During infection, H. parasuis has to reach the lung and survive the host pulmonary defenses before invading the blood stream . In the lung, bacteria have to confront alveolar macrophages, whose main roles include: ingestion of bacteria by phagocytosis, destruction of bacteria within phagolysosomes and recruitment of inflammatory cells to the site of infection via chemokines and acute-phase proteins .
Phagocytosis is a cytoskeleton-dependent process of engulfment of large particles, and macrophages could present a restricted number of phagocytic receptors that induce rearrangements in the actin cytoskeleton that lead to the internalization of the particle . Phagocytosis is a key mechanism used by macrophages to control virulent Pasteurellaceae, such as Pasteurella multocida, Haemophilus parasuis, Haemophilus influenzae, Actinobacillus pleuropneumoniae [13, 37–40]. In this study, the cd14 [41–44], hmox1 , fcgr2β  and abca1  genes, which were identified as DE genes, were also found to be involved in the phagocytosis. Meanwhile, the STRING analysis indicated that many DE gene encoded proteins could interact with CD14 and FCGR2β molecules, suggesting that PAMs may upregulate these genes to facilitate the phagocytosis of H. parasuis or other cells to play their immunological roles.
One of the important mechanisms used by macrophage to play its immunological functions is to kill bacteria by the activation and recruitment of antibacterial effectors to the phagolysosome . The fusion of phagosomes with lysosomes results in the formation of phagolysosomes [11, 36]. In our study, we found two DE genes that were related to the formation of phagolysosome, that is, smpd1  and coronin 1a [29–31]. Interestingly, many groups have reported that the CORONIN 1A could prevent lysosomal delivery and allow the bacteria to survive intracellularly [30, 32–34]. In the course of infection, H. parasuis has to survive from the host pulmonary defense, such as alveolar macrophages, to produce disease. In this way, the up-regulation of coronin 1a gene may facilitate the H. parasuis in producing the disease.
Interleukin-1 beta (IL-1β) is an important inflammation-associated gene that is up-regulated in many microarray experiments [11, 14, 16, 49]. Interestingly, Wilkinson et al reported that an increase in IL-1β gene expression is observed in H. parasuis-infected lungs . In our study, IL-1β was also up-regulated in H. parasuis-infected PAMs. Unsurprisingly, STRING analysis also revealed that many molecules encoded by up-regulated genes interact with IL-1β and form the IL-1β network. Meanwhile, the pathway analysis indicated that IL-1β is in some pathways, such as cytokine-cytokine receptor interaction (p = 2.18E-10), MAPK signaling pathway (p = 7.63E-04), and toll-like receptor signaling pathway (p = 1.93E-05). CCL5/RANTES plays an important role in regulating the movements of inflammatory cells to the infection sites [51, 52]. Many viruses, such as Japanese encephalitis virus (JEV) , respiratory syncytial virus (RSV) , influenza virus A  and porcine reproductive and respiratory syndrome virus (PRRSV)  have been shown to induce CCL5. In addition, some papers have reported that the CCL5 could be induced in macrophages by bacterial infections, such as Salmonella typhimurium infection, Streptococcus pyogenes infection and Lactobacillus rhamnosus infection [56, 57]. Interestingly, in our study, the up-regulation of CCL5 was observed in H. parasuis-infected PAMs, which suggested that CCL5 plays a role in the host response against H. parasuis infection. Thus, during the H. parasuis infection, the PAMs mount a powerful inflammatory response in an effort to clear this pathogen. Alternatively, the influx of inflammatory cells to the site of infection may provide additional host cells for H. parasuis infection. However, sustained or excessive production of inflammatory cytokines can have damaging consequences. To counterbalance inflammatory cytokines, anti-inflammatory cytokines are produced. Anti-inflammatory cytokines include interleukin 10 (IL-10), transforming growth factor β (TGF-β), and IL-1 receptor antagonist (IL-1RA) [11, 58, 59]. Wilkinson et al reported that the IL-1β and its antagonist, IL-1RA are both more highly expressed in "susceptible" animals challenged with H. parasuis . In our study, TGF-β, an anti-inflammatory cytokine, was increased in H. parasuis infection group. During H. parasuis infection, anti-inflammatory signals may decrease the potentially damaging effects of proinflammatory cytokines on host tissue.
