HSP70 Gene Expression Pattern in Peripheral Blood Mononuclear Cells during Different Seasons in Pandharpuri Buffaloes

Livestock plays an important role in rural economy through its contribution to food, employment generation and drought power. Buffalo has been the mainstay of rural economy in the Indian subcontinent and South East Asian countries. The world buffalo population is 204 million in forty two countries of which 97% population is confined to Asia and India (FAO, 2021). India ranks first in buffalo population in Asia and has 113.33 million buffaloes (DAHD, 2019). Buffalo is predominantly distributed in different regions of the country and well adopted to different agro climatic conditions due to its ability to sustain and thrive on poor quality roughages and better ability to resist tropical diseases. These buffaloes are concentrated in Pandharpur, North Solapur, South Solapur, Akkalkot, Sangola and Mangalvedha tehsils of Solapur district; Miraj, Walwa,  Tasgaon tehsils of Sangli district of Maharashtra state (NDDB, 2015). The region of Satara district is heterogeneous in its agro-ecology due to diversities in its physiographic and climatic profile and is located between 17°5' and 18°11' north latitudes and 78° 33' and 74° 54' east longitudes. Although this buffalo breed called “Pride of Western Maharashtra”, but full potential of this animal however still remained to be exploited for rural upliftment. Heat stress has various detrimental effects on livestock (Marai and Habeeb, 2010). Further, humidity becomes more important since it directly affects the evaporation rate. Therefore, the temperature humidity index (THI) becomes relevant under conditions of high temperature and high humidity. Temperature-humidity index (THI), a parameter that is extensively used to describe heat load on animal and humans, is a good indicator of stressful thermal climatic conditions (McDowell et al., 1976).

HSP70 is known to be a highly inducible chaperon and plays a key role to stabilize the native conformation of proteins and maintenance of cell survivability during thermal stress (Beckham et al., 2004). Particularly in mammals, exposure to hypothermia or hyperthermia has been related to morphological and physiological modifications. Heat shock proteins (HSPs) are multigene families that range in molecular size from 10-150 kDa and are found in all major cellular compartments (Patir and Upadhyay, 2010). They are highly conserved proteins present in all the cells of living organisms and are essential for cellular viability as these have major physiological roles in protein homeostasis. Dangi et al., (2012) revealed that 70-kDa heat shock protein family assists the folding of proteins upon translation in the cytosol of both prokaryotic and eukaryotic cells as determined by genetic and biochemical analyses.

Buffaloes are seasonally polyestrous with a marked reduction in their reproductive performance during summer in regions of high latitude. Relative expression of HSP70 genes varied markedly among the heat- and cold-adapted goat breeds with a moderate variation between breeds and showed a good response to increased or decreased ambient temperature (Banerjee et al., 2014, in caprine PBMCs (Gupta et al., 2013), leukocytes of buffalo (Pawar et al., 2014), Murrah buffalo (Mishra et al., (2010), dermal fibroblasts of cattle (Singh et al., 2014). In addition, it is speculated that heat shock stress effects on the early embryo directly and it also influences the development to the late embryo (Wee et al., 2008). Heat stress in farm animals, such as cattle and buffalo during summer and post-summer seasons is a problem for livestock producers. The effect of heat stress becomes pronounced when heat stress is accompanied with ambient humidity impairing the immune status, growth, production and reproductive performance of animals (Mishra et al., 2010). No studies have been designed so far to gain an insight into the seasonal variation effects on HSP70 gene expression profile in Pandharpuri buffalo. The present study is therefore, proposed to study HSP70 gene expression pattern in peripheral blood mononuclear cells.

Sample collection
 
The experiment was conducted on 10 apparently healthy Pandharpuri buffaloes above 2 years of age maintained under loose housing condition. The meteorological variables like temperature (dry bulb and wet bulb) and relative humidity were recorded for the Month of May (summer season), for the month of August (rainy season) and for the month of December (winter season) and were used for calculation of temperature humidity index (THI) as per the formula given by Madar et al., (2006). 5 ml blood samples from these animals were collected aseptically by jugular vein puncture during peak winter season, peak summer season and peak rainy season. Safety measures were taken to minimize the effect of ribonuclease during processing. All samples were processed within one hour of collection.
 
