Global profiling of lncRNAs-miRNAs-mRNAs reveals differential expression of coding genes and non-coding RNAs in the lung of beagle dogs at different stages of Toxocara canis infection

Highlights

• Global profiles of lncRNAs-miRNAs-mRNAs in the lungs of Beagle dogs infected with Toxocara canis were determined.

• Many altered RNAs were found in infected dogs, but the inflammatory responses remained balanced.

• The level of scgb1a1 increased more than 100 times in the three stages of infection.

• The competing endogenous RNA networks of DElncRNA-DEmiRNA-DEmRNA were constructed.

• New insights into the interaction between T. canis and the canine lung were provided.

Abstract

The roundworm Toxocara canis causes toxocariasis in dogs and larval migrans in humans. Better understanding of the lung response to T. canis infection could explain why T. canis must migrate to and undergoes part of its development inside the lung of the definitive host. In this study, we profiled the expression patterns of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in the lungs of Beagle dogs infected by T. canis, using high throughput RNA sequencing. At 24 h p.i., 1,012 lncRNAs, 393 mRNAs and 10 miRNAs were differentially expressed (DE). We also identified 883 DElncRNAs, 264 DEmRNAs and 20 DEmiRNAs at 96 h p.i., and 996 DElncRNAs, 342 DEmRNAs and eight DEmiRNAs at 36 days p.i., between infected and control dogs. Significant changes in the levels of expression of transcripts related to immune response and inflammation were associated with the antiparasitic response of the lung to T. canis. The remarkable increase in the expression of scgb1a1 at all time points after infection suggests the need for consistent moderation of the excessive inflammatory response. Also, upregulation of foxj1 at 24 h p.i., and downregulation of IL-1β and IL-21 at 96 h p.i., suggest an attenuation of the humoral immunity of infected dogs. These results indicate that T. canis pathogenesis in the lung is mediated through contributions from both pro-inflammatory and anti-inflammatory mechanisms. Competing endogenous RNA (ceRNA) network analysis revealed significant interactions between DElncRNAs, DEmiRNAs and DEmRNAs, and improved our understanding of the ceRNA regulatory mechanisms in the context of T. canis infection. These data provide comprehensive understanding of the regulatory networks that govern the lung response to T. canis infection and reveal new mechanistic insights into the interaction between the host and parasite during the course of T. canis infection in the canine.

Introduction

The ascarid roundworm Toxocara canis infects the small intestine of dogs and can be transmitted to other mammals including humans (Ma et al., 2018). This parasite is highly zoonotic and has been listed as one of the five neglected parasitic infections by the American Centers for Disease Control and Prevention (https://www.cdc.gov/parasites/npi/). Toxocara canis is widespread throughout the world and environmental contamination with T. canis eggs is very common in soil samples, especially in urban public parks (Chen et al., 2018, Fakhri et al., 2018). Toxocara canis has a complex life cycle and during its development the parasite encounters diverse and multiple physiological niches inside the host (Maizels, 2013). Following ingestion of infectious eggs by the canine definitive host, larvae are released from eggs in the intestine where they penetrate the intestinal wall and migrate to the liver, heart and lung. Larvae penetrate through the alveolar wall and migrate up to the trachea and pharynx, where they are swallowed and enter the intestine to complete their development into adult worms.

In the paratenic and accidental hosts, the pulmonary phase of the life cycle is missing; larvae migrate through the blood vessel and spread to various tissues where they become arrested at the L3 stage for extensive periods without reaching the trachea, which is a prerequisite route to re-enter the host digestive tract (Webster, 1958). This dichotomy in the parasite’s behaviour between the natural host and paratenic/accidental host raised an interesting question as to why T. canis requires a lung migratory phase during its development inside the definitive host, particularly if the parasite takes up residence in the definitive host’s intestine. Also, what role does the lung play during the pulmonary component of the T. canis lifecycle? (Craig and Scott, 2014). Impaired lung function due to T. canis infection manifests as coughing, eosinophilic pneumonia and asthma, and the severity of the respiratory manifestations correlates with the larval load in the lung (Kuzucu, 2006). Therefore, in this strategic anatomical location, lung tissues must play an eminent role in host defence by recognising and responding to T. canis invasion. On the other hand, T. canis has to deal with the lung defence mechanisms to ensure its own survival. The outcome of this host-parasite interaction determines the outcome of infection. How T. canis maintains its survival under these hostile circumstances is largely unknown.

Previous studies involving high-throughput genomic, transcriptomic and proteomic approaches have been performed in order to explore the systems biology of T. canis (Zhu et al., 2015, Zheng et al., 2020). Also, metabolomics showed that T. canis infection can alter some important metabolic pathways in the definitive host (Zheng et al., 2019). Additionally, microarray analysis revealed transcriptional differences in neurotoxocarosis caused by T. canis and Toxocara cati (Janecek et al., 2015). Despites these efforts, genomic information regarding the interaction between T. canis and the complex microenvironment of the host lung is lacking. Similarly, we do not have a detailed understanding of the extent of transcriptional regulation during lung infection, including temporal changes in expression of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs, and changes in transcription factors (TFs). These different types of regulatory information need to be simultaneously collected in order to reconstruct an accurate and detailed understanding of the canine lung response to T. canis infection.

In the present study, we investigated alterations in the expression of lncRNAs, miRNAs and mRNAs in the lungs of Beagle dogs infected by T. canis, at different stages of infection. Our data provided comprehensive information about T. canis infection-related gene expression trajectories and key regulators of specific immunological mechanisms that mediate the interaction between T. canis and the canine lung.