Multi-omics analysis reveals the impact of influenza a virus host adaptation on immune signatures in pig tracheal tissue.

Introduction: Influenza A virus (IAV) infection is a global respiratory disease, which annually leads to 3-5 million cases of severe illness, resulting in 290,000-650,000 deaths. Additionally, during the past century, four global IAV pandemics have claimed millions of human lives. The epithelial lining of the trachea plays a vital role during IAV infection, both as point of viral entry and replication as well as in the antiviral immune response. Tracheal tissue is generally inaccessible from human patients, which makes animal models crucial for the study of the tracheal host immune response. Method: In this study, pigs were inoculated with swine- or human-adapted H1N1 IAV to gain insight into how host adaptation of IAV shapes the innate immune response during infection. In-depth multi-omics analysis (global proteomics and RNA sequencing) of the host response in upper and lower tracheal tissue was conducted, and results were validated by microfluidic qPCR. Additionally, a subset of samples was selected for histopathological examination. Results: A classical innate antiviral immune response was induced in both upper and lower trachea after infection with either swine- or human-adapted IAV with upregulation of genes and higher abundance of proteins associated with viral infection and recognition, accompanied by a significant induction of interferon stimulated genes with corresponding higher proteins concentrations. Infection with the swine-adapted virus induced a much stronger immune response compared to infection with a human-adapted IAV strain in the lower trachea, which could be a consequence of a higher viral load and a higher degree of inflammation. Discussion: Central components of the JAK-STAT pathway, apoptosis, pyrimidine metabolism, and the cytoskeleton were significantly altered depending on infection with swine- or human-adapted virus and might be relevant mechanisms in relation to antiviral immunity against putative zoonotic IAV. Based on our findings, we hypothesize that during host adaptation, IAV evolve to modulate important host cell elements to favor viral infectivity and replication.

Pulmonary MicroRNA expression after heterologous challenge with swine influenza A virus (H1N2) in immunized and non-immunized pigs.

MicroRNAs (miRNAs) contribute to post-transcriptional modulation of the host response during influenza A virus (IAV) infection and may be involved in shaping disease severity. Differential disease severity was achieved in two groups of pigs by immunization of one group with a commercial swine IAV vaccine prior to heterologous IAV (H1N2) challenge of both groups. Lung tissue was harvested 1, 3, and 14 days after challenge and miRNA expression was quantified. Gene Ontology term enrichment analysis was employed to examine the functional relevance of genes potentially regulated by differentially expressed miRNAs in pigs with varying degrees of disease severity following IAV infection. Results suggested that the miRNA response associated with less severe disease may modulate host mechanisms essential for viral life cycle, e.g. transcription, translation, and protein trafficking. During more severe disease, miRNA-mediated regulation may focus on dampening virus-specific processes e.g. virion assembly and viral protein processing, and controlling host metabolism.

Association of serum and fecal microRNA profiles in cats with gastrointestinal cancer and chronic inflammatory enteropathy.

BACKGROUND: Differentiation of gastrointestinal cancer (GIC) from chronic inflammatory enteropathies (CIE) in cats can be challenging and often requires extensive diagnostic testing. MicroRNAs (miRNAs) have promise as non-invasive biomarkers in serum and feces for diagnosis of GIC. HYPOTHESIS/OBJECTIVES: Cats with GIC will have serum and fecal miRNA profiles that differ significantly from healthy cats and cats with CIE. Identify serum and fecal miRNAs with diagnostic potential for differentiation between cats with GIC and CIE as compared to healthy cats. ANIMALS: Ten healthy cats, 9 cats with CIE, and 10 cats with GIC; all client-owned. METHODS: Cats were recruited for an international multicenter observational prospective case-control study. Serum and feces were screened using small RNA sequencing for miRNAs that differed in abundance between cats with GIC and CIE, and healthy cats. Diagnostic biomarker potential of relevant miRNAs from small RNA sequencing and the literature was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Serum miR-223-3p was found to distinguish between cats with GIC and CIE with an area under the curve (AUC) of 0.9 (95% confidence interval [CI], 0.760-1.0), sensitivity of 90% (95% CI, 59.6-99.5%), and specificity of 77.8% (95% CI, 45.3-96.1%). Serum miR-223-3p likewise showed promise in differentiating a subgroup of cats with small cell lymphoma (SCL) from those with CIE. No fecal miRNAs could distinguish between cats with GIC and CIE. CONCLUSION AND CLINICAL IMPORTANCE: Serum miR-223-3p potentially may serve as a noninvasive diagnostic biomarker of GIC in cats, in addition to providing a much needed tool for the differentiation of CIE and SCL.

