key: cord-0007157-l4e4yc5g
authors: Bódi, Ildikó; Felföldi, Balázs; Minkó, Krisztina; Benyeda, Zsófia; Nagy, Nándor; Kiss, Anna L; Palya, Vilmos; Oláh, Imre
title: Effect of IBDV infection on the interfollicular epithelium of chicken bursa of Fabricius
date: 2018-11-27
journal: Poult Sci
DOI: 10.3382/ps/pey512
sha: 90e8a770e0bfa16a66efcdb3697d49e664346461
doc_id: 7157
cord_uid: l4e4yc5g

In the chicken bursa of Fabricius (BF), the interfollicular epithelium (IFE) consists of cylindrical- and cuboidal-shaped cells. Among the cylindrical-shaped epithelial cells, mucus-producing and caveolin-1 (Cav-1)-expressing cells can be distinguished. Occasionally, the cuboidal-shaped cells also express Cav-1, which suggests that they are precursors of both mucus-producing and Cav-1-expressing cells. Very virulent infectious bursal disease virus (IBDV) impedes the differentiation of Cav-1-expressing cells and shifts the differentiation of cuboidal cells towards mucus-producing cells. In control birds exclusively, the IFE surface shows a mucous membrane, but after IBDV infection, the surfaces of both IFE and FAE are also covered by a mucous membrane. After IBDV infection, the cells of FAE also produce mucus, providing evidence for cell transformation. In late postinfection (pi; 28 d pi), the Cav-1 expression returned in the IFE cells, whereas the follicle (the primary lymphoid organ) underwent atrophy. The appearance of the renewed Cav-1-positive cells is similar to that of the normal basal cell, but they randomly locate in different levels of IFE, suggesting the loss of epithelial polarity. Between days 2 and 7 pi, the Cav-1 expression in the endothelial cells of the cortico-medullary capillary web is variable, which may explain the hemorrhage in several infected birds. The IBDV infection stops the Cav-1 expression and subsequently the cholesterol efflux into the bursal lumen. In the infected birds, the high cholesterol level may further worsen the clinical syndrome of IBDV.

Bursa of Fabricius (BF) is a complex organ. (i) The follicle with the follicle-associated epithelium (FAE) is a primary lymphoid tissue, which is responsible for B-cell maturation and survival. (ii) A considerable number of interfollicular epithelial cells (IFE) express caveolin-1 (Cav-1) and contribute to cholesterol homeostasis by cholesterol efflux into the bursal lumen (Bódi et al., 2017) . (iii) The endothelial cells of the cortico-medullary (CM) capillary web also express Cav-1, which influences the bursal growth (Farsang et al., 2018) .

Cav-1 is an integral membrane protein, localized in the plasma membrane invaginations (caveolae), trans-Golgi-network (TGN), and vesicular structures of the cytoplasm (Rothberg et al., 1992; Couet et al., 2001; Goetz et al., 2008) . It is involved in numerous cellular and physiological functions C 2018 Poultry Science Association Inc. Received August 2, 2018. Accepted October 29, 2018. 1 Corresponding author: olah.imre@med.semmelweis-univ.hu (Parton and Simons, 2007; Feng et al., 2013) : intracellular cholesterol transport, cholesterol homeostasis and cholesterol efflux (Fielding and Fielding, 1995 van Deurs et al., 2003; Martin and Parton, 2005; Frank et al., 2006) , endocytosis (Pelkmans and Helenius, 2002; PU et al., 2002; Kiss, 2012) , cell migration (Navarro et al., 2004) , and cell differentiation (Ravid et al., 2006; Parton and Simons, 2007; Björnstad et al., 2014; Guan et al., 2016) . A large number of caveolae can be found in endothelial cells (van Deurs et al., 2003; Navarro et al., 2004) , adipocytes Guan et al., 2016) , fibroblasts , and type I pneumocytes (Jung et al., 2012; Björnstad et al., 2014) . Cav-1 can mediate simian virus 40 uptake through caveolae, and the internalized caveolae carry the virus into smooth-surfaced endoplasmic reticulum (Pelkmans et al., 2001) .

