Veterinary Immunology and Immunopathology 158 (2014) 182–188
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
TLR4 is constitutively expressed in chick thymic epithelial cells Hai-Bo Huang, Quan-Hang Xiang, Hui Wu, Abdur Rahman Ansari, Le Wen, Xiao-Hong Ge, Ji-Xiang Wang, Ke-Mei Peng, Hua-Zhen Liu ∗ Department of Anatomy, Histology and Embryology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 18 December 2013 Accepted 8 January 2014
Keywords: Chick Thymus development Toll-like receptor 4
a b s t r a c t Toll-like receptor 4 (TLR4) has been suggested to play a regulatory role in immune cell development; however, studies regarding the role of TLR4 in the development of the chick thymus are scarce. In this study, we investigated the distribution and expression pattern of TLR4 in normal chick thymi at different stages of development, in order to better understand the role of TLR4 in chick thymus development. We studied the thymi from 15 chicks, collected at days 7, 21 and 35 of age. The relative change in TLR4 mRNA expression in the chick thymus at different ages was determined by quantitative real-time PCR, and changes in protein expression were analyzed by immunohistochemistry and Western blotting. Furthermore, the distribution of TLR4 in the chick thymus was analyzed by immunohistochemistry, and compared with the distribution of TLR4 expression in juvenile female pigs (gilts). Our results indicated that TLR4 was constitutively expressed in the chick thymus. TLR4 was primarily expressed in the thymic cortico-medullary junction and the medulla, particularly in the epithelial cells of Hassall’s corpuscles. The mRNA and protein expression level of TLR4 increased in the thymus with increasing age (p < 0.05). Taken together, these results indicate that TLR4 is constitutively expressed by epithelial cells in the chick thymus, suggesting it may participate in thymic development by inducing factors affecting its development. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction Toll like receptors (TLRs) are evolutionarily conserved immune receptors that play important roles in the recognition of pathogens and endogenous “danger” signals that are critical for host immune defenses (Akira et al., 2001). To date, 13 murine TLRs and 10 human TLRs have been identified (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2002; Beutler et al., 2006). In chickens, 10 TLRs have been identified, including TLR1 (types 1 and 2), 3, 4, 5, 7, 15 and 21 (Keestra et al., 2013). TLR4 was the first
∗ Corresponding author. Tel.: +86 27 87286970; fax: +86 27 87280408. E-mail address: [email protected] (H.-Z. Liu).
TLR identified in humans and was subsequently found to be the specific receptor for recognition of lipopolysaccharide, the endotoxic component of gram-negative bacteria (Medzhitov and Janeway, 1997; Hoshino et al., 1999). In chickens, TLR4 has been identified and characterized at the molecular and functional level, and suggested to be linked to resistance to infection with Salmonella enterica serovar Typhimurium (Leveque et al., 2003). While previous studies have paid much attention to the function of TLR4 as sensor to trigger the immune system (Beutler, 2000, 2002), accumulating evidence also indicates that TLR4 may affect immune cell development. For example, activated TLR4 alters the differentiation of hematopoietic stem cells, yielding larger numbers of myeloid cells at the expense of lymphoid cells (Boiko and Borghesi, 2012). In
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H.-B. Huang et al. / Veterinary Immunology and Immunopathology 158 (2014) 182–188
addition, the maturation of myeloid cells (e.g. dendritic cells and macrophages) and lymphoid cells (e.g. B cells, T cells) can also be induced by the TLR4 signaling pathway (Michelsen et al., 2001; Pasare and Medzhitov, 2004; Hayashi et al., 2005; Benoit et al., 2008; Pufnock et al., 2011). The thymus is crucial for the development of T lymphocytes by providing an inductive microenvironment in which bone marrow-derived lymphoid progenitor cells undergo differentiation, selection and proliferation processes to develop into functional T-cells (Gameiro et al., 2010). TLR4 expression at the mRNA level has previously been shown in the chicken, mouse, pig and human thymus (Iwami et al., 2000; Bernasconi et al., 2005; Iqbal et al., 2005; Iournals, 2011). Furthermore, its expression level has been shown to vary with age during development, suggesting TLR4 may affect thymus development (Iournals, 2011), although the underlying mechanism is unknown. Studies regarding the expression of TLR4 in the chick thymus are rare; therefore, the present investigation was designed to study the temporal and cellular distribution of TLR4 expression in the developing chick thymus, with the goal of obtaining evidence that TLR4 functions in chick thymus development. 2. Materials and methods 2.1. Animals Animal procedures were performed according to protocols approved by Hubei Province, PR China Animal Care and Use Committee for Biological Studies. Fifteen 1-dayold broiler chicks (Cobb strain) were obtained from the Wuhan Zhengda chicken breeding company. The chicks were housed under conventional conditions without any vaccinations. Three pure-bred Meishan gilts, at the age of 14 days, were obtained from the Pig Breeding Farm of Huazhong Agricultural University, and used for comparative purposes in this study. 2.2. Tissue collection and preparation Thymi were collected from chicks at 7, 21 or 35 days of age (n = 5 in each group), and were fixed in 4% paraformaldehyde for morphological analysis, or frozen in liquid nitrogen and stored at −70 ◦ C for biomolecular analysis. Thymi collected from gilts were used only for morphological analysis and were processed identically to chick thymi. 2.3. Immunostaining Chick thymi collected at day 7 were used to determine the protein expression pattern of TLR4 and pancytokeratin (PCK); immunostaining was visualized by fluorescence and chromogenic markers. After 24 h of fixation, the thymic tissue was dehydrated, and embedded in paraffin wax. Next, 4-m tissue sections were cut using a Leica microtome (Leica Microsystems Nussloch GmbH, Nussloch, Germany), mounted on polylysine-coated slides (Boster, Wuhan, China) and stored at 4 ◦ C prior to staining. Sections
