Morphological and immunohistochemical phenotype of TCs in the intestinal bulb of Grass carp and their potential role in intestinal immunity

The current study investigated telocytes (TCs) in the intestinal bulb of Grass carp using light microscopy (LM), Transmission electron microscopy (TEM), scanning electron microscopy, and immunohistochemistry (IHC). By LM, TCs were distinguished by the typical morphological features that had a cell body and telopodes using HE, toluidine blue, methylene blue, Marsland silver stain, Grimelius’s silver nitrate, Giemsa, PAS, combined AB pH2,5/PAS, Crossmon’s and Mallory triple trichrome, Van Gieson stains, Verhoeff’s stain, Sudan black, osmic acid, performic acid with methylene blue and bromophenol blue. TCs were identified under the epithelium as an individual cell or formed a TCs sheath. They detected in the lamina propria, between muscle fibers, around the myenteric plexus and fibrous tissue. TCs acquired immunological features of endocrine cells that exhibited high affinity for silver stain, performic acid with methylene blue, Marsland stain, and immunohistochemical staining using chromogranin A. Sub epithelial TCs were closely related to the endocrine cells. TCs and their secretory activities were recognized using acridine orange. TCs were identified by IHC using CD34, CD117, S100-protein, desmin. TCs formed a3D network that established contact with macrophage, mast cells, dendritic cells, lymphocytes, smooth muscle fibers, fibroblast, Schwann cells and nerve fibers. In conclusion, the localization of TCs in relation to different types of immune cells indicated their potential role in the maintenance of intestinal immunity.

Acridine orange (fluorescent stain). The procedure was performed according to that of Hoff et al. modified by Refs. 27,[44][45][46][47][48] . Acridine Orange is a cationic dye and stains proteins-containing membranous vesicles including secretory vesicles, membrane bounded acidic compartments, and lysosomes that had an acidic nature. Acridine Orange exhibits a metachromatic reaction that associated with the liberation of green and red fluorescence. Acridine Orange reacts with the membrane bounded vesicles and appeared orange or red. Acridine Orange is used for the identification of secretory vesicles and lysosomes [49][50][51] . The stained sections analyzed using a Leitz DM 2500 microscope with the external fluorescent unit Leica EL 6000.
Immunohistochemistry staining (IHC) for CD34, CD117. Immunohistochemically staining was performed on paraffin sections .Antigen localization was achieved using combined with the avidin-biotin complex (ABC) technique 52 . Using the Reagent of Ultra Vision Detection System (Anti-Polyvalent, HRP/DAB (ready to use, TP-015-HD: Thermo Fischer Scientific TP-015HD) according to the manufacturer's instructions. The procedure according to a description of 53 as the following: Paraffin sections of (5 µm) were dewaxed by xylene, rehydrated by ascending grades of alcohols, and rinsed by PBS pH 7.4 (3 times for 5 min). Endogenous peroxidase was suppressed by using a hydrogen Peroxide block at room temperature. The sections were thoroughly washed by running tap water for an additional 10 min. To enhance antigen retrieval, the slides were treated with 10 mM sodium citrate buffer (Table 1) (pH 6.0) at a temperature reached 95-98 in a water bath for 20 min. The sections were cooled for 20 min from room temperature and subsequently were washed in PBS (pH 7.4, 3 times for 5 min). Block non-specific background staining was performed by using Ultra V block (Thermo Fisher scientific, UK. Lab Vision corporation; USA) for 5 min at room temperature. Ultra V block application did not exceed 10 min to avoid staining artifact). The sections were incubated with the primary antibodies (The used primary antibody, sources, dilutions, and time of incubation of each antibody are shown in (Table 2). Sections were washed using PBS (at pH 7.4, 3 times for 5 min). The Biotinylated secondary antibody was applied for one hour at room temperature. The (Table 2). Sectioned were washed in PBS (pH 7.4, 3 times for 5 min) and subsequently incubated with streptavidin-peroxidase complex ,Thermo Fisher Scientific, UK. Lab Vision corporation; USA) for10 min at room temperature. Visualization of the bound antibodies was performed using 1 drop of DAB plus chromogen to 2 mL of DAB plus substrate. The mixture was applied and incubated at room temperature for 5 min .The incubation processes were carried out in a humid chamber. Harris hematoxylin was used as counter stained for 30 s. The sections were dehydrated using ethanol and isopropanol I and II, cleared in xylene, and covered by DPX.
Use a Leitz Dialux 20 Microscope provided by a cannon digital camera (Cannon Power shot A95) to examine the stained section.
Immunohistochemical procedures of Desmin, S100 protein, chromogranin A. A Technique using the reagent of The DAKO En Vision TM + System, HRP peroxidase. The DAKO Envision TM + System, HRP is a two-step immunohistochemical staining technique 54 .
The procedure of staining according to 55 used the following protocol: sections (5 µm) of paraffin-embedded sections were dewaxed, rehydrated, and rinsed in PBS, pH 7.4 (3 times for 5 min). Endogenous peroxidase was inhibited by adding drops of 3% hydrogen peroxide in methanol at room temperature for 20 min followed by intense washing under running tap water for an additional 10 min. For antigen retrieval, slides were placed in Table 2. Identity, sources, and working dilution of antibodies used in immunohistochemical studies. Antibodies used that showed reactivity in fish species in past publication. www.nature.com/scientificreports/ 10 mM sodium citrate buffer (pH 6.0) ( Table 1) and heated to 95-98 in a water bath for 20 min, followed by cooling for 20 min at room temperature. Sections were then rinsed in PBS (pH 7.4, 3 times × 5 min). Sections were covered by adding drops of blocking serum (Dako) to cover the sections for 5 min at room temperature to block non-specific background staining. (Note: Do not exceed 10 min or there may be a reduction in the desired stain.). Sections were then incubated with the primary antibody (Table 2: Identity, sources, and working dilution of antibodies used in immunohistochemical studies). After incubation, slides were washed with PBS (pH 7.4, 3 times × 5 min). Followed by incubation for 30 min at room temperature with secondary antibody at room temperature. The slides were thereafter rinsed in PBS (pH 7.4, 3 times for 5 min) followed by Incubation for 5-10 min at room temperature with 3,3′-diaminobenzidine (DAB) + substrate-chromogen which results in a brown-colored precipitate at the antigen site. The sections were counterstained with Harris Hematoxylin were used as a counters stained for 30 s. The sections were dehydrated using ethanol alcohol 90%, and 100% II, cleared in xylene, and covered by DPX and I Leitz Dialux 20 Microscope provided by a cannon digital camera (Canon Power shot A95) was used to examine immunohistochemical staining. All Negative controls of the five markers were performed as the previous steps without adding the primary antibody.

