Chicken Macrophage Cell Research Articles

CircNFIC Balances Inflammation and Apoptosis by Sponging miR-30e-3p and Regulating DENND1B Expression.

Disordered inflammation and apoptosis are closely related to diseases, and inflammation can also promote cell apoptosis, where growing evidence has shown that circular RNAs (circRNAs) play important roles. Lipopolysaccharide (LPS) is the main component of the cytoderm of gram-negative bacterium, which can cause inflammatory responses in macrophages. We constructed an inflammatory model by exposing chicken macrophage cell lines (also known as HD11) to LPS for in vitro experiments. In this study, we validated a novel circRNA—circNFIC—which was dramatically up-regulated in tissues infected by coccidia and cells exposed to LPS. Besides, circNFIC could significantly promote the expression levels of pro-inflammation factors, including (IL-1β, TNFα, and IFNγ) and pro-apoptosis maker genes (caspase 3 and caspase 8) in HD11 exposed to LPS or not. In terms of mechanism, circNFIC exerted notable effects on DENND1B to regulate cell inflammation and apoptosis by sponging miR-30e-3p. The molecular functions played by miR-30e-3p and DENND1B have been explored, respectively. In addition, the effects of circNFIC knockdown suppressing the expression of pro-inflammatory and pro-apoptosis functions could be reversed by a miR-30e-3p inhibitor. On the whole, circNFIC promoted cell inflammation and apoptosis via the miR-30e-3p/DENND1B axis.

Modulatory Effects of Mercury (II) Chloride (HgCl2 ) on Chicken Macrophage and B-Lymphocyte Cell Lines with Viral-Like Challenges In Vitro.

Mercury (Hg) is a toxic trace metal ubiquitously distributed in the environment. Inorganic mercury (as HgCl2) can cause immunotoxicity in birds, but the mechanisms of action are still not fully resolved, especially with respect to responses to viral infections. To investigate the potential immunomodulatory effects of Hg2+ on specific cell types of the avian immune system, chicken macrophage (HD‐11) and B‐lymphocyte (DT40) cell lines were applied as in vitro models for the innate and adaptive immune systems, respectively. The cells were stimulated with synthetic double‐stranded RNA, which can be recognized by toll‐like receptor‐3 to mimic a viral infection. The Hg2+ showed concentration‐dependent cytotoxicity in both cell lines, with similar median effect concentrations at 30 µM. The cytotoxicity of Hg2+ was closely related to glutathione (GSH) depletion and reactive oxygen species induction, whereas the de novo synthesis of GSH acted as a primary protective strategy. Nitric oxide produced by activated macrophages was strongly inhibited by Hg2+, and was also influenced by cellular GSH levels. Cell proliferation, gene expression of microRNA‐155, and cellular IgM levels in B cells were decreased at noncytotoxic Hg2+ concentrations. The secretion of antiviral interferon‐α was induced by Hg2+ in both cell lines. Overall, our results suggest that Hg2+ exposure can cause immunomodulatory effects in birds by disrupting immune cell proliferation and cytokine production, and might result in disorders of the avian immune system. Environ Toxicol Chem 2021;40:2813–2824. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of Dietary Maltol on Innate Immunity, Gut Health, and Growth Performance of Broiler Chickens Challenged With Eimeria maxima.

