Increase of temperatures substantially above normal induces the synthesis of heat-shock proteins whose functional role is well known at the cellular level. Several studies have been directed towards the importance of heat-shock proteins for resistance of livestock and poultry to high-temperature stress and other environmental stressors.
The present study was undertaken to characterize chicken 70 kDa heat shock protein (HSP70) and study its expression level along with two of the most potent proinflammatory cytokines TNF-Î±, IL-1Î² at different temperatures (42°, 44°, 46° and 48° C) in vitro in six divergent strains/breeds of chickens (viz. Rhode Island Red (RIR-B and RIR-C strains), Dahlam Red, Punjab Red and White Leghorn (PL2 and Naked Neck strains).
The chicken (Punjab Red) Hsp70 amplicon was cloned and sequenced (Gen Bank Accession No: GU980869). Sequence comparison and phylogenetic analysis of Hsp70 revealed high degree of identity suggesting that Hsp70 is conserved among species and is under purifying selection.
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The expression profiling of Hsp70 and downstream cytokine genes was evaluated by quantitative Real Time PCR. The expression of Hsp70, TNF-Î±, IL-1Î² was found to increase from 42Â°C to 44Â°C and decrease from thereon, being lowest at 48Â°C. This may be attributed to the inability of the cells to survive high temperatures beyond 44Â°C as indicated by trypan blue dye exclusion test. Among the strains/breeds highest and lowest expression of Hsp70, TNF-Î±, and IL-1Î² was observed in RIR-C and Punjab Layer strain, respectively. The expression of these genes was observed to be higher in Red varieties (RIR-B,RIR-C, DR, PR) than the White varieties (NN, PL2). Among the Red varieties, the expression of Hsp70 in Punjab Red was higher than its parental breeds (RIR-B and Dahlam Red).
The results of the present study indicate that the expression of Hsp70 in chickens is affected by heat stress.
Gene expression in cells alters rapidly on abrupt exposure to heat stress. The cellular response to stress stimuli, both in vivo and in vitro, induces the synthesis of heat shock proteins (HSPs) and represses the expression of almost all the other genes (Morimoto et al 1990). HSPs are a family of highly homologous chaperones that limit the consequences of damage and facilitate cellular recovery. They are not only critical to cell survival to heat shock, but also are essential under normal growth conditions (Foster and Brown,1996; Mezquita et al, 2001).
HSPs are classified into 5 families according to their molecular weight (100, 90, 70, 60, and small HSP). The intracellular levels of Hsp70, the most abundant class of HSPs, indicate the degree of thermotolerance in terms of the ability of cells to become resistant to heat stress after a prior sub-lethal heat exposure (Morimoto et al 1994). HSPs are similar to the major histocompatibility complex (MHC) molecules in their ability to bind the antigenic fingerprint of cells. However, the HSPs are more promiscuous than the MHC molecules. The interaction of HSPs with the APCs leads to several peptide-independent activities like secretion of inflammatory cytokines, tumour-necrosis-Î± (TNF-Î±), interleukin-1Î² (IL-1Î²), IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by macrophages. In addition, HSPs have become useful models for studying transcriptional regulation, the stress response and molecular evolution (Srivastava, 2002). HSPs can and do exit the cells, interact with the cells of the immune system and exert immunoregulatory effects(Asea et al., 2002)
Poultry industry has been one of the rapidly growing sectors of Indian agriculture, with an average increase in egg and broiler production to the tune of 8 to 10% per annum in contrast to 1.5 to 2% per annum for agricultural crops. Heat stress has been recognized as one of the critical issues in further growth of poultry industry, since chickens are more sensitive to high ambient temperatures than other domestic animals (Geraert et al 1993). Increased environmental temperature reduces feed intake and efficiency, growth rate, egg shell-quality and survivability in chickens leading to huge loss of revenue. A developing country like India cannot afford to artificial temperature regulatory devices to ameliorate heat stress making it even more important to select birds that are thermotolerent. Limited studies have been conducted on expression profiling of Hsp70, NFÎºB, and proinflammatory cytokines, tumour-necrosis-Î± (TNF-Î±) and interleukin-1Î² (IL-1Î²) in divergent chicken germplasm in India.
The present study was undertaken to characterize chicken 70 kDa heat shock protein (Hsp70) and study its expression level along with NFÎºB and two of the most potent proinflammatory cytokines TNF-Î±, IL-1Î² at different temperatures (42°, 44°, 46° and 48° C) in vitro in six divergent strains/breeds of chickens (viz. Rhode Island Red (RIR-B and RIR-C strains), Dahlam Red, Punjab Red and White Leghorn (PL2 and Naked Neck strains).
