Background

Toxocara canis, a ubiquitous nematode in dogs, is responsible for the disease known as toxocariasis. Dogs infected by T. canis excrete eggs which contaminate the soil and the dog’s hair [1]. When T. canis eggs are ingested by the definitive canid host, the larvae hatch in the intestine, penetrate the intestinal wall and migrate to the mesenteric lymph nodes and reach the liver by 24 h post-infection (hpi). Subsequently, the larvae migrate to the lung at approximately 96 hpi following a cardio-pulmonary route. In the lungs, larvae break out into the alveoli and ascend through the trachea to the pharynx from where they are swallowed and enter the gastrointestinal tract to complete their development into adult roundworms [2].

Toxocara canis can also infect humans where the larvae migrate throughout the body without reaching the adult stage [3]. Clinical disease in humans can take several forms including visceral toxocariasis, ocular toxocariasis, neurotoxocariasis and covert toxocariasis [4]. Mice infected by T. canis exhibit pulmonary inflammation and increased levels of IgE, eosinophils and Th-2 type cytokines [5,6,7]. Migration of T. canis larvae through the livers can cause hepatic damage and inflammation [2]. A previous study showed the significant role of miRNAs in the pathogenesis of T. canis during the hepatic phase of parasite development in Beagle dogs [8].

The recent advances in understanding the molecular pathogenesis of T. canis infection in dogs has been facilitated by the application of high-throughput ‘omics’ approaches, including transcriptomics, proteomics and metabolomics [9]. For example, previous transcriptomic analysis allowed the identification of long noncoding RNA (lncRNA) and messenger RNA (mRNA) differentially expressed in the spleen [10] and lung [11] of Beagle dogs during T. canis infection. LncRNAs are cellular RNA transcripts with a length of > 200 nt [12] and regulate gene expression through a variety of mechanisms [13, 14]. They are involved in the regulation of diverse biological processes, including mediating host-parasite interaction [15,16,17].

Despite the advances in the understanding of the pathobiology of T. canis infection, the roles of lncRNAs in the hepatic immune-inflammatory response to infection are still incompletely defined. Therefore, we focused on the identification of lncRNAs and mRNAs involved in hepatic pathology associated with T. canis infection. In the present study, RNA-Seq analysis was performed to investigate the effect of T. canis infection on the expression of lncRNAs and mRNAs in the liver of Beagle dogs at different stages post-infection.

Methods

Ethics approval

The study was approved by the Research Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval no. 2018–015). All animals were under constant veterinary care during the study and were housed, fed and handled in strict compliance with accepted animal welfare guidelines.

Toxocara canis infection

The T. canis eggs were isolated from the uteri of female worms and incubated at 28 °C and 85–95% relative humidity for 28 days. Then, the embryonated eggs were stored at 4 °C (in 1% formalin solution) as previously described [18]. The study protocol used to establish T. canis infection was described previously [18]. Briefly, 6–7-week-old Beagle puppies (n = 18) were allocated to six groups, with three dogs per group [8]. Three infected groups were inoculated orally with 1 ml saline containing 300 T. canis embryonated eggs/puppy, whereas dogs in the three control groups were inoculated with 1 ml saline only. All dogs were in apparent good health prior to and at the start of the study. Additionally, thorough health observations of each dog were performed at least daily during the study.