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were deparaffinized in xylene and rehydrated in a graded series of ethanol. Antigen retrieval was performed using a microwave oven with a maximum delivered power of 700 W (MYA-2270M, Haier, Qindao, China). Sections were microwaved in citric acid buffer (pH 6.0) for 25 min (5 min at 700 W, 20 min at 116 W) and then allowed to cool down at room temperature. For immunofluorescence, background staining and autofluorescence were quenched using previously described methods (Casella et al., 2004). In brief, sections were treated with 0.1% sodium borohydride in PBS (30 min), followed by 5 min incubation in 0.5% Sudan black in 70% ethanol. Non-specific antibody binding was blocked by incubating sections with 3% bovine serum albumin (BSA) for 30 min at 37 ◦ C. Sections were then separately incubated with a mouse anti-PCK primary antibody (clone PCK-26; Boster; dilution 1:50) at 4 ◦ C overnight. The anti-PCK antibody (clone PCK-26) has previously been used to identify PCK expression in birds (Ciriaco et al., 1996, 1997). The next day, sections were washed and then incubated with a biotinylated goat anti-mouse IgG (Boster) for the anti-PCK antibody, for 20 min at 37 ◦ C. Immunostaining was visualized with a Cy3 conjugated streptavidin (Boster) for 5 min at 37 ◦ C. Finally, sections were washed and mounted with a coverslip. Immunostaining was detected using a conventional fluorescence microscope (Zeiss AX10, Göttingen, Germany). Negative control sections were prepared using the same method, omitting the primary antibody. The preparation of slides for immunohistochemistry was the same as described above. After antigen retrieval, sections were incubated with 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidases. Non-specific antibody binding was blocked by incubating sections with 3% bovine serum albumin (BSA) for 30 min at 37 ◦ C. Sections were then incubated with primary antibodies against PCK (described above) and two separate anti-TLR4 antibodies, at a dilution of 1:100. The rabbit anti-TLR4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was raised against a peptide near the C-terminus of human TLR4, and has previously been used to identify TLR4 expression in the chicken (Farnell et al., 2003; Subedi et al., 2007; Ozoe et al., 2009). The rabbit anti-TLR4 antibody (Boster) we also used was raised against a peptide near the N-terminus of human TLR4 (50–68 aa, DNLPFSTKNLDLSFNPLRH). Next, sections were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster) for 20 min at 37 ◦ C. Immunostaining was then visualized using diaminobenzidine (DAB) Chromogenic Kits (Boster), which produced yellow staining, and counterstaining was performed using hematoxylin. For co-expression analysis of TLR4 and PCK, a yellow DAB Chromogenic Kit (Boster) was used for TLR4, and blue DAB Chromogenic Kit (Boster) was used for PCK. Counterstaining was performed using hematoxylin for TLR4 and fast red for PCK, respectively. Finally, sections were washed, dried, dehydrated, cleared, and mounted with a coverslip. Staining was examined by light microscopy (Olympus BX51, Tokyo, Japan). Negative control sections were prepared using
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the same method, but without the addition of the primary antibody. The co-expression of TLR4 and PCK was determined by comparing their expression in serial sections. 2.4. Semi-quantitative analysis for the TLR4 protein in chick thymus with age Chick thymi collected at days 7, 21 and 35 were used to determine the protein expression pattern of TLR4 with an anti-TLR4 antibody (Boster, Wuhan, China) by immunohistochemistry. The relative optical density of staining in tissue sections was analyzed using the integrated optical density (IOD) ratio. This ratio was calculated by dividing the IOD with the total area of interest (comprising the cortico-medullary junction and the medulla). Briefly, three 6.3× magnification TIFF-format images from five individual chicks in each group were analyzed in a blinded manner. All of the images were taken using the same microscope and camera set. Image-Pro Plus 6.0 software (Media Cybernetics, USA) was used to calculate the IOD ratio for positive staining. 2.5. Quantitative real time PCR The mRNA expression of TLR4 was determined in the chick thymus at days 7, 21 and 35. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA,
USA), according to the manufacturer’s protocol. Extracted RNA was checked for quality by 0.8% agarose gel electrophoresis. To remove contaminating genomic DNA, 5 g of RNA was treated with 1 l (50 u/l) RNase-free DNase I (Fermentas, Opelstrasse, Germany) at 37 ◦ C for 30 min, then mixed with 1 l (50 mM/ml) EDTA and incubated at 60 ◦ C for 10 min to inhibit the DNase. The cDNA was synthesized by reverse transcription using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Opelstrasse, Germany), according to the manufacturer’s instructions. Subsequently, quantitative analysis of TLR4 expression was performed using real-time PCR. The level of TLR4 gene expression was normalized to the reference gene -actin. The primer sequences were as follows: TLR4, forward: 5 AAG CCA TGG AAG GCT GCT AGA C-3 , reverse: 5 -CCA CCC TGG ACT TGG ACC TCA-3 ; and -actin, forward: 5 -CAT CTT TCT TGG GTA TGG AGT C-3 , reverse: 5 -CAG GGA GGC CAG GAT AGA G-3 . Real-time PCR was performed in 20 l reaction mixtures containing 10 l SYBR Select Master Mix (Applied Biosystems), 0.3 l of each forward and reverse primer, and 2 l of template cDNA. Real-time PCR experiments were carried out using a LightCycler 480 (Roche, Mannheim, Germany) with 2 min at 95 ◦ C, followed by 40 cycles of amplification (20 s at 95 ◦ C, 20 s at 65 ◦ C, and 15 s at 72 ◦ C). PCR was performed in triplicate and gene expression levels were quantified using the Ct (Ct) value method (Livak and Schmittgen, 2001). Gene expression data are expressed as the mean ± SEM.
