Abstract
Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress.
Highlights
Thymus is the primary immune organ for T cell development in jawed vertebrates [1]
In normal developing ostrich thymus, the apoptotic cells mainly distributed in thymic cortex and their number increased with age (P < 0.1) (Fig 2A, 2B and 2D)
Thymus is a common target for nutritional disorder [2, 6], and excessive boron supplement was suggested to be harmful for immune organs [6, 13]
Summary
Thymus is the primary immune organ for T cell development in jawed vertebrates [1]. Normal thymus maintains distinct architecture including outer compact cortex where T cell precursors commit to T cell lineage and gain the ability to interact with self-antigen and central loose medulla where most autoreactive thymocytes are deleted. Multiple environmental stressors, such as pathogenic infection and imbalance of trace elements, can disrupt normal thymus organization and reduce organ size by acute depletion of immature CD4+CD8+ thymocytes in cortex, which are associated with impaired peripheral immune response [2, 6]. Excessive boron (> 400 mg/kg) has been reported to inhibit the development of multiple immune organs (eg, thymus, spleen) in chickens and rats [6, 13]. TLRs are expressed in the thymus of chickens and are suggested to participate in thymus development by inducing downstream factors [15, 16]. We hypothesized that TLRs may participate in the process of boron stress in thymus. The impact of chronical boron stress on thymus was assessed, and the expressions of TLR3 and TLR4 with their downstream factors were evaluated
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