In the present study we have mainly been interested in investigation of microRNAs involved in the progression of the APP infection rather than comparing infected versus healthy individuals. Furthermore, we decided to use infected and visually uninfected tissue from pigs infected with the bacterial pathogen Actinobacillus Pleuropneumoniae in order to minimize the genetic and phenotypic diversity between samples. We have used these samples to generate expression profiles of microRNA by high throughput. Working with infected tissue, which shows severe signs of advanced necrosis, is challenging because RNA degradation has to be taken into account. As expected, the annotation of the RNAseq data revealed a dominating presence of degradation products in the necrotic sample, regardless of the fact that all the samples included in the present study showed RNA quality indexes acceptable for gene expression studies (RQI > 6.0). The read length distribution in the visually unaffected sample is comparable to profiles generated in other studies ,  with the highest number of reads belonging to the 19-25nt microRNA range which suggests the reliability of the library construction and the sequencing. A meaningful expression profile can only be generated if the raw reads are normalized in accordance to the degree of degradation. To avoid letting rRNA and mRNA degradation products over-shadow the read counts of the miRNAs among the reads generated in the necrotic sample, the miRNAs from both samples were studied separately. Two different mapping methods have been used in this study: one with unique reads using Novoalign , and one with matches to maximum five genomic loci using Bowtie. The results generated by the two methods are in strong agreement with each other.
The sequencing data provide evidence of microRNA deregulation during lung infection with the bacterial pathogen. Both up- and down-regulation of microRNAs were found when comparing the expression in the necrotic versus visually unaffected tissue of the lung. A subset of the highly expressed and biologically relevant genes (13 miRNAs and one snoRNA) was subjected to RT-qPCR. In 10 out of 14 genes, RT-qPCR results supported the results obtained by deep-sequencing indicating that the data generated reflects a biologically meaningful profile. The disagreement featured in the remaining four microRNAs is most likely pointing towards sequencing bias accompanying RNAseq experiments rather than misevaluation of the expression by a specific and sensitive RT-qPCR.
The number of annotated microRNAs in domestic pig in the newest miRBase 18.0 is still much smaller than in other organisms, featuring 1527 entries for human, 741 for mouse and only 228 for pig. Multiple alignments to the pig genome and 17 other organisms were performed based on the assumption that the majority of miRNA sequences are conserved among species , . A total of 27 of the annotated 168 microRNAs matched hairpins annotated for other organisms than pig in miRBase and based on sequence homology they could be annotated in pig enriching the repertoire of porcine microRNAs by 27 homologous miRNAs. Furthermore, 11 novel microRNAs were found by search performed with miRDeep2 pipeline . Often, novel microRNAs are not represented by many read counts. An exception to that rule is one of the detected miRNAs, annotated at the negative strand of chromosome 5, called – miR-d5, which is represented by 5413 and 3690 read counts in necrotic and visually unaffected sample, respectively. Moreover, this novel microRNA was found in only two other organisms: cow and dolphin out of the 17 different reference genomes tested.
Studies on high throughput sequencing of small RNAs show that there are in most cases a few distinct microRNAs that constitute the majority of microRNA reads . Our study also reports highly abundant microRNA namely miR-143 which is represented by 65% and 49% of the miRNA reads in the visually unaffected and the necrotic sample, respectively. RT-qPCR validation of this microRNA did, not reflect such a high level of expression. Even more striking is the fact that this particular microRNA has not previously been described as extremely abundant in mammalian lung, neither has it been pointed out as a major regulator of immune response . A possible explanation for the high read count in both libraries could be that during the library construction an artifact has been introduced in the RNAseq experiment.
The second most abundant microRNA in both samples – miR-21 is involved in many biological scenarios [58–61]. In the present study, miR-21 shows down-regulation in the necrotic sample in comparison to the visually unaffected one, however supported by quite low p-values (Table 4). RT-qPCR performed on miR-21 detected the highest expression level of all the assayed microRNAs. No differential expression was found between the samples included in the RT-qPCR experiment, which could indicate an important role of miR-21 in the homeostasis of lung.
