Transcriptomic profiling of dorsal root ganglia in atopic and healthy dogs: A comparative RNA sequencing study with implications in cutaneous itch research

Abstract

Background

Itch is a common clinical sign in skin disorders. While the neural pathways of itch transmission from the skin to the brain are well understood in rodents, the same pathways in dogs remain unclear. The knowledge gap hinders the development of effective treatments for canine itch‐related disorders.

Hypothesis/Objectives

This study aimed to investigate the differential gene expression in the dorsal root ganglia (DRGs) between healthy and atopic dogs to identify specific molecules potentially involved in itch signalling and neuroinflammation in canine atopic dermatitis (cAD).

Animals

Two atopic and four healthy dogs.

Materials and Methods

DRGs were collected from atopic and healthy dogs to compare their transcriptional profiles using RNA sequencing.

Results

Principal component and heatmap analyses revealed two distinct clusters separating atopic from healthy dogs. Consistent with this observation, we identified 627 (543 upregulated and 84 downregulated) differentially expressed genes (DEGs) in atopic compared with healthy dogs. We further narrowed down our genes of interest to common DEGs in each atopic dog, which revealed 159 (132 upregulated and 27 downregulated) DEGs. Among these genes, when we focused on itch signalling–associated molecules, P2RY12, IL‐2RG, TLR1 and POSTN were significantly upregulated, while MRGPRD and LPAR3 were both significantly downregulated in atopic dogs compared with those in healthy dogs. Pathway analysis showed a significant upregulation of CREB signalling in neurons, myelination signalling and neuroinflammation signalling pathways in atopic dogs.

Conclusions and Clinical Relevance

Our study suggested that dysregulation of neuroinflammatory pathways might play a role in the pathomechanism of cAD as in humans.

Background – Itch is a common symptom in skin disorders. While the neural pathways of itch transmission from the skin to the brain are well‐understood in rodents, the same pathways in dogs remain unclear. The knowledge gap hinders the development of effective treatments for canine itch‐related disorders. Hypothesis/Objectives – This study aimed to investigate the differential gene expression in the dorsal root ganglia between healthy and atopic dogs to identify specific molecules potentially involved in itch signalling and neuroinflammation in canine atopic dermatitis (cAD). Conclusions and Clinical Relevance – Our study suggested that dysregulation of neuroinflammatory pathways might play a role in the pathomechanism of canine AD as in humans.

INTRODUCTION

Atopic dermatitis (AD) manifests as a common, often chronic, allergic skin disease in both humans and dogs, characterised by mild‐to‐severe pruritus and inflammatory skin lesions. ¹ , ² The pathogenesis of AD involves multiple factors. In 2023, the International Committee on Allergic Diseases of Animals (ICADA) revised the definition of canine AD (cAD) to reflect current knowledge. The revised definition states that cAD is a hereditary, typically pruritic and predominantly T‐cell‐driven inflammatory skin disease involving interplay between skin barrier abnormalities, allergen sensitisation and microbial dysbiosis. ³ This updated definition highlights the multifactorial nature of the disease, advocating for a multimodal approach to manage the disease's complex factors. However, a key pathogenic aspect of human AD remains unknown in cAD.

Recent studies in humans strongly support that neuroimmune pathways perpetuate atopic itch and skin inflammation, through cross‐talk between the nervous system, cutaneous immune system and keratinocytes. ⁴ , ⁵ Pruritogens, such as allergens and cytokines, bind to receptors that are present on primary afferent C‐fibre somatosensory neurons innervating the skin, initiating an atopic flare. ⁴ , ⁵ Activated neurons transmit signals from the skin to the brain through dorsal root ganglia (DRGs), where they are interpreted as itch. ⁴ , ⁵ Additionally, activated cutaneous sensory neurons release neuropeptides, such as substance P and calcitonin gene‐related peptide, from the cutaneous nerve endings into the skin, inducing vasodilation, cell recruitment and the release of pro‐inflammatory mediators from keratinocytes to fuel further inflammation, keratinocyte proliferation and epidermal thickening. ⁴ , ⁵ This cascade, termed ‘neurogenic inflammation’, is now recognised as a pivotal contributor to AD pathogenesis in humans. ⁴ , ⁵ While human and canine AD share similarities, the role of neuroimmune pathways in cAD is unexplored. Our objective was to identify key differences in gene expression in the DRGs between atopic and healthy dogs using RNA sequencing (RNA‐Seq), paving the way for novel therapeutic targets.