Electron microscopic examination.
A different small specimen about 3mm 3 thickness was carefully excised and fixed in a Karnovsky fixative 56 (Table 1) for preparation for scanning electron microscope and transmission electron microscope examination. propylene oxide (approximately 1:1, for 30 min), followed by Epon (for 3 h). Epon was prepared as follows: 5 mL of Epon 812 (Polysciences, Eppelheim, Germany) + 5 mL of Araldite + 12 mL of DDSA. Then, the Epon was mixed thoroughly by incubation in a shaker at 60 °C. Sample polymerization was performed using Epon mix and an accelerator (DMP30) (1.5%). The blocks were incubated for 3 days as follows: 60 °C on the first day, 70 °C on the second day, and 75 °C on the third day.
Ultrathin sections were obtained from semi-thin sections by a Reichert ultra-microtome. The sections (70 nm) were stained with uranyl acetate and lead citrate 61 and examined by JEOL100CX II transmission electron microscope at the Electron Microscopy Unit of Assiut University.
Semi-thin sections stained by PAS stain. Additional resin-embedded specimens were used in PAS stain. Resin sections were treated with a saturated alcoholic solution of sodium hydroxide for 3 min to dissolve the resin 62 . Then processed the methods as usual PAS stain after immersion in ethanol alcohol 100%, 90%, 80%, 70% for 3 min in each concentration. The protocol used in the preparation of semi-thin sections was performed according to references 8,32 .
Digital coloring of scanning and transmission electron microscopic images. We applied digital coloring for the transmission electron microscopic images using the Photo Filter 6.3.2 program to identify different types of cells and structures. Many authors previously used the methods 12,63-67 .
CMEIAS color segmentation (for supplementary images). Negative images were performed using CMEIAS Color Segmentation, an improved computing technology used to process color images by segmenting the foreground object of interest from the background 64 . This has been done by the following steps: open the image with CMEIAS Color Segmentation, then select "Process" from the menu items, and subsequently choose "Negative image" 68-71 . Ethical approval. The National ethics committee of Assiut University and veterinary authorities -from Assiut province, Egypt, were approved the method of work. "All methods were performed in accordance with the relevant guidelines and regulations".

