Domestication of dogs is primarily viewed as an evolutionary response to a new, human-dominated environment, influenced by artificial selective pressures exerted by humans (1). India harbors a diversified and well-integrated fauna of all Indigenous animals, including different breeds/strains/genetic groups of dogs, such as Mudhol Hound, Chippiparai, Kanni, etc. Native animals are those species that have their origin within a particular locality and, over time, developed in tandem with the prevailing climatic and environmental conditions of the Locality (69). These native dogs vary in conformation, exhibiting differential genetic predisposition to diseases and variable responses to infections (2). It is worth noting that molecular characterization of many of these genetic groups is still warranted. The Gaddi dog is an important breed and has recently been recognized by the National Bureau of Animal Genetic Resources (NBAGR)¹ (Accession number INDIA_DOG_0600_GADDI_19004). The Gaddi dogs or “Shepherds’ dogs” are one such breed originating from the northern parts of India, majorly Himachal Pradesh, Uttarakhand, Jammu, and Kashmir, and some parts of Punjab, used by the Gaddi tribe for guarding livestock in hilly terrains. Moreover, Gaddi dogs are ideal for transcriptomic studies since these populations of free-ranging dogs are pre-adapted to harsh climatic conditions and high altitudes. These Gaddi dogs, being “Free-ranging dogs” (FRDs), showed signs of diversifying selection in genes whose function is in reproduction, immunity, and chemosensory perception, suggesting adaptations for independent survival (3). Such adaptive characteristics may benefit dogs in human-altered landscapes through hybridization and gain enhanced immunity against environmental strains and diseases (4).

Blood transcriptomic profiling is a valuable tool for evaluating host immune responses to specific stimulants or pathogens (5). Peripheral blood mononuclear cells (PBMCs) are vital players in the immune system, crucial for defending against infections (6). PBMCs have been frequently employed as an ex vivo model for studying cytokine gene expression following stimulation with purified toll-like receptor (TLR) ligands or live probiotics (7, 8). MicroRNAs (miRNAs), small non-coding RNAs (~19–23 nucleotides), are essential regulators of protein expression and have significant roles in development, physiological processes, and disease. Their ability to interact dynamically with multiple target genes and rapidly alter expression in response to external stimuli underscores their importance in maintaining cellular homeostasis (9–11). Moreover, research reveals that miRNA expression is adjusted under stressful conditions to manage the stress response (12). In our lab, we conducted similar research investigating differentially expressed miRNAs in buffalo PBMCs exposed to various TLR ligands (13, 14, 77). RNA sequencing (RNA-seq) provides a high-throughput approach for comprehensive transcriptome analysis, offering detailed insights into gene expression (15, 72). Understanding miRNA-mRNA regulatory networks is critical for elucidating gene expression at the post-transcriptional level. However, in silico models for predicting miRNA-mRNA interactions often have limited accuracy and context-specific relevance. A more robust strategy involves integrating actual mRNA and miRNA transcriptomic and miRNAomic data with computational predictions (16, 17).

The present study was designed to explore the prominent differences in the transcriptome and miRNAome profiles of Indigenous Gaddi dogs and exotic Labrador retrievers because of their distinct origin and genetic background. This study was undertaken to elucidate the molecular profile of PBMCs under in vitro simulated viral and bacterial infections, using Toll-like receptor (TLR) ligands—TLR4, TLR3, and TLR9. First, literature on mRNA and miRNA responsive expression to these conditions in dogs is limited. Our study has addressed questions concerning breed-specific immune responses and the genetics involved in their adaptation, underscoring preservation and research on Indigenous dog breeds as holders of unique genetic heritage.

