Takahiro Nii

Effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokines and the subsequent recruitment of immunocompetent cells in the oviduct of laying and molting hens

The goal of this study was to examine whether lipopolysaccharide (LPS) induces the expression of proinflammatory cytokines and chemokines and recruits T cells in the lower part of the oviduct, and whether that response to LPS is different between the laying and molting phase. White Leghorn laying and molting hens were intravenously injected with saline (control) or LPS. The uterus and vagina of oviducts were collected 3 or 6 h after injection, and used for reverse transcription PCR analysis of IL-1β, IL-6, IL-8 (CXCLi2), and lymphotactin (Lptn), and for immunohistochemical analysis for the frequency of CD4+ and CD8+ T cells. The expressions of IL-1β, IL-6, and CXCLi2 in the uterus and that of IL-1β in the vagina were upregulated in response to LPS 3 or 6 h after injection in both laying and molting hens. The CXCLi2 expression in the vagina was upregulated by LPS in laying hens, whereas those effects of LPS were not significant in molting hens. Expression of Lptn showed a tendency to be downregulated after 3 h, with recovery by 6 h after LPS injection. The frequency of CD4+ T cells tended to increase in response to LPS after 6 h in the lamina propria of the uterus and vagina in both laying and molting hens. The CD8+ T cell frequencies in the lamina propria of the uterus and vagina of laying hens increased in response to LPS after 6 h. However, in the molting hens, LPS stimulation resulted in CD8+ T cell increase in the vagina only and not in the uterus. These results suggest that expressions of proinflammatory cytokines and CXCLi2 chemokine are upregulated in association with T cell recruitment in response to LPS in the lower part of the oviduct, although CD8+ T cells in the uterus may be depressed during the molting phase. These immunoresponses may play roles in the defense against infection of the oviduct.

Effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct

The aim of this study was to determine the mechanism by which the avian infectious bronchitis virus (IBV) affects eggshell formation. Attenuated IBV (aIBV group) or vehicle (control group) was injected into the oviductal magnum lumen of White Leghorn laying hens. The changes in the expression of genes related to eggshell formation (collagen types I and V, and CaBP-D28K), densities of cytotoxic cells (CD8(+) and TCR-γδ(+) T cells), and gene expression of molecules related to cytotoxic immunoreaction (B-NK, perforin, granzyme, and IL-2) and proinflammatory cytokines (IL-1β, IL-6 and IFN-γ) were examined by quantitative reverse transcriptase polymerase chain reaction or immunohistochemistry in the isthmus and uterus. Gene expression of IL-1β and IL-6receptors in the tubular gland cells of the isthmus and uterus was analyzed by reverse transcriptase polymerase chain reaction. Gene expression of collagen type I, but not collagen type V, in the isthmus and CaBP-D28K in the uterus was decreased in the aIBV group compared with that in the control. The frequencies of CD8(+) cells and TCR-γδ(+) T cells in the isthmus and uterus were significantly higher in the aIBV group than in the control group. The expression of cytotoxic molecular and proinflammatory cytokines was also higher in the aIBV group than in the control. The expression of IL-6 receptor, but not IL-1β receptor, was identified in the tubular gland cells in the isthmus and uterus. These results suggest that IBV infection causes disorder of eggshell formation by disturbing gene expression of collagen type I in the isthmus and CaBP-D28K in the uterus, probably via the effects of substances from cytotoxic cells and proinflammatory cytokines.

Expression of Toll-like receptors and effects of lipopolysaccharide on the expression of proinflammatory cytokines and chemokine in the testis and epididymis of roosters

The aim of this study was to determine the expression profiles of Toll-like receptors (TLR) in the testis and epididymis of rooster and whether the expression of IL-1β, IL-6, CXCLi2, and TLR-4 was affected by lipopolysaccharide (LPS), a TLR-4 ligand. Roosters were intravenously injected with LPS or phosphate-buffered saline. Testes and epididymis were collected before and after 3 or 6 h postinjection. Total RNA was isolated from those tissues and expression of TLR and proinflammatory cytokines was analyzed by reverse-transcription PCR and quantitative real-time PCR. Reverse-transcription PCR analysis revealed that 7 of the known 10 chicken TLR in the testis and 9 of 10 in the epididymis were expressed. Expression of TLR-4 was found in both tissues. Expression of TLR-4 was significantly upregulated by LPS in the testis but not in the epididymis. Injection with LPS upregulated the expression of IL-1β, IL-6, and CXCLi2 in the testis and epididymis by 3 to 6 h postinjection. However, injection with phosphate-buffered saline (control) did not affect their expression. These results suggest that proinflammatory cytokines and chemokine expression was upregulated by LPS probably through TLR-4 activation, and thus the reproductive tissues are comprehensively equipped to deal with a pathogenic insult.