MATERIALS AND METHODS

Ethics

Sample collection from atopic dogs was approved beforehand by our university's Institutional Animal Care and Use Committee (IACUC; ID no.: 18‐130‐B). Samples from healthy dogs were sourced from cadavers euthanised at the local shelters for population control purposes; thus, the IACUC approval was not deemed necessary.

Sample‐size calculation

Given the lack of previous studies comparing DRG gene expression between spontaneous AD and healthy individuals in any species, we conducted a power analysis based on a previous study using an interleukin (IL)‐31‐induced atopic‐like mouse model. ⁶ The analysis indicated that a minimum of three dogs per group would be required to achieve >80% power to detect the significant differences at a p‐value of 0.05. However, as a consequence of the limited availability of the DRG samples of atopic dogs, we were unable to meet this sample size requirement.

Animals

Samples were collected from two dogs in an atopic dog model colony (one intact female and one intact male, both aged 13 years) and four healthy dogs (two females [neuter status unknown] and two intact males); all were of young‐to‐middle age (their exact ages were not documented) and had no gross skin issues at the time of sample collection. The atopic dog model utilised in this study was an inbred line of laboratory Maltese‐beagle dogs that are known to spontaneously exhibit a high immunoglobulin (Ig)E response to food allergens and to develop cAD signs upon allergen challenge. These dogs also are easily experimentally sensitised to Dermatophagoides farinae (Df) house dust mite (HDM) allergen during early life stages, and reproducibly produce IgE against Df and develop atopic skin lesions following epicutaneous HDM provocations. ⁷ , ⁸ Dogs in this colony are maintained with restricted dietary management and reside in a controlled housing environment to prevent the development of spontaneous AD flares triggered by both food and environmental allergens. At the time of sample collection, both atopic dogs were already retired from laboratory duties yet remained in a controlled housing environment. Consequently, they had not been exposed to HDM allergens for several years (957 and 541 days for the female and male atopic dogs, respectively) and did not have active skin lesions at the time of sample collection. Hence, within the scope of this study, we designate them as atopic dogs rather than ‘dogs with cAD’.

Sample collection

We collected two cervical DGRs (the cluster of the cell bodies of sensory neurons) from each atopic dog (AD1‐C, AD1‐C, AD2‐C2 and AD2‐C4) and three cervical DRGs (Dog1‐C2‐4, Dog2‐C2‐4, Dog3‐C2‐4 and Dog4‐C2‐4) pooled together as one sample from each healthy dog. All samples were stored at −80°C until further processing.

Additionally, two extra cervical DRGs from one of the atopic dogs (AD1‐C#1 and AD1‐C#2) and one each DRG from two additional healthy dogs (Dog5‐C3 and Dog6‐C3) were collected for RNA in situ hybridisation (ISH). These collected samples were embedded in an optimal cutting temperature embedding medium (OTC Compound, catalogue no.: 4585; Fisher HealthCare;) and snap‐frozen on dry ice. The embedded samples were cryosectioned at 5 μm thick on the day of RNA ISH staining. All sections were arranged on the same slide to enable simultaneous staining, thereby minimising the potential for batch effects.

The signalments and locations of the samples are summarised in Table S1.

RNA isolation and RNA sequencing

Total RNA extraction from each DRG sample was conducted using an RNeasy Fibrous Tissue Mini Kit (reference no.: 74704; Qiagen) following the manufacturer's protocol. Following quality assurance and RNA library preparation, RNA‐Seq was carried out using the NextSeq 500 platform (Illumina). The detailed procedures and results for RNA extraction, RNA purity/integrity, library preparation and RNA‐Seq are described in Appendix S1. The raw sequences are publicly available under study PRJNA1148493.

