Effect on the splenocyte function of weaned piglets induced by continuous lipopolysaccharide injections

Pigs are one of the most economically important domestic animals in the world. During the weaning stage of piglets, stress problems often occur, which pose a great challenge to pig farm production (36). Stress is a set of non-specific responses that occur when the organism is stimulated by an internal or external stressor. Different stresses cause the body to produce specific and non-specific responses. Exogenous pathogens in the environment, such as bacteria and viruses, can infect the animal and cause an immune response. Under excessive stress, the immune system is activated, immune cells continuously release inflammatory cytokines in response to external stimuli, and immune allostasis is lost (38). Excessive stress can also result in heavy economic losses for the farmer; the stressed animal may exhibit a series of negative symptoms, including an increase in temperature and productivity-harming decreases in feeding rate and cessation of growth, and excessive stress may even lead to the animal’s death (20). Moderate stress, in contrast, promotes the adaptation of the organism to the environment and improves tolerance to adverse stress (1, 4).

Lipopolysaccharide (LPS), a component of the cell surface in Gram-negative bacteria, is the main pathogenic factor that causes an inflammatory response. When Gram-negative bacteria lyse and die, LPS is released and recognised by toll-like receptor 4 (TLR4) (39). After binding to TLR4, LPS initiates signalling along a series of intracellular pathways, promoting the selection of bioactive factors such as interleukin (IL)-2, IL-10, interferon gamma (IFN-γ) and nitric oxide, and resulting in an inflammatory response (12, 28).

The spleen is a major immune organ, which is populated by many types of immune cells such as T cells, B cells and macrophages (33). As the central site of the immune response, the spleen plays a vital role in both innate and adaptive immunity which is crucial for protection against pathogens (6). Many of the regulators in splenic response involve splenocyte proliferation. The proliferation ability of splenocytes is thus the most direct indicator of spleen immune function (41). Several active factors are involved besides the spleen in the immune response, such as nitric oxide (NO). Nitric oxide is an important messenger in the pathogenesis of inflammation, linking innate and adaptive immunity. When the immune function is activated, NO may mediate signalling, which is closely associated with the release of cytokines and T-cell differentiation (34, 38).

The relationship between stress and immune function is intricate. Moreover, the effect of stress-induced immune response varies depending on the species, pathway, duration and intensity (7). When an animal is faced with stress, the number and ratios of its blood leukocytes may change, secretion of cytokines may climb to abnormal levels and the ratio of subpopulation sizes after lymphocyte differentiation come into imbalance. These changes can significantly impact immune function (23). As an integral part of the immune function, splenocytes become involved when animals come under stress. However, relatively little research has been conducted on how splenocytes play a role in LPS-induced immune response. A stress model was established by continuous injection of LPS as presented by Zhou et al. (43). Such a model is also a popular one widely used in similar studies on stress and nutritional disturbances (37, 40). The aim of the study was to evaluate the effect on splenocyte function in a model of weaned piglets in which stress was induced by multiple low doses of LPS.

Populated by lymphocytes, macrophages and other immune cells, the spleen is an important site for generating the immune response to stimulation by antigens and is essential for total immune function (42). Lymphocytes are significant components of acquired immunity and serve as the primary effector cells of both cellular and humoral immunity. Splenic lymphocytes make up 25% of the body’s lymphocytes (32). Much research has revealed that changes in immune function involve the activation and proliferation of lymphocytes. Thus, the ability of lymphocytes to proliferate and differentiate is an important indicator of cellular immunity (41). Lymphocyte mitogens, including ConA and phytohaemagglutinin, are non-specific for lymphocyte activation. Lymphocyte activity and immune response can be accurately assessed by adding mitogens to induce lymphocytes to proliferate in vitro (19). In the present study, the proliferation of splenocytes after ConA stimulation was investigated to elucidate the effect of continuous LPS induction. The results showed a significant increase in the transformation ability of splenocytes in vitro from the 5^th day after the start of the LPS challenge. Proliferation ability is an important indicator reflecting the immune status of lymphocyte immunity (22). As an immune activator, LPS is the primary target of the immune system for recognising pathogens. It has been proved to mediate innate immunity by interacting with toll-like receptors on the surface of immune cells, releasing large amounts of cytokines while inducing the proliferation of lymphocytes as the primary specific immune response (12). The transformation ability of lymphocytes significantly increased and the immune system was activated in stressed pigs subjected to an LPS challenge (20). The microbial endotoxin induced the proliferation of pigs’ T lymphocytes and promoted the creation of IL-2 (22). As important messengers, cytokines such as IL-2, tumour necrosis factor alpha and IFN-γ are released to induce further proliferation and activation of immune cells, as exemplified in an experiment by Ma et al. (21) in which they administered ulinastatin to pancreatitic rats and analysed their splenocytes.

