This study reveals how ETEC’s heat-labile enterotoxin (LT) compromises porcine monocyte viability and immune function — providing new insights into the pathogen’s immunomodulatory strategies.
Authors:
Jinglin Ma, Hans Van der Weken, Leen Hermans, Matthias Dierick, Eric Cox and Bert Devriendt
Abstract
Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea in humans and animals, including pigs. Enterotoxins are important virulence factors for ETEC. Although much is known about the mechanism of enterotoxininduced diarrhoea, less is known about its efects on innate immune cells such as monocytes. Monocytes can diferentiate into macrophages and dendritic cells and play a pivotal role in bridging the innate and adaptive immune systems. Understanding the interaction between ETEC enterotoxins and monocytes can help in the development of more efective preventive and therapeutic strategies to combat this disease. In this study, we aimed to investigate the efects of heat labile enterotoxin (LT) and heat stable enterotoxin a (STa) produced by ETEC on porcine monocytes. Our results showed that STa did not afect the viability or efector functions of monocytes. LT, on the other hand, decreased the viability of monocytes. While LT did not alter the production of reactive oxygen species (ROS) by monocytes, it signifcantly reduced the production of ROS induced by phorbol 12-myristate 13-acetate (PMA). In addition, LT decreased the phagocytosis of E. coli by monocytes and enhanced the survival of intracellular ETEC. Furthermore, LT triggered the production of the cytokines IL-1β, IL-6 and TNF-α as well as the chemokines CCL-3 and CXCL-8. Together, our results show that, in contrast to STa, LT can cause monocyte death and disrupt monocyte immune efector functions, potentially acting as an immune evasion strategy to establish infection.
Keywords: Heat labile enterotoxin, heat stable enterotoxin, monocytes, immune evasion, pig
1 Introduction
Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea in children and travelers. ETEC infections also cause morbidity and mortality in farm animals, including piglets, leading to signifcant economic losses to pig farmers. In ETEC-induced diarrhea, the heat labile enterotoxin (LT) and heat stable enterotoxin (ST) secreted by ETEC play crucial roles. Upon secretion, LT enters host gut epithelial cells through binding with ganglioside M1 (GM1). Once internalized, LT activates adenylate cyclase, which results in an increase in the level of intracellular cyclic adenosine monophosphate (cAMP). The latter in turn activates protein kinase A (PKA), which modulates the activation of membrane ion channels, ultimately leading to the secretion of electrolytes and water into the intestinal lumen. To date, two distinct types of STs, namely, STa and STb, have been identifed. The initial phase of STa-induced diarrhoea involves the activation of guanylate cyclase C (GC-C) on gut epithelial cells. This leads to increased cyclic guanosine monophosphate (cGMP) levels and subsequent activation of cGMPdependent protein kinase II (cGMPKII). These molecular cascades induce the secretion of chloride and bicarbonate ions while inhibiting Na+ uptake, thereby causing diarrhoea. ETEC strains produce STs that not only induce secretory diarrhea but also modulate the expression of proinfammatory cytokines, chemokines, and other immune-related genes. Moreover, their impact on the innate immune function of enterocytes has been explored recently. For example, STa induces rapid and transient expression of the interleukins IL-33 and IL-1Ra in human gut epithelial cells. Additionally, compared with wild-type mice, mice defcient in the IL-33 receptor are less susceptible to STa. However, the impact of STs on the immune function of intestinal immune cells remains largely unexplored. In contrast to STs, LTs have been reported to activate human and murine immune cells and to enhance cellular and humoral immune responses to antigens. In-depth investigations have elucidated the underlying mechanisms involved. Some studies in mice have indicated that the enhancement of T-cell proliferation is linked to the functional activation of dendritic cells induced by LT. However, previous results suggest that the mechanism is complex and that various immune cells are involved in this immunomodulation process. For example, in a mouse model, LT administration increased the production of IL-1β by dendritic cells. On the other hand, another study
indicated that LT-IIa and LT-IIb could suppress the production of IL-1β induced by LPS in the human monocytic cell line THP-1. Furthermore, previous studies have indicated that LT can increase antigen uptake by murine dendritic cells, whereas a recent study demonstrated that LT inhibits the phagocytosis of ETEC by murine macrophages. Although the efect of LT on certain immune cell populations is well established, its efect on monocytes, precursors to macrophages and dendritic cells, remains poorly understood, particularly in pigs. Monocytes play pivotal roles as bridges between the innate and adaptive immune systems. Like various pattern recognition receptors, they can detect both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Upon recognizing these signals, monocytes become activated and undergo chemotaxis, migrating towards the infection site. Once they are present, they exhibit a certain degree of plasticity by diferentiating into either macrophages or dendritic cells, which are infuenced by local cues and signals. Moreover, even under steady-state conditions, there is a constant infux of blood monocytes into the gut, where they continuously replenish intestinal macrophages and dendritic cells. Tis versatility allows monocytes to contribute to gut homeostasis. Some of these monocytes, however, retain their characteristics and reside in the intestinal lamina propria [18]. In addition, monocytes are present within the small intestinal lumen in mice. When ETEC enters the small intestine or when the epithelial barrier is disrupted by ETEC, these resident monocytes are exposed to ETEC and its secreted enterotoxins. In response, monocytes engage their efector functions, including phagocytosis, reactive oxygen species (ROS) production, and the release of cytokines and chemokines, to eliminate pathogens. Moreover, monocytes can serve as antigen-presenting cells, possessing the ability to capture, process, and deliver antigens to T cells, thereby facilitating the coordination of the host immune response. Here, we investigated the efects of LT and STa on the function of porcine monocytes. Tese fndings contribute to a deeper understanding of immunoregulation by LT and STa.