Fig. 1. Immunohistochemical analysis of TLR4 in thymus of chick (7 days) and gilt (14 days) with primary antibodies from two separate companies. Both in the chick (A, B) and gilt thymus (C, D), TLR4-positive staining (yellow) was primarily found at the cortico-medullary junction and medulla, specifically in Hassall’s corpuscles. The antibody from Boster (A, C) and Santa Cruz (B, D) both yield a similar pattern of TLR4 expression. The black rectangle in panels A and B is enlarged in upper right corner of each panel. Abbreviations: cortex (Co), medulla (Me). Scale bar in A and B = 100 m; C and D = 50 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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2.6. Western blotting
2.7. Statistic analysis
Immunoblotting for TLR4 was performed according to standard protocols. Briefly, frozen chick thymus tissue was crushed in liquid nitrogen and homogenized in lysis buffer containing a protease inhibitor. The tissue supernatants were then vortexed for 20 s and incubated on ice for 30 min and then centrifuged at 12,000 × g for 5 min. The protein concentration in the supernatants was determined by the Bradford assay (Bio-Rad Protein Dye Reagent; Bio-Rad, Richmond, USA) against a BSA standard. Samples of soluble proteins (40 g) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% polyacrylamide gels. After separation, proteins were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, USA). The membrane was blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBS-T) plus 5% milk for 1 h before incubation overnight in the primary rabbit anti-human TLR4 (Boster, 1:400) or antihuman -actin (Santa Cruz, 1:1000). After washing five times in TBS-T, the membranes were incubated in the secondary antibody conjugated to peroxidase (1:3000) for 30 min. Immunopositive bands were visualized with an enhanced chemiluminescene (ECL) western blotting detection reagent (Goodbio technology Co., Ltd, Wuhan, China). Films were digitized at 1200 dpi (scanner Epson Perfection V300 Photo).
All the data were presented as mean ± SEM, with samples derived from 5 individuals in each group. Significant differences between groups were studied using a twotailed t-test. All analyses and graphic representations were performed with Prism software 6.0 (GraphPad Software, Inc., San Diego, USA). A p-value < 0.05 was considered statistically significant. 3. Results 3.1. Distribution of TLR4 expression in the chick and gilt thymus The expression of TLR4 in the chick and gilt thymus was examined by immunostaining and visualized with chromogenic kits (Fig. 1). We found that TLR4 was expressed in the chick thymus at different ages (Fig. 3). In chicks, our immunostaining revealed strong expression of TLR4 at the cortico-medullary junction and in the medulla of the thymus, especially in Hassall’s corpuscles, with no obvious staining in the cortex (Fig. 1), using anti-TLR4 antibody purchased from Boster (Fig. 1A) and Santa Cruz Biotechnology (Fig. 1B). In Hassall’s corpuscles, TLR4-positive cells had a stellate, annular or irregular shape (Fig. 1A and B), suggesting that TLR4-positive cells may be epitheliallike cells. Furthermore, we analyzed these two anti-TLR4
Fig. 2. Immunostaining analysis of TLR4 and PCK in chick thymus at day 7. PCK-positive staining (yellow) was primarily found at the cortico-medullary junction and cortex (A, B). Colocalization of PCK (blue; C) and TLR4 (yellow; D) was also studied in adjacent sections. Arrows in C and D indicate that the same cells that are immunostained for PCK are also stained for TLR4. The black rectangle in panels C and D is enlarged in upper right corner of each panel. Abbreviations: cortex (Co), medulla (Me). Scale bar in A and B = 50 m; C and D = 100 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Immunohistochemical analysis of TLR4 in chick thymus at days 7, 21 and 35. TLR4 positive staining (yellow) was consistently found at the corticomedullary junction and medulla in chick thymus at days 7 (A), 21 (B) and 35 (C). Immune staining was strongest in the 35 day old chick thymus (C). Scale bar in A, B and C = 200 m. Quantification of average integrated optical density (IOD) of TLR4 positive staining in the chick thymus (7 days group vs. 21 days group, p = 0.06; 7 days group versus 35 days group, p = 0.04, n = 5/group; D). All data are graphed as the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
antibodies in another species (gilt), and obtained the similar results. Specifically, TLR4 expression was located primarily at the cortico-medullary junction and in the medulla of the thymus, especially in Hassall’s corpuscles (Fig. 1C and D).