Sample collection and RNA isolation

At 12 h post-infection (hpi), 24 hpi and 36 days post-infection (dpi), one infected group and one control group of puppies were euthanized by intracardiac injection of potassium chloride (KCl) solution under general anesthesia using Zoletil™ 50 (Virbac, Carros, France). This procedure was performed by a well-trained veterinarian with good knowledge of the anesthetic techniques to ensure the suitability of the anesthetic depth prior to administration of KCl. The livers were dissected from each puppy and washed with saline to remove the blood from the liver surface. Twenty-five milligram sample from the left lateral hepatic lobe was collected from each animal and stored in liquid nitrogen. No visible pathological changes were observed in the liver because of low infection of T. canis eggs. The total RNA was extracted from the liver sample using TRIZOL (Life Technologies, Carlsbad, CA, USA). The hepatic tissue samples were homogenized using liquid nitrogen, followed by lysis using 1 ml of TRIZOL reagent. The RNA was then extracted using chloroform, and the aqueous phase was obtained by centrifugation at 11,500 g for 15 min at 4 °C. The aqueous phase was transferred to a new RNA-free EP tube, and RNA was precipitated using isopropanol. Finally, the RNA samples were washed with 75% ethanol. Genomic DNA was removed from the RNA samples using DNaseI (New England BioLabs, Ipswich, MA, USA). Then 1% agarose gel was used to detect any degradation and contamination in the extracted RNA. Then, the RNA concentration and integrity were measured, and only RNA with integrity number (RIN) ≥ 8 was used in sequencing analysis [19].

Library preparation and sequencing

Each lncRNA library was constructed with 3 μg rRNA-depleted RNA and was generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). The first-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). To select cDNA fragments of ~ 150 to 200 bp length, the library fragments were subjected to purification using the AMPure XP system (Beckman Coulter, Beverly, MA, USA). Subsequently, 3 μl USER Enzyme (NEB, Ipswich, USA) was applied to the size-selected cDNA fragments, which were ligated with adaptors, and the mixture was incubated at 37 °C for 15 min. The mixture was then heated to 95 °C for 5 min and subjected to amplification. The PCR was carried out using Phusion High-Fidelity DNA polymerase, with Universal PCR primers and Index (X) Primer. The PCR products were purified using the AMPure XP system, and the library quality was evaluated on the Agilent Bioanalyzer 2100 system [20]. The index-coded samples were clustered using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) on a cBot Cluster Generation System, following the manufacturer's instructions. Subsequently, the generated clusters were sequenced on an Illumina Hiseq 4000 platform, producing 150-bp paired-end reads for the libraries.

Identification of lncRNAs and mRNAs

The raw reads (raw data) of fastq format were obtained. Then, the adapter, poly-N and low-quality reads were removed to obtain the clean reads (clean data). The GC content was calculated to assess the quality of the data. The HISAT2 v2.0.4 was used to align clean reads to the Canis lupus familiaris reference genome [21]. StringTie (v1.3.1) was used to assemble the mapped reads [22]. Cuffdiff (v2.1.1) was used to calculate the FPKMs (fragments per kilobase of exon model per million fragments) of lncRNAs and mRNAs [23]. The splicing transcripts with exons ≥ 2 and lengths > 200 bp were screened for lncRNA transcripts. Three tools, CNCI (Coding-Non-Coding-Index) (v2), CPC (Coding Potential Calculator) (0.9-r2) and Pfam Scan (v1.3) (v20121028), were used to predict novel lncRNAs [24,25,26].

Differential expression analysis

The differential expression analysis at each infection stage was performed using DESeq 2 (1.14.1) [27]. P-value and fold change were set as criteria for identifying the differential transcripts for lncRNAs and mRNAs. P-value < 0.05 and log2 (fold change) |≥ 1 were considered as differentially expressed (DE) lncRNA and DEmRNA.

Functional analysis of DEmRNAs

Gene ontology (GO) annotation analysis of the DEmRNAs was performed using the GOseq R package [28], including biological process (BP), cellular component (CC) and molecular function (MF). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to map genes and identify the enriched signaling pathway using KOBAS 3.0 [29]. P-value < 0.05 was considered significant enrichment.

Target gene prediction and GO analysis of DElncRNA

LncRNAs change the expression of nearby genes; thus, the potential target genes of DElncRNAs were predicted according to the co-location (within the upstream and downstream 100 kb) of mRNAs-DElncRNAs. GO annotation analysis was performed using the potential target genes of DElncRNA to predict the functions of DElncRNA. P < 0.05 was considered significant enrichment. The network between DElncRNAs and DEmRNAs was visualized using Cytoscape v.3.5 [30].

