Neural activity has been shown to dominate multiple central and peripheral inflammatory responses and is essential for maintaining immune homeostasis [1, 2]. As an important neurotransmitter in the neuronal system, acetylcholine (Ach) is synthesized from choline and acetyl-CoA by choline acetyl transferase (ChAT), then is transported and released by vesicular acetylcholine transporter (VAChT) to bind to acetylcholine receptors (AChRs) to transmit neural signals . AChRs have been identified to be expressed in both neuronal and non-neuronal cells, mainly including muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs) [4, 5].
The mAChRs, composed of five main subtypes, M1, M2, M3, M4, and M5, are widely expressed in effector cells innervated by parasympathetic ganglionic fibers, including pancreatic cells , keratinocytes , intestinal epithelium  and most immune cells, such as T cells, B cells and monocytes . These finding suggested that non-neuronal cholinergic systems were involved in the regulation of immune cell function . A few studies have shown that compared with wild type, the expression of pro-inflammatory cytokines such as tumor necrosis factor (Tnf-α), Ifn-γ and Il-6 in splenocytes in mAChRs M1/M5 knockout mice were significantly changed [11, 12] and more antigen-specific antibodies were produced in M1/M5 mutant group . Based on static or in vitro experiments, these studies suggested that mAChRs could regulate immune responses at the cytokines level, but roles of mAChRs in the recruitment of immune cells, especially neutrophils, have rarely been reported, and its potential regulatory mechanisms were not been elucidated. Neutrophils are the most important member type of innate immune cells, accounting for about 40%-60% of the total number of leukocytes [13, 14], which is essential for regulating the inflammatory responses [15, 16]. As a model animal, the optical transparency of juveniles makes zebrafish an ideal model for in vivo imaging to study the neutrophils [17, 18]. Hence, this study intends to explore the effects of mAChRs on the recruitment of neutrophils and also the expression of cytokines in zebrafish.
There are two subtypes of arginases, arginase 1 (Arg1) and arginase 2 (Arg2), which are mainly distributed in organs such as liver, kidney and testes . Recent studies also showed that Arg1 and Arg2 were widely expressed in immune cells [20, 21] and found that the expression of Arg1 could be induced by inflammatory signals including interferons and transforming growth factor-beta (Tgf-β) . Given the role of Args in inflammatory response, we speculated that Args might be involved in the regulation of mAChRs on inflammation.
In this study, we used pharmacological methods to inhibit and activate mAChRs, and found that mAChRs could regulate neutrophils recruitment and cytokines expression using in vivo imaging in zebrafish. We further proved that Args, as a downstream protein of mAChRs, could participate in the regulation of inflammatory response in zebrafish. Here, we used zebrafish to provide additional evidence that cholinergic receptors regulate peripheral immune responses, deepening the link between nervous system and immune functions.