RNA‐seq data analyses

We performed the RNA‐Seq data analysis, principal component analysis (PCA) and heatmap analysis using the CLC genomic workbench v22 (Qiagen) with default parameters. Further elaboration on the data processing procedures is found in Appendix S1.

Differential expression analysis

Differential expression analysis between the atopic and healthy dogs (atopic versus healthy) was conducted using the differential expression for rna‐seq tool within the CLC genomic workbench (Qiagen). Criteria for significance included an absolute fold change (FC) greater than two, a false discovery rate (FDR) of ≤0.05, and a maximum group mean of reads per kilobase of exon per million mapped reads (max RPKM) of >1. The FCs were calculated by dividing gene expressions in atopic dogs by those in healthy dogs; thus, positive numbers indicate upregulation, and negative numbers indicate downregulation in atopic compared with healthy dogs.

RNA in situ hybridisation (RNAscope)

We performed a fluorescence ISH using the RNAscope method (Advanced Cell Diagnostics) to localise the expression of IL‐33 mRNA in DRGs from both atopic and healthy dogs. The RNAscope^T Multiplex Fluorescent V2 Assay (catalogue no.: 323110; Advanced Cell Diagnostics) was employed according to the manufacturer's protocol. Details regarding the probes used in the staining and corresponding fluorophores to visualise the signals are summarised in Table 1. A comprehensive protocol and the fluorescence microscope settings are described in Appendix S1.

TABLE 1.

RNAscope probes utilised for staining and corresponding fluorophores.

Target gene	Protein coding	Probe type	Channel no.	ACD catalogue no.	Fluorophores
IL‐33	Interleukin‐33	Target probe	1	484161	Opal 570 (red)
GFAP	Glial fibrillary acidic protein	Target probe	2	877971‐C2	Opal 520 (green)
TUBB3	Tubulin beta 3 class III	Target probe	3	1122091‐C3	Opal 690 (magenta)
Polr2a	Polymerase II polypeptide A	Positive control probe	1	323931	Opal 570 (red)
PPIB	Peptidylprolyl isomerase B	Positive control probe	2	323931	Opal 520 (green)
UBC	Ubiquitin C	Positive control probe	3	323931	Opal 690 (magenta)
dapB	Dihydrodipicolinate reductase	Negative control probe	1,2,3	321831	Opal 570, 520, 690

Ingenuity pathway analysis

Differentially expressed genes (DEGs) exhibiting our criteria of significant difference between groups were then subjected to canonical pathway analysis (CPA) using the ingenuity pathway analysis (IPA) program (Qiagen), aiming to predict up‐ or downregulated pathways in atopic compared with healthy dogs. To align with the purpose of this study, we restricted the analysis of pathways to neurotransmitters and other nervous system signalling. Statistical significance was determined by p‐values ≤0.05 and absolute z‐scores >2.

RESULTS

RNA‐Seq data analysis

The average number of sequencing reads obtained per sample was 132.0 million, with a range of 119.4 to 152.5 million reads. An average of 90% of the read pairs (range: 88%–91%) were mapped to the canine reference genome (CanFam3.1). A total of 19,989 genes were annotated in each DRG sample. The PCA plot illustrates the differences in gene expression profiles between atopic and healthy dog DRGs (Figure 1a). Different sexes in atopic samples also showed a distinct clustering (Figure 1a). However, because two samples from each sex were obtained from the same dog, it was unclear whether the difference was caused by sex or individual variability. Likewise, visualisation of the expression of genes across the samples by the heatmap revealed two distinct hierarchical clusters separating atopic from healthy dogs (Figure 1b).

FIGURE 1.

