The Molecular Mechanism Study Of Ostrich Chick Thymus In Response To Boron Stimulation Based On The RNA-Seq Technology

African ostriches have high economic values and biological importance. Ostrich chicks had a high mortality rate ranging from 10 to 50% in livestock farms, especially within the first 3 months of age, which usually resulted from exposure to various stressors, infections or diseases. In addition, ostriches are fast-growing birds, and the birds from the same batch often differ greatly in size, particularly during the first 3 months of age. One of the most important measures of ostrich chicks is growth development, which is usually balanced against the immune function. The thymus is an important lymphoid and immune organ, which grows considerably immediately after birth in response to postnatal antigen stimulation and is crucially involved in the differentiation of T lymphocytes. Boron is a kind of essential trace element in animal’s body, which has many biological functions. Boron could regulate the immune response in animals’ body, the appropriate boron dosage could promote the immune organ growth and development. In the present study, we chose the 1 day-old ostrich chicks as the experimental animals, and added the dose gradient boron(0, 40, 80, 160, 320, and 640 mg/L) in the drinking water, after 90 days, the ostrich chicks were killed and the thymuses were removed to do the following experiments. In this study, we would like to detect the regulation function of boron on ostrich immune organs, and to clarify the mechanism of action of boron on the immune response in ostrich thymuses. 1. The effect of boron on gene expression related to growth and development of ostrich chick thymusFoxn1 is necessary for the development of thymic epithelial cells, BMP2 and BMP4 which are regulated in T cells growth and development. All of these genes are important for maintaining the thymus homeostasis. The relationship between boron and thymus development, and effects of boron on the expression of Foxn1, BMP2 and BMP4 were investigated in the present study. The histological changes in thymus were observed by HE staining. Ostrich Foxn1 was sequenced by Race PCR method. The expression of Foxn1 was analyzed by immunohistochemistry and western blot, the expression of BMP2 and BMP4 were analyzed by immunofluorescence technique. TUNEL technique was used to label the apoptotic cells in ostrich thymus. The expression of cleaved caspase-3 was detected by immunohistochemistry. The mRNA levels of Foxn1,BMP2,and BMP4 were detected by qRT-PCR.The results are as following:(1) The effect of boron on the morphological structure of the ostrich chick thymus was detected by HE staining, as compared with control group, thymic cortex was histologically characterized by karyorrhexis of lymphocytes with active phagocytosis by macrophages, which created a prominent “starry-sky” appearance of the thymic cortex in boric acid supplemented groups, the lymphocytes inside the thymus lobules were reduced and exhausted, and thymus lobules were indistinct, especially in B320 group and B640 groups, whereas, thymic cortex was normal in group III and group IV as compared with control group. The structure of thymus was changed, as the numbers of thymic epithelial cells decreased and the cortical and medullary compartments were disrupted in B640 group.(2) To elucidate the effect of boron on cell apoptosis in thymus, we detected the apoptosis by TUNEL assay and immunohistochemistry. TUNEL-positive cells were mainly localized in the thymic medulla and faintly in thymic cortex. The control, B80, and B160 groups had very low levels of TUNEL-positive cells, while increased TUNEL staining in the thymus confirmed cell apoptosis with increasing dosage of boron.(3) The cleaved caspase-3 positive signals were markedly increased in B320 and B640 groups, high-dose of boron could activate the cleaved caspase-3 and induce the apoptosis in ostrich thymuses.(4) The sequence of the conservative middle segment of ostrich Foxn1 was 1477 bp by using the degenerate primer, and the sequences of the 5’ RACE and 3’ RACE of ostrich Foxn1 were 384 bp and 1050 bp by RACE-PCR analysis. The full-length sequence and encoded protein of ostrich Foxn1 were 2736 bp and 654 aa. Ostrich Foxn1 is highly conserved as compared with other species, which shared a 92.1%, 91.1%, 9 0.8%, 89.6%, 88.1%, and 83.5% identity with the peregrine falcon, saker falcon, budgerigar, rock pigeon, mallard and chicken, respectively.