Heat shock increases levels of reactive oxygen species,autophagy and apoptosis

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5 °C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39–41 °C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5 °C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42 °C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (<60 min) at 42 °C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42 °C increased levels of ROS. Increased levels of LC3 and AVOs at 42 °C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42 °C (<60 min) was mediated by ROS. Conversely, heat shock at 42 °C for longer times (1?3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40 °C, were resistant to activation of apoptosis at 42 °C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42 °C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.     

Synergistic induction of apoptosis and caspase-independent autophagic cell death by a combination of nitroxide Tempo and heat shock in human leukemia U937 cells

We have shown that heat stress or a superoxide dismutase mimic nitroxide, Tempo, induces apoptosis, while their combination causes nonapoptotic cell death; however, the underlying mechanism for this switch remains unclear. Here we identified for the first time that 10 mM Tempo present during heating at 44°C for 30 min rapidly induced autophagy in U937 leukemic cells in spite of Bax activation and mitochondrial outer membrane (MOM) permeabilization. This co-treatment inhibited the processing of heat-activated procaspases-2, -8, -9 and -3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy. The inactivation of caspases, a key event, could result from oxidation of active-site-CysSH of all caspases by a prooxidant oxo-ammonium cation, an intermediate derived Tempo during dismutation of heat-induced superoxide anion. In addition, the co-treatment caused mitochondrial calcium overloads, the mitochondrial inner membrane permeabilization, profound mitochondrial dysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction of autophagy. These autophagic cells underwent propidium iodide-positive necrosis in a delayed fashion, leading to the complete proliferative inhibition. Remarkably, ruthenium red and BAPTA, which interfere with mitochondrial calcium uptake, facilitated autophagic necrotic death. Cyclosporin A, which binds to cyclophilin D, had a similar necrotic effect. 3-Methyladenine facilitated the necrosis of autophagic cells. In contrast, 5 mM Tempo-44°C/10 min or 44°C/30 min induced Bax-mediated MOM permeabilization and caspase-dependent apoptosis more potently than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.     

Cloning and differential expression of three heat shock protein genes associated with thermal stress from the wolf spider Pardosa pseudoannulata (Araneae: Lycosidae)

Pardosa pseudoannulata is the main predatory natural enemy of crop pests in a paddy ecosystem. When P. pseudoannulata is exposed to unfavorable temperature conditions, the response of heat shock proteins could resist the damage, and is therefore, conducive to the organism’s rapid adaptation to the surrounding stress environment. In this study, we explored the roles of hsp70 and hsp90 genes in response to heat stress, using the rapid amplification of cDNA ends technique and cloned full-length cDNAs of Pphsp70, Pphsp83, and Pphsp90. The mRNA expression levels of the three genes under different temperature stresses (25, 28, 31, 34, 37, 40, and 43 °C) and with different duration stresses (4, 8, 12, 16, and 20 h) were analyzed by quantitative real-time polymerase chain reaction. The full-length cDNA of Pphsp70, Pphsp83, and Pphsp90 was 2331 base pair (bp), 2466 bp, and 2663 bp, respectively. Phylogenetic analysis of amino acid sequences of Pphsp70, Pphsp83, and Pphsp90 showed that the sequences had high homology with that of other spiders. The mRNA expression of all three genes was extremely significantly up-regulated at 43 °C. Moreover at 43 °C, the expression of all three genes in both female and male spiders at the duration of 4 h was the highest compared to that of other stress duration groups. Therefore, it can be inferred that the three genes of P. pseudoannulata play a crucial protective role in resistance in a high-temperature environment.     

Effect of salicylic acid on the development of induced Thermotolerance and induction of heat shock protein synthesis in the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cell culture

Salicylic acid (SA) could be involved in the development of tolerance to abiotic stresses, to heat shock in particular. Under normal conditions (26°C), treatment with SA improved the tolerance of heterotrophic Arabidopsis thaliana (L.) Heynh culture to severe heat shock (50°C). Under mild heat shock (37°C) inducing the development of thermotolerance, the presence of SA, in contrast, reduced the capability of arabidopsis cells to tolerate high temperature (50°C) and simultaneously suppressed induction of HSP synthesis (Hsp101 and Hsp17.6) important for the development of induced thermotolerance. Since SA suppressed cell respiration and activated the alternative pathway of electron transport, SA is supposed, by modulating mitochondria functions, to be an endogenous regulator of plant stress gene expression.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Does increased heat resistance result in higher susceptibility to predation? A test using Drosophila melanogaster selection and hardening