Principal component analysis (PCA) (a) and heatmap analysis (b). Both PCA (each plot represents a sample) and heatmap analysis (each column represents a sample) revealed a distinct separation between atopic and healthy dogs. Refer to Table S1 for sample IDs. F, female; MI, male intact.

Differential expression analysis

Using our predefined criteria, we identified 627 DEGs (543 upregulated and 84 downregulated genes) in the atopic DRG samples compared with healthy dogs. Owing to significant individual differences between the two atopic dogs detected by PCA, we focused on common DEGs in both atopic dogs when each was independently compared with healthy counterparts, revealing 159 DEGs (132 upregulated and 27 downregulated) (Figure 2; Table S2). To further investigate the mechanisms underlying itch sensation, we focused on 144 genes related to itch‐associated receptors, neurotransmitters, neuropeptides and signalling molecules (Table S3). ⁴ Among the common DEGs, P2RY12 (purinergic receptor P2Y12; FC = 4.54, FDR = 1.2E‐04, max RPKM = 1.37), IL‐2RG (IL‐2 receptor subunit gamma; FC = 3.78, FDR = 4.0E‐04, max RPKM = 1.47), TLR1 (toll‐like receptor 1; FC = 3.32, FDR = 1.2E‐04, max RPKM = 1.73) and POSTN (periostin; FC = 2.22, FDR = 6.7E‐03, max RPKM = 1.85) showed significant upregulation, while MRGPRD (MAS‐related GPR family member D; FC = −2.39, FDR = 3.5E‐03, max PRKM = 4.17) and LPAR3 (lysophosphatidic acid receptor 3; FC = −2.19, FDR = 7.63E‐05, max RPKM = 8.46) showed significant downregulation in both atopic dogs compared with those in healthy dogs (Figure 3). The remaining 138 genes showed no significant differences between atopic and healthy dogs.

FIGURE 2.

Number of common differentially expressed genes (DEGs). Our refined DEG analysis identified 159 common DEGs (132 upregulated and 27 downregulated) in atopic compared with healthy dogs. DRGs, dorsal root ganglia.

FIGURE 3.

Genes showing significant differences in atopic dogs compared with healthy dogs. Each plot represents the TPM value of each sample, with lines (−) indicating the median TPM of each group. Asterisks (*) indicate common DEGs that showed statistically significant differences at |FC| > 2, FDR ≦ 0.05, RPKM ≧ 1. Hashtags (#) indicate genes that did not fulfil all criteria for significant differences yet showed a tendency of difference (not common DEGs or 1.5 < |FC| < 2) that we still believe are interesting to point out. cAD, canine atopic dog; C‐DRG, cervical dorsal root ganglia; DEG, differentially expressed gene; FC, fold change; FDR, false discovery rate; RPKM, reads per kilobase of exon per million mapped reads; TPM, transcripts per million.

RNA in situ hybridisation (RNAscope)

Our preliminary RNA‐Seq analysis revealed low IL‐33 mRNA expression levels in healthy human, murine and feline DRGs, with mean transcript per million (TPM) values of 6.3, 19.6 and 5.5, respectively (data not yet published). By contrast, both healthy and atopic canine DRGs exhibited moderate expression of IL‐33, with mean TPM values of 267.0 and 436.2, respectively. Moreover, IL‐33 expression was higher in atopic than in healthy dogs (FC = 1.65, FDR = 3.61E‐03, max RPKM = 134.45, Figure 4a), although with FC <2. A mouse study showed IL‐33 protein production by satellite glial cells in DRGs, yet the cellular source of IL‐33 in canine DRGs is still unknown to the best of the authors' knowledge.

FIGURE 4.

mRNA expression levels of interleukin (IL)33 and its receptors by RNA‐Seq (a) and 3‐plex fluorescent RNAscope (b) in atopic and healthy dorsal root ganglia (DRGs). One each representative staining for atopic and healthy dogs showing IL‐33 signals (red) co‐expressed on GFAP (glial cell marker, green)‐positive cells but not on TUBB3 (neuron cell marker and magenta)‐positive cells. Conversely, the expression of its receptor (IL‐1RL1, red) was not detected either by RNA‐Seq or RNAscope in both atopic and healthy dogs. DAPI, 4′,6‐diamidino‐2‐phenylindole; GFAP, glial fibrillary acidic protein; TUBB3, tubulin beta 3 class III.