3.2. Colocalization of TLR4 and PCK in thymic epithelial cells To confirm whether TLR4 was expressed by thymic epithelial cells, the distribution of epithelial cells was
Fig. 4. Analysis of TLR4 mRNA and protein expression in chick thymus at days 7, 21 and 35 of age. (A) The quantification of TLR4 mRNA in the chick thymus at days 7 (n = 5); 21 (n = 5) and 21 (n = 5) using real-time PCR. All data are graphed as the mean ± SEM. (B) Western blotting analysis of TLR4 and -actin in chick thymus at days 7 and 35.
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assessed using an antibody against the epithelial cell marker PCK. We observed that epithelial cells strongly expressing PCK were found at the cortico-medullary junction and in the cortex, especially in Hassall’s corpuscles, and that there was no obvious positive signal in the central medulla (Fig. 2A and B). Comparison of TLR4 and PCK staining in serial sections revealed that TLR4 was coexpressed in PCK-positive epithelial cells in Hassall’s corpuscles (Fig. 2C and D). This result indicates that TLR4 is strongly expressed in the epithelial cells of Hassall’s corpuscles within the chick thymus. 3.3. Expression of TLR4 in the chick thymus with age To perform quantitative analysis of TLR4 expression in the chick thymus at different ages, we investigated TLR4 mRNA expression by real-time PCR and TLR4 protein expression by immunohistochemistry and Western blotting. Our results revealed that there was a trend for TLR4 expression increasing with age, with significant differences between TLR4 expression at day 7 and day 35 (p < 0.05; Figs. 3 and 4). These results clearly indicate that the mRNA and protein expression of TLR4 increases with age in the chick thymus. 4. Discussion In the present study, the distribution pattern of TLR4 in the thymus of the broiler chick and Meishan gilt was investigated for the first time. Our results show that TLR4 is expressed in the epithelial cells in the cortico-medullary junction and medulla of the thymus of the chick and gilt, and is particularly strongly expressed in Hassall’s corpuscles. It is known that thymic epithelial cells can guide the later development of T cells in the thymic corticomedullary junction and medulla, by providing self-antigens that affect the orientation of T cells during differentiation (Palmer, 2003; Stritesky et al., 2012), and other products (e.g. cytokines and chemokines) that induce T cell migration (Takahama, 2006). Based on their interaction with self-antigens, most high-affinity thymocytes undergo negative selection and die to preserve central tolerance, while a few high-affinity thymocytes undergo agonist selection and become self-reactive T cells (e.g. regulatory T cells, Tregs), whereas low-affinity thymocytes survive after negative selection and become classical naïve CD4 or CD8T cells (Palmer, 2003; Stritesky et al., 2012). Moreover, the thymic epithelial cells of Hassall’s corpuscles can express thymic stromal lymphopoietin that indirectly induce the maturation of CD4+CD25+ Tregs (Watanabe et al., 2005). This process is of clinical importance, as it is known that defects in the thymic stroma can result in immunodeficiency or autoimmunity (Fletcher et al., 2011). TLR4 can be stimulated by both endogenous and exogenous stress signals, such as endogenous fibronectin, fibrinogen, heparan and heat shock proteins (Okamura et al., 2001; Smiley et al., 2001; Johnson et al., 2002; Termeer et al., 2002; Tsan and Gao, 2004), as well as exogenous bacterial lipopolysaccharide (Medzhitov and
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Janeway, 1997; Hoshino et al., 1999). After activation, TLR4 can initiate a series of intracellular reactions via phosphorylation, ubiquitinylation and binding of downstream signaling molecules, such as IRAK1, IRAK2, IRAK4, TRAF6 and NEMO, and then causes nuclear location of transcription factors such as NF-B, which induce the expression of proinflammatory and antiinflammatory cytokines (Kawai and Akira, 2007). Based on the expression patterns that we detected, TLR4 may affect the later development of T cells, including Tregs, by mediating thymus epithelial cell-derived activities and inducing the release of cytokines. Furthermore, it is interesting to note that abnormal thymic development in human is associated with increased TLR4 expression (Bernasconi et al., 2005). We observed that the expression of TLR4 in the chick thymus increased with age during postnatal development. The thymus is a common target in various diseases, and intrathymic homeostasis and immune safety are important for normal thymus development (Savino, 2006). Neonatal vertebrates have limited ability to synthesize antibodies endogenously, and their humoral (antibody-mediated) immune defenses are based on maternally-derived antibodies, which disappear around the time of onset of active antibody production by the offspring. For example, in the chick (Gallus domesticus), maternal antibodies only persist for approximately 14 days after birth (Grindstaff et al., 2003). Therefore, increased TLR4 expression may affect chick thymus development, especially in the later stages of postnatal development, by inducing cytokines and other antimicrobial factors while maternal antibodies gradually leave the chick; however, the mechanisms that govern this process require further investigation. 5. Conclusions We found that TLR4 was strongly expressed in epithelial cells and was primarily distributed in the cortico-medullary junction and medulla, especially Hassall’s corpuscles. We also found that the mRNA expression level of TLR4 increased with age. Taken together, our study provides morphological and quantitative information regarding the potential role of TLR4 in chick thymus development, and indicates that TLR4 may participate in thymus development by inducing factors affecting their development. Conflict of interest The authors declare that there are no conflict of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30800808), the Fundamental Research Funds for the Central Universities (2010SC02, 2012ZYTS048, 2013YB09), and the National College Students Innovation Experiment Program (201210504005).