The effect of estrogen on the early cytotoxic response to IB virus infection in hen oviduct

The aim of this study was to determine whether the egg-laying phase and estrogen affect the induction of cytotoxic cells in response to avian infectious bronchitis (IB) virus at early stage of infection in the oviduct. Attenuated IB virus (aIBV group) or its vehicle (control group) was introduced to the oviductal magnum lumen of White Leghorn hens in the laying and molting phase, as well as molting hens injected with estradiol benzoate (M-EB hens) or corn oil (M-oil hens). Oviductal isthmus and uterus were collected 24h after injection. The frequency of CD8(+) and TCRγδ(+) T cells expression was examined by immunohistochemistry, followed by image analysis. The expression of the genes of toll-like receptor 7 (TLR7), natural killer cell receptor (BNK), cytotoxic substances (granzyme, perforin), and cytokines (CXCL12, CX3CL1, and IFNγ) were examined by real-time polymerase chain reaction analysis. The frequency of CD8(+) and TCRγδ(+) T cells in the isthmus, and CD8(+) cells in the uterus was significantly higher in the aIBV group compared to the control group of laying and M-EB hens. The expression of all the genes examined in this study in the isthmus, and CX3CL1 and IFNγ expression in the uterus was significantly higher in the aIBV group in the laying and M-EB hens. These results suggested that infection with IB virus causes an immune response involving the influx of cytotoxic cells and upregulation of cytokines in the isthmus and uterus at early stage of infection. This response was stronger during the laying phase compared to the molting phase, probably due to the effect of estrogen.

Innate immune functions in hen reproductive organs

The ovary and oviduct of the hen are susceptible to various pathogenic microorganisms, and infections in these organs may not only contaminate the eggs, but also disorder the egg formation. The immune function of the ovary and oviduct is essential to protect these tissues from infection as well as for the production of hygienic eggs. This paper reviews recent studies on the host defence system in the reproductive organs with reference to their innate immune functions, with emphasis on the important role of Toll-like receptors and avian β-defensins in this defence system.

Modulatory roles of proinflammatory cytokines on the expression of cathelicidins in the lower regions of the oviduct of laying hens

HighlightsThe PCR analysis confirmed that IL1B and IL6, their receptors, and CATH1, CATH2 and CATH3 were expressed in the mucosal tissues of the uterus and vagina.In the uterus, IL1B did not affect the expression of CATH1 and CATH2 in the uterus at any examined dose or incubation time, whereas CATH3 was significantly downregulated by incubation for 1.5 h with IL1B; IL6 stimulation of the uterus mucosal tissue downregulated the expression of CATH1 at 1.5 h of incubation. Whereas the expression of CATH3 was significantly upregulated by incubation for 1.5 h.In the vagina, IL1B stimulation upregulated the expression of CATH1 in a dose dependent manner, whereas the expression of CATH2 and CATH3 was also significantly upregulated by incubation for 1.5 h. IL6 stimulation of the vaginal cultured tissues did not affect the expression of all examined CATHs.From the current results, it is suggested that expression of CATH1, 2 and 3 in the vagina are upregulated by IL1B and CATH3 in the uterus is also upregulated by IL6. Therefore, the IL1B and IL6 synthesized in response to infection by the microbes may enhance the defense system of the oviductal mucosa by increasing the synthesis of CATHs. Abstract The current study aimed to confirm and examine the physiological roles of the proinflammatory cytokines IL1B and IL6 on the immune functions which mediated by cathelicidins (CATHs) in the uterus and vagina of laying hens. Snaps of the mucosal tissues of uterus and vagina were incubated in culture medium or chicken recombinant IL1B and IL6 for 1.5 h or 3 h before extraction of total RNA to be used for examination of IL1B and IL6, their receptors, and cathelicidins by semi‐quantitative PCR; and to examine the changes in cathelicidins expressions by real‐time PCR. PCR analysis confirmed that IL1B and IL6, their receptors, and CATH1‐3 were expressed in the mucosal tissues of the uterus and vagina. In uterus tissue, IL1B did not affect the expression of CATH1 and −2 at different doses and incubation time, whereas CATH3 was significantly downregulated by incubation with IL1B for 1.5 h. In the vaginal tissue, the expressed CATH1, −2 and −3 were significantly upregulated by incubation with IL1B for 1.5 h in a dose‐dependent manner. In uterus tissue, CATH1 expression was down‐regulate by IL6 incubation for 1.5 h, but not by 3 h however, CATH3 expression was significantly increased by incubation with IL6 for 1.5 h, but not for 3 h. In the vaginal tissues, all CATHs expression was not affected significantly by incubation with IL6. These current observations suggest that CATH1, −2 and −3 in the vagina are upregulated by IL1B, and CATH3 in the uterus is also upregulated by IL6. IL1B and IL6 synthesized in response to infection by the microbes may enhance the defense system in the oviduct mucosal tissues by increasing the synthesis of CATHs.

