe (Watson et al., 2015). On one hand, my analysis of the virulence of SIV shows that the three strains H3N2/2006, H1N1/1981, and H1N2/2000 cause disease in pigs as evidenced by a rise in the parameters dyspnea, rectal temperature, and virus load. Low virulence was determined for strain H1N1/2006 which showed humble dyspnea values and no increase in rectal temperature in experimentally infected pigs.
On the other hand, the determination of primary target cells in the PCLS infected by different SIV strains showed that the three subtypes of SIV infected both ciliated cells and mucus-producing cells, consistent with the previous study about the A/Bissendorf/IDT1864/2003 strain (Punyadarsaniya et al., 2011). A major determinant of influenza virus infection is the presence of sialic acid on susceptible host cells to which the viral hemagglutinin is able to bind. Recent reports have shown that both the α-2,3 and the α-2,6 sialic acid linkage types are present on the respiratory tract of pigs and the distribution of sialic acids along the airway was similar to that reported for humans (Nelli et al., 2010; Shinya et al., 2006); α-2,6-linked sialic acid was predominantly present in the trachea and bronchi while in bronchioles and alveoli, the amount of α-2,3-linked sialic acid was relatively increased (Nelli et al., 2010).
Interestingly, when PCLS were infected by high-virulent viruses of the H3N2 subtype, both the lobar bronchi and the terminal bronchioli were efficiently infected as evidenced by high virus titers at 24 hpi. By contrast, the low-virulent H1N1/2006 strain infected the terminal bronchioli more efficiently than the lobar bronchi with more than a 100-fold lower virus titer detected at 48 hpi. This indicates that the H1N1/2006 strain shows a preference to infect the epithelium of the lower airways. Swine influenza viruses generally have a preference for α-2,6-linked sialic acid, which facilitates infection of the upper respiratory tract where this linkage type predominates. It will be interesting in the future to determine whether the H1N1/2006 strain has a preference for α-2,3-linked sialic acid, e.g. by performing a glycan array analysis. For such a virus it would be difficult to reach the terminal bronchiole to initiate a successful infection. This would explain why the H1N1/2006 strain not only replicates less efficiently in PCLS than the strains of the H3N2 subtype but also shows a low virulence in experimentally infected pigs.
The five strains were also compared for their ability to induce a ciliostatic effect in infected PCLS, a parameter which cannot be determined in permanent respiratory epithelial cell lines in vitro. The ciliary beating can easily be observed under the light microscope and the absence of ciliary activity is assumed to indicate the death of ciliated cells (Punyadarsaniya et al., 2011; Rosner et al., 2014). I found that the infection by different strains resulted in a different extent of the ciliostatic effect. Strains of the H3N2 subtype caused a significant reduction (50%) of the ciliary activity at 48 hpi and the lowest ciliostatic effect was found with the H1N1 strain H1N1/2006. The H1N1/1981 strain showed an intermediate extent of the ciliostatic effect in comparison to the H3N2 strains and the H1N1/2006 strain. This result indicates that the ciliary activity may be a good predictor of the viral virulence.
Concerning the bacterial infection on primary cell cultures, a similar study about the infection by S. pneumoniae caused patchy damage to the organ culture of the human respiratory mucosa and a decreased ciliary beat frequency has been reported (Rayner et al., 1995). Two studies about infection by S. aureus and S. pneumoniae on human respiratory epithelial cells of ALI cultures have been described recently (Carey et al., 2015; Nguyen et al., 2015). So far, no study about the infection of porcine PCLS by bacterial pathogens has been described.
To analyze the virulence of S. suis with well-differentiated epithelial cells, PCLS were inoculated with a encapsulated S. suis strain 10, a nonencapsulated mutant (10cpsΔEF), or a
suilysin-deficient mutant (10Δsly). I found that all the mutants and the parental strain efficiently adhered to and colonized both well-differentiated ciliated cells and mucus on the luminal surface of bronchioli but only a very slight ciliostatic effect was observed in PCLS infected by either of the bacteria. The suilysin-deficient mutant 10Δsly showed the lowest ability of adherence to and colonization of bronchioli in comparison to the suilysin-positive strain and the nonencapsulated mutant. This result indicates that suilysin has a positive effect on the adherence and colonization properties of S. suis in well-differentiated respiratory epithelial cells which is consistent with previous reports that suilysin promotes adherence of S. suis to HEp-2 (Norton et al., 1999; Seitz et al., 2013).
Most interestingly, I found that the encapsulated strain adhered as efficiently as the nonencapsulated mutant, which differs from what others have observed using conventional immortalized cell lines (Benga et al., 2004; Seitz et al., 2013). This may be due to mixed cell types present in PCLS comprising not only mucus-secreting cells but also ciliated cells that are important for the ciliary “escalator” function of the airway epithelium (Wilson et al., 1996). During the S. suis infection, it was observed that streptococci were efficiently moved away by the ciliary beating as soon as they approached the luminal surface of bronchioli.
Hence, it was not easy for the streptococci to get close contact to the membrane of well-differentiated epithelial cells, in contrast to conventional immortalized cell lines which do not contain such a defense mechanism. Bronchial mucus which is secreted by goblet cells and submucosal glands is able to entrap aerocontaminants and bacteria, and the entrapment of bacteria by respiratory mucus and their elimination by the ciliary beating is the first stage in the defense of the human respiratory epithelium (Girod et al., 1992; Plotkowski et al., 1989). I observed different adherence patterns: the encapsulated S. suis strain adhered to both ciliated cells and mucus, whereas the nonencapsulated mutant, which showed a cluster-like adherence, preferentially bound to mucus. In natural infections, the bacteria which were entrapped by mucus may be transported out of the airway by the ciliary beating.
Tanabe et al. (2010) reported a positive correlation between the absence of capsule and biofilm formation in S. suis, and in S. pneumoniae, and the downregulation of the capsule cpsA gene during biofilm growth has been reported (Hall-Stoodley et al., 2008; Tanabe et al., 2010). In addition, mucin was found to enhance biofilm formation of other bacteria (Landry et al., 2006; Mothey et al., 2014). Hence, biofilms produced by S. suis adherent to mucin-producing cells may play a role in colonization, but this remains to be studied further. Taken
together, my results show that the role of the capsule (and regulation of its expression) in colonization and infection of mucosal surfaces should be reconsidered and further studied.
In conclusion, PCLS are a useful culture system for analyzing the different properties of swine influenza viruses; viral replication and ciliary activity were consistent with results of experimental pig infections; results obtained with PCLS may be used as an indicator to predict viral virulence. Furthermore, the PCLS system is suitable to study S. suis adherence to and colonization of primary respiratory epithelial cells. Therefore, PCLS is a promising model to analyze the effect of co-infection on well-differentiated respiratory epithelial cells by SIV and S. suis.