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
TLR4 is constitutively expressed in chick thymic epithelial cells Hai-Bo Huang, Quan-Hang Xiang, Hui Wu, Abdur Rahman Ansari, Le Wen, Xiao-Hong Ge, Ji-Xiang Wang, Ke-Mei Peng, Hua-Zhen Liu ∗ Department of Anatomy, Histology and Embryology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
a r t i c l e
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Article history: Received 19 June 2013 Received in revised form 18 December 2013 Accepted 8 January 2014
Keywords: Chick Thymus development Toll-like receptor 4
a b s t r a c t Toll-like receptor 4 (TLR4) has been suggested to play a regulatory role in immune cell development; however, studies regarding the role of TLR4 in the development of the chick thymus are scarce. In this study, we investigated the distribution and expression pattern of TLR4 in normal chick thymi at different stages of development, in order to better understand the role of TLR4 in chick thymus development. We studied the thymi from 15 chicks, collected at days 7, 21 and 35 of age. The relative change in TLR4 mRNA expression in the chick thymus at different ages was determined by quantitative real-time PCR, and changes in protein expression were analyzed by immunohistochemistry and Western blotting. Furthermore, the distribution of TLR4 in the chick thymus was analyzed by immunohistochemistry, and compared with the distribution of TLR4 expression in juvenile female pigs (gilts). Our results indicated that TLR4 was constitutively expressed in the chick thymus. TLR4 was primarily expressed in the thymic cortico-medullary junction and the medulla, particularly in the epithelial cells of Hassall’s corpuscles. The mRNA and protein expression level of TLR4 increased in the thymus with increasing age (p < 0.05). Taken together, these results indicate that TLR4 is constitutively expressed by epithelial cells in the chick thymus, suggesting it may participate in thymic development by inducing factors affecting its development. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.
1. Introduction Toll like receptors (TLRs) are evolutionarily conserved immune receptors that play important roles in the recognition of pathogens and endogenous “danger” signals that are critical for host immune defenses (Akira et al., 2001). To date, 13 murine TLRs and 10 human TLRs have been identified (Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2002; Beutler et al., 2006). In chickens, 10 TLRs have been identified, including TLR1 (types 1 and 2), 3, 4, 5, 7, 15 and 21 (Keestra et al., 2013). TLR4 was the first
∗ Corresponding author. Tel.: +86 27 87286970; fax: +86 27 87280408. E-mail address: [email protected] (H.-Z. Liu).
TLR identified in humans and was subsequently found to be the specific receptor for recognition of lipopolysaccharide, the endotoxic component of gram-negative bacteria (Medzhitov and Janeway, 1997; Hoshino et al., 1999). In chickens, TLR4 has been identified and characterized at the molecular and functional level, and suggested to be linked to resistance to infection with Salmonella enterica serovar Typhimurium (Leveque et al., 2003). While previous studies have paid much attention to the function of TLR4 as sensor to trigger the immune system (Beutler, 2000, 2002), accumulating evidence also indicates that TLR4 may affect immune cell development. For example, activated TLR4 alters the differentiation of hematopoietic stem cells, yielding larger numbers of myeloid cells at the expense of lymphoid cells (Boiko and Borghesi, 2012). In
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addition, the maturation of myeloid cells (e.g. dendritic cells and macrophages) and lymphoid cells (e.g. B cells, T cells) can also be induced by the TLR4 signaling pathway (Michelsen et al., 2001; Pasare and Medzhitov, 2004; Hayashi et al., 2005; Benoit et al., 2008; Pufnock et al., 2011). The thymus is crucial for the development of T lymphocytes by providing an inductive microenvironment in which bone marrow-derived lymphoid progenitor cells undergo differentiation, selection and proliferation processes to develop into functional T-cells (Gameiro et al., 2010). TLR4 expression at the mRNA level has previously been shown in the chicken, mouse, pig and human thymus (Iwami et al., 2000; Bernasconi et al., 2005; Iqbal et al., 2005; Iournals, 2011). Furthermore, its expression level has been shown to vary with age during development, suggesting TLR4 may affect thymus development (Iournals, 2011), although the underlying mechanism is unknown. Studies regarding the expression of TLR4 in the chick thymus are rare; therefore, the present investigation was designed to study the temporal and cellular distribution of TLR4 expression in the developing chick thymus, with the goal of obtaining evidence that TLR4 functions in chick thymus development. 2. Materials and methods 2.1. Animals Animal procedures were performed according to protocols approved by Hubei Province, PR China Animal Care and Use Committee for Biological Studies. Fifteen 1-dayold broiler chicks (Cobb strain) were obtained from the Wuhan Zhengda chicken breeding company. The chicks were housed under conventional conditions without any vaccinations. Three pure-bred Meishan gilts, at the age of 14 days, were obtained from the Pig Breeding Farm of Huazhong Agricultural University, and used for comparative purposes in this study. 2.2. Tissue collection and preparation Thymi were collected from chicks at 7, 21 or 35 days of age (n = 5 in each group), and were fixed in 4% paraformaldehyde for morphological analysis, or frozen in liquid nitrogen and stored at −70 ◦ C for biomolecular analysis. Thymi collected from gilts were used only for morphological analysis and were processed identically to chick thymi. 2.3. Immunostaining Chick thymi collected at day 7 were used to determine the protein expression pattern of TLR4 and pancytokeratin (PCK); immunostaining was visualized by fluorescence and chromogenic markers. After 24 h of fixation, the thymic tissue was dehydrated, and embedded in paraffin wax. Next, 4-m tissue sections were cut using a Leica microtome (Leica Microsystems Nussloch GmbH, Nussloch, Germany), mounted on polylysine-coated slides (Boster, Wuhan, China) and stored at 4 ◦ C prior to staining. Sections
