miR-146a Maintains Immune Tolerance of Kupffer Cells and Facilitates Hepatitis B Virus Persistence in Mice.

Kupffer cells (KCs), the largest tissue-resident macrophage population in the body, play a central role in maintaining a delicate balance between immune tolerance and immunity in the liver. However, the underlying molecular mechanism remains elusive. In this study, we show that KCs express high levels of miR-146a, which is under control of the PU.1 transcription factor. miR-146a deficiency promoted KCs differentiation toward a proinflammatory phenotype; conversely, miR-146a overexpression suppressed this phenotypic differentiation. We found that hepatitis B virus (HBV) persistence or HBV surface Ag treatment significantly upregulated miR-146a expression and thereby impaired polarization of KCs toward a proinflammatory phenotype. Furthermore, in an HBV carrier mouse model, KCs depletion by clodronate liposomes dramatically promoted HBV clearance and enhanced an HBV-specific hepatic CD8+ T cell and CD4+ T cell response. Consistent with this finding, miR-146a knockout mice cleared HBV faster and elicited a stronger adaptive antiviral immunity than wild-type mice. In vivo IL-12 blockade promoted HBV persistence and tempered the HBV-specific CTL response in the liver of miR-146a knockout mice. Taken together, our results identified miR-146a as a critical intrinsic regulator of an immunosuppressive phenotype in KCs under inflammatory stimuli, which may be beneficial in maintenance of liver homeostasis under physiological condition. Meanwhile, during HBV infection, miR-146a contributed to viral persistence by inhibiting KCs proinflammatory polarization, highlighting its potential as a therapeutic target in HBV infection.

GPC3 and PEG10 peptides associated with placental gp96 elicit specific T cell immunity against hepatocellular carcinoma.

The placenta and tumors can exhibit a shared expression profile of proto-oncogenes. The basis of placenta-derived heat shock protein gp96, which induces prophylactic and therapeutic T cell responses against cancer including hepatocellular carcinoma (HCC), remains unknown. Here, we identified the associated long peptides from human placental gp96 using matrix-assisted laser desorption/ionization-time-of-flight and mass spectrometry and analyzed the achieved proteins through disease enrichment analysis. We found that placental gp96 binds to numerous peptides derived from 73 proteins that could be enriched in multiple cancer types. Epitope-harboring peptides from glypican 3 (GPC3) and paternally expressed gene 10 (PEG10) were the major antigens mediating anti-HCC T cell immunity. Molecular docking analysis showed that the GPC3- and PEG10-derived peptides, mainly obtained from the cytotrophoblast layer of the mature placenta, bind to the lumenal channel and client-bound domain of the gp96 dimer. Immunization with bone marrow-derived dendritic cells pulsed with recombinant gp96-GPC3 or recombinant gp96-PEG10 peptide complex induced specific T cell responses, and T cell transfusion led to pronounced growth inhibition of HCC tumors in nude mice. We demonstrated that the chaperone gp96 can capture antigenic peptides as an efficient approach for defining tumor rejection oncoantigens in the placenta and provide a basis for developing GPC3 and PEG10 peptide-based vaccines against HCC. This study provides insight into the underlying mechanism of the antitumor response mediated by embryonic antigens from fetal tissues, and this will incite more studies to identify potential tumor rejection antigens from placenta.

Broadly Protective CD8+ T Cell Immunity to Highly Conserved Epitopes Elicited by Heat Shock Protein gp96-Adjuvanted Influenza Monovalent Split Vaccine.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection.IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

Fragile Watermark in Medical Image Based on Prime Number Distribution Theory.

The research of medical image in the field of anti-counterfeiting and authentication plays a crucial role in the development of hospital digitization. In this paper, a new fragile medical watermarking scheme is proposed using the prime number distribution theorem, chaotic mapping, and Hash. Firstly, an approximate pixel set is constructed according to the distribution of prime numbers, and then the parity of the pixel value is re-granted with chaotic mapping, and Hash. The embedding and extraction of the watermark are done by using logical operations to adjust the pixel value according to the parity of the pixel value and the number of prime numbers contained in the pixel value. The experimental results demonstrate that the proposed scheme is imperceptible, efficient, and safe. Compared with the existing methods, the proposed scheme achieves the binding of medical image and patient information, as well as exhibits excellent detection and positioning capability, and will have good application prospects in medical image content authentication and tampering detection.

Cellular gp96 upregulates AFP expression by blocking NR5A2 SUMOylation and ubiquitination in hepatocellular carcinoma.

Alpha-fetoprotein (AFP) is the most widely used biomarker for the diagnosis of hepatocellular carcinoma (HCC). However, a substantial proportion of HCC patients have either normal or marginally increased AFP levels in serum, and the underlying mechanisms are not fully understood. In the present study, we provided in vitro and in vivo evidence that heat shock protein gp96 promoted AFP expression at the transcriptional level in HCC. NR5A2 was identified as a key transcription factor for the AFP gene, and its stability was enhanced by gp96. A further mechanistic study by co-immunoprecipitation, GST pull-down, and molecular docking showed gp96 and the SUMO E3 ligase RanBP2 competitively binding to NR5A2 at the sites spanning from aa 507 to aa 539. The binding of gp96 inhibited SUMOylation, ubiquitination, and subsequent degradation of NR5A2. In addition, clinical analysis of HCC patients indicated that gp96 expression in tumors was positively correlated with serum AFP levels. Therefore, our study uncovered a novel mechanism that gp96 regulates the stability of its client proteins by directly affecting their SUMOylation and ubiquitination. These findings will help in designing more accurate AFP-based HCC diagnosis and progression monitoring approaches.