Innate antiviral immune response against infectious bronchitis virus and involvement of prostaglandin E2 in the uterine mucosa of laying hens

Infectious bronchitis virus (IBV) is an enveloped RNA virus that causes deformities in eggshells. The aim of this study was to investigate the innate immune response to IBV, and to determine whether prostaglandin (PG) E2, which is synthesized during inflammation, is involved in the innate immune response in the uterine mucosa. The effects of intra-oviductal inoculation with attenuated IBV (aIBV) on the expression of viral RNA recognition receptors and innate antiviral factors were examined by real-time PCR and immunohistochemistry, and on PGE2 levels by ELISA. Then, the effects of PGE2 on the expression of innate antiviral factors in cultured uterine mucosal cells were examined. The results showed that the expression of RNA virus pattern recognition receptors (TLR3, 7, and MDA5), antimicrobial peptides (avian β-defensins, including AvBD1, 2, 4-6 and cathelicidins, including CATH1 and 3), and interferons (IFNα, β, γ, λ) were upregulated, and the expression of cyclooxygenase 2 (PG synthase) and the level of PGE2 were increased in the uterine mucosa following aIBV inoculation. The number of AvBD2-positive cells in the mucosa also increased in response to aIBV. In cultured mucosal cells (mainly epithelial), the expression of AvBD4, 10-13 and IFNα, β, and λ was upregulated following incubation with 500 nM PGE2. These results suggest that the expression of viral RNA-recognition receptors, AvBDs, CATHs, and IFNs and PGE2 are induced by the IBV antigen, and that the expression of a different set of AvBDs is also induced by PGE2 in the cultured uterine mucosal cells. These antiviral factors may play a role in the protection of the uterine mucosa from IBV infection.

Effects of TLR Ligands on the Expression of Cytokines and Possible Role of NF κ B in its Process in the Theca of Chicken Follicles

The aim of this study was to determine the effects of Toll-like receptor (TLR) ligands on the expression of cytokines in chicken follicular theca and to investigate whether nuclear factor-κB (NFκB) was involved in their expression. The follicular theca was collected from the largest follicle of laying hens. In experiment 1, the expression of TLRs in the theca interna and externa was confirmed using RT-PCR. The theca tissues were then incubated with or without Pam3CSK4 (TLR2 ligand), poly I:C (TLR3 ligand), LPS (TLR4 ligand), flagellin (TLR5 ligand), R837 (TLR7 ligand), and CpG-ODN (TLR21 ligand) for 3 h, after which cytokine expression (IL-1β, IL-6, TNFSF15, CXCLi2, IFN-α, and IFN-β) was analyzed by real-time PCR. In experiment 2, the theca tissues were incubated in a medium containing Pam3CSK4, poly I:C, LPS, or CpG-ODN with or without BAY 11-7085 (an inhibitor of NFκB) for 3 h. The results of experiment 1 revealed that all TLRs, namely TLR1 (type 1 and 2), TLR2 (type 1 and 2), 3–5, 7, 15, and 21, were expressed in the follicular theca, although the PCR products of TLR1 (type 2) and TLR21 were faint. Moreover, Pam3CSK4 and LPS upregulated the expression of all detected cytokines, except for IFN-α, whose expression was not upregulated by LPS. Poly I:C upregulated the expression of IL-6, CXCLi2, and IFN-β, while CpG-ODN upregulated IL-1β. Flagellin and R837 did not significantly affect cytokine expression. In experiment 2, the expression of IL-1β, IL-6, CXCLi2 and IFN-β in tissues incubated with LPS was downregulated by BAY 11-7085. These results suggest that the innate immune system, including pattern recognition by TLRs and cytokine synthesis, occur in the theca; whereas, functions for recognition of bacterial patterns is more developed than that of viral ones.