Heat resistance of ectotherms can be increased both by plasticity and evolution, but these effects may have trade‐offs resulting from biotic interactions. Here, we test for predation costs in Drosophila melanogaster populations with altered heat resistance produced by adult hardening and directional selection for increased heat resistance. In addition, we also tested for genetic trade‐offs by testing heat resistance in lines that have evolved under increased predation risk. We show that while 35/37 °C hardening increases heat resistance as expected, it does not increase predation risk from jumping spiders or mantids; in fact, there was an indication that survival may have increased under predation following a triple 37 °C compared to a single 35 °C hardening treatment. Flies that survived a 39 °C selection cycle showed lower survival under predation, suggesting a predation cost of exposure to a more severe heat stress. There was, however, no correlated response to selection because survival did not differ between control and selected lines after selection was relaxed for one or two generations. In addition, lines selected for increased predation risk did not differ in heat resistance. Our findings suggest independent evolutionary responses to predation and heat as measured in laboratory assays, and no costs of heat hardening on susceptibility to predation.     

Trehalose protects wine yeast against oxidation under thermal stress

Accumulation of trehalose in yeasts has been suggested to be an important mechanism of tolerance against adverse stress conditions, particularly in thermal stress. However, under thermal stress, it is not clear if the mechanism of protection is related to its antioxidant role. In this study, a newly isolated wine yeast Saccharomyces cerevisia was used to examine the protective effect of trehalose against oxidation during thermal stress treatment. Cells were treated either with a mild heat treatment at 37°C (which leads to trehalose accumulation) or with a 50 mM trehalose solution and then exposed to a high temperature of 53°C. According to our results, mild heat treatment at 37°C and trehalose addition which promote accumulation of trehalose significantly increased cell survival upon exposure to thermal stress at 53°C which seems to be correlated with decrease in reactive oxygen species levels and lipid peroxidation. Trehalose could protect yeast from oxidative injuries under thermal stress.     

Mild hypothermia in rat with acute myocardial ischaemia-reperfusion injury complicating severe sepsis

Myocardial ischemia-reperfusion injury (MIRI) with concurrent severe sepsis has led to substantial mortality. Mild hypothermia (MHT) has been proved to have a therapeutic effect in either MIRI or severe sepsis, which suggests it might be beneficial for MIRI complicating severe sepsis. In this study, Sprague-Dawley rats with MIRI complicating severe sepsis were allotted in either MHT (33 ± 0.5°C) group or normothermia (NT, 37 ± 0.5°C) group; as control, rats receiving sham surgery and normal saline were kept at NT. After 2h of temperature maintenance, blood and heart tissue were acquired for detections. Lactate dehydrogenase (LDH) and MB isoenzyme of creatine kinase (CK-MB) in blood, triphenyl tetrazolium chloride and Evans blue staining, hematoxylin and eosin staining for myocardium were employed to detect myocardial damage. Tumor necrosis factor (TNF)-α and caspase-3 was performed by immunohistochemistry to exam myocardial inflammation and apoptosis. Detection of NADPH oxidase (NOX) 2 was for myocardial oxidative stress. In MHT group, systolic blood pressure was improved significantly compared with NT group. Myocardial infarct size, morphological change, LDH and CK-MB levels were attenuated compared to NT group. Moreover, less expressions of TNF-α, caspase-3 and NOX2 in MHT group were presented compared with NT group. MHT showed cardioprotection by improving cardiac dysfunction, reducing myocardial infarct size and attenuating myocardial injury, inflammation, apoptosis and oxidative stress.     

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with K_D ~ 1 μM. Proteins co-localize in the soluble fraction of the cell at 30–37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpA_His6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30–37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.     

Heat stress disrupts intestinal stem cell migration and differentiation along the crypt–villus axis through FAK signaling

During heat stress (HS), the intestinal epithelium suffers damage due to imbalance of tissue homeostasis. However, the specific mechanism by which intestinal stem cells (ISCs) migrate and differentiate along the crypt–villus axis to heal lesions upon insult is unclear. In our study, C57BL/6 mice and IPEC-J2 cells were subjected to normal ambient conditions (25 °C for 7 days in vivo and 37 °C for 18 h in vitro) or 41 °C. The results showed that HS impaired intestinal morphology and barrier function. The numbers of ISCs (SOX9⁺ cells), mitotic cells (PCNA⁺ cells), and differentiated cells (Paneth cells marked by lysozyme, absorptive cells marked by Villin, goblet cells marked by Mucin2, enteroendocrine cells marked by Chromogranin A, and tuft cells marked by DCAMKL1) were reduced under high temperature. Importantly, BrdU incorporation confirmed the decreased migration ability of jejunal epithelial cells exposed to 41 °C. Furthermore, intestinal organoids (IOs) expanded from jejunal crypt cells in the HS group exhibited greater growth disadvantages. Mechanistically, the occurrence of these phenotypes was accompanied by FAK/paxillin/F-actin signaling disruption in the jejunum. The fact that the FAK agonist ZINC40099027 reversed the HS-triggered inhibition of IPEC-J2 cell differentiation and migration further confirmed the dominant role of FAK in response to high-temperature conditions. Overall, the present investigation is the first to reveal a major role of FAK/paxillin/F-actin signaling in HS-induced ISC migration and differentiation along the crypt–villus axis, which indicates a new therapeutic target for intestinal epithelial regeneration after heat injuries.     