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were deparaffinized in xylene and rehydrated in a graded series of ethanol. Antigen retrieval was performed using a microwave oven with a maximum delivered power of 700 W (MYA-2270M, Haier, Qindao, China). Sections were microwaved in citric acid buffer (pH 6.0) for 25 min (5 min at 700 W, 20 min at 116 W) and then allowed to cool down at room temperature. For immunofluorescence, background staining and autofluorescence were quenched using previously described methods (Casella et al., 2004). In brief, sections were treated with 0.1% sodium borohydride in PBS (30 min), followed by 5 min incubation in 0.5% Sudan black in 70% ethanol. Non-specific antibody binding was blocked by incubating sections with 3% bovine serum albumin (BSA) for 30 min at 37 ◦ C. Sections were then separately incubated with a mouse anti-PCK primary antibody (clone PCK-26; Boster; dilution 1:50) at 4 ◦ C overnight. The anti-PCK antibody (clone PCK-26) has previously been used to identify PCK expression in birds (Ciriaco et al., 1996, 1997). The next day, sections were washed and then incubated with a biotinylated goat anti-mouse IgG (Boster) for the anti-PCK antibody, for 20 min at 37 ◦ C. Immunostaining was visualized with a Cy3 conjugated streptavidin (Boster) for 5 min at 37 ◦ C. Finally, sections were washed and mounted with a coverslip. Immunostaining was detected using a conventional fluorescence microscope (Zeiss AX10, Göttingen, Germany). Negative control sections were prepared using the same method, omitting the primary antibody. The preparation of slides for immunohistochemistry was the same as described above. After antigen retrieval, sections were incubated with 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidases. Non-specific antibody binding was blocked by incubating sections with 3% bovine serum albumin (BSA) for 30 min at 37 ◦ C. Sections were then incubated with primary antibodies against PCK (described above) and two separate anti-TLR4 antibodies, at a dilution of 1:100. The rabbit anti-TLR4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was raised against a peptide near the C-terminus of human TLR4, and has previously been used to identify TLR4 expression in the chicken (Farnell et al., 2003; Subedi et al., 2007; Ozoe et al., 2009). The rabbit anti-TLR4 antibody (Boster) we also used was raised against a peptide near the N-terminus of human TLR4 (50–68 aa, DNLPFSTKNLDLSFNPLRH). Next, sections were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster) for 20 min at 37 ◦ C. Immunostaining was then visualized using diaminobenzidine (DAB) Chromogenic Kits (Boster), which produced yellow staining, and counterstaining was performed using hematoxylin. For co-expression analysis of TLR4 and PCK, a yellow DAB Chromogenic Kit (Boster) was used for TLR4, and blue DAB Chromogenic Kit (Boster) was used for PCK. Counterstaining was performed using hematoxylin for TLR4 and fast red for PCK, respectively. Finally, sections were washed, dried, dehydrated, cleared, and mounted with a coverslip. Staining was examined by light microscopy (Olympus BX51, Tokyo, Japan). Negative control sections were prepared using
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the same method, but without the addition of the primary antibody. The co-expression of TLR4 and PCK was determined by comparing their expression in serial sections. 2.4. Semi-quantitative analysis for the TLR4 protein in chick thymus with age Chick thymi collected at days 7, 21 and 35 were used to determine the protein expression pattern of TLR4 with an anti-TLR4 antibody (Boster, Wuhan, China) by immunohistochemistry. The relative optical density of staining in tissue sections was analyzed using the integrated optical density (IOD) ratio. This ratio was calculated by dividing the IOD with the total area of interest (comprising the cortico-medullary junction and the medulla). Briefly, three 6.3× magnification TIFF-format images from five individual chicks in each group were analyzed in a blinded manner. All of the images were taken using the same microscope and camera set. Image-Pro Plus 6.0 software (Media Cybernetics, USA) was used to calculate the IOD ratio for positive staining. 2.5. Quantitative real time PCR The mRNA expression of TLR4 was determined in the chick thymus at days 7, 21 and 35. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA,
USA), according to the manufacturer’s protocol. Extracted RNA was checked for quality by 0.8% agarose gel electrophoresis. To remove contaminating genomic DNA, 5 g of RNA was treated with 1 l (50 u/l) RNase-free DNase I (Fermentas, Opelstrasse, Germany) at 37 ◦ C for 30 min, then mixed with 1 l (50 mM/ml) EDTA and incubated at 60 ◦ C for 10 min to inhibit the DNase. The cDNA was synthesized by reverse transcription using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Opelstrasse, Germany), according to the manufacturer’s instructions. Subsequently, quantitative analysis of TLR4 expression was performed using real-time PCR. The level of TLR4 gene expression was normalized to the reference gene -actin. The primer sequences were as follows: TLR4, forward: 5 AAG CCA TGG AAG GCT GCT AGA C-3 , reverse: 5 -CCA CCC TGG ACT TGG ACC TCA-3 ; and -actin, forward: 5 -CAT CTT TCT TGG GTA TGG AGT C-3 , reverse: 5 -CAG GGA GGC CAG GAT AGA G-3 . Real-time PCR was performed in 20 l reaction mixtures containing 10 l SYBR Select Master Mix (Applied Biosystems), 0.3 l of each forward and reverse primer, and 2 l of template cDNA. Real-time PCR experiments were carried out using a LightCycler 480 (Roche, Mannheim, Germany) with 2 min at 95 ◦ C, followed by 40 cycles of amplification (20 s at 95 ◦ C, 20 s at 65 ◦ C, and 15 s at 72 ◦ C). PCR was performed in triplicate and gene expression levels were quantified using the Ct (Ct) value method (Livak and Schmittgen, 2001). Gene expression data are expressed as the mean ± SEM.