Results
The current study investigated TCs in the intestinal bulb of the Grass carp. The intestinal bulb consisted of epithelium, lamina propria, muscular layer, serosa ( Fig. 2A). Light microscope (LM), Transmission electron microscope (TEM), Scanning electron microscope (SEM), and immunohistochemistry (IHC) recognized Telocytes (TCs). For light microscopic examination, their typical morphological features distinguished TCs that had a cell body contained the nucleus and telopodes. TCs recognized by H&E (Fig. 3A,B) and toluidine blue blue (Fig. 5A-F). They had high affinity for nervous tissue-specific staining; methylene blue ( Fig. 1A-E), Marsland silver stain ( Fig. 2A-G), Grimelius's silver nitrate impregnation (Fig. 4B,C) and Giemsa (Fig. 4E,F) (Fig. 3C), combined AB ph2.5 /PAS stain (Fig. 3D), took the affinity of PAS only. They had a strong affinity for collagen fiber-specific stains including Light green (Fig. 3E,F), Methyl blue (Fig. 3G). While Tc tend to stain by Verhoeff stain (Fig. 4D) and exhibited a reaction similar to elastic fibers rather than collagen fibers when stained by Combined Weigert counter by Van Gieson stain and Van Gieson (Fig. 4G,H). They were visible in paraffin sections stained by Sudan black (Fig. 3H) and osmic acid ( TCs acquired immunological features of endocrine cells. Relation between TCs and epithelial and interstitial endocrine cells was recognized using Grimelius's silver nitrate stain (Fig. 6A-c)., performic acid with methylene blue( (Fig. 6D), Marsland stain (Fig. 6G,H) and Immunohistochemical staining using chromogranin A (Fig. 6E,F,I).Chromogranin A positive TCs in the lamina propria (Fig. 6F,I). The Negative images were represented as a supplementary figure (supplementary Fig. 6).
TCs and their secretory activities were recognized using Acridine orange. They were distributed under the epithelium in the (Fig. 7D,F), in the lamina propria (Fig. 7A,C,E,G,H), in the muscular layer (Fig. 7B).
Localization of TCs in the intestinal blub of Grass carp was confirmed by Immunohistochemical staining. CD34 positive TCs were detected in the sub epithelial layer (Fig. 8A), around the blood vessels in the lamina propria (Fig. 8B). The Negative images were represented as a supplementary figure (supplementary Fig. 1).   (Fig. 9A,B), in the lamina propria (Fig. 9A,C,E,F), between MSF (Fig. 9G-I) and around the myenteric plexus. (Fig. 9D). The Negative images were represented as a supplementary figure (supplementary Fig. 2).
By Scanning electron microscope (SEM), Sub epithelial TC may form a continuous sheet (Fig. 13B,C). TCs were distributed in the lamina propria (Fig. 13A) where they connected to rodlet cells (Fig. 13D-F). TCs formed a network in the lamina propria ( Fig. 14A-C). They were also identified between muscle bundles (Fig. 14D-G, I) and in the serosa (Fig. 14H).
The relation of TCs with endocrine and immune cells was summarized in the illustration figure (Fig. 15).

