Evaluation and comparison of the constitutive expression levels of Toll-like receptors 2 , 3 and 7 in the peripheral blood mononuclear cells of Tharparkar and crossbred cattle

Aim: This study was undertaken to assess the differential expression levels of toll-like receptors (TLRs) 2, 3 and 7 in peripheral blood mononuclear cells (PBMCs) isolated from Tharparkar and Crossbred cattle belonging to different regions of India. Materials and Methods: PBMCs were isolated from blood samples of Tharparkar cattle from Indian Veterinary Research Institute (IVRI) farm (n=30); Suratgarh farm (n=61); Jaipur farm (n=8) and cross breed cattle from Jaipur (n=47). RNA was isolated from PBMCs and cDNA was synthesized using random hexamers. The expression profiles of TLR 2, 3 and 7 were estimated by real-time PCR and normalized to the expression of β-actin. Results: PBMCs of Tharparkar cattle from Suratgarh, exhibited a significantly higher (p<0.05) constitutive expression levels of TLR2, TLR3 and TLR7 genes as compared to Tharparkar cattle from IVRI or Jaipur as well as the crossbred cattle from Jaipur. PBMCs of crossbred cattle from Jaipur showed higher expression profiles of all the TLRs than Tharparkar cattle from Jaipur and IVRI. Conclusion: Our study indicates, expression levels of TLR2, TLR3 and TLR7 are significantly higher for Tharparkar cattle from Suratgarh than the cattle from Jaipur and IVRI and crossbred cattle from Jaipur. However, crossbred cattle from Jaipur showed higher basal expression levels of all the three TLRs than Tharparkar cattle from Jaipur and IVRI. Results also indicate that PBMCs of Tharparkar cattle show a regional variation in the expression pattern of TLRs.


Introduction
and tissues are suggestive of an individual's ability to respond to pathogens.Toll-like receptors (TLRs) recognize the invading The expression pattern and distribution of the pathogens through the detection of 29 highly TLRs have been shown to be characteristic of each conserved pathogen associated molecular patterns species [8][9][10].Over the last two decades there has been (PAMPs) derived from a wide range of pathogens [1] great progress in identifying TLRs in different species and activate the innate immune response [2].The term of farm animals and exploring their roles in the disease "Toll-like receptors" was proposed in 1997 for mamsusceptibility and resistance against the invading malian proteins structurally related to the "TOLL" cell pathogens.Various breeds of animal show differences surface receptor seen in the Drosophila larvae.TLRs in their susceptibility to infectious diseases, while some trigger the activation of adaptive immune system by breeds show a better resistance to a pathogen while promoting the presentation of antigens and upothers are susceptible to infectious diseases.Cattle also regulation of various cytokines and co-stimulatory show differences in their susceptibility/ resistance to molecules [3,4].Till date, at least 13 TLR members various invading organisms and this might be have been described in mammals [2,5,6].Although, attributed to differential expression profile of TLR TLR family proteins are well conserved, the different genes [11].TLR2, TLR3 and TLR7 detect lipoproteins, members of the TLR family have very distinct func-dsRNA and ssRNA respectively, and are involved in tions in the host defence.The main characteristics that defence against the bacterial and viral infections.The distinguish various TLRs are ligand specificity, signal constitutive expression levels of these TLRs in transduction pathways and sub cellular localization different breeds of animal may explain the differential [7].Basal TLR expression profiles in different cells susceptibility status of various breeds.Thus, this study was planned to assess the differential expression levels of TLR2, TLR3 and TLR7 in Tharparkar cattle from different regions and

Evaluation and comparison of the constitutive expression levels of Toll-like receptors 2, 3 and 7 in the peripheral blood mononuclear cells of Tharparkar and crossbred cattle
crossbred cattle from Jaipur.This information might of the β-actin gene and the relative expression of each

Isolation of peripheral blood mononuclear cells
PBMCs of Tharparkar cattle from Suratgarh, (PBMCs) in Tharparkar cattle and Crossbred cattle: exhibited a significantly higher (p<0.05)constitutive Blood was collected from the jugular vein using a expression levels of TLR2, TLR3 and TLR7 genes as sterile syringe containing heparin as an anticoagulant.
compared to Tharparkar cattle from IVRI or Jaipur as The blood was slowly layered over on an equal volume well as the crossbred cattle from Jaipur.PBMCs of of Ficoll Hypaque (Sigma, MO, USA) with specific crossbred cattle from Jaipur showed higher expression gravity 1.077 g/ml.It was then centrifuged at 600g for profiles of all the TLRs than Tharparkar cattle from 30 min and then the interface containing the PBMCs Jaipur and IVRI.The expression data of TLR2, TLR3 was collected and washed twice in sterile phosphate and TLR7 normalized to β-actin was found to be buffered saline (PBS).Their viability was determined 0.519±0.17,0.118±0.08,0.121±0.07(IVRI farm); by Trypan blue staining.