MATERIALS AND METHODS
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The chickens were obtained from the Poultry Breeding Farm of Department of Animal Breeding and Genetics, GADVASU, Ludhiana, India. The birds were given free access to water and feed, and were maintained in thermoneutral environment. Six birds belonging to the same age group and sex were randomly selected from each of the six divergent strains/breeds viz; Red varieties: Punjab Red (PR), Dahlem Red (DR), Rhode Island Red (RIR-B and RIR- C) and white varieties: Naked Neck (NN), PL2.
Collection of Blood
Blood samples were aseptically collected from the wing vein of each bird (using sterile 24G needle) in a sterile tube containing 0.5M EDTA (pH 7.4).
Cloning and sequencing of Hsp70
Leucocytes were isolated from fresh blood sample of Punjab Red strain by density gradient centrifugation using HISTOPAQUE-1119/1077 (Sigma-Aldrich, USA). Total RNA was isolated from the leucocytes using Trizol method (Invitrogen, Life Sciences, USA). RNA template with absorbance ratio (260/280) 1.9 to 2.0 was subjected to first strand cDNA synthesis. The extracted RNA was reverse transcribed using the Promega Reverse Transcription kit (Promega) according to manufacturer's instructions. Briefly, oligo dT primer (0.5 mg) was used to reverse transcribe 1 mg of RNA in the presence of dNTP's (250 mM), reverse transcriptase buffer (10 mMTris-HCl, 50 mMKCl, 0.1% Triton-X-100), AMV Reverse Transcriptase high conc. (15 units/mg) and RNasin Ribonuclease inhibitor (1 unit/ml) at 42° C for 60 min following inactivation at 99° C for 5 min.
The primer sequences 5'-GGGCACCACGTATTCTTG-3' and 5'-TGCCCAGCTCCTCC AG -3', respectively, were designed from the published sequence of Gallus gallus Hsp70 (Accession No.J02579; Morimoto et al, 1986). The amplification was carried out in 50 Âµl final volume containing 1.5mM MgCl2, 50mM Tris-HCl (pH 9.0 at 25oC), 15mM (NH4)2SO4 and 0.1% Triton-X; 0.4ÂµM of primers; 200ÂµM of each dNTP's and 1 unit of Pfu Taq Polymerase. After a denaturation at 94°C for 5 minutes, 35 cycles of amplification were performed as follows: denaturation at 94°C for 45 sec, annealing at 50°C for 45 sec and extension at 72°C for 90 sec. A final extension step of 10 min at 72oC ended the reaction
The PCR product was checked for quality and quantity, gel purified (QIAquick Gel extraction kit, QIAGEN, Germany) and cloned into the pUCmT - easy cloning vector and sequenced using automated dye-terminator cycle sequencing method with Ampli Taq DNA polymerase in ABI PRIZM 377 DNA sequencer (Perkin-Elmer). The fragment was sequenced at least twice with each primer to reduce possibility of sequencing artifacts (Gen Bank Accession no : GU980869)
The nucleotide sequence of chicken Hsp70 was subjected to nucleotide blast (Altschul et al., 1997) and from the top 50 hits some representative sequences were chosen for phylogenetic analysis using the unpaired group mean average (UPGMA), Neighbor-Joining and Maximum Parsimony programs in MEGA 4.0 (Tamura et al., 2007). The Boot-strapping option with 1000 replicates was used with the Dayhoff-PAM substitution model and with pair-wise deletion of the gaps/missing data.
The nucleotide sequences of HSP70 were used for estimation of the number of synonymous substitutions per synonymous site (dS) and the number of non-synonymous substitutions per non-synonymous site (dN). Estimates were computed using a maximum likelihood (ML) method (Goldman and Yang 1994) and several counting methods (Nei and Gojobori 1986; Yang and Nielsen 2000) implemented in the CODEML program of the PAML package Version 4.1 (Yang 2007). ML analysis was performed with runmode-2. Positive (dS<dN) or purifying (dS>dN) selection was tested with a codon-based z-test, using the Nei-Gojobori method (P-distance) in MEGA 4.0 (Tamura et al., 2007).