Evaporative heat loss in Bos taurus: Do different cattle breeds cope with heat stress in the same way?

The aim of this study was to compare two Portuguese (Alentejana and Mertolenga) and two exotic (Frisian and Limousine) cattle breeds in terms of the relationship between the increase in ambient temperature and the responses of the evaporative heat loss pathways and the effects on homeothermy. In the experiment, six heifers of the Alentejana, Frisian, and Mertolenga breeds and four heifers of the Limousine breed were used. The animals were placed in four temperature levels, the first one under thermoneutral conditions and the other ones with increase levels of thermal stress. When submitted to severe heat stress, the Frisian developed high thermal tachypnea (125 mov/min) and moderate sweating rates (117 g m⁻² h⁻¹), which did not prevent an increase in the rectal temperature (from 38.4 °C to 40.0 °C). Moderate increases in rectal temperature were observed in the Alentejana (from 38.8 °C to 39.4 °C) and Limousine (from 38.6 °C to 39.4 °C), especially in the period of highest heat stress. The Limousine showed moderate levels of tachypnea (101 mov/min) while showing the lowest sweating rates. The Alentejana showed significant increases in sweating rate (156 g m⁻² h⁻¹) that played a major role in homeothermy. The Mertolenga showed a superior stability of body temperature, even in the period of highest heat stress (from 38.5 °C to 39.1 °C). Uncommonly, the maintenance of homeothermy during moderate heat stress was achieved primarily by intense tachypnea (122 mov/min). The sweating rate remained abnormally low under conditions of moderate heat stress, rising significantly (110 g m⁻² h⁻¹) without evidence of stabilization, only when tendency for heat storage occurred. This unusual response of the evaporative heat loss pathways infers a different thermoregulatory strategy, suggesting a different adaptation to semi-arid environment and strong association with water metabolism.     

Antioxidant responses of the pest natural enemy Hylyphantes graminicola (Araneae: Linyphiidae) exposed to short-term heat stress

Temperature is a critical abiotic factor that causes physiological changes in arthropods. However, little is known about the effect of heat stress on the antioxidant responses of Araneae species. Hylyphantes graminicola is a dominant predator in many cropping systems in China. In the present study, the effect of short-term heat stress (36, 38, 40 or 42 °C) on the reactive oxygen species (ROS) levels, the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], peroxidases [POD] and glutathione-S-transferases GST]), total antioxidant capacity (TAC), malondialdehyde (MDA) concentrations and survival of H. graminicola spiderlings and adults were investigated. The results showed that H. graminicola adults had a significantly higher survival rate compared to spiderlings at 40 °C. The heat stress increased ROS contents in H. graminicola. The SOD, CAT, POD and GST activities increased in spiderlings and adults under heat stress. These data suggest a defensive function for these enzymes in alleviating oxidative damage. Specifically, SOD plays a key role in reducing the high level of superoxide radicals in spiderlings and adults. Moreover, the POD and CAT capabilities for scavenging H₂O₂ in spiderlings were similar, and CAT may play a more important role than POD in scavenging H₂O₂ in adults at 42 °C. The spiderling TAC increased significantly at 40 and 42 °C, and the adult TAC was stable at 36–40 °C but decreased at 42 °C. These data suggest that TAC was insufficient in H. graminicola adults under more severe stress conditions. These results further our understanding of the physiological response of Araneae species exposed to heat stress.     

Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos

Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12 °C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2 h followed by a 2 h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6 °C TS for 1 h every 6 days or +9 °C TS for 1 h every 6 days. Following a 2 h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12 °C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1 h TS and 2 h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development.     

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31°C for 16 h), a conventional in vitro heat shock (43°C for 30 min) or control conditions (37°C), then harvested immediately or allowed to recover for 3 h at 37°C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.     