(5) Foxn1-positive cells were mainly distributed in thymic medulla, faintly in thymic cortex. Foxn1-positive signals were increased markedly in the B80 group, whereas, Foxn1-positive signals were significantly decreased in the B640 group.(6) There was a boron dose-dependent effect of the mRNA levels of BMP2 and BMP4 in ostrich thymuses. The mRNA levels were significantly increased in B80 group, whereas, the mRNA levels were decreased markedly in B640 group.These results demonstrated that appropriate-dose of boron played a protective role in the thymus development, while high-dose of boron could damage the organ or even produced toxic effect. The high-dose boron induced thymus structure disruption, thymic cells apoptosis, and enhanced the expression of cleaved caspase-3, finally inhibited the growth and development of ostrich thymus. 80 mg/L dose of boron moderately increased the mRNA and protein levels of BMP2, BMP4, and Foxn1 in ostrich chick thymus. These factors could promote the differentiation and development of thymic epithelial cells, and involved in the T cells growth and development, enhance the ostrich chick body immunity. However, the high-dose of boron not only significantly inhibited the mRNA and protein levels of BMP2, BMP4, and Foxn1, but also resulted in the destruction and degeneration of ostrich thymus histological structure, as well as disruption in the cortical and medullary compartments, ultimately leading to the inhibition of the growth and development of thymic epithelial cells. 2. RNA-Seq analysis on ostrich chick thymus response to boronThe objective of this study was to construct a RNA-Seq tag profile to identify genes and pathways potentially related to immunity in the ostrich chick. Boron exposure induces an immune response in ostrich, but the underlying mechanism is not completely clear yet. Thus, transcriptomic data for ostriches are needed as an important resource to identify genes and to gain insights into the function of boron on the immune response of thymus. RNASeq analysis was performed using the Illumina technique to investigate differentially expressed genes in ostrich thymuses treated with different boric acid concentrations(0, 80, and 640 mg/L).The results of this study are shown below:(1) The raw reads in each library(control, B80, and B640) was 3.22, 3.43 and 3.20 billion. After filtering the low quality tags, the total number of clean reads in each library was 3.175, 3.380 and 3.158 billion, which correspond to 98.61%, 98.60%, 98.65% of raw reads, respectively.(2) In the control, B80 and B640 libraries, 11.93, 12.85, and 11.41 million reads, respectively,(which correspond to 75.14%, 76.04%, and 72.28%, respectively) were uniquely mapped to the ostrich genome.(3) Our results showed that 72%, 71% and 69% of the expressed genes had exhibited a sequencing coverage of 90-100% in the control, B80 and B640 libraries, respectively.(4) The variations in gene expression were analyzed by comparing the control with B640, B80 with B640, and again the control with B80 groups. A total of 2,044 genes, including 1,816 down-regulated and 228 up-regulated genes, were identified in the B640 group compared to control group. The total quantity of differentially expressed genes was 1,085, of which 863 were down-regulated and 222 were up-regulated between in the B80 and the B640 groups. There were 902 genes, of which 593 were down-regulated and 309 were upregulated in the B80 group compared with the control group.(5) We classified 6,260 DEGs into 7 profiles, of which 3, 807 were clustered into three profiles(P-value < 0.05). Profiles 0, 1, 3 exhibited significant clustering trends. Profile 0 represented genes for which the expression consistently decreased with increasing boron concentrations. Profile 1 represented the genes for which the expression initially decreased and then maintained invariability as the boron concentrations increased. Profile 3 represent genes for which expression were initially stable but then decreased.Profiles 0, 1 and 3 contained 1290, 1030, and 1487 differentially expressed genes, respectively.(6) The KEGG enrichment analysis revealed that the significantly enriched inflammation- and immunity-related pathways associated with the differentially expressed genes were significantly enriched in “Cytokinecytokine receptor interaction”, “Pathways in cancer”, “Calcium signaling pathway”, “MAPK signaling pathway”, and “Regulation of actin cytoskeleton”.