Quantitative real-time PCR (qRT-PCR) analysis

Seven DEmRNAs and three DElncRNAs were randomly chosen to verify the RNA-Seq results using qRT-PCR as described previously [11]. Briefly, the PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Tokyo, Japan) and lnRcute lncRNA cDNA first-chain synthesis kit (TianGen, Beijing, China) were used to synthesize the first-strand cDNA of the mRNA and lncRNA, respectively. Then, qRT-PCR was performed using a LightCycler480 (Roche, Basel, Switzerland). The amplification conditions for mRNAs were initial denaturation (95 °C for 10 min) followed by amplification and quantification program repeated for 40 cycles (95 °C for 30 s, 60 °C for 1 min, 60 °C for 1 min). The amplification condition of lncRNA included an initial denaturation (95 °C for 3 min) and 40 cycles of template denaturation at 94 °C for 5 s and annealing at 60 °C for 15 s.

The melting curve analysis was included in each reaction. The transcripts and primers are listed in Table 1. The ribosomal protein L13A was used as the housekeeping gene [11]. All reactions were repeated independently three times. The relative expression level was calculated by the 2−ΔΔCt method [31].

Table 1 Transcripts and primers used in the qRT-PCR analysis
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Results

RNA-sequencing data

In this study, 2,119,950,482 raw reads were obtained from liver samples of 18 puppies. We identified 2,068,104,062 clean reads and 310.22 Gb clean data. The averages of GC and Q30 in the clean reads were 52.47% and 93.73%, respectively (Table 2). After mapping to the C. lupus familiaris reference genome, the transcripts were divided into different sub-types. A total of 23,928 mRNAs, 883 annotated lncRNAs and 20,694 novel lncRNAs transcripts were identified.

Table 2 Summary of the characteristics of lncRNA libraries
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DElncRNAs and DEmRNAs

Compared to the control groups, a total of 876 DElncRNAs and 288 DEmRNAs were identified at 12 hpi; 906 DElncRNAs and 261 DEmRNAs were identified at 24 hpi; 876 DElncRNAs and 302 DEmRNAs were identified at 36 dpi (Fig. 1 and Additional file 1: Table S1). Also, 16 DEmRNAs (e.g. crp, dpp4 and gnas) were commonly detected at the three infection stages (Fig. 2 and Table 3), of which the expression level of C-reactive protein (crp) was significantly downregulated at 24 hpi and 36 dpi. In addition, the expression of dipeptidyl peptidase 4 (dpp4) was significantly upregulated at 12 hpi, reaching its maximum at 24 hpi, but was significantly downregulated at 36 dpi. The expression of G-protein alpha-subunit (gnas) was significantly increased at 24 hpi (Table 3). The expression profile of the DElncRNAs and DEmRNAs was confirmed by qRT-PCR (Additional file 2: Fig. S1).

Fig. 1
figure 1

Volcano plots showing the differentially expressed (DE) mRNAs (a–c) and DElnRNAs (d–f) at 12 hpi, 24 hpi and 36 dpi, respectively. The X-axis shows the log2 fold change of the DERNAs, while the Y-axis shows the corresponding -log10 P-value. Up- and downregulated RNAs are shown in red and blue, respectively. DERNAs that did not pass the threshold for the log fold change (not significant) are shown in gray

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Fig. 2
figure 2

Venn diagram of the differentially expressed mRNAs at 12 hpi, 24 hpi and 36 dpi

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Table 3 Common differentially expressed mRNAs in the dog’s liver at three stages post Toxocara canis infection
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GO annotation and KEGG pathway analysis of the DEmRNAs

A total of 482, 744 and 610 GO terms were significantly enriched at 12 hpi, 24 hpi and 36 dpi, respectively (Additional file 3: Table S2). At 12 hpi, most of the DEmRNAs were mainly associated with cellular processes (GO:0000902, GO:0016043, GO:0071840 and GO:0032989); at 24 hpi, DEmRNAs were mainly related to lipoprotein component (GO:0034364, GO:0034358 and GO:0032994); at 36 dpi, DEmRNAs were mainly involved in the regulation of growth of symbiont involved in interaction with host (GO:0044130, GO:0044146, GO:0044126, GO:0044144, GO:0044110, GO:0044116 and GO:0044117) (Additional file 3: Table S2).