Host autophagic degradation and associated symbiont loss in response to heat stress in the symbiotic anemone,Aiptasia pallida

Coral bleaching involves the loss of essential photosynthetic dinoflagellates (Symbiodinium sp.) from host gastrodermal cells in response to temperature or light stress. Although numerous potential cellular bleaching mechanisms have been proposed, there remains much uncertainty regarding which cellular events occur during early breakdown of the host–dinoflagellate symbiosis. In this study, transmission electron microscopy was used to conduct a detailed examination of symbiotic tissues of the tropical anemone Aiptasia pallida during early stages of host stress. Bleaching was induced by exposing specimens to a stress treatment of 32.5±0.5°C at 140±7 μ mol photons m^?2 s^?1 light intensity for 12 h, followed by 12 h at 24±1°C in darkness, repeated over a 48 h period. Ultrastructural examinations revealed numerous dense autophagic structures and associated cellular degradation in tentacle tissues after ~12 h of the stress treatment. Anemones treated with rapamycin, a known autophagy inducer, exhibited the same ultrastructural characteristics as heat‐stressed tissues, confirming that the structures observed during heat stress treatment were autophagic. In addition, symbionts appeared to be expelled from host cells via an apocrine‐like detachment mechanism from the apical ends of autophagic gastrodermal cells. This study provides the first ultrastructural evidence of host autophagic degradation during thermal stress in a cnidarian system and also supports earlier suggestions that autophagy is an active cellular mechanism during early stages of bleaching.     

UBQLN2 undergoes a reversible temperature-induced conformational switch that regulates binding with HSPA1B: ALS/FTD mutations cripple the switch but do not destroy HSPA1B binding

Here we present evidence, based on alterations of its intrinsic tryptophan fluorescence, that UBQLN2 protein undergoes a conformational switch when the temperature is raised from 37 °C to 42 °C. The switch is reset on restoration of the temperature. We speculate that the switch regulates UBQLN2 function in the heat shock response because elevation of the temperature from 37 °C to 42 °C dramatically increased in vitro binding between UBQLN2 and HSPA1B. Furthermore, restoration of the temperature to 37 °C decreased HSPA1B binding. By comparison to wild type (WT) UBQLN2, we found that all five ALS/FTD mutant UBQLN2 proteins we examined had attenuated alterations in tryptophan fluorescence when shifted to 42 °C, suggesting that the conformational switch is crippled in the mutants. Paradoxically, all five mutants bound similar amounts of HSPA1B compared to WT UBQLN2 protein at 42 °C, suggesting that either the conformational switch is not instrumental for HSPA1B binding, or that, although damaged, it is still functional. Comparison of the poly-ubiquitin chain binding revealed that WT UBQLN2 binds more avidly with K63 than with K48 chains. The avidity may explain the involvement of UBQLN2 in autophagy and cell signaling. Consistent with its function in autophagy, we found UBQLN2 binds directly with LC3, the autophagosomal-specific membrane-tethered protein. Finally, we provide evidence that WT UBQLN2 can homodimerize, and heterodimerize with WT UBQLN1. We show that ALS mutant P497S-UBQLN2 protein can oligomerize with either WT UBQLN1 or 2, providing a possible mechanism for how mutant UBQLN2 proteins could bind and inactivate UBQLN proteins, causing loss of function.     

Effect of in vitro zinc supplementation on HSPs expression and Interleukin 10 production in heat treated peripheral blood mononuclear cells of transition Sahiwal and Karan Fries cows

The changing climatic scenario with apprehended rise in global temperature is likely to affect the livestock adversely vis-à-vis production and reproduction. This has prompted more focus in addressing the unfavorable effects of thermal stress in livestock system. Presuming that the trace element zinc is indispensible for cellular antioxidant system and immune function, the present study was designed to investigate the effect of zinc treatment on heat stress alleviation and immune modulation in peripheral blood mononuclear cells (PBMC) of indigenous and crossbred transition cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were selected for the experiment. The blood samples were collected at −21, 0 and +21 days in relation to expected date of calving. The experiment was carried out in vitro after isolating PBMC from whole blood. The 48 h cultured PBMC were subjected to assorted levels of exposures viz. 37 °C, 42 °C to impose heat stress and 42 °C+zinc to alleviate heat stress and modulate immunity. The PBMC viability was 86%, 69% and 78%, respectively. The mRNA expression of heat shock proteins (HSP 40, 70 and 90α) and Interleukin-10 (IL-10) production varied between the two breeds vis-à-vis days and levels of exposure. The mRNA expression of HSP40 and HSP70 was significantly (P<0.05) higher in Karan Fries than the Sahiwal cows. Both the breeds showed maximum expression of HSP on the day of parturition, more so in KF than Sahiwal. There was a significant (P<0.05) difference in the HSP mRNA expression at different levels of exposure. Zinc treatment to heat stressed PBMC caused a significant (P<0.05) down regulation of HSP. For immune status, anti-inflammatory cytokine, IL-10 in the culture supernatant was accessed. The IL-10 was significantly (P<0.05) higher in Karan Fries (168.18±14.09 pg/ml) than the Sahiwal cows (147.24±11.82 pg/ml). The IL-10 concentration was highest on the day of calving. Zinc treatment reduced the IL-10 concentration. From the study, it could be concluded that the zinc supplementation in heat stressed PBMC can ameliorate thermal stress and modulate immune response which can act as a model for reducing heat stress during the periparturient period in tropical livestock.