Introduction

With global warming, heat stress caused huge economic losses in the poultry industry [1]. According to the statistics, heat stress has caused losses of 125–165 million dollars to the poultry industry in the United States [2]. Heat stress is the sum of non-specific responses of chickens when they are under an excessively high temperature exceeding their thermoregulatory capacity, and can be roughly divided into acute and chronic heat stress according to the environmental temperature and exposure time. Under a high temperature, acute heat stress in chickens will be induced in a short time, causing high levels of tissue-damage and mortality [3]. Due to the influence of global warming, the occurrence frequency of extreme heat weather in various regions is increasing, which is short-lived but can still cause a high level of mortality. For example, in the summer of 2012, a large number of deaths of laying hens were caused by experiencing three periods of temperatures exceeding 38℃ in a short time [4]. Some regions, especially the tropical desert areas, have extremely high temperatures causing several problems for local poultry industry. For example, the highest temperature in the Cairo area of Egypt exceeded 44℃ for five years in the past decade (https://rp5.ru). Additionally, in modern poultry industry, temperature is controlled at the cost of a lot of energy, in which unexpected events such as power outages can’t be completely avoided. When power outages occurred, the environmental temperature would rise sharply. A reported example was that the environmental temperature was as much as 55 °C after a power outage and the mortality of population experienced such an event was as high as 98% [5].

In fact, a series of studies have proved that heat stress could cause injuries to multiple tissues in chickens. For example, heat stress could cause mitochondrial damage and produce excess reactive oxygen species (ROS) [6], thus resulting in the overwhelmed liver buffering system and oxidative injuries to enzymes, cellular lipids and mitochondrial membranes [7]. Heat stress could cause myocardial fiber rupture [8] and up-regulation of multiple genes involved in cardiac contraction [9]. Additionally, heat stress caused reduced layers and structural abnormalities of spermatogenic cells in chicken testis [1); three genes were transcribed from intronic regions of HSPA8, including LOC112530276, LOC112530277 and LOC112530278; one gene was overlapped with HSPH (Table 1).

Fig. 1
figure 1

Comparative analysis of five visceral tissues between the control and heat-stress groups. (A) Summary of differentially expressed gene numbers in five visceral tissues between the control and heat-stress groups. (B) Distribution of fold changes of differentially expressed genes between the control and heat-stress groups. (C) Significant pathways enriched by differentially expressed genes between the control and heat-stress groups. (D) The number of differentially expressed genes from five visceral tissues involved in significant pathways

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Fig. 2
figure 2

Comparative analysis of differentially expressed genes in different tissues between the control and heat-stress groups

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Table 1 The 13 overlap** differentially expressed genes in five visceral tissues between the control and heat-stress groups
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Then, all DEGs were used for KEGG pathway enrichment analysis. The results showed that a total of 23 pathways exhibited significance, and 13 of these pathways, including cytokine-cytokine receptor interaction, signal transduction, metabolic pathway, neuronal system, and calcium signalling pathway, were shared in all tissues (Fig. 1C). These results indicated that acute heat stress could cause inflammatory responses broadly in every visceral tissue, and signal transduction and the nervous system might play an important regulatory role in response to acute heat stress, and metabolic homeostasis might also be remodelled under acute heat stress.

Transcriptome data of multiple tissues reveals that the lung is the main target tissue under acute heat stress

As mentioned above, the DEG number in the lung was much higher than that in other tissues, and the fold changes of DEGs were significantly greater than that of other tissues. In the shared pathways, the number of DEGs involved in the GPCR signalling pathway from the lung was significantly higher than that of other tissues (Fig. 1D), indicating the signalling transduction in the lung under acute heat stress was more active. The number of DEGs enriched in neural ligand-receptor interaction in the lung was significantly higher than that in other tissues (Fig. 1D), indicating that the connection between lung and nervous system under acute heat stress was more intimate than that of other tissues. Besides, the number of DEGs in the lung related to the metabolic pathway was higher than that in other tissues (Fig. 1D), suggesting a greater effect on the lung metabolic homeostasis under acute heat stress. The number of DEGs in the heart and lung involved in cytokine-cytokine receptor interaction was higher than that in other tissues (Fig. 1D), indicating a more severe inflammatory response in the heart and lung under acute heat stress.