Some GO terms related to immune response or inflammation were observed at three stages of infection (Additional file 4: Table S3 and Additional file 5: Fig. S2). At 12 hpi, 47 DEmRNAs were significantly enriched in 44 immune- or inflammation-related GO terms, and the top 30 terms were mainly associated with immune system process (GO:0002376), negative regulation of immune system process (GO:0002683), Toll-like receptor 3 signaling pathway (GO:0034138) and cell adhesion-mediated by integrin (GO:0033627) (Additional file 5: Fig. S2a). At 24 hpi, 49 DEmRNAs were significantly enriched in 47 immunity- or inflammation-related GO terms, such as immune system process (GO:0002376), immune response (GO:0006955), immune system development (GO:0002520), cytokine production (GO:0001816) and T cell activation (GO:0042110) (Additional file 5: Fig. S2b).

At 36 dpi, 45 DEmRNAs were significantly enriched in 63 immune- or inflammation-related GO terms that were mainly associated with leukocyte-mediated cytotoxicity (GO:0001909), antigen processing and presentation (GO:0019882) and regulation of leukocyte-mediated cytotoxicity (GO:0001910), and interleukin-8 production (GO:0032637) (Additional file 5: Fig. S2c). In addition, DEmRNA crp was significantly enriched in immune system process (GO:0002376), regulation of stress-activated MAPK cascade (GO:0032872), immune system development (GO:0002520), leukocyte mediated immunity (GO:0002443), cytokine production (GO:0001816), immune response (GO:0006955), leukocyte-mediated immunity (GO:0002443), lymphocyte-mediated immunity (GO:0002449), interleukin-8 production (GO:0032637), regulation of interleukin-8 production (GO:0032677) and interleukin-8 secretion (GO:0072606). The dpp4 was involved in the immune system process (GO:0002376), regulation of cytokine production (GO:0001817) and lymphocyte activation (GO:0046649) at three stages of infection (Fig. 3a–c).

Fig. 3
figure 3

Chord diagrams showing the gene ontology (GO) of the differentially expressed genes in the liver at 12 hpi (a), 24 hpi (b) and 36 dpi (c), respectively. The diagrams show the clustered genes and their assigned immune- or inflammation-related GO terms, which are connected by ribbons. On the left side of the chord plot, blue to red represents the log2 fold change. On the right side of the chord plot, different colors ranging from red to pink represent different GO terms

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The KEGG enrichment analysis at 12 hpi revealed that 10 DEmRNAs (e.g. tat) were significantly enriched in three pathways, including other types of O-glycan biosynthesis, cysteine and methionine metabolism, and protein digestion and absorption (Fig. 4a). At 24 hpi, 13 DEmRNAs (e.g. gnas, itgal) were significantly enriched in five pathways, including insulin secretion, viral myocarditis, salivary secretion, vascular smooth muscle contraction and thyroid hormone synthesis (Fig. 4b). At 36 dpi, 27 DEmRNAs (e.g. hap1, clta and dctn4) were significantly enriched in 12 pathways, including pentose and glucuronate interconversions, starch and sucrose metabolism, ascorbate and aldarate metabolism, endocrine and other factor-regulated calcium reabsorption, alcoholism, sulfur relay system, drug metabolism-other enzymes, chemical carcinogenesis, Huntington's disease, porphyrin and chlorophyll metabolism, long-term depression and circadian entrainment (Fig. 4c and Additional file 6: Table S4).