The cell cycle was examined to further investigate the effect of LPS on splenocyte proliferation ability. In the cell cycle, DNA preparation is in the G1 phase, DNA synthesis is in the S phase, mitosis preparation is in the G2 phase, and mitosis occurs in the M phase (16). Many studies have shown that the G0/G1 phase is the initiation point of cell proliferation, while there may be a blockage in the G1/S phase. When the G1 phase is blocked, the cells cannot enter the S phase for DNA replication, which affects the proliferation of lymphocytes (8). Without any such blockage, as the proportion of cells in the G1 phase decreases and the proportion of those in the S phase to G2 phase increases, cells progress through the cycle, which leads to cell proliferation (18). In the present findings, the proportion of LPS-treated splenocytes in the G0/G1 phase significantly decreased and the proportions in the S to G2 phases significantly increased, as did the PrI. Splenocyte proliferation and DNA synthesis are closely related to mitosis, and when splenocytes are induced into the G2/M phase, the number of cells increases significantly (9). The number of immune cells is critical to effective immune response (41). Numerous studies have shown that an increase in cells in the G0/G1 phase delays the development of the spleen and suppresses its immune function. Moreover, an increase in cells in the S to G2/M phases also indicates its immune dysfunction (11, 16, 25, 30).

Apoptosis is a programmed cell death mode and an important physiological process to maintain allostasis (10). The level of splenocyte apoptosis may to some extent reflect the immune status of the organism. Our results showed that after LPS stimulation, the proportion of early and late apoptotic cells decreased significantly and the proportion of normal cells increased significantly, which consolidated the number of splenocytes and suggested that the splenocytes were activated. In physiological conditions, lymphocyte proliferation and apoptosis are balanced to maintain a stable number of these cells (24). The interplay of lymphocyte proliferation and apoptosis is closely related to initiating and terminating the immune response. Baumann et al. (2) suggested that activated lymphocytes are resistant to apoptosis. Therefore, when the immune function is activated, the proliferation of immune cells is increased and apoptosis is inhibited. Many studies have shown that splenocyte apoptosis is suppressed when the immune status is activated. Conversely, immune suppression in the spleen is generally associated with excessive splenocyte apoptosis (15, 31). The experiment revealed a reduction in splenocyte apoptosis in piglets induced by LPS, and thus suggested that splenocyte viability increased and immune function was activated in piglets.

Nitric oxide is produced from L-arginine, catalysed by nitric oxide synthase (NOS), and plays a vital role in regulating vascular function, neurotransmission, inflammatory response and host defence (34). It can regulate immune function together with cytokines and its level reflects the development of diseases and the level of immunity (26). It was shown that most active immune cells, such as macrophages, monocytes and natural killer cells, can express inducible NOS (iNOS) and secrete NO. Moreover, the cytokines released in response to stress upregulate iNOS expression within immune cells and stimulate NO secretion, and this is the most critical pathway affecting NO expression (3). Hou et al. (13) found that the release of NO from intestinal epithelial cells increased after LPS stimulation in piglets, which caused damage to the intestinal mucosa. Jafari-Khataylou et al. (14) showed that NO was significantly increased in the serum of LPS-treated mice, while treatment with the hydroxyethylrutoside troxerutin significantly reduced NO levels and alleviated inflammatory symptoms. Tanaka et al. (35) isolated splenocytes from rats treated with LPS and found that LPS induced more inflammatory factors and NO production in those cells. In the present study, the NO release in the LPS group increased significantly on the 5^th and 9^th days, but did not change significantly on the 13^th day. The induction of stress by LPS probably triggers macrophages to express NO in large quantities, but the compound’s excessive concentration inhibited its sustained expression and protected the organism from its sustained effects (5). Nitric oxide has a direct cytotoxic effect on pathogens in non-specific immunity and also exerts a powerful immunomodulatory effect (34). A lower concentration of NO selectively induces the expression of IL-12, affecting lymphocyte proliferation and the balance of T helper 1 and 2 cells (27). Using iNOS knockout rats, Shen et al. (34) found that a deficiency of NO reduced the immune function of mice and made them more susceptible to pathogens. It was also demonstrated that NO was engaged in critical immunological processes such as apoptosis and autoimmune and infectious diseases (3).

Beginning on the 5^th day of the experiment, splenocytes from the LPS group were significantly more activated than those from the control group. A decrease in the ratio of cells in the resting phase and gap 1 phase, an increase in the ratio of cells in the synthesis, gap 2 and mitosis phases, a decrease in splenocyte apoptosis, and an upregulation of NO on the 5^th and 9^th days were observed, which may be attributed to the effect of spleen immunomodulation. This demonstrates the spleen’s cellular-level immune response to LPS induction in weaned piglets. In the future, further research is needed to investigate how splenocytes specifically regulate the action mode of the immune system at different stages of the piglet’s life.