Pathological section reveals severe inflammation and emphysema in the lung under acute heat stress

Additionally, we set heat-stress and heat-death groups to examine the lung injuries under acute heat stress. The heat-death time of these individuals ranged from 49 to 75 min and we randomly selected three individuals for further analysis with their heat-death time from 53 to 75 min (Table S3). We found the right lung index of the heat-death group was significantly higher than that of the control group (P < 0.01) (Fig. 3A), and the ratio between right lung weight after drying at 37℃ for 48 h and fresh weight of right lung in the heat-death group was lower than that of the control group (P = 0.17) (Fig. 3B). Compared to the control group (Fig. 3C, F, I), we detected excessive inflammatory cell infiltration in the lung epithelial mucosa of the heat-stress group (Fig. 3D) and heat-death group (Fig. 3E), mild emphysema in heat-stress group (Fig. 3G), severe emphysema and alveolar rupture in heat-death group (Fig. 3H), pulmonary haemorrhage in both groups (Fig. 3J and K). In short, our results revealed that acute heat stress could damage the normal structure of the lung, and cause severe inflammatory response, emphysema and pulmonary hemorrhage.

Fig. 3
figure 3

Pathological findings in the lung under acute heat stress. (A) the index of right lung weight divided by body weight between the control and heat-death groups. (B) the dry weight of right lung after drying at 37℃ for 48 h divided by its fresh weight. The different letters indicate significant difference between groups. (C-K) the representative photographs of HE-stained lungs, and their scale is 200 μm. (C) (F) and (I) show that pathological lung sections in the control group were normal. Severe inflammation was observed in the heat-stress group (D) and heat-death group (E). Emphysema was observed in the heat-stress group (G) and heat-death group (H), and alveolar ruptures were observed in the heat-death group (H). Pulmonary hemorrhage was observed in the heat-stress group (J) and heat-death group (K)

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Evidence reveals the potential mechanism of inflammation in the lung under acute heat stress

A total of 10 genes involved in cytokine-cytokine receptor interaction were differentially expressed in the heat-stress group, including BMP10, IL20RA, GDF2, GDF15, THPO, CNTFR, INHBA, CCR6, LIFR and BMP3 (Figure S3). In these 10 genes, all genes were up-regulated significantly in the heat-stress group except CCR6, LIFR and BMP3. CCR6, LIFR, IL20RA and CNTFR were selected to examine their expression in the heat-stress and heat-death groups because they have been reported to be pro-inflammatory genes. Besides, the other 3 genes, including FURIN, AvBD9 and BAIAP9, were also targeted for validation because they were reported to be related to the innate immune system. In these 7 genes used for validation, the expression trends of five genes in transcriptome data were consistent with the results of qRT-PCR (Table S2). Transcriptome data revealed that CCR6 and LIFR were significantly down-regulated with low-fold changes in the heat-stress group (Table S2). The results of qRT-PCR revealed their up-regulation, suggesting that we should be particularly careful about DEGs with low fold change when using the transcriptome data. All these genes for validation exhibited up-regulations in the heat-stress group, including 5 pro-inflammatory genes and 2 anti-inflammatory genes (Fig. 4). The 4 pro-inflammatory genes, including CNTFR, CCR6, LIFR and FURIN, were up-regulated both in the heat-stress and heat-death groups, with higher fold changes in the heat-death group than in the heat-stress group. The pro-inflammatory gene IL20RA also exhibited up-regulation in both groups, but its fold change in the heat-death group was lower than that in the heat-stress group. The anti-inflammatory gene AvBD9 exhibited significant up-regulation in the heat-stress group but down-regulation in the heat-death group with potential significance. Another anti-inflammatory gene BAIAP2 exhibited insignificant up-regulation in the heat-stress and heat-death groups.

