Localization of vasoactive intestinal peptide and toll‐like receptor 2 immunoreactive cells in endostyle of urochordate Styela plicata (Lesueur, 1823)

Abstract The endostyle is the first component of the ascidian digestive tract, it is shaped like a through and is located in the pharynx's ventral wall. This organ is divided longitudinally into nine zones that are parallel to each other. Each zone's cells are physically and functionally distinct. Support elements are found in zones 1, 3, and 5, while mucoproteins secreting elements related to the filtering function are found in zones 2, 4, and 6. Zones 7, 8, and 9, which are located in the lateral dorsal section of the endostyle, include cells with high iodine and peroxidase concentrations. Immunohistochemical technique using the following antibodies, Toll‐like receptor 2 (TLR‐2) and vasoactive intestinal peptide (VIP), and lectin histochemistry (WGA—wheat‐germagglutinin), were used in this investigation to define immune cells in the endostyle of Styela plicata (Lesueur, 1823). Our results demonstrate the presence of immune cells in the endostyle of S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates. Research highlights Immune cells positive to TLR‐2 and VIP in the endostyle of Styela plicata. Expression of WGA in several zones of endostyle. Use of comparative biology to improve the knowledge about immunology in ascidians.


| INTRODUCTION
The ascidians, also known as tunicates because of the characteristic tunic covering the whole organism, are marine invertebrates classified among the urochordates. These animals may be pelagic or sessile. Styela plicata (Lesueur, 1823) is a solitary benthic ascidian that represents a valid model of evolutionary study .
The endostyle, the initial part of the ascidian digestive tract, has a trough shape and is placed in the ventral wall of the pharynx. This organ plays an important immune function (Giacomelli et al., 2012) and is subdivided into nine different zones longitudinally parallel to each other (Hiruta et al., 2006). The cells of each zone are morphologically and functionally specialized (Aros & Viragh, 1969;Fujita & Nanba, 1971;Osugi et al., 2020) (Figure 1). Zones 1, 3, and 5 contain support elements, zones 2, 4, and 6 present mucoproteins secreting elements associated with the filtering function. Zones 7, 8, and 9, located in the lateral dorsal portion of the endostyle, show cells with high concentrations of iodine and peroxidase (Fujita & Sawano, 1979;Thorpe et al., 1972) and are considered to be homologous to thyroid follicles (Fujita & Sawano, 1979). The expression of several thyroid-associated genes in these areas supports this homology (Ogasawara et al., 1999;Ogasawara & Satou, 2003;Ristoratore et al., 1999). The endostyle represents a key structure in the chordates evolution (Bone et al., 2003;Petersen, 2007). The mucus produced by zones 1 and 4 together with the galactins produced by zones 2 and 4 (Vizzini et al., 2015), creates a mesh that plays the role of filtering food and furthermore acts as a first barrier against microbes and pathogens, such as mammalian mucus produced by goblet cells in the gut (Flood & Fiala-Medioni, 1981;Petersen, 2007). In addition, the endostyle shows a defense immune function against foreign agents using the oral and atrial (cloacal) siphon as preferential entry routes of microorganisms. In zone 8 a population of phagocytes is exposed to seawater. These sentinel cells can recognize and ingest foreign cells, preventing them from entering the pharynx. (Sasaki et al., 2009).
This study aimed to characterize immune cells in the endostyle using Toll like receptor 2 (TLR-2) and vasoactive intestinal peptide (VIP) antibodies, and lectin histochemistry (WGA).
VIP is a neuroimmune peptide present in different regions of the vertebrate intestine  and is also expressed in immune cells such as T and B cells, mast cells, and eosinophilic granulocytes (Alessio et al., 2020;Iwasaki et al., 2019). Neuropeptides are normally expressed in the mammalian digestive system, under physiological and pathological conditions . Several studies have shown the presence of neuropeptides, such as Neuropeptide Y, in S. plicata, produced by the hemocytes (Pestarino, 1992).
WGA is a haemagglutinating lectin present on phagocytic hemocytes (Cima et al., 2001), and morula cells (MCs), the predominant type of hemocytes (Ballarin & Cima, 2005). WGA lectin also stains modestly mucous cells and a brush-like boundary Lauriano et al., 2019). Moreover, WGA is involved in innate immune response (Hillyer & Christensen, 2002;Jeong et al., 2002), collaborating with epithelial barriers in cellular defense, and cooperates with pattern-recognition receptors to stimulate proinflammatory signaling cascades in the innate immune system, playing a key role in the interaction with Toll-like receptors (TLRs) (Unitt & Hornigold, 2011).

