Macrophage colony-stimulating factor (CSF1) is an essential growth factor to control the proliferation, differentiation and survival of cells of the macrophage lineage in vertebrates. We have previously produced a recombinant chicken CSF1-Fc fusion protein and administrated it to birds which produced a substantial expansion of tissue macrophage populations. To further study the biology of CSF1 in the chicken, here we generated anti-chicken CSF1 antibodies (ROS-AV181 and 183) using CSF1-Fc as an immunogen. The specific binding of each monoclonal antibody was confirmed by ELISA, Western blotting and immunohistochemistry on tissue sections. Using the anti-CSF1 antibodies, we show that chicken bone marrow derived macrophages (BMDM) express CSF1 on their surface, and that the level appears to be regulated further by exogenous CSF1. By capture ELISA circulating CSF1 levels increased transiently in both layer and broiler embryos around the day of hatch. The levels of CSF1 in broilers was higher than in layers during the first week after hatch. Antibody ROS-AV183 was able to block CSF1 biological activity in vitro and treatment of hatchlings using this neutralising antibody in vivo impacted on some tissue macrophage populations, but not blood monocytes. After anti-CSF1 treatment, CSF1R-transgene reporter expressing cells were reduced in the bursa of Fabricius and cecal tonsil and TIM4+ Kupffer cells in the liver were almost completely ablated. Anti-CSF1 treatment also produced a reduction in overall bone density, trabecular volume and TRAP+ osteoclasts. Our novel neutralising antibody provides a new tool to study the roles of CSF1 in birds.

Regulation and function of macrophage colony-stimulating factor (CSF1) in the chicken immune system.

// Qian Zhang 1,2 , Yasir Waqas 1 , Ping Yang 1 , Xuejing Sun 1 , Yi Liu 1 , Nisar Ahmed 1 , Bing Chen 1 , Quanfu Li 1 , Lisi Hu 1 , Yufei Huang 1 , Hong Chen 1 , Bing Hu 3 and Qiusheng Chen 1 1 Laboratory of Animal Cell Biology and Embryology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China 2 Key Laboratory of Antibody Techniques of Ministry of Health, Nanjing Medical University, Nanjing, China 3 Biological experiment and Teaching Center, College of Life Sciences, Nanjing Agricultural University, Nanjing, China Correspondence to: Qiusheng Chen, email: // Keywords : lymphocyte homing; chicken spleen; HEV; sheathed capillary; adhesion molecules; Immunology and Microbiology Section, Immune response, Immunity Received : May 22, 2016 Accepted : December 27, 2016 Published : January 04, 2017 Abstract The immune function of the chicken spleen depends on its different compartments of red and white pulps, but little is known about the mechanism underlying lymphocyte homing towards the different compartments. In the present study, the role of lymphocyte homing in the chicken spleen was investigated during lipopolysaccharide (LPS) stimulation. Morphological analysis demonstrated the cuboidal endothelial cells of the splenic sheathed capillary facilitated the passage of lymphocyte homing to the chicken spleen. The tissue-specific adhesion molecules- vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MADCAM-1) expressed on the sheathed capillary, which suggested the high endothelial venule (HEV)-like vessels of the chicken spleen. Electron microscope analysis showed LPS activated the endothelium of the sheathed capillary and recruited lymphocytes to the chicken spleen. Transferring of 5, 6- carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeled lymphocytes depicted the rout of lymphocyte homing to the compartments of the chicken spleen was from the white pulp to the red pulp. Furthermore, the mRNA and protein levels of adhesion molecular integrin β1 and VCAM-1 increased after LPS stimulation. The mechanism underlying the integrin β1 and VCAM-1 during LPS stimulation might be associated with the integrin linked kinase (ILK)- dependent regulation of protein kinase B (PKB/AKT). This study firstly shows lymphocyte homing in the chicken spleen after LPS-induced inflammation. These results contribute to our knowledge of comparative immunology and provide a better means for investigating the pharmacological strategies concerning the possible role of lymphocyte homing in inflammation and immunological reactions in infectious disease.

/pdf/cytological-study-on-the-regulation-of-lymphocyte-homing-in-7smiviilk3.pdf

Cytological study on the regulation of lymphocyte homing in the chicken spleen during LPS stimulation.

To identify the existence and composition of the blood-spleen barrier (BSB) in ducks, the microanatomical structures of the duck spleen were investigated by light and transmission electron microscopy, silver staining, enzymatic histochemistry and intravenous injection of ink. The endothelial cells of the sheathed capillaries were cuboidal-shaped and surrounded by an ellipsoid consisting of reticular cells, similar to high endothelial venules (HEVs). After ink injection, carbon particles were initially restricted to the ellipsoid and later trapped in the periellipsoidal lymphatic sheaths (PELS), and then transferred to the periarteriolar lymphatic sheaths (PALS) and splenic nodules over time. Reticular fibers were primarily distributed at the basement membrane of the sheathed capillaries and the periphery of the PELS. Macrophages were primarily distributed at the border between red pulp and PELS. These results suggested that the BSB was present in the ellipsoid and PELS and consisted of the mechanical barrier composed of endothelial cells of the sheathed capillaries, reticular cells and reticular fibers and the biological barrier composed of ellipsoid-associated macrophages. In conclusion, the BSB was identified in the duck spleen for the first time, including cuboidal endothelial cells, ellipsoid-associated macrophages, reticular cells and fibers, and resisting circulating pathogen invasions. The study of BSB in ducks provides a theoretical foundation for the structural composition of the avian immune system.

The novel histological evidence of the blood-spleen barrier in duck (Anas platyrhynchos).