Discussion
The current study investigated the distribution, morphology, immunohistochemical characterization of TCs in the intestinal bulb of Grass carp. We distinguished TCs using a light microscope that had typical morphological features that had a cell body contained the nucleus and telopodes. TCs affinity for nervous tissue-specific staining (methylene blue, Marsland silver stain , silver impregnation, and Giemsa), collagen fiber-specific stains (Crossmon trichrome, Mallory triple, www.nature.com/scientificreports/ Weigert Van Gieson), polysaccharides-specific stain (PAS,AB pH2.5/PAS) and protein stain (the bromophenol blue) is discussed by Ref. 8 . Methylene blue, Marsland silver stain , silver impregnation stained cytoskeletal elements in the TCs. Cytoskeletal inclusions are stained by methylene blue 72 , Marsland silver stain 73 , silver impregnation 74 . TCs exhibited a strong affinity for carbohydrate staining such as PAS and combined PAS/AB, which revealed neutral polysaccharides. Telocytes had the affinity of protein-detecting stains. They stained positive for bromophenol blue and performic acid with Methylene blue. Proteomic profile analysis is performed for lung TCs, which categorized the functional implications of protein, involved in metabolic processes, cellular processes including cell communication, cytokinesis, cellular component movement, cell cycle. Some proteins implicated in developmental processes including anatomical structure, morphogenesis, mesoderm development, system development, and ectoderm development. Other proteins are linked to localization processes such as vesicle mediated transport, protein transport, and ion transport 3 . TCs had strong staining affinity for Sudan black and osmic acid. This may regard to the lipid components of the TCs cell membrane.
In the current study, TCs were located under the epithelium as an individual cell or formed TCs in the lamina propria, between muscle fibers, between muscle bundles, around the myenteric plexus and in the fibrous tissue. The distribution of TCs in the intestinal bulb of the Grass carp was similar to other tubular organs in mammals. In the bovine uterine tubes, TCs are located under the epithelium forming a subepithelial sheath as well as three other sheath; an outer perimuscular, inner perimuscular and intramuscular sheath. TCs are also distributed in the lamina propria, between the SMF and in the serosa 8 . In chicken ileum, ICC subtypes are mentioned according to location. ICC-MY surrounds the myenteric ganglia. ICC-DMP organized in the deep muscular plexus parallel to the circular muscle bundles. ICC-LP is located in the lamina propria 75   www.nature.com/scientificreports/ of the submucosal plexus (ICC-SMP) occur in the submucosa and submucosal plexus, respectively. ICC of the subserosa locates in the subserosa 76,77 . CD-34 is frequently used as a marker for TCs in mammalian and fish species 17,27 . CD34 is a transmembrane phosphoglycoprotein that commonly identified in hematopoietic stem cells and is detected in other progenitor cells such as interstitial cell progenitors, muscle satellite cells, epithelial progenitors, corneal keratocytes, and vascular endothelial progenitors 78 .
TCs expressed chromogranin A that is a highly acidic secretory glycoprotein and is expressed by most neuroendocrine cells. Chromogranin A is closely associated and packed with neurotransmitter peptides and      Moreover, in vitro studies support TCs role in the regulation of immune response via the paracrine pathway. Uterine TCs have a major role in the activation of peritoneal macrophages. Mouse peritoneal macrophages acquired abundant pseudopodia and cytoplasmic secretory granules when co-cultured with TCs. Macrophages increase the section of several cytokines including TNF-α, IL1-R1, and IL-10, but not TGF-β1, IL-1β, IL-23α, and   www.nature.com/scientificreports/ IL-18, suggesting potential role in immunoregulatory and immunosurveillance mechanism 82 . TCs implicated to triggers tissue changes in dendritic-linked immune disease 83 . C-kit or CD117 is strong markers of stem cells 84,85 . CD117 interacts with the ligand stem cell factor to stimulate cellular differentiation, proliferation, chemotaxis, cell adhesion, and apoptosis 86 . Telocytes are commonly express c-kit. c-kit is a member of the transmembrane kinase receptor. c-kit plays a critical role in TCs functions 87 . C-kit tyrosine kinase is involved in the transduction of intracellular signaling 88 . Telocytes are categorized depending on c-kit immunoaffinity. Some TCs were CD34-positive/c-kit-negative in the human bladder 89 .
TCs had high affinity for immunostaining of cytoskeletal components, particularly the intermediate filaments desmin, and S-100 protein. The term S-10 protein is used based on the physical properties of the protein that is soluble in a 100% saturated solution of ammonium sulfate at neutral pH 90 . S-100 proteins are a member of Ca +2 -binding proteins. They function as Ca +2 sensor proteins and promote Ca +2 signal transduction. S-100 proteins have intracellular and extracellular functions. They combine into specific proteins in cells, e.g., desmin, tubulin, and others 91 . S-100 proteins bind to different types of cell receptors 92,93 . S-100 proteins are implicated in the regulation of cell dynamics and shape through the interaction of cytoskeletal proteins including microfilaments, microtubules, and intermediate filaments 94,95 . They interact with proteins that act as protein Players that regulate the secretory pathway 93 . Thus, S-100 proteins may participate in the regulation of the TC morphology and the secretory functions of TCS. TCS express desmin in the intestinal bulb of the Grass carp. Desmin is a member of the intermediate filament. As the other cytoskeletal elements, desmin has a supportive function to the cells 96 . TCs express desmin in the uterine tube of bovine 8 .

Conclusion
The current study presented the relation of TCs with different types of immune cells in the intestinal bulb of the Grass carp. We suggest that TCs may have an essential role to maintain intestinal immunity. TCs share histochemical and immunological features of endocrine cells. This may reveal that TCS had endocrine properties. Further studies should investigate TCs proteins profile linked to endocrine function.

Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary Information files.