RNAscope ISH showed strong IL‐33 mRNA expression in both healthy and atopic DRGs (Figure 4b). Notably, IL‐33 mRNA signals were mainly expressed on GFAP‐positive satellite glial cells, with insufficient presence on TUBB3‐positive cells to support the expression of IL‐33 mRNA on neurons. Owing to challenges in cell segregation, the exact count of positive cells was not feasible. By contrast, IL‐1RL1, a subunit of the IL‐33 receptor, was not detected by either RNA‐Seq or RNAscope (Figure 4a,b).

Ingenuity pathway analysis

Canonical pathway analysis on neurotransmitters and other nervous system signalling unveiled significant upregulation of cAMP responsive element binding protein (CREB) signalling in neurons (z = 2.50, p = 6.16E‐05), myelination signalling pathway (z = 2.45, p = 2.31E‐02) and neuroinflammation signalling pathway (z = 2.12, p = 2.98E‐04; Figure 5). Table S4 presents the associated DEGs for each pathway.

FIGURE 5.

Ingenuity pathway analysis on neurotransmitters and other nervous system signalling. Based on our criteria of significant difference at p < 0.05 (−log [p‐value] > 1.3) and |z‐score| > 2, we observed upregulation of CREB signalling in neurons, and the myelination signalling and neuroinflammation signalling pathways. Darker colours correlate with higher z‐score. CREB, cAMP responsive element binding protein.

DISCUSSION

Recent studies have implicated neuroimmune pathways in AD pathogenesis, yet their role in cAD is still unknown. In this study, we compared the transcriptomic profiles of DRGs between atopic and healthy dogs to identify molecules involved in cAD and neuroinflammation. Specifically, we aimed to investigate the expression of neuroimmune axis‐related genes. Notably, the atopic dogs in this study did not have active skin lesions when samples were collected, ensuring that the observed differences in gene expression are not secondary to skin inflammation. We found significant differences in the expressions of P2Y12, IL‐2RG, TLR1, POSTN, MRGPRD, LPAR3 and, potentially IL‐33, in atopic DRGs compared with healthy ones.

P2 purinergic receptors are activated by nucleotides, such as ATP, ADP, UTP and UDP, which are abundant in the nervous system and other tissues. ⁹ ATP, besides being an energy source, also functions as a pruriceptive neurotransmitter, initiating and sustaining neuronal excitability, and contributing to neuroinflammation. ¹⁰ , ¹¹ , ¹² , ¹³ In a mouse model of type 2 diabetes mellitus (DM), silencing or inhibiting P2Y12 alleviated chronic itching and reduced reactive oxygen species, NLRP3 inflammasome, IL‐1β and IL‐18. ¹³ In our study, P2Y12 was significantly upregulated in atopic dogs, suggesting a potential role in atopic itch. Additionally, we observed upregulation of NLRP3 (FC = 3.09, FDR = 2.25E‐4, max RPKM = 0.996) and IL‐18 (FC = 5.07, FDR = 1.22E‐6, max RPKM = 3.50; Table S2) consistent with findings in a pruritic DM mouse model. ¹³ The exact role of P2Y12 in canine AD remains unclear and needs further investigation.