Fig. 4
figure 4

Scatter plots showing KEGG pathway enrichment results of the differentially expressed (DE) mRNAs. The top 20 enriched pathways at 12 hpi (a), 24 hpi (b) and 36 dpi (c), respectively. The X-axis labels denote an enrichment score (-log10 P-value), and the Y-axis labels represent the names of KEGG pathways. The rich factor represents the number of DEmRNAs enriched in a pathway to the total number of annotated genes in that pathway. The greater the value of the rich factor is, the greater the degree of KEGG pathway enrichment

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Co-localization of DElncRNAs

At 12 hpi, 63 DElncRNAs were located in the vicinity of 47 DEmRNAs. Among these DElncRNAs, LNC_015175, LNC_015166, LNC_015177, LNC_015172, LNC_015165 and LNC_015176 were located in the vicinity of the c9 gene. The LNC_008755 and LNC_010262 were located in the vicinity of fbxl2 and atg101 genes, respectively. The transcriptional level of LNC_001408 located in the vicinity of the il1r1 and il1rl1 gene was upregulated. The LNC_001412 was located in the vicinity of the il1r2 and was downregulated (Additional file 7: Table S5 and Additional file 8: Fig. S3a).

At 24 hpi, 73 DElncRNAs were found in the adjacent area of 55 DEmRNAs. The LNC_015756 was located in the adjacent area of the apolipoprotein A4 (apoa4) and il1a genes and significantly downregulated (Additional file 8: Fig. S3b). Also, the LNC_008220, LNC_008226, LNC_008050 and LNC_008052, which were located in the vicinity of the saa1 gene, were significantly decreased at 24 hpi and 36 dpi. The expression of LNC_006670, located in the vicinity of nod2 gene and cyld gene, was significantly upregulated. The LNC_002735 is located with the adjacency of the btnl2 gene (Additional file 8: Fig. S3b, c).

At 36 dpi, 58 DElncRNAs were found within the up- or downstream of 52 DEmRNAs (Additional file 7: Table S5 and Additional file 8: Fig. S3), including the LNC_013524 that was located in the vicinity of il1rap gene, and were significantly downregulated. The expression levels of LNC_005671 and LNC_011661, which were located with in the vicinity of trim44 and otop1, respectively, were significantly upregulated. The LNC_007354 was located in the vicinity of the tlr9 gene and was significantly upregulated. The transcription levels of LNC_011050 and LNC_011052, which were located in the vicinity of the pdcd4 gene, were significantly increased. The LNC_005105 and LNC_005401 were located in the vicinity of the il1rn gene and were significantly up- and downregulated at 36 dpi, respectively (Additional file 8: Fig. S3c).

GO annotation analysis of DElncRNAs

GO enrichment analyses of the potential target genes (co-localization) revealed that 482, 744 and 610 GO terms were significantly enriched at 12 hpi, 24 hpi and 36 dpi, respectively (Additional file 9: Table S6). In addition, some immune- or inflammation-related terms of DElncRNAs were observed at three infection stages, and the top 30 GO terms are shown in Additional file 10: Fig. S4. Among those GO terms, at 12 hpi, 156 target mRNAs (e.g. fbxl2 and atg101) of DElncRNAs (e.g. 181 DElncRNAs) were significantly enriched in 24 immune- or inflammation-related GO terms (e.g. autophagy) (Fig. 5). At 24 hpi, 178 target mRNAs (e.g. nod2 and cyld) of DElncRNAs (including 188 DElncRNAs) were significantly enriched in 68 immune- or inflammation-related GO terms (e.g. innate immune response process). At 36 dpi, 428 target mRNAs (e.g. trim44, otop1, tlr9 and pdcd4) of DElncRNAs (e.g. 367 DElncRNAs) were significantly enriched in 42 immune- or inflammation-related GO terms (e.g. response to cytokine stimulus and inflammatory response) (Additional file 11: Table S7).

Fig. 5
figure 5

Alluvial plots showing partial immune- or inflammation-related GO terms of target genes of the differentially expressed (DE) lncRNAs at three stages of infection. The GO terms, target genes and DElncRNAs are shown on the left, middle and right side of the plot, respectively

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Discussion

Genomics, transcriptomics and proteomics approaches have been previously used to obtain insight into the pathogenesis of T. canis [9]. In the present study, we profiled the changes in the expression of mRNAs and lncRNAs in the liver of Beagle dogs during T. canis infection.