Fig. 1. Immunohistochemical analysis of TLR4 in thymus of chick (7 days) and gilt (14 days) with primary antibodies from two separate companies. Both in the chick (A, B) and gilt thymus (C, D), TLR4-positive staining (yellow) was primarily found at the cortico-medullary junction and medulla, specifically in Hassall’s corpuscles. The antibody from Boster (A, C) and Santa Cruz (B, D) both yield a similar pattern of TLR4 expression. The black rectangle in panels A and B is enlarged in upper right corner of each panel. Abbreviations: cortex (Co), medulla (Me). Scale bar in A and B = 100 m; C and D = 50 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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2.6. Western blotting
2.7. Statistic analysis
Immunoblotting for TLR4 was performed according to standard protocols. Briefly, frozen chick thymus tissue was crushed in liquid nitrogen and homogenized in lysis buffer containing a protease inhibitor. The tissue supernatants were then vortexed for 20 s and incubated on ice for 30 min and then centrifuged at 12,000 × g for 5 min. The protein concentration in the supernatants was determined by the Bradford assay (Bio-Rad Protein Dye Reagent; Bio-Rad, Richmond, USA) against a BSA standard. Samples of soluble proteins (40 g) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% polyacrylamide gels. After separation, proteins were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, USA). The membrane was blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBS-T) plus 5% milk for 1 h before incubation overnight in the primary rabbit anti-human TLR4 (Boster, 1:400) or antihuman -actin (Santa Cruz, 1:1000). After washing five times in TBS-T, the membranes were incubated in the secondary antibody conjugated to peroxidase (1:3000) for 30 min. Immunopositive bands were visualized with an enhanced chemiluminescene (ECL) western blotting detection reagent (Goodbio technology Co., Ltd, Wuhan, China). Films were digitized at 1200 dpi (scanner Epson Perfection V300 Photo).
All the data were presented as mean ± SEM, with samples derived from 5 individuals in each group. Significant differences between groups were studied using a twotailed t-test. All analyses and graphic representations were performed with Prism software 6.0 (GraphPad Software, Inc., San Diego, USA). A p-value < 0.05 was considered statistically significant. 3. Results 3.1. Distribution of TLR4 expression in the chick and gilt thymus The expression of TLR4 in the chick and gilt thymus was examined by immunostaining and visualized with chromogenic kits (Fig. 1). We found that TLR4 was expressed in the chick thymus at different ages (Fig. 3). In chicks, our immunostaining revealed strong expression of TLR4 at the cortico-medullary junction and in the medulla of the thymus, especially in Hassall’s corpuscles, with no obvious staining in the cortex (Fig. 1), using anti-TLR4 antibody purchased from Boster (Fig. 1A) and Santa Cruz Biotechnology (Fig. 1B). In Hassall’s corpuscles, TLR4-positive cells had a stellate, annular or irregular shape (Fig. 1A and B), suggesting that TLR4-positive cells may be epitheliallike cells. Furthermore, we analyzed these two anti-TLR4
Fig. 2. Immunostaining analysis of TLR4 and PCK in chick thymus at day 7. PCK-positive staining (yellow) was primarily found at the cortico-medullary junction and cortex (A, B). Colocalization of PCK (blue; C) and TLR4 (yellow; D) was also studied in adjacent sections. Arrows in C and D indicate that the same cells that are immunostained for PCK are also stained for TLR4. The black rectangle in panels C and D is enlarged in upper right corner of each panel. Abbreviations: cortex (Co), medulla (Me). Scale bar in A and B = 50 m; C and D = 100 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Immunohistochemical analysis of TLR4 in chick thymus at days 7, 21 and 35. TLR4 positive staining (yellow) was consistently found at the corticomedullary junction and medulla in chick thymus at days 7 (A), 21 (B) and 35 (C). Immune staining was strongest in the 35 day old chick thymus (C). Scale bar in A, B and C = 200 m. Quantification of average integrated optical density (IOD) of TLR4 positive staining in the chick thymus (7 days group vs. 21 days group, p = 0.06; 7 days group versus 35 days group, p = 0.04, n = 5/group; D). All data are graphed as the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
antibodies in another species (gilt), and obtained the similar results. Specifically, TLR4 expression was located primarily at the cortico-medullary junction and in the medulla of the thymus, especially in Hassall’s corpuscles (Fig. 1C and D).