Two studies were conducted to evaluate the effects of maltol as a postbiotic on innate immunity, gut health, and enteric infection. In the first study, an in vitro culture system was used to evaluate the effects of maltol on the innate immune response of chicken macrophage cells (CMC), gut integrity of chicken intestinal epithelial cells (IEC), anti-parasitic activity against Eimeria maxima, and differentiation of quail muscle cells (QMC) and primary chicken embryonic muscle cells (PMC). All cells seeded in the 24-well plates were treated with maltol at concentrations of 0.1, 1.0, and 10.0 μg. CMC and IEC were stimulated by lipopolysaccharide to induce an innate immune response, and QMC and PMC were treated with 0.5 and 2% fetal bovine serum, respectively. After 18 h of incubation, pro-inflammatory cytokines, tight junction proteins (TJPs), and muscle cell growth markers were measured. In the second study, the dietary effect of maltol was evaluated on disease parameters in broiler chickens infected with E. maxima. Eighty male 1-day-old broiler chickens were allocated into the following four treatment groups: (1) Control group without infection, (2) Basal diet with E. maxima, (3) High maltol (HI; 10.0 mg /kg feed) with E. maxima, and (4) Low maltol (LO; 1.0 mg/kg feed) with E. maxima. Body weights (BW) were measured on days 0, 7, 14, 20, and 22. All chickens except the CON group were orally infected with 104 E. maxima per chicken on day 14. Jejunum samples were collected for gut lesion scoring, and the gene expression of cytokines and TJPs. Data was analyzed using PROC MIXED in SAS. In vitro, maltol not only increased TJPs in IEC and cytokines in the LPS-stimulated CMC but also showed direct cytotoxicity against sporozoites of E. maxima. In vivo, the HI group improved the BW, reduced the gut lesion scores and fecal oocyst shedding, and decreased jejunal TNFSF15 and IL-1β expression in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary maltol in the enhancement of growth performance, gut health, and coccidiosis resistance and the applicability of maltol as a postbiotic for the replacement of antibiotic growth promoters in commercial poultry production.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Abstract Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed a sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast was used to immunize the mice. Five mAbs which specifically recognize chIFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing assay to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were identified as optimum pairs of antibodies for detection of chIFN-α. The sandwich ELISA effectively detected native chicken IFN-α in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus (VSV) was evaluated in chicken embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize the activity of chicken IFN-α against VSV were identified. The newly developed antigen capture sandwich ELISA for the detection of chIFN-α will be a useful tool to monitor IFN-α production in chickens during viral infections.

Polarization of avian macrophages upon avian flavivirus infection

Avian Tembusu virus (TMUV) is a newly emerging avian pathogenic flavivirus that spreads rapidly, has an expanding host range and undergoes cross-species transmission. Our previous study identified avian monocytes/macrophages as the key targets of TMUV infection, since the infection of host monocytes/macrophages was crucial for the replication, transmission, and pathogenesis of TMUV. The polarization of host macrophages determines the functional phenotypes of macrophages; however, the effect of TMUV infection on macrophage polarization remains unclear. Here, we analysed the expression spectra of the marker genes of macrophage polarization upon TMUV infection in the HD11 chicken macrophage cell line and primary monocytes/macrophages isolated from the peripheral blood of specific pathogen-free (SPF) chickens and ducks. We found that viral replication mainly induced M1 marker genes and triggered nitric oxide (NO) release at different levels, suggesting that TMUV infection led mainly to host macrophages polarizing into the classically activated (M1) type. The NO that was increased upon infection did not function as an antiviral agent against TMUV, since the replication of TMUV in HD11 cells was not affected by the addition of an organic NO donor. Furthermore, upon TMUV infection, polarized HD11 cells exhibited increased migration but reduced phagocytosis, as evidenced by scratch assay and neutral red uptake assay, respectively. Our present study characterized the polarization of host monocytes/macrophages upon TMUV infection, which may lay a foundation for further research on the immune escape mechanism and pathogenic mechanism of TMUV.

Optimized Detoxification of a Live Attenuated Vaccine Strain (SG9R) to Improve Vaccine Strategy against Fowl Typhoid.

The live attenuated vaccine strain, SG9R, has been used against fowl typhoid worldwide, but it can revert to the pathogenic smooth strain owing to single nucleotide changes such as nonsense mutations in the rfaJ gene. As SG9R possesses an intact Salmonella plasmid with virulence genes, it exhibits dormant pathogenicity and can cause fowl typhoid in young chicks and stressed or immunocompromised brown egg-laying hens. To tackle these issues, we knocked out the rfaJ gene of SG9R (named Safe-9R) to eliminate the reversion risk and generated detoxified strains of Safe-9R by knocking out lpxL, lpxM, pagP, and phoP/phoQ genes to attenuate the virulence. Among the knockout strains, live ΔlpxL- (Dtx-9RL) and ΔlpxM-9R (Dtx-9RM) strains induced remarkably less expression of inflammatory cytokines in chicken macrophage cells, and oil emulsion (OE) Dtx-9RL did not cause body weight loss in chicks. Live Dtx-9RM exhibited efficacy against field strain challenge in one week without any bacterial re-isolation, while the un-detoxified strains showed the development of severe liver lesions and re-isolation of challenged strains. Thus, SG9R was optimally detoxified by knockout of lpxL and lpxM, and Dtx-9RL and Dtx-9RM might be applicable as OE and live vaccines, respectively, to prevent fowl typhoid irrespective of the age of chickens.