Fig. 4
figure 4

Relative expression level of several inflammatory genes by qRT-PCR. Among these genes, CNTFR, CCR6, LIFR, FURIN and IL20RA belong to pro-inflammatory genes, and AvBD9 and BAIAP2 belong to anti-inflammatory genes. Expression levels were normalized against that of the GAPDH gene. Values are means (n = 3) and error bars represent the standard deviation. Different letters above the bars indicate a significant difference in different groups

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Discussion

Chicken has no sweat glands and is covered with feathers, thus chicken has a higher temperature than mammals. Thus, chickens are more susceptible to heat stress than mammals when under high temperatures. Many previous studies have found that heat stress could induce multiple tissue injuries [6, 7, Total RNA extraction, RNA-Seq and DEGs identification

To examine the tissue injuries under heat stress, 3 samples of the control and heat-stress groups were randomly selected to perform RNA-Seq. Total RNA was extracted by TRIzol reagent based on the manufacturer’s instruction, whose quality was examined by NanoDrop and Agilent 2100. The mRNA was isolated by Oligo (dT)-attached magnetic beads, and randomly fragmented in a fragmentation buffer. First-strand cDNA was synthesized with the fragmented mRNA as a template and random hexamers as primers, followed by second-strand synthesis with the addition of PCR buffer, dNTPs, RNase H and DNA polymerase I, and cDNA was purified with AMPure XP beads. Double-strand cDNA was subjected to the end repair, and a cDNA library was obtained by certain rounds of PCR. A total of 36 libraries were sequenced on Illumina HiSeq 2500 platform (Illumina Inc., San Diego, USA) according to Illumina’s RNA-Seq instructions to obtain 150-bp paired-end reads for further study.

The low-quality reads and adapters were removed with Trimmomatic-0.39 (Bolger, et al., 2014). High-quality reads were aligned to the reference genome (GRCg6a) by hisat2 [34], and gene count was generated by HTSeq [35]. Differentially expressed genes (DEGs) were detected by DESeq2 package [36]. The genes with P-value < 0.05 and |Log2 (fold change)| >1 were defined as DEGs. DEGs were used to examine their enriched pathways with KOBAS 3.0 [37]. For the most important pathways, the raw images were obtained from KEGG [38,39,40], and the related significant gene information was highlighted using Pathview[41].

Histopathological examination

The samples fixed in 4% formaldehyde solution (National Pharmaceutical Group Chemical Reagent Co., Ltd., China) were paraffin-embedded, and 3 μm-thick sections of the paraffin embedding samples were obtained and used for hematoxylin-eosin (H&E) staining. We examined all the histological slides with an Olympus light microscope and digitized images with a Nikon DS-U3 camera (Olympus Corporation, Tokyo, Japan) control unit connected to a Nikon Eclipse CI upright optical microscope.

Quantitative real-time PCR (qRT-PCR)

We performed qRT-PCR technology with 3 samples of the control, heat-stress and heat-death groups to quantify the expressions of key genes and verify the reliability of RNA-Seq. Total RNA from every sample was converted into cDNA using HiScript Reverse Transcriptase (Vazyme Biotech Co., Ltd., Nan**g, China), and cDNA was used for qRT-PCR with GAPDH as the internal control gene. qRT-PCR was performed with SYBR Green I kit (Vazyme Biotech Co., Ltd., Nan**g, China), with 1.6ul cDNA and 0.2ul of forward and reversed primers in a final volume of 10ul. The samples were centrifuged briefly and run on the PCR machine using the program ( 95℃ for 5 min, 95℃ for 30s, 58℃ for 30s, 72℃ for 30s, 72℃ for 5 min and 25℃ for 1 min) in triplicate. The relative gene expression levels were determined using the 2–ΔΔCt method, the mean value of three technological replications was used to represent the expression of the individual and three biological replications of different groups was used to estimate the difference in different groups. We used LSD.test function to estimate the difference in gene expression in different groups. The primer sequences of genes for validation are represented in Table 2.

Table 2 Primer sequences used in qRT-PCR
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