PBMCs isolation
 
Dilution of whole blood was done in phosphate buffer saline (PBS, pH=7.4) in the ratio of 1:1. PBMCs were isolated by using HiSepTM Lymphocyte separation media 1077 (LSM) (Himedia). LSM was aseptically transferred to a 15 ml clean centrifuge tube and overlaid with diluted blood to produce a clean interface between the two layers. The mixture was centrifuged at 1500 rpm for 30 min. at room temperature. PBMCs fraction from the interface was collected gently. Further centrifugation was done for washing the cells with phosphate buffer saline (PBS) (pH 7.4). Red blood cells lysis buffer was added to PBMCs pallet, mixed well and centrifuged. Supernatant was discarded and washing was repeated twice. Finally, PBMCs pellet was re-suspended and transferred to a sterile DEPC micro-centrifuge tube.
 
Total RNA extraction and quality determination
 
Total RNA was isolated using Trizol reagent (SRL). Trizol reagent and chloroform were added to PBMCs pellet and mixed gently followed by centrifugation at 12,000 rpm for 15 min at 4°C. The initial upper aqueous layer was aliquoted into sterile microcentrifuge tubes and equal volume of ice cold isopropanol was added, vortexed gently followed by centrifugation at 12,000 rpm for 12 min. 4°C. Pellet was washed twice with 75% ethanol by centrifugation at 7500 rpm for 5 min at 4°C. The isolated total RNA was stored in nuclease free water at 4°C (Qiagen, India). Purity of RNA was checked by using NanoDrop spectrophotometer (ARGLabs). 2 μl of dissolved RNA was added to find out the ratios of O.D. at 260 nm and 280 nm. Quality of RNA was assessed by electrophoresis on a denaturing agarose (1.5% w/v gel). 30 ml of 1.5% agarose gel was used along with 4 μl Ethidium bromide for staining of the bands. The RNA suspension was further processed for cDNA preparation.
 
Reverse Transcription and Quantitative Real-Time PCR
 
1 μg of total RNA were reversed transcribed to complementary DNA (cDNA) using cDNA synthesis kit (Fermentas) according to manufacture instructions. First strand cDNA was confirmed by amplification of GAPDH gene.
 
Primers
 
Primers were designed for HSP70 by the Integrated DNA Technologies (IDT) using Beacon software. The sequences and expected polymerase chain reaction (PCR) product lengths are shown in Table 1.



Quantitative Real-time PCR (qPCR) was performed with Invitrogen Sybr green ®Supermix kit. The qPCR conditions were as follows, initial denaturation at 95°C for 30 s, annealing at 58°C for 10 s and lastly extension at 72°C for 15 s for 35 cycles. No template control (NTC) was placed for gene quantification for checking the contamination in the reaction components other than the cDNA. After the run has ended, cycle threshold (Ct) values and amplification plot for all determined factors were acquired by using the “dissociation curve” method of the real time machine (Applied Biosystem, USA). The specificity of real time PCR products were checked by analysis of melting temperature (Tm) of the product obtained from dissociation or melting curve and by 1.5% agarose gel electrophoresis to verify the exact amplicon size. Relative expression of PCR product was determined by the equation suggested by Pfaffl (2001). The relative expression of HSP70 gene with GAPDH as reference gene was determined in Pandharpuri buffalo using Pfaffl method (2001) and with using Completely Randomized Design cited by Snedecor and Cochran (1980). The significance of analysis was determined at probability levels of 95 per cent (P<0.05).
 

Temperature-humidity index
 
Mean values of temperature humidity index (THI) during different seasons of the study period are presented in the Table 2.



During the study period, Mean values of THI  in summer, rainy and winter season was found to be 76.97±0.38, 73.32±0.38 and 68.81± 0.49. The highest THI was recorded during the peak summer season and lowest THI was recorded during the peak winter season. The THI recorded was significantly (P<0.05) higher in peak summer season as compared to the rainy and winter season. THI of summer, rainy and winter seasons differed significantly (P<0.05). Our results are consistent with the observation made by Baumgard et al., (2006) in dairy cattles, who reported that THI > 72 was the point at which a dairy cow starts to decrease productivity as in high temperature. In this experiment, THI of 72 can be achieved at moderate temperatures if relative humidity is high. Further, effect of heat on dairy cattle maintenance and milk production is heavily influenced by relative humidity.
 
Relative expression profile of HSP70 gene
 
The expression of HSP70 gene showed temperature sensitivity and seasonal variation. Relative expression of HSP70 gene varied markedly among different seasons (Fig 1).



