Rong Li's research while affiliated with Southern Medical University and other places

Well-orchestrated transcriptional programs in intestinal epithelial cells (IECs) are essential for maintenance of optimal mucosal barrier functions, whereas the contribution of elongation-related mechanisms to barrier function remains unknown. Here, a combination of genetic and genomic approaches defined a critical role of IEC-intrinsic negative elongation factor (NELF) complex in maintenance of epithelial homeostasis. By direct occupancy at endogenous gene loci, NELF sustained expression of a subset of genes related to junctional integrity. As a result, epithelial NELF deficiency results in subdued levels of these junction-related genes and excessive IEC necroptosis in vivo secondary to commensal microbial invasion. In a colitis model, NELF-deficient mice exhibited severely impaired barrier integrity characterized by increased intestinal permeability and significantly exacerbated intestinal inflammation with lethal consequences. Our findings reveal the protective function of the NELF complex against intestinal damage and inflammation and suggest that elongation represents a biologically important step in defining IEC transcriptome.

CD4 protein is a single chain transmembrane glycoprotein and has a broad functionality beyond cell-mediated immunity. In this study, we cloned the full-length coding sequence (CDS) of duck CD4 (duCD4) and analyzed its sequence and structure, and expression levels in several tissues. It consists of 1449 nucleotides and encodes a 482 amino acid protein. The putative protein of duCD4 consisted of an N-terminal signal peptide, three immunoglobulins and one immunoglobulins-like domain in its central, one terminal transmembrane region, and a C-terminal domain of the CD4 T cell receptor. The duCD4 also has the typical signature “CXC” of CD4s. The multiple sequence alignment suggests duCD4 has four potential N-glycosylation sites and the phylogenetic analysis suggests duCD4 shares greater similarity with avian than other vertebrates. Quantitative real-time PCR analysis showed that duCD4 mRNA transcripts are widely distributed in the healthy cherry valley duck, and the highest level in the thymus. During the virus infection, the obvious change of duCD4 expression was observed in the spleen, lung and brain, which suggesting that duCD4 could be involved in the host's immune response to multiple types of viruses. Our research studied the characterization, tissue distribution, and antiviral immune responses of duCD4.

Abstract High-mobility group box 1 protein (HMGB1) shows endogenous damage-associated molecular patterns (DAMPs) and is also an early warning protein that activates the body’s innate immune system. Here, the full-length coding sequence of HMGB1 was cloned from the spleen of Cherry Valley duck and analyzed. We find that duck HMGB1(duHMGB1) is mostly located in the nucleus of duck embryo fibroblast (DEF) cells under normal conditions but released into the cytoplasm after lipopolysaccharide (LPS) stimulation. Knocking-down or overexpressing duHMGB1 had no effect on the baseline apoptosis rate of DEF cells. However, overexpression increased weakly apoptosis after LPS activation. In addition, overexpression strongly activated the IFN-I/IRF7 signaling pathway in DEF cells and significantly increased the transcriptional level of numerous pattern recognition receptors (PRRs), pro-inflammatory cytokines (IL-6, TNF-α), IFNs and antiviral molecules (OAS, PKR, Mx) starting from 48 h post-transfection. Overexpression of duHMGB1 strongly impacted duck virus replication, either by inhibiting it from the first stage of infection for novel duck reovirus (NDRV) and at late stage for duck Tembusu virus (DTMUV) or duck plague virus (DPV), or promoting replication at early stage for DTMUV and DPV infection. Importantly, data from duHMGB1 overexpression and knockdown experiments, time-dependent DEF cells transcriptional immune responses suggest that duHMGB1 and RIG-I receptor might cooperate to promote the expression of antiviral proteins after NDRV infection, as a potential mechanism of duHMGB1-mediated antiviral activity.