Vascular system is a dynamic organ, which is able to react and adapt rapidly to internal and external demands. Capillaries are important vessels; hence, most of the exchange of water, water-soluble substances, and respiratory gases takes place through the capillary wall. Transcytosis in the endothelial cells (van Deurs et al., 2003; Navarro et al., 2004) is one of the fundamental 3464 processes by which the endothelial cells contribute to the communication between blood and tissues. The endothelial transcytosis is a basic cytological function that is carried out by caveolae. Cav-1 binds cholesterol in 1 to 1 ratio; therefore, the changes in the Cav-1 expression influence the cholesterol homeostasis. The excess free cholesterol is toxic (Warner et al., 1995; Fielding and Fielding, 1997) ; therefore, the cholesterol efflux from the IFE could be essential for the bird. The middle plical vessel (MPV) of BF branches off to form interfollicular arterioles, from which the CM capillary web offshoots (Schoenwolf et al., 1981; Abbate et al., 2007) for supplying the follicular cortex and medulla. Serious infection of infectious bursa disease virus (IBDV) may result in hemorrhage in the BF, and the CM capillary web is possibly the major site of bursal hemorrhage.

The aim of this study was to determine how the IBDV infection influences the Cav-1 expression and mucusproducing cells of the IFE and endothelial cells of the CM capillary web.

Three-week-old White Leghorn layer type chickens (sex: mixed) of SPF status (Charles River) were used in the test. The chickens were reared under standard conditions on deep litter; feed (sterilized broiler grower mix) and water (tap water) were available ad libitum. The study was conducted in compliance with the provisions of Directive 2010/63/EU, Hungarian Act No. XXVIII/1998, and the Hungarian Governmental Decree No. PE/EA/151-4/2018 (I.29.) and with the permission of the Hungarian competent authority.

IDBV infection was carried out using a very virulent field isolate (D407/02/04/TR). The virus suspension was diluted in steril phoshate buffer (PBS) and at a dose of 3.0 IgEID50/chicken in 0,2 ml PBS was applied orally. Tissue samples were collected 36 h, 2, 4, 7, 14, 21, and 28 d postinfection (pi). The chickens were humanly euthanized by CO 2 inhalation.

Anti-IBDV monoclonal antibody 5A10 was received from Ceva-Phylaxia Ltd. (Budapest, Hungary). The antibody was produced against VP2 protein of IBDV. Polyclonal rabbit anti-Cav-1 was obtained from BD Transduction Laboratories (610406, BD Biosciences, San Jose, CA).

Tissue samples were embedded in liver and frozen in liquid nitrogen. The 10 μm cryostat sections were fixed in cold acetone for 10 min and immunostained, as discussed (Bódi et al., 2017) . Briefly, the sections were incubated with primary antibodies for 45 min, followed by an isotype-specific biotinylated secondary antibody. The binding sites of primary antibodies were detected by 4-chloro-naphtol (C8890, Sigma-Aldrich, Budapest, Hungary).

Tissue samples were fixed in 4% paraformaldehyde for 60 min. After fixation, the tissue was washed 3 times in PBS and embedded in 7.5% gelatin. Fifty-micrometer-thick gelatin-embedded sections were cut with cryomicrotome and collected in PBS under a free-floating condition. For blocking the endogenous peroxidase, the sections were treated by 3% H 2 O 2 (H1009, Sigma-Aldrich, Budapest, Hungary) for 30 min. After 30-min washing, anti Cav-1 antiserum diluted in PBS (1:200) containing 1% bovine serumalbumin was used for 2 h followed by the use of the avidin-biotin complex (1:100; Vectastain Elite PK-6100, Vector 115 Laboratories, Burlingame, CA) and biotin-conjugated goat anti-rabbit secondary antibody (1:200; BA-1100, Burlingame, CA) for 2 h. The immunoperoxidase reaction was developed with 3,3diaminobenzidine (DAB; D8001, Sigma-Aldrich, Budapest, Hungary). After immunohistochemistry, the sections were post-fixed in 1% osmium tetroxide (23310-10, Polysciences Inc., Warrington, PA) for 10 min to preserve DAB staining. After dehydration in graded ethanol, the tissue sections were embedded in Polybed/Araldite 6500 (00552, Polysciences Inc. Warrington, PA) mixture and polymerized for 48 h at 60 • C. The ultrathin sections were contrasted with uranyl acetate and lead citrate and studied with a Hitachi electron microscope, type H-7600.