Transcriptional Host Responses to Infection with Streptococcus suis in a Porcine Precision-Cut Lung Slice Model: Between-Strain Differences Suggest Association with Virulence Potential.

Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.

Association of fecal and serum microRNA profiles with gastrointestinal cancer and chronic inflammatory enteropathy in dogs.

BACKGROUND: Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum microRNAs (miRNAs) have been proposed as diagnostic and prognostic markers of GI disease in humans and dogs. HYPOTHESIS/OBJECTIVES: Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. AIMS: (a) identify miRNAs that differentiate GIC from CIE, (b) use high-throughput reverse transcription quantitative real-time PCR (RT-qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs. ANIMALS: Twenty-four dogs with GIC, 10 dogs with CIE, and 10 healthy dogs, all client-owned. METHODS: An international multicenter observational prospective case-control study. Small RNA sequencing was used to identify fecal and serum miRNAs, and RT-qPCR was used to establish fecal and serum miRNA panels with the potential to distinguish GIC from CIE. RESULTS: The best diagnostic performance for distinguishing GIC from CIE was fecal miR-451 (AUC: 0.955, sensitivity: 86.4%, specificity: 100%), miR-223 (AUC: 0.918, sensitivity: 90.9%, specificity: 80%), and miR-27a (AUC: 0.868, sensitivity: 81.8%, specificity: 90%) and serum miR-20b (AUC: 0.905, sensitivity: 90.5%, specificity: 90%), miR-148a-3p (AUC: 0.924, sensitivity: 85.7%, specificity: 90%), and miR-652 (AUC: 0.943, sensitivity: 90.5%, specificity: 90%). Slightly improved diagnostic performance was achieved when combining fecal miR-451 and miR-223 (AUC: 0.973, sensitivity: 95.5%, specificity: 90%). CONCLUSIONS AND CLINICAL IMPORTANCE: When used as part of a diagnostic RT-qPCR panel, the abovementioned miRNAs have the potential to function as noninvasive biomarkers for the differentiation of GIC and CIE in dogs.

Urinary microRNAome in healthy cats and cats with pyelonephritis or other urological conditions.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have been found in urine and have shown diagnostic potential in human nephropathies. Here, we aimed to characterize, for the first time, the feline urinary miRNAome and explore the use of urinary miRNA profiles as non-invasive biomarkers for feline pyelonephritis (PN). Thirty-eight cats were included in a prospective case-control study and classified in five groups: healthy Control cats (n = 11), cats with PN (n = 10), cats with subclinical bacteriuria or cystitis (SB/C, n = 5), cats with ureteral obstruction (n = 7) and cats with chronic kidney disease (n = 5). By small RNA sequencing we identified 212 miRNAs in cat urine, including annotated (n = 137) and putative novel (n = 75) miRNAs. The 15 most highly abundant urinary miRNAs accounted for nearly 71% of all detected miRNAs, most of which were previously identified in feline kidney. Ninety-nine differentially abundant (DA) miRNAs were identified when comparing Control cats to cats with urological conditions and 102 DA miRNAs when comparing PN to other urological conditions. Tissue clustering analysis revealed that the majority of urine samples clustered close to kidney, which confirm the likely cellular origin of the secreted urinary miRNAs. Relevant DA miRNAs were verified by quantitative real-time PCR (qPCR). Eighteen miRNAs discriminated Control cats from cats with a urological condition. Of those, seven miRNAs were DA by both RNAseq and qPCR methods between Control and PN cats (miR-125b-5p, miR-27a-3p, miR-21-5p, miR-27b-3p, miR-125a-5p, miR-17-5p and miR-23a-3p) or DA between Control and SB/C cats (miR-125b-5p). Six additional miRNAs (miR-30b-5p, miR-30c, miR-30e-5p, miR-27a-3p, miR-27b-39 and miR-222) relevant for discriminating PN from other urological conditions were identified by qPCR alone (n = 4) or by both methods (n = 2) (P<0.05). This panel of 13 miRNAs has potential as non-invasive urinary biomarkers for diagnostic of PN and other urological conditions in cats.