A strain of Bacillus subtilis (B. subtilis) BYS2 was previously isolated from Mount Tai, which is located in Tai’an City in the Shandong Province of China. The strain was then stored in the Environmental Microbiology Laboratory at Shandong Agricultural University. To evaluate the effect of the bacterium preparation in broiler production, we fed the bacterium (106 CFU/g) to 1-day-old broilers and continued this feeding for 6 weeks to analyze its effect on growth and immune performance. We found that the average weight of the bacterium-fed group increased by 17.19% at weeks 5 compared to the control group (P < 0.05). The height of the villi in the duodenum and jejunum and the ratio of villi to crypt were significantly increased in the bacterium-fed group at weeks 5 (P < 0.05). Also, the IgG in the serum of broilers in the experimental group increased by 31.60% (P < 0.05) and IgM 30.52% (P < 0.05) compared with those in the control group. The expressions of the major pattern recognition receptors (PRRs), antiviral proteins, pro-inflammatory cytokines, and β-defensins were significantly higher than those in the control group (P < 0.05). Meanwhile, the bursa immune organ indices of broilers in the experimental group were significantly higher than those in the control group (P < 0.05). Also, after 5 weeks of continuous feeding, when infected with Escherichia coli (E. coli) O1K1 and Newcastle disease virus (NDV) F48E8, the content of bacteria and virus in tissues and organs of the experimental group decreased significantly, and the survival rate of infected chickens increased by 31.1% and 17.7%, respectively (P < 0.05). These results show that the anti-infective B. subtilis BYS2 could, to some extent, replace antibiotics to promote growth, improve innate immunity, and enhance disease resistance in broilers.

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation. Macrophage activation is integral to innate immunity and inflammation, and involves transcriptome remodeling leading to the rapid upregulation of pro- and anti-inflammatory effector genes. Here the authors show that the negative elongation factor (NELF) complex controls the transcription of anti-inflammatory genes through Pol II pause release.

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report widespread promoter-proximal RNA polymerase II pausing in resting macrophages, marked by broad co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

Background: Although several studies have revealed that the sensitivity of ducklings to duck Tembusu virus (DTMUV) was related to age, however, DTMUV was originally isolated from egg-laying ducks, and the ovary was the target organ of this virus. Cherry Valley breeding ducks aged 15- and 55-week-old (they are reserve breeding ducks and the normal egg-laying breeding ducks, respectively) were infected with DTMUV, using intramuscular injection, to study the effect of age-related difference on the pathogenicity of DTMUV in breeding ducks. Results: Examinations of clinical symptoms, gross and microscopic lesions, viral loads, cytokines and serum neutralizing antibodies were performed. Results showed that obvious clinical symptoms, such as depression, ruffled feathers, ataxia and egg-laying drop were observed in the 55-week-old laying ducks, with five ducks dying at 5-7 days post infection (dpi). The 15-week-old ducks showed slight symptoms during infection. Gross lesions were severe and characterized by the congestion, hemorrhage and swelling of some organs in the 55-week-old ducks, including the hemorrhage of endocardium, hepatomegaly, splenomegaly, oviduct hemorrhage, hyperemia and deformation of the ovary. Mild endocardial hemorrhage and hepatosplenomegaly were observed in the 15-week-old ducks. Similarly, there was a significant difference in microscopic lesions between the two groups. The older ducks displayed severe microscopic lesions, specifically in the hemorrhage, interstitial inflammatory cell infiltration of the endocardium, typical viral encephalitis and hemorrhage in the ovary. But on the whole, the 15-week-old ducks showed milder lesions. Viral loads in tissues of the older group were significantly higher than those of the younger group. The levels of interferon (IFN)-γ, interleukin (IL)-2 and neutralizing antibody in the 15-week-old ducks were higher than in the 55-week-old ducks at the early stage of the DTMUV infection, suggesting the immune response in the younger ducks to DTMUV was stronger than in the older ducks. Conclusions: These results demonstrated that age-related differences in susceptibility to DTMUV in breeding ducks was significant, with 55-week-old egg-laying ducks being more susceptible to DTMUV than 15-week-old reserve breeding ducks.

