Lead exposure induced inflammation in bursa of Fabricius of Japanese quail (C. japonica) via NF-κB pathway activation and Wnt/β-catenin signaling inhibition
Graphical abstract
Structural injury indicated inflammation in bursa of Fabricius (BF). Increased apoptosis and oxidative stress also cause BF inflammation. Moreover, wingless integrated (Wnt) signaling inhibition and Nuclear Factor kappa B (NF-κB) pathway activation stimulated inflammatory responses. BF inflammation induced by Pb exposure resulted in reduction of lymphocyte, immunoglobulin and complement levels.
Introduction
The human-induced lead (Pb) pollution caused extensive health problems to people and it is regarded as one of ten chemicals of major public health concern by WHO [59]. Birds are also seriously affected by lead contamination. Lead poisoning not only causes unnecessary death of birds, but also severely harms the welfare of large numbers of wild birds [45,46]. Lead has negative impacts on multiple organs and systems of birds including nervous system, hematopoietic system, reproductive system, renal system and digestive system [2,42].
Avian immune system is also the target of lead toxicology [60]. Pb exposure can affect most components of the immune system of birds [13]. Lead not only affects innate immunity such as macrophages, neutrophils and natural killer cells, but also impacts on induced immunity such as T-lymphocyte and B-lymphocyte [13,57]. For instance, lead alters the process of T-lymphocyte-driven B-cell maturation, and lead exposure reduces serum immunoglobulin levels [5,13]. Moreover, lead exposure results in changes of T-helper 1 (Th1) cytokine and T-helper 2 (Th2) cytokine production [13]. Usually, lead exposure caused immunosuppression by causing histopathological damages, lymphoid organ weight loss, growth retard, immune imbalance, and immune related genetic pathway disruption [26,57]. However, lead exposure also showed stimulatory immune function [43].
As one of primary lymphoid organs, the bursa of Fabricius (BF) is globular, sac-like structure that attaches to the bird's cloaca [48]. The BF originates in the cloaca, and its wall retains four layers including mucosa, submucosa, muscularis, and tunica adventitia [48]. There are many lymphoid nodules in the mucosa, which is rich in a large number of lymphocytes. Lymphoid nodules can be divided into cortex, middle layer and medulla. Mature B-lymphocytes are mainly distributed in the cortex [48]. BF is a unique immune organ of birds and it plays an essential role in B-lymphocytes amplification, proliferation and differentiation and antibody production [8]. This organ is composed of 98% of B-lymphocytes and is mainly involved in humoral immunity of birds [25]. All avian immunoglobulins are produced by B lymphocytes. Immunoglobulins in birds mainly include immunoglobulin G (IgG), M (IgM), A (IgA) and D (IgD), and IgG is the most abundant and most important immunoglobulin in serum, while IgM is less abundant in poultry and it plays a dominant role in the primary immune response and is the earliest immunoglobulin in the primary immune response [28]. Previous studies showed lead exposure can cause BF structural damages, immunoglobulin decrease, and induced immunotoxicity [27]. However, the molecular mechanism of lead causing BF immunotoxicity is still unclear.
As one of transcription factors, nuclear factor kappa B (NF-κB) mediates various biological process including cell growth, body development and immune functions. In particular, NF-κB is involve in inflammatory responses [55]. Activation of NF-κB signaling pathway can induce the expression of a large number of inflammatory genes, including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL − 1β), interleukin 8 (IL-8), prostaglandin-endoperoxide synthase 2 (COX-2), thereby inducing inflammatory responses [36,66]. NF-κB is recognized as a central mediator of proinflammatory gene induction and function [36]. Recent studies revealed that the stimulation of NF-κB signaling by NH3 exposure could lead to the inflammation in BF and thymus of chicken and the activation of NF-κB signaling by Pb exposure may induce splenic necrosis in chicken [51,64]. Moreover, wingless integrated (Wnt) signaling pathway may interact with NF-κB signaling and they both play important roles in inflammation stimulation or suppression [22,24,40].
