Lead exposure induced inflammation in bursa of Fabricius of Japanese quail (C. japonica) via NF-κB pathway activation and Wnt/β-catenin signaling inhibition

https://doi.org/10.1016/j.jinorgbio.2021.111587Get rights and content

Highlights

  • Lead can induce developing immunotoxicology in Japanese quails.

  • Lead caused developmental retard and size reduction in bursa of Fabricius.

  • The decrease of immunoglobulins and complement indicated immunosuppression by lead.

  • Structural damage and oxidative stress implied bursa inflammation by lead exposure.

  • Lead could trigger inflammatory responses in bursa via Nuclear Factor κB signaling pathway.

Abstract

Bursa of Fabricius (BF), one of primary lymphoid organ, is unique to birds. Meanwhile, lead (Pb) is well known for its high toxicology to birds. Therefore, this study aimed to examine the chronic toxic effects of lead exposure on BF in Japanese quails (C. japonica) and the underlying mechanism of lead immunotoxicity. One-week old male quails were exposed to 0 ppm, 50 ppm, 500 ppm and 1000 ppm Pb concentrations by drinking water for four weeks. The results showed that Pb accumulation in BF increased in a dose dependent way. The growth and development of BF was retarded in 500 ppm and 1000 ppm Pb groups. The number of lymphocytes was decreased and the release of immunoglobulin G and M (IgG, IgM), complement 3 and 4 (C3, C4) was inhibited by Pb exposure. Lead exposure also caused oxidative stress and increasing apoptosis in BF. Moreover, histopathological damages characterized by inflammatory hyperemia and inflammatory cell infiltration and ultrastructural injury featured by mitochondrial vacuole, cristae fracture and chromatin concentration were found in BF of 500 ppm and 1000 ppm Pb groups. Furthermore, RNA sequencing based transcriptomic analysis revealed that molecular signaling and functional pathways in BF were disrupted by lead exposure. In addition, the activation of Nuclear Factor kappa B (NF-κB) pathway while the inhibition of wingless integrated/catenin beta 1 (Wnt/β-catenin) signaling by Pb exposure were confirmed by quantitative real-time PCR (qPCR). Our study may benefit to understand potential mechanistic pathways of developmental immunotoxicology under Pb stress.

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.

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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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