3.2. Colocalization of TLR4 and PCK in thymic epithelial cells To confirm whether TLR4 was expressed by thymic epithelial cells, the distribution of epithelial cells was
Fig. 4. Analysis of TLR4 mRNA and protein expression in chick thymus at days 7, 21 and 35 of age. (A) The quantification of TLR4 mRNA in the chick thymus at days 7 (n = 5); 21 (n = 5) and 21 (n = 5) using real-time PCR. All data are graphed as the mean ± SEM. (B) Western blotting analysis of TLR4 and -actin in chick thymus at days 7 and 35.
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assessed using an antibody against the epithelial cell marker PCK. We observed that epithelial cells strongly expressing PCK were found at the cortico-medullary junction and in the cortex, especially in Hassall’s corpuscles, and that there was no obvious positive signal in the central medulla (Fig. 2A and B). Comparison of TLR4 and PCK staining in serial sections revealed that TLR4 was coexpressed in PCK-positive epithelial cells in Hassall’s corpuscles (Fig. 2C and D). This result indicates that TLR4 is strongly expressed in the epithelial cells of Hassall’s corpuscles within the chick thymus. 3.3. Expression of TLR4 in the chick thymus with age To perform quantitative analysis of TLR4 expression in the chick thymus at different ages, we investigated TLR4 mRNA expression by real-time PCR and TLR4 protein expression by immunohistochemistry and Western blotting. Our results revealed that there was a trend for TLR4 expression increasing with age, with significant differences between TLR4 expression at day 7 and day 35 (p < 0.05; Figs. 3 and 4). These results clearly indicate that the mRNA and protein expression of TLR4 increases with age in the chick thymus. 4. Discussion In the present study, the distribution pattern of TLR4 in the thymus of the broiler chick and Meishan gilt was investigated for the first time. Our results show that TLR4 is expressed in the epithelial cells in the cortico-medullary junction and medulla of the thymus of the chick and gilt, and is particularly strongly expressed in Hassall’s corpuscles. It is known that thymic epithelial cells can guide the later development of T cells in the thymic corticomedullary junction and medulla, by providing self-antigens that affect the orientation of T cells during differentiation (Palmer, 2003; Stritesky et al., 2012), and other products (e.g. cytokines and chemokines) that induce T cell migration (Takahama, 2006). Based on their interaction with self-antigens, most high-affinity thymocytes undergo negative selection and die to preserve central tolerance, while a few high-affinity thymocytes undergo agonist selection and become self-reactive T cells (e.g. regulatory T cells, Tregs), whereas low-affinity thymocytes survive after negative selection and become classical naïve CD4 or CD8T cells (Palmer, 2003; Stritesky et al., 2012). Moreover, the thymic epithelial cells of Hassall’s corpuscles can express thymic stromal lymphopoietin that indirectly induce the maturation of CD4+CD25+ Tregs (Watanabe et al., 2005). This process is of clinical importance, as it is known that defects in the thymic stroma can result in immunodeficiency or autoimmunity (Fletcher et al., 2011). TLR4 can be stimulated by both endogenous and exogenous stress signals, such as endogenous fibronectin, fibrinogen, heparan and heat shock proteins (Okamura et al., 2001; Smiley et al., 2001; Johnson et al., 2002; Termeer et al., 2002; Tsan and Gao, 2004), as well as exogenous bacterial lipopolysaccharide (Medzhitov and
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Janeway, 1997; Hoshino et al., 1999). After activation, TLR4 can initiate a series of intracellular reactions via phosphorylation, ubiquitinylation and binding of downstream signaling molecules, such as IRAK1, IRAK2, IRAK4, TRAF6 and NEMO, and then causes nuclear location of transcription factors such as NF-B, which induce the expression of proinflammatory and antiinflammatory cytokines (Kawai and Akira, 2007). Based on the expression patterns that we detected, TLR4 may affect the later development of T cells, including Tregs, by mediating thymus epithelial cell-derived activities and inducing the release of cytokines. Furthermore, it is interesting to note that abnormal thymic development in human is associated with increased TLR4 expression (Bernasconi et al., 2005). We observed that the expression of TLR4 in the chick thymus increased with age during postnatal development. The thymus is a common target in various diseases, and intrathymic homeostasis and immune safety are important for normal thymus development (Savino, 2006). Neonatal vertebrates have limited ability to synthesize antibodies endogenously, and their humoral (antibody-mediated) immune defenses are based on maternally-derived antibodies, which disappear around the time of onset of active antibody production by the offspring. For example, in the chick (Gallus domesticus), maternal antibodies only persist for approximately 14 days after birth (Grindstaff et al., 2003). Therefore, increased TLR4 expression may affect chick thymus development, especially in the later stages of postnatal development, by inducing cytokines and other antimicrobial factors while maternal antibodies gradually leave the chick; however, the mechanisms that govern this process require further investigation. 5. Conclusions We found that TLR4 was strongly expressed in epithelial cells and was primarily distributed in the cortico-medullary junction and medulla, especially Hassall’s corpuscles. We also found that the mRNA expression level of TLR4 increased with age. Taken together, our study provides morphological and quantitative information regarding the potential role of TLR4 in chick thymus development, and indicates that TLR4 may participate in thymus development by inducing factors affecting their development. Conflict of interest The authors declare that there are no conflict of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30800808), the Fundamental Research Funds for the Central Universities (2010SC02, 2012ZYTS048, 2013YB09), and the National College Students Innovation Experiment Program (201210504005).
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