IL‐4 and IL‐13, key T‐helper 2 (Th2) cytokines, play a central role in both human and canine AD. ¹ , ¹⁴ They induce scratching behaviour in mice and enhance neuronal responses to pruritogenic stimuli in human DRG neurons, ¹⁵ , ¹⁶ indicating their involvement in itch induction. However, their effects on itch in dogs are not well understood. We found significant upregulation of IL‐2RG, a receptor subunit of IL‐4 and other cytokines, in atopic DRGs. IL‐4R (a shared receptor subunit for IL‐4 and IL‐13) and IL‐13RA1 (a receptor subunit for IL‐13) showed elevated expression, yet did not meet our predefined criteria of significant difference, in DRGs of atopic compared with healthy dogs. Notably, our atopic dogs did not have active atopic flare‐ups when samples were collected, implying that the upregulation was not the result of elevated levels of IL‐4 and IL‐13 in the skin. Instead, these findings suggest increased susceptibility to IL‐4 and IL‐13 stimuli.

Emerging evidence suggests that keratinocyte TLRs contribute to human AD by stimulating cytokine release and modulating tight junctions. ¹⁷ In mice, TLR3, TLR4 and TLR7 on DRG neurons regulate itch sensation in AD pathogenesis. ¹⁸ Here, we found significant upregulation of TLR1 in atopic dogs, which recognises bacterial lipoprotein and glycolipids in complex with TLR2. Remarkably, a recent genome‐wide association study identified TLR1 as a candidate gene associated with cAD in Labrador retrievers. ¹⁹ Further research is needed to understand the role of TLR1 in sensory neurons in atopic dogs.

Periostin, an extracellular matrix protein, plays a pivotal role in tissue remodelling and repair. ²⁰ Additionally, it has been implicated in chronic allergic inflammation in human AD patients and can directly activate itch sensory neurons, triggering itch behaviours across species, including dogs. ²¹ , ²² While periostin expression is elevated in the skin of dogs with cAD, ²³ , ²⁴ , ²⁵ the present study is the first to demonstrate significant upregulation of POSTN mRNA in DRGs of atopic compared with healthy dogs. ITGB3, a receptor subunit, also showed upregulation in atopic DRGs, although it did not meet our criteria for significant difference owing to substantial individual difference between the two atopic dogs. In mice, periostin is produced by satellite glial cells in DRGs and facilitates the migration of Schwann cells. ²⁶ However, the cellular source of periostin in canine DRGs is unidentified. Further exploration is warranted to elucidate the role of periostin in DRGs in human and canine AD.

MrgprD is a G protein‐coupled receptor, and nerve fibres expressing MrgprD terminate as free nerve endings in the epidermis. ²⁷ Additionally, MrgprD is associated with histamine‐independent chronic itch in humans. ²⁸ , ²⁹ We found significantly decreased MRGPRD expression in atopic dogs, suggesting a potential adaptive protective response against pruritogenic stimuli. Further investigation is needed to confirm this hypothesis. Notably, canine MrgprD is unresponsive to histamine, similar to humans, ³⁰ yet its functional equivalence to human MrgprD remains uncertain owing to limited homology (76.4%).

LPARs (LPAR1‐6) are a family of G protein‐coupled receptors activated by lysophosphatidic acid (LPA), a bioactive phospholipid that is produced during the synthesis of cell membranes. ³¹ LPA has been linked to pruritus in human cholestatic patients through LPAR5 receptor activation. ³² However, pruritus associated with systemic diseases, such as cholestatic disorders, has not been reported in canine patients. Although LPAR5 expression data were unavailable owing to its absence in the canine reference genome, we noted a significant downregulation of LPAR3 in atopic dogs. While LPAR5 is widely acknowledged for its role in itch in humans, direct evidence of LPAR3's involvement in itch or AD remains elusive. It is interesting to note that a recent mouse study utilising single‐cell RNA‐Seq demonstrated that LPAR3 and LPAR5 were exclusively expressed on one of the three discrete populations of nonpeptidergic nociceptor DRG neurons (NP1) alongside other itch receptors, channels and neuropeptides, suggesting a potential role for LPAR3 in chronic itch, including AD. ¹⁵ , ³³ However, it is still unclear whether this classification applies to dogs. Additionally, we lack a satisfactory explanation for the significant downregulation, instead of its upregulation, of LPAR3 in atopic DRGs. In the aforementioned study, MRGPRD was likewise found to be exclusively expressed within the NP1 neuron population. ³³ This decline in both MRDPRD and LPAR3 expression in our atopic DRG samples might be caused by a reduction in the NP1 neuron population. Further investigation is required to validate this hypothesis and uncover its underlying cause.