Macrophage also effectively controls bacterial infection by producing of reactive species such as oxygen species and nitric oxide (NO). Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths and tumor cells. Unsurprisingly, H. parasuis infection could cause up-regulated expression of a large set of genes involved in the nitric oxide production. These genes were: spr, rora, klrk1, sod2 and il-1β [60–66]. The up-regulated genes related to the nitric oxide production may contribute to the PAM for confronting H. parasuis infection.
The DE genes that are related to phagocytosis, formation of phagolysosome, chemokines production, and nitric oxide production may help us to better understand the complicated mechanisms by which PAMs play their functions. Another highlight of our study is the new identified candidate genes that may be implicated in the pathogenesis of Glässer's disease. These genes could help to screen the potential host agents for reducing the prevalence of H. parasuis and further understand the molecular pathogenesis associated with H. parasuis infection in pigs. These genes are: s100a4, s100a6, caveolin 2 and ppp1r13l.
S100 A4 and S100 A6 belong to the S100 family that contained 2 EF-hand calcium-binding motifs [23, 27]. Two of S100 family genes (S100 calcium-binding protein A8 and A9) are dramatically up-regulated in spleen and lung following H. parasuis infection . Meanwhile, many other S100 family genes are up-regulated following different bacterial and viral infection [16, 18, 67, 68], suggesting that the S100 family genes play roles in the immune response to infections. In our study, the S100 calcium-binding protein A4 and A6 were up-regulated after H. parasuis infection when determined by microarray and qPCR. Further immunostimulation analysis indicated that the mRNA levels of S100 calcium-binding protein A4 (S100A4) and S100 calcium-binding protein A6 (S100A6) in porcine PK-15 cells increased within 48 h and were sustained after administration of LPS and Poly (I:C) respectively. We also found that the s100a4 and s100a6 genes were up-regulated in lungs, spleen and lymph nodes in H. parasuis infected pigs. Interestingly, the kidney fibrosis [24–26, 69], liver fibrosis [70, 71], lung fibrosis [72–74], cardiac fibrosis [23, 75, 76] and peritoneal fibrosis  are found to be related to the expression of s100a4. Glässer's disease is characterized mainly by fibrinous polyserositis, meningitis, and arthritis. In this way, we hypothesized that the increase expression of s100a4 may underlie fibrosis during H. parasuis infection in pigs. Meanwhile, some reports indicated that s100a6 plays roles in cell proliferation and signaling transduction [27, 28]. Therefore, the s100a4 and s100a6 genes could be two novel genes related to H. parasuis infection.
Caveolins are the major components and protein markers of caveolae that are 50-100 nm invaginations of membrane. The caveolin gene family includes three members in vertebrates, caveolin-1, caveolin-2 and caveolin-3, of which caveolin 1 and caveolin 2 have been detected in mouse macrophages [35, 78]. Caveolin 1 molecule is related to H. parasuis infection . Caveolin 2, which localizes to the Golgi complex but redistributes to plasma membrane, caveolae and rafts when co-expressed with caveolin 1, is a potential key molecule related to the Pseudomonas infection causing pneumonia in patients with cystic fibrosis and other immunocompromising conditions [79, 80]. In our study, the caveolin 2 gene was highly expressed in PAM isolated from the H. parasuis serovar 5 challenged group. Therefore, in addition to the caveolin 1 gene, the caveolin 2 gene may be a novel candidate gene related to H. parasuis infection.
The NF-kappa B (NF-κB) signaling pathway is important in signal transduction during the innate immune response . NF-κB signaling relies on the targeting of IκB (inhibitor of NF-κB) subunit to the proteasome to allow NF-κB to translocate from the cytosol to the nucleus where it activates gene transcription . The PPP1R13L is mentioned as a novel inhibitor of NF-κB . In our study, microarray and qPCR analysis indicated that the mRNA of PPP1R13L was down-regulated significantly compared to control. The IPA network indicated that the PPP1R13L could directly or indirectly interacts with many molecules, such as micro RNAs, transcriptions, enzymes, and cytokines (Additional file 8), suggesting that ppp1r13l gene is an innate immune related gene that plays a role in PAM during H. parasuis infection. The detailed mechanism of ppp1r13l gene in NF-κB signaling pathway in H. parasuis infected PAM needs further studies.