Five-micron-thick paraffin sections were stained with rutin Hematoxylin-eosin staining and modified Movat pentachrome staining (Bancroft and Gamble 2002) .

Images were processed using Adobe Photoshop.

The IFE of the BF is a pseudostratified epithelium, which consists of cuboidal-shaped basal cells and cylindrical-shaped cells. Among the cylindrical-shaped cells, mucus-secreting ( Figure 2a) and Cav-1-positive cells can be distinguished (Figure 1a) . The Movat pentachrome staining shows the mucus in the apical portion of the IFE cells and the continuous mucous membrane on the surface epithelium of the IFE (Figure 2a) . The cells of the FAE do not express Cav-1 (Figure 1a) , and the mucous membrane is absent in the FAE (Figure 2a) . In the FAE, the absence of Cav-1 suggests the lack of Cav-1-mediated endocytosis by the cells. The shape of the Cav-1-positive cell looks like a "tennis-racket" (Figure 1b) . The "handle" of the racket starts close to the basal lamina, and below the nucleus, the "handle" splits and surrounds the nucleus and possibly the TGN above the nucleus (Figure 1c) . The cytoplasm reveals a fine granulated reaction product (Figure 1b) . Occasionally, the basal cell also expresses Cav-1 (Figure 1b and d) . The Cav-1 appearance in the cuboidal-shaped basal cells suggests that these cells could be the precursor of both types of cylindricalshaped cells; for example, they function as "epithelial progenitor or stem cells," although mitosis in these cells cannot be found; however, their number decreases with age, and in late IBDV infection (Figure 2g ).

The follicular cortex expresses a very fine Cav-1positive network (Figure 1f ), which may be related to the cortical reticular cells (Figure 1e and g) . The transmission micrograph shows coated vesicle-formation in the reticular cell's membrane (Figure 1g ).

The effect of Gumboro virus infection on the primary lymphoid organ of the BF is well-documented, both morphologically (Käufer and Weiss, 1976; Panisup et al., 1988; Ramm et al., 1991; Vervelde and Davison, 1997) and immunologically (Naqi et al., 1983; Rautenschlein et al., 2007; Ingrao et al., 2013) . However, no information was available about the effect of viral infection on the cells of IFE. After 2 d pi, the α-Gumboro immunostaining shows high variability among the bursal follicles (Figure 2d) . A considerable number of follicles do not show α-Gumboro staining, while in the positive follicles, the infected cells are localized in one or two rings. In the case of one ring, the α-Gumboropositive cells are located in the follicular medulla, close to the CM border. If there are two α-Gumboro-positive rings, the outer ring is in the cortex, also close to the CM border (Figure 2d ). Between the two α-Gumboropositive rings, there are scattered positive cells, which migrate between the cortex and medulla (Figure 2d ). In this stage of pi, there is no significant alternation in the Cav-1-positive IFE cells. Between 2 and 7 d pi, the Cav-1 expression is variable in the endothelial cells of the CM capillary web, but the number of Cav-1-positive IFE cells drastically decreased by day 4 pi (Figure 3a) .

The variability in the histopathological signs of follicular infection does not reflect in the Cav-1 expression of the IFE cells, which suggests that the Cav-1 expression is independent of the primary lymphoid organ (FAE+follicle). The virus infection might have hindered the differentiating capacity of basal cells into Cav-1positive cells. The differentiation is possibly shifted towards the mucus-secreting cell because in later stages of the infection, the surface epithelium is highly folded and consists of mucus-secreting cells (Figure 2c) .

In control birds, the mucous membrane-as one of the subjects of innate immunity-covers the surface of the IFE, but not of the FAE (Figure 2a) . The FAE constructs 10% of the bursal surface. The number of FAE is 8,000 to 12,000/bursa, which appear as small islands in the IFE (Oláh and Glick, 1978) . A question has been raised: How does the FAE exclude the mucous membrane? Is it possible that the viscosity of a mucous membrane is so high, "sticky" that it prevents its spreading on the bursal surface? Or the cells of FAE have a peculiar capacity to keep away the mucous substance?