Peripheral Leucocyte isolation and Heat shock
Blood samples collected from the experimental birds (n=6 per group) were subjected to leukocyte separation by centrifugation through Histopaque1119/1077 (Sigma-Aldrich, USA). The isolated leucocytes were washed thrice with Hanks' balanced salt solution (HBSS) and re-suspended in Eagle's methionine-free minimum essential medium. The cell concentration was adjusted to 1Ã- 107/ml and subjected to different incubation temperatures in vitro (42Â°, 44Â°, 46Â° and 48Â° C for 1hour) in triplicates.
Quantitative Reverse Transcriptase PCR for Real Time PCR
The total RNA was reverse transcribed to cDNA, using QuantiTect Reverse Transcription Kit (QIAGEN, Germany), according to the manufacturer's instruction. Quantitative Real-Time PCR was performed by TaqMan assay using Applied Biosystems 7500 system. The Real Time PCR reaction was carried out in 20 Âµl final volume containing 1X TaqMan master mix, 900nM of each primer and 200nM of probe and template cDNA (2 Î¼l equivalent to 100 ng). The detail of the probe-primer mix is shown in Table 1. Cycle threshold (Ct) values were calculated using the SDS software v.2.3 using automatic baseline settings and a threshold of 0.2. Since a Ct value of 35 represents single molecule template detection, Ct values > 35 were considered to be below the detection level of the assay (Guthrie et al,2008).The PCR efficiency (E) was calculated according to the formula: E = 2-1/slope(Î²c). The data was normalized by subtracting the Ct value of endogenous control gene (18S) from the corresponding Ct value for the target gene resulting in the Î”Ct value which was used for relative quantification of gene expression. As there is an inverse correlation between Î”Ct and gene expression level, lower Î”Ct values were associated with increased mRNA expression. For relative quantification, Î”Î”Ct for a treatment-gene combination is calculated by taking treatment-gene combination with lowest Î”Ct as a calibrator.
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The Statistical Analysis System 9.2 (SAS Institute Inc., Cary, NC, USA) procedure Proc Mixed was used for performing simple linear regression for each of the treatment-gene combinations to estimate the slope (Î²c) and Proc Glm was used to find significant difference in slope between treatment-gene combinations. The estimate of the slope gives percentage efficiency ( (Yuan et al,2006). Several effects namely, the effect of treatment, gene, and replicates were taken as quantitative variables with Ct number relating to these multiple effects and their interactions, to run a multiple regression model using Proc mixed.
A fragment of 1643 bp of chicken Hsp70 was amplified, cloned and sequenced. The sequence showed 96% identity with Gallus gallus (original sequence), 93% with Coturnix japonica (Japanese quail) and Numida meleagris (Guinea fowl), 89% with Anser cygnoides (Goose), 88% with Anas platyrhynchus (Duck) and Taeniopygia guttata (Zebra finch), 79% with Mus musculus(Mouse), Oryctolagus cuniculus (Rabbit), Pongo abelii (Orangutan), Sus scrofa (Pig), Canis familiaris (Dog), Ornithorhynchus anatinus (Platypus), Capra hircus (Goat), Homo sapiens (Man) and Pan troglodytes(Chimpanzee), 78% with Callithrix jacchus (Monkey), Monodelphis domesticus (Opossum) and Bos Taurus (European cattle). On phylogenetic analysis, the neighbor joining tree (Fig 1) was with relatively higher bootstrap values providing clear resolution of all the nodes. Gallus gallus sequence clustered in the avian class away from the mammalian class, closest to reptilia and egg laying mammal (Ornithorhynchus anaticus), and farthest from the primate group (Homo sapiens, Callithrix jacchus , Pan troglodytes, Pongo abelii). In the mammalian class there was clear clustering of different orders at distinct sub nodes. The order artiodactyla (Bos taurus, Bos indicus, Bubalus bubalis,Ovis aries,Capra hircus and Sus scrofa) clustered away from the orders primates (Homo sapiens, Macaca mulatta, Gorilla gorilla, Pan troglodytes, Pongo pygmaeus, Theropithecus gelada, Cercopithecus mona and Cercocebus agilis), rodentia (Mus musculus, Rattus norvegicus), marsupalia (monodelphus domesticus) and carnivora (Canis familiaris). In the bovidae family of the order artiodactyla the ruminants (Bos taurus, Bos indicus, Bubalus bubalis,Ovis aries and Capra hircus) clustered away from the nonruminants group(Sus scrofa).