Function of the DEmRNAs at three infection stages

GO enrichment analysis showed that many DEmRNAs were related to immune processes at the early stages of infection (12 and 24 hpi). However, at the late stage of infection (36 dpi), many DEmRNAs were related to leukocyte-mediated processes. This difference may be related to the migration of the larvae in Beagle dogs during T. canis infection. The crp is a marker of inflammation and tissue damage and is involved in the immune process and regulation of interleukin-8 (il-8) [32]. The downregulation of crp at 36 dpi suggests that T. canis reduces inflammation in the dog’s liver at the late stage of infection. The crp has been used to monitor the development of heartworm disease in dogs [33], suggesting that the abnormal expression of crp may be used as a potential biomarker for the early detection of toxocariasis. In the present study, dpp4 was significantly upregulated at the early stages of infection. The dpp4 is ubiquitously expressed in the liver and other tissues and is considered a co-stimulatory molecule to control the differentiation and immune responses of T cells [34]. These findings suggest that T. canis could induce the immune responses of the dog liver at the early infection stage, and dpp4 may be associated with immune response in the liver.

KEGG analysis showed that tat was significantly increased at 12 hpi and was involved in cysteine and methionine metabolism pathways. Methionine is an essential amino acid, which regulates the metabolic processes, innate immune system and digestive function in mammals [35]. This suggests that tat may modulate the innate immune response of livers in dogs infected by T. canis. However, the exact role of tat in the hepatic innate immune process during T. canis infection remains to be investigated. gnas and itgal were enriched in the insulin secretion pathway and viral myocarditis pathway, respectively, at 24 hpi. Increasing evidence suggests that the immune system can play a crucial role in tuning metabolic homeostasis. For example, the immune system regulates the endocrine function of islets of Langerhans cells [36] and can stimulate the production of beta-cell insulin under steady state [36]. The gnas-activating mutations can form a rare subgroup of inflammatory liver tumors [37]. Among the gastric foveolar metaplasia lesions, gnas mutations are more common in lesions without active inflammation [38]. The gnas was significantly increased at 24 hpi, suggesting that gnas is likely involved in the liver inflammatory and immune response induced by T. canis infection. Myocarditis is caused by viral infections and/or post-viral immune-mediated responses [39]. The itgal is related to inflammation and apoptosis [40] and was significantly downregulated at 24 hpi. These findings indicate that itgal plays a role in the inflammatory reactions in the liver during T. canis infection. In this study, hap1, clta and dctn4 genes were significantly upregulated and related to Huntington's disease pathway at 36 dpi. Huntington's disease is an autosomal-dominant neurodegenerative disease caused by the expansion of CAG repeats in the Huntingtin (htt) gene [41]. Mutant huntingtin protein (mHTT) can promote cell-autonomous immune activation when it is highly expressed in immune cells [42]. Inflammation has been suggested as an important feature of Huntington’s disease [43]. Hence, we speculate that these genes in the puppies’ livers may promote immune and inflammatory responses of puppies to T. canis infection.

Co-localization analysis of DElncRNAs and DEmRNAs

We detected multiple mRNAs whose regulation was mediated by several lncRNAs at all three infection stages, suggesting a complex regulatory relationship between DElncRNAs and DEmRNAs (Additional file 7: Table S5). The complement c9 plays a crucial role in the innate immune response to pathogens [44]. The c9 is the major component to form a pore-like structure, which is characteristic of the fully formed membrane attack complex [45]. The membrane attack complex forms on the cell membrane and plays an important role in innate immune response [46]. From the DElncRNA-mRNA co-located network, we found the target gene c9 was regulated by LNC_015175, LNC_015166 and LNC_015177. The target gene c9 was significantly decreased at 12 hpi, as well as the transcriptional level of LNC_015175, LNC_015166 and LNC_015177. These results reveal that these DElncRNAs might be associated with the immune response of the liver to T. canis infection at an early stage.