extracted with chloroform and precipitated with
The innate immune system is essential for host isopropanol.The RNA pellet was briefly washed with defence and is responsible for early detection of 70% ethanol and resuspended in RNase free water invading pathogen.Effector mechanisms of innate (Invitrogen, CA, USA).Upon isolation of RNA, cDNA immunity are activated immediately after infection, synthesis was carried out by using random hexamer with the aim to control the replication of the infecting primers (Fermentas, MD, USA).
pathogen at the site of the entrance.The discovery of Real-time PCR quantification (qRT-PCR): Expression toll-like receptors (TLRs), particularly on the cells of levels of mRNA of TLR 2, TLR 3 and TLR 7 were innate arm of immune system [15,16] and their critical analyzed by Real-time PCR using the QuantiTect role in providing immunity has aroused intense SYBR Green qPCR system (Qiagen, CA, USA) on Icuriosity in the scientific arena in understanding the cycler (Bio-Rad, CA, USA) using gene specific functions of the immune system [17,18].In Cattle, ten primers.Beta actin gene was used as the reference gene TLRs (1-10) have recently been reported [19,20].

(endogenous control). The primers used in the study
Overall, bovine TLRs seem to show a greater sequence were designed by Becon Designer software (Table-1).
homology to humans than to mice [11,20,21].Real-time PCR was carried out in triplicate for each In the present study, we evaluated the expression sample in a total volume of 20 µl consisting of 2 µl profiles of TLR 2, TLR3 and TLR7 genes in the cDNA, 10 µl of Affimetrix SYBR Green Master Mix PBMCs of Tharparkar cattle from different regions and (USB, CA, USA), primers 0.5 µl each (20 pm/µl).Realcrossbred cattle from Jaipur.TLR2 is an extracellular time PCR cyclic conditions were 95ºC for 2 min, 95ºC receptor and implicated in the recognition of Gram-30 sec, 51ºC for 30 sec, 72ºC for 15 sec, followed by 40 positive bacterial components, bacterial lipoproteins, cycles.The final step was to obtain a melt curve for the and zymosan [22,23].TLR2-deficient mice displayed PCR products to determine the specificity of the impaired cytokine production in response to amplicons.Expression levels of the TLR 2, TLR3 and Staphylococcus aureus peptidoglycon preparation and TLR7 genes were normalized relative to the expression mycoplasmal lipopeptide [24].TLR3 and TLR7 are present on intracellular compartments and ligands for gens.Higher expression of TLRs reflects a better these TLRs, are double-stranded RNA (Poly I:C -innate immune response and disease resistance.Our Polyinosinic : polycytidylic acid) and single-stranded study reveals the higher expression of TLR2, TLR3 and TLR7 in the PBMCs of Tharparkar cattle from RNA, suggesting their role in the detection of RNA Suratgarh in comparison to crossbred cattle.However, viruses [6].
Tharparkar cattle from Jaipur and IVRI did not show We have found that all TLRs, under investigation, the same pattern.Crossbred cattle from Jaipur exhibited are expressed constitutively in the PBMCs of a better immune competence than the Tharparkar Tharparkar and crossbred cattle; however, PBMCs of cattle.Keeping in view the complexity of the innate Tharparkar cattle from Suratgarh, expressed higher immune system, further investigations are required to mRNA transcripts of all three TLRs than that of explain these differences in the expression levels of PBMCs from Tharparkar cattle from Jaipur or IVRI TLRs in Tharparkar and crossbred cattle from different and crossbred cattle (Figure -1).Further, though regions.The knowledge generated will help us to Tharparkar cattle from Suratgarh showed significantly recognize important alleles involved in disease resistance higher expression levels of TLR2, TLR3 and TLR7 and in designing selective breeding programmes for than crossbred from Jaipur, cattle from IVRI and efficient control and prevention of diseases.Jaipur, did not show the same pattern and expressed very low levels of all the three TLRs than crossbred All the experiments were run in Materials and Methods triplicates.The results of three independent experiments were reported as mean + SE.As the normality Ethical approval: Blood samples used in this study test failed, the Mann-Whitney nonparametric test was were collected from Indian Veterinary Research Institute done in JMP 9 (SAS Institute Inc., Cary, NC, USA).(IVRI) farm, Suratgarh farm and Jaipur farm after the approval of Institutional Animal Ethics Committee.

Table - 1
: List of primers used in the study