After IBDV infection, the cells of FAE may have undergone transformation to be mucus-producing cells, and the mucous membrane appears on the surface of the transformed cells (Figure 2b) . The "sticky" state of the mucous membrane may be supported by the finding that the mucous-negative FAE cells are devoid of a mu-cous membrane (Figure 2b ). At this stage of infection, the FAE consists of two kinds of cells: (i) the sui generis FAE cells and (ii) mucus-producing transformed cells. The first one is devoid of a mucous membrane, and the second one reveals a mucous membrane. After IBDV infection, the regeneration capacity of bursal follicles may be influenced by the proportion of the two kinds of cells.

The mucous substance appears in the follicular medulla, but not in the follicular cortex, which may be produced by medullary epithelial reticular cells, or the cells of the FAE released the mucous substance into the intercellular space of medulla? Future studies should provide an answer to numerous questions, which touch the innate immunity of BF.

By day 4 pi, the α-Gumboro-positive cells of the inner ring are scattered over the medulla (Figure 2e ). The interfollicular connective tissue does not show α-Gumboro-positive cells. There is a significant decrease in the number of Cav-1-positive IFE cells, and the majority of cells show only the "head" of tennis racket (Figure 3a) . The Cav-1-positive IFE cell releases cholesterol into the bursal lumen (Bódi et al., 2017) . The decreased number of Cav-1-positive cells suggests that the cholesterol efflux is also diminished or ceased. High cholesterol level is toxic for the bird, which may worsen the clinical syndrome of IBDV. The IBDV infection temporary stopped the differentiation of new Cav-1-positive cells. The drastic decrease in the number of Cav-1-positive cells took place by 4 d pi, which may allow us to estimate the differentiation time of Cav-1 and possibly also the mucin-secreting cells from the basal cells. The 4 to 5 d of differentiation time may be confirmed by the finding that a considerable number of Cav-1-positive cells emerged between 21 and 28 d pi. At day 7 pi, in the majority of follicles, the medulla and cortex were cleared from Gumboro-positive cells, but many virus-containing cells appeared in the interfollicular connective tissue (Figure 2f ). In the IFE cells, Cav-1 expression can be seen only occasionally (Figure 3b) . Parallel with the drastically decreased Cav-1-positive IFE cells, the number of the secretory cells increased, and almost every cylindrical-shaped cell is mucous positive (Figure 2g ). In some places, the capillary web seems to be defective (not complete around the follicular medulla; Figure 3b ). Possibly, the endothelial cells lost their capability for caveola formation. The absence of caveola formation in the endothelial cell of the capillary web results in the disappearance of transcytosis, which subsequently damages the communication between blood and bursal follicles. This phenomenon may result in bursal hemorrhage. At 21 d pi, Cav-1 expression may be found in few IFE cells and in the rest of the CM capillary web (Figure 3c ). By day 28 pi, the follicular organization cannot be recognized, but many IFE cells again produced Cav-1 (Figure 3d and e). The shape of these Cav-1-positive cells looks similar to that of normal basal cells, and they appeared in different levels of the epithelium. By day 28 pi, these randomly localized Cav-1-positive cells in the IFE suggest that the IFE lost its polarity, which may be maintained by the primary lymphoid organ (FAE+follicle). This observation shows that Cav-1 expression is not re-established in the former Cav-1-positive cells, but the differentiation capability of basal cells might have been regained following the infection. In the MPV, the Cav-1 expression became strong, but the CM capillary web disappeared (Figure 3d ).

The atrophy of lymphoid tissues of BF and the return of Cav-1 expression in the IFE cells support the hypothesis that the BF is a complex organ (Bódi et al., 2017) . The IFE consists of histologically and functionally three types of cells, and the differentiation and function of these cells are influenced by IBDV infection. (i) Mucus-secreting cells produce a mucous membrane that contributes to the innate immunity. After IBDV infection, the innate immunity overlaps the atrophying primary lymphoid organ. (ii) Cav-1-expressing cells contribute to cholesterol homeostasis of the bird by cholesterol efflux. Cholesterol efflux is ceased by IBDV infection. (iii) Basal cell, which serves as an epithelial progenitor cell, and the IBDV infection shift the differentiation towards mucous-producing cells.

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