India possesses a rich diversity of Indigenous dog breeds, which have evolved in tandem with local climatic and environmental conditions over time (2). Numerous studies highlight the disease resistance and robust immune responses exhibited by Indigenous animals. According to (39), native breeds generally exhibit higher disease resistance than non-native or exotic breeds due to their adaptation to local environmental stressors (40). This resilience is primarily attributed to genetic traits shaped by natural selection, enabling greater tolerance to pathogens and reduced dependence on intensive management (41). Gaddi dogs, a free-ranging breed traditionally used by the Gaddi tribe for livestock guarding in mountainous regions, are pre-adapted to harsh climatic conditions and high altitudes. Such adaptive traits may confer advantages in human-altered environments through hybridization, potentially enhancing immunity against environmental pathogens (4). The official recognition of the Gaddi dog breed by the National Bureau of Animal Genetic Resources (NBAGR; See footnote 1; Accession number INDIA_DOG_0600_GADDI_19004) marks a significant step toward preserving and promoting this indigenous breed. Recognition validates its distinct genetic identity, ensuring focused conservation efforts, and breeding programs. Investigating Gaddi dogs’ transcriptomic and miRNAome profiles and comparing them with the widely owned Labrador Retriever has yielded valuable insights contributing to the existing knowledge base.

Cultured PBMCs were divided into three treatment groups to simulate bacterial and viral infections, along with a control group. During the treatment period, the PBMCs in the stimulated groups showed rapid division and dense growth, while the control group exhibited a lower cell count. The cells were further processed for mRNA and miRNA extraction. The mRNA and miRNA sequencing of Labrador and Gaddi dog PBMCs simulated with various TLR ligands {Control vs. Poly I:C (TLR-3), Control vs. LPS (TLR-4), and Control vs. CpG ODN(TLR-9)} was done using Illumina NovaSeq-6000 platform. Raw reads were generated, and considering the dog genome size of approximately 2.5 GB (82) with a sequencing depth of 15x, it was estimated that 5–10% of the cellular RNA is coding RNA (mRNA). Thus, the expected raw data per sample was around 4 GB. However, the Gaddi dog PBMCs in the Control, Poly I:C, and CpG groups produced 12.13 GB, 11.98 GB, and 9.17 GB of data, respectively (Table 2). In the differential expression analysis across Labrador and Gaddi dog groups, distinct patterns emerged. For Labradors, significant DEGs were observed in all comparisons, with the Ctrl vs. Poly I:C group revealing 5,476 significant DEGs out of 18,954 total, the Ctrl vs. LPS group showing 5,621 significant DEGs out of 19,381, and the Ctrl vs. CpG ODN group identifying 4,978 significant DEGs out of 19,822. In Gaddi dogs, the Ctrl vs. Poly I:C comparison identified 1,482 significant DEGs out of 22,251 total, the Ctrl vs. LPS group had 3,645 significant DEGs out of 22,800, and the Ctrl vs. CpG ODN group showed 2,416 significant DEGs out of 22,463. The variation in DEG counts between the breeds highlights differences in their immune responses to the same stimuli, indicating breed-specific molecular mechanisms.