In this study, LNC_015756 was located in the vicinity of apoa4 gene and was significantly downregulated at 24 hpi. apoa4 is a member of the apolipoprotein family of lipid-binding proteins, which is involved in the regulation of the immune responses [47,48,49]. apoa4 reduces the secretion of pro-inflammatory cytokines in human peripheral blood mononuclear cells [50], suggesting that apoa4 may be involved in an anti-inflammatory feedback loop. It was also considered an anti-inflammatory agent in the immune system. Another gene, il-1α (IL1A), migrates from the nucleus to the cytosol; when the cell is exposed to a necrotic signal, the lysates of these cells are highly inflammatory [51]. il-1α also evokes significant inflammatory reactions via the IL-1R1 from necrotic cells [52]. The transcriptional level of il1a was significantly increased at 24 hpi and regulated by LNC_015756. These findings suggest that LNC_015756 may play a pro-inflammatory role in the liver of puppies to counter T. canis infection. Serum amyloid A (saa) includes saa1, saa2, saa3 and saa4, which can be used as a diagnostic or prognostic marker for many diseases [53, 54]. Each of the saa genes encodes a different protein such as saa1-4, of which saa1 and saa2 are induced by inflammatory cytokines. When an infectious or inflammatory stimulus persists, the liver produces more saa to counter infection [53]. The saa1 was significantly downregulated and regulated by LNC_008220, LNC_008226, LNC_008050 and LNC_008052 at 24 hpi and 36 dpi. We speculate that T. canis larvae reduce inflammatory cytokines at 24 hpi and 36 dpi to facilitate its migration in the liver by downregulating these LncRNAs and saa1 gene. We also found that LNC_013524 regulates target gene il1rap at 36 dpi. Il-1racp (IL1RAP) and il-33 play important roles in the innate immune response [55]. When il-33 binds to ST2 and il-1racp, the il-33 receptor complex is activated and recruits the adaptor molecule myeloid differentiation factor 88 (MyD88) [56]. Myd88 plays an important role in early innate immune responses during coronavirus-induced encephalomyelitis [57]. Il1rap and the LNC_013524 were significantly downregulated at 36 dpi. LNC_013524 may participate in the innate immunity of liver during T. canis infection, and the downregulation of LNC_013524 may compromise the innate immune response, therefore increasing T. canis survival at a late stage of infection.

Functions of DElncRNAs at three stages of infection

Autophagy is involved in multiple processes of innate and adaptive immune responses in mammalian cells, and with the help of autophagy adaptor proteins, it can trigger a series of immune responses through pattern recognition receptors (PRRs) [58]. These processes play an essential role in eliminating intracellular microorganisms [58]. Moreover, autophagy influences the adaptive immune response by regulating homeostasis of antigen-presenting cells [59]. In this study, the LNC_008755 and LNC_010262 were significantly downregulated and regulated fbxl2 and atg101 genes that were enriched in autophagy at 12 hpi. These findings suggest that LNC_008755 and LNC_010262 may be related to the hepatic immune responses to T. canis infection. The il-1 family of ligands and receptors is related to acute and chronic inflammation. Anti-inflammatory members of the il-1 family can affect disease severity [60]. The il1r1, il1rl1 and il1r2 were regulated by LNC_001408 and LNC_001412, which participate in interleukin-1 receptor activity term at 12 hpi. LNC_001408 was significantly increased and LNC_001412 significantly decreased at 12 hpi. These findings indicate that expression of LNC_001408 and LNC_001412 may be related to T. canis infection-triggered inflammation in puppy liver at an early stage of T. canis infection.