IL‐33 is constitutively expressed as a nuclear protein in epithelial tissues across organs, such as the lung, stomach and skin. ³⁴ It is released extracellularly upon tissue damage, cell death or cell stress, acting as an endogenous danger signal (alarmin). ³⁴ Accumulative evidence suggests that IL‐33 is involved in human and canine AD. ³⁵ , ³⁶ However, a recent Phase 2b clinical trial in human AD using a humanised anti‐human IL‐33 monoclonal antibody (etokimab) failed to demonstrate treatment benefits over the placebo control group (NCT03533751), highlighting our incomplete understanding of the precise role of IL‐33 in AD pathogenesis. We found a higher IL‐33 expression in atopic DRGs compared with the healthy control group, although it did not meet our significance criteria. Notably, this is the first report showing IL‐33 expression in DRGs of both healthy dogs and those with cAD. Interestingly, unlike dogs, our preliminary RNA‐Seq analysis found very low IL‐33 in healthy human, murine and feline DRGs, suggesting the uniqueness of IL‐33 expression in canine DRGs. Although anti‐IL‐33 therapy failed in human AD, it may still be a therapeutic target in cAD, especially if targeting IL‐33 in DRGs, where its role may differ from that in human AD. Furthermore, our RNAscope staining showed that IL‐33 is expressed on satellite glial cells in canine DRGs, regulating neuronal homeostasis and promoting regenerative growth in sensory neurons. ³⁷ , ³⁸ This finding aligns with a report in mouse DRGs, ³⁹ and our study is the first to identify the cellular source of IL‐33 in canine DRGs. Although the IL‐33 receptor expression has been detected in human and murine DRGs, ¹⁶ , ⁴⁰ our RNA‐Seq analysis and RNAscope staining failed to detect the IL‐33 receptors mRNA expression in canine DRGs. This suggests a puzzling absence of cells capable of receiving the IL‐33 signal. Further investigation is needed to determine the recipient cells of the IL‐33 and to understand its role in canine DRGs.

In our study, CPA using IPA predicted the upregulation of three nerve system‐related pathways. CREB signalling activation in neurons leads to various biological responses, such as neuronal excitation and proliferation. Myelin, a lipid‐rich sheath enveloping long axons, is generated by Schwann cells in DRGs. The significance of upregulated CREB and myelination signalling in atopic DRGs is still unknown. Neuroinflammatory signalling plays a critical role in maintaining nervous system homeostasis, functioning in the removal of damaging agents and clearance of injured neural tissues. Although neuronal damage is unlikely in atopic dogs, upregulation of the neuroinflammatory signalling pathway may still occur as a consequence of the potential activation of neuroimmune circuits between the nervous system and the cutaneous immune system, potentially associated with these findings.

This study encountered several limitations, with the most prominent being the small sample size. Samples from atopic dogs were sourced from only two individuals, resulting in a total of four samples. Moreover, we found significant individual variability in gene expression between the two dogs, still leaving a question about whether this discrepancy stemmed from inherent individual differences or potentially from sex‐specific factors. To address individual variation, we focused on identifying common DEGs. However, this approach may still lead to overestimation or underestimation of true DEGs. This experimental dog model utilised in this study was an inbred line of laboratory Maltese‐beagle dogs, whose lineage traces back approximately 25 years to ancestors diagnosed with cAD. Given the unique characteristics of this colony and extremely limited opportunities for DRG collection, expanding the sample size proved challenging. Furthermore, while three DRGs were pooled for analysis in healthy dogs, two individual DRGs were collected and analysed separately for each atopic dog, and this decision to process the atopic dog samples separately was based on the valuable nature of the samples and the need to minimise the risk of losing entire samples if processing or sequencing errors occurred. Additionally, RNA‐Seq analysis does not allow for averaging TPM values of samples from the same dog as these values are calculated based on the total gene expressions of each individual sample, not the dog, which introduces pseudo‐replication in the atopic group. We recognise that this decision may have impacted the statistical analysis.