The mRNA Seq analysis in Labrador dog PBMCs across all the treatments has shown common enrichment of Th1 and Th17 terms, and the interplay between them is associated with signaling pathways that are prominent in immune-related response (42). In the Control vs. Poly I:C group of Labrador-, The Hematopoietic Cell Lineage pathway showed an increase in genes like CD2 and CD8B, suggesting boosted development and function of immune cells. A study by (43) is a cell adhesion molecule primarily expressed on T cells and natural killer (NK) cells, where it interacts with CD58 (LFA-3) on antigen-presenting cells (APCs). The Human T-cell Leukemia Virus 1 Infection pathway involved genes such as STAT5A and IL2RB, highlighting a heightened immune response against viral infections. Th1 and Th2 Cell Differentiation and Th17 Cell Differentiation pathways, featuring genes like STAT5A and CD3G, pointed to enhanced specific immune responses (44–46). Zhang et al. (47) highlighted the critical regulation of Th1 and Th2 cells, emphasizing that any imbalance in their function could contribute to various immunopathological conditions, including allergies, immune deficiencies, and lymphomas. The CD3E gene is vital for TCR-CD3 complex assembly, stability, and surface expression, impacting T-cell proliferation and cytokine production. These genes are involved in crucial pathways such as TCR signaling, PD-L1/PD-1 checkpoint pathways, and hematopoietic cell lineage, emphasizing their role in immune homeostasis and immune cell development. CD3E deficiencies can cause severe immunodeficiencies, demonstrating its importance in maintaining immune function. Their involvement in checkpoint pathways highlights their relevance in cancer immune evasion (78). Understanding CD3E and CD3G functions offers insights into therapeutic targeting in immune-related diseases and cancer. Further research could reveal strategies for modulating immune responses in conditions with compromised T-cell function (48). Moreover, the ITK gene plays a pivotal role in several key immune pathways, particularly the T-cell receptor (TCR) signaling pathway, chemokine signaling pathway, and leukocyte trans endothelial migration. In the TCR signaling pathway, ITK is activated upon TCR engagement and recruited to the plasma membrane, where it phosphorylates phospholipase C gamma 1 (PLCγ1). This phosphorylation generates secondary messengers such as DAG and IP3, initiating downstream signaling cascades involving MAPK, NF-κB, and calcium signaling, ultimately driving cytokine production, T-cell survival, proliferation, and differentiation (49, 50). ITK also influences the chemokine signaling pathway, critical for T-cell migration and homing. Its activity supports cytoskeletal rearrangement and cell adhesion, promoting efficient chemotaxis to sites of infection and inflammation. Additionally, ITK is integral to leukocyte transendothelial migration, regulating the signaling required for leukocytes to traverse the endothelial barrier into tissues. This process is vital for immune surveillance and response, as ITK coordinates with other molecules to facilitate proper leukocyte adhesion and transmigration (51). The IL2RB gene plays a central role in immune regulation, participating in pathways such as Cytokine-Cytokine Receptor Interaction, Viral Protein Interaction with Cytokine and Cytokine Receptor, Endocytosis, PI3K-Akt Signaling, JAK-STAT Signaling, and Th1/Th2 Cell Differentiation. These pathways highlight its importance in both routine immune function and pathogen response. IL2RB is particularly crucial for Th1 and Th2 cell differentiation, influencing whether the immune response is cell-mediated (Th1) or humoral (Th2). Additionally, its involvement in the JAK–STAT pathway underscores its significance in cytokine responses, vital for immune function and inflammation control (52). The FCGRT gene encodes the neonatal Fc receptor (FcRn), essential for IgG transport and recycling in dogs and other mammals. FcRn extends the half-life of IgG and albumin, ensuring sustained antibody levels for immune protection by neutralizing pathogens and toxins (53). The SLPI gene encodes a serine protease inhibitor that protects tissues during inflammation by inhibiting enzymes like elastase, with antimicrobial properties contributing to tissue protection during inflammatory responses (Gene Cards).

The Gaddi dog PBMCs indicated few immune-related responses across the treatment groups. In the Control vs. Poly I:C group, the enrichment of the Adaptive Immune System pathway highlights the involvement of key genes like SNAP23, LCP2, ICOS, RNF111, and LRRC41, which are essential for immune responses and T cell activation, suggesting a robust adaptive immune response triggered by Poly I:C (54, 55). The pathways related to Membrane Trafficking and Vesicle-mediated Transport are also significantly represented, involving genes such as STX16, SNAP23, TBC1D15, and NAPG. These pathways are critical for intracellular transport and distribution of proteins and other molecules, which are essential for cellular responses to external stimuli, including immune challenges (56). Moreover, the T Cell Receptor Signaling Pathway is emphasized by the upregulation of NFATC2, PPP2R5A, LCP2, and ICOS, indicating enhanced T cell receptor signaling necessary for T cell activation and differentiation during an immune response (57). Additionally, the Efferocytosis pathway, involving genes like RXRA, TGFBRAP1, ATP2A2, and NFATC2, is also enriched, underscoring its relevance in the immune response (58). The DEGs in the control vs. LPS, group of Gaddi dogs highlight key components of the JAK–STAT signaling pathway and related processes. This pathway is critical for regulating cellular activities such as growth, differentiation, and programmed cell death. The JAK–STAT signaling pathway is notably emphasized in dogs through the KEGG pathway (cfa04630), which underlines its importance in canine cellular communication. According to Kuo et al. (59), lipopolysaccharide (LPS) influences the JAK/STAT pathway by activating Toll-like receptor 4 (TLR4), which then triggers the JAK/STAT cascade. This activation results in elevated production of proinflammatory cytokines, like I6, further highlighting the pathway’s role in mediating inflammation and hypertrophy in cardiac cells. Likewise, CpG treatment has revealed pathways enrichment like The Cytokine-cytokine receptor interaction pathway, featuring genes like CSF3 and IL1R2, which play a vital role in intercellular communication during immune responses (76). The Neurotrophin signaling pathway, involving genes such as MAPK8 and FOXO3, is crucial for neural development and function. Additionally, the FoxO signaling pathway, including genes like FOXO3 and SGK1, is essential for cellular homeostasis and stress resistance (83). A study by Zhang et al. (84) highlights the importance of these pathways in immune responses. For instance, CSF3 is crucial for the proliferation, differentiation, and survival of granulocytes, which are key players in the innate immune response. IL1R2 functions as a decoy receptor, modulating the activity of I1, a major inflammatory mediator. Research indicates that the cytokine-cytokine receptor interaction pathway is fundamental for coordinating the immune system’s response to various stimuli and maintaining immune homeostasis (60).