(7) The KEGG enrichment analysis between the paired samples(control-B80, B80-B640, control-B640) showed that boron could also regulate the Toll like receptor signaling pathway, B/T cells receptor signaling pathways and apoptosis pathway.Results from this study provide comprehensive gene expression information at the transcriptional levels that enhanced our understanding of the molecular mechanisms of boron on the ostrich immune system. These results demonstrated that boron could regulate the ostrich thymus immunity mainly through the MAPK signaling pathway, Calcium signaling pathway, B/T cells receptor signaling pathways, apoptosis pathway and pathway in cancer. 3. The regulation effect of boron on immunity, inflammation and growth development in ostrich chick(1) The regulation effect of boron on MAPK signaling pathway: In the B80 group, the expression of specific genes were increased, whereas others remained unchanged compared to the control group. 24 out of the 27 differentially expressed genes exhibited downregulated trends, of which 3 exhibited upregulated trends in the B640 group. Boron could regulate the Ras/ERK, JNK, and p38 MAPK signaling pathway, however, we did not observe any genes that were differentially expressed in the ERK5 signaling pathway. The western blot results showed that the ostrich thymuses in the 80 mg/L of boric acid group exhibited increased expression levels of p-ERK, p-JNK and p-p38 compared to the control group, the expression levels of p-ERK, p-JNK and p-p38 were gradually attenuated in a boron dose-dependent manner after 80 mg/L, reaching the lowest levels at 640 mg/L. The immune response to boron is mediated by the MAPK signaling pathway in the ostrich thymus.(2) The regulation effect of boron on calcium signaling pathway: There was 11 differentially expressed genes enrichment in calcium signaling pathway, Boron mainly regulated the activity of Calcineurin(CaN) and calcium/calmodulin-dependent protein kinase(CaMK). Boron regulated the two subunits(PPP3CA, PPP3R1) of the CaN, mainly regulated the activity of PPP3R1. In addition, boron could regulate the downstream of transcription factor NFAT and MEF2 C and involved in the Calcium-Calcineurin-NAFT signaling pathway.(3) The regulation effect of boron on B/T cells receptor signaling pathways: There were 7 differentially expressed genes enrichment in B cell receptor signaling pathway, and 11 differentially expressed genes enrichment in T cell receptor signaling pathway. B/T cells receptor signaling pathways could mediate the activity of the Regulation of actin cytoskeleton pathway, the MAPK signaling pathway, Ca N-NFAT signaling pathway and PI3K-AKT signaling pathway. All of these pathways interact with each other, for regulating the ostrich immunity. According to the KEGG enrichment and real-time PCR results, boron could regulate the activity of the PI3 K kinase and involved in the PI3K-AKT signaling pathway. Boron regulated the activity of the B/T cells receptor signaling pathways closely related with the activity of MAPK, calcium, and PI3K-AKT signaling pathway.(4) The regulation effect of boron on the Toll like receptor signaling pathway: There were 11 differentially expressed genes enrichment in toll like receptor signaling pathway. Boron not only regulated the MyD88-dependent signaling pathway but it also regulated the MyD88-independent signaling pathway(TLR3 signaling pathway). Moreover, high-dose of boron played a negative regulation in toll like receptor signaling pathway, which could inhibit the activity of toll like receptors, and induce the ostrich immune function.(5) The regulation effect of boron on the heat shock proteins(Hsp): Heat shock proteins have a close relationship with apoptosis, which are anti-apoptosis protein. Hsp70 is the represent member in the Hsp family, and Hsp40 is a co-chaperone of Hsp70. High-dose boron significantly inhibited the protein levels of Hsp70 and Hsp40 in ostrich chick thymuses. The low levels of Hsp70 and Hsp40 could inhibit the anti-apoptosis effect and the immune response ability in ostrich.(6) The regulation effect of boron on the apoptosis and cancer pathways: Boron mainly regulated the Trail-induced extrinsic apoptosis signaling pathway, and regulated the activity of the anti-apoptosis proteins: FLIP and IAP. However, Boron had little effect on the caspase family, but is could regulate the pathway in cancer, mainly regulate the Wnt, PI3K-AKT, MAPK and the cytokine-cytokine receptor interaction signaling pathways in the cancer pathway. The regulation of boron on these signaling pathways is an elaborated and complex process, all of these pathways were likely to be involved in the cell proliferation, differentiation, and apoptosis in ostrich chicks.