Analysis of chicken macrophage functions and gene expressions following infectious bronchitis virus M41 infection

Infectious bronchitis virus (IBV) is a pathogenic coronavirus with high morbidity and mortality in chicken breeding. Macrophages with normal biofunctions are essential for host immune responses. In this study, the HD11 chicken macrophage cell line and chicken peripheral blood mononuclear cell-derived macrophages (PBMCs-Mφ) were infected with IBV at multiplicity of infection (MOI) of 10. The dynamic changes of their biofunctions, including cell viability, pathogen elimination function, phagocytic ability, and gene expressions of related proteins/mediators in innate and acquired immunity, inflammation, autophagy and apoptosis were analyzed. Results showed that IBV infection decreased chicken macrophage viability and phagocytic ability, and increased pathogen elimination function. Moreover, IBV augmented the gene expressions of most related proteins in macrophages involved in multiple host bioprocesses, and the dynamic changes of gene expressions had a close relationship with virus replication. Among them, MHCII, Fc receptor, TLR3, IFN-α, CCL4, MIF, IL-1β, IL-6, and iNOS showed significantly higher expressions in IBV-infected cells. However, TLR7, MyD88, MDA5, IFN-γ, MHCII, Fc receptor, MARCO, CD36, MIF, XCL1, CXCL12, TNF-α, iNOS, and IL-10 showed early decreased expressions. Overall, chicken macrophages play an important role in host innate and acquired immune responses to resist IBV infection, despite early damage or suppression. Moreover, the IBV-induced autophagy and apoptosis might participate in the virus-host cell interaction which is attributed to the biological process.

Molecular characterization and functional analysis of Eimeria tenella citrate synthase.

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.

Component Identification and Functional Analysis of Outer Membrane Vesicles Released by Avibacterium paragallinarum.

Avibacterium paragallinarum, the causative agent of infectious coryza, is known to release outer membrane vesicles (OMVs). In the present study, we investigated the composition, bioactivities, and functional properties of the OMVs of A. paragallinarum. Following extraction and purification, the OMVs were observed to be spherical in shape, with diameters ranging from 20 to 300 nm. The vesicles contained endotoxin as well as genomic DNA. The molecular weights of the OMV-contained protein fragments were mostly concentrated at 65 and 15 kDa. The components of the OMV proteins were mainly various functional enzymes (e.g., ATP-dependent RNA helicase), structural components (e.g., streptomycin B receptor and membrane protein), and some hypothetical proteins with unknown functions. The expression levels of inflammation-related factors, such as interleukin (IL)-2, IL-6, IL-1β, IL-10, and inducible nitric oxide synthase (iNOs), were significantly upregulated in chicken macrophage cells HD11 incubated with OMVs. Serum IgG antibodies were measured after two intramuscular injections of an OMV-based vaccine into specific pathogen-free (SPF) chickens. The vaccinated chickens were then challenged by A. paragallinarum of homologous and heterologous serovars. It was noted that the vaccinated chickens produced immunoglobulin G (IgG) antibodies against A. paragallinarum. The OMVs conferred an acceptable level of protection (70%), defined as an absence of colonization and of clinical signs, against the homologous strain (serovar A), while the cross-protection against heterologous challenge with serovars B and C was much weaker. However, the OMVS did provide significant protection against clinical signs for all three serovars. Overall, this study laid a foundation for further unraveling the functional roles of OMVs released by A. paragallinarum.