The spleen is a separate organ of the teleost, playing an essential role in immune reactions. The morphology of the spleen is different from the fish species. Little knowledge about the spleen structure and the blood splenic barrier (BSB) in Nile tilapia has been reported. To address this issue, we studied the histology of the spleen and the BSB in healthy Nile tilapia. The morphology of the spleen was observed, then H&E staining, modified Jame’s staining, and ultrastructural techniques were performed to portion the spleen into three subregions and analyze the location of components and fibers. Thereafter, vital staining of Nile tilapia with Trypan blue was conducted to elucidate the composition and function of BSB. Histologically, the spleen could be divided into three subregions (inner, middle, and outer). The venules, clumps of lymphocytes, and vessels were separately characterized features of the outer, middle, and inner layers. Post injection, Trypan blue was intercepted in the endotheliocytes of ellipsoids in the middle layer (i.p.) or was deposited to the reticular fibers surrounding the ellipsoids (i.v.). Additionally, the amount of Trypan blue was shown to be positively correlated to that of the Acid phosphatase expressed. In conclusion, the spleen could be portioned into three subregions, and the BSB lay in the middle layer, composed of the cuboidal-shaped endotheliocytes and the surrounding reticular fibers of the ellipsoid capillaries. The present study enriched the research of immune tissues and system in tilapia and provided reference for the study of spleen in other fish species.

Morphology of the Spleen in Oreochromis niloticus: Splenic Subregions and the Blood-Spleen Barrier.

miRNAs are small non-coding regulatory RNAs that play key roles in diverse biological processes. In this study, we used the Solexa sequencing technique to profile miRNAs in breeder cock testes to illustrate their functions. A total of 663 co-expressed miRNAs and 3,180 co-expressed piRNAs were detected in three libraries. Based on Mir-X™ miRNA qRT-PCR, three miRNAs representing low, medium and high expression levels according to the sequencing results were selected randomly to validate the miRNAs' expression profiles. Results suggested that the miRNA expression profiles data could represent actual miRNA expression levels. Moreover, target genes prediction of the co-expressed miRNAs and further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed, which revealed that some candidate miRNAs were involved in the regulation of the spermatogenesis process, spermatozoa function and testicular metabolism. In conclusion, we provided a useful resource for further elucidation of the miRNAs' regulatory role in spermatogenesis, contributing to a preliminary database for functional and molecular mechanistic studies in testicular metabolism, spermatogenesis and other testes functions.

miRNA and piRNA expression profiles of breeder cock testes detected by next-generation sequencing.

To identify the existence and composition of the blood–spleen barrier (BSB) in chickens, the microanatomical features of the spleen were investigated by light and transmission electron microscopy, intravenous injection of ink, acid phosphatase reaction, and silver impregnation. The results showed that the white pulp in chicken spleen consists of lymphoid nodules, periarteriolar lymphatic sheaths (PALS) and periellipsoidal lymphatic sheaths (PELS). There was no evidence for the presence of a marginal zone. The splenic ellipsoid was a unique structure, which functioned as a barrier for filtering and phagocytosis. Uptake of carbon particles was limited to the ellipsoid and PELS, 60 min after injection of carbon particles. Reticular fibres were densely distributed in the ellipsoid and extended into the PELS. Ellipsoid-associated cells (EACs), reticular cells and macrophages were acid phosphatase positive. The sheathed capillaries, surrounded by the ellipsoid, were similar to high endothelial venules (HEVs). These findings suggest that the BSB of chickens is present in the ellipsoid and PELS, protecting the spleen from invasion from circulating pathogens. The BSB was a reticular framework, between the arterial and venous vessels, which included cuboidal-shaped endothelial cells, supporting cells, EACs, macrophages, reticular cells and fibres. Lymphocyte migration into the spleen parenchyma is most likely via the HEV-like vessels. These research findings contribute to better understanding of avian immunology and provide an insight into evolutionary differences in the immune system.

Identification and structural composition of the blood-spleen barrier in chickens

Objective: Information regarding the development of the enteric nervous system (ENS) is important for understanding the functional abnormalities of the gut. Because fertilized chicken eggs provide easy access to embryos, chicken models have been widely used to study embryonic development of myenteric plexus; however, no study has been focused on the postnatal period. The aim of this study was to perform a qualitative and quantitative analysis of the nitrergic neurons in the myenteric plexus of developing chickens in the postnatal period. Methods: Whole-mount preparations of the myenteric plexus were made in 7-d, 15-d, and 40-d old (adult) chickens of either sex (n=15). The myenteric plexus was studied after nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry using light microscopy, digital photography, and Image-Pro Plus 6.0 software. The numbers of positively stained neurons and ganglia were counted in the duodenum, jejunum, ileum, caecum, and colon in the different age groups. Data were expressed as mean±standard deviation (SD), and statistical analysis was performed using a one-way analysis of variance (ANOVA) test. Results: The positively stained neurons showed various morphologies and staining intensities, and formed bead-shaped and U-shaped arrangements in the myenteric plexus. The densities of neurons and ganglia increased with age. However, the number of positive neurons per ganglion increased. The number of NADPH-d-positive neurons was highest in the colon, followed by the ileum, the jejunum, the duodenum, and the caeca in all age groups. Conclusions: Developmental changes in the myenteric plexus of chickens continue in the postnatal period, indicating that the maturation process of the gastrointestinal function is gradual. In addition, no significant difference is happening among different intestinal segments during postnatal development, suggesting that the function of different intestinal segments had been determined after birth.

Quantitative changes of nitrergic neurons during postnatal development of chicken myenteric plexus

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Identification and structural composition of the blood-spleen barrier in chickens

Quantitative changes of nitrergic neurons during postnatal development of chicken myenteric plexus