| Animals
Samples of adult specimens of S. plicata used in this study were collected from the natural oriented reserve of "Capo Peloro"

| Statistical analysis
For each sample, 5 sections and 10 fields were investigated to generate data for statistical analysis. Subjectively, the fields were chosen based on the cell's positivity reaction. The ImageJ software was used to examine each field (Schneider et al., 2012). After converting the acquired image to 8 bits, a "Threshold" filter and a mask were used to pick cells and remove the background. The cells were then counted using the "Analyze particles" plug-in. ANOVA was used to determine the statistical significance of the positive cells number respectively for TLR2, VIP, and WGA. SigmaPlot version 14.0 was used to perform statistical analyses (Systat Software, San Jose, CA). The information gathered was reported as median values with a SD (Δs). To compare regularly distributed data, two-tailed t tests were utilized, and Mann-Whitney rank-sum tests were used to analyze non-normally distributed data. Values of p below .05 were judged statistically significant in this order: *p ≤ .01, **p ≤ .02, ***p ≤ .03, ****p ≤ .04, *****p ≤ .05.  (Table 1).

| RESULTS
Statistical analysis confirms a significant number of positive cells for TLR2, VIP, and WGA in the endostyle zones, especially in the 6 and 8 zones (Table 2, Figure 4).

| DISCUSSION
The immune response is mediated by circulating effector cells. Hemocytes, or immunocytes, include professional phagocytes (Franchi et al., 2011;Jimenez-Merino et al., 2019) and cytotoxic hemocytes, able to induce oxidative stress (Ballarin & Cima, 2005). These cytotoxic cells contain phenoloxidase (PO) (POCCs) and have a berry-like morphology, called morula cells (MCs), and account for more than 50% of circulating hemocytes (Cammarata et al., 2008;Parrinello et al., 2003). Cytochemical analyses have shown high levels of polyphenols in the vacuoles of these cells. These phenolic compounds play a key role in the cytotoxicity of these hemocytes and act as substrates for POs. Polyphenols are compounds with antibacterial, anti-inflammatory, antioxidant, and immunostimulant activity Alesci, Nicosia, Fumia, et al., 2022;Capillo et al., 2018;Fumia et al., 2021). Several studies have shown that an ethanol or methanol extract of ascidian has antibacterial, antimicrobial, anti-inflammatory, and antioxidant activity, assuming that these phenolic compounds are involved in the immune response of tunicates (Asayesh et al., 2021;Carletti et al., 2020;Elya & Edawati, 2018).
In the present study, we have marked endostyle zones cells of F I G U R E 2 (a) May-Grünwald-Giemsa, magnification Â40, scale bar 50 μm. Endostyle is bathed by cells flowing through its breasts, with macrophages organized into islands next to it. The digestive system and heart are located near its rear end. Endostyle is outlined at the front end. A longitudinal section of the endostyle, lymphocyte cells, and macrophages can be seen in the breast. (b) AB/pas 2.5, magnification Â40, scale bar 50 μm. Histochemical stain shows positive mucosal cells in zone 2, 4, and 6 (arrows), confirming that these zones are responsible for mucous secretion (Rosental et al., 2020). Zones 2, 4, and 6 within it produce mucus, as shown by our data with AB/PAS staining.

The ascidian hemocytes involved in immune responses
(immunocytes) represent the largest fraction of circulating hemocytes (Franchi & Ballarin, 2017). They include phagocytes and cytotoxic cells.
At the molecular level TLR1 is expressed in both phagocytes and MCs as a member of the TLR receptor family, actively involved in self/nonself recognition (Goldstein et al., 2021;Peronato et al., 2020). The oral and atrial (cloacal) siphon are preferential entry routes for microorganisms. In zone 8 a population of phagocytes is exposed to seawater. These sentinel cells can recognize and ingest foreign cells, preventing them from entering the pharynx (Sasaki et al., 2009). In the endostyle, as well as in the immunocytes, genes for the Toll-like and mannose-binding lectin receptors (MBLs) are transcribed, following the important role of immunosurveillance of the food tract (Franchi & Ballarin, 2017).
Our F I G U R E 4 Graphic of statistical data of S. plicata (esophagus, stomach, and intestine) (Pestarino, 1982) but not in the pharynx. We have characterized VIP in ascidian endostyle for the first time, showing labeled immune cells in zones 3, 6, 7, 8, and 9. Zone 8 of the endostyle contains TLR-positive, VIP-positive, and WGA-positive cells, confirming that cell populations of this zone do play a role in the innate immunity of these animals.

| CONCLUSIONS
In conclusion, our results demonstrating the presence of immune cells in the endostyle of S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates. TLR2 and VIP play in ascidians a key role in adaptive immune response, as in mammals. Therefore, this animal model allows the study of the cellular and molecular processes that orchestrate innate immune responses. This information can be translated into human immunity, with a particular impact on improving therapeutic strategies for stem cells, tissues, and organ transplantation. In addition, the immune defenses of tunicates have made them a potential source of natural drug resources with great potential for pharmacological applications.

ACKNOWLEDGMENTS
The authors are grateful to all the researchers whom we cited in this article for their significant and valuable research.
Open Access Funding provided by Universita degli Studi di Messina within the CRUI-CARE Agreement.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request ORCID Gioele Capillo https://orcid.org/0000-0002-2002-1138