In the present study, the miRNAome profile of Labrador- and Gaddi dog PBMC was also explored across the three treatment groups and a control group. The miRNA expression analysis in Labrador and Gaddi samples revealed clear differences. Labrador samples had a higher number of expressed miRNAs, predominantly novel. The L-Control group had 2,025 miRNAs (185 known, 1,840 novel), while L-Poly I:C had the most at 2,248 miRNAs (154 known, 2,094 novels).

L-LPS and L-CpG showed 2,068 and 1,946 miRNAs, respectively, with most being novel. In contrast, Gaddi samples had fewer miRNAs, with the G-Control showing 1,259 miRNAs (194 known, 1,065 novel), while G-Poly I:C, G-LPS, and G-CpG had 1,011, 176, and 829 miRNAs, respectively, showing particularly low expression under treatment conditions.

In the analysis of differentially expressed miRNAs across various conditions, 175 miRNAs were common across all treatment groups in Labrador- and Gaddi dogs, with several showing consistent regulation in multiple comparisons. Notably, miR-204, miR-676, miR-106a, miR-335, and miR-132 were DE Gaddi dog PBMCs across in comparisons, indicating their involvement in broad immune responses. Similarly, miR-182, miR-203, and miR-98 were commonly regulated between Control vs. CpG and Control vs. LPS, suggesting their roles in response modulation. miR-196a and miR-144 appeared in comparisons involving Control-Poly I:C, while miR-152 and miR-1 were found across Control-LPS and Control vs. Poly I:C. miR-449a and miR-193b were highlighted in comparisons involving Control vs. CpG, Control vs. CpG, and Control vs. LPS, pointing to their involvement in inflammation. miR-551a and miR-1249 were identified in Control vs. CpG, Control vs. LPS, and Control vs. Poly I:C, reflecting their roles in various stimuli responses. In Gaddi dog PBMCs, miR-582 was uniquely regulated across all treatments. It plays a critical role in early B cell development by inhibiting excessive proliferation of pre-B cells and targeting Hif1α and Rictor involved in mTORC2 signaling (85). For Labrador dog PBMCs, miR-551a and miR-1249 were uniquely expressed. miR-551a functions as an oncogene in head and neck squamous cell carcinoma by targeting GLIPR2 mRNA, which modulates autophagy (61). miR-1249, released by NK cells, induces macrophage apoptosis by targeting anti-apoptotic genes and modulating apoptosis pathways (27, 73).