The immunomodulatory effect of cathelicidin-B1 on chicken macrophages

Cathelicidins (CATHs) play an important role in the innate immune response against microbial infections. Among the four chicken cathelicidins, CATH-B1 is studied the least. In this study, the effect of CATH-B1 on the macrophage response towards avian pathogenic E. coli (APEC) and bacterial ligands was investigated. Our results show that APEC induced CATH-B1 gene expression in both a chicken macrophage cell line (HD11 cells) and primary macrophages, while expression of the other three CATHs was virtually unaffected. While the antimicrobial activity of CATH-B1 is very low under cell culture conditions, it enhanced bacterial phagocytosis by macrophages. Interestingly, CATH-B1 downregulated APEC-induced gene expression of pro-inflammatory cytokines (IFN-β, IL-1β, IL-6 and IL-8) in primary macrophages. In addition, CATH-B1 pre-incubated macrophages showed a significantly higher gene expression of IL-10 after APEC challenge, indicating an overall anti-inflammatory profile for CATH-B1. Using isothermal titration calorimetry (ITC), CATH-B1 was shown to bind LPS. This suggests that CATH-B1 reduces toll like receptor (TLR) 4 dependent activation by APEC which may partly explain the decreased production of pro-inflammatory cytokines by macrophages. On the contrary, direct binding of CATH-B1 to ODN-2006 enhanced the TLR21 dependent activation of macrophages as measured by nitric oxide production. In conclusion, our results show for the first time that CATH-B1 has several immunomodulatory activities and thereby could be an important factor in the chicken immune response.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were determined and a standard curve was established using recombinant chIFN-α. The sandwich ELISA effectively detected an increased IFN-α production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C), and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize its activity were identified. The newly developed antigen sandwich ELISA developed in this study will be a useful tool to monitor IFN-α production in chickens.

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1β, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Chicken avian β-defensin 8 modulates immune response via the mitogen-activated protein kinase signaling pathways in a chicken macrophage cell line

Defensins are antimicrobial peptides composed of 3 conserved disulfide bridges, a β-sheet, and both hydrophobic and cationic amino acids. In this study, we aimed to demonstrate the immunomodulation role of avian β-defensin 8 (AvBD8) in a chicken macrophage cell line. Chicken AvBD8 stimulated the expression of proinflammatory cytokines (IL-1β, interferon gamma, and IL-12p40) and chemokines (CCL4, CXCL13, and CCL20) in macrophages. Furthermore, by Western blotting and immunocytochemistry, we confirmed that AvBD8 activated the mitogen-activated protein kinase signaling pathway via extracellular regulated kinases 1/2 and p38 signaling molecules. Overall, AvBD8 plays a crucial role in host defense as not only an antimicrobial peptide but also an immunomodulator by activating the mitogen-activated protein kinase signaling pathway and inducing the expression of proinflammatory cytokines and chemokines.

Immunomodulatory effects of avian β-defensin 5 in chicken macrophage cell line

Antimicrobial peptides (AMPs) protect host from pathogens as first line of defense. Especially, β-defensins shows antimicrobial activity and immune modulation effects. Avian species also have β-defensins as avian β-defensins (AvBDs) from AvBD1 to AvBD14. In this study, we characterized chicken AvBD5 and demonstrated its immune modulatory functions in chicken macrophage cell line (HD11). Chicken AvBD5 is composed of a signal, pro, and mature peptides containing one α-helix, four β-sheet, and three disulfide bonds. Here, we also showed that chicken AvBD5 induced Th1, Th2, and Th17 cytokines in chicken macrophage cell line and stimulated MAPK signaling pathways through ERK1/2 and p38 molecules. In addition, AvBD5 stimulated MyD88 and CD40 to regulate immune systems. Taken together, chicken AvBD5 can modulate host immune systems by inducing cytokines expression and stimulating MAPK signaling pathway.

NLRC5 Serves as a Pro-viral Factor During Influenza Virus Infection in Chicken Macrophages.

Avian influenza viruses (AIVs) cause major economic losses to the global poultry industry. Many host factors have been identified that act as regulators of the inflammatory response and virus replication in influenza A virus (IAV) infected cells including nucleotide-binding oligomerization domain (NOD) like receptor (NLR) family proteins. Evidence is emerging that NLRC5, the largest NLR member, is a regulator of host immune responses against invading pathogens including viruses; however, its role in the avian immune system and AIV pathogenesis has not been fully explored. In this study, we found that NLRC5 is activated by a range of low and highly pathogenic AIVs in primary chicken lung cells and a chicken macrophage cell line. Further, siRNA mediated NLRC5 knockdown in chicken macrophages resulted in a significant reduction in AIV replication which was associated with the upregulation of genes associated with activated NFκB signaling pathway. The knockdown of NLRC5 enhanced the expression of genes known to be associated with viral defense and decreased innate cytokine gene expression following AIV infection. Overall, our investigation strongly suggests that NLRC5 is a pro-viral factor during IAV infection in chicken and may contribute to pathogenesis through innate cytokine regulation. Further studies are warranted to investigate the IAV protein(s) that may regulate activation of NLRC5.