RNA sequencing (RNA-Seq) is an indispensable sequencing method for transcriptome sequencing. It can be more accurately quantified, has a wider detection range, higher reproducibility and more reliable analysis, and has been widely used in the study of toxicity mechanism [67]. Meanwhile, RNA-Seq can simultaneously analyze gene expression changes in quails exposed to various lead doses in one experiment to reveal transcriptional expression [58].
Therefore, we used a toxicological model species Japanese quail to determine the potential molecular mechanisms of immunotoxic effects of lead on BF by combining RNA-Seq analysis, histological technique, biochemical analysis and quantitative real-time PCR (qPCR) skills. We tried to find inflammatory changes of BF by microstructural and ultrastructural analysis, and then we endeavored to detect immunological function alterations such as immunoglobulin and complement levels and antioxidant enzymes activities. We wanted to examine the potential molecular pathways and critical genes associated with BF immune responses to Pb stress. We tried to verify the mRNA expression of genes involved in Wnt signaling pathway and NF-κB signaling pathway to determine molecular inflammatory responses in BF caused by Pb exposure. The study will benefit for understanding avian immune responses to lead stress and it also provides valuable suggestions for wildlife conservation under the influence of heavy metal.
Section snippets
Animal husbandry and chronic experiment
One hundred and twenty newly male hatchlings were bought from local quail farm in Xi'an, Shaanxi Province, China (108°47′27″, 34°03′52″). We only used male birds to avoid possible gender difference in BF development. These quails were transferred into brooding box for one-week acclimation at 30 °C. Then these one-week-old birds were randomly separated into 4 cultivation boxes 130 × 68 × 18 cm (length × width × height) with 30 birds in each box. The stocking density of quails referred to
Pb accumulation in BF
The average Pb accumulation in BF exposed to control, 50, 500 and 1000 ppm of Pb were 0.022 ± 0.011, 0.134 ± 0.003, 0.392 ± 0.003, and 1.413 ± 0.003 mg/kg respectively. The Pb content in the BF of 50, 500 and 1000 ppm groups were significantly higher than that in control group (p < 0.01). The Pb accumulation in BF increased in a dose-dependent way (Fig. 1A).
Body measurements and bursa index
After 4 weeks of lead exposure, the quails in 500 ppm and 1000 ppm Pb groups showed listlessness, emaciation, anorexia, behavioral inertia
Discussion
Our study showed cumulative lead induced immunotoxicology in Japanese quails. Pb accumulation in BF increased in a dose dependent way. High lead dose (500 ppm and 1000 ppm) did cause body size reduction, wing length difference, BF underdevelopment, histopathological damages and ultrastructural impairment. The significant wing length difference between left wing and right wing suggested increasing fluctuating asymmetry, and the asymmetry indicated lower fitness and developmental instability
Conclusions
Pb induced functional immunosuppression in BF of Japanese quails by developmental delay, lymphocyte decrease, IgM, IgG, C3 and C4 inhibition. Moreover, Pb caused structural injury, oxidative stress and increasing apoptosis in BF. Furthermore, Pb led to inflammatory responses in BF by triggering NF-κB pathway through suppressing Wnt/β-catenin signaling pathway. This study lays a foundation on avian immunotoxicology by Pb exposure and provides insight into the potential mechanism of Pb toxicology.
Declaration of Competing Interest
All the authors declared no competing interest.
Acknowledgement
We are grateful for Ms. Yuhan Gao and Yuan Yang and Mr. Wangdong Zheng for experimental assistance and Ms. Yu He, Jing Ya, and Xuan Li for valuable suggestions about the manuscript. The work was supported by National Natural Science Foundation of China (No. 33372201).
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2022, Journal of Inorganic BiochemistryCitation Excerpt :The specific procedure for the determination of Pb in the thymus was based on previous study [14]. The thymus tissue was prepared into 6 μm paraffin sections and stained by hematoxylin-eosin (HE) for microstructural survey and the protocol followed previous study [14,48]. Moreover, thymus samples for ultrastructural observation were quickly placed into an electron microscope fixative solution (Servicebio, G1102) and thymus was cut into approximately 1mm3 pieces using a double-sided blade.