Notably, both nod2 and cyld were enriched in the immune response process and could be regulated by LNC_006670. nod1 and nod2 play fundamental and pleiotropic roles in host defense against infection [61]. CYLD lysine 63 deubiquitinase (cyld) plays a crucial role in regulating IFN receptor signaling [62]. LNC_006670 was significantly increased at 24 hpi. Hence, we speculate that upregulation of LNC_006670 in the liver may promote the immune response of puppies against T. canis infection via regulating the expression of nod2 and cyld. Also, btnl2 was regulated by LNC_002735 and was enriched in the MyD88-dependent Toll-like receptor signaling pathway at 24 hpi. During Trypanosoma cruzi infection, MyD88 plays an important role in the development of a robust Th1 response [63]. MyD88 is an elementary adaptor molecule for most Toll-like receptors (TLRs), which can mediate the induction of inflammatory cytokines via nuclear factor κB (NF-κB) [64]. TLRs can also mediate infection-induced inflammation and sterile inflammation via endogenous molecules [65]. Btnl2, as a negative co-stimulatory molecule, has an important role in inflammatory disease [66]. LNC_006670 was significantly downregulated at 24 hpi, indicating that downregulation of LNC_002735 may regulate btnl2 to mediate the inflammatory response of the puppies’ liver.

At 36 dpi, the target genes trim44 and otop1 were regulated by LNC_005671 and LNC_011661, respectively, and were enriched in the response to cytokine stimulus. Cytokines mediate and control the inflammatory and immune responses [67]. Trim44 regulates the virus-triggered immune response via enhancing the stability of VISA [68]. Otop1 (Otopetrin 1) as a unique target of cytokine signaling can reduce the adipose tissue inflammation caused by obesity [69]. LNC_005671 and LNC_011661 were significantly upregulated at 36 dpi, suggesting that upregulation of these lncRNAs could enhance immune response and weaken tissue inflammation against T. canis infection in the puppies’ liver at late stages of infection. Tlr9 and pdcd4 were enriched in the inflammatory response and were regulated by LNC_007354, LNC_011050 and LNC_011052, respectively. Tlr9 activates inflammatory factors such as il-1 and il-8, increasing immune inhibitory factor il-10 secretion and MMP-2 expression to enhance the invasion and metastasis of lung cancer cells [70, 71]. The LNC_007354 was significantly upregulated at 36 dpi. LNC_007354 regulates tlr9 to activate pro-inflammatory factors and increases immune inhibitory factors to promote the invasion and migration of T. canis larvae. Pdcd4 (programmed cell death 4) may suppress the activation of inflammatory macrophages via NF-kB and mitogen-activated protein kinase (MAPK) signaling in atherosclerosis [72]. The deficiency of pdcd4 significantly increases the level of pro-inflammatory cytokines, such as il-6 and lipopolysaccharide/D-galactosamine, leading to acute liver injury, colitis and colorectal cancer [73, 74]. LNC_011050 and LNC_011052 were significantly upregulated at 36 dpi, suggesting that they may play roles in suppressing pro-inflammatory cytokines and liver injury repair post T. canis infection.

The il1rn was enriched in the interleukin-1 receptor binding and was regulated by LNC_005105 and LNC_005401, which were significantly up- and downregulated at 36 dpi, respectively. The mutation of il1rn (interleukin 1 receptor antagonist) can lead to a deficiency of the il-1-receptor antagonist causing an autosomal recessive autoinflammatory disease [75]. The il1rn gene encodes interleukin-1 receptor antagonist (il-1ra), which is a potent anti-inflammatory cytokine that competitively inhibits stimulation by inflammatory mediators via binding to il-1r1 and preventing the recruitment of il-1racp [76]. The altered expression of LNC_005105 and LNC_005401 may be associated with the release of anti-inflammatory cytokines that mediate the healing of the liver pathology at the late stage of infection.

Conclusions

We performed a comprehensive analysis of lncRNA and mRNA expression patterns in the liver of Beagle dogs infected by T. canis at three stages after infection. The functional enrichment analysis showed that many DEmRNAs are associated with the immune responses of the puppies’ liver at an early stage of infection. Co-localization analysis suggested that some DElncRNAs might play an anti-inflammatory role in in the liver during T. canis infection. These findings provided new insight into the transcriptomic basis of the interaction between T. canis and the definitive canine host. Future studies should focus on investigation of the exact roles of the identified DElncRNAs and miRNAs in the innate/adaptive immune response of dogs to T. canis infection.