The 12 microRNA candidates assayed by RT-qPCR were investigated for target predictions in order to provide more insight into the biological pathways where the different microRNAs could play a role. The conservative predictions indeed anticipated a number of very promising, biologically interesting interactions. One of the major factors involved in detection of pathogens and initiation of inflammatory response are TLRs. Moreover, a rapid immune response is triggered upon pathogen invasion by the presence of LPSs – a major component of the gram negative APP membranes. After binding of LPS to the TLR4/MD2/CD14 receptor complex, signalling pathways lead to the activation of nuclear factor κB (NF-κB) that further regulates a large set of genes critical to cell proliferation, inflammation and innate immunity . Literature reports a number of microRNAs, mainly miR-21, miR-155, miR-146a, miR-223 to be important regulators of the protein coding genes involved in the above mentioned pathways , , . Studies on microRNA expression in bacterial infections of various sources similarly point towards the same microRNAs , , ,  as the key players in the host response. We found 17 microRNAs up regulated in necrotic versus visually unaffected sample, while the protein coding study on the same biological material found that the highest number of deregulated genes was in the necrotic area . The microRNAs showing remarkably high expression level in our study namely miR-21 is a powerful anti-inflammatory regulator, which by direct targeting of a PDCD4 gene inhibits the pro-inflammatory regulator NF-kB. Moreover, the levels of anti-inflammatory cytokine IL-10 are increased upon miR-21 up-regulation . However, our study detected an extremely high expression of miR-21 in all the samples, regardless the infection status. Similar results of high expression of miR-21 have been found in viral infection of porcine dendritic cells .
Two targets of miR-21: Interleukin I Beta (IL1β) and tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), which is rapidly induced by the TNF were assayed by RT-qPCR on mRNA in another APP infection study in pigs . In the study of Mortensen and collaborators IL1β was up-regulated in infected samples and TNFAIP3 was repressed. Ideally, we would expect an up-regulation of microRNA while mRNA is attenuated and vice versa. However, the lack of such correlation is not necessarily contradictive to the nature of microRNA mediated regulation. A single mRNA can be fine-tuned by multiple microRNAs and this regulation is a very complex, multi-factorial and time dependent network and possibly samples taken at different time points of infection would result in slightly different profiles.
The following key player in control of immunity and inflammation, miR-155, showed rather contradicting results to those existing in the literature ; ; . In our RT-qPCR study, miR-155 was significantly down-regulated under pathogen triggered inflammation and so was mRNA coding for the miR-155 target IFN-γ in the same animals .
The 5b APP serotype used in the present study is highly virulent. It has been show that the lethality of pleuropneumonia in pigs is not necessarily caused by ventilator failure  but by a septic shock-like condition, which develops when, immunological and inflammatory responses are unbalanced and uncontrolled release of cytokines occurs . Such dramatic disturbance in regulation of immune response could be the explanation for the surprisingly reversed miR-155 expression profile.
Another microRNA candidate namely miR-223 was investigated in the present study. We indeed detected a remarkable, gradually increasing expression of miR-223 with infection progression, in infected samples in comparison to the control. Moreover, Matsushima and co-workers (2011) found miR-223 to be the only up-regulated microRNA in Helicobacter pylori-infected gastric mucosa. TLR4 and TLR3 pathways are possibly targeted by miR-223 .
Nearly all up-regulated microRNAs in lung tissue from infected pigs target IL-6R and/or IL6ST, in addition miR-144 and miR-146a 5p targets mRNA coding for IL-6. IL-6 was found to be highly up-regulated in the same animals  14–18 h after experimental infection. We might speculate that expression of this pro-inflammatory cytokine as well as its receptor will be tightly regulated in order to avoid host mediated tissue damage. Unpublished time-course studies of mRNA from lung tissue reveal IL-6 first to be highly up-regulated and then to be significant down-regulated somewhere between 12 and 24 h after infection with APP (Skovgaard, personal communication) MicroRNA miR-148a and miR-126 are also mentioned in the inflammation related literature with miR-148a having implication in function of primary bronchial epithelial cells  and miR-126 in chronic asthma where initial increase in expression of this microRNA was found . The miR-148a similarly to miR-146a-5p was found to target mRNA encoding IRAK1. Our most confident prediction for miR-148a is a SMAD2 gene involved in regulation of cell growth and proliferation - two processes which are rather silenced during stressful bacterial infection. This corresponds to great up-regulation of miR-148a in infected tissue. Interestingly, Interleukin 21 receptor (IL21R) is regulated by miR-126. Interleukin 21 is yet another immunoregulatory cytokine though to be involved in transition from innate to adaptive immunity. The remaining microRNA candidates namely: miR-15a, -142-5p, -144, -451 and −664 were not previously described as important in response to infectious pathogen lung infection. Nevertheless, we provide a very distinct and significant expression profiles supported by multiple target predictions for those microRNAs, which suggest that they might be a new coming group of microRNAs involved in inflammation regulatory networks.