Changes in the Expression of Avian β-defensins (AvBDs) and Proinflammatory Cytokines and Localization of AvBD2 in the Intestine of Broiler Embryos and Chicks during Growth

The aim of this study was to determine the changes in the expression of avian β-defensins (AvBDs) and proinflammatory cytokines and localization of AvBD2 in the intestine of broiler embryos and chicks during growth. The ileum and cecum of embryonic day 19 (ED19) and of day-old (D0) and 7-day-old (D7) chicks were collected. Gene expression levels of 10 AvBDs (AvBD1–8, 10, and 12) and proinflammatory cytokines (IL-1β, -6, and -8) were analyzed using real-time PCR, and the localization of AvBD2 was examined by immunohistochemistry. Gene expression levels of AvBD1, 2, 6, and 7 in the ileum and of AvBD1 and 4 in the cecum were higher on ED19 than on D7. The expression of AvBD10 in the ileum was higher on D0 than on ED19, whereas the expression levels of AvBD8 and 10 in the cecum were higher on D0 than on ED19, and that of AvBD10 decreased on D7. The expression levels of IL-1β, -6, and -8 in the ileum were higher on D7 than on ED19. The expression levels of IL-1β, -6, and -8 in the cecum were higher on D0 than on ED19, and that of IL-1β and -6 declined on D7. AvBD2-positive cells were localized in the lamina propria beneath epithelial cells of villi and crypts. The number of positive cells in the cecum mucosa was greater on D0 than on ED19 and D7. In conclusion, we suggest that AvBDs are expressed in the ileum and cecum of embryos and chicks at high levels before or just after hatching and decrease by D7. The expression of proinflammatory cytokines in the ileum increases with growth until D7, but is the highest in the cecum around hatching. These AvBDs and proinflammatory cytokines may play roles in host defense in the intestinal mucosa of embryos and neonatal chicks.

Expression of pro- and anti-inflammatory cytokines and chemokines during the ovulatory cycle and effects of aging on their expression in the uterine mucosa of laying hens

Abstract The aim of this study was to examine whether cytokines and chemokines expressed in the uterine mucosa play a role in the process of eggshell formation in the chicken uterus. Changes in the expression levels of pro‐ and anti‐inflammatory cytokines and chemokines in the uterine mucosa during an ovulatory cycle (experiment 1) and effects of aging on their expression (experiment 2) were examined. In experiment 1, the expression of the pro‐inflammatory cytokines IL1&bgr;, IL6, TNFSF15, and IFN&ggr;, and a chemokine CX3CL1 was found to increase during eggshell biomineralization (16 h following oviposition), while anti‐inflammatory TGF&bgr;2 expression was found to increase at 4 h following oviposition. In experiment 2, a higher expression of the anti‐inflammatory cytokines TGF&bgr;2 and TGF&bgr;3, and chemokines CXCLi2 and CX3CL1, was observed in aged hens than in young hens. A significantly higher number of macrophages and CD8+ T cells were observed in the uterine tissue of aged hens than in young hens. Furthermore, the expression of adhesion molecules associated with leukocytic infiltration was found to be higher in aged hens than in young hens. We conclude that the eggshell formation process may be affected by the pro‐ and anti‐inflammatory cytokines and chemokines. The balanced expressions of these molecules might be disrupted in aged hens.