Interleukin-dependent modulation of the expression of MHC class I and MHC class II genes in chicken HD11 cells

Interleukins (ILs) regulate cell surface antigens known as activation markers, which have distinct functional roles. However, the regulation of major histocompatibility complex (MHC) class I, MHC class II, and related genes by cytokines in chickens is not well understood. In the present study, we evaluated the influence of certain recently discovered chicken interleukins—i.e., IL-11, IL-12B, IL-17A, IL-17B, IL-26, and IL-34—on the expression and regulation of genes related to MHC class I, MHC class II, and the associated proteins in an HD11 chicken macrophage cell line. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemical, and flow cytometric analyses to assess dose- and time-dependent expression in the HD11 cell line and found that the ILs induced MHC class I, MHC class II, and associated protein. As NF-κB is actively involved in cell activation and is constitutively activated in many immune cells, we also determined whether NF-κB regulates MHC class I, MHC class II, and related gene expression in the HD11 cell line. The NF-κB inhibitor sulfasalazine (Sz) dose-dependently inhibited MHC class I and MHC class II in the HD11 cell line. Sz also downregulated the expression of MHC class I, MHC class II, and the associated proteins in the IL-induced HD11 cell line. The expression of MHC class I, MHC class II, and associated genes was accompanied by the Sz-sensitive degradation of the p65 (RelA) and p50 subunits of NF-κB and IκBα. Our results indicate that the different effects of each IL on the expression of genes related to MHC class I, MHC class II, and the associated proteins are involved with the regulation of the dose and duration of antigenic peptide presentation and, thus, also influence Th1, Th2, and Th17 production.

Cytotoxicity studies of Fe3O4 nanoparticles in chicken macrophage cells.

Magnetic Fe3O4 nanoparticles (Fe3O4-NPs) have been widely investigated for their biomedical applications. The main purpose of this study was to evaluate the cytotoxic effects of different sizes of Fe3O4-NPs in chicken macrophage cells (HD11). Experimental groups based on three sizes of Fe3O4-NPs (60, 120 and 250 nm) were created, and the Fe3O4-NPs were added to the cells at different doses according to the experimental group. The cell activity, oxidative index (malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS)), apoptosis and pro-inflammatory cytokine secretion level were detected to analyse the cytotoxic effects of Fe3O4-NPs of different sizes in HD11 cells. The results revealed that the cell viability of the 60 nm Fe3O4-NPs group was lower than those of the 120 and 250 nm groups when the same concentration of Fe3O4-NPs was added. No significant difference in MDA was observed among the three Fe3O4-NP groups. The SOD level and ROS production of the 60 nm group were significantly greater than those of the 120 and 250 nm groups. Furthermore, the highest levels of apoptosis and pro-inflammatory cytokine secretion were caused by the 60 nm Fe3O4-NPs. In conclusion, the smaller Fe3O4-NPs produced stronger cytotoxicity in chicken macrophage cells, and the cytotoxic effects may be related to the oxidative stress and apoptosis induced by increased ROS production as well as the increased expression of pro-inflammatory cytokines.

Association of SPOP Expression with the Immune Response to Salmonella Infection in Chickens.