Nucleotide substitutions in genes coding for proteins can be either synonymous (do not change amino acid) , alternatively called silent substitutions, or non-synonymous(changes amino acid).Usually, most non-synonymous changes would be expected to be eliminated by purifying selection, but under certain conditions Darwanian selection may lead to their retention. Purifying selection is clearly evidenced by the fact that mutations that alter the amino acid sequence, which in many cases presumably have a deleterious effect, have gone to fixation at a much lower rate than those that do not. Traditionally, this is expressed in terms of the ratio of non-synonymous (dN) to synonymous (dS) substitutions, dN/dS, where dS is here used as an index of the rate of unconstrained, neutral evolution. When dN/dS is less than 1, the usual interpretation is that negative selection has taken place on non-synonymous substitutions resulting in purifying selection. When dN/dS is greater than 1, positive selection is likely to have accelerated the rate of fixation of non-synonymous substitutions. Purifying selection is the conservative force in molecular evolution, whereas positive selection is the diversifying force that drives molecular adaptation (Ellegran,2005). In the present study, the dS was significantly (P<0.01) higher than the dN in the analyses of Hsp70 nucleotide sequences suggesting that Hsp70 is under purifying selection (Table 2). The gene encoding Hsp70 is not under positive selection and no advantageous mutations have played an important role in evolutionary adaptation.
Expression of Hsp70, IL-1Î² and TNFÎ± mRNA under heat stress:
Heat stressed induced levels of Hsp70, IL-1Î² and TNFÎ± expression relative to the basal level (42Â°C) expression is shown in Table 2 and Figure 2, 3 & 4 among different breeds/strains. The expression of Hsp70, IL-1Î² and TNFÎ± in peripheral leucocytes exposed in-vitro at 44Â°C, was elevated significantly (P â‰¤ 0.01) by 3.43, 2.35 and 1.21 folds, respectively, on an average over the basal level. Expression of these genes depressed at 46Â°C and was least at 48Â°C because the cells were not able to survive at the higher temperatures. The percentage of viable cells was significantly lower at 48Â°C than at 46Â°C and 44Â°C indicating that the cells were not able to survive high temperatures in all breeds/strains. Among the six strains/breeds of chickens, the RIR-C showed the lowest basal (42Â°C) expression of HSP70, IL-1Î² and TNFÎ± and therefore was chosen as calibrator for comparison of expression among breeds (Fig 5). The expression profile of Hsp70, IL-1Î² and TNFÎ± showed a clear pattern of difference between the Red and the White plumage varieties: the Red varieties (RIR-B, DR, PR) had higher level of expression than the White varieties (NN, PL2). The colored varieties exhibited about 60%, 70% and 30% higher expression of Hsp70 , IL-1Î² and TNFÎ± at 44Â°C,respectively, as compared to the white varieties.
On comparing the expression levels of HSP70, IL1Î² and TNF Î± within each breed/strain RIR-C significantly (P â‰¤ 0.05) showed higher expression followed by Dahlam Red, RIR-B, Punjab Red, Naked Neck and Punjab layer, at increased temperature (44Â°C) compared to the basal level (42Â°C) (Fig). The expression of Hsp70, IL-1Î² and TNFÎ± in peripheral leucocytes exposed invitro at 440C, was elevated by 7.8, 8.0 and 1.17 folds, in RIR-C; 4.5, 1.07 and 0.87 folds, in Dahlam Red; 2.75, 1.35 and 1.17 folds, in RIR B; 2.06, 2.0 and 1.98 folds, in Punjab Red; 1.78, 1.2 and 1.76 folds, in Naked Neck and 1.53, 0.50 and 0.34 folds, in Punjab layer, respectively, over the basal level.
Correlation between HSP70 and inflammatory cytokines
The Hsp70 level was correlated with the levels of TNFÎ± (r=0.943) and IL-1Î² (r=0.964).The highest correlation was between the two early cytokines (TNFÎ± and IL-1Î²) (r=0.994). The positive correlations between Hsp70, TNFÎ± and IL-1Î² were statistically significant (P<0.01).
Hsp70 gene is highly conserved and is under purifying selection
Selection modulates gene-sequence-evolution in different ways, by constraining potential changes of amino acid sequences (purifying selection) or by favouring new and adaptive genetic variants (positive selection). The number of non-synonymous differences in a pair of protein-coding sequences is used to quantify the mode and strength of selection. To control for regional variation in substitution rates the proportionate number of non-synonymous differences (dN) is divided by the proportionate number of synonymous differences (dS). The resulting ratio (dN/dS) is a widely used indicator for functional divergence to identify particular genes that underwent positive selection. Investigating the number of synonymous and non-synonymous substitutions may therefore provide information about the degree of selection operating on a system. Positive selection pressure acting on protein-coding sequences is usually inferred when the rate of non synonymous substitution is greater than the synonymous rate.Â High dN/dS ratios have been found for several genes that undergo rapid adaptive evolutionary change. The present study revealed that there exists less variability in sequence of Hsp70 cDNA among divergent species, which signifies that Hsp70 is highly conserved among species (Pelham 1982; Morimoto et al 1986; Gaviol et al, 2008) and is under purifying selection.