Punicalagin promotes autophagic degradation of human papillomavirus E6 and E7 proteins in cervical cancer through the ROS-JNK-BCL2 pathway.

Punicalagin, which is derived from pomegranate peel, is reported to exert growth-inhibitory effects against various cancers. However, the underlying mechanisms have not been elucidated. Human papillomavirus (HPV), a major oncovirus, utilizes the host autophagic machinery to support its replication. Here, punicalagin markedly downregulated the levels of the major HPV oncoproteins E6 and E7 in cervical cancer cells through the autophagy-lysosome system. Additionally, punicalagin activated the reactive oxygen species (ROS)-JNK pathway and promoted the phosphorylation of BCL2, which led to the dissociation of BCL2 from BECN1 and the induction of autophagy. Treatment with autophagy and JNK inhibitors or ROS scavengers mitigated the punicalagin-induced degradation of E6 and E7. Moreover, the knockout of ATG5 using the clustered regularly interspaced palindrome repeat/Cas 9 system mitigated the punicalagin-induced downregulation of E6/E7. This indicated that punicalagin-induced degradation of E6 and E7 was dependent on autophagy. The results of in vivo studies demonstrated that punicalagin efficiently inhibits cervical cancer growth. In conclusion, this study elucidated a mechanism of punicalagin-induced autophagic degradation of E6 and E7. It will enable the future applications of punicalagin as a therapeutic for HPV-induced cervical cancer.

Plasma gp96 is a Novel Predictive Biomarker for Severe COVID-19.

Early and effective identification of severe coronavirus disease 2019 (COVID-19) may allow us to improve the outcomes of associated severe acute respiratory illness with fever and respiratory symptoms. This study analyzed plasma concentrations of heat shock protein gp96 in nonsevere (including mild and typical) and severe (including severe and critical) patients with COVID-19 to evaluate its potential as a predictive and prognostic biomarker for disease severity. Plasma gp96 levels that were positively correlated with interleukin-6 (IL-6) levels were significantly elevated in COVID-19 patients admitted to the hospital but not in non-COVID-19 patients with less severe respiratory impairment. Meanwhile, significantly higher gp96 levels were observed in severe than nonsevere patients. Moreover, the continuous decline of plasma gp96 levels predicted disease remission and recovery, whereas its persistently high levels indicated poor prognosis in COVID-19 patients during hospitalization. Finally, monocytes were identified as the major IL-6 producers under exogenous gp96 stimulation. Our results demonstrate that plasma gp96 may be a useful predictive and prognostic biomarker for disease severity and outcome of COVID-19. IMPORTANCE Early and effective identification of severe COVID-19 may allow us to improve the outcomes of associated severe acute respiratory illness with fever and respiratory symptoms. Some heat shock proteins (Hsps) are released during oxidative stress, cytotoxic injury, and viral infection and behave as danger-associated molecular patterns (DAMPs). This study analyzed plasma concentrations of Hsp gp96 in nonsevere and severe patients with COVID-19. Significantly higher plasma gp96 levels were observed in severe than those in nonsevere patients, and its persistently high levels indicated poor prognosis in COVID-19 patients. The results demonstrate that plasma gp96 may be a useful predictive and prognostic biomarker for disease severity and outcome of COVID-19.

Induction of Foxp3 and activation of Tregs by HSP gp96 for treatment of autoimmune diseases.

Upregulation and stabilization of Foxp3 expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, gp96 immunization showed obvious therapeutic effects in a Lyn -/- mouse model of systemic lupus erythematosus. Moreover, gp96 alleviated the initiation and progression of MOG-induced experimental autoimmune encephalomyelitis. Immunization of gp96 increased Treg frequency, expansion, and suppressive function. Gene expression profiling identified the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody-induced and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases.

Biological characteristics and genome analysis of a novel phage vB_KpnP_IME279 infecting Klebsiella pneumoniae.

Klebsiella pneumoniae (family Enterobacteriaceae) is a gram-negative bacterium that has strong pathogenicity to humans and can cause sepsis, pneumonia, and urinary tract infection. In recent years, the unreasonable use of antibacterial drugs has led to an increase in drug-resistant strains of K. pneumoniae, a serious threat to public health. Bacteriophages, viruses that infect bacteria, are ubiquitous in the natural environment. They are considered to be the most promising substitute for antibiotics because of their high specificity, high efficiency, high safety, low cost, and short development cycle. In this study, a novel phage designated vB_KpnP_IME279 was successfully isolated from hospital sewage using a multidrug-resistant strain of K. pneumoniae as an indicator. A one-step growth curve showed that vB_KpnP_IME279 has a burst size of 140 plaque-forming units/cell and a latent period of 20 min at its optimal multiplicity of infection (MOI = 0.1). Phage vB_KpnP_IME279 survives in a wide pH range between 3 and 11 and is stable at temperatures ranging from 40 to 60 °C. Ten of the 20 strains of K. pneumoniae including the host bacteria were lysed by the phage vB_KpnP_IME279, and the multilocus sequence typing and wzi typing of the 10 strains were ST11, ST37, ST375, wzi209, wzi52, and wzi72, respectively. The genome of vB_KpnP_IME279 is 42,518 bp long with a G + C content of 59.3%. Electron microscopic observation showed that the phage belongs to the family Podoviridae. BLASTN alignment showed that the genome of the phage has low similarity with currently known phages. The evolutionary relationship between phage vB_KpnP_IME279 and other Podoviridae was analyzed using a phylogenetic tree based on sequences of phage major capsid protein and indicates that the phage vB_KpnP_IME279 belongs to the Podoviridae subfamily. These data enhance understanding of K. pneumoniae phages and will help in development of treatments for multidrug-resistant bacteria using phages.