Statistical analysis revealed a significant variation between different seasons (P<0.01) for all HSP70 gene expression. The expression of HSP70 gene was significantly ( P<0.05) higher in summer season as compared to the rainy and winter season and the relative mRNA expression of HSP70 was very low in winter season. Amplification plots and multicomponent plot for all the reactions of HSP70 gene expression was analyzed to check unspecific binding, primer dimer formation or secondary structure formation. Single peak in all experiments during qPCR signified that the primers were highly specific to the target and there was not any primer dimmers formation (Picture 1) and amplification plot for HSP70 gene and GAPDH expression depicted in Picture 2 and confirmation of qPCR products on 1.5% agarose gel electrophoresis showed in Picture 3.







The expression studies indicated that of HSP70 gene was up regulated during both summer and rainy seasons whereas HSP70 gene was observed to be down regulated during winter season. An increased mRNA expression in Pandharpuri buffalo during summer season was found to be more than 2 fold and 5 fold than in rainy and winter seasons respectively (Fig 1). During summer season, HSP70 gene expression was found statistically significant (P>0.05) with compared to rainy season.

In our study, it has been observed that the expression of HSP70 was significantly higher during the summer season as compared to the winter season in Pandharpuri buffalo, which might play an important role in thermal stress tolerance against harsh environmental conditions. Thermal stress induces differential gene expression and biochemical response at the cellular level. Our findings corroborates with the reports of Dangi et al., (2012) who reported that individuals exposed to stress elicit HSP response in the cells of various organs and higher expression of HSP70 at the tissue level provides protection to cells during chronic heat stress. Investigations in the present study are corroborated by Parmar et al., (2015) who reported HSP70 gene expression pattern in Sahiwal cows during different. Our research findings corroborate with the previous studies, where thermal stress induced rise in HSP70 expression in caprine PBMCs (Gupta et al., 2013), leukocytes of buffalo (Pawar et al., 2014), in thigh muscle and colon tissue of Ghungroo and Large White Yorkshire (Parkunan et al., 2015), Murrah buffalo (Mishra et al., (2010), dermal fibroblasts of cattle (Singh et al., 2014), in bovine ovary (Velazquez et al., 2011) and bull sperms (Rajoriya et al., 2014). Heat stress reduces the efficiency of animal production leading to multibillion dollar losses to global animal agriculture (Bernabucci et al., 2010). Further, Collier et al., (2008) reported that cellular responses to heat stress include activation of heat shock transcription factor 1. In the present study, increased expression pattern of HSP70 gene in summer season than that of rainy and winter seasons in Pandharpuri buffalo may be due to the fact that HSPs provide signaling to the immune system to encourage increased killing of pathogenic bacteria by neutrophils and macrophages and other innate immune cells against invading bacteria. The result obtained in this experiment confirms the results of earlier studies by Kapila et al., (2013) on HSP70 gene expression pattern in buffalo and suggested that heat stress condition in summer showed immediate induction in their expression after heat shock and remained up regulated after exposure to 42°C for one hour.

The findings of present study suggested that expression of HSP70 is influenced by the THI of the season and its up-regulation during high THI may play a crucial role in providing defence against thermal injury at cellular level. In our study, the expression of HSP70 gene in Pandharpuri buffalo was dependent on heat stress during summer season at 41.10 ±0.32°C and 86.00±2.06% RH under present finding. This heat stress may be due to high environmental temperature influence on HSP70 gene expression in these buffaloes. Pandharpuri buffalo being an indigenous breed of western Maharashtra, it is well developed defence mechanism involving the maintenance of high constitutive level of HSP70 gene in their PBMC as a mechanism for the protection against summer periods of extreme heat stress and hence, the present studies on Pandharpuri buffalo indicated that HSP70 gene induction takes place due to heat exposure during summer season.

The expression of HSP70 gene in Pandharpuri buffalo during summer seasons was found to be more than 2 fold and 5 fold than that of the rainy and winter seasons respectively. Hence, it is considered that HSP is most sensitive and reliable marker of heat stress in this breed. Further, it has been suggested that HSP70 gene influenced by THI values and shows possible involvement to ameliorate deleterious effect of thermal stress so as to maintain cellular integrity and homeostasis. HSP70 gene expression in these breed has significant implication in development of strategies to cope with challenges with climate change.

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