Galectin-1, as a typical animal galactose-binding protein, it is found on the cell surface and in the extracellular matrix. Cloning the full-length coding sequence of galectin-1 from the spleens of Cherry Valley ducks revealed that the coding sequence of duck galectin-1 (duGal-1) comprises 405 bp, encoding 134 amino acids. Homologic analysis revealed its amino acid sequence is most identical to that of Anas platyrhynchos (98.8%) followed by Gallus gallus. Quantitative real-time PCR analysis indicated that duGal-1 mRNA is broadly expressed in healthy Cherry Valley duck tissues, primarily in the heart and trachea but minimally in the lung and skin. Meanwhile, the duGal-1 expression is slightly upregulated in the infected liver and spleen. Furthermore, the expression levels of ISGs (Mx, PKR, OAS) and some cytokines such as IFN-α, IL-1β, IL-2, are up-regulated to varying degrees after overexpression the duGal-1, In contrast, Knockdown of duGal-1 found that the expression levels of ISGs and some inflammatory cytokines were down-regulated. Antiviral assay showed that duGal-1 could inhibit viral replications early during infection. This is the first study of the cloning, tissue distribution, and antiviral immune responses of duGal-1, and findings imply it is involved in the early stages of antiviral innate immune responses to duck plague virus infections in ducks.

The polymerase acidic (PA) protein is the third subunit of the influenza A virus polymerase. In recent years, studies have shown that PA plays an important role in overcoming the host species barrier and host adaptation of the avian influenza virus (AIV). The objective of this study was to elucidate the role of the PA subunit on the replication and airborne transmission of the H9N2 subtype AIV. By reverse genetics, a reassortant rSD01-PA was derived from the H9N2 subtype AIV A/Chicken/Shandong/01/2008 (SD01) by introducing the PA gene from the pandemic influenza A H1N1 virus A/swine/Shandong/07/2011 (SD07). Specific pathogen-free (SPF) chickens and guinea pigs were selected as the animal models for replication and aerosol transmission studies. Results show that rSD01-PA lost the ability of airborne transmission among SPF chickens because of the single substitution of the PA gene. However, rSD01-PA could infect guinea pigs through direct contact, while the parental strain SD01 could not, even though the infection of rSD01-PA could not be achieved through aerosol. In summary, our results indicate that the protein encoded by the PA gene plays a key role in replication and airborne transmission of the H9N2 subtype AIV.

The nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that is involved in host innate immunity and located in the cytoplasm. In the present study, the full-length cDNA of Cherry Valley duck NLRP3 (duNLRP3) (2,805 bp encode 935 amino acids) was firstly cloned from the spleen of healthy Cherry Valley ducks, and the phylogenetic tree indicated that the duNLRP3 has the closest relationship with Anas platyrhynchos in the bird branch. According to quantitative real-time PCR analysis, the duNLRP3 mRNA has a broad expression spectrum in healthy Cherry Valley duck tissues, and the highest expression is in the pancreas. There was significant up-regulation of duNLRP3 mRNA expression in the liver and down-regulation in the spleen after infection with avian pathogenic Escherichia coli (APEC) O1K1, especially at 3 days after the infection. Ducks hatched from NLRP3-lentiviral vector-injected eggs had significantly higher duNLRP3 mRNA expression in the liver, spleen, brain, and cecum, which are tissues usually with lower background expression. The mRNA expression levels of inflammatory cytokines IL-1β, IL-18, and TNF-α significantly increased after the APEC infection in those tissues. The bacterial content in the liver and spleen decreased significantly compared with the NC-lentiviral vector-injected ducks. In addition, in the duck embryo fibroblasts, both of the overexpression and knockdown of duNLRP3 can trigger the innate immune response during the E. coli infection. Specifically, overexpression induced antibacterial activation, and knockdown reduced the antibacterial activity of the host cells. The IL-1β, IL-18, and TNF-α mRNA expressions showed up-regulation or down-regulation. The results demonstrate that duNLRP3 has a certain antibacterial activity during E. coli infection. These findings also contribute to better understanding the importance of duNLRP3 in regulating the inflammatory response and the innate immune system of ducks.