The differential expression of miR-196a and miR-144 in Labrador PBMCs following Poly I:C stimulation highlights their distinct roles in immune response modulation. miR-196a has been implicated in antiviral defenses and cellular responses to viral infections (86, 87). This suggests that miR-196a could play a role in the early immune response to viral mimics like Poly I:C by enhancing antiviral pathways and bolstering the cell’s defense against viral threats. Meanwhile, miR-144 is involved in the regulation of the NF-κB signaling pathway by targeting and suppressing IκBα expression, as shown in studies by Yang et al. (88) and Zhang et al. (89). By inhibiting IκBα, miR-144 facilitates the activation of p65, a key player in the NF-κB pathway, thereby amplifying NF-κB signaling. This pathway is critical for regulating immune and inflammatory responses, leading to the expression of pro-inflammatory cytokines and other immune-related genes essential for a robust immune response. The findings suggest that miR-144’s enhancement of NF-κB signaling plays a vital role in controlling the inflammatory response triggered by Poly I:C. Together, miR-196a and miR-144 appear to coordinate the regulation of antiviral and inflammatory responses in Labrador-PBMCs—miR-196a by directly managing viral defense and miR-144 by modulating the downstream inflammatory processes for a balanced immune reaction. The research Bamunuarachchi et al. (62) demonstrates that miR-144 impairs the host’s antiviral response by suppressing TRAF6 levels, leading to reduced activation of the IRF7 pathway. In vivo studies showed that ablation of miR-144 enhanced resistance to influenza virus infection, indicating its role as a negative regulator of antiviral immunity. Additionally, LPS treatment uniquely regulates cfa-miR-152 and cfa-miR-1. cfa-miR-152 is a tumor suppressor and negatively regulates immune responses by decreasing inflammatory cytokine and type I IFN production in TLR-activated dendritic cells (DCs) (71). It also inhibits the maturation, activation, and function of DCs (80). In contrast, cfa-miR-1 influences critical genes like HSP60, KLF4, and HAND2, which are essential for the physiological functions of smooth and skeletal muscles. This miRNA is also involved in the pathogenesis of various disorders and is dysregulated in several cancers, including gastric, colorectal, breast, prostate, and lung (63, 70, 90).

In Gaddi dog PBMCs, 184 miRNAs were shared across all three treatment groups, while only three miRNAs (cfa-miR-98, 182, and 203) were commonly shared between the Control vs. CpG and Control vs. LPS comparisons. miR-98 regulates SOCS1 and SOCS4, key inhibitors in the JAK–STAT pathway, thus controlling cytokine signaling (75, 91). Targeting SOCS allows JAK–STAT activation, promoting immune cell survival and inflammation, making it relevant in inflammatory disorders (92). miR-182 is primarily linked to cancer and apoptosis, with studies showing it induces apoptosis in rats (93). miR-203 has been associated with combating Canine Atopic Dermatitis (94). In control vs. CpG comparison, three unique miRNAs were identified three unique miRNAs were identified in the Control vs. CpG comparison (cfa-miR-449a, 193b, and 206). miR-449a is involved in immune responses by targeting HDAC1, suppressing IFN-β expression, which is crucial for antiviral defense (95). It also regulates cell cycle arrest and apoptosis. miR-206 plays a role in modulating inflammation by regulating neutrophil recruitment in infections and alleviating inflammatory injury via the JAK2/STAT3 pathway, relevant in sepsis and other inflammatory conditions (96, 97).

The unique miRNA in Gaddi dog PBMC across all the treatment groups was cfa-mir-582. A study by (85) showed that miR-582 plays an important role in early B cell development. We have found that miR-582 is essential for inhibiting the excessive proliferation of pre-B cells during B cell development in BM. There is a negative regulator of B-cell proliferation and survival. It achieves this by targeting and downregulating Hif1α and Rictor, which are involved in mTORC2 signaling and metabolism. Its downregulation in the Gaddi PBMC indicates the B-cell proliferation against the simulations.

Likewise, two DE miRNAs were unique in Labrador dog PBMC across all the treatment groups including cfa-mir-551a and cfa-mir-1249. In a study Karanam et al. (61), miR-551a is an oncogene in head and neck squamous cell carcinoma (HNSCC). It promotes tumor growth, migration, and invasion. It is associated with poor survival miR-551a directly targets GLIPR2 mRNA, which is a negative regulator of autophagy, and its overexpression leads to reduced GLIPR2 levels, thereby modulating autophagy and affecting the cancer cells’ response to treatment. miR-551a is a microRNA implicated in various biological processes across different animal species. In bovine studies, miR-551a was upregulated in non-pregnant cows’ oviductal epithelial cells, suggesting a role in reproductive physiology (64). In porcine research, miR-551a was highly expressed in milk exosomes, indicating potential involvement in immune system development and fundamental physiological processes (65). These findings suggest that miR-551a plays diverse roles in animal biology, including reproductive functions, immune system regulation, and cancer progression.