Simple SummarySalmonella frequently causes human illness via the consumption of contaminated meat or eggs. At present, studies about how the host immune response against Salmonella is regulated are limited. Speckle-type POZ (poxvirus and zinc finger) protein (SPOP) is a specific adaptor of Cul3-based ubiquitin ligase, which catalyzes the ubiquitination and degrades the substrates. However, its role in the immune response is still unknown. Therefore, this study measured expression of SPOP and the proinflammatory cytokines interleukin-1β and interleukin-8 in chicken macrophage cells stimulated with a bacterial substitute and assessed their relationship using the quantitative polymerase chain reaction. We then validated the results in chickens infected with Salmonella. Notably, SPOP expression gradually decreased and then gradually increased in cells after challenging the bacterial substitute, indicating its potential involvement in the regulation of the immune response. Additionally, SPOP expression was negatively correlated with expression of interleukin 1β and interleukin-8 both in vivo and in vitro. More importantly, SPOP expression was related to immunoglobulin (Ig) A production and bacterial loads in chickens infected with Salmonella. These results indicate that SPOP could be a potential marker of the immune response in chickens.Salmonellosis is a zoonosis that is not only harmful to the health of poultry but also poses a threat to human health. Although many measures have been put in place to reduce morbidity, they have not provided satisfactory results. Therefore, it is necessary to clarify the immune mechanisms involved in improving the resistance of chickens against Salmonella. BTB (Broad-complex Tramtrack and Bric-a-brac) Speckle-type POZ (poxvirus and zinc finger) protein (SPOP) regulates protein expression by promoting substrate ubiquitination and degradation. The correlation between SPOP expression and the immune response has not been fully described. Therefore, the aim of this study was to clarify this relationship. In vitro, we stimulated chicken macrophage cells (HD11) with lipopolysaccharide, then analyzed the correlation between SPOP and IL1β or IL8 expression using quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we infected 7-days-old chickens with Salmonella Typhimurium, then analyzed the association between SPOP expression and the immune response, including IL1β and IL8 expression, IgA production, and bacterial loads. We found that SPOP may participate in the regulation of the immune response in macrophage cells. SPOP expression was negatively correlated with IL-1β and IL-8 expression both in vivo and in vitro. SPOP expression was also negatively related to bacterial loads and immunoglobulin (Ig) A production. These results indicate that SPOP may have important functions in the response to Salmonella infection.

MicroRNA gga-miR-200a-3p modulates immune response via MAPK signaling pathway in chicken afflicted with necrotic enteritis

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to host immune response as post-transcriptional regulation. The current study investigated the biological role of the chicken (Gallus gallus) microRNA-200a-3p (gga-miR-200a-3p), using 2 necrotic enteritis (NE) afflicted genetically disparate chicken lines, 6.3 and 7.2, as well as the mechanisms underlying the fundamental signaling pathways in chicken. The expression of gga-miR-200a-3p in the intestinal mucosal layer of NE-induced chickens, was found to be upregulated during NE infection in the disease-susceptible chicken line 7.2. To validate the target genes, we performed an overexpression analysis of gga-miR-200a-3p using chemically synthesized oligonucleotides identical to gga-miR-200a-3p, reporter gene analysis including luciferase reporter assay, and a dual fluorescence reporter assay in cultured HD11 chicken macrophage cell lines. Gga-miR-200a-3p was observed to be a direct transcriptional repressor of ZAK, MAP2K4, and TGFβ2 that are involved in mitogen-activated protein kinase (MAPK) pathway by targeting the 3′-UTR of their transcripts. Besides, gga-miR-200a-3p may indirectly affect the expression of protein kinases including p38 and ERK1/2 at both transcriptional and translational levels, suggesting that this miRNA may function as an important regulator of the MAPK signaling pathway. Proinflammatory cytokines consisting of IL-1β, IFN-γ, IL-12p40, IL-17A, and LITAF belonging to Th1 and Th17-type cytokines, were upregulated upon gga-miR-200a-3p overexpression. These findings have enhanced our knowledge of the immune function of gga-miR-200a-3p mediating the chicken immune response via regulation of the MAPK signaling pathway and indicate that this miRNA may serve as an important biomarker of diseases in domestic animals.

Chicken CATH-2 Increases Antigen Presentation Markers on Chicken Monocytes and Macrophages.

: BackgroundCathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells.ObjectiveThe aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37.MethodsChicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor C-type 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads.ResultsCATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, markers for antigen presentation, on primary chicken monocytes, whereas LL-37 did not. D-CATH-2 also increased the MRC1 and MHC-II expression if a chicken macrophage cell line (HD11 cells) was used. In addition, LPS-induced NO production by HD11 cells was inhibited by CATH-2 and D-CATH-2.ConclusionThese results are a clear indication that CATH-2 (and D-CATH-2) affect the activation state of monocytes and macrophages, which leads to optimization of the innate immune response and enhancement of the adaptive immune response.