Innate antiviral responses in porcine nasal mucosal explants inoculated with influenza A virus are comparable with responses in respiratory tissues after viral infection.

Nasal mucosal explant (NEs) cultured at an air-liquid interface mimics in vivo conditions more accurately than monolayer cultures of respiratory cell linesor primary cells cultured in flat-bottom microtiter wells. NEs might be relevant for studies of host-pathogen interactions and antiviral immune responses after infection with respiratory viruses, including influenza and corona viruses. Pigs are natural hosts for swine influenza A virus (IAV) but are also susceptible to IAV from humans, emphasizing the relevance of porcine NEs in the study of IAV infection. Therefore, we performed fundamental characterization and study of innate antiviral responses in porcine NEs using microfluidic high-throughput quantitative real-time PCR (qPCR) to generate expression profiles of host genes involved in inflammation, apoptosis, and antiviral immune responses in mock inoculated and IAV infected porcine NEs. Handling and culturing of the explants ex vivo had a significant impact on gene expression compared to freshly harvested tissue. Upregulation (2-43 fold) of genes involved in inflammation, including IL1A and IL6, and apoptosis, including FAS and CASP3, and downregulation of genes involved in viral recognition (MDA5 (IFIH1)), interferon response (IFNA), and response to virus (OAS1, IFIT1, MX1) was observed. However, by comparing time-matched mock and virus infected NEs, transcription of viral pattern recognition receptors (RIG-I (DDX58), MDA5 (IFIH1), TLR3) and type I and III interferons (IFNB1, IL28B (IFNL3)) were upregulated 2-16 fold in IAV-infected NEs. Furthermore, several interferon-stimulated genes including MX1, MX2, OAS, OASL, CXCL10, and ISG15 was observed to increase 2-26 fold in response to IAV inoculation. NE expression levels of key genes involved in antiviral responses including IL28B (IFNL3), CXCL10, and OASL was highly comparable to expression levels found in respiratory tissues including nasal mucosa and lung after infection of pigs with the same influenza virus isolate.

Piglet innate immune response to Streptococcus suis colonization is modulated by the virulence of the strain.

Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of piglets by S. suis is very common and occurs early in life. The clinical outcome of infection is influenced by the virulence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohistochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inoculation. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites, with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response in piglets that appears to be related to the virulence potential of the colonizing strain.

AU Content in the MicroRNA Sequence Influences its Stability after Heat Treatment.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules which regulate gene expression post-transcriptionally and are involved in a multitude of cellular processes. MiRNAs are known to be very stable compared to messenger RNAs (mRNAs), making them excellent candidates as biomarkers for disease. Recently, studies have suggested that miRNA stability in formalin fixed samples might depend on their nucleotide composition. OBJECTIVE: To explore the stability of a panel of miRNAs isolated from porcine blood and lung tissue after heat and enzyme treatment. METHOD: Porcine RNA isolated from lung tissue and blood leukocytes was used for this study. RNA samples were exposed to heat treatment and RNAse A digestion. The levels of selected miRNAs were measured by means of qPCR before and after heat and enzyme treatment. RESULTS: Fourteen miRNAs were successfully analysed, and they were found to degrade differently after exposure to heat or RNAse A. MiRNAs with <60% of adenine (A) and uracil (U) in their sequence were found to be more stable. CONCLUSION: This is the first study showing that different miRNAs isolated from lung tissue display unequal stability after heat treatment, probably based on their nucleotide composition, highlighting the importance of considering the miRNA sequence when investigating their value as biomarkers.

Animal Models for Influenza A Virus Infection Incorporating the Involvement of Innate Host Defenses: Enhanced Translational Value of the Porcine Model.