Another limitation was the age range difference between atopic and healthy groups. While the exact ages of healthy dogs were not recorded, they were presumed to be adults based on their physical examination. By contrast, both atopic dogs were 13 years old and considered senior. Thus, the observed difference in gene expression in atopic dogs could potentially be attributed to age‐related changes. In humans, the elderly population is more susceptible to chronic itch, which can stem from various pathophysiological mechanisms, including age‐related degeneration of central or peripheral nerves. ⁴¹ , ⁴² For instance, aged mice (24 months old) were more susceptible to mechanically evoked itch compared with young mice (2 months old). ⁴³ This increased in susceptibility is a result of the apoptosis of neuropeptide Y‐producing neurons in the spinal dorsal horn, which counteract mechanical itch. ⁴³ However, NPY (FC = 2.5, FDR = 0.34 and max RPKM = 0.72) expression did not differ significantly in atopic dogs. The impact of senile neuropathic change on chronic itch has yet to be investigated in companion animals. Additional transcriptome comparison using age‐matched healthy dogs is warranted to mitigate potential age‐related DEGs. However, despite several limitations, the use of atopic samples from dogs, combined with high throughput sequencing, provides unprecedented resources from a naturally occurring disease model, revealing molecules that may inform future research.

Finally, it is important to note that this study did not investigate gene expression differences during atopic flares, as the atopic dogs involved did not exhibit active skin lesions when samples were collected, although the microscopical skin inflammation was not examined owing to the lack of skin samples. As genes may be upregulated after initial atopic stimuli, owing to animal welfare concerns, we could not obtain DRG samples from an experimentally induced canine atopic model. Likewise, sampling from client‐owned dogs with spontaneous cAD was challenging because these dogs are not typically necropsied. Although some genes may have been underestimated in this study, we believe that characterising gene expression in atopic dogs without active atopic flares can aid future research.

In conclusion, our analysis revealed distinct differential expression patterns in DRG transcriptional profiles of atopic versus healthy dogs, suggesting that neuroimmune pathways are involved in cAD, similar to human findings. Notably, genes, such as P2Y12, IL‐2RG, TLR1, POSTN, MRGPRD, LPAR3 and IL‐33, warrant further investigation. These results provide a foundation for future studies to explore the functional implications of these genes in cAD potentially leading to new therapies.

AUTHOR CONTRIBUTIONS

Chie Tamamoto‐Mochizuki: Conceptualization; methodology; software; data curation; investigation; validation; formal analysis; visualization; writing – original draft; writing – review and editing. Santosh K. Mishra: Conceptualization; methodology; validation; supervision; funding acquisition; project administration; resources; writing – review and editing.

FUNDING INFORMATION

Self‐funded.

CONFLICT OF INTEREST STATEMENT

No conflicts of interest have been declared.

Supporting information

APPENDIX S1. Detailed description of RNA sequencing, data analysis and RNAscope.

TABLE S1. Signalments and locations of the DRG samples utilised in this study.

TABLE S2. Common 159 DEGs in both atopic dogs compared with healthy counterparts.

TABLE S3. Genes associated with itch‐associated receptors, neurotransmitters, neuropeptides and signalling molecules selected for our detailed investigation.

TABLE S4. Associated DEGs for each IPA pathway.

Tamamoto‐Mochizuki C, Mishra SK. Transcriptomic profiling of dorsal root ganglia in atopic and healthy dogs: A comparative RNA sequencing study with implications in cutaneous itch research. Vet Dermatol. 2025;36:401–411. 10.1111/vde.13324

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in NIH Genbank at https://www.ncbi.nlm.nih.gov/genbank/, reference number PRJNA1148493.