Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (<i>Bubalus Bubalis</i>)

<div><p>The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several <i>in-vitro</i> cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family <i>viz</i>., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes <i>viz</i>., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several <i>HSP</i> (<i>HSP40</i>, <i>HSP60</i>, <i>HSP70</i>, <i>HSP90</i>, and <i>HSPB1</i>), apoptotic (<i>Bax</i> and <i>Bcl2</i>), immune (<i>IL6</i>, <i>TNFα and NF-kβ</i>) and oxidative stress (<i>GPX1</i> and <i>DUSP1</i>) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load.</p></div

Immunocytostaining for expression of cytoskeletal markers in buffalo MECs A) Fluorescent image of Cytokeratin 18 showing intermediate filaments running in bundles with interconnections between cells, B) Light image of Cytokeratin 18, C) Fluorescent image of buffalo MECs stained for Vimentin showing filament degradation, D) Light image of Vimentin.

<p>Immunocytostaining for expression of cytoskeletal markers in buffalo MECs A) Fluorescent image of Cytokeratin 18 showing intermediate filaments running in bundles with interconnections between cells, B) Light image of Cytokeratin 18, C) Fluorescent image of buffalo MECs stained for Vimentin showing filament degradation, D) Light image of Vimentin.</p

Venn diagram showing number of DEG at 30 min, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h with respect to unstressed (CTR) at fold change > = 3.0.

<p>Venn diagram showing number of DEG at 30 min, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h with respect to unstressed (CTR) at fold change > = 3.0.</p

Cell viability pattern in heat stressed buffalo MECs using trypan blue dye exclusion method.

<p>CTR-unstressed (control) MECs.</p

List of top 50 genes down-regulated in heat stressed buffalo MECs (Fold change > = 3.0).

<p>List of top 50 genes down-regulated in heat stressed buffalo MECs (Fold change > = 3.0).</p

Correction: Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (<i>Bubalus Bubalis</i>)

Correction: Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (<i>Bubalus Bubalis</i>

Cytokines & Inflammatory response pathway; shows the significant affected genes (yellow color) across all time points post heat stress.

<p>Cytokines & Inflammatory response pathway; shows the significant affected genes (yellow color) across all time points post heat stress.</p

List of significant GO terms obtained from REVIGO analysis.

<p>List of significant GO terms obtained from REVIGO analysis.</p

Evaluation of cellular proliferation in unstressed (control) and heat stressed buffalo MECs using MTT assay.

<p>CTR-unstressed (control); TRT- heat stress treated MECs.</p

Bar graph showing up- and down-regulated genes at each time point (Fold change 3) in heat stressed buffalo MECs.

<p>Bar graph showing up- and down-regulated genes at each time point (Fold change 3) in heat stressed buffalo MECs.</p