Influenza is a viral respiratory disease having a major impact on public health. Influenza A virus (IAV) usually causes mild transitory disease in humans. However, in specific groups of individuals such as severely obese, the elderly, and individuals with underlying inflammatory conditions, IAV can cause severe illness or death. In this review, relevant small and large animal models for human IAV infection, including the pig, ferret, and mouse, are discussed. The focus is on the pig as a large animal model for human IAV infection as well as on the associated innate immune response. Pigs are natural hosts for the same IAV subtypes as humans, they develop clinical disease mirroring human symptoms, they have similar lung anatomy, and their respiratory physiology and immune responses to IAV infection are remarkably similar to what is observed in humans. The pig model shows high face and target validity for human IAV infection, making it suitable for modeling many aspects of influenza, including increased risk of severe disease and impaired vaccine response due to underlying pathologies such as low-grade inflammation. Comparative analysis of proteins involved in viral pattern recognition, interferon responses, and regulation of interferon-stimulated genes reveals a significantly higher degree of similarity between pig, ferret, and human compared with mice. It is concluded that the pig is a promising animal model displaying substantial human translational value with the ability to provide essential insights into IAV infection, pathogenesis, and immunity.

IFN-λ and microRNAs are important modulators of the pulmonary innate immune response against influenza A (H1N2) infection in pigs.

The innate immune system is paramount in the response to and clearance of influenza A virus (IAV) infection in non-immune individuals. Known factors include type I and III interferons and antiviral pathogen recognition receptors, and the cascades of antiviral and pro- and anti-inflammatory gene expression they induce. MicroRNAs (miRNAs) are increasingly recognized to participate in post-transcriptional modulation of these responses, but the temporal dynamics of how these players of the antiviral innate immune response collaborate to combat infection remain poorly characterized. We quantified the expression of miRNAs and protein coding genes in the lungs of pigs 1, 3, and 14 days after challenge with swine IAV (H1N2). Through RT-qPCR we observed a 400-fold relative increase in IFN-λ3 gene expression on day 1 after challenge, and a strong interferon-mediated antiviral response was observed on days 1 and 3 accompanied by up-regulation of genes related to the pro-inflammatory response and apoptosis. Using small RNA sequencing and qPCR validation we found 27 miRNAs that were differentially expressed after challenge, with the highest number of regulated miRNAs observed on day 3. In contrast, the number of protein coding genes found to be regulated due to IAV infection peaked on day 1. Pulmonary miRNAs may thus be aimed at fine-tuning the initial rapid inflammatory response after IAV infection. Specifically, we found five miRNAs (ssc-miR-15a, ssc-miR-18a, ssc-miR-21, ssc-miR-29b, and hsa-miR-590-3p)-four known porcine miRNAs and one novel porcine miRNA candidate-to be potential modulators of viral pathogen recognition and apoptosis. A total of 11 miRNAs remained differentially expressed 14 days after challenge, at which point the infection had cleared. In conclusion, the results suggested a role for miRNAs both during acute infection as well as later, with the potential to influence lung homeostasis and susceptibility to secondary infections in the lungs of pigs after IAV infection.

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells.

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.

Late regulation of immune genes and microRNAs in circulating leukocytes in a pig model of influenza A (H1N2) infection.

MicroRNAs (miRNAs) are a class of short regulatory RNA molecules which are implicated in modulating gene expression. Levels of circulating, cell-associated miRNAs in response to influenza A virus (IAV) infection has received limited attention so far. To further understand the temporal dynamics and biological implications of miRNA regulation in circulating leukocytes, we collected blood samples before and after (1, 3, and 14 days) IAV challenge of pigs. Differential expression of miRNAs and innate immune factor mRNA transcripts was analysed using RT-qPCR. A total of 20 miRNAs were regulated after IAV challenge, with the highest number of regulated miRNAs seen on day 14 after infection at which time the infection was cleared. Targets of the regulated miRNAs included genes involved in apoptosis and cell cycle regulation. Significant regulation of both miRNAs and mRNA transcripts at 14 days after challenge points to a protracted effect of IAV infection, potentially affecting the host's ability to respond to secondary infections. In conclusion, experimental IAV infection of pigs demonstrated the dynamic nature of miRNA and mRNA regulation in circulating leukocytes during and after infection, and revealed the need for further investigation of the potential immunosuppressing effect of miRNA and innate immune signaling after IAV infection.

Concurrent host-pathogen gene expression in the lungs of pigs challenged with Actinobacillus pleuropneumoniae.

BACKGROUND: Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. RESULTS: Host and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen. CONCLUSIONS: Microbial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.