Real Time data analysis
Two mathematical models are very widely applied for real time data analysis (i) the efficiency calibrated model (ii) the Î”Î”Ct model (Yuan et al, 2006). Ct number is first plotted against logarithm cDNA input, and the slope of the plot is calculated to determine the amplification efficiency. The efficiency-calibrated model is a more generalized Î”Î”Ct model. Î”Î”Ct model can be derived from the efficiency-calibrated model, if both target and reference genes reach their highest PCR amplification efficiency. In this study, the efficiency of all four genes ((Hsp70, TNFÎ± and IL-1Î²-target genes),(18sRNA-reference genes)) was not significantly (P â‰¥ 0.05) different from 2 in different treatment-gene combinations and therefore, data analysis was done by using Î”Î”Ct model.
Expression of Hsp70 under heat stress
High temperatures trigger massive synthesis of heat shock proteins that fold heat denatured proteins and block caspase dependent apoptosis, permitting repair and thereby thwarts cellular death. However, extreme exposure to heat overwhelms the capacity of intracellular stress proteins and triggers death. Synthesis of Hsp70 is rapidly induced in lymphocytes, with increase in temperature (2°C - 3°C) (Morimoto and Fodor, 1984) and is found to be 10 to 24 times higher than that of basal level in the tissues like muscle, liver and brain, following induced heat shock to 44°C (Zhen et al., 2006), which signifies the self-protection mechanism to adapt the animal to the environmental stress. In chicken and turkey poults expression of Hsp70 initially increased with increase in temperature and depressed at 47°C and abolished at 49°C because the cells were not able to survive high temperature (Wang and Edens, 1998). The expression level of Hsp70 is tissue-dependent in chicken embryos subjected to heat stress (Givisiez et al, 2001) and a positive correlation is reported between environmental temperature and Hsp70 levels in liver of five days old Naked Neck chicken (Dionello et al, 1998). The expression of Hsp70 is also dependant on ambient temperature and time of exposure to heat stress (Gabriel et al, 1996). In the present study the heat stressed leukocytes in vitro showed differential expression of HSP70 at different incubation temperatures among different strains/breeds of chickens. The expression levels of HSP70 gene increased at 44°C but gradually decreased at 46°C and 48°C in different strains/breeds as the viability of cells decreased with increase in temperatures.
Correlated expression of Hsp70, TNF-Î± and IL-1Î²
Hsp70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca(2+) flux, activate NF-kappaB, and up-regulate the expression of pro-inflammatory cytokines, TNF-Î±, IL1Î², in human monocytes. (Asea et al, 2002). The binding of HSP70 results in a broad non-specific immunostimulation by initiating the secretion of different proinflammatory cytokines.(Radons and Multhoff 2005; Basu et al, 2000;). In the present study with the increase of Hsp70 there was a proportional increase in the level of IL-1and TNF-Î±. Considering the positive correlation between Hsp70 and TNF-Î±, IL1Î² we postulate a representative role for HSP70 as a cytoprotectant under stress and suggestÂ that Hsp70-induced pro-inflammatory cytokine production may be mediated via the MyD88/IRAK/NF-kappaB signal transduction pathway to transduce its proinflammatory signal under stress Fig 6. NF-ÎºB is a major transcription factor that regulates genes responsible for both theÂ innateÂ andÂ adaptive immune response.
The Hsp70 is highly conserved among the species and is under purifying selection. Highest expression of Hsp70, TNF-Î± and IL-1 occurred at 44Â°C with a gradual decrease at higher temperatures because of the decrease in viability of the at high temperatures. The Red varieties (RIR-B, DR, PR) had higher level of expression than the White varieties (NN, PL2) indicating the suitability of the red varieties to withstand elevated temperatures. With increase of Hsp70 there was a proportional increase in the level of IL-1Î² and TNF-Î± indicating the involvement of MyD88/IRAK/NF-kappaB signal transduction pathway.