miR-1249, found in exosomes released by natural killer (NK) cells and alveolar epithelial cells (AECs), has a role in regulating immune responses and modulating macrophage functions. In the context of NK cells, miR-1249 induces apoptosis in macrophages by targeting anti-apoptotic genes and modulating key components of the apoptosis machinery (66). This results in increased macrophage cell death, contributing to immune regulation. Similarly, miR-1249-5p derived from AECs is transferred to macrophages in response to influenza A virus infection. This exosomal transfer of miR-1249-5p promotes the release of pro-inflammatory cytokines such as TNF-α and IL-6 by inhibiting the expression of solute carrier family four-member 1 (SLC4A1), which activates the NF-κB signaling pathway (74). These dual functions of miR-1249 underscore its role in controlling immune cell interactions, apoptosis, and inflammation, suggesting its potential as a therapeutic target in diseases involving immune dysregulation and infection.

miR-1249, found in exosomes released by natural killer (NK) cells, induces apoptosis in macrophages. When macrophages internalize these NK cell-derived exosomes, the miR-1249 within them can promote cell death. This occurs through several mechanisms: miR-1249 may target and downregulate anti-apoptotic genes, reduce the expression of proteins that prevent apoptosis, or modulate pro-apoptotic pathways by influencing key components of the apoptosis machinery. Additionally, miR-1249 can affect cellular stress responses, such as those related to inflammation and oxidative stress, further contributing to macrophage apoptosis. This process highlights miR-1249’s role in regulating immune cell interactions and its potential as a target for therapeutic strategies (53). miR-1249-5p, derived from alveolar epithelial cells (AECs), plays a role in immune regulation during lung infections. In response to influenza A virus infection, miR-1249-5p is transferred via exosomes from lung epithelial cells to macrophages. This transfer promotes the release of pro-inflammatory cytokines TNF-α and IL-6 by suppressing the expression of solute carrier family four member 1 (SLC4A1), activating the NF-κB signaling pathway (67). The unique miRNAs, miR-551a and miR-1249, in the Gaddi dog breed, offer a valuable opportunity to explore their regulatory roles in gene expression, specifically in dogs. Studying these miRNAs could reveal insights into breed-specific adaptations, immune responses, and resistance to environmental stressors or diseases.

Insights on differential systems biology on Labrador home-owned and exotic origin contrarily Gaddi dogs, reared by locals of the sub-Himalayan region to guard and herd livestock, and are free-range dogs. Hence, the evolution of the immunological systems must have some differences. The mRNA-Seq analysis of Labrador PBMCs revealed consistent enrichment of Th1, Th2, and Th17 differentiation pathways across all treatment groups. In contrast, Gaddi dog PBMCs also showed enrichment in Th1 and Th17 differentiation pathways and inflammatory pathways, chemokine and cytokine signaling, apoptosis, cell proliferation, and T-cell activation pathways across the treatment groups. These findings suggest a heightened immune response in Gaddi dogs against various TLR ligands. The miRNA-Seq analysis identified a significant overlap in miRNA Between the two breeds, predominantly regulating transcription factors, the cell cycle, autophagy, TLR signaling, and cytokine and chemokine pathways. However, breed-specific miRNAs were observed: Labradors exhibited cfa-miR-196a, miR-144, miR-204, miR-676, miR-106a, miR-335, and miR-132 across treatment groups, whereas Gaddi dogs showed cfa-miR-449a, miR-193b, miR-206, miR-551a, and miR-1249. Moreover, a study by Guo et al. (68) explored the role of miR-1249 in intervertebral disc degeneration (IDD) and being used as a target treatment for IDD. These findings underscore the distinct transcriptomic and miRNAomic landscapes between the two genetics groups, reflecting their diverse immunogenetic adaptations and potential disease resistance mechanisms based on pathway analysis.

We present the inaugural atlas of mRNA and miRNA expression in TLR ligand-stimulated PBMCs of Labrador (exotic) and Gaddi (Indigenous) dogs. This reference will support future functional studies and challenge experiments to identify disease-associated genes and immune pathways. Additionally, it provides a maiden report on differential transcriptomes, contributing to breed recognition of the Gaddi dog.